Optimization of process variables by central composite design for the immobilization of urease enzyme on functionalized gold nanoparticles for various applications.

PubMed

Talat, Mahe; Singh, Ashwani Kumar; Srivastava, O N

2011-08-01

In the present study, enzyme urease has been immobilized on amine-functionalized gold nanoparticles (AuNPs). AuNPs were synthesized using natural precursor, i.e., clove extract and amine functionalized through 0.004 M L: -cysteine. Enzyme (urease) was extracted and purified from the vegetable waste, i.e., seeds of pumpkin to apparent homogeneity (sp. activity 353 U/mg protein). FTIR spectroscopy and transmission electron microscopy was used to characterize the immobilized enzyme. The immobilized enzyme exhibited enhanced activity as compared with the enzyme in the solution, especially, at lower enzyme concentration. Based on the evaluation of activity assay of the immobilized enzyme, it was found that the immobilized enzyme was quite stable for about a month and could successfully be used even after eight cycles having enzyme activity of about 47%. In addition to this central composite design (CCD) with the help of MINITAB version 15 Software was utilized to optimize the process variables viz., pH and temperature affecting the enzyme activity upon immobilization on AuNPs. The results predicted by the design were found in good agreement (R2 = 96.38%) with the experimental results indicating the applicability of proposed model. The multiple regression analysis and ANOVA showed the individual and cumulative effect of pH and temperature on enzyme activity indicating that the activity increased with the increase of pH up to 7.5 and temperature 75 °C. The effects of each variables represented by main effect plot, 3D surface plot, isoresponse contour plot and optimized plot were helpful in predicting results by performing a limited set of experiments.

Genetically Engineered Materials for Biofuels Production

NASA Astrophysics Data System (ADS)

Raab, Michael

2012-02-01

Agrivida, Inc., is an agricultural biotechnology company developing industrial crop feedstocks for the fuel and chemical industries. Agrivida's crops have improved processing traits that enable efficient, low cost conversion of the crops' cellulosic components into fermentable sugars. Currently, pretreatment and enzymatic conversion of the major cell wall components, cellulose and hemicellulose, into fermentable sugars is the most expensive processing step that prevents widespread adoption of biomass in biofuels processes. To lower production costs we are consolidating pretreatment and enzyme production within the crop. In this strategy, transgenic plants express engineered cell wall degrading enzymes in an inactive form, which can be reactivated after harvest. We have engineered protein elements that disrupt enzyme activity during normal plant growth. Upon exposure to specific processing conditions, the engineered enzymes are converted into their active forms. This mechanism significantly lowers pretreatment costs and enzyme loadings (>75% reduction) below those currently available to the industry.

Enzymes in Fish and Seafood Processing

PubMed Central

Fernandes, Pedro

2016-01-01

Enzymes have been used for the production and processing of fish and seafood for several centuries in an empirical manner. In recent decades, a growing trend toward a rational and controlled application of enzymes for such goals has emerged. Underlying such pattern are, among others, the increasingly wider array of enzyme activities and enzyme sources, improved enzyme formulations, and enhanced requirements for cost-effective and environmentally friendly processes. The better use of enzyme action in fish- and seafood-related application has had a significant impact on fish-related industry. Thus, new products have surfaced, product quality has improved, more sustainable processes have been developed, and innovative and reliable analytical techniques have been implemented. Recent development in these fields are presented and discussed, and prospective developments are suggested. PMID:27458583

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

PubMed Central

Santiago, Margarita; Ramírez-Sarmiento, César A.; Zamora, Ricardo A.; Parra, Loreto P.

2016-01-01

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications. PMID:27667987

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes.

PubMed

Santiago, Margarita; Ramírez-Sarmiento, César A; Zamora, Ricardo A; Parra, Loreto P

2016-01-01

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis.

PubMed

Florencio, Camila; Cunha, Fernanda M; Badino, Alberto C; Farinas, Cristiane S; Ximenes, Eduardo; Ladisch, Michael R

2016-08-01

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei. Copyright © 2016 Elsevier Inc. All rights reserved.

Measuring potential denitrification enzyme activity rates using the membrane inlet mass spectrometer

EPA Science Inventory

The denitrification enzyme activity (DEA) assay, provides a quantitative assessment of the multi enzyme, biological process of reactive nitrogen removal via the reduction of N03 to N2. Measured in soil, usually under non limiting carbon and nitrate concentrations, this short ter...

Recycling cellulases for cellulosic ethanol production at industrial relevant conditions: potential and temperature dependency at high solid processes.

PubMed

Lindedam, Jane; Haven, Mai Østergaard; Chylenski, Piotr; Jørgensen, Henning; Felby, Claus

2013-11-01

Different versions of two commercial cellulases were tested for their recyclability of enzymatic activity at high dry matter processes (12% or 25% DM). Recyclability was assessed by measuring remaining enzyme activity in fermentation broth and the ability of enzymes to hydrolyse fresh, pretreated wheat straw. Industrial conditions were used to study the impact of hydrolysis temperature (40 or 50°C) and residence time on recyclability. Enzyme recycling at 12% DM indicated that hydrolysis at 50°C, though ideal for ethanol yield, should be kept short or carried out at lower temperature to preserve enzymatic activity. Best results for enzyme recycling at 25% DM was 59% and 41% of original enzyme load for a Celluclast:Novozyme188 mixture and a modern cellulase preparation, respectively. However, issues with stability of enzymes and their strong adsorption to residual solids still pose a challenge for applicable methods in enzyme recycling. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enzyme leaps fuel antichemotaxis

PubMed Central

Jee, Ah-Young; Dutta, Sandipan; Cho, Yoon-Kyoung

2018-01-01

There is mounting evidence that enzyme diffusivity is enhanced when the enzyme is catalytically active. Here, using superresolution microscopy [stimulated emission-depletion fluorescence correlation spectroscopy (STED-FCS)], we show that active enzymes migrate spontaneously in the direction of lower substrate concentration (“antichemotaxis”) by a process analogous to the run-and-tumble foraging strategy of swimming microorganisms and our theory quantifies the mechanism. The two enzymes studied, urease and acetylcholinesterase, display two families of transit times through subdiffraction-sized focus spots, a diffusive mode and a ballistic mode, and the latter transit time is close to the inverse rate of catalytic turnover. This biochemical information-processing algorithm may be useful to design synthetic self-propelled swimmers and nanoparticles relevant to active materials. Executed by molecules lacking the decision-making circuitry of microorganisms, antichemotaxis by this run-and-tumble process offers the biological function to homogenize product concentration, which could be significant in situations when the reactant concentration varies from spot to spot. PMID:29255047

Encapsulation of alpha-amylase into starch-based biomaterials: an enzymatic approach to tailor their degradation rate.

PubMed

Azevedo, Helena S; Reis, Rui L

2009-10-01

This paper reports the effect of alpha-amylase encapsulation on the degradation rate of a starch-based biomaterial. The encapsulation method consisted in mixing a thermostable alpha-amylase with a blend of corn starch and polycaprolactone (SPCL), which were processed by compression moulding to produce circular disks. The presence of water was avoided to keep the water activity low and consequently to minimize the enzyme activity during the encapsulation process. No degradation of the starch matrix occurred during processing and storage (the encapsulated enzyme remained inactive due to the absence of water), since no significant amount of reducing sugars was detected in solution. After the encapsulation process, the released enzyme activity from the SPCL disks after 28days was found to be 40% comparatively to the free enzyme (unprocessed). Degradation studies on SPCL disks, with alpha-amylase encapsulated or free in solution, showed no significant differences on the degradation behaviour between both conditions. This indicates that alpha-amylase enzyme was successfully encapsulated with almost full retention of its enzymatic activity and the encapsulation of alpha-amylase clearly accelerates the degradation rate of the SPCL disks, when compared with the enzyme-free disks. The results obtained in this work show that degradation kinetics of the starch polymer can be controlled by the amount of encapsulated alpha-amylase into the matrix.

[Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

PubMed

Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

2015-09-01

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

Transformation of cyclodextrin glucanotransferase (CGTase) from aqueous suspension to fine solid particles via electrospraying.

PubMed

Saallah, Suryani; Naim, M Nazli; Mokhtar, Mohd Noriznan; Abu Bakar, Noor Fitrah; Gen, Masao; Lenggoro, I Wuled

2014-10-01

In this study, the potential of electrohydrodynamic atomization or electrospraying to produce nanometer-order CGTase particles from aqueous suspension was demonstrated. CGTase enzyme was prepared in acetate buffer solution (1% v/v), followed by electrospraying in stable Taylor cone-jet mode. The deposits were collected on aluminium foil (collector) at variable distances from the tip of spraying needle, ranging from 10 to 25 cm. The Coulomb fission that occurs during electrospraying process successfully transformed the enzyme to the solid state without any functional group deterioration. The functional group verification was conducted by FTIR analysis. Comparison between the deposit and the as-received enzyme in dry state indicates almost identical spectra. By increasing the distance of the collector from the needle tip, the average particle size of the solidified enzyme was reduced from 200±117 nm to 75±34 nm. The average particle sizes produced from the droplet fission were in agreement with the scaling law models. Enzyme activity analysis showed that the enzyme retained its initial activity after the electrospraying process. The enzyme particles collected at the longest distance (25 cm) demonstrated the highest enzyme activity, which indicates that the activity was controlled by the enzyme particle size. Copyright © 2014 Elsevier Inc. All rights reserved.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE PAGES

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; ...

2015-12-18

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A; Zhao, Lishan; Cayouette, Michelle H

2015-11-04

The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

2015-09-08

The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

PubMed Central

Ishii, Satoshi; Chang, Hui-Hwa; Kawasaki, Kunito; Yasuda, Kayo; Wu, Hui-Li; Garman, Scott C.; Fan, Jian-Qiang

2007-01-01

Fabry disease is a lysosomal storage disorder caused by the deficiency of α-Gal A (α-galactosidase A) activity. In order to understand the molecular mechanism underlying α-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal Km and Vmax values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) α-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q α-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant α-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant α-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations. PMID:17555407

Effects of dietary lead acetate on hepatic detoxication enzyme activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagstaff, D.J.

1979-12-01

Lead-containing compounds usually inhibit enzymic and metabolic processes. This inhibition is presumed to be the mechanism of intoxication by these compounds. Inhibition of detoxication activities of liver microsomal enzymes could be particularly detrimental because the toxicity of many different substances would be increased. Exposure of experimental animals to lead compounds in several studies has been associated with depressed activity of hepatic microsomal enzymes, reduced levels of hepatic cytochrome P-450, reduced levels of hepatic microsomal protein, and prolonged hexobarbital sleep times. The present report contains observations that under certain experimental conditions there is stimulated hepatic meicrosomal enzyme activity in rats fedmore » lead acetate.« less

Quality-related enzymes in fruit and vegetable products: effects of novel food processing technologies, part 1: high-pressure processing.

PubMed

Terefe, Netsanet Shiferaw; Buckow, Roman; Versteeg, Cornelis

2014-01-01

The activity of endogenous deteriorative enzymes together with microbial growth (with associated enzymatic activity) and/or other non-enzymatic (usually oxidative) reactions considerably shorten the shelf life of fruits and vegetable products. Thermal processing is commonly used by the food industry for enzyme and microbial inactivation and is generally effective in this regard. However, thermal processing may cause undesirable changes in product's sensory as well as nutritional attributes. Over the last 20 years, there has been a great deal of interest shown by both the food industry and academia in exploring alternative food processing technologies that use minimal heat and/or preservatives. One of the technologies that have been investigated in this context is high-pressure processing (HPP). This review deals with HPP focusing on its effectiveness for controlling quality-degrading enzymes in horticultural products. The scientific literature on the effects of HPP on plant enzymes, mechanism of action, and intrinsic and extrinsic factors that influence the effectiveness of HPP for controlling plant enzymes is critically reviewed. HPP inactivates vegetative microbial cells at ambient temperature conditions, resulting in a very high retention of the nutritional and sensory characteristics of the fresh product. Enzymes such as polyphenol oxidase (PPO), peroxidase (POD), and pectin methylesterase (PME) are highly resistant to HPP and are at most partially inactivated under commercially feasible conditions, although their sensitivity towards pressure depends on their origin as well as their environment. Polygalacturonase (PG) and lipoxygenase (LOX) on the other hand are relatively more pressure sensitive and can be substantially inactivated by HPP at commercially feasible conditions. The retention and activation of enzymes such as PME by HPP can be beneficially used for improving the texture and other quality attributes of processed horticultural products as well as for creating novel structures that are not feasible with thermal processing.

Biocatalytic material comprising multilayer enzyme coated fiber

DOEpatents

Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

2009-11-03

The present invention relates generally to high stability, high activity biocatalytic materials and processes for using the same. The materials comprise enzyme aggregate coatings having high biocatalytic activity and stability useful in heterogeneous environment. These new materials provide a new biocatalytic immobilized enzyme system with applications in bioconversion, bioremediation, biosensors, and biofuel cells.

Silicate enhanced enzymatic dehairing: a new lime-sulfide-free process for cowhides.

PubMed

Saravanabhavan, Subramani; Thanikaivelan, Palanisamy; Rao, Jonnalagadda Raghava; Nair, Balachandran Unni

2005-05-15

A conventional dehairing process with sodium sulfide and lime is a major source of the pollution from the tanning industry. In other words, conventional dehairing processes degrade the hair to the extent that it cannot be recovered; thus, these processes become a major contributor to wastewater pollution. In this study, an attempt has been made to develop a lime and sulfide-free dehairing process using a commercial enzyme formulation with the activation of a silicate salt. A dip and pile method of application has been standardized. The amount of enzyme and sodium metasilicate has also been optimized based on complete removal of hair. Enhancement of enzyme activity by the addition of silicate has been demonstrated through activity measurements. Hair removal is found to be complete using scanning electron microscope analysis. Strength and bulk properties of the experimental leathers are comparable to that of control leathers. The process enjoys a significant reduction in chemical oxygen demand (COD) and total solids (TS) by 53 and 26%, respectively. More importantly, the application of enzyme for dehairing results in an 8% area increase in the final leather. Also, the process is proven to be techno-economically feasible.

An active recombinant cocoonase from the silkworm Bombyx mori: bleaching, degumming and sericin degrading activities.

PubMed

Unajak, Sasimanas; Aroonluke, Suradet; Promboon, Amornrat

2015-04-01

Cocoonase is a serine protease produced by silk moths and used for softening the cocoons so that they can escape. Degumming is one of the important steps in silk processing. This research aimed to produce an active recombinant Bombyx mori cocoonase (BmCoc) for the silk degumming process. A recombinant BmCoc was successfully expressed in a Pichia pastoris system. The purified enzyme showed specific activity of 227 U mg(-1) protein, 2.4-fold purification, 95% yield and a molecular weight of 26 kDa. The enzyme exhibited optimal temperature at 40 °C and optimal pH at 8, and showed thermal stability at 25-45 °C and pH stability at 5-9. The recombinant enzyme exhibited sericin degumming ability and color bleaching characteristics, and did not affect the fibroin fiber. The enzyme also degraded sericin substrate with a product size about 30-70 kDa. In this study, we successfully produced the active recombinant BmCoc in P. pastoris with promising functions for the Thai silk degumming process, which includes degumming, sericin degrading and color bleaching activities. Our data clearly indicated that the recombinant enzyme had proteolytic activity on sericin but not on fibroin proteins. The recombinant BmCoc has proven to be suitable for numerous applications in the silk industry. © 2014 Society of Chemical Industry.

Technology Prospecting on Enzymes: Application, Marketing and Engineering

PubMed Central

Li, Shuang; Yang, Xiaofeng; Yang, Shuai; Zhu, Muzi; Wang, Xiaoning

2012-01-01

Enzymes are protein molecules functioning as specialized catalysts for chemical reactions. They have contributed greatly to the traditional and modern chemical industry by improving existing processes. In this article, we first give a survey of representative industrial applications of enzymes, focusing on the technical applications, feed industry, food processing and cosmetic products. The recent important developments and applications of enzymes in industry are reviewed. Then large efforts are dedicated to the worldwide enzyme market from the demand and production perspectives. Special attention is laid on the Chinese enzyme market. Although enzyme applications are being developed in full swing, breakthroughs are needed to overcome their weaknesses in maintaining activities during the catalytic processes. Strategies of metagomic analysis, cell surface display technology and cell-free system might give valuable solutions in novel enzyme exploiting and enzyme engineering. PMID:24688658

Integrating Proteomics and Enzyme Kinetics Reveals Tissue-Specific Types of the Glycolytic and Gluconeogenic Pathways.

PubMed

Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

2015-08-07

Glycolysis is the core metabolic pathway supplying energy to cells. Whereas the vast majority of studies focus on specific aspects of the process, global analyses characterizing simultaneously all enzymes involved in the process are scarce. Here, we demonstrate that quantitative label- and standard-free proteomics allows accurate determination of titers of metabolic enzymes and enables simultaneous measurements of titers and maximal enzymatic activities (Amax) of all glycolytic enzymes and the gluconeogenic fructose 1,6-bisphosphatase in mouse brain, liver and muscle. Despite occurrence of tissue-specific isoenzymes bearing different kinetic properties, the enzyme titers often correlated well with the Amax values. To provide a more general picture of energy metabolism, we analyzed titers of the enzymes in additional 7 mouse organs and in human cells. Across the analyzed samples, we identified two basic profiles: a "fast glucose uptake" one in brain and heart, and a "gluconeogenic rich" one occurring in liver. In skeletal muscles and other organs, we found intermediate profiles. Obtained data highlighted the glucose-flux-limiting role of hexokinase which activity was always 10- to 100-fold lower than the average activity of all other glycolytic enzymes. A parallel determination of enzyme titers and maximal enzymatic activities allowed determination of kcat values without enzyme purification. Results of our in-depth proteomic analysis of the mouse organs did not support the concepts of regulation of glycolysis by lysine acetylation.

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

PubMed Central

Jackson, Colin R.; Tyler, Heather L.; Millar, Justin J.

2013-01-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample. PMID:24121617

Determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays.

PubMed

Jackson, Colin R; Tyler, Heather L; Millar, Justin J

2013-10-01

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.

PubMed

Staab, J F; Ginkel, D L; Rosenberg, G B; Munford, R S

1994-09-23

Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.

Neofunctionalization of a duplicate hatching enzyme gene during the evolution of teleost fishes.

PubMed

Sano, Kaori; Kawaguchi, Mari; Watanabe, Satoshi; Yasumasu, Shigeki

2014-10-19

Duplication and subsequent neofunctionalization of the teleostean hatching enzyme gene occurred in the common ancestor of Euteleostei and Otocephala, producing two genes belonging to different phylogenetic clades (clade I and II). In euteleosts, the clade I enzyme inherited the activity of the ancestral enzyme of swelling the egg envelope by cleavage of the N-terminal region of egg envelope proteins. The clade II enzyme gained two specific cleavage sites, N-ZPd and mid-ZPd but lost the ancestral activity. Thus, euteleostean clade II enzymes assumed a new function; solubilization of the egg envelope by the cooperative action with clade I enzyme. However, in Otocephala, the clade II gene was lost during evolution. Consequently, in a late group of Otocephala, only the clade I enzyme is present to swell the egg envelope. We evaluated the egg envelope digestion properties of clade I and II enzymes in Gonorynchiformes, an early diverging group of Otocephala, using milkfish, and compared their digestion with those of other fishes. Finally, we propose a hypothesis of the neofunctionalization process. The milkfish clade II enzyme cleaved N-ZPd but not mid-ZPd, and did not cause solubilization of the egg envelope. We conclude that neofunctionalization is incomplete in the otocephalan clade II enzymes. Comparison of clade I and clade II enzyme characteristics implies that the specificity of the clade II enzymes gradually changed during evolution after the duplication event, and that a change in substrate was required for the addition of the mid-ZPd site and loss of activity at the N-terminal region. We infer the process of neofunctionalization of the clade II enzyme after duplication of the gene. The ancestral clade II gene gained N-ZPd cleavage activity in the common ancestral lineage of the Euteleostei and Otocephala. Subsequently, acquisition of cleavage activity at the mid-ZPd site and loss of cleavage activity in the N-terminal region occurred during the evolution of Euteleostei, but not of Otocephala. The clade II enzyme provides an example of the development of a neofunctional gene for which the substrate, the egg envelope protein, has adapted to a gradual change in the specificity of the corresponding enzyme.

Processing of poultry feathers by alkaline keratin hydrolyzing enzyme from Serratia sp. HPC 1383.

PubMed

Khardenavis, Anshuman A; Kapley, Atya; Purohit, Hemant J

2009-04-01

The present study describes the production and characterization of a feather hydrolyzing enzyme by Serratia sp. HPC 1383 isolated from tannery sludge, which was identified by the ability to form clear zones around colonies on milk agar plates. The proteolytic activity was expressed in terms of the micromoles of tyrosine released from substrate casein per ml per min (U/mL min). Induction of the inoculum with protein was essential to stimulate higher activity of the enzyme, with 0.03% feathermeal in the inoculum resulting in increased enzyme activity (45U/mL) that further increased to 90U/mL when 3d old inoculum was used. The highest enzyme activity, 130U/mL, was observed in the presence of 0.2% yeast extract. The optimum assay temperature and pH for the enzyme were found to be 60 degrees C and 10.0, respectively. The enzyme had a half-life of 10min at 60 degrees C, which improved slightly to 18min in presence of 1mM Ca(2+). Inhibition of the enzyme by phenylmethyl sulfonyl fluoride (PMSF) indicated that the enzyme was a serine protease. The enzyme was also partially inhibited (39%) by the reducing agent beta-mercaptoethanol and by divalent metal ions such as Zn(2+) (41% inhibition). However, Ca(2+) and Fe(2+) resulted in increases in enzyme activity of 15% and 26%, respectively. The kinetic constants of the keratinase were found to be 3.84 microM (K(m)) and 108.7 microM/mLmin (V(max)). These results suggest that this extracellular keratinase may be a useful alternative and eco-friendly route for handling the abundant amount of waste feathers or for applications in other industrial processes.

Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house

PubMed Central

Kovacs, Krisztina; Macrelli, Stefano; Szakacs, George; Zacchi, Guido

2009-01-01

Background Improvement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process. Results Lignocellulolytic enzyme complexes were produced by the mutant Trichoderma atroviride TUB F-1663 on three different steam-pretreated lignocellulosic substrates, namely spruce, wheat straw and sugarcane bagasse. Filter paper activities of the enzymes produced on the three materials were very similar, while β-glucosidase and hemicellulase activities were more dependent on the nature of the substrate. Hydrolysis of the enzyme preparations investigated produced similar glucose yields. However, the enzymes produced in-house proved to degrade the xylan and the xylose oligomers less efficiently than a commercial mixture of cellulase and β-glucosidase. Furthermore, accumulation of xylose oligomers was observed when the TUB F-1663 supernatants were applied to xylan-containing substrates, probably due to the low β-xylosidase activity of the enzymes. The efficiency of the enzymes produced in-house was enhanced by supplementation with extra commercial β-glucosidase and β-xylosidase. When the hydrolytic capacities of various mixtures of a commercial cellulase and a T. atroviride supernatant produced in the lab were investigated at the same enzyme loading, the glucose yield appeared to be correlated with the β-glucosidase activity, while the xylose yield seemed to be correlated with the β-xylosidase level in the mixtures. Conclusion Enzyme supernatants produced by the mutant T. atroviride TUB F-1663 on various pretreated lignocellulosic substrates have good filter paper activity values combined with high levels of β-glucosidase activities, leading to cellulose conversion in the enzymatic hydrolysis that is as efficient as with a commercial cellulase mixture. On the other hand, in order to achieve good xylan conversion, the supernatants produced by the mutant have to be supplemented with additional β-xylosidase activity. PMID:19580644

The Secretory Response of Rat Peritoneal Mast Cells on Exposure to Mineral Fibers.

PubMed

Borelli, Violetta; Trevisan, Elisa; Francesca, Vita; Zabucchi, Giuliano

2018-01-10

Exposure to mineral fibers is of substantial relevance to human health. A key event in exposure is the interaction with inflammatory cells and the subsequent generation of pro-inflammatory factors. Mast cells (MCs) have been shown to interact with titanium oxide (TiO₂) and asbestos fibers. In this study, we compared the response of rat peritoneal MCs challenged with the asbestos crocidolite and nanowires of TiO₂ to that induced by wollastonite employed as a control fiber. Rat peritoneal MCs (RPMCs), isolated from peritoneal lavage, were incubated in the presence of mineral fibers. The quantities of secreted enzymes were evaluated together with the activity of fiber-associated enzymes. The ultrastructural morphology of fiber-interacting RPMCs was analyzed with electron microscopy. Asbestos and TiO₂ stimulate MC secretion. Secreted enzymes bind to fibers and exhibit higher activity. TiO₂ and wollastonite bind and improve enzyme activity, but to a lesser degree than crocidolite. (1) Mineral fibers are able to stimulate the mast cell secretory process by both active (during membrane interaction) and/or passive (during membrane penetration) interaction; (2) fibers can be found to be associated with secreted enzymes-this process appears to create long-lasting pro-inflammatory environments and may represent the active contribution of MCs in maintaining the inflammatory process; (3) MCs and their enzymes should be considered as a therapeutic target in the pathogenesis of asbestos-induced lung inflammation; and (4) MCs can contribute to the inflammatory effect associated with selected engineered nanomaterials, such as TiO₂ nanoparticles.

Purification and Characterization of a Highly Efficient Calcium-Independent α-Amylase from Talaromyces pinophilus 1-95

PubMed Central

Xian, Liang; Wang, Fei; Luo, Xiang; Feng, Yu-Liang; Feng, Jia-Xun

2015-01-01

Alpha-amylase is a very important enzyme in the starch conversion process. Most of the α-amylases are calcium-dependent and exhibit poor performance in the simultaneous saccharification and fermentation process of industrial bioethanol production that uses starch as feedstock. In this study, an extracellular amylolytic enzyme was purified from the culture broth of newly isolated Talaromyces pinophilus strain 1-95. The purified amylolytic enzyme, with an apparent molecular weight of 58 kDa on SDS-PAGE, hydrolyzed maltopentaose, maltohexaose, and maltoheptaose into mainly maltose and maltotriose and minor amount of glucose, confirming the endo-acting mode of the enzyme, and hence, was named Talaromyces pinophilus α-amylase (TpAA). TpAA was most active at pH 4.0–5.0 (with the temperature held at 37°C) and 55°C (at pH 5.0), and stable within the pH range of 5.0–9.5 (at 4°C) and below 45°C (at pH 5.0). Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2+-independent. TpAA displayed higher enzyme activity toward malto-oligosaccharides and dextrin than other previously reported α-amylases. This highly active Ca2+-independent α-amylase may have potential applications in starch-to-ethanol conversion process. PMID:25811759

A thermodynamic and theoretical view for enzyme regulation.

PubMed

Zhao, Qinyi

2015-01-01

Precise regulation is fundamental to the proper functioning of enzymes in a cell. Current opinions about this, such as allosteric regulation and dynamic contribution to enzyme regulation, are experimental models and substantially empirical. Here we proposed a theoretical and thermodynamic model of enzyme regulation. The main idea is that enzyme regulation is processed via the regulation of abundance of active conformation in the reaction buffer. The theoretical foundation, experimental evidence, and experimental criteria to test our model are discussed and reviewed. We conclude that basic principles of enzyme regulation are laws of protein thermodynamics and it can be analyzed using the concept of distribution curve of active conformations of enzymes.

A molecular dynamics study of the complete binding process of meropenem to New Delhi metallo-β-lactamase 1.

PubMed

Duan, Juan; Hu, Chuncai; Guo, Jiafan; Guo, Lianxian; Sun, Jia; Zhao, Zuguo

2018-02-28

The mechanism of substrate hydrolysis of New Delhi metallo-β-lactamase 1 (NDM-1) has been reported, but the process in which NDM-1 captures and transports the substrate into its active center remains unknown. In this study, we investigated the process of the substrate entry into the NDM-1 activity center through long unguided molecular dynamics simulations using meropenem as the substrate. A total of 550 individual simulations were performed, each of which for 200 ns, and 110 of them showed enzyme-substrate binding events. The results reveal three categories of relatively persistent and noteworthy enzyme-substrate binding configurations, which we call configurations A, B, and C. We performed binding free energy calculations of the enzyme-substrate complexes of different configurations using the molecular mechanics Poisson-Boltzmann surface area method. The role of each residue of the active site in binding the substrate was investigated using energy decomposition analysis. The simulated trajectories provide a continuous atomic-level view of the entire binding process, revealing potentially valuable regions where the enzyme and the substrate interact persistently and five possible pathways of the substrate entering into the active center, which were validated using well-tempered metadynamics. These findings provide important insights into the binding mechanism of meropenem to NDM-1, which may provide new prospects for the design of novel metallo-β-lactamase inhibitors and enzyme-resistant antibiotics.

Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

PubMed

Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

2014-12-12

The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibiting the HIV Integration Process: Past, Present, and the Future

PubMed Central

2013-01-01

HIV integrase (IN) catalyzes the insertion into the genome of the infected human cell of viral DNA produced by the retrotranscription process. The discovery of raltegravir validated the existence of the IN, which is a new target in the field of anti-HIV drug research. The mechanism of catalysis of IN is depicted, and the characteristics of the inhibitors of the catalytic site of this viral enzyme are reported. The role played by the resistance is elucidated, as well as the possibility of bypassing this problem. New approaches to block the integration process are depicted as future perspectives, such as development of allosteric IN inhibitors, dual inhibitors targeting both IN and other enzymes, inhibitors of enzymes that activate IN, activators of IN activity, as well as a gene therapy approach. PMID:24025027

Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses*

PubMed Central

Jiang, Rui; Kim, Eun-Hye; Gong, Ji-Hee; Kwon, Hyun-Mi; Kim, Chan-Hee; Ryu, Kyoung-Hwa; Park, Ji-Won; Kurokawa, Kenji; Zhang, Jinghai; Gubb, David; Lee, Bok-Luel

2009-01-01

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and β-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins. PMID:19858208

Redox-initiated hydrogel system for detection and real-time imaging of cellulolytic enzyme activity.

PubMed

Malinowska, Klara H; Verdorfer, Tobias; Meinhold, Aylin; Milles, Lukas F; Funk, Victor; Gaub, Hermann E; Nash, Michael A

2014-10-01

Understanding the process of biomass degradation by cellulolytic enzymes is of urgent importance for biofuel and chemical production. Optimizing pretreatment conditions and improving enzyme formulations both require assays to quantify saccharification products on solid substrates. Typically, such assays are performed using freely diffusing fluorophores or dyes that measure reducing polysaccharide chain ends. These methods have thus far not allowed spatial localization of hydrolysis activity to specific substrate locations with identifiable morphological features. Here we describe a hydrogel reagent signaling (HyReS) system that amplifies saccharification products and initiates crosslinking of a hydrogel that localizes to locations of cellulose hydrolysis, allowing for imaging of the degradation process in real time. Optical detection of the gel in a rapid parallel format on synthetic and natural pretreated solid substrates was used to quantify activity of T. emersonii and T. reesei enzyme cocktails. When combined with total internal reflection fluorescence microscopy and AFM imaging, the reagent system provided a means to visualize enzyme activity in real-time with high spatial resolution (<2 μm). These results demonstrate the versatility of the HyReS system in detecting cellulolytic enzyme activity and suggest new opportunities in real-time chemical imaging of biomass depolymerization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biosynthesis and processing of cathepsin K in cultured human osteoclasts.

PubMed

Rieman, D J; McClung, H A; Dodds, R A; Hwang, S M; Holmes, M W; James, I E; Drake, F H; Gowen, M

2001-03-01

Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous enzyme in osteoclasts. In this study, we show that mature cat K protein and enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature enzyme occurred in a time-dependent manner. Subsequently, the level of mature enzyme decreased. Little or no cat K was observed in the culture media at any timepoint. Pretreatment of osteoclasts with either chloroquine or monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with N-glycosidase F, suggesting the presence of high-mannose-containing oligosaccharides. Treatment of osteoclasts with the PI3-kinase inhibitor, Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of proenzyme into the culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases. Furthermore, cat K is not secreted as a proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active enzyme into the resorption lacunae.

Kinetic study of Candida antarctica lipase B immobilization using poly(methyl methacrylate) nanoparticles obtained by miniemulsion polymerization as support.

PubMed

Valério, Alexsandra; Nicoletti, Gabrieli; Cipolatti, Eliane P; Ninow, Jorge L; Araújo, Pedro H H; Sayer, Cláudia; de Oliveira, Débora

2015-03-01

With the objective to obtain immobilized Candida antarctica lipase B (CalB) with good activity and improved utilization rate, this study evaluated the influence of enzyme and crodamol concentrations and initiator type on the CalB enzyme immobilization in nanoparticles consisting of poly(methyl methacrylate) (PMMA) obtained by miniemulsion polymerization. The kinetic study of immobilized CalB enzyme in PMMA nanoparticles was evaluated in terms of monomer conversion, particle size, zeta potential, and relative activity. The optimum immobilization condition for CalB was compared with free enzyme in the p-NPL hydrolysis activity measurement. Results showed a higher CalB enzyme stability after 20 hydrolysis cycles compared with free CalB enzyme; in particular, the relative immobilized enzyme activity was maintained up to 40%. In conclusion, PMMA nanoparticles proved to be a good support for the CalB enzyme immobilization and may be used as a feasible alternative catalyst in industrial processes.

Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase

NASA Astrophysics Data System (ADS)

Konwarh, Rocktotpal; Karak, Niranjan; Rai, Sudhir Kumar; Mukherjee, Ashis Kumar

2009-06-01

Nanotechnology holds the prospect for avant-garde changes to improve the performance of materials in various sectors. The domain of enzyme biotechnology is no exception. Immobilization of industrially important enzymes onto nanomaterials, with improved performance, would pave the way to myriad application-based commercialization. Keratinase produced by Bacillus subtilis was immobilized onto poly(ethylene glycol)-supported Fe3O4 superparamagnetic nanoparticles. The optimization process showed that the highest enzyme activity was noted when immobilized onto cyanamide-activated PEG-assisted MNP prepared under conditions of 25 °C and pH 7.2 of the reaction mixture before addition of H2O2 (3% w/w), 2% (w/v) PEG6000 and 0.062:1 molar ratio of PEG to FeCl2·4H2O. Further statistical optimization using response surface methodology yielded an R2 value that could explain more than 94% of the sample variations. Along with the magnetization studies, the immobilization of the enzyme onto the PEG-assisted MNP was characterized by UV, XRD, FTIR and TEM. The immobilization process had resulted in an almost fourfold increase in the enzyme activity over the free enzyme. Furthermore, the immobilized enzyme exhibited a significant thermostability, storage stability and recyclability. The leather-industry-oriented application of the immobilized enzyme was tested for the dehairing of goat-skin.

Cathepsin B is not the processing enzyme for mouse prorenin.

PubMed

Mercure, Chantal; Lacombe, Marie-Josée; Khazaie, Khashayarsha; Reudelhuber, Timothy L

2010-05-01

Renin, an aspartyl protease that catalyzes the rate-limiting step in the renin-angiotensin system (RAS), is proteolytically activated by a second protease [referred to as the prorenin processing enzyme (PPE)] before its secretion from the juxtaglomerular cells of the kidney. Although several enzymes are capable of activating renin in vitro, the leading candidate for the PPE in the kidney is cathepsin B (CTSB) due to is colocalization with the renin precursor (prorenin) in juxtaglomerular cell granules and because of its site-selective activation of human prorenin both in vitro and in transfected tissue culture cell models. To verify the role of CTSB in prorenin processing in vivo, we tested the ability of CTSB-deficient (CTSB-/-) mice to generate active renin. CTSB-/- mice do not exhibit any overt symptoms (renal malformation, preweaning mortality) typical of an RAS deficiency and have normal levels of circulating active renin, which, like those in control animals, rise more than 15-fold in response to pharmacologic inhibition of the RAS. The mature renin enzyme detected in kidney lysates of CTSB-/- mice migrates at the same apparent molecular weight as that in control mice, and the processing to active renin is not affected by chloroquine treatment of the animals. Finally, the distribution and morphology of renin-producing cells in the kidney is normal in CTSB-/- mice. In conclusion, CTSB-deficient mice exhibit no differences compared with controls in their ability to generate active renin, and our results do not support CTSB as the PPE in mice.

Computational Steroidogenesis Model To Predict Biochemical Responses to Endocrine Active Chemicals: Model Development and Cross Validation

EPA Science Inventory

Steroids, which have an important role in a wide range of physiological processes, are synthesized primarily in the gonads and adrenal glands through a series of enzyme-mediated reactions. The activity of steroidogenic enzymes can be altered by a variety of endocrine active chem...

Nutraceutical composition of Zizyphus mauritiana Lamk (Indian ber): effect of enzyme-assisted processing.

PubMed

Koley, Tanmay Kumar; Walia, Shweta; Nath, Prerna; Awasthi, O P; Kaur, Charanjit

2011-05-01

Zizyphus (Indian ber) is an excellent source of several phenolic compounds. The effect of two cell wall degrading enzymes, namely pectinase and viscozyme, on the nutraceutical composition of Zizyphus juice was investigated in the present study. Enzyme assisted processing significantly (P < 0.05) improved the juice yield, total soluble solids, total phenolics and total antioxidant activity (AOX). There was significant increase in recovery of antioxidants, to the tune of 70.51%, 66%, and 45% respectively in ascorbic acid, total phenolics and total flavonoids through viscozyme. The in-vitro total AOX of juice extracted via enzyme-assisted processing was 20.9 and 15.59 μmol Trolox/ml in ferric-reducing antioxidant power and cupric-reducing antioxidant capacity assays, respectively. There was 41% increase in AOX of juice extracted with enzyme over straight pressed juice. Results indicate that enzyme-assisted processing can significantly improve the functional properties of the Zizyphus juice.

Optimization of Enzyme Co-Immobilization with Sodium Alginate and Glutaraldehyde-Activated Chitosan Beads.

PubMed

Gür, Sinem Diken; İdil, Neslihan; Aksöz, Nilüfer

2018-02-01

In this study, two different materials-alginate and glutaraldehyde-activated chitosan beads-were used for the co-immobilization of α-amylase, protease, and pectinase. Firstly, optimization of multienzyme immobilization with Na alginate beads was carried out. Optimum Na alginate and CaCl 2 concentration were found to be 2.5% and 0.1 M, respectively, and optimal enzyme loading ratio was determined as 2:1:0.02 for pectinase, protease, and α-amylase, respectively. Next, the immobilization of multiple enzymes on glutaraldehyde-activated chitosan beads was optimized (3% chitosan concentration, 0.25% glutaraldehyde with 3 h of activation and 3 h of coupling time). While co-immobilization was successfully performed with both materials, the specific activities of enzymes were found to be higher for the enzymes co-immobilized with glutaraldehyde-activated chitosan beads. In this process, glutaraldehyde was acting as a spacer arm. SEM and FTIR were used for the characterization of activated chitosan beads. Moreover, pectinase and α-amylase enzymes immobilized with chitosan beads were also found to have higher activity than their free forms. Three different enzymes were co-immobilized with these two materials for the first time in this study.

Evaluation of free and immobilized Aspergillus niger NRC1ami pectinase applicable in industrial processes.

PubMed

Esawy, Mona A; Gamal, Amira A; Kamel, Zeinat; Ismail, Abdel-Mohsen S; Abdel-Fattah, Ahmed F

2013-02-15

The Aspergillus niger NRC1ami pectinase was evaluated according to its hydrolysis efficiency of dry untreated orange peels (UOP), HCl-treated orange peels and NaOH-treated orange peels (HOP and NOP). Pectinase was entrapped in polyvinyl alcohol (PVA) sponge and the optimum pH and temperature of the free and immobilized enzymes were shifted from 4, 40 °C to 6, 50 °C respectively. The study of pH stability of free and immobilized pectinase showed that the immobilization process protected the enzyme strongly from severe alkaline pHs. The immobilization process improved the enzyme thermal stability to great instant. The unique feature of the immobilization process is its ability to solve the orange juice haze problem completely. Immobilized enzyme was reused 12 times in orange juice clarification with 9% activity loss from the original activity. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) of the partially purified form were significantly changed after immobilization. Copyright © 2012 Elsevier Ltd. All rights reserved.

ENZYMATIC PROCESSES USED BY PLANTS TO DEGRADE ORGANIC COMPOUNDS

EPA Science Inventory

This is a review of recent plant enzyme systems that have been studied in uptake and transformation of organic contaminants. General procedures of plant preparation and enzyme isolation are covered. Six plant enzyme systems have been investigated for activity with selected pollut...

Evaluation of possible reasons for the low phenylalanine ammonia lyase activity in cellulose nitrate membrane microcapsules.

PubMed

Habibi-Moini, S; D'mello, A P

2001-03-14

Microencapsulated phenylalanine ammonia lyase (PAL) exhibits a marked reduction in activity compared to the activity of the free enzyme in pH 8.5 Tris buffer. The purpose of this investigation was to evaluate the contribution of incomplete entrapment, the internal environment of cellulose nitrate membrane microcapsules, the diffusional barrier of the membrane and the microcapsulation process to the low activity of encapsulated PAL. A solution of PAL and 10% w/v hemoglobin was incorporated into cellulose nitrate membrane microcapsules. Hemoglobin incorporation was used as a surrogate marker of PAL entrapment. Using 14C hemoglobin, the encapsulation efficiency was determined to be 70% and suggested that incomplete entrapment might partially account for the low activity of encapsulated PAL. The effect of the internal environment of the microcapsule (10% hemoglobin solution) on PAL activity was evaluated by comparing enzyme activity in 10% w/v hemoglobin solution and pH 8.5 Tris buffer. Similar K(M) and V(max) values of PAL in the two media indicated that the internal environment of the microcapsule did not contribute to the reduction in activity of the encapsulated enzyme. The contribution of a membrane diffusional barrier was determined by breaking the putative barrier and measuring PAL activity in intact and broken microcapsules. Similar activity of PAL in these two conditions is evidence for the lack of a diffusional barrier. The effect of the microencapsulation process on PAL activity was evaluated by comparing K(M) and V(max) of free and encapsulated PAL. Similar K(M) values in these two media suggested that the process did not affect the conformation of PAL. However, encapsulated PAL had a 50% lower V(max) value compared to free PAL, which showed that the microencapsulation process deactivated a substantial proportion of the enzyme.

Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

2012-01-24

The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Enzyme Activities at Different Stages of Plant Biomass Decomposition in Three Species of Fungus-Growing Termites

PubMed Central

Pedersen, Kristine S. K.; Aanen, Duur K.

2017-01-01

ABSTRACT Fungus-growing termites rely on mutualistic fungi of the genus Termitomyces and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced in situ. Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites. IMPORTANCE Fungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished. PMID:29269491

Enzyme Activities at Different Stages of Plant Biomass Decomposition in Three Species of Fungus-Growing Termites.

PubMed

da Costa, Rafael R; Hu, Haofu; Pilgaard, Bo; Vreeburg, Sabine M E; Schückel, Julia; Pedersen, Kristine S K; Kračun, Stjepan K; Busk, Peter K; Harholt, Jesper; Sapountzis, Panagiotis; Lange, Lene; Aanen, Duur K; Poulsen, Michael

2018-03-01

Fungus-growing termites rely on mutualistic fungi of the genus Termitomyces and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced in situ Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites. IMPORTANCE Fungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished. Copyright © 2018 da Costa et al.

Effect of immobile isolated enzymes from rumen liquid by using alginate matrices on the bay leaf extraction

NASA Astrophysics Data System (ADS)

Paramita, Vita; Yulianto, Mohammad Endy; Yohana, Eflita; Arifan, Fahmi; Hanifah, Amjad, Muhammad Taqiyuddin

2015-12-01

This research aims to develop the enzymatically of bay leaves phytochemical extraction process. The novelty and the main innovations of this research is the development of extraction process by using enzymatic extractor and isolate the enzymes from rumen liquid to shift the equilibrium phase, increase the extraction rate and increase the extraction yield. The activity of rumen liquid enzyme was represented by the activity of cellulase and protease. The analyze of total flavonoid content was performed by using UV-Vis Spectrofometry. The activity of immobilized enzyme of cellulase (0.08±0.00 U/ml) was lower than the un-immobilized one (0.23±0.00 U/ml). However, there was no difference activity of the immobilized (0.75±0.00 U/ml) and un-immobilized (0.76±0.01 U/ml) of protease. The model of mass transfer of un-immobilized enzyme can be fitted on the experimental data, however the model of mass transfer of immobilized enzyme did not match with the experimental data. The mass transfer coefficient of enzymatic extraction flavonoids bay leaf without immobilization was 0.17167 s-1 which greater than the reported value of obtained KLa from extraction by using electric heating.

In-vitro engineering of novel bioactivity in the natural enzymes

NASA Astrophysics Data System (ADS)

Tiwari, Vishvanath

2016-10-01

Enzymes catalyze various biochemical functions with high efficiency and specificity. In-vitro design of the enzyme leads to novel bioactivity in this natural biomolecule that give answers of some vital questions like crucial residues in binding with substrate, molecular evolution, cofactor specificity etc. Enzyme engineering technology involves directed evolution, rational designing, semi-rational designing and structure-based designing using chemical modifications. Similarly, combined computational and in-vitro evolution approaches together help in artificial designing of novel bioactivity in the natural enzyme. DNA shuffling, error prone PCR and staggered extension process are used to artificially redesign active site of enzyme, which can alter its efficiency and specificity. Modifications of the enzyme can lead to the discovery of new path of molecular evolution, designing of efficient enzymes, locating active sites and crucial residues, shift in substrate and cofactor specificity. The methods and thermodynamics of in-vitro designing of the enzyme are also discussed. Similarly, engineered thermophilic and psychrophilic enzymes attain substrate specificity and activity of mesophilic enzymes that may also be beneficial for industry and therapeutics.

Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

PubMed

Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

2017-11-22

Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

The Role of Cholesterol Utilization in a Computational Adrenal Steroidogenesis Model to Improve Predictability of Biochemical Responses to Endocrine Active Chemicals

EPA Science Inventory

Steroids, which have an important role in a wide range of physiological processes, are synthesized primarily in the gonads and adrenal glands through a series of enzyme-mediated reactions. The activity of steroidogenic enzymes can be altered by a variety of endocrine active chem...

Computational Investigations of Trichoderma Reesei Cel7A Suggest New Routes for Enzyme Activity Improvements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beckham, G. T.; Payne, C. M.; Bu, L.

2012-01-01

The Trichoderma reesei Family 7 cellulase (Cel7A) is a key industrial enzyme in the production of biofuels from lignocellulosic biomass. It is a multi-modular enzyme with a Family 1 carbohydrate-binding module, a flexible O-glycosylated linker, and a large catalytic domain. We have used simulation to elucidate new functions for the 3 sub-domains, which suggests new routes to increase the activity of this central enzyme. These findings include new roles for glycosylation, which we have shown can be used to tune the binding affinity. We have also examined the structures of the catalytically-active complex of Cel7A and its non-processive counterpart, Cel7B,more » engaged on cellulose, which suggests allosteric mechanisms involved in chain binding when these cellulases are complexed on cellulose. Our computational results also suggest that product inhibition varies significantly between Cel7A and Cel7B, and we offer a molecular-level explanation for this observation. Finally, we discuss simulations of the absolute and relative binding free energy of cellulose ligands and various mutations along the CD tunnel, which will affect processivity and the ability of Cel7A (and related enzymes) to digest cellulose. These results highlight new considerations in protein engineering for processive and non-processive cellulases for production of lignocellulosic biofuels.« less

Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew

Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations inmore » heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.« less

Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage.

PubMed

Bechman, Allison; Phillips, Robert D; Chen, Jinru

2012-06-01

Koji are solid-state fermentation products made by inoculating steamed grains with the spores of fungi, particularly Aspergillus spp. This research was undertaken to identify the fermentation and storage conditions optimal for the production and maintenance of selected hydrolytic enzymes, such as α-amlyase and protease, in koji. Steamed rice and barley were inoculated with 2 × 10 ¹¹ Aspergillus oryzae spores per kilogram of grains and fermented for 118 h in a growth chamber at 28 to 32 °C with controlled relative humidities. Samples were drawn periodically during fermentation and storage at -20, 4, or 32 °C, and α-amylase and protease activity, mold counts, a(w), moisture contents, and pH of collected samples were determined. It was observed that the a(w), moisture contents, and pH of the koji were influenced by the duration of fermentation and temperature of storage. The α-amylase activity of both koji increased as the populations of A. oryzae increased during the exponential growth phase. The enzyme activity of barley koji was significantly higher than that of rice koji, reaching a peak activity of 211.87 or 116.57 U at 46 and 58 h, respectively, into the fermentation process. The enzyme activity in both products started to decrease once the mold culture entered the stationary growth phase. The protease activities of both koji were low and remained relatively stable during fermentation and storage. These results suggest that rice and barley koji can be used as sources of α-amylase and desired enzyme activity can be achieved by controlling the fermentation and storage conditions. Amylases and proteases are 2 important hydrolytic enzymes. In the food industry, these enzymes are used to break down starches and proteins while reducing the viscosity of foods. Although amylases and proteases are found in plants and animals, commercial enzymes are often produced using bacteria or molds through solid state fermentation, which is designed to use natural microbial process to produce enzymes in a controlled environment. A properly produced and maintained koji with a high hydrolytic enzyme activity can serve as an important source of the enzymes for the food industry. © 2012 Institute of Food Technologists®

Marine Sponges and Bacteria as Challenging Sources of Enzyme Inhibitors for Pharmacological Applications

PubMed Central

Ruocco, Nadia; Costantini, Susan; Palumbo, Flora; Costantini, Maria

2017-01-01

Enzymes play key roles in different cellular processes, for example, in signal transduction, cell differentiation and proliferation, metabolic processes, DNA damage repair, apoptosis, and response to stress. A deregulation of enzymes has been considered one of the first causes of several diseases, including cancers. In the last several years, enzyme inhibitors, being good candidates as drugs in the pathogenic processes, have received an increasing amount of attention for their potential application in pharmacology. The marine environment is considered a challenging source of enzyme inhibitors for pharmacological applications. In this review, we report on secondary metabolites with enzyme inhibitory activity, focusing our attention on marine sponges and bacteria as promising sources. In the case of sponges, we only reported the kinase inhibitors, because this class was the most representative isolated so far from these marine organisms. PMID:28604647

Relationship between sol-gel conditions and enzyme stability: a case study with β-galactosidase/silica biocatalyst for whey hydrolysis.

PubMed

Escobar, Sindy; Bernal, Claudia; Mesa, Monica

2015-01-01

The sol-gel process has been very useful for preparing active and stable biocatalysts, with the possibility of being reused. Especially those based on silica are well known. However, the study of the enzyme behavior during this process is not well understood until now and more, if the surfactant is involved in the synthesis mixture. This work is devoted to the encapsulation of β-galactosidase from Bacillus circulans in silica by sol-gel process, assisted by non-ionic Triton X-100 surfactant. The correlation between enzyme activity results for the β-galactosidase in three different environments (soluble in buffered aqueous reference solution, in the silica sol, and entrapment on the silica matrix) explains the enzyme behavior under stress conditions offered by the silica sol composition and gelation conditions. A stable β-galactosidase/silica biocatalyst is obtained using sodium silicate, which is a cheap source of silica, in the presence of non-ionic Triton X-100, which avoids the enzyme deactivation, even at 40 °C. The obtained biocatalyst is used in the whey hydrolysis for obtaining high value products from this waste. The preservation of the enzyme stability, which is one of the most important challenges on the enzyme immobilization through the silica sol-gel, is achieved in this study.

Combining an optical resonance biosensor with enzyme activity kinetics to understand protein adsorption and denaturation.

PubMed

Wilson, Kerry A; Finch, Craig A; Anderson, Phillip; Vollmer, Frank; Hickman, James J

2015-01-01

Understanding protein adsorption and resultant conformation changes on modified and unmodified silicon dioxide surfaces is a subject of keen interest in biosensors, microfluidic systems and for medical diagnostics. However, it has been proven difficult to investigate the kinetics of the adsorption process on these surfaces as well as understand the topic of the denaturation of proteins and its effect on enzyme activity. A highly sensitive optical whispering gallery mode (WGM) resonator was used to study a catalytic enzyme's adsorption processes on different silane modified glass substrates (plain glass control, DETA, 13 F, and SiPEG). The WGM sensor was able to obtain high resolution kinetic data of glucose oxidase (GO) adsorption with sensitivity of adsorption better than that possible with SPR. The kinetic data, in combination with a functional assay of the enzyme activity, was used to test hypotheses on adsorption mechanisms. By fitting numerical models to the WGM sensograms for protein adsorption, and by confirming numerical predictions of enzyme activity in a separate assay, we were able to identify mechanisms for GO adsorption on different alkylsilanes and infer information about the adsorption of protein on nanostructured surfaces. Copyright © 2014 Elsevier Ltd. All rights reserved.

Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hara-Nishimura, I.; Nishimura, M.

1987-10-01

The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulse-labeled with (/sup 35/S)methionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, ..gamma.. and delta. The pH optimum for the vacuole-mediated conversion was at pH 5.0. The proteolytic processing of proglobulin bymore » the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg/sup 2 +/, and Cu/sup 2 +/, but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles.« less

Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

PubMed

Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D

2014-12-01

Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes.

PubMed

Mattossovich, Rosanna; Iacono, Roberta; Cangiano, Giuseppina; Cobucci-Ponzano, Beatrice; Isticato, Rachele; Moracci, Marco; Ricca, Ezio

2017-11-28

The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-D-xylans to remove successive D-xylose residues from the non-reducing termini. We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.

Simultaneous determination of multiple soil enzyme activities for soil health-biogeochemical indexes

USDA-ARS?s Scientific Manuscript database

Enzyme activities (EAs) are soil health indicators of changes in decomposition processes due to management and the crop(s) affecting the quantity and quality of plant residues and nutrients entering the soil. More commonly assessed soil EAs can provide information of reactions where plant available ...

[Effect of melaxen on free radical processes intensity and some antioxidant enzymes activity in rats liver and blood serum under type 2 diabetes mellitus].

PubMed

Verevkin, A N; Popova, T N; Agarkov, A A; Semenikhina, A V

2015-01-01

The effect of melaxen on free radical processes and activity of superoxide dismutase and catalase in rats with type 2 diabetes mellitus (T2DM) has been investigated. It was established that melaxen administration to diabetic rats caused a decrease of the intensity of free radical processes as evidenced a decrease of the lipid peroxidation primary products content and biochemiluminescence parameters. The activity of the antioxidant enzymes changed towards normal values. These effects were probably induced by the correction of the melatonin level at the result of the melaxen action.

Digestive enzyme activities are higher in the shortfin mako shark, Isurus oxyrinchus, than in ectothermic sharks as a result of visceral endothermy.

PubMed

Newton, Kyle C; Wraith, James; Dickson, Kathryn A

2015-08-01

Lamnid sharks are regionally endothermic fishes that maintain visceral temperatures elevated above the ambient water temperature. Visceral endothermy is thought to increase rates of digestion and food processing and allow thermal niche expansion. We tested the hypothesis that, at in vivo temperatures, the endothermic shortfin mako shark, Isurus oxyrinchus, has higher specific activities of three digestive enzymes-gastric pepsin and pancreatic trypsin and lipase-than the thresher shark, Alopias vulpinus, and the blue shark, Prionace glauca, neither of which can maintain elevated visceral temperatures. Homogenized stomach or pancreas tissue obtained from sharks collected by pelagic longline was incubated at both 15 and 25 °C, at saturating substrate concentrations, to quantify tissue enzymatic activity. The mako had significantly higher enzyme activities at 25 °C than did the thresher and blue sharks at 15 °C. This difference was not a simple temperature effect, because at 25 °C the mako had higher trypsin activity than the blue shark and higher activities for all enzymes than the thresher shark. We also hypothesized that the thermal coefficient, or Q 10 value, would be higher for the mako shark than for the thresher and blue sharks because of its more stable visceral temperature. However, the mako and thresher sharks had similar Q 10 values for all enzymes, perhaps because of their closer phylogenetic relationship. The higher in vivo digestive enzyme activities in the mako shark should result in higher rates of food processing and may represent a selective advantage of regional visceral endothermy.

An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation.

PubMed

Huston, Scott; Collins, John; Sun, Fangfang; Zhang, Ting; Vaden, Timothy D; Zhang, Y-H Percival; Fu, Jinglin

2018-05-01

A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its β-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Enzymes immobilized in mesoporous silica: a physical-chemical perspective.

PubMed

Carlsson, Nils; Gustafsson, Hanna; Thörn, Christian; Olsson, Lisbeth; Holmberg, Krister; Åkerman, Björn

2014-03-01

Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at the molecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials. © 2013 Elsevier B.V. All rights reserved.

The dual exo/endo-type mode and the effect of ionic strength on the mode of catalysis of chitinase 60 (CHI60) from Serratia sp. TU09 and its mutants.

PubMed

Kuttiyawong, K; Nakapong, S; Pichyangkura, R

2008-11-03

Mutations of the tryptophan residues in the tryptophan-track of the N-terminal domain (W33F/Y and W69F/Y) and in the catalytic domain (W245F/Y) of Serratia sp. TU09 Chitinase 60 (CHI60) were constructed, as single and double point substitutions to either phenylalanine or tyrosine. The enzyme-substrate interaction and mode of catalysis, exo/endo-type, of wild type CHI60 and mutant enzymes on soluble (partially N-acetylated chitin), amorphous (colloidal chitin), and crystalline (β-chitin) substrates were studied. All CHI60 mutants exhibited a reduced substrate binding activity on colloidal chitin. CHI60 possesses a dual mode of catalysis with both exo- and endo-type activities allowing the enzyme to work efficiently on various substrate types. CHI60 preferentially uses the endo-type mode on soluble and amorphous substrates and the exo-type mode on crystalline substrate. However, the prevalent mode of hydrolysis mediated by CHI60 is regulated by ionic strength. Slightly elevated ionic strength, 0.1-0.2M NaCl, which promotes enzyme-substrate interactions, enhances CHI60 hydrolytic activity on amorphous substrate and, interestingly, on partially N-acetylated chitin. High ionic strength, 0.5-2.0M NaCl, prevents the enzyme from dissociating from amorphous substrate, occupying the enzyme in an enzyme-substrate non-productive complex. However, on crystalline substrates, the activity of CHI60 was only inhibited approximately 50% at high ionic strength, suggesting that the enzyme hydrolyzes crystalline substrates with an exo-type mode processively while remaining tightly bound to the substrate. Moreover, substitution of Trp-33 to either phenylalanine or tyrosine reduced the activity of the enzyme at high ionic strength, suggesting an important role of Trp-33 on enzyme processivity.

Structure-Activity Relations In Enzymes: An Application Of IR-ATR Modulation Spectroscopy

NASA Astrophysics Data System (ADS)

Fringeli, Urs P.; Ahlstrom, Peter; Vincenz, Claudius; Fringeli, Marianna

1985-12-01

Relations between structure and specific activity in immobilized acetylcholinesterase (ACNE) have been studied by means of pH- and Ca++-modulation technique combined with attenuated total reflection (ATR) infrared (IR) spectroscopy and enzyme activity measurement. Periodic modulation of pH and Ca++-concentration enabled a periodic on-off switching of about 40% of the total enzyme activity. It was found that about 0.5 to 1% of the amino acids were involved in this process. These 15 to 30 amino acids assumed antiparallel pleated sheet structure in the inhibited state and random and/or helical structure in the activated state.

Extracellular Enzyme Activity Profile in a Chemically Enhanced Water Accommodated Fraction of Surrogate Oil: Toward Understanding Microbial Activities After the Deepwater Horizon Oil Spill

PubMed Central

Kamalanathan, Manoj; Xu, Chen; Schwehr, Kathy; Bretherton, Laura; Beaver, Morgan; Doyle, Shawn M.; Genzer, Jennifer; Hillhouse, Jessica; Sylvan, Jason B.; Santschi, Peter; Quigg, Antonietta

2018-01-01

Extracellular enzymes and extracellular polymeric substances (EPS) play a key role in overall microbial activity, growth and survival in the ocean. EPS, being amphiphilic in nature, can act as biological surfactant in an oil spill situation. Extracellular enzymes help microbes to digest and utilize fractions of organic matter, including EPS, which can stimulate growth and enhance microbial activity. These natural processes might have been altered during the 2010 Deepwater Horizon oil spill due to the presence of hydrocarbon and dispersant. This study aims to investigate the role of bacterial extracellular enzymes during exposure to hydrocarbons and dispersant. Mesocosm studies were conducted using a water accommodated fraction of oil mixed with the chemical dispersant, Corexit (CEWAF) in seawater collected from two different locations in the Gulf of Mexico and corresponding controls (no additions). Activities of five extracellular enzymes typically found in the EPS secreted by the microbial community – α- and β-glucosidase, lipase, alkaline phosphatase, leucine amino-peptidase – were measured using fluorogenic substrates in three different layers of the mesocosm tanks (surface, water column and bottom). Enhanced EPS production and extracellular enzyme activities were observed in the CEWAF treatment compared to the Control. Higher bacterial and micro-aggregate counts were also observed in the CEWAF treatment compared to Controls. Bacterial genera in the order Alteromonadaceae were the most abundant bacterial 16S rRNA amplicons recovered. Genomes of Alteromonadaceae commonly have alkaline phosphatase and leucine aminopeptidase, therefore they may contribute significantly to the measured enzyme activities. Only Alteromonadaceae and Pseudomonadaceae among bacteria detected here have higher percentage of genes for lipase. Piscirickettsiaceae was abundant; genomes from this order commonly have genes for leucine aminopeptidase. Overall, this study provides insights into the alteration to the microbial processes such as EPS and extracellular enzyme production, and to the microbial community, when exposed to the mixture of oil and dispersant. PMID:29740422

Endo-beta-N-acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol.

PubMed

Suzuki, Tadashi; Yano, Keiichi; Sugimoto, Seiji; Kitajima, Ken; Lennarz, William J; Inoue, Sadako; Inoue, Yasuo; Emori, Yasufumi

2002-07-23

Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-beta-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein. The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.

Expression of three Trichoderma reesei cellulase genes in Saccharomyces pastorianus for the development of a two-step process of hydrolysis and fermentation of cellulose.

PubMed

Fitzpatrick, J; Kricka, W; James, T C; Bond, U

2014-07-01

To compare the production of recombinant cellulase enzymes in two Saccharomyces species so as to ascertain the most suitable heterologous host for the degradation of cellulose-based biomass and its conversion into bioethanol. cDNA copies of genes representing the three major classes of cellulases (Endoglucanases, Cellobiohydrolases and β-glucosidases) from Trichoderma reesei were expressed in Saccharomyces pastorianus and Saccharomyces cerevisiae. The recombinant enzymes were secreted by the yeast hosts into the medium and were shown to act in synergy to hydrolyse cellulose. The conditions required to achieve maximum release of glucose from cellulose by the recombinant enzymes were defined and the activity of the recombinant enzymes was compared to a commercial cocktail of T. reesei cellulases. We demonstrate that significantly higher levels of cellulase activity were achieved by expression of the genes in S. pastorianus compared to S. cerevisiae. Hydrolysis of cellulose by the combined activity of the recombinant enzymes was significantly better at 50°C than at 30°C, the temperature used for mesophilic yeast fermentations, reflecting the known temperature profiles of the native enzymes. The results demonstrate that host choice is important for the heterologous production of cellulases. On the basis of the low activity of the T. reesei recombinant enzymes at fermentation temperatures, we propose a two-step process for the hydrolysis of cellulose and its fermentation into alcohol using cellulases produced in situ. © 2014 The Society for Applied Microbiology.

Enzyme activities by indicator of quality in organic soil

NASA Astrophysics Data System (ADS)

Raigon Jiménez, Mo; Fita, Ana Delores; Rodriguez Burruezo, Adrián

2016-04-01

The analytical determination of biochemical parameters, as soil enzyme activities and those related to the microbial biomass is growing importance by biological indicator in soil science studies. The metabolic activity in soil is responsible of important processes such as mineralization and humification of organic matter. These biological reactions will affect other key processes involved with elements like carbon, nitrogen and phosphorus , and all transformations related in soil microbial biomass. The determination of biochemical parameters is useful in studies carried out on organic soil where microbial processes that are key to their conservation can be analyzed through parameters of the metabolic activity of these soils. The main objective of this work is to apply analytical methodologies of enzyme activities in soil collections of different physicochemical characteristics. There have been selective sampling of natural soils, organic farming soils, conventional farming soils and urban soils. The soils have been properly identified conserved at 4 ° C until analysis. The enzyme activities determinations have been: catalase, urease, cellulase, dehydrogenase and alkaline phosphatase, which bring together a representative group of biological transformations that occur in the soil environment. The results indicate that for natural and agronomic soil collections, the values of the enzymatic activities are within the ranges established for forestry and agricultural soils. Organic soils are generally higher level of enzymatic, regardless activity of the enzyme involved. Soil near an urban area, levels of activities have been significantly reduced. The vegetation cover applied to organic soils, results in greater enzymatic activity. So the quality of these soils, defined as the ability to maintain their biological productivity is increased with the use of cover crops, whether or spontaneous species. The practice of cover based on legumes could be used as an ideal choice for the recovery of degraded soils, because these soils have the highest levels of enzymatic activities.

Enzyme Analysis to Determine Glucose Content

NASA Astrophysics Data System (ADS)

Carpenter, Charles; Ward, Robert E.

Enzyme analysis is used for many purposes in food science and technology. Enzyme activity is used to indicate adequate processing, to assess enzyme preparations, and to measure constituents of foods that are enzyme substrates. In this experiment, the glucose content of corn syrup solids is determined using the enzymes, glucose oxidase and peroxidase. Glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide (H2O2), which then reacts with a dye in the presence of peroxidase to give a stable colored product.

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

PubMed

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

Studies on the production of alkaline α-amylase from Bacillus subtilis CB-18.

PubMed

Nwokoro, Ogbonnaya; Anthonia, Odiase

2015-01-01

Amylases are among the main enzymes used in food and other industries. They hydrolyse starch molecules into polymers composing glucose units. Amylases have potential applications in a number of industrial processes including foods and pharmaceutical industries. Alkaline α-amylase has the potential of hydrolysing starch under alkaline pH and is useful in the starch and textile industries and as an ingredient of detergents. Amylases are produced from plants, however, microbial production processes have dominated applications in the industries. Optimization of microbial production processes can result in improved enzyme yields. Amylase activity was assayed by incubating the enzyme solution (0.5 ml) with 1% soluble starch (0.5 ml) in 0.1 M Tris/HCl buffer (pH 8.5). After 30 minutes, the reaction was stopped by the addition of 4 mL of 3,5-dinitrosalicylic acid (DNS) reagent then heated for 10 min in boiling water bath and cooled in a refrigerator. Absorbance readings were used to estimate the units of enzyme activity from glucose standard curve. Hydrolysed native starches from cassava, rice, corn, coco yam, maize and potato and soluble starch were adjusted to pH 8.5 prior to incubation with crude enzyme solution. Reducing sugars produced were therefore determined. The effect of pH on enzyme activity of the alkaline α-amylase was determined by using buffer solutions of different pH (potassium phosphate buffer, 6.0-7.0; Tris-HCl buffer 7.5 to 9.0 and carbonate/bicarbonate buffer, pH 9.5-11) for enzyme assay. The pH stability profile of the enzyme was determined by incubating 0.5 ml of α-amylase enzyme in 0.1 M Tris/HCl buffer (pH 8.5) and 0.5 ml of 1% (w/v) soluble starch (Merck) in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h in various buffers. The effect of temperature on enzyme activity was studied by incubating 0.5 mL of the enzyme solution contained in the test tube and 0.5 mL of 1% soluble starch (Merck) solution prepared in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h at various temperatures (25, 30, 35, 40, 45, 50, 55 and 60°C) in a thermo static water bath. The reactions were stopped by adding DNS reagent. The enzyme activity was therefore determined. Thermal stability was studied by incubating 0.5 ml of enzyme solution in 0.1 M Tris/HCl buffer (pH 8.5) and 0.5 ml of 1% (w/v) soluble starch (Merck) in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h at various temperatures (20, 30, 40, 50, 60 and 70°C) for 60 min. The enzyme displayed optimal activity at pH 8.0 at which it produced maximum specific activity of 34.3 units/mg protein. Maximum stability was at pH 8.0 to 9.0. Maximum activity was observed at temperature of 50°C while thermo stability of the enzyme was observed at 40-50°C. The enzyme displayed a wide range of activities on starch and caused the release of 5.86, 4.75, 5.98, 3.44, 3.96, 8.84 mg/mL reducing sugar from cassava, potato, cocoyam, corn, rice and soluble starch respectively. This investigation reports some biochemical characterization of alkaline α-amylase from Bacillus subtilis CB-18. The substrate specificities of this enzyme on various starches suggested that the alkaline α-amylase enzyme had combined activities on raw and soluble starch.

Malabsorption syndrome observed in the common octopus Octopus vulgaris infected with Aggregata octopiana (Protista: Apicomplexa).

PubMed

Gestal, C; Páez de la Cadena, M; Pascual, S

2002-08-15

Octopus vulgaris infected with Aggregata octopiana were collected from an open-water culture system in the Ría of Aldán (NW Spain). Digestive tract infection values were determined with the use of a Neubauer chamber by counting the number of A. octopiana sporocysts. After determining enzyme activity values by the colorimetric Api-Zym system Biomerieux, one representative enzyme of glycosidases, peptid hydrolases and phosphoric hydrolases showing high activity was spectrophotometrically analysed. The enzymes were maltase and leucine-aminopeptidase (LAP) involved in the absorption process, and acid phosphatase, a lysosomic enzyme, respectively. Enzymatic activity of maltase and LAP decreased significantly, with increased sporocyst counts. However, acid phosphatase activity increased with severity of infection, indicating the presence of degradative enzymes from phagocytic cells in the infected area. A detrimental effect on gastrointestinal function may result from a decrease or malfunction of absorption enzymes. The results suggest a malabsorption syndrome resulting from parasitic infection.

Comparison of Free and Immobilized L-asparaginase Synthesized by Gamma-Irradiated Penicillium cyclopium.

PubMed

El-Refai, Heba A; Shafei, Mona S; Mostafa, Hanan; El-Refai, Abdel-Monem H; Araby, Eman M; El-Beih, Fawkia M; Easa, Saadia M; Gomaa, Sanaa K

2016-01-01

Gamma irradiation is used on Penicillium cyclopium in order to obtain mutant cells of high L-asparaginase productivity. Using gamma irradiation dose of 4 KGy, P. cyclopium cells yielded L-asparaginase with extracellular enzyme activity of 210.8 ± 3 U/ml, and specific activity of 752.5 ± 1.5 U/mg protein, which are 1.75 and 1.53 times, respectively, the activity of the wild strain. The enzyme was partially purified by 40-60% acetone precipitation. L-asparaginase was immobilized onto Amberlite IR-120 by ionic binding. Both free and immobilized enzymes exhibited maximum activity at pH 8 and 40 degrees C. The immobilization process improved the enzyme thermal stability significantly. The immobilized enzyme remained 100% active at temperatures up to 60 degrees C, while the free asparaginase was less tolerant to high temperatures. The immobilized enzyme was more stable at pH 9.0 for 50 min, retaining 70% of its relative activity. The maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) of the free form were significantly changed after immobilization. The K(m) value for immobilized L-asparaginase was about 1.3 times higher than that of free enzyme. The ions K+, Ba2+ and Na+ showed stimulatory effect on enzyme activity with percentages of 110%, 109% and 106% respectively.

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

PubMed

Nadar, Shamraja S; Rathod, Virendra K

2017-08-22

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (k m and V max ) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system

PubMed Central

Desguin, Benoît; Goffin, Philippe; Viaene, Eric; Kleerebezem, Michiel; Martin-Diaconescu, Vlad; Maroney, Michael J; Declercq, Jean-Paul; Soumillion, Patrice; Hols, Pascal

2014-01-01

Racemases catalyze the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel α/β fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent enzymes. PMID:24710389

[Importance of the 11β-hydroxysteroid dehydrogenase enzyme in clinical disorders].

PubMed

Feldman, Karolina; Likó, István; Nagy, Zsolt; Szappanos, Agnes; Grolmusz, Vince Kornél; Tóth, Miklós; Rácz, Károly; Patócs, Attila

2013-02-24

Glucocorticoids play an important role in the regulation of carbohydrate and amino acid metabolism, they modulate the function of the immune system, and contribute to stress response. Increased and decreased production of glucocorticoids causes specific diseases. In addition to systemic hypo- or hypercortisolism, alteration of local synthesis and metabolism of cortisol may result in tissue-specific hypo- or hypercortisolism. One of the key enzymes participating in the local synthesis and metabolism of cortisol is the 11β-hydroxysteroid dehydrogenase enzyme. Two isoforms, type 1 and type 2 enzymes are located in the endoplasmic reticulum and catalyze the interconversion of hormonally active cortisol and inactive cortisone. The type 1 enzyme mainly works as an activator, and it is responsible for the generation of cortisol from cortisone in liver, adipose tissue, brain and bone. The gene encoding this enzyme is located on chromosome 1. The authors review the physiological and pathophysiological processes related to the function of the type 1 11β-hydroxysteroid dehydrogenase enzyme. They summarize the potential significance of polymorphic variants of the enzyme in clinical diseases as well as knowledge related to inhibitors of enzyme activity. Although further studies are still needed, inhibition of the enzyme activity may prove to be an effective tool for the treatment of several diseases such as obesity, osteoporosis and type 2 diabetes.

A meta-analysis of soil exoenzyme responses to simulated climate change

NASA Astrophysics Data System (ADS)

Gebhardt, M.; Espinosa, N. J.; Blankinship, J. C.; Gallery, R. E.

2017-12-01

Microorganisms produce extracellular enzymes to decompose plant matter and drive biogeochemical transformations in soils. Climate change factors, such as warming and altered precipitation patterns, can impact enzyme activity through both direct and indirect mechanisms. Although many individual studies have examined how soil exoenzyme activities respond to climate change manipulations, there is disagreement surrounding the direction of these responses. We performed a synthesis of published studies to examine the influence of warming and altered precipitation on microbial exoenzyme activity. We found that warming increased enzyme activity with a more pronounced effect for oxidative relative to hydrolytic enzymes. Reduced precipitation consistently decreased exoenzyme activity. These responses, however, varied by season, biome, and enzyme type. The majority of studies fitting our criteria (e.g., experiments lasting a minimum of one growing season, paired treatments and controls) were located in North America and Europe. Inferences from this analysis therefore exclude many important ecosystems such as hyper-arid, wetlands, and artic systems. Carbon degrading enzyme activities were less sensitive to climate change manipulations when compared to phosphorus and nitrogen degrading enzyme activities. Linking enzyme activity to biogeochemical processes requires concomitant measurements of organic and inorganic carbon pools, mineralogy, nutrients, microbial biomass and community structure, and heterotrophic respiration within individual studies. Furthermore, linking these parameters to climate and environmental factors will require a comprehensive and consistent inclusion of biotic and abiotic variables among researchers and experiments. Globally, soils contain the largest carbon pools. Understanding the impacts of large-scale perturbations on soil enzyme activity will help to constrain predictions on the fate of biogeochemical transformations and improve model projections.

In Vitro Assembly of Catalase*

PubMed Central

Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars

2014-01-01

Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process. PMID:25148685

An approach to determine multiple enzyme activities in the same soil sample for soil health-biogeochemical indexes

USDA-ARS?s Scientific Manuscript database

Enzyme activities (EAs) are soil health indicators of changes in decomposition processes due to management and the crop(s) affecting the quantity and quality of plant residues and nutrients entering the soil. More commonly assessed soil EAs can provide information of reactions where plant available ...

Complex enzyme hydrolysis releases antioxidative phenolics from rice bran.

PubMed

Liu, Lei; Wen, Wei; Zhang, Ruifen; Wei, Zhencheng; Deng, Yuanyuan; Xiao, Juan; Zhang, Mingwei

2017-01-01

In this study, phenolic profiles and antioxidant activity of rice bran were analyzed following successive treatment by gelatinization, liquefaction and complex enzyme hydrolysis. Compared with gelatinization alone, liquefaction slightly increased the total amount of phenolics and antioxidant activity as measured by ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Complex enzyme hydrolysis significantly increased the total phenolics, flavonoids, FRAP and ORAC by 46.24%, 79.13%, 159.14% and 41.98%, respectively, compared to gelatinization alone. Furthermore, ten individual phenolics present in free or soluble conjugate forms were also analyzed following enzymatic processing. Ferulic acid experienced the largest release, followed by protocatechuic acid and then quercetin. Interestingly, a major proportion of phenolics existed as soluble conjugates, rather than free form. Overall, complex enzyme hydrolysis releases phenolics, thus increasing the antioxidant activity of rice bran extract. This study provides useful information for processing rice bran into functional beverage rich in phenolics. Copyright © 2016 Elsevier Ltd. All rights reserved.

In Situ Observation of Chymotrypsin Catalytic Activity Change Actuated by Nonheating Low-Frequency Magnetic Field.

PubMed

Efremova, Maria V; Veselov, Maxim M; Barulin, Alexander V; Gribanovsky, Sergey L; Le-Deygen, Irina M; Uporov, Igor V; Kudryashova, Elena V; Sokolsky-Papkov, Marina; Majouga, Alexander G; Golovin, Yuri I; Kabanov, Alexander V; Klyachko, Natalia L

2018-04-24

Magnetomechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using a nonheating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out in situ during exposure to the magnetic field. The first approach involves α-chymotrypsin-GM-MNPs conjugates, in which the enzyme undergoes mechanical deformations with the reorientation of the MNPs under LF MF (16-410 Hz frequency, 88 mT flux density). Such mechanical deformations result in conformational changes in α-chymotrypsin structure, as confirmed by infrared spectroscopy and molecular modeling, and lead to a 63% decrease of enzyme initial activity. The second approach involves an α-chymotrypsin-GM-MNPs/trypsin inhibitor-GM-MNPs complex, in which the activity of the enzyme is partially inhibited. In this case the reorientation of MNPs in the field leads to disruption of the enzyme-inhibitor complex and an almost 2-fold increase of enzyme activity. The results further demonstrate the utility of magnetomechanical actuation at the nanoscale for the remote modulation of biochemical reactions.

DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

PubMed Central

Bernard, Mark A.; Wang, Leyu; Tachado, Souvenir D.

2015-01-01

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K m=74 nM) with substrate inhibition kinetics (K i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy. PMID:25793518

Engineering Cellulase Enzymes for Bioenergy

NASA Astrophysics Data System (ADS)

Atreya, Meera Elizabeth

Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current methods. Protein engineering targets to improve cellulases include reducing enzyme inhibition, improving inter-enzyme synergy, and increasing enzyme thermotolerance. Ameliorating enzyme inhibition could improve catalytic activity and thus the speed of conversion from biomass to fermentable sugars. Improved enzyme synergy could reduce the enzyme loading required to achieve equivalent biomass conversion. Finally, thermostable enzymes could enable more biomass to be processed at a time, due to high temperatures decreasing the viscosity of biomass slurries. A high-temperature enzyme saccharification reaction could also decrease the risk of contamination in the resulting concentrated sugar solution. Throughout my PhD, I have explored research projects broadly across all of these topics, with the most success in addressing the issue of enzyme inhibition. Cellulase enzyme Cel7A is the most abundant cellulase employed by natural systems for cellulose hydrolysis. Cellobiohydrolase enzymes like Cel7A break down cellulose into cellobiose (two glucose molecules). Unfortunately, upon cleavage, this product molecule interferes with continued hydrolysis activity of Cel7A; the strong binding of cellobiose in the active site can obstruct the enzyme from processing down the cellulase chain. This phenomenon, known as product inhibition, is a bottleneck to efficient biomass breakdown. Using insights from computational protein modeling studies, I experimentally generated and tested mutant Cel7A enzymes for improved tolerance to cellobiose. Indeed, this strategy yielded Cel7A enzymes exhibiting reduced product inhibition, including some mutants completely impervious to cellobiose. The improvements in tolerance to cellobiose, however, resulted in an overall reduction of enzyme activity for the mutants tested. Nevertheless, my findings substantiated computational reports with experimental evidence and pinpointed an amino acid residue in the Cel7A product binding site that is of interest for follow-up mutational studies. My goal was to improve the effective catalytic activity of cellulase enzymes in industrially-relevant conditions (such as in the presence of high concentrations of cellobiose or at elevated temperatures). The insights gained from my work on enzyme inhibition may inform future efforts to address this important issue. More efficient enzymes should reduce the amount of these proteins needed to break down cellulose to glucose. This, in turn, should decrease the price of the resulting biofuel making it more cost-competitive with fossil fuels and thus encouraging adoption of renewable transportation fuels that reduce our greenhouse gas emissions.

Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.

PubMed

Fonseca, L P; Cabral, J M

1996-01-01

Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes*

PubMed Central

Kuusk, Silja; Sørlie, Morten; Väljamäe, Priit

2015-01-01

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the 14C-labeled crystalline polymeric substrates 14C chitin nanowhiskers and 14C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki(obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki(obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation. PMID:25767120

The predominant molecular state of bound enzyme determines the strength and type of product inhibition in the hydrolysis of recalcitrant polysaccharides by processive enzymes.

PubMed

Kuusk, Silja; Sørlie, Morten; Väljamäe, Priit

2015-05-01

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the (14)C-labeled crystalline polymeric substrates (14)C chitin nanowhiskers and (14)C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki (obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki (obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Statistical optimization of arsenic biosorption by microbial enzyme via Ca-alginate beads.

PubMed

Banerjee, Suchetana; Banerjee, Anindita; Sarkar, Priyabrata

2018-04-16

Bioremediation of arsenic using green technology via microbial enzymes has attracted scientists due to its simplicity and cost effectiveness. Statistical optimization of arsenate bioremediation was conducted by the enzyme arsenate reductase extracted from arsenic tolerant bacterium Pseudomonas alcaligenes. Response surface methodology based on Box-Behnken design matrix was performed to determine the optimal operational conditions of a multivariable system and their interactive effects on the bioremediation process. The highest biosorptive activity of 96.2 µg gm -1 of beads was achieved under optimized conditions (pH = 7.0; As (V) concentration = 1000 ppb; time = 2 h). SEM analysis showed the morphological changes on the surface of enzyme immobilized gluteraldehyde crosslinked Ca-alginate beads. The immobilized enzyme retained its activity for 8 cycles. ANOVA with a high correlation coefficient (R 2 > 0.99) and lower "Prob > F"value (<0.0001) corroborated the second-order polynomial model for the biosorption process. This study on the adsorptive removal of As (V) by enzyme-loaded biosorbent revealed a possible way of its application in large scale treatment of As (V)-contaminated water bodies.

Screening and optimization of pectin lyase and polygalacturonase activity from ginseng pathogen Cylindrocarpon Destructans

PubMed Central

Sathiyaraj, Gayathri; Srinivasan, Sathiyaraj; Kim, Ho-Bin; Subramaniyam, Sathiyamoorthy; Lee, Ok Ran; Kim, Yeon-Ju; Yang, Deok Chun

2011-01-01

Cylindrocarpon destructans isolated from ginseng field was found to produce pectinolytic enzymes. A Taguchi’s orthogonal array experimental design was applied to optimize the preliminary production of polygalacturonase (PG) and pectin lyase (PL) using submerged culture condition. This method was applied to evaluate the significant parameters for the production of enzymes. The process variables were pH, pectin concentration, incubation time and temperature. Optimization of process parameters resulted in high levels of enzyme (PG and PL) production after ten days of incubation at a pH of 5.0 at 25°C in the presence of 1.5% pectin. Among different nitrogen sources, urea and peptone showed high production of PG and PL, respectively. The enzyme production and mycelial growth seems to have direct influence on the culture conditions; therefore, at stationary state high enzyme production and mycelial growth were obtained than agitation state. Along with this, optimization of enzyme activity was also determined using various physiological parameters like, temperature, incubation time and pH. Taguchi’s data was also analyzed using one step ANOVA statistical method. PMID:24031695

Enhanced stability and chemical resistance of a new nanoscale biocatalyst for accelerating CO2 absorption into a carbonate solution.

PubMed

Zhang, Shihan; Lu, Hong; Lu, Yongqi

2013-12-03

A novel potassium-carbonate-based absorption process is currently being developed to reduce the energy consumption when capturing CO2 from coal combustion flue gas. The process employs the enzyme carbonic anhydrase (CA) as a catalyst to accelerate the rate of CO2 absorption. This study focused on the immobilization of a new variant of the CA enzyme onto a new group of nonporous nanoparticles to improve the enzyme's thermal stability and its chemical resistance to major impurities from the flue gas. The CA enzyme was manufactured at the pilot scale by a leading enzyme company. As carrier materials, two different batches of SiO2-ZrO2 composite nanoparticles and one batch of silica nanoparticle were synthesized using a flame spray pyrolysis method. Classic Danckwerts absorption theory with reaction was applied to determine the kinetics of the immobilized enzymes for CO2 absorption. The immobilized enzymes retained 56-88% of their original activity in a K2CO3/KHCO3 solution over a 60-day test period at 50 °C, compared with a 30% activity retention for their free CA enzyme counterpart. The immobilized CA enzymes also revealed improved chemical stability. The inactivation kinetics of the free and immobilized CA enzymes in the K2CO3/KHCO3 solution were experimentally quantified.

The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases.

PubMed

Hassa, Paul O; Hottiger, Michael O

2008-01-01

Poly-ADP-ribose metabolism plays a mayor role in a wide range of biological processes, such as maintenance of genomic stability, transcriptional regulation, energy metabolism and cell death. Poly-ADP-ribose polymerases (PARPs) are an ancient family of enzymes, as evidenced by the poly-ADP-ribosylating activities reported in dinoflagellates and archaebacteria and by the identification of Parp-like genes in eubacterial and archaeabacterial genomes. Six genes encoding "bona fide" PARP enzymes have been identified in mammalians: PARP1, PARP2, PARP3, PARP4/vPARP, PARP5/Tankyrases-1 and PARP6/Tankyrases-2. The best studied of these enzymes PARP1 plays a primary role in the process of poly-ADP-ribosylation. PARP1-mediated poly-ADP-ribosylation has been implicated in the pathogenesis of cancer, inflammatory and neurodegenerative disorders. This review will summarize the novel findings and concepts for PARP enzymes and their poly-ADP-ribosylation activity in the regulation of physiological and pathophysiological processes. A special focus is placed on the proposed molecular mechanisms involved in these processes, such as signaling, regulation of telomere dynamics, remodeling of chromatin structure and transcriptional regulation. A potential functional cross talk between PARP family members and other NAD+-consuming enzymes is discussed.

Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging.

PubMed

Chyan, Wen; Raines, Ronald T

2018-06-20

Fluorogenic probes, small-molecule sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are powerful tools for chemical biology. Those probes that respond to enzymatic activity illuminate the complex dynamics of biological processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. Here, we review recent advances in enzyme-activated fluorogenic probes for biological imaging. We organize our survey by enzyme classification, with emphasis on fluorophore masking strategies, modes of enzymatic activation, and the breadth of current and future applications. Key challenges such as probe selectivity and spectroscopic requirements are described alongside of therapeutic, diagnostic, and theranostic opportunities.

Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons

NASA Technical Reports Server (NTRS)

Chalmers, G. R.; Edgerton, V. R.

1989-01-01

The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.

Enzyme clustering accelerates processing of intermediates through metabolic channeling

PubMed Central

Castellana, Michele; Wilson, Maxwell Z.; Xu, Yifan; Joshi, Preeti; Cristea, Ileana M.; Rabinowitz, Joshua D.; Gitai, Zemer; Wingreen, Ned S.

2015-01-01

We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation. PMID:25262299

Potential Applications of Immobilized β-Galactosidase in Food Processing Industries

PubMed Central

Panesar, Parmjit S.; Kumari, Shweta; Panesar, Reeba

2010-01-01

The enzyme β-galactosidase can be obtained from a wide variety of sources such as microorganisms, plants, and animals. The use of β-galactosidase for the hydrolysis of lactose in milk and whey is one of the promising enzymatic applications in food and dairy processing industries. The enzyme can be used in either soluble or immobilized forms but the soluble enzyme can be used only for batch processes and the immobilized form has the advantage of being used in batch wise as well as in continuous operation. Immobilization has been found to be convenient method to make enzyme thermostable and to prevent the loss of enzyme activity. This review has been focused on the different types of techniques used for the immobilization of β-galactosidase and its potential applications in food industry. PMID:21234407

Effect of acute hypoxic shock on the rat brain morphology and tripeptidyl peptidase I activity.

PubMed

Petrova, Emilia B; Dimitrova, Mashenka B; Ivanov, Ivaylo P; Pavlova, Velichka G; Dimitrova, Stella G; Kadiysky, Dimitar S

2016-06-01

Hypoxic events are known to cause substantial damage to the hippocampus, cerebellum and striatum. The impact of hypoxic shock on other brain parts is not sufficiently studied. Recent studies show that tripeptidyl peptidase I (TPPI) activity in fish is altered after a hypoxic stress pointing out at a possible enzyme involvement in response to hypoxia. Similar studies are not performed in mammals. In this work, the effect of sodium nitrite-induced acute hypoxic shock on the rat brain was studied at different post-treatment periods. Morphological changes in cerebral cortex, cerebellum, medulla oblongata, thalamus, mesencephalon and pons were assessed using silver-copper impregnation for neurodegeneration. TPPI activity was biochemically assayed and localized by enzyme histochemistry. Although less vulnerable to oxidative stress, the studied brain areas showed different histopathological changes, such as neuronal loss and tissue vacuolization, dilatation of the smallest capillaries and impairment of neuronal processes. TPPI activity was strictly regulated following the hypoxic stress. It was found to increase 12-24h post-treatment, then decreased followed by a slow process of recovery. The enzyme histochemistry revealed a temporary enzyme deficiency in all types of neurons. These findings indicate a possible involvement of the enzyme in rat brain response to hypoxic stress. Copyright © 2016 Elsevier GmbH. All rights reserved.

Influence of Different Forest System Management Practices on Leaf Litter Decomposition Rates, Nutrient Dynamics and the Activity of Ligninolytic Enzymes: A Case Study from Central European Forests

PubMed Central

Schulz, Elke; Schloter, Michael; Buscot, François; Hofrichter, Martin; Krüger, Dirk

2014-01-01

Leaf litter decomposition is the key ecological process that determines the sustainability of managed forest ecosystems, however very few studies hitherto have investigated this process with respect to silvicultural management practices. The aims of the present study were to investigate the effects of forest management practices on leaf litter decomposition rates, nutrient dynamics (C, N, Mg, K, Ca, P) and the activity of ligninolytic enzymes. We approached these questions using a 473 day long litterbag experiment. We found that age-class beech and spruce forests (high forest management intensity) had significantly higher decomposition rates and nutrient release (most nutrients) than unmanaged deciduous forest reserves (P<0.05). The site with near-to-nature forest management (low forest management intensity) exhibited no significant differences in litter decomposition rate, C release, lignin decomposition, and C/N, lignin/N and ligninolytic enzyme patterns compared to the unmanaged deciduous forest reserves, but most nutrient dynamics examined in this study were significantly faster under such near-to-nature forest management practices. Analyzing the activities of ligninolytic enzymes provided evidence that different forest system management practices affect litter decomposition by changing microbial enzyme activities, at least over the investigated time frame of 473 days (laccase, P<0.0001; manganese peroxidase (MnP), P = 0.0260). Our results also indicate that lignin decomposition is the rate limiting step in leaf litter decomposition and that MnP is one of the key oxidative enzymes of litter degradation. We demonstrate here that forest system management practices can significantly affect important ecological processes and services such as decomposition and nutrient cycling. PMID:24699676

Influence of different forest system management practices on leaf litter decomposition rates, nutrient dynamics and the activity of ligninolytic enzymes: a case study from central European forests.

PubMed

Purahong, Witoon; Kapturska, Danuta; Pecyna, Marek J; Schulz, Elke; Schloter, Michael; Buscot, François; Hofrichter, Martin; Krüger, Dirk

2014-01-01

Leaf litter decomposition is the key ecological process that determines the sustainability of managed forest ecosystems, however very few studies hitherto have investigated this process with respect to silvicultural management practices. The aims of the present study were to investigate the effects of forest management practices on leaf litter decomposition rates, nutrient dynamics (C, N, Mg, K, Ca, P) and the activity of ligninolytic enzymes. We approached these questions using a 473 day long litterbag experiment. We found that age-class beech and spruce forests (high forest management intensity) had significantly higher decomposition rates and nutrient release (most nutrients) than unmanaged deciduous forest reserves (P<0.05). The site with near-to-nature forest management (low forest management intensity) exhibited no significant differences in litter decomposition rate, C release, lignin decomposition, and C/N, lignin/N and ligninolytic enzyme patterns compared to the unmanaged deciduous forest reserves, but most nutrient dynamics examined in this study were significantly faster under such near-to-nature forest management practices. Analyzing the activities of ligninolytic enzymes provided evidence that different forest system management practices affect litter decomposition by changing microbial enzyme activities, at least over the investigated time frame of 473 days (laccase, P<0.0001; manganese peroxidase (MnP), P = 0.0260). Our results also indicate that lignin decomposition is the rate limiting step in leaf litter decomposition and that MnP is one of the key oxidative enzymes of litter degradation. We demonstrate here that forest system management practices can significantly affect important ecological processes and services such as decomposition and nutrient cycling.

Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.

PubMed

Gao, En-Feng; Kang, Kyung Lhi; Kim, Jeong Hee

2014-06-01

Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields.

Cellulase stability, adsorption/desorption profiles and recycling during successive cycles of hydrolysis and fermentation of wheat straw.

PubMed

Rodrigues, Ana Cristina; Felby, Claus; Gama, Miguel

2014-03-01

The potential of enzymes recycling after hydrolysis and fermentation of wheat straw under a variety of conditions was investigated, monitoring the activity of the enzymes in the solid and liquid fractions, using low molecular weight substrates. A significant amount of active enzymes could be recovered by recycling the liquid phase. In the early stage of the process, enzyme adsorb to the substrate, then gradually returning to the solution as the saccharification proceeds. At 50°C, normally regarded as an acceptable operational temperature for saccharification, the enzymes (Celluclast) significantly undergo thermal deactivation. The hydrolysis yield and enzyme recycling efficiency in consecutive recycling rounds can be increased by using high enzyme loadings and moderate temperatures. Indeed, the amount of enzymes in the liquid phase increased with its thermostability and hydrolytic efficiency. This study contributes towards developing effective enzymes recycling strategies and helping to reduce the enzyme costs on bioethanol production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

PubMed

Hrycay, E G; Bandiera, S M

2009-12-01

The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

The Algicidal Fungus Trametes versicolor F21a Eliminating Blue Algae via Genes Encoding Degradation Enzymes and Metabolic Pathways Revealed by Transcriptomic Analysis.

PubMed

Dai, Wei; Chen, Xiaolin; Wang, Xuewen; Xu, Zimu; Gao, Xueyan; Jiang, Chaosheng; Deng, Ruining; Han, Guomin

2018-01-01

The molecular mechanism underlying the elimination of algal cells by fungal mycelia has not been fully understood. Here, we applied transcriptomic analysis to investigate the gene expression and regulation at time courses of Trametes versicolor F21a during the algicidal process. The obtained results showed that a total of 193, 332, 545, and 742 differentially expressed genes were identified at 0, 6, 12, and 30 h during the algicidal process, respectively. The gene ontology terms were enriched into glucan 1,4-α-glucosidase activity, hydrolase activity, lipase activity, and endopeptidase activity. The KEGG pathways were enriched in degradation and metabolism pathways including Glycolysis/Gluconeogenesis, Pyruvate metabolism, the Biosynthesis of amino acids, etc. The total expression levels of all Carbohydrate-Active enZYmes (CAZyme) genes for the saccharide metabolism were increased by two folds relative to the control. AA5, GH18, GH5, GH79, GH128, and PL8 were the top six significantly up-regulated modules among 43 detected CAZyme modules. Four available homologous decomposition enzymes of other species could partially inhibit the growth of algal cells. The facts suggest that the algicidal mode of T. versicolor F21a might be associated with decomposition enzymes and several metabolic pathways. The obtained results provide a new candidate way to control algal bloom by application of decomposition enzymes in the future.

Digestive enzymes of the Californian two-spot octopus, Octopus bimaculoides (Pickford and McConnaughey, 1949).

PubMed

Ibarra-García, Laura Elizabeth; Tovar-Ramírez, Dariel; Rosas, Carlos; Campa-Córdova, Ángel Isidro; Mazón-Suástegui, José Manuel

2018-01-01

Octopus bimaculoides is an important commercially fished species in the California Peninsula with aquaculture potential; however, to date limited information is available regarding its digestive physiology. The objective of this study was focused on biochemically characterizing the main enzymes involved in the digestion of O. bimaculoides. Optimum pH, temperature and thermostability were determined for amylases, lipases, trypsin and chymotrypsin; optimum pH and protease inhibitor effect were assessed for acidic and alkaline proteases, and the effect of divalent ions on trypsin and chymotrypsin activity was evaluated in enzymatic extracts from the digestive (DG) and salivary glands (SG) and crop gastric juices (GJ). High amylase activity was detected in GD and GJ whereas this activity is very low in other cephalopods. Salivary glands had the greatest activity in most of the enzyme groups, showing the importance of this organ in digestion. Optimum pH was different depending on the organ and enzyme analyzed. The optimum pH in DG was 3 showing the predominance of acidic proteases in the digestion process. All enzymes were resistant and stable at high temperatures in contrast with other marine species. Trypsin and chymotrypsin activity were highly incremented with the presence of Mg 2+ , Co 2+ , Cu 2+ and Zn 2+ in some tissues. The inhibitor assay showed the importance of serine proteases, metalloproteases and aspartic proteases in the digestive process of this species. This study is the first in assessing the main digestive enzymes of O. bimaculoides and in remarking the importance of other digestive enzyme groups besides proteases in octopuses. Copyright © 2017 Elsevier Inc. All rights reserved.

Magnetic cross-linked enzyme aggregates (CLEAs): a novel concept towards carrier free immobilization of lignocellulolytic enzymes.

PubMed

Bhattacharya, Abhishek; Pletschke, Brett I

2014-01-01

The enzymatic conversion of lignocellulosic biomass into biofuels has been identified as an excellent strategy to generate clean energy. However, the current process is cost-intensive as an effective immobilization approach to reuse the enzyme(s) has been a major challenge. The present study introduces the concept and application of novel magnetic cross-linked enzyme aggregates (mag-CLEAs). Both mag-CLEAs and calcium-mag-CLEAs (Ca-mag-CLEAs) exhibited a 1.35 fold higher xylanase activity compared to the free enzyme and retained more than 80.0% and 90.0% activity, respectively, after 136h of incubation at 50°C, compared to 50% activity retained by CLEAs. A 7.4 and 9.0 fold higher sugar release from lime-pretreated and NH4OH pre-treated sugar bagasse, respectively, was achieved with Ca-mag-CLEAs compared to the free enzymes. The present study promotes the successful application of mag-CLEAs and Ca-mag-CLEAs as carrier free immobilized enzymes for the effective hydrolysis of lignocellulolytic biomass and associated biofuel feedstocks. Copyright © 2014 Elsevier Inc. All rights reserved.

Chitosan-based biocatalytic nanoparticles for pollutant removal from wastewater.

PubMed

Alarcón-Payán, Dulce A; Koyani, Rina D; Vazquez-Duhalt, Rafael

2017-05-01

Chitosan, a renewable biopolymer has the prospective applications in different fields due to its gelation capacity. Nanoconfiguration of chitosan through ionotropic gelation to encapsulate enzymatic activity offers numerous potential applications. In the present study, the preparation and characterization of chitosan nanoparticles loaded with versatile peroxidase are reported. Their performance in bioremediation process and the resistance enhancement against natural microbial biodegradation were studied. The average diameter of enzymatic nanoparticles was 120nm and showed a high enzyme loading capacity. The kinetic parameters of nanoparticles exhibited a slightly lower catalytic activity (k cat ), similar affinity constant (Km) for hydrogen peroxide and higher Km value for the phenolic compound when compared with the free enzyme. The enzymatic nanoparticles showed higher thermostability and the same pH activity profile than those from free enzyme. Ten phenolic compounds, including pesticides, halogenated compounds, endocrine disruptors and antibacterials were transformed by the enzymatic nanoparticles. The transformation rate was lower than those obtained with free enzyme suggesting mass transfer limitations. But very importantly, the enzymatic nanoparticles showed a significant increase of the operational stability in real conditions of wastewater treatment process. Moreover, chemical modification of nanoparticles with different aldehydes still enhanced the operational stability of nanoparticulated enzymes. This enhancement of stability in real conditions and the potential use of biocatalytic nanoparticles in bioremediation processes are discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Application of ionic liquids based enzyme-assisted extraction of chlorogenic acid from Eucommia ulmoides leaves.

PubMed

Liu, Tingting; Sui, Xiaoyu; Li, Li; Zhang, Jie; Liang, Xin; Li, Wenjing; Zhang, Honglian; Fu, Shuang

2016-01-15

A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions. Copyright © 2015 Elsevier B.V. All rights reserved.

A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

PubMed

Pompey, Justine M; Foda, Bardees; Singh, Upinder

2015-01-01

Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System

PubMed Central

Singh, Upinder

2015-01-01

Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway. PMID:26230096

Novel optimization strategy for tannase production through a modified solid-state fermentation system.

PubMed

Wu, Changzheng; Zhang, Feng; Li, Lijun; Jiang, Zhedong; Ni, Hui; Xiao, Anfeng

2018-01-01

High amounts of insoluble substrates exist in the traditional solid-state fermentation (SSF) system. The presence of these substrates complicates the determination of microbial biomass. Thus, enzyme activity is used as the sole index for the optimization of the traditional SSF system, and the relationship between microbial growth and enzyme synthesis is always ignored. This study was conducted to address this deficiency. All soluble nutrients from tea stalk were extracted using water. The aqueous extract was then mixed with polyurethane sponge to establish a modified SSF system, which was then used to conduct tannase production. With this system, biomass, enzyme activity, and enzyme productivity could be measured rationally and accurately. Thus, the association between biomass and enzyme activity could be easily identified, and the shortcomings of traditional SSF could be addressed. Different carbon and nitrogen sources exerted different effects on microbial growth and enzyme production. Single-factor experiments showed that glucose and yeast extract greatly improved microbial biomass accumulation and that tannin and (NH 4 ) 2 SO 4 efficiently promoted enzyme productivity. Then, these four factors were optimized through response surface methodology. Tannase activity reached 19.22 U/gds when the added amounts of tannin, glucose, (NH 4 ) 2 SO 4 , and yeast extract were 7.49, 8.11, 9.26, and 2.25%, respectively. Tannase activity under the optimized process conditions was 6.36 times higher than that under the initial process conditions. The optimized parameters were directly applied to the traditional tea stalk SSF system. Tannase activity reached 245 U/gds, which is 2.9 times higher than our previously reported value. In this study, a modified SSF system was established to address the shortcomings of the traditional SSF system. Analysis revealed that enzymatic activity and microbial biomass are closely related, and different carbon and nitrogen sources have different effects on microbial growth and enzyme production. The maximal tannase activity was obtained under the optimal combination of nutrient sources that enhances cell growth and tannase accumulation. Moreover, tannase production through the traditional tea stalk SSF was markedly improved when the optimized parameters were applied. This work provides an innovative approach to bioproduction research through SSF.

Growth, sucrose synthase, and invertase activities of developing Phaseolus vulgaris L. fruits

Treesearch

Shi-Jean S. Sung; W.J. Sheih; D.R. Geiger; C.C. Black

1994-01-01

Activities of sucrose-cleaving enzymes, acid and neutral invertase and sucrose synthase, were measured in pods and seeds of developing snap bean (Phaseolus vulgaris L.) fruits, and compared with 14C-import, elongation and dry weight accumulation. The data supports the association of specific sucrose-cleaving enzymes with the specific processes that occur in the...

De Novo Enzyme Design Using Rosetta3

PubMed Central

Richter, Florian; Leaver-Fay, Andrew; Khare, Sagar D.; Bjelic, Sinisa; Baker, David

2011-01-01

The Rosetta de novo enzyme design protocol has been used to design enzyme catalysts for a variety of chemical reactions, and in principle can be applied to any arbitrary chemical reaction of interest, The process has four stages: 1) choice of a catalytic mechanism and corresponding minimal model active site, 2) identification of sites in a set of scaffold proteins where this minimal active site can be realized, 3) optimization of the identities of the surrounding residues for stabilizing interactions with the transition state and primary catalytic residues, and 4) evaluation and ranking the resulting designed sequences. Stages two through four of this process can be carried out with the Rosetta package, while stage one needs to be done externally. Here, we demonstrate how to carry out the Rosetta enzyme design protocol from start to end in detail using for illustration the triosephosphate isomerase reaction. PMID:21603656

Biochemical and Molecular Characterization of a Serine Keratinase from Brevibacillus brevis US575 with Promising Keratin-Biodegradation and Hide-Dehairing Activities

PubMed Central

Jaouadi, Nadia Zaraî; Rekik, Hatem; Badis, Abdelmalek; Trabelsi, Sahar; Belhoul, Mouna; Yahiaoui, Amina Benkiar; Aicha, Houda Ben; Toumi, Abdessatar; Bejar, Samir; Jaouadi, Bassem

2013-01-01

Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solid (TSS) loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS) newly isolated from Brevibacillus brevis strain US575. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca2+. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS) were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals used for the dehairing of rabbit, goat, sheep and bovine hides in the leather processing industry. PMID:24146914

Evaluation of pectinolytic activities for oenological uses from psychrotrophic yeasts.

PubMed

Sahay, S; Hamid, B; Singh, P; Ranjan, K; Chauhan, D; Rana, R S; Chaurse, V K

2013-08-01

Of the twenty-three morphotypes of yeasts isolated from soil capable of utilizing pectin as sole carbon source at 6°C, two yeast isolates, one psychrotolerant (PT1) and one psychrophilic (SPY11), were selected according to their ability to secrete pectinolytic enzymes under some oenological conditions (temperature 6 and 12°C and pH 3.5) and ability or inability to grow above 20°C, respectively. As compared to their optimal activity, the three pectinolytic enzymes viz., pectin methyl esterase (PME), endopolygalacturonase (endo-PG) and exopolygalacturonase (exo-PG) isolated and assayed at pH 3.5 from PT1 were found to retain 39, 60 and 60% activity at 12°C and 40, 79 and 74% activity at 28°C, respectively. Likewise, the enzymes PME and endo-PG at pH 3.5 from SPY11 displayed 46 and 86% activity at 12°C and 50 and 60% activity at 28°C, respectively. All these enzymes showed 20-90% of residual activity at pH 3.5 and 6°C. The yeast isolates PT1 and SPY11 were identified as Rhodotorula mucilaginosa and Cystofilobasidium capitatum, respectively, on the basis of morphological, physiological and molecular characteristics. This study presents the first report on pectinolytic activities under major oenological conditions from psychrotolerant isolate R. mucilaginosa PT1 and psychrophilic isolate C. capitatum SPY11. The cold-active pectinolytic enzymes (PME, endo-PG and exo-PG) from the newly isolated and identified psychrophilic yeast Cystofilobasidium capitatum SPY11 and psychrotolerant yeast Rhodotorula mucilaginosa PT1that exhibited 50-80% of their optimum activity under some major oenological conditions pH (3.5) and temperatures (6 and 12°C) could be applied to wine production and juice clarification at low temperature. The psychrotrophic yeasts themselves could be applied to cold process for the production of enzymes thus saving cost of energy and protecting process from contamination. © 2013 The Society for Applied Microbiology.

Alginate/polymethacrylate copolymer microparticles for the intestinal delivery of enzymes.

PubMed

Scocca, Sarah; Faustini, Massimo; Villani, Simona; Munari, Eleonora; Conte, Ubaldo; Russo, Vincenzo; Riccardi, Alessia; Vigo, Daniele; Torre, Maria Luisa

2007-04-01

Proteins administered orally must pass through the gastric environment in order to reach their site of absorption in the intestine. How to protect these exogenously administered proteins from the damaging effects of gastric acid and pepsin proteolytic activity, which often induce irreversible structural and functional alterations to the molecules, is an intriguing challenge. Another problem is the physical and chemical instability of proteins during some technological processes, which often involve the use of organic solvents or high temperatures. In this study we investigated the use of alginate microparticles containing one of two enzymes, an enteric polymer and a lyoprotectant for the intestinal delivery of proteins. The two enzymes tested in this protein delivery system were lactate dehydrogenase and alpha-amylase: the former was chosen because of its sensitivity to denaturation, the latter for its relevance in nutrition and medicine. A sodium alginate aqueous solution containing the enteric polymer, a lyoprotectant and the enzyme was either extruded or sprayed into a calcium chloride solution, with the resultant formation of beads and microspheres which were freeze-dried. About 90% of the enzyme activity was maintained during the process of loading the proteins into the microparticles and the subsequent freeze-drying process. The stability of the encapsulated enzyme in an acid medium and the enzymatic activity in an intestinal environment were then investigated by a dissolution test. This consisted of exposing the microparticles to simulated gastric fluid (pH 1.2) for 2 hours and to simulated intestinal fluid (pH 7.5+/-0.1) for 1 hour. The morphology of the microparticles did not change in the acid environment, whereas they completely dissolved within 3 min in the simulated intestinal fluid. Residual enzymatic activity after the test remained satisfactory for both enzymes. In conclusion, these microparticle systems offer promise for applications in human and veterinary medicine as well as in human and animal nutrition.

A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes

PubMed Central

Mulder, Monique P.C.; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N.; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C.O.; Schulman, Brenda A.; Komander, David; Neefjes, Jacques; Oualid, Farid El; Ovaa, Huib

2016-01-01

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe ‘hops’ and ‘traps’ catalytically active ubiquitin-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades. PMID:27182664

Enzymatic processing of pigmented and non pigmented rice bran on changes in oryzanol, polyphenols and antioxidant activity.

PubMed

Prabhu, Ashish A; Jayadeep, A

2015-10-01

Bran from different rice varieties is a treasure of nutrients and nutraceuticals, and its use is limited due to the poor sensory and functional properties. Application of enzymes can alter the functional and phytochemical properties. So the effect of endo-xylanase, cellulase and their combination on microstructural, nutraceutical and antioxidant properties of pigmented (Jyothi) and non-pigmented (IR64) rice bran were investigated. Scanning electron micrograph revealed micro structural changes in fibre structures on processing. All the enzymatic processing methods resulted in an increase in the content of oryzanol, soluble, bound and total polyphenols, flavonoid and tannin. It also showed an increase in the bioactivity with respect to free radical scavenging activity and total antioxidant activity. However, extent of the increase in bio-actives varied with the type of bran and enzyme application method. Endo-xylanase showed higher percentage difference compared to controls of Jyothi and IR64 bran extracts respectively in the content of the bound (10 & 19 %) and total (20 & 14 %) polyphenols. Combination of both the enzymes resulted in higher percentage increase of bioactive components and properties. It resulted in greater percentage difference compared to controls of Jyothi and IR64 extracts respectively in the content of soluble (58 & 17 %) and total (21 & 14 %) polyphenols, flavonoids (12 & 38 %), γ-oryzanol (10 & 12 %), free radical scavenging activity (64 & 30 %) and total antioxidant activity (82 & 136 %). It may be concluded that enzymatic bio-processing of bran with cellulose and hemicellulose degrading enzymes can improve its nutraceutical properties, and it may be used for development of functional foods.

Gold core/Ceria shell-based redox active nanozyme mimicking the biological multienzyme complex phenomenon

DOE PAGES

Bhagat, Stuti; Srikanth Vallabani, NV; Shutthanandan, Vaithiyalingam; ...

2017-12-02

Catalytically active individual gold (Au) and cerium oxide (CeO 2) nanoparticles (NPs) are well known to exhibit specific enzyme-like activities, such as natural catalase, oxidase, superoxide dismutase, and peroxidase enzymes. Our activities have been maneuvered to design several biological applications such as immunoassays, glucose detection, radiation and free radical protection and tissue engineering. In biological systems, multienzyme complexes are involved in catalyzing important reactions of essential metabolic processes such as respiration, biomolecule synthesis, and photosynthesis. It is well known that metabolic processes linked with multienzyme complexes offer several advantages over reactions catalyzed by individual enzymes. A functional nanozyme depicting multienzymemore » like properties has eluded the researchers in the nanoscience community for the past few decades. Here, we have designed a functional multienzyme in the form of Gold (core)-CeO 2 (shell) nanoparticles (Au/CeO 2 CSNPs) exhibiting excellent peroxidase, catalase, and superoxide dismutase enzyme-like activities that are controlled simply by tuning the pH. The reaction kinetic parameters reveal that the peroxidase-like activity of this core-shell nanozyme is comparable to natural horseradish peroxidase (HRP) enzyme. Unlike peroxidase-like activity exhibited by other nanomaterials, Au/CeO 2 CSNPs showed a decrease in hydroxyl radical formation, suggesting that the biocatalytic reactions are performed by efficient electron transfers. A significant enzyme-like activity of this core-shell nanoparticle was conserved at extreme pH (2 – 11) and temperatures (up to 90 °C), clearly suggesting the superiority over natural enzymes. Further, the utility of peroxidase-like activity of this core-shell nanoparticles was extended for the detection of glucose, which showed a linear range of detection between (100 µM – 1 mM). It is hypothesized that the proximity of the redox potentials of Au+/Au and Ce (III)/Ce (IV) may result in a redox couple promoting the multienzyme activity of core-shell nanoparticles. Au/CeO 2 CSNPs may open new directions for development of single platform sensors in multiple biosensing applications.« less

Gold core/Ceria shell-based redox active nanozyme mimicking the biological multienzyme complex phenomenon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhagat, Stuti; Srikanth Vallabani, NV; Shutthanandan, Vaithiyalingam

Catalytically active individual gold (Au) and cerium oxide (CeO 2) nanoparticles (NPs) are well known to exhibit specific enzyme-like activities, such as natural catalase, oxidase, superoxide dismutase, and peroxidase enzymes. Our activities have been maneuvered to design several biological applications such as immunoassays, glucose detection, radiation and free radical protection and tissue engineering. In biological systems, multienzyme complexes are involved in catalyzing important reactions of essential metabolic processes such as respiration, biomolecule synthesis, and photosynthesis. It is well known that metabolic processes linked with multienzyme complexes offer several advantages over reactions catalyzed by individual enzymes. A functional nanozyme depicting multienzymemore » like properties has eluded the researchers in the nanoscience community for the past few decades. Here, we have designed a functional multienzyme in the form of Gold (core)-CeO 2 (shell) nanoparticles (Au/CeO 2 CSNPs) exhibiting excellent peroxidase, catalase, and superoxide dismutase enzyme-like activities that are controlled simply by tuning the pH. The reaction kinetic parameters reveal that the peroxidase-like activity of this core-shell nanozyme is comparable to natural horseradish peroxidase (HRP) enzyme. Unlike peroxidase-like activity exhibited by other nanomaterials, Au/CeO 2 CSNPs showed a decrease in hydroxyl radical formation, suggesting that the biocatalytic reactions are performed by efficient electron transfers. A significant enzyme-like activity of this core-shell nanoparticle was conserved at extreme pH (2 – 11) and temperatures (up to 90 °C), clearly suggesting the superiority over natural enzymes. Further, the utility of peroxidase-like activity of this core-shell nanoparticles was extended for the detection of glucose, which showed a linear range of detection between (100 µM – 1 mM). It is hypothesized that the proximity of the redox potentials of Au+/Au and Ce (III)/Ce (IV) may result in a redox couple promoting the multienzyme activity of core-shell nanoparticles. Au/CeO 2 CSNPs may open new directions for development of single platform sensors in multiple biosensing applications.« less

Mineralogical impact on long-term patterns of soil nitrogen and phosphorus enzyme activities

NASA Astrophysics Data System (ADS)

Mikutta, Robert; Turner, Stephanie; Meyer-Stüve, Sandra; Guggenberger, Georg; Dohrmann, Reiner; Schippers, Axel

2014-05-01

Soil chronosequences provide a unique opportunity to study microbial activity over time in mineralogical diverse soils of different ages. The main objective of this study was to test the effect of mineralogical properties, nutrient and organic matter availability over whole soil pro-files on the abundance and activity of the microbial communities. We focused on microbio-logical processes involved in nitrogen and phosphorus cycling at the 120,000-year Franz Josef soil chronosequence. Microbial abundances (microbial biomass and total cell counts) and enzyme activities (protease, urease, aminopeptidase, and phosphatase) were determined and related to nutrient contents and mineralogical soil properties. Both, microbial abundances and enzyme activities decreased with soil depth at all sites. In the organic layers, microbial biomass and the activities of N-hydrolyzing enzymes showed their maximum at the intermediate-aged sites, corresponding to a high aboveground biomass. In contrast, the phosphatase activity increased with site age. The activities of N-hydrolyzing enzymes were positively correlated with total carbon and nitrogen contents, whereas the phosphatase activity was negatively correlated with the phosphorus content. In the mineral soil, the enzyme activities were generally low, thus reflecting the presence of strongly sorbing minerals. Sub-strate-normalized enzyme activities correlated negatively to clay content as well as poorly crystalline Al and Fe oxyhydroxides, supporting the view that the evolution of reactive sec-ondary mineral phases alters the activity of the microbial communities by constraining sub-strate availability. Our data suggest a strong mineralogical influence on nutrient cycling par-ticularly in subsoil environments.

Purification and properties of a novel ferricyanide-linked xanthine dehydrogenase from Pseudomonas putida 40.

PubMed Central

Woolfolk, C A

1985-01-01

The isolation of a xanthine dehydrogenase from Pseudomonas putida 40 which utilizes ferricyanide as an electron acceptor at high efficiency is presented. The new activity is separate from the NAD+ and oxygen-utilizing activities of the same organism but displays a broad pattern for reducing substrates typical of those of previously studied xanthine-oxidizing enzymes. Unlike the previously studied enzymes, the new enzyme appears to lack flavin but possess heme and is resistant to cyanide treatment. However, sensitivity of the purified enzyme to methanol and the selective elimination of the activity when tungstate is added to certain growth media suggest a role for molybdenum. The enzyme is subject to a selective proteolytic action during processing which is not accompanied by denaturation or loss of activity and which is minimized by the continuous exposure of the activity to EDTA and phenylmethylsulfonyl fluoride. Electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate suggests that the enzyme is constructed of subunits with a molecular weight of approximately 72,000. Electrophoresis under native conditions of a purified enzyme previously exposed to magnesium ion reveals a series of major and minor activity bands which display some selectivity toward both electron donors and acceptors. An analysis of the effect of gel concentration on this pattern suggests that the enzyme forms a series of charge and size isomers with a pair of trimeric forms predominating. Comparison of the rate of sedimentation of the enzyme in sucrose gradients with its elution profile from standardized Sepharose 6B columns suggests a molecular weight of 255,000 for the major form of the native enzyme. Images PMID:3860496

Lignocellulose-Degrading Microbial Communities in Landfill Sites Represent a Repository of Unexplored Biomass-Degrading Diversity.

PubMed

Ransom-Jones, Emma; McCarthy, Alan J; Haldenby, Sam; Doonan, James; McDonald, James E

2017-01-01

The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, to address the paucity of information on biomass-degrading microbial diversity beyond the gastrointestinal tract, cellulose (cotton) "baits" were incubated in landfill leachate microcosms to enrich the landfill cellulolytic microbial community for taxonomic and functional characterization. Metagenome and 16S rRNA gene amplicon sequencing demonstrated the dominance of Firmicutes , Bacteroidetes , Spirochaetes , and Fibrobacteres in the landfill cellulolytic community. Functional metagenome analysis revealed 8,371 carbohydrate active enzymes (CAZymes) belonging to 244 CAZyme families. In addition to observing biomass-degrading enzymes of anaerobic bacterial "cellulosome" systems of members of the Firmicutes , we report the first detection of the Fibrobacter cellulase system and the Bacteroidetes polysaccharide utilization locus (PUL) in landfill sites. These data provide evidence for the presence of multiple mechanisms of biomass degradation in the landfill microbiome and highlight the extraordinary functional diversity of landfill microorganisms as a rich source of biomass-degrading enzymes of potential biotechnological significance. IMPORTANCE The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, we identified Firmicutes , Spirochaetes , and Fibrobacteres as key phyla in the landfill cellulolytic community, detecting 8,371 carbohydrate active enzymes (CAZymes) that represent at least three of the recognized strategies for cellulose decomposition. These data highlight substantial hydrolytic enzyme diversity in landfill sites as a source of new enzymes for biomass conversion.

Lignocellulose-Degrading Microbial Communities in Landfill Sites Represent a Repository of Unexplored Biomass-Degrading Diversity

PubMed Central

Ransom-Jones, Emma; McCarthy, Alan J.; Haldenby, Sam; Doonan, James

2017-01-01

ABSTRACT The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, to address the paucity of information on biomass-degrading microbial diversity beyond the gastrointestinal tract, cellulose (cotton) “baits” were incubated in landfill leachate microcosms to enrich the landfill cellulolytic microbial community for taxonomic and functional characterization. Metagenome and 16S rRNA gene amplicon sequencing demonstrated the dominance of Firmicutes, Bacteroidetes, Spirochaetes, and Fibrobacteres in the landfill cellulolytic community. Functional metagenome analysis revealed 8,371 carbohydrate active enzymes (CAZymes) belonging to 244 CAZyme families. In addition to observing biomass-degrading enzymes of anaerobic bacterial “cellulosome” systems of members of the Firmicutes, we report the first detection of the Fibrobacter cellulase system and the Bacteroidetes polysaccharide utilization locus (PUL) in landfill sites. These data provide evidence for the presence of multiple mechanisms of biomass degradation in the landfill microbiome and highlight the extraordinary functional diversity of landfill microorganisms as a rich source of biomass-degrading enzymes of potential biotechnological significance. IMPORTANCE The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, we identified Firmicutes, Spirochaetes, and Fibrobacteres as key phyla in the landfill cellulolytic community, detecting 8,371 carbohydrate active enzymes (CAZymes) that represent at least three of the recognized strategies for cellulose decomposition. These data highlight substantial hydrolytic enzyme diversity in landfill sites as a source of new enzymes for biomass conversion. PMID:28776044

Homology to peptide pattern for annotation of carbohydrate-active enzymes and prediction of function.

PubMed

Busk, P K; Pilgaard, B; Lezyk, M J; Meyer, A S; Lange, L

2017-04-12

Carbohydrate-active enzymes are found in all organisms and participate in key biological processes. These enzymes are classified in 274 families in the CAZy database but the sequence diversity within each family makes it a major task to identify new family members and to provide basis for prediction of enzyme function. A fast and reliable method for de novo annotation of genes encoding carbohydrate-active enzymes is to identify conserved peptides in the curated enzyme families followed by matching of the conserved peptides to the sequence of interest as demonstrated for the glycosyl hydrolase and the lytic polysaccharide monooxygenase families. This approach not only assigns the enzymes to families but also provides functional prediction of the enzymes with high accuracy. We identified conserved peptides for all enzyme families in the CAZy database with Peptide Pattern Recognition. The conserved peptides were matched to protein sequence for de novo annotation and functional prediction of carbohydrate-active enzymes with the Hotpep method. Annotation of protein sequences from 12 bacterial and 16 fungal genomes to families with Hotpep had an accuracy of 0.84 (measured as F1-score) compared to semiautomatic annotation by the CAZy database whereas the dbCAN HMM-based method had an accuracy of 0.77 with optimized parameters. Furthermore, Hotpep provided a functional prediction with 86% accuracy for the annotated genes. Hotpep is available as a stand-alone application for MS Windows. Hotpep is a state-of-the-art method for automatic annotation and functional prediction of carbohydrate-active enzymes.

Determinants on an efficient cellulase recycling process for the production of bioethanol from recycled paper sludge under high solid loadings.

PubMed

Gomes, Daniel; Gama, Miguel; Domingues, Lucília

2018-01-01

In spite of the continuous efforts and investments in the last decades, lignocellulosic ethanol is still not economically competitive with fossil fuels. Optimization is still required in different parts of the process. Namely, the cost effective usage of enzymes has been pursued by different strategies, one of them being recycling. Cellulase recycling was analyzed on recycled paper sludge (RPS) conversion into bioethanol under intensified conditions. Different cocktails were studied regarding thermostability, hydrolysis efficiency, distribution in the multiphasic system and recovery from solid. Celluclast showed inferior stability at higher temperatures (45-55 °C), nevertheless its performance at moderate temperatures (40 °C) was slightly superior to other cocktails (ACCELLERASE ® 1500 and Cellic ® CTec2). Celluclast distribution in the solid-liquid medium was also more favorable, enabling to recover 88% of final activity at the end of the process. A central composite design studied the influence of solid concentration and enzyme dosage on RPS conversion by Celluclast. Solids concentration showed a significant positive effect on glucose production, no major limitations being found from utilizing high amounts of solids under the studied conditions. Increasing enzyme loading from 20 to 30 FPU/g cellulose had no significant effect on sugars production, suggesting that 22% solids and 20 FPU/g cellulose are the best operational conditions towards an intensified process. Applying these, a system of multiple rounds of hydrolysis with enzyme recycling was implemented, allowing to maintain the steady levels of enzyme activity with only 50% of enzyme on each recycling stage. Additionally, interesting levels of solid conversion (70-81%) were also achieved, leading to considerable improvements on glucose and ethanol production comparatively with the reports available so far (3.4- and 3.8-fold, respectively). Enzyme recycling viability depends on enzyme distribution between the solid and liquid phases at the end of hydrolysis, as well as enzymes thermostability. Both are critical features to be observed for a judicious choice of enzyme cocktail. This work demonstrates that enzyme recycling in intensified biomass degradation can be achieved through simple means. The process is possibly much more effective at larger scale, hence novel enzyme formulations favoring this possibility should be developed for industrial usage.

Extracellular Enzyme Composition and Functional Characteristics of Aspergillus niger An-76 Induced by Food Processing Byproducts and Based on Integrated Functional Omics.

PubMed

Liu, Lin; Gong, Weili; Sun, Xiaomeng; Chen, Guanjun; Wang, Lushan

2018-02-07

Byproducts of food processing can be utilized for the production of high-value-added enzyme cocktails. In this study, we utilized integrated functional omics technology to analyze composition and functional characteristics of extracellular enzymes produced by Aspergillus niger grown on food processing byproducts. The results showed that oligosaccharides constituted by arabinose, xylose, and glucose in wheat bran were able to efficiently induce the production of extracellular enzymes of A. niger. Compared with other substrates, wheat bran was more effective at inducing the secretion of β-glucosidases from GH1 and GH3 families, as well as >50% of proteases from A1-family aspartic proteases. Compared with proteins induced by single wheat bran or soybean dregs, the protein yield induced by their mixture was doubled, and the time required to reach peak enzyme activity was shortened by 25%. This study provided a technical platform for the complex formulation of various substrates and functional analysis of extracellular enzymes.

Simultaneous measurement of two enzyme activities using infrared spectroscopy: A comparative evaluation of PARAFAC, TUCKER and N-PLS modeling.

PubMed

Baum, Andreas; Hansen, Per Waaben; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

2013-08-06

Enzymes are used in many processes to release fermentable sugars for green production of biofuel, or the refinery of biomass for extraction of functional food ingredients such as pectin or prebiotic oligosaccharides. The complex biomasses may, however, require a multitude of specific enzymes which are active on specific substrates generating a multitude of products. In this paper we use the plant polymer, pectin, to present a method to quantify enzyme activity of two pectolytic enzymes by monitoring their superimposed spectral evolutions simultaneously. The data is analyzed by three chemometric multiway methods, namely PARAFAC, TUCKER3 and N-PLS, to establish simultaneous enzyme activity assays for pectin lyase and pectin methyl esterase. Correlation coefficients Rpred(2) for prediction test sets are 0.48, 0.96 and 0.96 for pectin lyase and 0.70, 0.89 and 0.89 for pectin methyl esterase, respectively. The retrieved models are compared and prediction test sets show that especially TUCKER3 performs well, even in comparison to the supervised regression method N-PLS. Copyright © 2013 Elsevier B.V. All rights reserved.

Optimization of ultrasound-assisted extraction of pectinase enzyme from guava (Psidium guajava) peel: Enzyme recovery, specific activity, temperature, and storage stability.

PubMed

Amid, Mehrnoush; Murshid, Fara Syazana; Manap, Mohd Yazid; Islam Sarker, Zaidul

2016-01-01

This study aimed to investigate the effects of the ultrasound-assisted extraction conditions on the yield, specific activity, temperature, and storage stability of the pectinase enzyme from guava peel. The ultrasound variables studied were sonication time (10-30 min), ultrasound temperature (30-50 °C), pH (2.0-8.0), and solvent-to-sample ratio (2:1 mL/g to 6:1 mL/g). The main goal was to optimize the ultrasound-assisted extraction conditions to maximize the recovery of pectinase from guava peel with the most desirable enzyme-specific activity and stability. Under the optimum conditions, a high yield (96.2%), good specific activity (18.2 U/mg), temperature stability (88.3%), and storage stability (90.3%) of the extracted enzyme were achieved. The optimal conditions were 20 min sonication time, 40 °C temperature, at pH 5.0, using a 4:1 mL/g solvent-to-sample ratio. The study demonstrated that optimization of ultrasound-assisted process conditions for the enzyme extraction could improve the enzymatic characteristics and yield of the enzyme.

Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

NASA Astrophysics Data System (ADS)

Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

2016-07-01

The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

Production and characterization of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean.

PubMed

Stamford, T L; Stamford, N P; Coelho, L C; Araújo, J M

2001-01-01

Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications.

Printable enzyme-embedded materials for methane to methanol conversion

DOE PAGES

Blanchette, Craig D.; Knipe, Jennifer M.; Stolaroff, Joshuah K.; ...

2016-06-15

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scalemore » structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas–liquid reactions.« less

Printable enzyme-embedded materials for methane to methanol conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blanchette, Craig D.; Knipe, Jennifer M.; Stolaroff, Joshuah K.

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scalemore » structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas–liquid reactions.« less

Stabilization of surface-immobilized enzymes using grafted polymers

NASA Astrophysics Data System (ADS)

Moskovitz, Yevgeny; Srebnik, Simcha

2004-03-01

Vast research efforts focus on improving the biocompatibility and biofunctionality of surfaces for artificial implants and organs. A relatively successful approach involves grafting of polymer (usually PEG) on the artificial surface, which significantly improves its biocompatibility. In addition, positioning enzymes on or in the vicinity of the surface can significantly enhance bioseparation processes. However, the catalytic activity of the anchored enzyme is often drastically impaired by the nonnatural environment, leading to loss of activity and denaturation. We study protein adsorption and stabilization by grafted polymers using a mean-field lattice model. The model protein is designed as a compact HP with a specific bulk conformation reproducing a catalytic cleft of natural enzymes. Using hydrophilic grafted polymers of tailored length and density, we show that the conformation as well as hydrophobic and active centers of the model enzyme can be restored. This research is inspired by the problem of biocompatibility and biofunctionality of surfaces for artificial implants and organs.

Printable enzyme-embedded materials for methane to methanol conversion

PubMed Central

Blanchette, Craig D.; Knipe, Jennifer M.; Stolaroff, Joshuah K.; DeOtte, Joshua R.; Oakdale, James S.; Maiti, Amitesh; Lenhardt, Jeremy M.; Sirajuddin, Sarah; Rosenzweig, Amy C.; Baker, Sarah E.

2016-01-01

An industrial process for the selective activation of methane under mild conditions would be highly valuable for controlling emissions to the environment and for utilizing vast new sources of natural gas. The only selective catalysts for methane activation and conversion to methanol under mild conditions are methane monooxygenases (MMOs) found in methanotrophic bacteria; however, these enzymes are not amenable to standard enzyme immobilization approaches. Using particulate methane monooxygenase (pMMO), we create a biocatalytic polymer material that converts methane to methanol. We demonstrate embedding the material within a silicone lattice to create mechanically robust, gas-permeable membranes, and direct printing of micron-scale structures with controlled geometry. Remarkably, the enzymes retain up to 100% activity in the polymer construct. The printed enzyme-embedded polymer motif is highly flexible for future development and should be useful in a wide range of applications, especially those involving gas–liquid reactions. PMID:27301270

[Hydrogen production and enzyme activity of acidophilic strain X-29 at different C/N ratio].

PubMed

Li, Qiu-bo; Xing, De-feng; Ren, Nan-qi; Zhao, Li-hua; Song, Ye-ying

2006-04-01

Some fermentative bacteria can produce hydrogen by utilizing carbohydrate and other kinds of organic compounds as substrates. Hydrogen production was also determined by both the limiting of growth and related enzyme activity in energy metabolism. Carbon and nitrogen are needed for the growth and metabolism of microorganisms. In addition, the carbon/nitrogen (C/N) ratio can influence the material metabolized and the energy produced. In order to improve the hydrogen production efficiency of the bacteria, we analyzed the effect of different C/N ratios on hydrogen production and the related enzyme activities in the acidophilic strain X-29 using batch test. The results indicate that the differences in the metabolism level and enzyme activity are obvious at different C/N ratios. Although the difference in liquid fermentative products produced per unit of biomass is not obvious, hydrogen production is enhanced at a specifically determined ratio. At a C/N ratio of 14 the accumulative hydrogen yield of strain X-29 reaches the maximum, 2210.9 mL/g. At different C/N ratios, the expression of hydrogenase activity vary; the activity of hydrogenase decrease quickly after reaching a maximum along with the fermentation process, but the time of expression is short. The activity of alcohol dehydrogenase (ADH) tend to stabilize after reaching a peak along with the fermentation process, the difference in expression activity is little, and the expression period is long at different C/N ratios. At a C/N ratio of 14 hydrogenase and ADH reach the maximum 2.88 micromol x (min x mg)(-1) and 33.2 micromol x (min x mg)(-1), respectively. It is shown that the C/N ratio has an important effect on enhancing hydrogen production and enzyme activity.

Modelling of different enzyme productions by solid-state fermentation on several agro-industrial residues.

PubMed

Diaz, Ana Belen; Blandino, Ana; Webb, Colin; Caro, Ildefonso

2016-11-01

A simple kinetic model, with only three fitting parameters, for several enzyme productions in Petri dishes by solid-state fermentation is proposed in this paper, which may be a valuable tool for simulation of this type of processes. Basically, the model is able to predict temporal fungal enzyme production by solid-state fermentation on complex substrates, maximum enzyme activity expected and time at which these maxima are reached. In this work, several fermentations in solid state were performed in Petri dishes, using four filamentous fungi grown on different agro-industrial residues, measuring xylanase, exo-polygalacturonase, cellulose and laccase activities over time. Regression coefficients after fitting experimental data to the proposed model turned out to be quite high in all cases. In fact, these results are very interesting considering, on the one hand, the simplicity of the model and, on the other hand, that enzyme activities correspond to different enzymes, produced by different fungi on different substrates.

Enzymatic dynamics into the Eisenia fetida (Savigny, 1826) gut during vermicomposting of coffee husk and market waste in a tropical environment.

PubMed

Ordoñez-Arévalo, Berenice; Guillén-Navarro, Karina; Huerta, Esperanza; Cuevas, Raúl; Calixto-Romo, M Angeles

2018-01-01

Epigeic worms modify microbial communities through their digestive processes, thereby influencing the decomposition of organic matter in vermicomposting systems. Nevertheless, the enzyme dynamics within the gut of tropically adapted earthworms is unknown, and the enzymes involved have not been simultaneously studied. The activities of 19 hydrolytic enzymes within three different sections of the intestine of Eisenia fetida were determined over a fasting period and at 24 h and 30, 60, and 90 days of vermicomposting, and data were evaluated by multivariate analyses. There were found positive correlations between the maximal activity of glycosyl hydrolases and one esterase with the anterior intestine (coincident with the reduction of hemicellulose in the substrate) and the activity of the protease α-chymotrypsin with posterior intestine. The results suggest that activities of enzymes change in a coordinated manner within each gut section, probably influenced by selective microbial enzyme enrichment and by the availability of nutrients throughout vermicomposting.

Data-Driven Microbial Modeling for Soil Carbon Decomposition and Stabilization

NASA Astrophysics Data System (ADS)

Luo, Yiqi; Chen, Ji; Chen, Yizhao; Feng, Wenting

2017-04-01

Microorganisms have long been known to catalyze almost all the soil organic carbon (SOC) transformation processes (e.g., decomposition, stabilization, and mineralization). Representing microbial processes in Earth system models (ESMs) has the potential to improve projections of SOC dynamics. We have recently examined (1) relationships of microbial functions with environmental factors and (2) microbial regulations of decomposition and other key soil processes. According to three lines of evidence, we have developed a data-driven enzyme (DENZY) model to simulate soil microbial decomposition and stabilization. First, our meta-analysis of 64 published field studies showed that field experimental warming significantly increased soil microbial communities abundance, which is negatively correlated with the mean annual temperature. The negative correlation indicates that warming had stronger effects in colder than warmer regions. Second, we found that the SOC decomposition, especially the transfer between labile SOC and protected SOC, is nonlinearly regulated by soil texture parameters, such as sand and silt contents. Third, we conducted a global analysis of the C-degrading enzyme activities, soil respiration, and SOC content under N addition. Our results show that N addition has contrasting effects on cellulase (hydrolytic C-degrading enzymes) and ligninase (oxidative C-degrading enzymes) activities. N-enhanced cellulase activity contributes to the minor stimulation of soil respiration whereas N-induced repression on ligninase activity drives soil C sequestration. Our analysis links the microbial extracellular C-degrading enzymes to the SOC dynamics at ecosystem scales across scores of experimental sites around the world. It offers direct evidence that N-induced changes in microbial community and physiology play fundamental roles in controlling the soil C cycle. Built upon those three lines of empirical evidence, the DENZY model includes two enzyme pools and explicitly characterizes two classes of extracellular enzyme activities: one that degrades organic molecules containing both C and N (e.g., chitin or protein) and another that degrades only C (e.g., cellulose). The DENZY model assumes that the microbes allocate resources to different enzyme pools so as to exactly satisfy microbial CN ratio stoichiometry in response to changes in climate conditions and soil attributes. The DENZY model can simulate differential effects of nitrogen fertilization on the two groups of enzymes and thus soil respiration and SOC dynamics. We will select field experimental sites to test the DENZY model. With increasing amounts of available observations and data synthesis, this DENZY model will be better parameterized and have a potential to reveal how responses of microbial enzymes to environmental changes regulate soil carbon decomposition and stabilization.

Thermal denaturation: is solid-state fermentation really a good technology for the production of enzymes?

PubMed

Muller dos Santos, Marcelo; Souza da Rosa, Alexandre; Dal'Boit, Silvia; Mitchell, David A; Krieger, Nadia

2004-07-01

The potential for thermal denaturation to cause enzyme losses during solid-state fermentation processes for the production of enzymes was examined, using the protease of Penicillium fellutanum as a model system. The frequency factor and activation energies for the first-order denaturation of this enzyme were determined as 3.447 x 10(59) h(-1) and 364,070 Jmol(-1), respectively. These values were incorporated into a mathematical model of enzyme deactivation, which was used to investigate the consequences of subjecting this protease to temporal temperature profiles reported in the literature for mid-height in a 34 cm high packed-bed bioreactor of 150 mm diameter. In this literature source, temperature profiles were measured for 5, 15 and 25 liters per minute of air and enzyme activities were measured as a function of time. The enzyme activity profiles predicted by the model were distributed similarly, one relative to the other, as had been found in the experimental study, with substantial amounts of denaturation being predicted when the substrate temperature exceeded 40 degrees C, which occurred at the lower two airflow rates. A mathematical model of a well-mixed bioreactor was used to explore the difficulties that would be faced at large scale. It suggests that even with airflows as high as one volume per volume per minute, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation. This work highlights the extra care that must be taken in scaling up solid-state fermentation processes for the production of thermolabile products. Copyright 2003 Elsevier Ltd.

Neuraminidase as an enzymatic marker for detecting airborne Influenza virus and other viruses.

PubMed

Turgeon, Nathalie; Toulouse, Marie-Josée; Ho, Jim; Li, Dongqing; Duchaine, Caroline

2017-02-01

Little information is available regarding the effectiveness of air samplers to collect viruses and regarding the effects of sampling processes on viral integrity. The neuraminidase enzyme is present on the surface of viruses that are of agricultural and medical importance. It has been demonstrated that viruses carrying this enzyme can be detected using commercial substrates without having to process the sample by methods such as RNA extraction. This project aims at evaluating the effects of 3 aerosol-sampling devices on the neuraminidase enzyme activity of airborne viruses. The purified neuraminidase enzymes from Clostridium perfringens, a strain of Influenza A (H1N1) virus, the FluMist influenza vaccine, and the Newcastle disease virus were used as models. The neuraminidase models were aerosolized in aerosol chambers and sampled with 3 different air samplers (SKC BioSampler, 3-piece cassettes with polycarbonate filters, and Coriolis μ) to assess the effect on neuraminidase enzyme activity. Our results demonstrated that Influenza virus and Newcastle disease virus neuraminidase enzymes are resistant to aerosolization and sampling with all air samplers tested. Moreover, we demonstrated that the enzymatic neuraminidase assay is as sensitive as RT-qPCR for detecting low concentrations of Influenza virus and Newcastle disease virus. Therefore, given the sensitivity of the assay and its compatibility with air sampling methods, viruses carrying the neuraminidase enzyme can be rapidly detected from air samples using neuraminidase activity assay without having to preprocess the samples.

Determining the safety of enzymes used in animal feed.

PubMed

Pariza, Michael W; Cook, Mark

2010-04-01

The purpose of this paper is to provide guidance for evaluating the safety of enzyme preparations used in animal feed. Feed enzymes are typically added to animal feed to increase nutrient bioavailability by acting on feed components prior to or after consumption, i.e., within the gastrointestinal tract. In contrast, food processing enzymes are generally used during processing and then inactivated or removed prior to consumption. The enzymes used in both applications are almost always impure mixtures of active enzyme and other metabolites from the production strain, hence similar safety evaluation procedures for both are warranted. We propose that the primary consideration should be the safety of the production strain and that the decision tree mechanism developed previously for food processing enzymes (Pariza and Johnson, 2001) is appropriate for determining the safety of feed enzymes. Thoroughly characterized non-pathogenic, non-toxigenic microbial strains with a history of safe use in enzyme manufacture are also logical candidates for generating safe strain lineages, from which additional strains may be derived via genetic modification by traditional and non-traditional strategies. For new feed enzyme products derived from a safe strain lineage, it is important to ensure a sufficiently high safety margin for the intended use, and that the product complies with appropriate specifications for chemical and microbial contamination. Copyright 2009 Elsevier Inc. All rights reserved.

Liposomes as protective capsules for active silica sol-gel biocomposite synthesis.

PubMed

Li, Ye; Yip, Wai Tak

2005-09-21

Using liposome to shield an enzyme from hostile chemical environments during the sol-gel formation process has resulted in a novel approach to synthesizing silica sol-gel biocomposite materials. By reporting the encapsulation of horseradish peroxidase and firefly luciferase, we demonstrate that this new protocol can produce silica biocomposites that are more active than trapping the enzymes directly into hydrogels.

Combining an Optical Resonance Biosensor with Enzyme Activity Kinetics to Understand Protein Adsorption and Denaturation

PubMed Central

Wilson, Kerry A.; Finch, Craig A.; Anderson, Phillip; Vollmer, Frank; Hickman, James J.

2014-01-01

Understanding protein adsorption and resultant conformation changes on modified and unmodified silicon dioxide surfaces is a subject of keen interest in biosensors, microfluidic systems and for medical diagnostics. However, it has been proven difficult to investigate the kinetics of the adsorption process on these surfaces as well as understand the topic of the denaturation of proteins and its effect on enzyme activity. A highly sensitive optical whispering gallery mode (WGM) resonator was used to study a catalytic enzyme’s adsorption processes on different silane modified glass substrates (plain glass control, DETA, 13F, and SiPEG). The WGM sensor was able to obtain high resolution kinetic data of glucose oxidase (GO) adsorption with sensitivity of adsorption better than that possible with SPR. The kinetic data, in combination with a functional assay of the enzyme activity, was used to test hypotheses on adsorption mechanisms. By fitting numerical models to the WGM sensograms for protein adsorption, and by confirming numerical predictions of enzyme activity in a separate assay, we were able to identify mechanisms for GO adsorption on different alkylsilanes and infer information about the adsorption of protein on nanostructured surfaces. PMID:25453976

Versatile de novo enzyme activity in capsid proteins from an engineered M13 bacteriophage library.

PubMed

Casey, John P; Barbero, Roberto J; Heldman, Nimrod; Belcher, Angela M

2014-11-26

Biocatalysis has grown rapidly in recent decades as a solution to the evolving demands of industrial chemical processes. Mounting environmental pressures and shifting supply chains underscore the need for novel chemical activities, while rapid biotechnological progress has greatly increased the utility of enzymatic methods. Enzymes, though capable of high catalytic efficiency and remarkable reaction selectivity, still suffer from relative instability, high costs of scaling, and functional inflexibility. Herein, we developed a biochemical platform for engineering de novo semisynthetic enzymes, functionally modular and widely stable, based on the M13 bacteriophage. The hydrolytic bacteriophage described in this paper catalyzes a range of carboxylic esters, is active from 25 to 80 °C, and demonstrates greater efficiency in DMSO than in water. The platform complements biocatalysts with characteristics of heterogeneous catalysis, yielding high-surface area, thermostable biochemical structures readily adaptable to reactions in myriad solvents. As the viral structure ensures semisynthetic enzymes remain linked to the genetic sequences responsible for catalysis, future work will tailor the biocatalysts to high-demand synthetic processes by evolving new activities, utilizing high-throughput screening technology and harnessing M13's multifunctionality.

Structure and Function of Viral Deubiquitinating Enzymes.

PubMed

Bailey-Elkin, Ben A; Knaap, Robert C M; Kikkert, Marjolein; Mark, Brian L

2017-11-10

Post-translational modification of cellular proteins by ubiquitin regulates numerous cellular processes, including innate and adaptive immune responses. Ubiquitin-mediated control over these processes can be reversed by cellular deubiquitinating enzymes (DUBs), which remove ubiquitin from cellular targets and depolymerize polyubiquitin chains. The importance of protein ubiquitination to host immunity has been underscored by the discovery of viruses that encode proteases with deubiquitinating activity, many of which have been demonstrated to actively corrupt cellular ubiquitin-dependent processes to suppress innate antiviral responses and promote viral replication. DUBs have now been identified in diverse viral lineages, and their characterization is providing valuable insights into virus biology and the role of the ubiquitin system in host antiviral mechanisms. Here, we provide an overview of the structural biology of these fascinating viral enzymes and their role innate immune evasion and viral replication. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

PubMed

Chen, Hongzhang; Shao, Meixue; Li, Hongqiang

2014-03-05

The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r=0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation. Copyright © 2013 Elsevier Inc. All rights reserved.

[Definition of parameters of the condition of oxidizing stress in smooth muscle cells under influence of exogenous nitroso-glutatyon in vitro].

PubMed

Kapilevich, L V; Nosarev, A V; D'iakova, E Iu; Andrushkevich, V V; Nasedkina, A K; Nosareva, O L; Davlet'iarova, K V; Ogorodova, L M; Kovalev, I V; Baskakov, M B; Medvedev, M A

2007-08-01

Influence of exogenous nitroso-glutatyon on intensity of oxidizing processes in smooth muscles of colon and bronchial tubes in intact and atopic sensitised porpoises (guinea pigs) was studied. In sensitised porpoises, antioxidant protection has been initially reduced against the background of increased maintenance of products of oxidizing that reflects a picture of oxidizing damage and can be associated with an inflammatory process. In incubation with nitroso-glutatyon, a decrease in activities of syperoxiddismutase and catalase is marked and, in sensitised animals, this effect has been expressed to a lesser degree. Syperoxiddismutase and catalase are antioxidant for the enzymes participating in protection of cells from free-radical damage. A dose-dependence decrease in activity catalase and syperoxiddismutase is defined by a parity of the enzymes participating in disintegration of nitrosoglutatyon and the enzymes which have kept antioxidant activity.

Metrological aspects of enzyme production

NASA Astrophysics Data System (ADS)

Kerber, T. M.; Dellamora-Ortiz, G. M.; Pereira-Meirelles, F. V.

2010-05-01

Enzymes are frequently used in biotechnology to carry out specific biological reactions, either in industrial processes or for the production of bioproducts and drugs. Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified applications. Lipase production by microorganisms is described in several published papers; however, none of them refer to metrological evaluation and the estimation of the uncertainty in measurement. Moreover, few of them refer to process optimization through experimental design. The objectives of this work were to enhance lipase production in shaken-flasks with Yarrowia lipolytica cells employing experimental design and to evaluate the uncertainty in measurement of lipase activity. The highest lipolytic activity obtained was about three- and fivefold higher than the reported activities of CRMs BCR-693 and BCR-694, respectively. Lipase production by Y. lipolytica cells aiming the classification as certified reference material is recommended after further purification and stability studies.

In vitro assembly of catalase.

PubMed

Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars

2014-10-10

Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Histochemical investigation of the activity of oxidoreductases in the skin lesions of lepromatous leprosy patients*

PubMed Central

Žuravleva, G. F.

1972-01-01

This paper reports an investigation of the activity of three basic groups of oxidoreductases in lepromatous leprosy: specific dehydrogenases, flavoprotein enzymes, and cytochrome oxidase. The activity of the enzymes was studied before treatment, at various stages of treatment during exacerbations, and in the stage of regression. The data obtained are of importance for evaluating metabolic process in the cells of the specific infiltrates and the dermal connective tissue in leprosy, for determining the nature and intensity of the inflammatory process, and for control purposes in cases of regression. ImagesFig. 4Fig. 5Fig. 6Fig. 1Fig. 2Fig. 3 PMID:4342274

Expression and characterization of fifteen Rhizopus oryzae 99-880 polygalacturonase enzymes in Pichia pastoris.

PubMed

Mertens, Jeffrey A; Bowman, Michael J

2011-04-01

Polygalacturonase (PG) enzymes hydrolyze the long polygalacturonic acid chains found in the smooth regions of pectin. Interest in this enzyme class continues due to their ability to macerate tissues of economically important crops and their use in a number of industrial processes. Rhizopus oryzae has a large PG gene family with 15 of 18 genes encoding unique active enzymes. The PG enzymes, 12 endo-PG and 3 exo-galacturonases, were expressed in Pichia pastoris and purified enabling biochemical characterization to gain insight into the maintenance of this large gene family within the Rhizopus genome. The 15 PG enzymes have a pH optima ranging from 4.0 to 5.0. Temperature optima of the 15 PG enzymes vary from 30 to 40 °C. While the pH and temperature optima do little to separate the enzymes, the specific activity of the enzymes is highly variable ranging from over 200 to less than 1 μmol/min/mg. A general pattern related to the groupings found in the phylogentic tree was visible with the group containing the exo-PG enzymes demonstrating the lowest specific activity. Finally, the progress curves of the PG enzymes, contained within the phylogenetic group that includes the exo-PG enzymes, acting on trigalacturonic acid lend additional support to the idea that the ancestral form of PG in Rhizopus is endolytic and exolytic function evolved later.

Improved enzymatic hydrolysis of microcrystalline cellulose (Avicel PH101) by polyethylene glycol addition.

PubMed

Ouyang, Jia; Dong, Zhenwei; Song, Xiangyang; Lee, Xin; Chen, Mu; Yong, Qiang

2010-09-01

The effects of additives on hydrolysis of microcrystalline cellulose (Avicel PH101) were examined using commercial cellulose-degrading enzymes (Celluclast 1.5L and Novozyme 188). Polyethylene glycol 4000 (PEG4000) was the most effective additive tested. When PEG4000 was added at 0.05 g/g glucan, the conversion of Avicel PH101 increased 91% (from 41.1% to 78.9%). The cellulase activity of Celluclast 1.5L increased 27.5% with PEG4000 addition. A positive effect on enzyme stabilities of Celluclast 1.5L and Novozyme 188 also occurred with PEG4000 addition. During hydrolysis process, significant changes in free protein concentration and cellulase activity were observed on Avicel PH101. More than 90% of the original enzyme activity remained in the solution after 48 h hydrolysis. Thus, PEG4000 addition is an efficient method to enhance digestibility of cellulosic materials and make enzyme recovery possible and valuable. This provides an opportunity of decreasing the operational cost of the hydrolysis process. (c) 2010 Elsevier Ltd. All rights reserved.

Fermentation and complex enzyme hydrolysis enhance total phenolics and antioxidant activity of aqueous solution from rice bran pretreated by steaming with α-amylase.

PubMed

Liu, Lei; Zhang, Ruifen; Deng, Yuanyuan; Zhang, Yan; Xiao, Juan; Huang, Fei; Wen, Wei; Zhang, Mingwei

2017-04-15

In this study, rice bran was successively steamed with α-amylase, fermented with lactic acid bacteria, and hydrolyzed with complex enzymes. The changes in phenolic profiles and antioxidant activities of the corresponding aqueous solutions from three stages were investigated. Compared to the first stage, fermentation and complex enzyme hydrolysis significantly increased the total phenolics, total flavonoids, total FRAP and ORAC values by 59.2%, 56.6%, 73.6% and 45.4%, respectively. Twelve individual phenolics present in free or soluble conjugate forms were also analyzed during the processing. Ferulic acid was released in the highest amount among different phenolics followed by protocatechuic acid. Moreover, a major proportion of phenolics existed as soluble conjugates. The results showed that fermentation and complex enzyme hydrolysis enhanced total phenolics and antioxidant activities of aqueous solution from rice bran pretreated by steaming with α-amylase. This research could provide basis for the processing of rice bran beverage rich in phenolics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Deciphering the Mode of Action of the Processive Polysaccharide Modifying Enzyme Dermatan Sulfate Epimerase 1 by Hydrogen-Deuterium Exchange Mass Spectrometry.

PubMed

Tykesson, Emil; Mao, Yang; Maccarana, Marco; Pu, Yi; Gao, Jinshan; Lin, Cheng; Zaia, Joseph; Westergren-Thorsson, Gunilla; Ellervik, Ulf; Malmström, Lars; Malmström, Anders

2016-02-01

Distinct from template-directed biosynthesis of nucleic acids and proteins, the enzymatic synthesis of heterogeneous polysaccharides is a complex process that is difficult to study using common analytical tools. Therefore, the mode of action and processivity of those enzymes are largely unknown. Dermatan sulfate epimerase 1 (DS-epi1) is the predominant enzyme during the formation of iduronic acid residues in the glycosaminoglycan dermatan sulfate. Using recombinant DS-epi1 as a model enzyme, we describe a tandem mass spectrometry-based method to study the mode of action of polysaccharide processing enzymes. The enzyme action on the substrate was monitored by hydrogen-deuterium exchange mass spectrometry and the sequence information was then fed into mathematical models with two different assumptions of the mode of action for the enzyme: processive reducing end to non-reducing end, and processive non-reducing end to reducing end. Model data was scored by correlation to experimental data and it was found that DS-epi1 attacks its substrate on a random position, followed by a processive mode of modification towards the non-reducing end and that the substrate affinity of the enzyme is negatively affected by each additional epimerization event. It could also be shown that the smallest active substrate was the reducing end uronic acid in a tetrasaccharide and that octasaccharides and longer oligosaccharides were optimal substrates. The method of using tandem mass spectrometry to generate sequence information of the complex enzymatic products in combination with in silico modeling can be potentially applied to study the mode of action of other enzymes involved in polysaccharide biosynthesis.

Ultra-high-pressure processing improves proteolysis and release of bioactive peptides with activation activities on alcohol metabolic enzymes in vitro from mushroom foot protein.

PubMed

Zhao, Rui-Jie; Huo, Chun-Yan; Qian, Yang; Ren, Di-Feng; Lu, Jun

2017-09-15

This study was to find an effective process to extract bioactive peptides from mushroom foot and determine their effects on activation of alcohol metabolic enzymes in vitro. The optimum extraction assisted by ultra-high-pressure processing of mushroom foot peptides was obtained with a pressure of 400MPa and a processing time of 10min. After ultrafiltration, peptides with molecular weight of 0-3kDa had the highest activity to activate alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) by 70.79% and 71.35%, respectively. Following dextran gel chromatography, two peaks (p-I and p-II) appeared and the activation activities on ADH and ALDH of p-I were 72.00% and 73.43%, both higher than p-II. Nine peptides were found in p-I as determined by LC-MS/MS, and two of them (IPLH and IPIVLL) were synthesized. IPLH activated ADH and ALDH by 42.7% and 29.2% respectively, which were higher than IPIVLL. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Effects of snow pack on soil nitrogen transformation enzyme activities in a subalpine Abies faxioniana forest of western Sichuan, China].

PubMed

Xiong, Li; Xu, Zhen-Feng; Wu, Fu-Zhong; Yang, Wan-Qin; Yin, Rui; Li, Zhi-Ping; Gou, Xiao-Lin; Tang, Shi-Shan

2014-05-01

This study characterized the dynamics of the activities of urease, nitrate reductase and nitrite reductase in both soil organic layer and mineral soil layer under three depths of snow pack (deep snowpack, moderate snowpack and shallow snowpack) over the three critical periods (snow formed period, snow stable period, and snow melt period) in the subalpine Abies faxoniana forest of western Sichuan in the winter of 2012 and 2013. Throughout the winter, soil temperature under deep snowpack increased by 46.2% and 26.2%, respectively in comparison with moderate snowpack and shallow snowpack. In general, the three nitrogen-related soil enzyme activities under shallow snowpack were 0.8 to 3.9 times of those under deep snowpack during the winter. In the beginning and thawing periods of seasonal snow pack, shallow snowpack significantly increased the activities of urease, nitrate and nitrite reductase enzyme in both soil organic layer and mineral soil layer. Although the activities of the studied enzymes in soil organic layer and mineral soil layer were observed to be higher than those under deep- and moderate snowpacks in deep winter, no significant difference was found under the three snow packs. Meanwhile, the effects of snowpack on the activities of the measured enzymes were related with season, soil layer and enzyme type. Significant variations of the activities of nitrogen-related enzymes were found in three critical periods over the winter, and the three measured soil enzymes were significantly higher in organic layer than in mineral layer. In addition, the activities of the three measured soil enzymes were closely related with temperature and moisture in soils. In conclusion, the decrease of snow pack induced by winter warming might increase the activities of soil enzymes related with nitrogen transformation and further stimulate the process of wintertime nitrogen transformation in soils of the subalpine forest.

An active second dihydrofolate reductase enzyme is not a feature of rat and mouse, but they do have activity in their mitochondria.

PubMed

Hughes, Linda; Carton, Robert; Minguzzi, Stefano; McEntee, Gráinne; Deinum, Eva E; O'Connell, Mary J; Parle-McDermott, Anne

2015-07-08

The identification of a second functional dihydrofolate reductase enzyme in humans, DHFRL1, led us to consider whether this is also a feature of rodents. We demonstrate that dihydrofolate reductase activity is also a feature of the mitochondria in both rat and mouse but this is not due to a second enzyme. While our phylogenetic analysis revealed that RNA-mediated DHFR duplication events did occur across the mammal tree, the duplicates in brown rat and mouse are likely to be processed pseudogenes. Humans have evolved the need for two separate enzymes while laboratory rats and mice have just one. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Demystifying O-GlcNAcylation: hints from peptide substrates.

PubMed

Shi, Jie; Ruijtenbeek, Rob; Pieters, Roland J

2018-03-22

O-GlcNAcylation, analogous to phosphorylation, is an essential post-translational modification of proteins at Ser/Thr residues with a single β-N-acetylglucosamine moiety. This dynamic protein modification regulates many fundamental cellular processes and its deregulation has been linked to chronic diseases such as cancer, diabetes and neurodegenerative disorders. Reversible attachment and removal of O-GlcNAc is governed only by O-GlcNAc transferase and O-GlcNAcase, respectively. Peptide substrates, derived from natural O-GlcNAcylation targets, function in the catalytic cores of these two enzymes by maintaining interactions between enzyme and substrate, which makes them ideal models for the study of O-GlcNAcylation and deglycosylation. These peptides provide valuable tools for a deeper understanding of O-GlcNAc processing enzymes. By taking advantage of peptide chemistry, recent progress in the study of activity and regulatory mechanisms of these two enzymes has advanced our understanding of their fundamental specificities as well as their potential as therapeutic targets. Hence, this review summarizes the recent achievements on this modification studied at the peptide level, focusing on enzyme activity, enzyme specificity, direct function, site-specific antibodies and peptide substrate-inspired inhibitors.

Influence of very low doses of mediators on fungal laccase activity - nonlinearity beyond imagination

PubMed Central

Malarczyk, Elzbieta; Kochmanska-Rdest, Janina; Jarosz-Wilkolazka, Anna

2009-01-01

Laccase, an enzyme responsible for aerobic transformations of natural phenolics, in industrial applications requires the presence of low-molecular substances known as mediators, which accelerate oxidation processes. However, the use of mediators is limited by their toxicity and the high costs of exploitation. The activation of extracellular laccase in growing fungal culture with highly diluted mediators, ABTS and HBT is described. Two high laccase-producing fungal strains, Trametes versicolor and Cerrena unicolor, were used in this study as a source of enzyme. Selected dilutions of the mediators significantly increased the activity of extracellular laccase during 14 days of cultivation what was distinctly visible in PAGE technique and in colorimetric tests. The same mediator dilutions increased demethylation properties of laccase, which was demonstrated during incubation of enzyme with veratric acid. It was established that the activation effect was assigned to specific dilutions of mediators. Our dose-response dilution process smoothly passes into the range of action of homeopathic dilutions and is of interest for homeopaths. PMID:19732425

Enzymology under global change: organic nitrogen turnover in alpine and sub-Arctic soils.

PubMed

Weedon, James T; Aerts, Rien; Kowalchuk, George A; van Bodegom, Peter M

2011-01-01

Understanding global change impacts on the globally important carbon storage in alpine, Arctic and sub-Arctic soils requires knowledge of the mechanisms underlying the balance between plant primary productivity and decomposition. Given that nitrogen availability limits both processes, understanding the response of the soil nitrogen cycle to shifts in temperature and other global change factors is crucial for predicting the fate of cold biome carbon stores. Measurements of soil enzyme activities at different positions of the nitrogen cycling network are an important tool for this purpose. We review a selection of studies that provide data on potential enzyme activities across natural, seasonal and experimental gradients in cold biomes. Responses of enzyme activities to increased nitrogen availability and temperature are diverse and seasonal dynamics are often larger than differences due to experimental treatments, suggesting that enzyme expression is regulated by a combination of interacting factors reflecting both nutrient supply and demand. The extrapolation from potential enzyme activities to prediction of elemental nitrogen fluxes under field conditions remains challenging. Progress in molecular '-omics' approaches may eventually facilitate deeper understanding of the links between soil microbial community structure and biogeochemical fluxes. In the meantime, accounting for effects of the soil spatial structure and in situ variations in pH and temperature, better mapping of the network of enzymatic processes and the identification of rate-limiting steps under different conditions should advance our ability to predict nitrogen fluxes.

Microbial enzymes with special characteristics for biotechnological applications.

PubMed

Nigam, Poonam Singh

2013-08-23

This article overviews the enzymes produced by microorganisms, which have been extensively studied worldwide for their isolation, purification and characterization of their specific properties. Researchers have isolated specific microorganisms from extreme sources under extreme culture conditions, with the objective that such isolated microbes would possess the capability to bio-synthesize special enzymes. Various Bio-industries require enzymes possessing special characteristics for their applications in processing of substrates and raw materials. The microbial enzymes act as bio-catalysts to perform reactions in bio-processes in an economical and environmentally-friendly way as opposed to the use of chemical catalysts. The special characteristics of enzymes are exploited for their commercial interest and industrial applications, which include: thermotolerance, thermophilic nature, tolerance to a varied range of pH, stability of enzyme activity over a range of temperature and pH, and other harsh reaction conditions. Such enzymes have proven their utility in bio-industries such as food, leather, textiles, animal feed, and in bio-conversions and bio-remediations.

Production and purification of amylolytic enzymes for saccharification of microalgal biomass.

PubMed

Rodrigues, Éllen Francine; Ficanha, Aline Matuella Moreira; Dallago, Rogério Marcos; Treichel, Helen; Reinehr, Christian Oliveira; Machado, Tainara Paula; Nunes, Greice Borges; Colla, Luciane Maria

2017-02-01

The aim of this study was the production of amylolytic enzymes by solid state or submerged fermentations (SSF or SF, respectively), followed by purification using chemical process or microfiltration and immobilization of purified enzymes in a polyurethane support. The free and immobilized enzymes obtained were used to evaluate enzymatic hydrolysis of the polysaccharides of Spirulina. Microfiltration of the crude extracts resulted in an increase in their specific activity and thermal stability at 40°C and 50°C for 24h, as compared to extracts obtained by SSF and SF. Immobilization of polyurethane purified enzyme produced yields of 332% and 205% for the enzymes obtained by SF and SSF, respectively. Free or immobilized enzymes favor the generation of fermentable sugar, being the application of the purified and immobilized enzymes in the hydrolysis of microalgal polysaccharides considered a promising alternative towards development of the bioethanol production process from microalgal biomass. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of Pectinase from Bacillus subtilis Strain Btk 27 and Its Potential Application in Removal of Mucilage from Coffee Beans.

PubMed

Oumer, Oliyad Jeilu; Abate, Dawit

2017-01-01

The demand for enzymes in the global market is projected to rise at a fast pace in recent years. There has been a great increase in industrial applications of pectinase owing to their significant biotechnological uses. For applying enzymes at industrial scale primary it is important to know the features of the enzyme. Thus, this study was undertaken with aims of characterizing the pectinase enzyme from Bacillus subtilis strain Btk27 and proving its potential application in demucilisation of coffee. In this study, the maximum pectinase activity was achieved at pH 7.5 and 50°C. Also, the enzyme activity was found stimulated with Mg2+ and Ca2+ metal ions. Moreover, it was stable on EDTA, Trixton-100, Tween 80, and Tween 20. Since Bacillus subtilis strain Btk27 was stable in most surfactants and inhibitors it could be applicable in various industries whenever pectin degradation is needed. The enzyme K m and V max values were identified as 1.879 mg/ml and 149.6 U, respectively. The potential application of the enzyme for coffee processing was studied, and it is found that complete removal of mucilage from coffee beans within 24 hours of treatment indicates the potential application in coffee processing.

Characterization of Pectinase from Bacillus subtilis Strain Btk 27 and Its Potential Application in Removal of Mucilage from Coffee Beans

PubMed Central

Abate, Dawit

2017-01-01

The demand for enzymes in the global market is projected to rise at a fast pace in recent years. There has been a great increase in industrial applications of pectinase owing to their significant biotechnological uses. For applying enzymes at industrial scale primary it is important to know the features of the enzyme. Thus, this study was undertaken with aims of characterizing the pectinase enzyme from Bacillus subtilis strain Btk27 and proving its potential application in demucilisation of coffee. In this study, the maximum pectinase activity was achieved at pH 7.5 and 50°C. Also, the enzyme activity was found stimulated with Mg2+ and Ca2+ metal ions. Moreover, it was stable on EDTA, Trixton-100, Tween 80, and Tween 20. Since Bacillus subtilis strain Btk27 was stable in most surfactants and inhibitors it could be applicable in various industries whenever pectin degradation is needed. The enzyme Km and Vmax values were identified as 1.879 mg/ml and 149.6 U, respectively. The potential application of the enzyme for coffee processing was studied, and it is found that complete removal of mucilage from coffee beans within 24 hours of treatment indicates the potential application in coffee processing. PMID:29085675

A structural model of PpoA derived from SAXS-analysis-implications for substrate conversion.

PubMed

Koch, Christian; Tria, Giancarlo; Fielding, Alistair J; Brodhun, Florian; Valerius, Oliver; Feussner, Kirstin; Braus, Gerhard H; Svergun, Dmitri I; Bennati, Marina; Feussner, Ivo

2013-09-01

In plants and mammals, oxylipins may be synthesized via multi step processes that consist of dioxygenation and isomerization of the intermediately formed hydroperoxy fatty acid. These processes are typically catalyzed by two distinct enzyme classes: dioxygenases and cytochrome P450 enzymes. In ascomycetes biosynthesis of oxylipins may proceed by a similar two-step pathway. An important difference, however, is that both enzymatic activities may be combined in a single bifunctional enzyme. These types of enzymes are named Psi-factor producing oxygenases (Ppo). Here, the spatial organization of the two domains of PpoA from Aspergillus nidulans was analyzed by small-angle X-ray scattering and the obtained data show that the enzyme exhibits a relatively flat trimeric shape. Atomic structures of the single domains were obtained by template-based structure prediction and docked into the enzyme envelope of the low resolution structure obtained by SAXS. EPR-based distance measurements between the tyrosyl radicals formed in the activated dioxygenase domain of the enzyme supported the trimeric structure obtained from SAXS and the previous assignment of Tyr374 as radical-site in PpoA. Furthermore, two phenylalanine residues in the cytochrome P450 domain were shown to modulate the specificity of hydroperoxy fatty acid rearrangement. Copyright © 2013 Elsevier B.V. All rights reserved.

The structure and function of Alzheimer's gamma secretase enzyme complex.

PubMed

Krishnaswamy, Sudarsan; Verdile, Giuseppe; Groth, David; Kanyenda, Limbikani; Martins, Ralph N

2009-01-01

The production and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterizes Alzheimer's disease (AD). A multi-subunit enzyme complex, referred to as gamma (gamma) secretase, plays a pivotal role in the generation of Abeta from its parent molecule, the amyloid precursor protein (APP). Four core components (presenilin, nicastrin, aph-1, and pen-2) interact in a high-molecular-weight complex to perform intramembrane proteolysis on a number of membrane-bound proteins, including APP and Notch. Inhibitors and modulators of this enzyme have been assessed for their therapeutic benefit in AD. However, although these agents reduce Abeta levels, the majority have been shown to have severe side effects in pre-clinical animal studies, most likely due to the enzymes role in processing other proteins involved in normal cellular function. Current research is directed at understanding this enzyme and, in particular, at elucidating the roles that each of the core proteins plays in its function. In addition, a number of interacting proteins that are not components of gamma-secretase also appear to play important roles in modulating enzyme activity. This review will discuss the structural and functional complexity of the gamma-secretase enzyme and the effects of inhibiting its activity.

Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

NASA Astrophysics Data System (ADS)

Gottschalk, Leda M. F.; Bon, Elba P. S.; Nobrega, Ronaldo

It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.

The Algicidal Fungus Trametes versicolor F21a Eliminating Blue Algae via Genes Encoding Degradation Enzymes and Metabolic Pathways Revealed by Transcriptomic Analysis

PubMed Central

Dai, Wei; Chen, Xiaolin; Wang, Xuewen; Xu, Zimu; Gao, Xueyan; Jiang, Chaosheng; Deng, Ruining; Han, Guomin

2018-01-01

The molecular mechanism underlying the elimination of algal cells by fungal mycelia has not been fully understood. Here, we applied transcriptomic analysis to investigate the gene expression and regulation at time courses of Trametes versicolor F21a during the algicidal process. The obtained results showed that a total of 193, 332, 545, and 742 differentially expressed genes were identified at 0, 6, 12, and 30 h during the algicidal process, respectively. The gene ontology terms were enriched into glucan 1,4-α-glucosidase activity, hydrolase activity, lipase activity, and endopeptidase activity. The KEGG pathways were enriched in degradation and metabolism pathways including Glycolysis/Gluconeogenesis, Pyruvate metabolism, the Biosynthesis of amino acids, etc. The total expression levels of all Carbohydrate-Active enZYmes (CAZyme) genes for the saccharide metabolism were increased by two folds relative to the control. AA5, GH18, GH5, GH79, GH128, and PL8 were the top six significantly up-regulated modules among 43 detected CAZyme modules. Four available homologous decomposition enzymes of other species could partially inhibit the growth of algal cells. The facts suggest that the algicidal mode of T. versicolor F21a might be associated with decomposition enzymes and several metabolic pathways. The obtained results provide a new candidate way to control algal bloom by application of decomposition enzymes in the future. PMID:29755442

Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

NASA Astrophysics Data System (ADS)

Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

2015-04-01

Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse change. Key words: Andosols, β-glucosidase, Cellobiohydrolase, Chitinase, Phosphatase, Mt. Kilimanjaro

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolasemore » activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific glycosyl hydrolase activities. It remains unclear whether loss of endocellulase activity to lignin binding is problematic for biomass conversion.« less

Molecular Basis of Impaired Glycogen Metabolism during Ischemic Stroke and Hypoxia

PubMed Central

Hossain, Mohammed Iqbal; Roulston, Carli Lorraine; Stapleton, David Ian

2014-01-01

Background Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke. Results Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia. Conclusion Our study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity. PMID:24858129

Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity[S

PubMed Central

Jaishy, Bharat; Zhang, Quanjiang; Chung, Heaseung S.; Riehle, Christian; Soto, Jamie; Jenkins, Stephen; Abel, Patrick; Cowart, L. Ashley; Van Eyk, Jennifer E.; Abel, E. Dale

2015-01-01

Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs. PMID:25529920

Heparanase: a target for drug discovery in cancer and inflammation

PubMed Central

McKenzie, E A

2007-01-01

The remodelling of the extracellular matrix (ECM) has been shown to be highly upregulated in cancer and inflammation and is critically linked to the processes of invasion and metastasis. One of the key enzymes involved in specifically degrading the heparan sulphate (HS) component of the ECM is the endo-β-glucuronidase enzyme heparanase. Processing of HS by heparanase releases both a host of bioactive growth factors anchored within the mesh of the ECM as well as defined fragments of HS capable of promoting cellular proliferation. The finding that heparanase is elevated in a wide variety of tumor types and is subsequently linked to the development of pathological processes has led to an explosion of therapeutic strategies to inhibit its enzyme activity. So far only one compound, the sulphated oligosaccharide PI88, which both inhibits heparanase activity and has effects on growth factor binding has reached clinical trials where it has shown to have promising efficacy. The scene has clearly been set however for a new generation of compounds, either specific to the enzyme or with dual roles, to emerge from the lab and enter the clinic. The aim of this review is to describe the current drug discovery status of small molecule, sugar and neutralising antibody inhibitors of heparanase enzyme activity. Potential strategies will also be discussed on the selection of suitable biomarker strategies for specific monitoring of in vivo heparanase inhibition which will be crucial for both animal model and clinical trial testing. PMID:17339837

Hyaluronan degrading silica nanoparticles for skin cancer therapy

NASA Astrophysics Data System (ADS)

Scodeller, P.; Catalano, P. N.; Salguero, N.; Duran, H.; Wolosiuk, A.; Soler-Illia, G. J. A. A.

2013-09-01

We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human melanoma bearing mice and compared with the non-immobilized enzyme, on the basis of equal enzymatic activity. Alcian Blue staining of A375 tumors indicated large overexpression of hyaluronan. At the end of the experiment, tumor volume reduction with SiNP-immobilized Hyal was significantly enhanced compared to non-immobilized Hyal. Field emission scanning electron microscopy (FE-SEM) images together with energy dispersive X-ray spectroscopy (EDS) spectra confirmed the presence of SiNP on the tumor. We mean a proof of concept: this extracellular matrix (ECM) degrading enzyme, immobilized on SiNP, is a more effective local adjuvant of cancer drugs than the non-immobilized enzyme. This could prove useful in future therapies using other or a combination of ECM degrading enzymes.We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human melanoma bearing mice and compared with the non-immobilized enzyme, on the basis of equal enzymatic activity. Alcian Blue staining of A375 tumors indicated large overexpression of hyaluronan. At the end of the experiment, tumor volume reduction with SiNP-immobilized Hyal was significantly enhanced compared to non-immobilized Hyal. Field emission scanning electron microscopy (FE-SEM) images together with energy dispersive X-ray spectroscopy (EDS) spectra confirmed the presence of SiNP on the tumor. We mean a proof of concept: this extracellular matrix (ECM) degrading enzyme, immobilized on SiNP, is a more effective local adjuvant of cancer drugs than the non-immobilized enzyme. This could prove useful in future therapies using other or a combination of ECM degrading enzymes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr02787b

From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

PubMed

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-10

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

Characterisation of a starch-hydrolysing enzyme of Aspergillus niger.

PubMed

Suresh, C; Dubey, A K; Srikanta, S; Kumar, S U; Karanth, N G

1999-05-01

A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 degrees C and glucose, maltose and maltodextrins at 70 degrees C as primary products, suggested significant applications for the enzyme in starch-processing industries.

Virulence-Associated Enzymes of Cryptococcus neoformans

PubMed Central

Almeida, Fausto; Wolf, Julie M.

2015-01-01

Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review focuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans. The enzymatic suite of C. neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalian host. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology. PMID:26453651

Proteases and nucleases involved in the biphasic digestion process of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae).

PubMed

Lomate, Purushottam R; Bonning, Bryony C

2018-07-01

Management of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), an invasive, agricultural pest in the United States, has presented significant challenges. This polyphagous insect uses both extra-oral and gut-based digestion thwarting protein- or nucleotide-based control strategies. The objective of this study was to biochemically characterize the digestive enzymes (proteases and nucleases) from the saliva, salivary gland and the gut of H. halys. Enzyme profiles for the two tissues and saliva radically differ: The pH optimum for proteases in the gut was six, with cysteine proteases predominant. In contrast, the alkaline pH optima for protease activity in the salivary gland (8-10) and saliva (7) reflected abundant serine protease and cathepsin activities. RNase enzymes were most abundant in saliva, while dsRNase and DNase activities were higher in the salivary gland and saliva compared to those in the gut. These very different enzyme profiles highlight the biphasic digestive system used by this invasive species for efficient processing of plant nutrients. Knowledge of H. halys digestive physiology will allow for counteractive measures targeting digestive enzymes or for appropriate protection of protein- or nucleotide-based management options targeting this pest. © 2018 Wiley Periodicals, Inc.

Functionalized Anodic Aluminum Oxide Membrane–Electrode System for Enzyme Immobilization

PubMed Central

2015-01-01

A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min–1 on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min–1. UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process. PMID:25025628

Conformational Transition Pathway in the Activation Process of Allosteric Glucokinase

PubMed Central

Shi, Ting; Zhao, Yaxue; Chen, Yingyi; Li, Xiaobai; Liu, Xinyi; Huang, Zhimin; Zhang, Jian

2013-01-01

Glucokinase (GK) is a glycolytic enzyme that plays an important role in regulating blood glucose level, thus acting as a potentially attractive target for drug discovery in the treatment of diabetes of the young type 2 and persistent hyperinsulinemic hypoglycemia of infancy. To characterize the activation mechanism of GK from the super-open state (inactive state) to the closed state (active state), a series of conventional molecular dynamics (MD) and targeted MD (TMD) simulations were performed on this enzyme. Conventional MD simulation showed a specific conformational ensemble of GK when the enzyme is inactive. Seven TMD simulations depicted a reliably conformational transition pathway of GK from the inactive state to the active state, and the components important to the conformational change of GK were identified by analyzing the detailed structures of the TMD trajectories. In combination with the inactivation process, our findings showed that the whole conformational pathway for the activation-inactivation-activation of GK is a one-direction circulation, and the active state is less stable than the inactive state in the circulation. Additionally, glucose was demonstrated to gradually modulate its binding pose with the help of residues in the large domain and connecting region of GK during the activation process. Furthermore, the obtained energy barriers were used to explain the preexisting equilibrium and the slow binding kinetic process of the substrate by GK. The simulated results are in accordance with the recent findings from the mutagenesis experiments and kinetic analyses. Our observations reveal a complicated conformational process in the allosteric protein, resulting in new knowledge about the delicate mechanisms for allosteric biological macromolecules that will be useful in drug design for targeting allosteric proteins. PMID:23409066

Improved bread-baking process using Saccharomyces cerevisiae displayed with engineered cyclodextrin glucanotransferase.

PubMed

Shim, Jae-Hoon; Seo, Nam-Seok; Roh, Sun-Ah; Kim, Jung-Wan; Cha, Hyunju; Park, Kwan-Hwa

2007-06-13

A bread-baking process was developed using a potential novel enzyme, cyclodextrin glucanotransferase[3-18] (CGTase[3-18]), that had previously been engineered to have enhanced hydrolyzing activity with little cyclodextrin (CD) formation activity toward starch. CGTase[3-18] was primarily manipulated to be displayed on the cell surface of Saccharomyces cerevisiae. S. cerevisiae carrying pdeltaCGT integrated into the chromosome exhibited starch-hydrolyzing activity at the same optimal pH and temperature as the free enzyme. Volumes of the bread loaves and rice cakes prepared using S. cerevisiae/pdeltaCGT increased by 20% and 45%, respectively, with no detectable CD. Retrogradation rates of the bread and rice cakes decreased significantly during storage. In comparison to the wild type, S. cerevisiae/pdeltaCGT showed improved viability during four freeze-thaw cycles. The results indicated that CGTase[3-18] displayed on the surface of yeast hydrolyzed starch to glucose and maltose that can be used more efficiently for yeast fermentation. Therefore, display of an antistaling enzyme on the cell surface of yeast has potential for enhancing the baking process.

Chitin-Lignin Material as a Novel Matrix for Enzyme Immobilization

PubMed Central

Zdarta, Jakub; Klapiszewski, Łukasz; Wysokowski, Marcin; Norman, Małgorzata; Kołodziejczak-Radzimska, Agnieszka; Moszyński, Dariusz; Ehrlich, Hermann; Maciejewski, Hieronim; Stelling, Allison L.; Jesionowski, Teofil

2015-01-01

Innovative materials were made via the combination of chitin and lignin, and the immobilization of lipase from Aspergillus niger. Analysis by techniques including FTIR, XPS and 13C CP MAS NMR confirmed the effective immobilization of the enzyme on the surface of the composite support. The electrokinetic properties of the resulting systems were also determined. Results obtained from elemental analysis and by the Bradford method enabled the determination of optimum parameters for the immobilization process. Based on the hydrolysis reaction of para-nitrophenyl palmitate, a determination was made of the catalytic activity, thermal and pH stability, and reusability. The systems with immobilized enzymes were found to have a hydrolytic activity of 5.72 mU, and increased thermal and pH stability compared with the native lipase. The products were also shown to retain approximately 80% of their initial catalytic activity, even after 20 reaction cycles. The immobilization process, using a cheap, non-toxic matrix of natural origin, leads to systems with potential applications in wastewater remediation processes and in biosensors. PMID:25903282

Optimal immobilization of β-galactosidase onto κ-carrageenan gel beads using response surface methodology and its applications.

PubMed

Elnashar, Magdy M; Awad, Ghada E; Hassan, Mohamed E; Mohy Eldin, Mohamed S; Haroun, Bakry M; El-Diwany, Ahmed I

2014-01-01

β-Galactosidase (β-gal) was immobilized by covalent binding on novel κ-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 2(2) full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55 °C compared to the free enzyme. The apparent K(m) after immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocity Vmax was 131.2 μ mol · min(-1) while it was 177.1 μ mol · min(-1) for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of the κ-carrageenan beads loaded with β -galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.

Strategies for discovery and improvement of enzyme function: state of the art and opportunities.

PubMed

Kaul, Praveen; Asano, Yasuhisa

2012-01-01

Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non-conventional media, using new substrates for old enzymes, active-site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities. © 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Evaluation of the energy efficiency of enzyme fermentation by mechanistic modeling.

PubMed

Albaek, Mads O; Gernaey, Krist V; Hansen, Morten S; Stocks, Stuart M

2012-04-01

Modeling biotechnological processes is key to obtaining increased productivity and efficiency. Particularly crucial to successful modeling of such systems is the coupling of the physical transport phenomena and the biological activity in one model. We have applied a model for the expression of cellulosic enzymes by the filamentous fungus Trichoderma reesei and found excellent agreement with experimental data. The most influential factor was demonstrated to be viscosity and its influence on mass transfer. Not surprisingly, the biological model is also shown to have high influence on the model prediction. At different rates of agitation and aeration as well as headspace pressure, we can predict the energy efficiency of oxygen transfer, a key process parameter for economical production of industrial enzymes. An inverse relationship between the productivity and energy efficiency of the process was found. This modeling approach can be used by manufacturers to evaluate the enzyme fermentation process for a range of different process conditions with regard to energy efficiency. Copyright © 2011 Wiley Periodicals, Inc.

Horseradish peroxidase-nanoclay hybrid particles of high functional and colloidal stability.

PubMed

Pavlovic, Marko; Rouster, Paul; Somosi, Zoltan; Szilagyi, Istvan

2018-08-15

Highly stable dispersions of enzyme-clay nanohybrids of excellent horseradish peroxidase activity were developed. Layered double hydroxide nanoclay was synthesized and functionalized with heparin polyelectrolyte to immobilize the horseradish peroxidase enzyme. The formation of a saturated heparin layer on the platelets led to charge inversion of the positively charged bare nanoclay and to highly stable aqueous dispersions. Great affinity of the enzyme to the surface modified platelets resulted in strong horseradish peroxidase adsorption through electrostatic and hydrophobic interactions as well as hydrogen bonding network and prevented enzyme leakage from the obtained material. The enzyme kept its functional integrity upon immobilization and showed excellent activity in decomposition of hydrogen peroxide and oxidation of an aromatic compound in the test reactions. In addition, remarkable long term functional stability of the enzyme-nanoclay hybrid was observed making the developed colloidal system a promising antioxidant candidate in biomedical treatments and industrial processes. Copyright © 2018 Elsevier Inc. All rights reserved.

Kinetics of leather dyeing pretreated with enzymes: role of acid protease.

PubMed

Kanth, Swarna Vinodh; Venba, Rajangam; Jayakumar, Gladstone Christopher; Chandrababu, Narasimhan Kannan

2009-04-01

In the present investigation, kinetics of dyeing involving pretreatment with acid protease has been presented. Application of acid protease in dyeing process resulted in increased absorption and diffusion of dye into the leather matrix. Enzyme treatment at 1% concentration, 60 min duration and 50 degrees C resulted in maximum of 98% dye exhaustion and increased absorption rate constants. The final exhaustion (C(infinity)) for the best fit of CI Acid Black 194 dye has been 98.5% with K and r2 values from the modified Cegarra-Puente isotherm as 0.1033 and 0.0631. CI Acid Black 194 being a 2:1 metal complex acid dye exhibited higher absorption rate than the acid dye CI Acid Black 210. A reduction in 50% activation energy calculated from Arrhenius equation has been observed in enzyme assisted dyeing process of both the dyes that substantiates enhanced dye absorption. The absorption rate constant calculated with modified Cegarra-Puente equation confirm higher rate constants and faster kinetics for enzyme assisted dyeing process. Enzyme treated leather exhibited richness of color and shade when compared with control. The present study substantiates the essential role of enzyme pretreatment as an eco-friendly leather dyeing process.

Variations in organic carbon, aggregation, and enzyme activities of gangue-fly ash-reconstructed soils with sludge and arbuscular mycorrhizal fungi during 6-year reclamation.

PubMed

Yin, Ningning; Zhang, Zhen; Wang, Liping; Qian, Kuimei

2016-09-01

Mining activities can cause drastic disturbances in soil properties, which adversely affect the nutrient cycling and soil environment. As a result, many efforts have been made to explore suitable reclamation strategies that can be applied to accelerate ecology restoration. In this study, we reconstructed mine soils with fly ash, gangue, sludge, planted ryegrass, and inoculated arbuscular mycorrhizal fungi (AMF) in Pangzhuang mine of Xuzhou during 2009 to 2015. The soil aggregation process, enzyme activities (i.e., invertase, urease and acid phosphatase activities), soil organic carbon (SOC) as well as other soil nutrients such as nitrogen, phosphorus, and potassium contents of the reconstructed mine soils were monitored during 6-year reclamation. The integrated application of sludge and AMF led to a promising reclamation performance of mining areas, in which soil aggregate stability, enzyme activities, SOC, and ryegrass biomass were effectively enhanced. The micro-aggregates (< 0.25 mm) decreased with the increase of macro-aggregates (> 0.25 mm) during the reclamation, indicating that macro-aggregates were gradually formed from micro-aggregates during the pedogenesis of reconstructed mine soils. The correlation analysis shows that SOC contents in aggregate fraction of 0.25∼0.5 mm were correlated with aggregate distribution and enzyme activities. Enzyme activities, however, were not significantly correlated with aggregate distribution. The outcomes from the present study could enrich our understanding on soil property changes in pedogenesis process of reconstructed mine soils, and meanwhile, the employment of sludge combined with AMF is suggested to be an effective alternative for the mine soil reclamation.

Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

NASA Astrophysics Data System (ADS)

Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

2015-12-01

We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

Enzyme processing of textiles in reverse micellar solution.

PubMed

Sawada, K; Ueda, M

2001-08-23

Scouring of cotton using pectinase enzyme, bioscouring, in reverse micellar system was studied. The effectiveness of bioscouring was evaluated by measuring weight loss of cotton, analyzing pectin and cotton wax remaining and by wetness testing. Pectinase enzyme showed excellent activity even in organic media, and the effectiveness of scouring was equivalent or better than that achieved by conventional alkaline process or bioscouring in aqueous media. Enzymatic modification of wool using protease enzyme in the same system was also studied. It has found that felting property and tensile strength of wool fabrics treated by protease in reverse micellar system were superior to those in aqueous media. Possibilities of utilization of the same system for the subsequent textile dyeing process were also investigated. It was found that cotton and polyester fabrics were dyed satisfactorily by reverse micellar system compared to conventional aqueous system.

The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders

PubMed Central

Auchus, Richard J.

2011-01-01

Steroidogenesis entails processes by which cholesterol is converted to biologically active steroid hormones. Whereas most endocrine texts discuss adrenal, ovarian, testicular, placental, and other steroidogenic processes in a gland-specific fashion, steroidogenesis is better understood as a single process that is repeated in each gland with cell-type-specific variations on a single theme. Thus, understanding steroidogenesis is rooted in an understanding of the biochemistry of the various steroidogenic enzymes and cofactors and the genes that encode them. The first and rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone by a single enzyme, P450scc (CYP11A1), but this enzymatically complex step is subject to multiple regulatory mechanisms, yielding finely tuned quantitative regulation. Qualitative regulation determining the type of steroid to be produced is mediated by many enzymes and cofactors. Steroidogenic enzymes fall into two groups: cytochrome P450 enzymes and hydroxysteroid dehydrogenases. A cytochrome P450 may be either type 1 (in mitochondria) or type 2 (in endoplasmic reticulum), and a hydroxysteroid dehydrogenase may belong to either the aldo-keto reductase or short-chain dehydrogenase/reductase families. The activities of these enzymes are modulated by posttranslational modifications and by cofactors, especially electron-donating redox partners. The elucidation of the precise roles of these various enzymes and cofactors has been greatly facilitated by identifying the genetic bases of rare disorders of steroidogenesis. Some enzymes not principally involved in steroidogenesis may also catalyze extraglandular steroidogenesis, modulating the phenotype expected to result from some mutations. Understanding steroidogenesis is of fundamental importance to understanding disorders of sexual differentiation, reproduction, fertility, hypertension, obesity, and physiological homeostasis. PMID:21051590

"Trojan Horse" strategy for deconstruction of biomass for biofuels production.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sinclair, Michael B.; Hadi, Masood Z.; Timlin, Jerilyn Ann

2008-08-01

Production of renewable biofuels to displace fossil fuels currently consumed in the transportation sector is a pressing multi-agency national priority. Currently, nearly all fuel ethanol is produced from corn-derived starch. Dedicated 'energy crops' and agricultural waste are preferred long-term solutions for renewable, cheap, and globally available biofuels as they avoid some of the market pressures and secondary greenhouse gas emission challenges currently facing corn ethanol. These sources of lignocellulosic biomass are converted to fermentable sugars using a variety of chemical and thermochemical pretreatments, which disrupt cellulose and lignin cross-links, allowing exogenously added recombinant microbial enzymes to more efficiently hydrolyze themore » cellulose for 'deconstruction' into glucose. This process is plagued with inefficiencies, primarily due to the recalcitrance of cellulosic biomass, mass transfer issues during deconstruction, and low activity of recombinant deconstruction enzymes. Costs are also high due to the requirement for enzymes and reagents, and energy-intensive and cumbersome pretreatment steps. One potential solution to these problems is found in synthetic biology; they propose to engineer plants that self-produce a suite of cellulase enzymes targeted to the apoplast for cleaving the linkages between lignin and cellulosic fibers; the genes encoding the degradation enzymes, also known as cellulases, are obtained from extremophilic organisms that grow at high temperatures (60-100 C) and acidic pH levels (<5). These enzymes will remain inactive during the life cycle of the plant but become active during hydrothermal pretreatment i.e., elevated temperatures. Deconstruction can be integrated into a one-step process, thereby increasing efficiency (cellulose-cellulase mass-transfer rates) and reducing costs. The proposed disruptive technologies address biomass deconstruction processes by developing transgenic plants encoding a suite of enzymes used in cellulosic deconstruction. The unique aspects of this technology are the rationally engineered, highly productive extremophilic enzymes, targeted to specific cellular locations (apoplast) and their dormancy during normal plant proliferation, which become Trojan horses during pretreatment conditions. They have been leveraging established Sandia's enzyme-engineering and imaging capabilities. Their technical approach not only targets the recalcitrance and mass-transfer problem during biomass degradation but also eliminates the costs associated with industrial-scale production of microbial enzymes added during processing.« less

Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production.

PubMed

Peng, Qing; Wang, Xu; Shang, Meng; Huang, Jinjin; Guan, Guohua; Li, Ying; Shi, Bo

2014-01-04

Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF. A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with K(m) and V(max) values of 1.148 mM and 3497 μmol∙min⁻¹∙mg⁻¹, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes. The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production.

Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production

PubMed Central

2014-01-01

Background Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF. Results A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with Km and Vmax values of 1.148 mM and 3497 μmol∙min-1∙mg-1, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes. Conclusions The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production. PMID:24387764

EFFECTS OF FIVE DIVERSE LIGNOCELLULOSIC DIETS ON DIGESTIVE ENZYME BIOCHEMISTRY IN THE TERMITE Reticulitermes flavipes.

PubMed

Karl, Zachary J; Scharf, Michael E

2015-10-01

Termites have recently drawn much attention as models for biomass processing, mainly due to their lignocellulose digestion capabilities and mutualisms with cellulolytic gut symbionts. This research used the lower termite Reticulitermes flavipes to investigate gut enzyme activity changes in response to feeding on five diverse lignocellulosic diets (cellulose filter paper [FP], pine wood [PW], beech wood xylan [X], corn stover [CS], and soybean residue [SB]). Our objectives were to compare whole-gut digestive enzyme activity and host versus symbiont contributions to enzyme activity after feeding on these diets. Our hypothesis was that enzyme activities would vary among diets as an adaptive mechanism enabling termites and symbiota to optimally utilize variable resources. Results support our "diet-adaptation" hypothesis and further indicate that, in most cases, host contributions are greater than those of symbionts with respect to the enzymes and activities studied. The results obtained thus provide indications as to which types of transcriptomic resources, termite or symbiont, are most relevant for developing recombinant enzyme cocktails tailored to specific feedstocks. With regard to the agricultural feedstocks tested (CS and SB), our results suggest endoglucanase and exoglucanase (cellobiohydrolase) activities are most relevant for CS breakdown; whereas endoglucanase and xylosidase activities are relevant for SB breakdown. However, other unexplored activities than those tested may also be important for breakdown of these two feedstocks. These findings provide new protein-level insights into diet adaptation by termites, and also complement host-symbiont metatranscriptomic studies that have been completed for R. flavipes after FP, PW, CS, and SB feeding. © 2015 Wiley Periodicals, Inc.

Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

PubMed

Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

2015-12-11

Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Delivery of Formulated Industrial Enzymes with Acoustic Technology.

PubMed

Hwang, Jennifer Dorcas; Ortiz-Maldonado, Mariliz; Paramonov, Sergey

2016-02-01

Industrial enzymes are instrumental in many applications, including carbohydrate processing, fabric and household care, biofuels, food, and animal nutrition, among others. Enzymes have to be active and stable not only in harsh application conditions, but also during shipment and storage. In protein stability studies, formulated concentrated enzyme solutions are frequently diluted gravimetrically prior to enzyme activity measurements, making it challenging to move toward more high-throughput techniques using conventional robotic equipment. Current assay methods pose difficulties when measuring highly concentrated proteins. For example, plastic pipette tips can introduce error because proteins adsorb to the tip surface, despite the presence of detergents, decreasing precision and overall efficiency of protein activity assays. Acoustic liquid handling technology, frequently used for various dilute small-molecule assays, may overcome such problems. Originally shown to effectively deliver dilute solutions of small molecules, this technology is used here as an effective alternative to the aforementioned challenge with viscous concentrated protein solutions. Because the acoustic liquid handler transfers nanoliter quantities of liquids without using pipette tips and without sample loss, it rapidly and uniformly prepares assay plates for enzyme activity measurements within minutes. This increased efficiency transforms the nature of enzyme stability studies toward high precision and throughput. © 2015 Society for Laboratory Automation and Screening.

Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides.

PubMed

Do Rego, Jean Luc; Seong, Jae Young; Burel, Delphine; Leprince, Jerôme; Luu-The, Van; Tsutsui, Kazuyoshi; Tonon, Marie-Christine; Pelletier, Georges; Vaudry, Hubert

2009-08-01

Neuroactive steroids synthesized in neuronal tissue, referred to as neurosteroids, are implicated in proliferation, differentiation, activity and survival of nerve cells. Neurosteroids are also involved in the control of a number of behavioral, neuroendocrine and metabolic processes such as regulation of food intake, locomotor activity, sexual activity, aggressiveness, anxiety, depression, body temperature and blood pressure. In this article, we summarize the current knowledge regarding the existence, neuroanatomical distribution and biological activity of the enzymes responsible for the biosynthesis of neurosteroids in the brain of vertebrates, and we review the neuronal mechanisms that control the activity of these enzymes. The observation that the activity of key steroidogenic enzymes is finely tuned by various neurotransmitters and neuropeptides strongly suggests that some of the central effects of these neuromodulators may be mediated via the regulation of neurosteroid production.

Enzymatic detection of As(III) in aqueous solution using alginate immobilized pumpkin urease: optimization of process variables by response surface methodology.

PubMed

Talat, Mahe; Prakash, Om; Hasan, S H

2009-10-01

Urease immobilized on alginate was utilized to detect and quantify As(3+) in aqueous solution. Urease from the seeds of pumpkin (vegetable waste) was purified to apparent homogeneity by heat treatment and gel filtration (Sephadex G-200). Further enzyme was entrapped in 3.5% alginate beads. Urea hydrolysis by enzyme revealed a clear dependence on the concentration and interaction time of As(3+). The process variables effecting the quantitation of As(3+) was investigated using central composite design with Minitab 15 software. The predicted results were found in good agreement (R(2)=96.71%) with experimental results indicating the applicability of proposed model. The multiple regression analysis and ANOVA showed that enzyme activity decreased with increase of As(3+) concentration and interaction time. 3D plot and contour plot between As(3+) concentration and interaction time was helpful to predict residual activity of enzyme for a particular As(3+) at a particular time.

Efficient production of L-asparaginase from Bacillus licheniformis with low-glutaminase activity: optimization, scale up and acrylamide degradation studies.

PubMed

Mahajan, Richi V; Saran, Saurabh; Kameswaran, Karthikeya; Kumar, Vinod; Saxena, R K

2012-12-01

L-Asparaginase has potential as an anti-cancer drug and for prevention of acrylamide formation in fried and baked foods. Production of the enzyme by Bacillus licheniformis (RAM-8) was optimized by process engineering using a statistical modeling approach and a maximum yield of 32.26 IU/ml was achieved. The L-asparaginase exhibited glutaminase activity of only 0.8 IU/ml and would therefore be less prone to cause the side effects associated with asparaginase therapy compared to enzyme preparations with higher glutaminase activities. When production was carried out in a 30-L bioreactor, enzyme production reached 29.94 IU/ml in 15 h. The enzyme inhibited poly-acrylamide formation in 10% acrylamide solution and reduced acrylamide formation in fried potatoes by 80%. Copyright © 2012 Elsevier Ltd. All rights reserved.

Sol-gel encapsulation of pullulanase in the presence of hybrid magnetic (Fe3O4-chitosan) nanoparticles improves thermal and operational stability.

PubMed

Long, Jie; Li, Xingfei; Zhan, Xiaobei; Xu, Xueming; Tian, Yaoqi; Xie, Zhengjun; Jin, Zhengyu

2017-06-01

Pullulanase was sol-gel encapsulated in the presence of magnetic chitosan/Fe 3 O 4 nanoparticles. The resulting immobilized pullulanase was characterized by scanning electron microscopy, vibrating sample magnetometry, Fourier transform infrared spectroscopy and thermogravimetric analysis. The results showed that the addition of pullulanase created a more regular surface on the sol-gel matrix and an enhanced magnetic response to an applied magnetic field. The maximal activity retention (83.9%) and specific activity (291.7 U/mg) of the immobilized pullulanase were observed under optimized conditions including an octyltriethoxysilane:tetraethoxysilane (OTES:TEOS) ratio of 1:2 and enzyme concentration of 0.484 mg/mL sol. The immobilized enzyme exhibited good thermal stability. When the temperature was above 60 °C, the immobilized pullulanase showed significantly higher activity than the free enzyme (p < 0.01); enzyme immobilized by simple sol-gel encapsulation and co-immobilized by crosslinking-encapsulation retained 52 and 69% of their initial activity after 5 h at 62 °C, respectively, compared to 11% for the free enzyme. Moreover, the stability of the pullulanase was improved by crosslinking-encapsulation, as the enzyme retained more than 85 and 81% of its original activity after 5 and 6 consecutive reuses, respectively, compared to 80 and 72% of its original activity for simple sol-gel encapsulated enzymes. This indicated the leakage of enzyme molecules through the pores of the gel was substantially abated by cross-linking. Such immobilized pullulanase provides high stability and ease of enzyme recovery, characteristics that are advantageous for applications in the food industry that involve continuous starch processing.

The lifestyle of prokaryotic organisms influences the repertoire of promiscuous enzymes.

PubMed

Martínez-Núñez, Mario Alberto; Rodríguez-Vázquez, Katya; Pérez-Rueda, Ernesto

2015-09-01

The metabolism of microbial organisms and its diversity are partly the result of an adaptation process to the characteristics of the environments that they inhabit. In this work, we analyze the influence of lifestyle on the content of promiscuous enzymes in 761 nonredundant bacterial and archaeal genomes. Promiscuous enzymes were defined as those proteins whose catalytic activities are defined by two or more different Enzyme Commission (E.C.) numbers. The genomes analyzed were categorized into four lifestyles for their exhaustive comparisons: free-living, extremophiles, pathogens, and intracellular. From these analyses we found that free-living organisms have larger genomes and an enrichment of promiscuous enzymes. In contrast, intracellular organisms showed smaller genomes and the lesser proportion of promiscuous enzymes. On the basis of our data, we show that the proportion of promiscuous enzymes in an organism is mainly influenced by the lifestyle, where fluctuating environments promote its emergence. Finally, we evidenced that duplication processes occur preferentially in metabolism of free-living and extremophiles species. © 2015 Wiley Periodicals, Inc.

Bioavailability of minerals in legumes.

PubMed

Sandberg, Ann-Sofie

2002-12-01

The mineral content of legumes is generally high, but the bioavailability is poor due to the presence of phytate, which is a main inhibitor of Fe and Zn absorption. Some legumes also contain considerable amounts of Fe-binding polyphenols inhibiting Fe absorption. Furthermore, soya protein per se has an inhibiting effect on Fe absorption. Efficient removal of phytate, and probably also polyphenols, can be obtained by enzymatic degradation during food processing, either by increasing the activity of the naturally occurring plant phytases and polyphenol degrading enzymes, or by addition of enzyme preparations. Biological food processing techniques that increase the activity of the native enzymes are soaking, germination, hydrothermal treatment and fermentation. Food processing can be optimized towards highest phytate degradation provided that the optimal conditions for phytase activity in the plant is known. In contrast to cereals, some legumes have highest phytate degradation at neutral or alkaline pH. Addition of microbial enzyme preparations seems to be the most efficient for complete degradation during processing. Fe and Zn absorption have been shown to be low from legume-based diets. It has also been demonstrated that nutritional Fe deficiency reaches its greatest prevalence in populations subsisting on cereal- and legume-based diets. However, in a balanced diet containing animal protein a high intake of legumes is not considered a risk in terms of mineral supply. Furthermore, once phytate, and in certain legumes polyphenols, is degraded, legumes would become good sources of Fe and Zn as the content of these minerals is high.

Enhancement of proteolytic enzyme activity excreted from Bacillus stearothermophilus for a thermophilic aerobic digestion process.

PubMed

Kim, Young-Kee; Bae, Jin-Hye; Oh, Byung-Keun; Lee, Won Hong; Choi, Jeong-Woo

2002-04-01

Proteolysis is one of the main enzymatic reactions involved in waste activated sludge (WAS) digestion. In this study, proteases excreted from Bacillus stearothermophilus (ATCC 31197) were classified, and an enhancement of protease activity was achieved using economical chemical additives for WAS digestion. Proteases excreted from B. stearothermophilus were classified into two families: serine and metallo-proteases. Various metal ions were investigated as additives which could potentially enhance protease activity. It was observed that Ca2+ and Fe2+ could markedly activate these enzymes. These results were applied to thermophilic aerobic digestion (TAD) of industrial WAS using B. stearothermophilus. The addition of these divalent ions enhanced the degradation performance of the TAD process in terms of reducing the total suspended solids (TSSs), the dissolved organic carbon (DOC) content, and the intracellular and extracellular protein concentrations. The best result, with respect to protein reduction in a digestion experiment, was obtained by the addition of 2 mM Ca2+. Therefore, a proposed TAD process activated by calcium addition can be successfully used for industrial and municipal WAS digestion to the upgrading of TAD process performance.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adhikari, B.; Pellegrino, J.; Stickel, J.

We are currently evaluating the feasibility of performing continuous enzymatic hydrolysis of lignocellulosic biomass to product sugars using a membrane-assisted reaction/separation process. The overarching technical goals are to continuously remove the sugars—this lowers product feedback inhibition—retain and recycle active enzyme, and continuously recover the co-product of lignin. Experimental d d d currently evaluating the feasibility of performing continuous enzymatic hydrolysis of lignocellulosic biomass to product sugars using a membrane-assisted reaction/separation process. The overarching technical goals are to continuously remove the sugars -- this lowers product feedback inhibition --retain and recycle active enzyme, and continuously recover the co-product of lignin.

Exploring the specific features of interfacial enzymology based on lipase studies.

PubMed

Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

2006-09-01

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

A bacterial hydrogen-dependent CO2 reductase forms filamentous structures.

PubMed

Schuchmann, Kai; Vonck, Janet; Müller, Volker

2016-04-01

Interconversion of CO2 and formic acid is an important reaction in bacteria. A novel enzyme complex that directly utilizes molecular hydrogen as electron donor for the reversible reduction of CO2 has recently been identified in the Wood-Ljungdahl pathway of an acetogenic bacterium. This pathway is utilized for carbon fixation as well as energy conservation. Here we describe the further characterization of the quaternary structure of this enzyme complex and the unexpected behavior of this enzyme in polymerizing into filamentous structures. Polymerization of metabolic enzymes into similar structures has been observed only in rare cases but the increasing number of examples point towards a more general characteristic of enzyme functioning. Polymerization of the purified enzyme into ordered filaments of more than 0.1 μm in length was only dependent on the presence of divalent cations. Polymerization was a reversible process and connected to the enzymatic activity of the oxygen-sensitive enzyme with the filamentous form being the most active state. © 2016 Federation of European Biochemical Societies.

The dynamic basis of energy transduction in enzymes.

PubMed

Somogyi, B; Welch, G R; Damjanovich, S

1984-09-06

The most important idea underlying our treatment herein is the unity of the enzyme molecule and the medium. Appreciation of this relationship is vital, if enzymology is to graduate from its present reductionistic status to a more holistic posture. Enzymes are biological entities firstly, and isolated objects of physicochemical analysis secondly. Perhaps the most crucial 'biological lesson', particularly apropos of enzymes in intermediary metabolism, concerns the 'cytosociology' of enzyme action in vivo [94,128]. The natural habitat of many enzymes in the living cell is far different from that in bulk aqueous solution in vitro. In order to obtain a real grasp of the nature of enzyme function, one must ultimately couch enzymology in concepts emerging from contemporary cell biology [95]. Notwithstanding, analysis precedes synthesis; and one must needs begin with the individual enzyme molecule. The trenchant efforts of the physical chemist and the organic chemist have produced a wealth of information on the nature of the binding and catalytic events at the enzyme active site. While it is not yet possible to explain precisely the complete sequence of events in the catalytic process, nevertheless, the basic mechanisms by which enzymes effect catalysis (i.e., reduce activation energy) now seem apparent [81,129]. The new frontier is to be found, in exploring the dynamic role of the protein matrix [17]. Not only does the protein provide the 3-D scaffolding for active-site processes, but, more importantly, it serves as the local solvent for the bound chemical subsystem. Thus, the dynamical aspects of enzyme catalysis (for thermally based systems) must arise from the fluctuational properties of the protein molecule. This notion is the common denominator in all of the models in subsection IIC. It is the anisotropic nature of this fluctuational behavior, which would characterize the energy-transduction phenomenon leading to localized catalytic events at the active-site. In Section III we attempted to show that all of the various enzyme models contribute pieces to a single, all-embracing jig-saw puzzle. Some models focus on the dynamical properties of the protein per se, whereas others deal with the stochastic aspects of protein-solvent interaction. The two approaches are complementary, as are mutually interlocking pieces of a puzzle. The ultimate picture depicted by this 'jig-saw puzzle' is still somewhat vague--owing to the present paucity of empirical information on protein motions.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of Pectate Lyase by Penicillium viridicatum RFC3 in Solid-State and Submerged Fermentation.

PubMed

Ferreira, Viviani; da Silva, Roberto; Silva, Dênis; Gomes, Eleni

2010-01-01

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500 U mL(-1) or 300 Ug(-1) of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme from SSF and designated PL I, II, III, IV, V, and VI, while five peaks were obtained from crude enzyme from SmF and labeled PL I', II', III', IV', and VII'. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35 degrees C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45 degrees C. Crude enzyme from SmF and PL III' showed thermophilic profiles of activity, with maximum activity at 60 and 55 degrees C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0-10.0 and PL III was most stable in the pH range 4.0-7.0. Crude enzyme from SmF retained 70%-80% of its maximum activity in the acid-neutral pH range (4.0-7.0), but PIII showed high stability at alkaline pH (7.5-9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55 degrees C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes.

Enzyme-Responsive Nanomaterials for Controlled Drug Delivery

PubMed Central

Hu, Quanyin; Katti, Prateek S.; Gu, Zhen

2015-01-01

Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials for controlled drug release have achieved significant development and been studied as an important class of drug delivery devices in nanomedicine. In this review, we describe enzymes such as proteases, phospholipase and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area. PMID:25251024

Enzyme-responsive nanomaterials for controlled drug delivery

NASA Astrophysics Data System (ADS)

Hu, Quanyin; Katti, Prateek S.; Gu, Zhen

2014-10-01

Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials used for controlled drug release have achieved significant development and have been studied as an important class of drug delivery strategies in nanomedicine. In this review, we describe enzymes such as proteases, phospholipases and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area.

Enzyme-MOF (metal-organic framework) composites.

PubMed

Lian, Xizhen; Fang, Yu; Joseph, Elizabeth; Wang, Qi; Li, Jialuo; Banerjee, Sayan; Lollar, Christina; Wang, Xuan; Zhou, Hong-Cai

2017-06-06

The ex vivo application of enzymes in various processes, especially via enzyme immobilization techniques, has been extensively studied in recent years in order to enhance the recyclability of enzymes, to minimize enzyme contamination in the product, and to explore novel horizons for enzymes in biomedical applications. Possessing remarkable amenability in structural design of the frameworks as well as almost unparalelled surface tunability, Metal-Organic Frameworks (MOFs) have been gaining popularity as candidates for enzyme immobilization platforms. Many MOF-enzyme composites have achieved unprecedented results, far outperforming free enzymes in many aspects. This review summarizes recent developments of MOF-enzyme composites with special emphasis on preparative techniques and the synergistic effects of enzymes and MOFs. The applications of MOF-enzyme composites, primarily in transferation, catalysis and sensing, are presented as well. The enhancement of enzymatic activity of the composites over free enzymes in biologically incompatible conditions is emphasized in many cases.

Laccase produced by a thermotolerant strain of Trametes trogii LK13

PubMed Central

Yan, Jinping; Chen, Yuhui; Niu, Jiezhen; Chen, Daidi; Chagan, Irbis

2015-01-01

Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%–50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g −1 ) along with low cellulose (2 U g −1 ) and xylanase (140 U g −1 ) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes. PMID:26221089

'Enzyme Test Bench': A biochemical application of the multi-rate modeling

NASA Astrophysics Data System (ADS)

Rachinskiy, K.; Schultze, H.; Boy, M.; Büchs, J.

2008-11-01

In the expanding field of 'white biotechnology' enzymes are frequently applied to catalyze the biochemical reaction from a resource material to a valuable product. Evolutionary designed to catalyze the metabolism in any life form, they selectively accelerate complex reactions under physiological conditions. Modern techniques, such as directed evolution, have been developed to satisfy the increasing demand on enzymes. Applying these techniques together with rational protein design, we aim at improving of enzymes' activity, selectivity and stability. To tap the full potential of these techniques, it is essential to combine them with adequate screening methods. Nowadays a great number of high throughput colorimetric and fluorescent enzyme assays are applied to measure the initial enzyme activity with high throughput. However, the prediction of enzyme long term stability within short experiments is still a challenge. A new high throughput technique for enzyme characterization with specific attention to the long term stability, called 'Enzyme Test Bench', is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests conducted under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimum non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The results of the characterization, based on micro liter format experiments of hours, are in good agreement with the results of long term experiments in 1L format. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature. The presented article gives a successful example for the application of multi-rate modeling, experimental design and parameter estimation within biochemical engineering. At the same time, it shows the limitations of the methods at the state of the art and addresses the current problems to the applied mathematics community.

Cost-efficient entrapment of β-glucosidase in nanoscale latex and silicone polymeric thin films for use as stable biocatalysts.

PubMed

Javed, Muhammad Rizwan; Buthe, Andreas; Rashid, Muhammad Hamid; Wang, Ping

2016-01-01

β-Glucosidase is an ubiquitous enzyme which has enormous biotechnological applications. Its deficiency in natural enzyme preparations is often overcome by exogenous supplementation, which further increases the enzyme utilization cost. Enzyme immobilization offers a potential solution through enzyme recycling and easy recovery. In the present work Aspergillus niger β-glucosidase is immobilized within nanoscale polymeric materials (polyurethane, latex and silicone), through entrapment, and subsequently coated onto a fibrous support. Highest apparent activity (90 U g(-1) polymer) was observed with latex, while highest entrapment efficiency (93%) was observed for the silicone matrix. Immobilization resulted in the thermo-stabilization of the β-glucosidase with an increase in optimum temperature and activation energy for cellobiose hydrolysis. Supplementation to cellulases also resulted in an increased cellulose hydrolysis, while retaining more than 70% functional stability. Hence, the current study describes novel preparations of immobilized β-glucosidase as highly stable and active catalysts for industrial food- and bio-processing applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

DOE PAGES

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; ...

2017-09-11

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Computational Model of Adrenal Steroidogenesis to Predict Biochemical Response to Endocrine Disruptors

EPA Science Inventory

Steroids, which have an important role in a wide range of physiological processes, are synthesized primarily in the gonads and adrenal glands through a series of enzyme mediated reactions. The activity of steroidogenic enzymes can be altered by various endocrine disrupters (ED), ...

Multiple enzyme activities of flavivirus proteins.

PubMed

Padmanabhan, R; Mueller, N; Reichert, E; Yon, C; Teramoto, T; Kono, Y; Takhampunya, R; Ubol, S; Pattabiraman, N; Falgout, B; Ganesh, V K; Murthy, K

2006-01-01

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.

Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

PubMed

Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

2013-05-10

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of Three L-Asparaginases from Maritime Pine (Pinus pinaster Ait.).

PubMed

Van Kerckhoven, Sonia H; de la Torre, Fernando N; Cañas, Rafael A; Avila, Concepción; Cantón, Francisco R; Cánovas, Francisco M

2017-01-01

Asparaginases (ASPG, EC 3.5.1.1) catalyze the hydrolysis of the amide group of L-asparagine producing L-aspartate and ammonium. Three ASPG, PpASPG1, PpASPG2, and PpASPG3, have been identified in the transcriptome of maritime pine ( Pinus pinaster Ait.) that were transiently expressed in Nicotiana benthamiana by agroinfection. The three recombinant proteins were processed in planta to active enzymes and it was found that all mature forms exhibited double activity asparaginase/isoaspartyl dipeptidase but only PpASPG1 was able to catalyze efficiently L-asparagine hydrolysis. PpASPG1 contains a variable region of 77 amino acids that is critical for proteolytic processing of the precursor and is retained in the mature enzyme. Furthermore, the functional analysis of deletion mutants demonstrated that this protein fragment is required for specific recognition of the substrate and favors enzyme stability. Potassium has a limited effect on the activation of maritime pine ASPG what is consistent with the lack of a critical residue essential for interaction of cation. Taken together, the results presented here highlight the specific features of ASPG from conifers when compared to the enzymes from angiosperms.

Characterization of Three L-Asparaginases from Maritime Pine (Pinus pinaster Ait.)

PubMed Central

Van Kerckhoven, Sonia H.; de la Torre, Fernando N.; Cañas, Rafael A.; Avila, Concepción; Cantón, Francisco R.; Cánovas, Francisco M.

2017-01-01

Asparaginases (ASPG, EC 3.5.1.1) catalyze the hydrolysis of the amide group of L-asparagine producing L-aspartate and ammonium. Three ASPG, PpASPG1, PpASPG2, and PpASPG3, have been identified in the transcriptome of maritime pine (Pinus pinaster Ait.) that were transiently expressed in Nicotiana benthamiana by agroinfection. The three recombinant proteins were processed in planta to active enzymes and it was found that all mature forms exhibited double activity asparaginase/isoaspartyl dipeptidase but only PpASPG1 was able to catalyze efficiently L-asparagine hydrolysis. PpASPG1 contains a variable region of 77 amino acids that is critical for proteolytic processing of the precursor and is retained in the mature enzyme. Furthermore, the functional analysis of deletion mutants demonstrated that this protein fragment is required for specific recognition of the substrate and favors enzyme stability. Potassium has a limited effect on the activation of maritime pine ASPG what is consistent with the lack of a critical residue essential for interaction of cation. Taken together, the results presented here highlight the specific features of ASPG from conifers when compared to the enzymes from angiosperms. PMID:28690619

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

PubMed Central

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-01

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

Specific glutaryl-CoA dehydrogenating activity is deficient in cultured fibroblasts from glutaric aciduria patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyman, D.B.; Tanaka, K.

Patients with glutaric aciduria (GA) have greatly increased urinary excretion of glutarate. Their leukocyte and fibroblast sonicates have deficient ability to produce /sup 14/CO2 from (1,5-/sup 14/C)glutaryl-CoA, an enzymatic process with two sequential reaction steps, dehydrogenation and decarboxylation. In normal individuals, it is not known whether these two reaction steps require one or two enzymes, and currently it is assumed that a single enzyme, glutaryl-CoA dehydrogenase (GDH), carries out these two reactions. Since GA patients also excrete increased amounts of 3-hydroxyglutarate and glutaconate in urine, it was thought that glutaryl-CoA in these patients may be dehydrogenated but not decarboxylated. Wemore » developed a new assay specific for glutaryl-CoA dehydrogenation which measures enzyme-catalyzed tritium release from (2,3,4-3H)glutaryl-CoA, and we studied the glutaryl-CoA dehydrogenating activity in cultured normal human fibroblasts and those from patients with GA. The Michaelis constant (Km) of normal human fibroblast GDH for (2,3,4-3H)glutaryl-CoA was 5.9 microM, and activity was severely inhibited by (methylenecyclopropyl)acetyl-CoA at low concentrations. Sonicates from all five GA fibroblast lines examined showed 2-9% of control glutaryl-CoA dehydrogenating activity, corresponding to the deficient 14CO2 releasing activity. These results indicate either that the conversion of glutaryl-CoA to crotonyl-CoA is accomplished by two enzymes, and patients with GA are deficient in the activity of the first component, or alternatively, that this process is carried out by a single enzyme which is deficient in these patients. It is unlikely that urinary glutaconate and 3-hydroxyglutarate in GA patients are produced via GDH.« less

Variation in soil enzyme activity as a function of vegetation amount, type, and spatial structure in fire-prone Mediterranean shrublands.

PubMed

Mayor, Ángeles G; Goirán, Silvana B; Vallejo, V Ramón; Bautista, Susana

2016-12-15

Fire-prone Mediterranean shrublands may be seriously threatened by land degradation due to progressive opening of the vegetation cover driven by increasing drought and fire recurrence. However, information about the consequences of this opening process for critical ecosystem functions is scant. In this work, we studied the influence of vegetation amount, type, and spatial pattern in the variation of extracellular soil enzyme activity (acid phosphatase, β-glucosidase, and urease) in fire-prone shrublands in eastern Spain. Soil was sampled in vegetation-patch and open-interpatch microsites in 15 shrubland sites affected by large wildfires in 1991. On average, the activities of the three enzymes were 1.5 (β-glucosidase and urease) to 1.7 (acid phosphatase) times higher in soils under vegetation patches than in adjacent interpatches. In addition, phosphatase activity for both microsites significantly decreased with the fragmentation of the vegetation. This result was attributed to a lower influence of roots -the main source of acid phosphatase- in the bigger interpatches of the sites with lower patch cover, and to feedbacks between vegetation pattern, redistribution of resources, and soil quality during post-fire vegetation dynamics. Phosphatase activity was also 1.2 times higher in patches of resprouter plants than in patches of non-resprouters, probably due to the faster post-fire recovery and older age of resprouter patches in these fire-prone ecosystems. The influence on the studied enzymes of topographic and climatic factors acting at the landscape scale was insignificant. According to our results, variations in the cover, pattern, and composition of vegetation patches may have profound impacts on soil enzyme activity and associated nutrient cycling processes in fire-prone Mediterranean shrublands, particularly in those related to phosphorus. Copyright © 2016 Elsevier B.V. All rights reserved.

Tracking dynamics of plant biomass composting by changes in substrate structure, microbial community, and enzyme activity

PubMed Central

2012-01-01

Background Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars. However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process. Results In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera) wood-chips and mown lawn grass clippings (85:15 in dry-weight) and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed. Conclusion The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP) and solid-state fermentation for the production of cellulolytic enzymes and biofuels. PMID:22490508

Tracking Dynamics of Plant Biomass Composting by Changes in Substrate Structure, Microbial Community, and Enzyme Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, H.; Tucker, M. P.; Baker, J. O.

2012-04-01

Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars. However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process. In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera) wood-chips and mown lawn grass clippings (85:15 in dry-weight) and used as amore » model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed. The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP) and solid-state fermentation for the production of cellulolytic enzymes and biofuels.« less

High pressure, temperature and time-dependent effects on enzymatic and microbial properties of fresh sugarcane juice.

PubMed

Chauhan, O P; Ravi, N; Roopa, N; Kumar, Sumeet; Raju, P S

2017-11-01

Efficacy of variable high pressure, temperature and time on the browning causing enzymes and microbial activities, which are major spoilage factors during preservation of sugarcane juice, was studied. The juice was processed at 200-600 MPa pressure for 2-8 min at 40 and 60 °C and their effect on polyphenol oxidase and peroxidase as well as microbiological quality in terms of total plate count, yeast and molds and total coliforms was studied. Application of high pressures were found to cause significant decrease in enzymatic and microbial activities. The effects were found to be significantly more pronounced at 60 °C as compared to 40 °C. Process time also caused significant ( p  < 0.05) negative effect on microbial and enzyme activities. The sugarcane juice treated at 600 MPa for 6 min at 60 °C was found sufficient to inactivate the microbial counts completely. Whereas, enzymes were found to be completely inactivated in the samples processed at 600 MPa for 8 min at 60 °C. A pressure of 600 MPa at 60 °C for 8 min could be applied during commercial preservation of sugarcane juice for getting complete inactivation of browning causing enzymes and spoilage causing microorganisms.

A model system for targeted drug release triggered by biomolecular signals logically processed through enzyme logic networks.

PubMed

Mailloux, Shay; Halámek, Jan; Katz, Evgeny

2014-03-07

A new Sense-and-Act system was realized by the integration of a biocomputing system, performing analytical processes, with a signal-responsive electrode. A drug-mimicking release process was triggered by biomolecular signals processed by different logic networks, including three concatenated AND logic gates or a 3-input OR logic gate. Biocatalytically produced NADH, controlled by various combinations of input signals, was used to activate the electrochemical system. A biocatalytic electrode associated with signal-processing "biocomputing" systems was electrically connected to another electrode coated with a polymer film, which was dissolved upon the formation of negative potential releasing entrapped drug-mimicking species, an enzyme-antibody conjugate, operating as a model for targeted immune-delivery and consequent "prodrug" activation. The system offers great versatility for future applications in controlled drug release and personalized medicine.

[Effect of Cu2+ and Zn2+ ions in Ca-ATPase activity isolated from Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) larvae].

PubMed

Dias, Decivaldo S; Coelho, Milton V

2007-01-01

ATPases, an important target of insecticides, are enzymes that hydrolyze ATP and use the energy released in that process to accomplish some type of cellular work. Pachymerus nucleorum (Fabricius) larvae possess an ATPase, that presents high Ca-ATPase activity, but no Mg-ATPase activity. In the present study, the effect of zinc and copper ions in the activity Ca-ATPase of that enzyme was tested. More than 90% of the Ca-ATPase activity was inhibited in 0.5 mM of copper ions or 0.25 mM of zinc ions. In the presence of EDTA, but not in the absence, the inhibition by zinc was reverted with the increase of calcium concentration. The inhibition by copper ions was not reverted in the presence or absence of EDTA. The Ca-ATPase was not inhibited by treatment of the ATPase fraction with copper, suggesting that the copper ion does not bind directly to the enzyme. The results suggest that zinc and copper ions form a complex with ATP and bind to the enzyme inhibiting its Ca-ATPase activity.

Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity.

PubMed

Kutcherlapati, S N Raju; Yeole, Niranjan; Jana, Tushar

2016-02-01

A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27-30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea-urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27-30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses. Copyright © 2015 Elsevier Inc. All rights reserved.

Bio-conversion of apple pomace into ethanol and acetic acid: Enzymatic hydrolysis and fermentation.

PubMed

Parmar, Indu; Rupasinghe, H P Vasantha

2013-02-01

Enzymatic hydrolysis of cellulose present in apple pomace was investigated using process variables such as enzyme activity of commercial cellulase, pectinase and β-glucosidase, temperature, pH, time, pre-treatments and end product separation. The interaction of enzyme activity, temperature, pH and time had a significant effect (P<0.05) on release of glucose. Optimal conditions of enzymatic saccharification were: enzyme activity of cellulase, 43units; pectinase, 183units; β-glucosidase, 41units/g dry matter (DM); temperature, 40°C; pH 4.0 and time, 24h. The sugars were fermented using Saccharomyces cerevisae yielding 19.0g ethanol/100g DM. Further bio-conversion using Acetobacter aceti resulted in the production of acetic acid at a concentration of 61.4g/100g DM. The present study demonstrates an improved process of enzymatic hydrolysis of apple pomace to yield sugars and concomitant bioconversion to produce ethanol and acetic acid. Copyright © 2012 Elsevier Ltd. All rights reserved.

Physical and chemical properties of Nam Prig Noom, a Thai green-chili paste, following ultra-high pressure and thermal processes

NASA Astrophysics Data System (ADS)

Apichartsrangkoon, Arunee; Srisajjalertwaja, Siriwan; Chaikham, Pittaya; Hirun, Sathira

2013-03-01

A study of processing green-chili pastes (Nam Prig Noom) by pressurization (100-600 MPa/30-50°C/20 min), pasteurization (90°C/3-5 min) or sterilization (121°C/4 min), subsequently, their physical, biochemical and microbiological qualities as well as the sensory acceptance were assessed. It was found that pressure at low levels (100-300 MPa) could improve activities of enzyme peroxidase (POD), polyphenoloxidase (PPO) and lypoxygenase (LOX) in the chili paste by more than 100%, while pressures above 500 or 300 MPa combined with heat would significantly inactivate these enzyme activities. Both color parameters and enzyme activities illustrated that though some enzymatic browning occurred with the pressurized products indicated by b* (yellowish) parameter, the magnitude of these browning was still milder than those thermally treated products indicated by-a* (greenness) and L (lightness) parameters, presumably as a consequence of the Maillard reaction. Moreover, the sensory scores were found in accordance with color parameters, firmness and capsaicin contents.

Noncovalent Ubiquitin Interactions Regulate the Catalytic Activity of Ubiquitin Writers.

PubMed

Wright, Joshua D; Mace, Peter D; Day, Catherine L

2016-11-01

Covalent modification of substrate proteins with ubiquitin is the end result of an intricate network of protein-protein interactions. The inherent ability of the E1, E2, and E3 proteins of the ubiquitylation cascade (the ubiquitin writers) to interact with ubiquitin facilitates this process. Importantly, contact between ubiquitin and the E2/E3 writers is required for catalysis and the assembly of chains of a given linkage. However, ubiquitin is also an activator of ubiquitin-writing enzymes, with many recent studies highlighting the ability of ubiquitin to regulate activity and substrate modification. Here, we review the interactions between ubiquitin-writing enzymes and regulatory ubiquitin molecules that promote activity, and highlight the potential of these interactions to promote processive ubiquitin transfer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phosphotriesterase-magnetic nanoparticles bioconjugates with improved enzyme activity in a biocatalytic membrane reactor.

PubMed

Gebreyohannes, Abaynesh Yihdego; Mazzei, Rosalinda; Yahia Marei Abdelrahim, Mohamed; Vitola, Giuseppe; Porzio, Elena; Manco, Giuseppe; Barboiu, Mihail; Giorno, Lidietta

2018-05-24

The need to find alternative bioremediation solutions for organophosphate degradation pushed the research to develop technologies based on organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membrane seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer and provide microenvironment with tuned physico-chemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme (SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other class of enzymes and related application.

Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal.

PubMed

Khatri, Bhim Prakash; Bhattarai, Tribikram; Shrestha, Sangita; Maharjan, Jyoti

2015-01-01

Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30-70 °C) and pH (6.2-9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

Effects of aqueous eucalyptus extracts on seed germination, seedling growth and activities of peroxidase and polyphenoloxidase in three wheat cultivar seedlings (Triticum aestivum L.).

PubMed

Ziaebrahimi, L; Khavari-Nejad, R A; Fahimi, H; Nejadsatari, T

2007-10-01

Evaluation of allelopathic effects of this plant on other near cultivations especially wheat is the aim of this study. Effects of water extracts of eucalyptus leaves examined on germination and growth of three wheat cultivar seeds and seedlings. Results showed that: germination percentage strongly decreased, leaf and root lengths also affected and dry and wet weights of both roots and shoots showed similar change patterns. Activities of peroxidase and polyphenoloxidase as antioxidant enzymes in roots and shoots measured. Activity of peroxidases increased in stress conditions and roots showed more increased enzyme activity than leaves. Activity of polyphenoloxidases increased only in one of three cultivars and again roots showed more activity of this enzyme in response to eucalyptus extract. Suggest that detoxification process were conducted mainly in roots of seedlings.

The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

PubMed

Cellini, Barbara; Montioli, Riccardo; Oppici, Elisa; Astegno, Alessandra; Voltattorni, Carla Borri

2014-02-01

The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Optimization of moistening solution concentration on xylanase activity in solid state fermentation from oil palm empty fruit bunches

NASA Astrophysics Data System (ADS)

Mardawati, Efri; Parlan; Rialita, Tita; Nurhadi, Bambang

2018-03-01

Xylanase is an enzyme used in the industrial world, including food industry. Xylanase can be utilized as a 1,4-β-xylosidic endo-hydrolysis catalyst of xylanase, a hemicellulose component for obtaining a xylose monomer. This study aims to determine the optimum concentration of the fermentation medium using Response Surface Method (RSM) in the production of xylanase enzyme from oil palm empty fruit bunches (OPEFB) through solid state fermentation process. The variables varied in this study used factor A (ammonium sulphate concentration 1.0-2.0 g/L), B (concentration of potassium dihydrogen phosphate 1.5-2.5 g/L) and C (urea concentration 0.2 – 0.5 g/L). The data was analysed by using Design Expert version 10.0.1.0 especially CCD with total 17 running including 3 times replicated of canter point. Trichoderma viride was used for the process production of xylanase enzyme. The ratio between substrate and moistening solution used was 0.63 g / mL with temperature of 32.80C, 60 h incubation time. The analysis of enzyme activity was done by DNS method with 1% xylan as substrate. Analysis of protein content in enzyme was done by Bradford method. The optimum of moistening solution concentration in this fermentation was obtained. They are, the ammonium sulphate concentration of 1.5 g/L, potassium dihydrogen phosphate 2.0 g/L and urea 0.35 g/L with activity of 684.70 U/mL, specific activity enzyme xylanase 6261.58 U/mg, protein content 0.1093 U/mg, the model was validated using experiment design with perfect reliability value 0.96.

Using ultrasound technology for the inactivation and thermal sensitization of peroxidase in green coconut water.

PubMed

Rojas, Meliza Lindsay; Trevilin, Júlia Hellmeister; Funcia, Eduardo Dos Santos; Gut, Jorge Andrey Wilhelms; Augusto, Pedro Esteves Duarte

2017-05-01

Green coconut water has unique nutritional and sensorial qualities. Despite the different technologies already studied, its enzymatic stability is still challenging. This study evaluated the use of ultrasound technology (US) for inactivating/sensitizing coconut water peroxidase (POD). The effect of both US application alone and as a pre-treatment to thermal processing was evaluated. The enzyme activity during US processing was reduced 27% after 30min (286W/L, 20kHz), demonstrating its high resistance. The thermal inactivation was described by the Weibull model under non-isothermal conditions. The enzyme became sensitized to heat after US pre-treatment. Further, the use of US resulted in more uniform heat resistance. The results suggest that US is a good technology for sensitizing enzymes before thermal processing (even for an enzyme with high thermal resistance). Therefore, the use of this technology could decrease the undesirable effects of long times and/or the high temperatures of the conventional thermal processing. Copyright © 2016 Elsevier B.V. All rights reserved.

The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme.

PubMed Central

González Flecha, F L; Castello, P R; Caride, A J; Gagliardino, J J; Rossi, J P

1993-01-01

In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein. PMID:8393658

Cold and Hot Extremozymes: Industrial Relevance and Current Trends

PubMed Central

Sarmiento, Felipe; Peralta, Rocío; Blamey, Jenny M.

2015-01-01

The development of enzymes for industrial applications relies heavily on the use of microorganisms. The intrinsic properties of microbial enzymes, e.g., consistency, reproducibility, and high yields along with many others, have pushed their introduction into a wide range of products and industrial processes. Extremophilic microorganisms represent an underutilized and innovative source of novel enzymes. These microorganisms have developed unique mechanisms and molecular means to cope with extreme temperatures, acidic and basic pH, high salinity, high radiation, low water activity, and high metal concentrations among other environmental conditions. Extremophile-derived enzymes, or extremozymes, are able to catalyze chemical reactions under harsh conditions, like those found in industrial processes, which were previously not thought to be conducive for enzymatic activity. Due to their optimal activity and stability under extreme conditions, extremozymes offer new catalytic alternatives for current industrial applications. These extremozymes also represent the cornerstone for the development of environmentally friendly, efficient, and sustainable industrial technologies. Many advances in industrial biocatalysis have been achieved in recent years; however, the potential of biocatalysis through the use of extremozymes is far from being fully realized. In this article, the adaptations and significance of psychrophilic, thermophilic, and hyperthermophilic enzymes, and their applications in selected industrial markets will be reviewed. Also, the current challenges in the development and mass production of extremozymes as well as future prospects and trends for their biotechnological application will be discussed. PMID:26539430

Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV.

PubMed

Vijayaraghavan, Ponnuswamy; Vijayan, Aija; Arun, Arumugaperumal; Jenisha, John Kennady; Vincent, Samuel Gnana Prakash

2012-01-01

Cow dung, a cheap and easily available source of energy, was used as the substrate for the production of alkaline protease by solid-state fermentation using the Bacillus subtilis strain VV. In order to achieve the maximum yield of this enzyme, the following optimum process parameters are needed: fermentation period (72 h), pH (10.0), moisture content (140%), inoculum (25%), temperature (30-40°C), carbon source (2% (w/w) maltose) and nitrogen source (1% (w/w) urea). The protease was stable over a broad temperature range (30-50°C) and pH (8.0-10.0), with maximum activity at 50°C and pH 10.0. Among the divalent ions tested, Ca(2+) (0.01 M) increased enzyme activity. The purified protease, after being subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was found to have a molecular mass of 38.5 kDa. The enzyme was solvent-and surfactant-stable and showed activity even after 24 h incubation along with various commercially available detergents. This enzyme possessed dehairing properties for animal hide after 16 h of incubation at room temperature. From these results it is evident that cow dung is a potential substrate for the production of a detergent-stable, dehairing protease by B. subtilis. This enzyme has a lot of potential applications in the detergent and leather-processing industries.

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4.

PubMed

Fang, Shujun; Chang, Jie; Lee, Yong Seok; Guo, Weiliang; Choi, Yong Lark; Zhou, Yongcan

2014-01-01

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure-function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.

Identification of Residues Involved in Substrate Specificity and Cytotoxicity of Two Closely Related Cutinases from Mycobacterium tuberculosis

PubMed Central

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production. PMID:23843969

Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.

PubMed

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

Engineering Neprilysin Activity and Specificity to Create a Novel Therapeutic for Alzheimer’s Disease

PubMed Central

Webster, Carl I.; Burrell, Matthew; Olsson, Lise-Lotte; Fowler, Susan B.; Digby, Sarah; Sandercock, Alan; Snijder, Arjan; Tebbe, Jan; Haupts, Ulrich; Grudzinska, Joanna; Jermutus, Lutz; Andersson, Christin

2014-01-01

Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer’s disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer’s disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1–40, 1–42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1–40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer’s disease. PMID:25089527

The autodigestion hypothesis: Proteolytic receptor cleavage in rheological and cardiovascular cell dysfunction1

PubMed Central

Schmid-Schönbein, Geert W.

2017-01-01

Transformation of circulating leukocytes from a dormant into an activated state with changing rheological properties leads to a major shift of their behavior in the microcirculation. Low levels of pseudopod formation or expression of adhesion molecules facilitate relatively free passage through microvessels while activated leukocytes with pseudopods and enhanced levels of adhesion membrane proteins become trapped in microvessels, attach to the endothelium and migrate into the tissue. The transformation of leukocytes into an activated state is seen in many diseases. While mechanisms for activation due to infections, tissue trauma, as well as non-physiological biochemical or biophysical exposures are well recognized, the mechanisms for activation in many diseases have not been conclusively liked to these traditional mechanisms and remain unknown. We summarize our recent evidence suggesting a major and surprising role of digestive enzymes in the small intestine as root causes for leukocyte activation and microvascular disturbances. During normal digestion of food digestive enzymes are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. When permeability of this barrier increases, these powerful degrading enzymes leak into the wall of the intestine and into the systemic circulation. Leakage of digestive enzymes occurs for example in physiological shock and multi-organ failure. Entry of digestive enzymes into the wall of the small intestine leads to degradation of the intestinal tissue in an autodigestion process. The digestive enzymes and tissue/food fragments generate not only activate leukocytes but also cause numerous cell dysfunctions. For example, proteolytic destruction of membrane receptors, plasma proteins and other biomolecules occurs. We conclude that escape of digestive enzymes from the intestinal track serves as a major source of cell dysfunction, morbidity and even mortality, including abnormal leukocyte activation seen in rheological studies. PMID:28269737

Insights into the activity change of spore photoproduct lyase induced by mutations at a peripheral glycine residue

NASA Astrophysics Data System (ADS)

Yang, Linlin; Li, Lei

2017-03-01

UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. This mechanism is consistent with the recently solved SPL structure that shows all three residues are located in proximity and thus able to participate in the radical transfer process during the enzyme catalysis. In contrast, an earlier in vivo mutational study identified a glycine to arginine mutation at the position 168 on the B. subtilis SPL that was later found to be > 15 Å away from the enzyme active site. This mutation appears to abolish the enzyme activity because endospores carrying this mutant were sensitive to UV light. To understand the molecular basis for this rendered enzyme activity, we constructed two SPL mutations G168A and G168R, examined their repair of dinucleotide SP TpT, and found that both mutants exhibit reduced enzyme activity. Comparing with the wildtype (WT) SPL enzyme, the G168A mutant slows down the SP TpT repair by 3 4 fold while the G168R mutant by 80 fold. Both mutants exhibit a smaller apparent (DV) kinetic isotope effect (KIE) but a bigger competitive (DV/K) KIE than that by the WT SPL. Moreover, the G168R mutant also produces a large portion of the abortive repair product TpT-SO2-; the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate. All these data imply that the mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process.

North Carolina Biomolecular Engineering and Materials Applications Center (NC-BEMAC).

DTIC Science & Technology

1987-12-29

enzyme has been replaced with cobalt(II). A further objective was to investigate Co2 activation by low molecular weight transition metal complexes as...Characterization of Low Molecular Weight Metal Complexes as Potential Models for IBio-Catalytic Processes. A number of transit ion met~~il oom~pi cxe; hive...binding, the enzyme suffered loss of activity during radiation polymerization. When covalent binding was u:sed it was necessary to introduce suitably

Microbial Activity and Silica Degradation in Rice Straw

NASA Astrophysics Data System (ADS)

Kim, Esther Jin-kyung

Abundantly available agricultural residues like rice straw have the potential to be feedstocks for bioethanol production. Developing optimized conditions for rice straw deconstruction is a key step toward utilizing the biomass to its full potential. One challenge associated with conversion of rice straw to bioenergy is its high silica content as high silica erodes machinery. Another obstacle is the availability of enzymes that hydrolyze polymers in rice straw under industrially relevant conditions. Microbial communities that colonize compost may be a source of enzymes for bioconversion of lignocellulose to products because composting systems operate under thermophilic and high solids conditions that have been shown to be commercially relevant. Compost microbial communities enriched on rice straw could provide insight into a more targeted source of enzymes for the breakdown of rice straw polysaccharides and silica. Because rice straw is low in nitrogen it is important to understand the impact of nitrogen concentrations on the production of enzyme activity by the microbial community. This study aims to address this issue by developing a method to measure microbial silica-degrading activity and measure the effect of nitrogen amendment to rice straw on microbial activity and extracted enzyme activity during a high-solids, thermophilic incubation. An assay was developed to measure silica-degrading enzyme or silicase activity. This process included identifying methods of enzyme extraction from rice straw, identifying a model substrate for the assay, and optimizing measurement techniques. Rice straw incubations were conducted with five different levels of nitrogen added to the biomass. Microbial activity was measured by respiration and enzyme activity. A microbial community analysis was performed to understand the shift in community structure with different treatments. With increased levels of nitrogen, respiration and cellulose and hemicellulose degrading activity increased. Silicase activity did not change across nitrogen treatments despite a shift in microbial community with varied nitrogen concentration. Samples treated with different nitrogen concentrations had similar levels of diversity, however the microbial community composition differed with added nitrogen. The results demonstrated that adding nitrogen to rice straw during thermophilic decomposition nurtured a more active microbial community and promoted enzyme secretion thus improving the ability to discover enzymes for rice straw deconstruction. These results can inform future experiments for cultivating a unique, thriving compost-derived microbial community that can successfully decompose rice straw. Understanding the silicase activity of microorganisms may alleviate the challenges associated with silica in various feedstocks.

Bio-functionalization of conductive textile materials with redox enzymes

NASA Astrophysics Data System (ADS)

Kahoush, M.; Behary, N.; Cayla, A.; Nierstrasz, V.

2017-10-01

In recent years, immobilization of oxidoreductase enzymes on electrically conductive materials has played an important role in the development of sustainable bio-technologies. Immobilization process allows the re-use of these bio-catalysts in their final applications. In this study, different methods of immobilizing redox enzymes on conductive textile materials were used to produce bio-functionalized electrodes. These electrodes can be used for bio-processes and bio-sensing in eco-designed applications in domains such as medicine and pollution control. However, the main challenge facing the stability and durability of these electrodes is the maintenance of the enzymatic activity after the immobilization. Hence, preventing the enzyme’s denaturation and leaching is a critical factor for the success of the immobilization processes.

[Effect of Low-Intensity 900 MHz Frequency Electromagnetic Radiation on Rat Brain Enzyme Activities Linked to Energy Metabolism].

PubMed

Petrosyan, M S; Nersesova, L S; Gazaryants, M G; Meliksetyan, G O; Malakyan, M G; Bajinyan, S A; Akopian, J I

2015-01-01

The research deals with the effect of low-intensity 900 MHz frequency electromagnetic radiation (EMR), power density 25 μW/cm2, on the following rat brain and blood serum enzyme activities: creatine kinase (CK), playing a central role in the process of storing and distributing the cell energy, as well as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) that play a key role in providing the conjunction of carbohydrate and amino acid metabolism. The comparative analysis of the changes in the enzyme activity studied at different times following the two-hour single, as well as fractional, radiation equivalent of the total time showed that the most radiosensitive enzyme is the brain creatine kinase, which may then be recommended as a marker of the radio frequency radiation impact. According to the analysis of the changing dynamics of the CK, ALT and AST activity level, with time these changes acquire the adaptive character and are directed to compensate the damaged cell energy metabolism.

Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

PubMed

Ostafe, Raluca; Prodanovic, Radivoje; Nazor, Jovana; Fischer, Rainer

2014-03-20

Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

Controlling enzymatic activity by immobilization on graphene oxide

NASA Astrophysics Data System (ADS)

Bolibok, Paulina; Wiśniewski, Marek; Roszek, Katarzyna; Terzyk, Artur P.

2017-04-01

In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells' viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.

Impact of limited solvent capacity on metabolic rate, enzyme activities, and metabolite concentrations of S. cerevisiae glycolysis.

PubMed

Vazquez, Alexei; de Menezes, Marcio A; Barabási, Albert-László; Oltvai, Zoltan N

2008-10-01

The cell's cytoplasm is crowded by its various molecular components, resulting in a limited solvent capacity for the allocation of new proteins, thus constraining various cellular processes such as metabolism. Here we study the impact of the limited solvent capacity constraint on the metabolic rate, enzyme activities, and metabolite concentrations using a computational model of Saccharomyces cerevisiae glycolysis as a case study. We show that given the limited solvent capacity constraint, the optimal enzyme activities and the metabolite concentrations necessary to achieve a maximum rate of glycolysis are in agreement with their experimentally measured values. Furthermore, the predicted maximum glycolytic rate determined by the solvent capacity constraint is close to that measured in vivo. These results indicate that the limited solvent capacity is a relevant constraint acting on S. cerevisiae at physiological growth conditions, and that a full kinetic model together with the limited solvent capacity constraint can be used to predict both metabolite concentrations and enzyme activities in vivo.

Impact of Limited Solvent Capacity on Metabolic Rate, Enzyme Activities, and Metabolite Concentrations of S. cerevisiae Glycolysis

PubMed Central

Vazquez, Alexei; de Menezes, Marcio A.; Barabási, Albert-László; Oltvai, Zoltan N.

2008-01-01

The cell's cytoplasm is crowded by its various molecular components, resulting in a limited solvent capacity for the allocation of new proteins, thus constraining various cellular processes such as metabolism. Here we study the impact of the limited solvent capacity constraint on the metabolic rate, enzyme activities, and metabolite concentrations using a computational model of Saccharomyces cerevisiae glycolysis as a case study. We show that given the limited solvent capacity constraint, the optimal enzyme activities and the metabolite concentrations necessary to achieve a maximum rate of glycolysis are in agreement with their experimentally measured values. Furthermore, the predicted maximum glycolytic rate determined by the solvent capacity constraint is close to that measured in vivo. These results indicate that the limited solvent capacity is a relevant constraint acting on S. cerevisiae at physiological growth conditions, and that a full kinetic model together with the limited solvent capacity constraint can be used to predict both metabolite concentrations and enzyme activities in vivo. PMID:18846199

Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter

PubMed Central

Huang, Yuhong; Yi, Zhuolin; Jin, Yanling; Huang, Mengjun; He, Kaize; Liu, Dayu; Luo, Huibo; Zhao, Dong; He, Hui; Fang, Yang; Zhao, Hai

2017-01-01

Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products. PMID:28955318

Lead nitrate-induced development of hypercholesterolemia in rats: sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis.

PubMed

Kojima, Misaki; Masui, Toshimitsu; Nemoto, Kiyomitsu; Degawa, Masakuni

2004-12-01

Changes in the gene expressions of hepatic enzymes responsible for cholesterol homeostasis were examined during the process of lead nitrate (LN)-induced development of hypercholesterolemia in male rats. Total cholesterol levels in the liver and serum were significantly increased at 3-72 h and 12-72 h, respectively, after LN-treatment (100 micromol/kg, i.v.). Despite the development of hypercholesterolemia, the genes for hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and other enzymes (FPPS, farnesyl diphosphate synthase; SQS, squalene synthase; CYP51, lanosterol 14alpha-demethylase) responsible for cholesterol biosynthesis were activated at 3-24 h and 12-18 h, respectively. On the other hand, the gene expression of cholesterol 7alpha-hydroxylase (CYP7A1), a catabolic enzyme of cholesterol, was remarkably suppressed at 3-72 h. The gene expression levels of cytokines interleukin-1beta (IL-1beta) and TNF-alpha, which activate the HMGR gene and suppress the CYP7A1 gene, were significantly increased at 1-3 h and 3-24 h, respectively. Furthermore, gene activation of SREBP-2, a gene activator of several cholesterogenic enzymes, occurred before the gene activations of FPPS, SQS and CYP51. This is the first report demonstrating sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis in LN-treated male rats. The mechanisms for the altered-gene expressions of hepatic enzymes in LN-treated rats are discussed.

Enzyme-triggered compound release using functionalized antimicrobial peptide derivatives† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04435b Click here for additional data file.

PubMed Central

Kashibe, Masayoshi; Matsumoto, Kengo; Hori, Yuichiro

2017-01-01

Controlled release is one of the key technologies for medical innovation, and many stimulus-responsive nanocarriers have been developed to utilize this technology. Enzyme activity is one of the most useful stimuli, because many enzymes are specifically activated in diseased tissues. However, controlled release stimulated by enzyme activity has not been frequently reported. One of the reasons for this is the lack of versatility of carriers. Most of the reported stimulus-responsive systems involve a sophisticated design and a complicated process for the synthesis of stimulus-responsive nanocarrier components. The purpose of this study was to develop versatile controlled release systems triggered by various stimuli, including enzyme activity, without modifying the nanocarrier components. We developed two controlled release systems, both of which comprised a liposome as the nanocarrier and a membrane-damaging peptide, temporin L (TL), and its derivatives as the release-controllers. One system utilized branched peptides for proteases, and the other utilized phosphopeptides for phosphatases. In our systems, the target enzymes converted the non-membrane-damaging TL derivatives into membrane-damaging peptides and released the liposome inclusion. We demonstrated the use of our antimicrobial peptide-based controlled release systems for different enzymes and showed the promise of this technology as a novel theranostic tool. PMID:28451373

Monitoring N2O Production Using a cNOR Modeled Active Site

NASA Astrophysics Data System (ADS)

Griffiths, Z. G.; Hegg, E. L.; Finders, C.; Haslun, J. A.

2017-12-01

Nitrous oxide (N2O) is a potent greenhouse gas with a 100-year global warming potential 265-296 times greater than carbon dioxide (CO2). It is the leading contributor to ozone depletion and can persist in the stratosphere for approximately 114 years. Hence, understanding the sources of atmospheric N2O emissions is critical to remediating the effects of climate change. Agricultural activities are the largest contributor to N2O emissions in the U.S. with microbial nitrification and denitrification as the dominating soil processes. The enzyme cytochrome c nitric oxide reductase (cNOR) is involved in bacterial denitrification. It is often difficult to study the enzymes involved in biotic N2O production, hence, model enzymes are a useful tool. The enzyme I107EFeBMb, a sperm whale myoglobin derivative, models the active site of cNOR and was used to simulate the anaerobic reduction of NO to N2O by cNOR. Dithionite was used to induce the catalytic activity of I107EFeBMb by reducing the enzyme. However, dithionite is a strong reductant that is capable of reducing NO to N2O directly. Therefore, the dithionite-enzyme mixture was passed through a size-exclusion column to isolate the reduced enzyme. This reduced and purified enzyme was then utilized to investigate N2O production from NO. This project will provide both an enzymatic and abiotic model to study N2O production.

Characterization of a feruloyl esterase from Aspergillus terreus facilitates the division of fungal enzymes from Carbohydrate Esterase family 1 of the carbohydrate-active enzymes (CAZy) database.

PubMed

Mäkelä, Miia R; Dilokpimol, Adiphol; Koskela, Salla M; Kuuskeri, Jaana; de Vries, Ronald P; Hildén, Kristiina

2018-04-26

Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell-wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and prediction of function by sequence similarity. Recently, an increasing number of fungal FAEs have been biochemically characterized, owing to their potential in various biotechnological applications and multitude of candidate FAEs in fungal genomes. However, only part of the fungal FAEs are included in Carbohydrate Esterase family 1 (CE1) of the carbohydrate-active enzymes (CAZy) database. In this work, we performed a phylogenetic analysis that divided the fungal members of CE1 into five subfamilies of which three contained characterized enzymes with conserved activities. Conservation within one of the subfamilies was confirmed by characterization of an additional CE1 enzyme from Aspergillus terreus. Recombinant A. terreus FaeD (AtFaeD) showed broad specificity towards synthetic methyl and ethyl esters, and released ferulic acid from plant biomass substrates, demonstrating its true FAE activity and interesting features as potential biocatalyst. The subfamily division of the fungal CE1 members enables more efficient selection of candidate enzymes for biotechnological processes. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Lys98 Substitution in Human AP Endonuclease 1 Affects the Kinetic Mechanism of Enzyme Action in Base Excision and Nucleotide Incision Repair Pathways

PubMed Central

Timofeyeva, Nadezhda A.; Koval, Vladimir V.; Ishchenko, Alexander A.; Saparbaev, Murat K.; Fedorova, Olga S.

2011-01-01

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme in the base excision repair (BER) and nucleotide incision repair (NIR) pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt) protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5′-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (F, tetrahydrofuran) containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU) containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg2+ concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP) site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage of DHU- and AP-substrates. PMID:21912662

Biomolecular hybrid material and process for preparing same and uses for same

DOEpatents

Kim, Jungbae [Richland, WA

2010-11-23

Disclosed is a composition and method for fabricating novel hybrid materials comprised of, e.g., carbon nanotubes (CNTs) and crosslinked enzyme clusters (CECs). In one method, enzyme-CNT hybrids are prepared by precipitation of enzymes which are subsequently crosslinked, yielding crosslinked enzyme clusters (CECs) on the surface of the CNTs. The CEC-enzyme-CNT hybrids exhibit high activity per unit area or mass as well as improved enzyme stability and longevity over hybrid materials known in the art. The CECs in the disclosed materials permit multilayer biocatalytic coatings to be applied to surfaces providing hybrid materials suitable for use in, e.g., biocatalytic applications and devices as described herein.

Catalytic activity of enzymes immobilized on AlGaN /GaN solution gate field-effect transistors

NASA Astrophysics Data System (ADS)

Baur, B.; Howgate, J.; von Ribbeck, H.-G.; Gawlina, Y.; Bandalo, V.; Steinhoff, G.; Stutzmann, M.; Eickhoff, M.

2006-10-01

Enzyme-modified field-effect transistors (EnFETs) were prepared by immobilization of penicillinase on AlGaN /GaN solution gate field-effect transistors. The influence of the immobilization process on enzyme functionality was analyzed by comparing covalent immobilization and physisorption. Covalent immobilization by Schiff base formation on GaN surfaces modified with an aminopropyltriethoxysilane monolayer exhibits high reproducibility with respect to the enzyme/substrate affinity. Reductive amination of the Schiff base bonds to secondary amines significantly increases the stability of the enzyme layer. Electronic characterization of the EnFET response to penicillin G indicates that covalent immobilization leads to the formation of an enzyme (sub)monolayer.

Increased Activity of Rhizosphere and Hyphosphere Enzymes under Elevated CO2 in a Loblolly Pine Stand

NASA Astrophysics Data System (ADS)

Meier, I.; Phillips, R.

2012-12-01

The stimulatory effect of elevated atmospheric CO2 under global climate change on forest productivity has been predicted to decrease over time as pools of available N in soil become depleted, but empirical support for such progressive N limitation has been lacking. Increased N acquisition from soil depleted in inorganic nitrogen requires stimulation of the microbial processing of organic N, possibly through increasing C supply to soil by plant roots or mycorrhizal hyphae. Increases in (mycorr)rhizosphere C fluxes could stimulate microbes to produce extra-cellular enzymes that release N from SOM, feeding back from soil microsites to ecosystem-scale processes. We investigated the influence of elevated CO2 on root exudation and soil enzyme activity at the Duke Forest FACE site, USA, where loblolly pine (Pinus taeda L.) stands have been exposed to elevated CO2 for 14 years and N fertilization for five years. In each plot, root boxes containing acetate windows were installed in 2008. Two years after installation, we collected soils adjacent to root tips (the rhizosphere), hyphal tips (the hyphosphere) and bulk soil. We measured in situ root exudation rates from intact pine roots. Study objectives were to analyze (i) the influence of atmospheric CO2 on root exudation and extra-cellular enzyme activities, (ii) the influence of soil N availability in regulating these activities, and (iii) the relationship between the activities of enzymes involved in N cycling in soils and gross N transformations at soil microsites. Elevated atmospheric CO2 significantly increased the activity of β-1-4-N-acetylglucosaminidase (NAG) in the rhizosphere by almost 2.5 times (39 to 95 nmol h-1 g-1), and 1.6fold in the hyphosphere relative to ambient plots. NAG is an enzyme involved in the degradation of chitin from the cell walls of soil organisms, releasing absorbable forms of nitrogen. The activity of peroxidase, which degrades aromatic C compounds of SOM, increased significantly in the hyphosphere of stands exposed to elevated CO2. Nitrogen fertilization diminished this effect of elevated CO2 on enzyme activities at microsites. Our results show that the metabolism of microbial communities is shifted to the decomposition of organic N under elevated atmospheric CO2, presumably stimulated by N limitation and increased root C exudation.

Microbial Extracellular Enzyme Activity and Community Assembly Processes Post Fire Disturbance Amanda Labrado, University of Texas at El Paso; Emily B. Graham, University of Colorado Boulder; Joseph E. Knelman, University of Colorado Boulder; Scott Ferrenberg, University of Colorado Boulder; Diana R. Nemergut, University of Colorado Boulder

NASA Astrophysics Data System (ADS)

Labrdo, A.; Knelman, J. E.; Graham, E. B.; Ferrenberg, S.; Nemergut, D. R.

2013-12-01

Microbes control major biogeochemical cycles and can directly impact the carbon, nitrogen, and phosphorus pools and fluxes of soils. However, many questions remain regarding when and where data on microbial community structure are necessary to accurately predict biogeochemical processes. In particular, it is unknown how shifts in microbial assembly processes may relate to changes in the relationship between community structure and ecosystem function. Here, we examine soil microbial community assembly processes and extracellular enzyme activity (EEA) at 4-weeks and 16-weeks after the Fourmile Canyon Fire in Boulder, CO in order to determine the effects of disturbance on community assembly and EEA. Microbial community structure was determined from 16S rRNA gene pyrosequencing, edaphic properties were determined using standard biogeochemical assays, and extracellular enzyme activity for β-1, 4-glucosidase (BG) and β-1, 4-N-acetylglucosaminidase (NAG) enzymes were determined using fluorimetric assays. Stepwise linear regressions were used to determine the effects of microbial community structure and edaphic factors on EEA. We determined that in 4-week post fire samples EEA was only correlated with microbial predictors. However, we observed a shift with 16-week samples in which EEA was significantly related to edaphic predictors. Null derivation analysis of community assembly revealed that communities in the 4-week samples were more neutrally assembled than communities in the 16-week samples. Together, these results support a conceptual model in which the relationship between edaphic factors and ecosystem processes is somewhat decoupled in more neutrally assembled communities, and data on microbial community structure is important to most accurately predict function.

Halophilic Bacteria as a Source of Novel Hydrolytic Enzymes

PubMed Central

de Lourdes Moreno, María; Pérez, Dolores; García, María Teresa; Mellado, Encarnación

2013-01-01

Hydrolases constitute a class of enzymes widely distributed in nature from bacteria to higher eukaryotes. The halotolerance of many enzymes derived from halophilic bacteria can be exploited wherever enzymatic transformations are required to function under physical and chemical conditions, such as in the presence of organic solvents and extremes in temperature and salt content. In recent years, different screening programs have been performed in saline habitats in order to isolate and characterize novel enzymatic activities with different properties to those of conventional enzymes. Several halophilic hydrolases have been described, including amylases, lipases and proteases, and then used for biotechnological applications. Moreover, the discovery of biopolymer-degrading enzymes offers a new solution for the treatment of oilfield waste, where high temperature and salinity are typically found, while providing valuable information about heterotrophic processes in saline environments. In this work, we describe the results obtained in different screening programs specially focused on the diversity of halophiles showing hydrolytic activities in saline and hypersaline habitats, including the description of enzymes with special biochemical properties. The intracellular lipolytic enzyme LipBL, produced by the moderately halophilic bacterium Marinobacter lipolyticus, showed advantages over other lipases, being an enzyme active over a wide range of pH values and temperatures. The immobilized LipBL derivatives obtained and tested in regio- and enantioselective reactions, showed an excellent behavior in the production of free polyunsaturated fatty acids (PUFAs). On the other hand, the extremely halophilic bacterium, Salicola marasensis sp. IC10 showing lipase and protease activities, was studied for its ability to produce promising enzymes in terms of its resistance to temperature and salinity. PMID:25371331

Screening for Cellulase Encoding Clones in Metagenomic Libraries.

PubMed

Ilmberger, Nele; Streit, Wolfgang R

2017-01-01

For modern biotechnology there is a steady need to identify novel enzymes. In biotechnological applications, however, enzymes often must function under extreme and nonnatural conditions (i.e., in the presence of solvents, high temperature and/or at extreme pH values). Cellulases have many industrial applications from the generation of bioethanol, a realistic long-term energy source, to the finishing of textiles. These industrial processes require cellulolytic activity under a wide range of pH, temperature, and ionic conditions, and they are usually carried out by mixtures of cellulases. Investigation of the broad diversity of cellulolytic enzymes involved in the natural degradation of cellulose is necessary for optimizing these processes.

Enzyme dehydration using Microglassification™ preserves the protein's structure and function.

PubMed

Aniket; Gaul, David A; Bitterfield, Deborah L; Su, Jonathan T; Li, Victoria M; Singh, Ishita; Morton, Jackson; Needham, David

2015-02-01

Controlled enzyme dehydration using a new processing technique of Microglassification™ has been investigated. Aqueous solution microdroplets of lysozyme, α-chymotrypsin, catalase, and horseradish peroxidase were dehydrated in n-pentanol, n-octanol, n-decanol, triacetin, or butyl lactate, and changes in their structure and function were analyzed upon rehydration. Water solubility and microdroplet dissolution rate in each solvent decreased in the order: butyl lactate > n-pentanol > triacetin > n-octanol > n-decanol. Enzymes Microglassified™ in n-pentanol retained higher activity (93%-98%) than n-octanol (78%-85%) or n-decanol (75%-89%), whereas those Microglassified™ in triacetin (36%-75%) and butyl lactate (48%-79%) retained markedly lower activity. FTIR spectroscopy analyses showed α-helix to β-sheet transformation for all enzymes upon Microglassification™, reflecting a loss of bound water in the dried state; however, the enzymes reverted to native-like conformation upon rehydration. Accelerated stressed-storage tests using Microglassified™ lysozyme showed a significant (p < 0.01) decrease in enzymatic activity from 46,560 ± 2736 to 31,060 ± 4327 units/mg after 3 months of incubation; however, it was comparable to the activity of the lyophilized formulation throughout the test period. These results establish Microglassification™ as a viable technique for enzyme preservation without affecting its structure or function. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Proteolytic and amylolytic enzymes from a newly isolated Bacillus mojavensis SA: Characterization and applications as laundry detergent additive and in leather processing.

PubMed

Hammami, Amal; Fakhfakh, Nahed; Abdelhedi, Ola; Nasri, Moncef; Bayoudh, Ahmed

2018-03-01

The present work aims to study the simultaneous production of highly alkaline proteases and thermostable α-amylases by a newly isolated bacterium Bacillus mojavensis SA. The optimum pH and temperature of amylase activity were 9.0 and 55°C, respectively, while those of the proteolytic activity were 12.0 and 60°C, respectively. Both α-amylase and protease enzymes showed a high stability towards a wide range of pH and temperature. Furthermore, SA crude enzymes were relatively stable towards non-ionic (Tween 20, Tween 80 and Triton X-100) and anionic (SDS) surfactants, as well as oxidizing agents. Both activities were improved by the presence of polyethylene glycol 4000 and glycerol. Additionally, the crude enzymes showed excellent stability against various solid and liquid detergents. Wash performance analysis revealed that the SA crude enzymes exhibited a remarkable efficiency in the removal of a variety type of stains, such as blood, chocolate, coffee and oil. On the other side, SA proteases revealed a potential dehairing activity of animal hide without chemical assistance or fibrous proteins hydrolysis. Thus, considering their promising properties, B. mojavensis SA crude enzymes could be used in several biotechnological bioprocesses. Copyright © 2017 Elsevier B.V. All rights reserved.

Extracellular lipase of an entomopathogenic fungus effecting larvae of a scale insect.

PubMed

Ali, Shaukat; Ren, Shunxiang; Huang, Zhen

2014-11-01

Lipases play an important role in the infection process of entomopathogenic fungi by hydrolyzing the ester bonds of lipoproteins, fats and waxes present on the insect surface and in the body. Here we report the purification and characterization of an extracellular lipase from Isaria fumosorosea. The enzyme was purified (138.46-fold) in three steps using (NH4 )2 SO4 precipitation followed by DEAE-cellulose and Sephadex G-100 column chromatography. The molecular weight of purified enzyme was determined to be 31 KDa by SDS-PAGE. The optimum temperature and pH for enzyme activity were 35 °C and 7.0, respectively, using p-nitrophenylpalmitate as the substrate. Lipolytic activity was enhanced in the presence of Ca(+2) , Mg(+2) , Na(+) , and NH4 (+) salts, while Zn(+2) , Fe(+2) , and Cu(+2) inhibited enzyme activity. The enzyme displayed broad substrate specificity with the highest activity observed for coconut oil and p-nitrophenyl carprate. Topical co-application of purified lipase with fungal conidial suspensions decreased the median survival time (ST50 ) of Dysmicoccus neobrevipes nymphs as compared to the fungus alone. Our results indicate that an extracellular lipase produced by I. fumosorosea can be exploited for development of enzyme-based insect management. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing the Role of N-Linked Glycans in the Stability and Activity of Fungal Cellobiohydrolases by Mutational Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adney, W. S.; Jeoh, T.; Beckham, G. T.

2009-01-01

The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many fungal species produce a glycoside hydrolase family 7 (Cel7A) cellobiohydrolase, a class of enzymes that catalytically process from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10, 2008) that expression of T. reesei cellobiohydrolase I in a commonlymore » used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120 h compared to the heterologously expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after 120 h compared to the wild type P. funiculosum enzyme expressed in A. niger var. awamori. Overall, this study demonstrates that 'tuning' enzyme glycosylation for expression from heterologous expression hosts is essential for generating engineered enzymes with optimal stability and activity.« less

On the distributive patterns of ATPase activity and its functional significance in retinae of certain birds.

PubMed

Tewari, H B; Tyagi, H R

1977-01-01

The present study incorporates the details of distribution of adenosine triphosphatase amongst the various constituents of retinae of Passer, Psittacula, Streptopelia and Athene. The outer segments in all the cases are intensely positive for the enzyme. This is the part where the light strikes first and initiates the visual processes. The nuclear layers are also positive for the enzyme activity. It is interesting to note that inner plexiform layers show clear-out demarcations of various sub-synaptic layers in all the birds except Psittacula. The ganglion cells and optic nerve fibres are also positive for the enzyme.

Effects of multiple enzyme-substrate interactions in basic units of cellular signal processing

NASA Astrophysics Data System (ADS)

Seaton, D. D.; Krishnan, J.

2012-08-01

Covalent modification cycles are a ubiquitous feature of cellular signalling networks. In these systems, the interaction of an active enzyme with the unmodified form of its substrate is essential for signalling to occur. However, this interaction is not necessarily the only enzyme-substrate interaction possible. In this paper, we analyse the behaviour of a basic model of signalling in which additional, non-essential enzyme-substrate interactions are possible. These interactions include those between the inactive form of an enzyme and its substrate, and between the active form of an enzyme and its product. We find that these additional interactions can result in increased sensitivity and biphasic responses, respectively. The dynamics of the responses are also significantly altered by the presence of additional interactions. Finally, we evaluate the consequences of these interactions in two variations of our basic model, involving double modification of substrate and scaffold-mediated signalling, respectively. We conclude that the molecular details of protein-protein interactions are important in determining the signalling properties of enzymatic signalling pathways.

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

PubMed Central

Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

2011-01-01

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications

PubMed Central

2014-01-01

Background Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. Results A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. Conclusions The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications. PMID:24485248

Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications.

PubMed

Li, Chunfang; Du, Miaofen; Cheng, Bin; Wang, Lushan; Liu, Xinqiang; Ma, Cuiqing; Yang, Chunyu; Xu, Ping

2014-01-31

Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

PubMed Central

Dougherty, W G; Semler, B L

1993-01-01

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates

PubMed Central

Carvalho, Ana Caroline Lustosa de Melo; Fonseca, Thiago de Sousa; de Mattos, Marcos Carlos; de Oliveira, Maria da Conceição Ferreira; de Lemos, Telma Leda Gomes; Molinari, Francesco; Romano, Diego; Serra, Immacolata

2015-01-01

Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase. PMID:26690428

Carotene-degrading activities from Bjerkandera adusta possess an application in detergent industries.

PubMed

Linke, Diana; Leonhardt, Robin; Eisele, Nadine; Petersen, Laura M; Riemer, Stephanie; Nimtz, Manfred; Berger, Ralf G

2015-06-01

Four extracellular enzymes, a versatile peroxidase, a manganese peroxidase, a dye-decolorizing peroxidase and a lignin peroxidase were discovered in liquid cultures of the basidiomycete Bjerkandera adusta. All of them cleaved β-carotene effectively. Expression was enhanced in the presence of β-carotene or Coomassie Brilliant Blue and peaked after 7-9 days. The monomeric proteins were purified by ion exchange and size exclusion chromatography and exhibited molecular masses of 41, 43, 51 and 43 kDa, respectively. The coding sequences showed homologies from 61 to 89 % to peroxidases from other basidiomycetes. The novel enzymes retained strong activity even in the absence of hydrogen peroxide and at alkaline pH. De-staining of fabrics using detergent-tolerant enzymes may help to save the most important bio-resources, energy and water, in washing processes and led to green processes in textile cleaning.

Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification

NASA Astrophysics Data System (ADS)

Longoria, Adriana; Tinoco, Raunel; Torres, Eduardo

An overview of enzyme technology applied to peroxidases is made. Immobilization on organic, inorganic, and hybrid supports; chemical modification of amino acids and heme group; and genetic modification by site-directed and random mutagenesis are included. Different strategies that were carried out to improve peroxidase performance in terms of stability, selectivity, and catalytic activity are analyzed. Immobilization of peroxidases on inorganic and organic materials enhances the tolerance of peroxidases toward the conditions normally found in many industrial processes, such as the presence of an organic solvent and high temperature. In addition, it is shown that immobilization helps to increase the Total Turnover Number at levels high enough to justify the use of a peroxidase-based biocatalyst in a synthesis process. Chemical modification of peroxidases produces modified enzymes with higher thermostability and wider substrate variability. Finally, through mutagenesis approaches, it is possible to produce modified peroxidases capable of oxidizing nonnatural substrates with high catalytic activity and affinity.

Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization.

PubMed

Johnson, Patrick A; Park, Hee Joon; Driscoll, Ashley J

2011-01-01

Immobilized enzymes are drawing significant attention for potential commercial applications as biocatalysts by reducing operational expenses and by increasing process utilization of the enzymes. Typically, immobilized enzymes have greater thermal and operational stability at various pH values, ionic strengths and are more resistant to denaturation that the soluble native form of the enzyme. Also, immobilized enzymes can be recycled by utilizing the physical or chemical properties of the supporting material. Magnetic nanoparticles provide advantages as the supporting material for immobilized enzymes over competing materials such as: higher surface area that allows for greater enzyme loading, lower mass transfer resistance, less fouling effect, and selective, nonchemical separation from the reaction mixture by an applied a magnetic field. Various surface modifications of magnetic nanoparticles, such as silanization, carbodiimide activation, and PEG or PVA spacing, aid in the binding of single or multienzyme systems to the particles, while cross-linking using glutaraldehyde can also stabilize the attached enzymes.

Use of Pressure Activation in Food Quality Improvement.

PubMed

Shigematsu, Toru

2015-01-01

Beside intensive studies on inactivation microorganisms by high hydrostatic pressure (HP) for food storage, pressure effects on property of food materials have also been studied based on knowledge in pressure effect on biomolecules. Pressure effects on biological membranes and mass transfer in cellular biological materials and on enzyme activity would give an idea that HP treatment can introduce two types of activations into food materials: improved mass transfer and enzyme activity. Studies focusing on these pressure activations on food materials were then reviewed. Rice flour with an exclusively fine mean particle size and small starch damage was obtained due to improved water absorption properties and/or enzyme activity by HP. HP treatment increased of free amino acids and γ-aminobutyric acid (GABA) in rice and soybeans due to improved proteolysis and amino acid metabolism. Improvement of antioxidant activity and alteration of polyphenolic-compounds composition in food materials were also demonstrated by HP treatment. The HP-induced activations on food materials could contribute towards processing technologies for food quality improvement.

Sucrose-Metabolizing Enzymes in Transport Tissues and Adjacent Sink Structures in Developing Citrus Fruit 1

PubMed Central

Lowell, Cadance A.; Tomlinson, Patricia T.; Koch, Karen E.

1989-01-01

Juice tissues of citrus lack phloem; therefore, photosynthates enroute to juice sacs exit the vascular system on the surface of each segment. Areas of extensive phloem unloading and transport (vascular bundles + segment epidermis) can thus be separated from those of assimilate storage (juice sacs) and adjacent tissues where both processes occur (peel). Sugar composition, dry weight accumulation, and activities of four sucrose-metabolizing enzymes (soluble and cell-wall-bound acid invertase, alkaline invertase, sucrose synthase, and sucrose phosphate synthase) were measured in these transport and sink tissues of grapefruit (Citrus paradisi Macf.) to determine more clearly whether a given enzyme appeared to be more directly associated with assimilate transport versus deposition or utilization. Results were compared at three developmental stages. Activity of sucrose (per gram fresh weight and per milligram protein) extracted from zones of extensive phloem unloading and transport was significantly greater than from adjacent sink tissues during the stages (II and III) when juice sacs grow most rapidly. In stage II fruit, activity of sucrose synthase also significantly surpassed that of all other sucrose-metabolizing enzymes in extracts from the transport tissues (vascular bundles + segment epidermis). In contrast, sucrose phosphate synthase and alkaline invertase at this stage of growth were the most active enzymes from adjacent, rapidly growing, phloem-free sink tissues (juice sacs). Activity of these two enzymes in extracts from juice sacs was significantly greater than that form the transport tissues (vascular bundles + segment epidermis). Soluble acid invertase was the most active enzyme in extracts from all tissues of very young fruit (stage I), including nonvascular regions, but nearly disappeared prior to the onset of juice sac sugar accumulation. The physiological function of high sucrose synthase activity in the transport tissues during rapid sucrose import remains to be determined. PMID:16666942

Purification and characterization of a protease produced by Bacillus megaterium RRM2: application in detergent and dehairing industries.

PubMed

Rajkumar, Renganathan; Jayappriyan, Kothilmozhian Ranishree; Rengasamy, Ramasamy

2011-12-01

An alkaline serine protease produced by Bacillus megaterium RRM2 isolated from the red alga, Kappaphycus alvarezii (Doty) Doty ex Silva was studied for the first time and the same analyzed for the production of protease in the present study. Identification of the bacterium was done on the basis of both biochemical analysis and by 16S rDNA sequence analysis. The extracellular protease obtained from B. megaterium RRM2 was purified by a three-step process involving ammonium sulphate precipitation, gel filtration (Sephadex G100) and Q-Sepharose column chromatography. The purity was found to be 30.6-fold with a specific activity of 3591.5 U/mg protein with a molecular weight of 27 kDa. The metal ions Ca(2+), Mg(2+), K(+) and Na(+) marginally enhanced the activity of the purified enzyme while Hg(2+), Cu(2+), Fe(2+), CO(2+) and Zn(2+), had reduced the activity. The enzyme was found to be active in the pH range of 9.0-10.0 and remained active up to 60 °C. Phenyl Methyl Sulfonyl Fluoride (PMSF) inhibited the enzyme activity, thus, confirming that this enzyme is an alkaline serine protease. Likewise, DTT also inhibited the enzyme thus confirming the disulfide nature of the enzyme. The enzyme exhibited a high degree of tolerance to Sodium Dodecyl Sulphate (SDS). The partially purified protease when used as an additive in the commercial detergents was found to be a suitable source for washing clothes especially those stained with blood. Further, it showed good dehairing activity within a short duration in goat skin without affecting its collagen component. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioremediation of Industrial Waste Through Enzyme Producing Marine Microorganisms.

PubMed

Sivaperumal, P; Kamala, K; Rajaram, R

Bioremediation process using microorganisms is a kind of nature-friendly and cost-effective clean green technology. Recently, biodegradation of industrial wastes using enzymes from marine microorganisms has been reported worldwide. The prospectus research activity in remediation area would contribute toward the development of advanced bioprocess technology. To minimize industrial wastes, marine enzymes could constitute a novel alternative in terms of waste treatment. Nowadays, the evidence on the mechanisms of bioremediation-related enzymes from marine microorganisms has been extensively studied. This review also will provide information about enzymes from various marine microorganisms and their complexity in the biodegradation of comprehensive range of industrial wastes. © 2017 Elsevier Inc. All rights reserved.

Nitrogen Deposition Reduces Decomposition Rates Through Shifts in Microbial Community Composition and Function

NASA Astrophysics Data System (ADS)

Waldrop, M.; Zak, D.; Sinsabaugh, R.

2002-12-01

Atmospheric nitrogen (N) deposition may alter soil biological activity in northern hardwood forests by repressing phenol oxidase enzyme activity and altering microbial community composition, thereby slowing decomposition and increasing the export of phenolic compounds. We tested this hypothesis by adding 13C-labelled cellobiose, vanillin, and catechol to control and N fertilized soils (30 and 80 kg ha-1) collected from three forests; two dominated by Acer Saccharum and one dominated by Quercus Alba and Quercus Velutina. While N deposition increased total microbial respiration, it decreased soil oxidative enzyme activities, resulting in slower degradation rates of all compounds, and larger DOC pools. This effect was larger in the oak forest, where fungi dominate C-cycling processes. DNA and 13C-phospolipid analyses showed that N addition altered the fungal community and reduced the activity of fungal and bacterial populations in soil, potentially explaining reduced soil enzyme activities and incomplete decomposition.

The Regulation of a Post-Translational Peptide Acetyltransferase: Strategies for Selectively Modifying the Biological Activity of Neural and Endocrine Peptides

DTIC Science & Technology

1988-02-01

quantitatively miror pathway. Only two of the enzymes which process 8-endorphin have been firmly identified, peptide acetyltransferase and... quantitatively minor. This implied that perhaps peptide acetyltransferase is not a critical determinant of the bioactivity of B-endorphin in brain. If so...provided us with a more difinitive understanding of the role of processing enzyme regulation in the overall biochemical and cellular response of the

Lipoamidase activity in normal and mutagenized pancreatic cholesterol esterase (bile salt-stimulated lipase).

PubMed Central

Hui, D Y; Hayakawa, K; Oizumi, J

1993-01-01

Purified human milk lipoamidase was digested with endoproteinase Lys-C and the digested peptides were subjected to gasphase microsequence analysis. The sequencing of three isolated peptides of human milk lipoamidase revealed the identity of this protein with human milk bile salt-stimulated lipase (pancreatic cholesterol esterase). The identity of the cholesterol esterase with lipoamidase was confirmed by expressing a recombinant form of rat pancreatic cholesterol esterase and testing for lipoamidase activity of the recombinant protein. The results showed that the recombinant cholesterol esterase displayed both lipolytic and lipoamidase activities and was capable of hydrolysing triacetin and lipoyl-4-aminobenzoate (LPAB). The mechanisms of the esterase and amidase activities of the enzyme were further tested by determining enzyme activity in a mutagenized cholesterol esterase with a His435-->Gln435 substitution. This mutation has been shown previously to abolish enzyme activity against esterase substrates [DiPersio, Fontaine and Hui (1991) J. Biol. Chem. 266, 4033-4036]. We showed that the mutagenized protein was effective in hydrolysing the amidase substrate LPAB and displayed similar enzyme kinetics to those of the native enzyme. These data indicate that the mechanism for the cholesterol esterase hydrolysis of lipoamides is different from that of the hydrolysis of substrates with an ester linkage. The presence of an enzyme in the gastrointestinal tract capable of both ester and amide hydrolysis suggests an important role for this protein in the digestion and absorption processes. PMID:8471055

Applicability of API ZYM to capture seasonal and spatial variabilities in lake and river sediments.

PubMed

Patel, Drashti; Gismondi, Renee; Alsaffar, Ali; Tiquia-Arashiro, Sonia M

2018-05-02

Waters draining into a lake carry with them much of the suspended sediment that is transported by rivers and streams from the local drainage basin. The organic matter processing in the sediments is executed by heterotrophic microbial communities, whose activities may vary spatially and temporally. Thus, to capture and evaluate some of these variabilities in the sediments, we sampled six sites: three from the St. Clair River and three from Lake St. Clair in spring, summer, fall, and winter of 2016. At all sites and dates, we investigated the spatial and temporal variations in 19 extracellular enzyme activities using API ZYM. Our results indicated that a broad range of enzymes were found to be active in the sediments. Phosphatases, lipases, and esterases were synthesized most intensively by the sediment microbial communities. No consistent difference was found between the lake and sediment samples. Differences were more obvious between sites and seasons. Sites with the highest metabolic (enzyme) diversity reflected the capacity of the sediment microbial communities to breakdown a broader range of substrates and may be linked to differences in river and lake water quality. The seasonal variability of the enzymes activities was governed by the variations of environmental factors caused by anthropogenic and terrestrial inputs, and provides information for a better understanding of the dynamics of sediment organic matter of the river and lake ecosystems. The experimental results suggest that API ZYM is a simple and rapid enzyme assay procedure to evaluate natural processes in ecosystems and their changes.

Thermophilic enzymes and their applications in biocatalysis: a robust aldo-keto reductase.

PubMed

Willies, Simon; Isupov, Misha; Littlechild, Jennifer

2010-09-01

Extremophiles are providing a good source of novel robust enzymes for use in biocatalysis for the synthesis of new drugs. This is particularly true for the enzymes from thermophilic organisms which are more robust than their mesophilic counterparts to the conditions required for industrial bio-processes. This paper describes a new aldo-keto reductase enzyme from a thermophilic eubacteria, Thermotoga maritima which can be used for the production of primary alcohols. The enzyme has been cloned and over-expressed in Escherichia coli and has been purified and subjected to full biochemical characterization. The aldo-keto reductase can be used for production of primary alcohols using substrates including benzaldehyde, 1,2,3,6-tetrahydrobenzaldehyde and para-anisaldehyde. It is stable up to 80 degrees C, retaining over 60% activity for 5 hours at this temperature. The enzyme at pH 6.5 showed a preference for the forward, carbonyl reduction. The enzyme showed moderate stability with organic solvents, and retained 70% activity in 20% (v/v) isopropanol or DMSO. These properties are favourable for its potential industrial applications.

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue.

PubMed

Nanduri, Bindu; Shack, Leslie A; Rai, Aswathy N; Epperson, William B; Baumgartner, Wes; Schmidt, Ty B; Edelmann, Mariola J

2016-12-15

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics. Copyright © 2016 Elsevier Inc. All rights reserved.

Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

PubMed

Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

2011-02-01

A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry. Copyright © 2010 Elsevier Ltd. All rights reserved.

Towards complete hydrolysis of soy flour carbohydrates by enzyme mixtures for protein enrichment: A modeling approach.

PubMed

Loman, Abdullah Al; Ju, Lu-Kwang

2016-05-01

Soy protein is a well-known nutritional supplement in proteinaceous food and animal feed. However, soybeans contain complex carbohydrate. Selective carbohydrate removal by enzymes could increase the protein content and remove the indigestibility of soy products for inclusion in animal feed. Complete hydrolysis of soy flour carbohydrates is challenging due to the presence of proteins and different types of non-structural polysaccharides. This study is designed to guide complex enzyme mixture required for hydrolysis of all types of soy flour carbohydrates. Enzyme broths from Aspergillus niger, Aspergillus aculeatus and Trichoderma reesei fermentations were evaluated in this study for soy carbohydrate hydrolysis. The resultant hydrolysate was measured for solubilized carbohydrate by both total carbohydrate and reducing sugar analyses. Conversion data attained after 48h hydrolysis were first fitted with models to determine the maximum fractions of carbohydrate hydrolyzable by each enzyme group, i.e., cellulase, xylanase, pectinase and α-galactosidase. Kinetic models were then developed to describe the increasing conversions over time under different enzyme activities and process conditions. The models showed high fidelity in predicting soy carbohydrate hydrolysis over broad ranges of soy flour loading (5-25%) and enzyme activities: per g soy flour, cellulase, 0.04-30 FPU; xylanase, 3.5-618U; pectinase, 0.03-120U; and α-galactosidase, 0.01-60U. The models are valuable in guiding the development and production of optimal enzyme mixtures toward hydrolysis of all types of carbohydrates present in soy flour and in optimizing the design and operation of hydrolysis reactor and process. Copyright © 2016 Elsevier Inc. All rights reserved.

Combined effect of three insect growth regulators on the digestive enzymatic profiles of Callosobruchus maculatus (Coleoptera: Bruchidae).

PubMed

Khatter, Najat Aly; Abuldahb, Faten Farid

2011-12-01

Insect growth regulators (IGRs) are insecticides that mimic insect produced hormones by regulatingdevelopmental process. Theyhave little or no mammalian toxicity, and are considered reduced-risk insecticides that are often exempt from tolerance requirements of regulatory agencies. IGRs, especially, chlorfluazuron, hydroprene and hexaflumuron (benzoylphenylurea) are currently studied because of possibility of using in stored products protection. Many of IGRs compounds usedin insect pests control are known to affect digestive enzymes. Chlorfluazuron, hydroprene and hexaflumuronwere tested topically at doses of 0.25%, 0.5%&1% for chlorfluazuron and hydroprene and 0.5, 1 & 2 microg/ml of hexaflumuron to investigate its effects on the activities of the digestive enzymes protease, amylase and lipase in Callosobruchusmaculatus larvae, which were affected by IGRs individually and in combination. When combined, the effect was more sever at low concentration. There were statistically significant differences (P < or = 0.05) in enzyme activities in combined and individual treatments. Combination three IGRs caused a two-fold decrease in enzyme activity even at reduced concentration. Clear dose-response relationships were established with respect to enzyme activity. A synergistic effect of IGRs was found by combination of low doses. These effects are most pronounced in early instars.

Bioprocessing of wheat bran for the production of lignocellulolytic enzyme cocktail by Cotylidia pannosa under submerged conditions.

PubMed

Sharma, Deepika; Garlapat, Vijay Kumar; Goel, Gunjan

2016-04-02

Characterization and production of efficient lignocellulytic enzyme cocktails for biomass conversion is the need for biofuel industry. The present investigation reports the modeling and optimization studies of lignocellulolytic enzyme cocktail production by Cotylidia pannosa under submerged conditions. The predominant enzyme activities of cellulase, xylanase and laccase were produced in the cocktail through submerged conditions using wheat bran as a substrate. A central composite design approach was utilized to model the production process using temperature, pH, incubation time and agitation as input variables with the goal of optimizing the output variables namely cellulase, xylanase and laccase activities. The effect of individual, square and interaction terms on cellulase, xylanase and laccase activities were depicted through the non-linear regression equations with significant R(2) and P-values. An optimized value of 20 U/ml, 17 U/ml and 13 U/ml of cellulase, xylanase and laccase activities, respectively, were obtained with a media pH of 5.0 in 77 h at 31C, 140 rpm using wheatbran as a substrate. Overall, the present study introduces a fungal strain, capable of producing lignocellulolytic enzyme cocktail for subsequent applications in biofuel industry.

Bioprocessing of wheat bran for the production of lignocellulolytic enzyme cocktail by Cotylidia pannosa under submerged conditions

PubMed Central

Sharma, Deepika; Garlapat, Vijay Kumar; Goel, Gunjan

2016-01-01

ABSTRACT Characterization and production of efficient lignocellulytic enzyme cocktails for biomass conversion is the need for biofuel industry. The present investigation reports the modeling and optimization studies of lignocellulolytic enzyme cocktail production by Cotylidia pannosa under submerged conditions. The predominant enzyme activities of cellulase, xylanase and laccase were produced in the cocktail through submerged conditions using wheat bran as a substrate. A central composite design approach was utilized to model the production process using temperature, pH, incubation time and agitation as input variables with the goal of optimizing the output variables namely cellulase, xylanase and laccase activities. The effect of individual, square and interaction terms on cellulase, xylanase and laccase activities were depicted through the non-linear regression equations with significant R2 and P-values. An optimized value of 20 U/ml, 17 U/ml and 13 U/ml of cellulase, xylanase and laccase activities, respectively, were obtained with a media pH of 5.0 in 77 h at 31C, 140 rpm using wheatbran as a substrate. Overall, the present study introduces a fungal strain, capable of producing lignocellulolytic enzyme cocktail for subsequent applications in biofuel industry. PMID:26941214

Agricultural waste from the tequila industry as substrate for the production of commercially important enzymes.

PubMed

Huitron, C; Perez, R; Sanchez, A E; Lappe, P; Rocha Zavaleta, L

2008-01-01

Approximately 1 million tons of Agave tequilana plants are processed annually by the Mexican Tequila industry generating vast amounts of agricultural waste. The aim of this study was to investigate the potential use of Agave tequilana waste as substrate for the production of commercially important enzymes. Two strains of Aspergillus niger (CH-A-2010 and CH-A-2016), isolated from agave fields, were found to grow and propagate in submerged cultures using Agave tequilana waste as substrate. Isolates showed simultaneous extracellular inulinase, xylanase, pectinase, and cellulase activities. Aspergillus CH-A-2010 showed the highest production of inulinase activity (1.48 U/ml), whereas Aspergillus niger CH-A-2016 produced the highest xylanase (1.52 U/ml) and endo-pectinase (2.7U/ml) activities. In both cases production of enzyme activities was significantly higher on Agave tequilana waste than that observed on lemon peel and specific polymeric carbohydrates. Enzymatic hydrolysis of raw A. tequilana stems and leaves, by enzymes secreted by the isolates yielded maximum concentrations of reducing sugars of 28.2 g/l, and 9.9 g/l respectively. In conclusion, Agave tequilana waste can be utilized as substrate for the production of important biotechnological enzymes.

Production of Pectate Lyase by Penicillium viridicatum RFC3 in Solid-State and Submerged Fermentation

PubMed Central

Ferreira, Viviani; da Silva, Roberto; Silva, Dênis; Gomes, Eleni

2010-01-01

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500 U  mL−1 or 300 Ug−1 of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme from SSF and designated PL I, II, III, IV, V, and VI, while five peaks were obtained from crude enzyme from SmF and labeled PL  I′, II′, III′, IV′, and VII′. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35°C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45°C. Crude enzyme from SmF and PL III′ showed thermophilic profiles of activity, with maximum activity at 60 and 55°C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0–10.0 and PL III was most stable in the pH range 4.0–7.0. Crude enzyme from SmF retained 70%–80% of its maximum activity in the acid-neutral pH range (4.0–7.0), but PIII showed high stability at alkaline pH (7.5–9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55°C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes. PMID:20689719

The role of electrostatic interactions in protease surface diffusion and the consequence for interfacial biocatalysis.

PubMed

Feller, Bob E; Kellis, James T; Cascão-Pereira, Luis G; Robertson, Channing R; Frank, Curtis W

2010-12-21

This study examines the influence of electrostatic interactions on enzyme surface diffusion and the contribution of diffusion to interfacial biocatalysis. Surface diffusion, adsorption, and reaction were investigated on an immobilized bovine serum albumin (BSA) multilayer substrate over a range of solution ionic strength values. Interfacial charge of the enzyme and substrate surface was maintained by performing the measurements at a fixed pH; therefore, electrostatic interactions were manipulated by changing the ionic strength. The interfacial processes were investigated using a combination of techniques: fluorescence recovery after photobleaching, surface plasmon resonance, and surface plasmon fluorescence spectroscopy. We used an enzyme charge ladder with a net charge ranging from -2 to +4 with respect to the parent to systematically probe the contribution of electrostatics in interfacial enzyme biocatalysis on a charged substrate. The correlation between reaction rate and adsorption was determined for each charge variant within the ladder, each of which displayed a maximum rate at an intermediate surface concentration. Both the maximum reaction rate and adsorption value at which this maximum rate occurs increased in magnitude for the more positive variants. In addition, the specific enzyme activity increased as the level of adsorption decreased, and for the lowest adsorption values, the specific enzyme activity was enhanced compared to the trend at higher surface concentrations. At a fixed level of adsorption, the specific enzyme activity increased with positive enzyme charge; however, this effect offers diminishing returns as the enzyme becomes more highly charged. We examined the effect of electrostatic interactions on surface diffusion. As the binding affinity was reduced by increasing the solution ionic strength, thus weakening electrostatic interaction, the rate of surface diffusion increased considerably. The enhancement in specific activity achieved at the lowest adsorption values is explained by the substantial rise in surface diffusion at high ionic strength due to decreased interactions with the surface. Overall, knowledge of the electrostatic interactions can be used to control surface parameters such as surface concentration and surface diffusion, which intimately correlate with surface biocatalysis. We propose that the maximum reaction rate results from a balance between adsorption and surface diffusion. The above finding suggests enzyme engineering and process design strategies for improving interfacial biocatalysis in industrial, pharmaceutical, and food applications.

Electrostatic steering and ionic tethering in enzyme-ligand binding: insights from simulations.

PubMed

Wade, R C; Gabdoulline, R R; Lüdemann, S K; Lounnas, V

1998-05-26

To bind at an enzyme's active site, a ligand must diffuse or be transported to the enzyme's surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and beta-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as "ionic tethering." We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme's surroundings even when the substrate is nonpolar.

Investigation on the Influence of Bio-catalytic Enzyme Produced from Fruit and Vegetable Waste on Palm Oil Mill Effluent

NASA Astrophysics Data System (ADS)

Rasit, Nazaitulshila; Chee Kuan, Ooi

2018-04-01

Pre-consumer waste from supermarkets, such as vegetables and fruits dreg are always discarded as solid waste and disposed to landfill. Implementing waste recovery method as a form of waste management strategy will reduce the amount of waste disposed. One of the ways to achieve this goal is through fermentation of the pre-consumer supermarket waste to produce a solution known as garbage enzyme. This study has been conducted to produce and characterize biocatalytic garbage enzyme and to evaluate its influence on palm oil mill effluent as a pre-treatment process before further biological process takes place. Garbage enzyme was produced by three-month long fermentation of a mixture of molasses, pre-consumer supermarket residues, and water in the ratio of 1:3:10. Subsequently, the characterization of enzyme was conducted based on pH, total solids (TS), total suspended solids (TSS), total dissolved solids (TDS), chemical oxygen demand (COD), and enzyme activities. The influence of produced enzyme was evaluated on oil & grease (O&G), TSS and COD of palm oil mill effluent (POME). Different levels of dilution of garbage enzyme to POME samples (5%, 10%, 15%) were explored as pre-treatment (duration of six days) and the results showed that the garbage enzyme contained bio-catalytic enzyme such as amylase, protease, and lipase. The pre-treatment showed removal of 90% of O&G in 15% dilution of garbage enzyme. Meanwhile, reduction of TSS and COD in dilution of 10% garbage enzyme were measured at 50% and 25% respectively. The findings of this study are important to analyse the effectiveness of pre-treatment for further improvement of anaerobic treatment process of POME, especially during hydrolysis stage.

Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater

PubMed Central

Obayashi, Yumiko; Wei Bong, Chui; Suzuki, Satoru

2017-01-01

Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities) in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution) DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube), protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the materials and method for measurements should be carefully selected in order to accurately determine the activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems; especially, low protein binding materials should be chosen to use at overall processes of the measurement. PMID:29067013

Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater.

PubMed

Obayashi, Yumiko; Wei Bong, Chui; Suzuki, Satoru

2017-01-01

Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities) in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution) DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube), protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the materials and method for measurements should be carefully selected in order to accurately determine the activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems; especially, low protein binding materials should be chosen to use at overall processes of the measurement.

Effects of combined pressure and temperature on enzymes related to quality of fruits and vegetables: from kinetic information to process engineering aspects.

PubMed

Ludikhuyze, L; Van Loey, A; Indrawati; Smout, C; Hendrickx, M

2003-01-01

Throughout the last decade, high pressure technology has been shown to offer great potential to the food processing and preservation industry in delivering safe and high quality products. Implementation of this new technology will be largely facilitated when a scientific basis to assess quantitatively the impact of high pressure processes on food safety and quality becomes available. Besides, quantitative data on the effects of pressure and temperature on safety and quality aspects of foods are indispensable for design and evaluation of optimal high pressure processes, i.e., processes resulting in maximal quality retention within the constraints of the required reduction of microbial load and enzyme activity. Indeed it has to be stressed that new technologies should deliver, apart from the promised quality improvement, an equivalent or preferably enhanced level of safety. The present paper will give an overview from a quantitative point of view of the combined effects of pressure and temperature on enzymes related to quality of fruits and vegetables. Complete kinetic characterization of the inactivation of the individual enzymes will be discussed, as well as the use of integrated kinetic information in process engineering.

Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme

PubMed Central

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; Holton, James M.; Charles, Emeric; O’Connell, Mitchell R.; Doudna, Jennifer A.

2018-01-01

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR-Cas systems include the Cas13a enzyme, an RNA-activated ribonuclease (RNase) capable of crRNA processing and single-stranded RNA degradation upon target transcript binding. Here we present the 2.0 Å resolution crystal structure of a crRNA-bound L. bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define for the first time the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked prior to target RNA recognition, with implications for both bacterial immunity and diagnostic applications. PMID:28892041

Functional diversity of carbohydrate-active enzymes enabling a bacterium to ferment plant biomass.

PubMed

Boutard, Magali; Cerisy, Tristan; Nogue, Pierre-Yves; Alberti, Adriana; Weissenbach, Jean; Salanoubat, Marcel; Tolonen, Andrew C

2014-11-01

Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF

DOE PAGES

Park, Kiyoung; Li, Ning; Kwak, Yeonju; ...

2017-05-01

Binuclear non-heme iron enzymes activate O 2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O 2. Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reactionmore » shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. As a result, this activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.« less

Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Kiyoung; Li, Ning; Kwak, Yeonju

Binuclear non-heme iron enzymes activate O 2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O 2. Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reactionmore » shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. As a result, this activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.« less

Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

PubMed Central

Lewańczuk, Marcin; Koźlecki, Tomasz; Liesiene, Jolanta; Bryjak, Jolanta

2016-01-01

Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH2), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity. PMID:27711193

Celluclast and Cellic® CTec2: Saccharification/fermentation of wheat straw, solid-liquid partition and potential of enzyme recycling by alkaline washing.

PubMed

Rodrigues, Ana Cristina; Haven, Mai Østergaard; Lindedam, Jane; Felby, Claus; Gama, Miguel

2015-11-01

The hydrolysis/fermentation of wheat straw and the adsorption/desorption/deactivation of cellulases were studied using Cellic(®) CTec2 (Cellic) and Celluclast mixed with Novozyme 188. The distribution of enzymes - cellobiohydrolase I (Cel7A), endoglucanase I (Cel7B) and β-glucosidase - of the two formulations between the residual substrate and supernatant during the course of enzymatic hydrolysis and fermentation was investigated. The potential of recyclability using alkaline wash was also studied. The efficiency of hydrolysis with an enzyme load of 10 FPU/g cellulose reached >98% using Cellic(®) CTec2, while for Celluclast a conversion of 52% and 81%, was observed without and with β-glucosidase supplementation, respectively. The decrease of Cellic(®) CTec2 activity observed along the process was related to deactivation of Cel7A rather than of Cel7B and β-glucosidase. The adsorption/desorption profiles during hydrolysis/fermentation revealed that a large fraction of active enzymes remained adsorbed to the solid residue throughout the process. Surprisingly, this was the case of Cel7A and β-glucosidase from Cellic, which remained adsorbed to the solid fraction along the entire process. Alkaline washing was used to recover the enzymes from the solid residue. This method allowed efficient recovery of Celluclast enzymes; however, this may be achieved only when minor amounts of cellulose remain present. Regarding the Cellic formulation, neither the presence of cellulose nor lignin restricted an efficient desorption of the enzymes at alkaline pH. This work shows that the recycling strategy must be customized for each particular formulation, since the enzymes found e.g. in Cellic and Celluclast bear quite different behaviour regarding the solid-liquid distribution, stability and cellulose and lignin affinity. Copyright © 2015 Elsevier Inc. All rights reserved.

Affinity alkylation of the active site of C/sub 21/ steroid side-chain cleavage cytochrome P-450 from neonatal porcine testis: a unique cysteine residue alkylated by 17-(bromoacetoxy)progesterone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onoda, M.; Haniu, M.; Yanagibashi, K.

1987-01-27

The affinity alkylating progesterone analogue 17-(bromoacetoxy)progesterone has been used to label the active site of a microsomal cytochrome P-450 enzyme from neonatal pig testis. The enzyme causes removal of the C/sub 20/ and C/sub 21/ side chains from the substrates progesterone and pregnenolone by catalyzing both 17-hydroxylase and C/sub 17,20/-lyase reactions, which produce the corresponding C/sub 1//sup 9/ steroidal precursors of testosterone. The progesterone analogue causes simultaneous inactivation of the two catalytic activities of the enzyme by a first-order kinetic process that obeys saturation kinetics. Progesterone and 17-hydroxyprogesterone each protect the enzyme against inactivation. The progesterone analogue is a competitivemore » inhibitor of the enzyme with K/sub i/ values of 8.4 ..mu..M and 7.8 ..mu..M for progesterone and 17-hydroxyprogesterone, respectively. The enzyme inactivation and kinetic data are consistent with a theory proposing that the analogue and the two substrates compete for the same active site. The radioactive analogue 17-((/sup 14/C)bromoacetoxy)progesterone causes inactivation of the enzyme with incorporation of 1.5-2.2 mol of the analogue per mole of inactivated enzyme. When this experiment is carried out in the presence of a substrate, then 0.9-1.2 mol of radioactive analogue is incorporated per mole of inactivated enzyme. The data suggest that the analogue can bind to two different sites, one of which is related to the catalytic site. Radiolabeled enzyme samples, from reactions of the /sup 14/C-labeled analogue with the enzyme alone or with enzyme in the presence of a substrate, were subjected to amino acid analysis and also in tryptic digestion and peptide mapping.« less

Biocatalysis for the production of industrial products and functional foods from rice and other agricultural produce.

PubMed

Akoh, Casimir C; Chang, Shu-Wei; Lee, Guan-Chiun; Shaw, Jei-Fu

2008-11-26

Many industrial products and functional foods can be obtained from cheap and renewable raw agricultural materials. For example, starch can be converted to bioethanol as biofuel to reduce the current demand for petroleum or fossil fuel energy. On the other hand, starch can also be converted to useful functional ingredients, such as high fructose and high maltose syrups, wine, glucose, and trehalose. The conversion process involves fermentation by microorganisms and use of biocatalysts such as hydrolases of the amylase superfamily. Amylases catalyze the process of liquefaction and saccharification of starch. It is possible to perform complete hydrolysis of starch by using the fusion product of both linear and debranching thermostable enzymes. This will result in saving energy otherwise needed for cooling before the next enzyme can act on the substrate, if a sequential process is utilized. Recombinant enzyme technology, protein engineering, and enzyme immobilization are powerful tools available to enhance the activity of enzymes, lower the cost of enzyme through large scale production in a heterologous host, increase their thermostability, improve pH stability, enhance their productivity, and hence making it competitive with the chemical processes involved in starch hydrolysis and conversions. This review emphasizes the potential of using biocatalysis for the production of useful industrial products and functional foods from cheap agricultural produce and transgenic plants. Rice was selected as a typical example to illustrate many applications of biocatalysis in converting low-value agricultural produce to high-value commercial food and industrial products. The greatest advantages of using enzymes for food processing and for industrial production of biobased products are their environmental friendliness and consumer acceptance as being a natural process.

Trial watch – inhibiting PARP enzymes for anticancer therapy

PubMed Central

Sistigu, Antonella; Manic, Gwenola; Obrist, Florine; Vitale, Ilio

2016-01-01

ABSTRACT Poly(ADP-ribose) polymerases (PARPs) are a members of family of enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) and/or mono(ADP-ribosyl)ation (MARylation), two post-translational protein modifications involved in crucial cellular processes including (but not limited to) the DNA damage response (DDR). PARP1, the most abundant family member, is a nuclear protein that is activated upon sensing distinct types of DNA damage and contributes to their resolution by PARylating multiple DDR players. Recent evidence suggests that, along with DDR, activated PARP1 mediates a series of prosurvival and proapoptotic processes aimed at preserving genomic stability. Despite this potential oncosuppressive role, upregulation and/or overactivation of PARP1 or other PARP enzymes has been reported in a variety of human neoplasms. Over the last few decades, several pharmacologic inhibitors of PARP1 and PARP2 have been assessed in preclinical and clinical studies showing potent antineoplastic activity, particularly against homologous recombination (HR)-deficient ovarian and breast cancers. In this Trial Watch, we describe the impact of PARP enzymes and PARylation in cancer, discuss the mechanism of cancer cell killing by PARP1 inactivation, and summarize the results of recent clinical studies aimed at evaluating the safety and therapeutic profile of PARP inhibitors in cancer patients. PMID:27308587

Transcription of lignocellulose-decomposition associated genes, enzyme activities and production of ethanol upon bioconversion of waste substrate by Phlebia radiata.

PubMed

Mäkinen, Mari A; Risulainen, Netta; Mattila, Hans; Lundell, Taina K

2018-05-04

Previously identified twelve plant cell wall degradation-associated genes of the white rot fungus Phlebia radiata were studied by RT-qPCR in semi-aerobic solid-state cultures on lignocellulose waste material, and on glucose-containing reference medium. Wood-decay-involved enzyme activities and ethanol production were followed to elucidate both the degradative and fermentative processes. On the waste lignocellulose substrate, P. radiata carbohydrate-active enzyme (CAZy) genes encoding cellulolytic and hemicellulolytic activities were significantly upregulated whereas genes involved in lignin modification displayed a more complex response. Two lignin peroxidase genes were differentially expressed on waste lignocellulose compared to glucose medium, whereas three manganese peroxidase-encoding genes were less affected. On the contrary, highly significant difference was noticed for three cellulolytic genes (cbhI_1, eg1, bgl1) with higher expression levels on the lignocellulose substrate than on glucose. This indicates expression of the wood-attacking degradative enzyme system by the fungus also on the recycled, waste core board material. During the second week of cultivation, ethanol production increased on the core board to 0.24 g/L, and extracellular activities against cellulose, xylan, and lignin were detected. Sugar release from the solid lignocellulose resulted with concomitant accumulation of ethanol as fermentation product. Our findings confirm that the fungus activates its white rot decay system also on industrially processed lignocellulose adopted as growth substrate, and under semi-aerobic cultivation conditions. Thus, P. radiata is a good candidate for lignocellulose-based renewable biotechnology to make biofuels and biocompounds from materials with less value for recycling or manufacturing.

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

PubMed

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

Molecular Imaging of Hydrolytic Enzymes Using PET and SPECT

PubMed Central

Rempel, Brian P.; Price, Eric W.

2017-01-01

Hydrolytic enzymes are a large class of biological catalysts that play a vital role in a plethora of critical biochemical processes required to maintain human health. However, the expression and/or activity of these important enzymes can change in many different diseases and therefore represent exciting targets for the development of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radiotracers. This review focuses on recently reported radiolabeled substrates, reversible inhibitors, and irreversible inhibitors investigated as PET and SPECT tracers for imaging hydrolytic enzymes. By learning from the most successful examples of tracer development for hydrolytic enzymes, it appears that an early focus on careful enzyme kinetics and cell-based studies are key factors for identifying potentially useful new molecular imaging agents. PMID:28927325

Molecular Imaging of Hydrolytic Enzymes Using PET and SPECT.

PubMed

Rempel, Brian P; Price, Eric W; Phenix, Christopher P

2017-01-01

Hydrolytic enzymes are a large class of biological catalysts that play a vital role in a plethora of critical biochemical processes required to maintain human health. However, the expression and/or activity of these important enzymes can change in many different diseases and therefore represent exciting targets for the development of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radiotracers. This review focuses on recently reported radiolabeled substrates, reversible inhibitors, and irreversible inhibitors investigated as PET and SPECT tracers for imaging hydrolytic enzymes. By learning from the most successful examples of tracer development for hydrolytic enzymes, it appears that an early focus on careful enzyme kinetics and cell-based studies are key factors for identifying potentially useful new molecular imaging agents.

Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): Influence of body size and temperature

PubMed Central

Marchand, Adrien; Turies, Cyril; Kerambrun, Elodie; Palluel, Olivier; Bado-Nilles, Anne; Beaudouin, Rémy; Porcher, Jean-Marc; Geffard, Alain; Dedourge-Geffard, Odile

2018-01-01

Determining digestive enzyme activity is of potential interest to obtain and understand valuable information about fish digestive physiology, since digestion is an elementary process of fish metabolism. We described for the first time (i) three digestive enzymes: amylase, trypsin and intestinal alkaline phosphatase (IAP), and (ii) three gut morphometric parameters: relative gut length (RGL), relative gut mass (RGM) and Zihler’s index (ZI) in threespine stickleback (Gasterosteus aculeatus), and we studied the effect of temperature and body size on these parameters. When mimicking seasonal variation in temperature, body size had no effect on digestive enzyme activity. The highest levels of amylase and trypsin activity were observed at 18°C, while the highest IAP activity was recorded at 20°C. When sticklebacks were exposed to three constant temperatures (16, 18 and 21°C), a temporal effect correlated to fish growth was observed with inverse evolution patterns between amylase activity and the activities of trypsin and IAP. Temperature (in both experiments) had no effect on morphometric parameters. However, a temporal variation was recorded for both RGM (in the second experiment) and ZI (in both experiments), and the later was correlated to fish body mass. PMID:29614133

Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): Influence of body size and temperature.

PubMed

Hani, Younes Mohamed Ismail; Marchand, Adrien; Turies, Cyril; Kerambrun, Elodie; Palluel, Olivier; Bado-Nilles, Anne; Beaudouin, Rémy; Porcher, Jean-Marc; Geffard, Alain; Dedourge-Geffard, Odile

2018-01-01

Determining digestive enzyme activity is of potential interest to obtain and understand valuable information about fish digestive physiology, since digestion is an elementary process of fish metabolism. We described for the first time (i) three digestive enzymes: amylase, trypsin and intestinal alkaline phosphatase (IAP), and (ii) three gut morphometric parameters: relative gut length (RGL), relative gut mass (RGM) and Zihler's index (ZI) in threespine stickleback (Gasterosteus aculeatus), and we studied the effect of temperature and body size on these parameters. When mimicking seasonal variation in temperature, body size had no effect on digestive enzyme activity. The highest levels of amylase and trypsin activity were observed at 18°C, while the highest IAP activity was recorded at 20°C. When sticklebacks were exposed to three constant temperatures (16, 18 and 21°C), a temporal effect correlated to fish growth was observed with inverse evolution patterns between amylase activity and the activities of trypsin and IAP. Temperature (in both experiments) had no effect on morphometric parameters. However, a temporal variation was recorded for both RGM (in the second experiment) and ZI (in both experiments), and the later was correlated to fish body mass.

[Intensity of pentose phosphate metabolism of carbohydrates in various brain areas in normal and starved animals].

PubMed

Kerimov, B F

2002-01-01

The activities of key enzymes of pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G-6 PD) and 6-phosphogluconate dehydrogenase (6-PGD), were studied in cytoplasmatic fractions of brain cortical (limbic, orbital, sensorimotor cortex) and subcortical (myelencefalon, mesencefalon, hypothalamus) structures of rats subjected to starvation for 1, 2, 3, 5 and 7 days. Short-term starvation (1-3 days) caused activation of 6-GPD and 6-PGD both in cortical and subcortical structures. Long-term starvation for 5-7 days caused a decrease of activities of the pentose phosphate pathway enzymes in all studied structures. It is suggested that enzymes of pentose phosphate pathway in nervous tissues are functionally and metabolically related to glutathione system and during starvation they indirectly participate in the regulation lipid peroxidation processes.

Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes.

PubMed

Wei, Ren; Oeser, Thorsten; Zimmermann, Wolfgang

2014-01-01

Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented. © 2014 Elsevier Inc. All rights reserved.

[Enzymatic characteristics of peroxidase from Chrysanthemum morifolium cv. Bo-ju].

PubMed

Zhu, Yu-Yun; Lyu, Xin-Lin; Li, Xiang-Wei; Zhang, Dong; Dong, Li-Hua; Zhu, Jing-Jing; Wang, Zhi-Min; Zhang, Jin-Zhen

2018-04-01

The enzymatic browning is one of the main reasons for affecting the quality of medicinal flowers. In the process of chrysanthemum harvesting and processing, improper treatment will lead to the browning and severely impact the appearance and quality of chrysanthemum. Peroxidase enzyme is one of the oxidoreductases that cause enzymatic browning of fresh chrysanthemum. The enzymatic characteristics of peroxidase (POD) in chrysanthemum were studied in this paper. In this experiment, the effects of different reaction substrates and their concentrations, PH value of buffer and reaction temperatures on the activity of POD enzyme were investigated. The results showed that the optimal substrate of POD was guaiacol, and the optimal concentration of POD was 50 mmol·L⁻¹. The optimal pH value and reaction temperature were 4.4 and 30-35 °C, respectively. Michaelis-Menten equation was obtained to express the kinetics of enzyme-catalyzed reaction of POD, Km=0.193 mol·L⁻¹, Vmax=0.329 D·min⁻¹. In addition, the results of POD enzyme thermal stability test showed that the POD enzyme activity was inhibited when being treated at 80 °C for 4 min or at 100 °C for 2 min. The above results were of practical significance to reveal the enzymatic browning mechanism, control the enzymatic browning and improve the quality of chrysanthemum, and can also provide the basis for the harvesting and processing of medicinal materials containing polyphenols. Copyright© by the Chinese Pharmaceutical Association.

[Role of nitric oxide as a regulator of cell processes in the formation of multiple organ failure].

PubMed

Riabov, G A; Azisov, Iu M

2001-01-01

Main aspects of functional activity of nitric oxide (NO) are discussed. Physicochemical properties of NO, routes of its formation in man, and mechanism of its effects on physiological processes are described. In human body NO is formed as a result of activity of a specific enzyme, nitric oxide synthase. Three isoforms of the enzyme are known: neuronal, inducible, and endothelial. NO regulates vascular tone, cell adhesion, neurotransmission, bronchodilatation, and platelet aggregation. NO can protect and damage cells under different conditions. The effect of NO can be direct and mediated. Mechanisms of vasodilating effect of NO and of its effect on apoptosis are discussed. The role of NO in regulation of the functional activity of hepatocytes is described. Regulation of NO level in human organism is discussed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maiti, Tushar K.; Permaul, Michelle; Boudreaux, David A.

Ubiquitin carboxy-terminal hydrolase L5 (UCHL5) is a proteasome-associated deubiquitinating enzyme, which, along with RPN11 and USP14, is known to carry out deubiquitination on proteasome. As a member of the ubiquitin carboxy-terminal hydrolase (UCH) family, UCHL5 is unusual because, unlike UCHL1 and UCHL3, it can process polyubiquitin chain. However, it does so only when it is bound to the proteasome; in its free form, it is capable of releasing only relatively small leaving groups from the C-terminus of ubiquitin. Such a behavior might suggest at least two catalytically distinct forms of the enzyme, an apo form incapable of chain processing activity,more » and a proteasome-induced activated form capable of cleaving polyubiquitin chain. Through the crystal structure analysis of two truncated constructs representing the catalytic domain (UCH domain) of this enzyme, we were able to visualize a state of this enzyme that we interpret as its inactive form, because the catalytic cysteine appears to be in an unproductive orientation. While this work was in progress, the structure of a different construct representing the UCH domain was reported; however, in that work the structure reported was that of an inactive mutant [catalytic Cys to Ala; Nishio K et al. (2009) Biochem Biophys Res Commun390, 855-860], which precluded the observation that we are reporting here. Additionally, our structures reveal conformationally dynamic parts of the enzyme that may play a role in the structural transition to the more active form.« less

Identification of a highly active tannase enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum.

PubMed

Tomás-Cortázar, Julen; Plaza-Vinuesa, Laura; de Las Rivas, Blanca; Lavín, José Luis; Barriales, Diego; Abecia, Leticia; Mancheño, José Miguel; Aransay, Ana M; Muñoz, Rosario; Anguita, Juan; Rodríguez, Héctor

2018-02-26

Tannases are tannin-degrading enzymes that have been described in fungi and bacteria as an adaptative mechanism to overcome the stress conditions associated with the presence of these phenolic compounds. We have identified and expressed in E. coli a tannase from the oral microbiota member Fusobacterium nucleatum subs. polymorphum (TanB Fnp ). TanB Fnp is the first tannase identified in an oral pathogen. Sequence analyses revealed that it is closely related to other bacterial tannases. The enzyme exhibits biochemical properties that make it an interesting target for industrial use. TanB Fnp has one of the highest specific activities of all bacterial tannases described to date and shows optimal biochemical properties such as a high thermal stability: the enzyme keeps 100% of its activity after prolonged incubations at different temperatures up to 45 °C. TanB Fnp also shows a wide temperature range of activity, maintaining above 80% of its maximum activity between 22 and 55 °C. The use of a panel of 27 esters of phenolic acids demonstrated activity of TanB Fnp only against esters of gallic and protocatechuic acid, including tannic acid, gallocatechin gallate and epigallocatechin gallate. Overall, TanB Fnp possesses biochemical properties that make the enzyme potentially useful in biotechnological applications. We have identified and characterized a metabolic enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum. The biochemical properties of TanB Fnp suggest that it has a major role in the breakdown of complex food tannins during oral processing. Our results also provide some clues regarding its possible participation on bacterial survival in the oral cavity. Furthermore, the characteristics of this enzyme make it of potential interest for industrial use.

Geometric and electronic structure contributions to function in non-heme iron enzymes.

PubMed

Solomon, Edward I; Light, Kenneth M; Liu, Lei V; Srnec, Martin; Wong, Shaun D

2013-11-19

Mononuclear non-heme Fe (NHFe) enzymes play key roles in DNA repair, the biosynthesis of antibiotics, the response to hypoxia, cancer therapy, and many other biological processes. These enzymes catalyze a diverse range of oxidation reactions, including hydroxylation, halogenation, ring closure, desaturation, and electrophilic aromatic substitution (EAS). Most of these enzymes use an Fe(II) site to activate dioxygen, but traditional spectroscopic methods have not allowed researchers to insightfully probe these ferrous active sites. We have developed a methodology that provides detailed geometric and electronic structure insights into these NHFe(II) active sites. Using these data, we have defined a general mechanistic strategy that many of these enzymes use: they control O2 activation (and limit autoxidation and self-hydroxylation) by allowing Fe(II) coordination unsaturation only in the presence of cosubstrates. Depending on the type of enzyme, O2 activation either involves a 2e(-) reduced Fe(III)-OOH intermediate or a 4e(-) reduced Fe(IV)═O intermediate. Nuclear resonance vibrational spectroscopy (NRVS) has provided the geometric structure of these intermediates, and magnetic circular dichroism (MCD) has defined the frontier molecular orbitals (FMOs), the electronic structure that controls reactivity. This Account emphasizes that experimental spectroscopy is critical in evaluating the results of electronic structure calculations. Therefore these data are a key mechanistic bridge between structure and reactivity. For the Fe(III)-OOH intermediates, the anticancer drug activated bleomycin (BLM) acts as the non-heme Fe analog of compound 0 in heme (e.g., P450) chemistry. However BLM shows different reactivity: the low-spin (LS) Fe(III)-OOH can directly abstract a H atom from DNA. The LS and high-spin (HS) Fe(III)-OOHs have fundamentally different transition states. The LS transition state goes through a hydroxyl radical, but the HS transition state is activated for EAS without O-O cleavage. This activation is important in one class of NHFe enzymes that utilizes a HS Fe(III)-OOH intermediate in dioxygenation. For Fe(IV)═O intermediates, the LS form has a π-type FMO activated for attack perpendicular to the Fe-O bond. However, the HS form (present in the NHFe enzymes) has a π FMO activated perpendicular to the Fe-O bond and a σ FMO positioned along the Fe-O bond. For the NHFe enzymes, the presence of π and σ FMOs enables enzymatic control in determining the type of reactivity: EAS or H-atom extraction for one substrate with different enzymes and halogenation or hydroxylation for one enzyme with different substrates.

The susceptibility of soil enzymes to inhibition by leaf litter tannins is dependent on the tannin chemistry, enzyme class and vegetation history.

PubMed

Triebwasser, Daniella J; Tharayil, Nishanth; Preston, Caroline M; Gerard, Patrick D

2012-12-01

By inhibiting soil enzymes, tannins play an important role in soil carbon (C) and nitrogen (N) mineralization. The role of tannin chemistry in this inhibitory process, in conjunction with enzyme classes and isoforms, is less well understood. Here, we compared the inhibition efficiencies of mixed tannins (MTs, mostly limited to angiosperms) and condensed tannins (CTs, produced mostly by gymnosperms) against the potential activity of β-glucosidase (BG), N-acetyl-glucosaminidase (NAG), and peroxidase in two soils that differed in their vegetation histories. Compared with CTs, MTs exhibited 50% more inhibition of almond (Prunus dulcis) BG activity and greater inhibition of the potential NAG activity in the gymnosperm-acclimatized soils. CTs exhibited lower BG inhibition in the angiosperm-acclimated soils, whereas both types of tannins exhibited higher peroxidase inhibition in the angiosperm soils than in gymnosperm soils. At all of the tested tannin concentrations, irrespective of the tannin type and site history, the potential peroxidase activity was inhibited two-fold more than the hydrolase activity and was positively associated with the redox-buffering efficiency of tannins. Our finding that the inhibitory activities and mechanisms of MTs and CTs are dependent on the vegetative history and enzyme class is novel and furthers our understanding of the role of tannins and soil isoenzymes in decomposition. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft.

PubMed

Valimberti, Ilaria; Tiberti, Matteo; Lambrughi, Matteo; Sarcevic, Boris; Papaleo, Elena

2015-10-14

Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation.

First report of urease activity in the novel systemic fungal pathogen Emergomyces africanus: a comparison with the neurotrope Cryptococcus neoformans.

PubMed

Lerm, Barbra; Kenyon, Chris; Schwartz, Ilan S; Kroukamp, Heinrich; de Witt, Riaan; Govender, Nelesh P; de Hoog, G Sybren; Botha, Alfred

2017-11-01

Cryptococcus neoformans is an opportunistic pathogen responsible for the AIDS-defining illness, cryptococcal meningitis. During the disease process, entry of cryptococcal cells into the brain is facilitated by virulence factors that include urease enzyme activity. A novel species of an Emmonsia-like fungus, recently named Emergomyces africanus, was identified as a cause of disseminated mycosis in HIV-infected persons in South Africa. However, in contrast to C. neoformans, the enzymes produced by this fungus, some of which may be involved in pathogenesis, have not been described. Using a clinical isolate of C. neoformans as a reference, the study aim was to confirm, characterise and quantify urease activity in E. africanus clinical isolates. Urease activity was tested using Christensen's urea agar, after which the presence of a urease gene in the genome of E. africanus was confirmed using gene sequence analysis. Subsequent evaluation of colorimetric enzyme assay data, using Michaelis-Menten enzyme kinetics, revealed similarities between the substrate affinity of the urease enzyme produced by E. africanus (Km ca. 26.0 mM) and that of C. neoformans (Km ca. 20.6 mM). However, the addition of 2.5 g/l urea to the culture medium stimulated urease activity of E. africanus, whereas nutrient limitation notably increased cryptococcal urease activity. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Proteomic analysis reveals large amounts of decomposition enzymes and major metabolic pathways involved in algicidal process of Trametes versicolor F21a.

PubMed

Gao, Xueyan; Wang, Congyan; Dai, Wei; Ren, Shenrong; Tao, Fang; He, Xingbing; Han, Guomin; Wang, Wei

2017-06-20

A recent algicidal mode indicates that fungal mycelia can wrap and eliminate almost all co-cultivated algal cells within a short time span. However, the underlying molecular mechanism is rarely understood. We applied proteomic analysis to investigate the algicidal process of Trametes versicolor F21a and identified 3,754 fungal proteins. Of these, 30 fungal enzymes with endo- or exoglycosidase activities such as β-1,3-glucanase, α-galactosidase, α-glucosidase, alginate lyase and chondroitin lyase were significantly up-regulated. These proteins belong to Glycoside Hydrolases, Auxiliary Activities, Carbohydrate Esterases and Polysaccharide Lyases, suggesting that these enzymes may degrade lipopolysaccharides, peptidoglycans and alginic acid of algal cells. Additionally, peptidase, exonuclease, manganese peroxidase and cytochrome c peroxidase, which decompose proteins and DNA or convert other small molecules of algal cells, could be other major decomposition enzymes. Gene Ontology and KEGG pathway enrichment analysis demonstrated that pyruvate metabolism and tricarboxylic acid cycle pathways play a critical role in response to adverse environment via increasing energy production to synthesize lytic enzymes or uptake molecules. Carbon metabolism, selenocompound metabolism, sulfur assimilation and metabolism, as well as several amino acid biosynthesis pathways could play vital roles in the synthesis of nutrients required by fungal mycelia.

An Improved Ultrasensitive Enzyme-Linked Immunosorbent Assay Using Hydrangea-Like Antibody-Enzyme-Inorganic Three-in-One Nanocomposites.

PubMed

Wei, Tianxiang; Du, Dan; Zhu, Mei-Jun; Lin, Yuehe; Dai, Zhihui

2016-03-01

Protein-inorganic nanoflowers, composed of protein and copper(II) phosphate (Cu3(PO4)2), have recently grabbed people's attention. Because the synthetic method requires no organic solvent and because of the distinct hierarchical nanostructure, protein-inorganic nanoflowers display enhanced catalytic activity and stability and would be a promising tool in biocatalytical processes and biological and biomedical fields. In this work, we first coimmobilized the enzyme, antibody, and Cu3(PO4)2 into a three-in-one hybrid protein-inorganic nanoflower to enable it to possess dual functions: (1) the antibody portion retains the ability to specifically capture the corresponding antigen; (2) the nanoflower has enhanced enzymatic activity and stability to produce an amplified signal. The prepared antibody-enzyme-inorganic nanoflower was first applied in an enzyme-linked immunosorbent assay to serve as a novel enzyme-labeled antibody for Escherichia coli O157:H7 (E. coli O157:H7) determination. The detection limit is 60 CFU L(-1), which is far superior to commercial ELISA systems. The three-in-one antibody (anti-E. coli O157:H7 antibody)-enzyme (horseradish peroxidase)-inorganic (Cu3(PO4)2) nanoflower has some advantages over commercial enzyme-antibody conjugates. First, it is much easier to prepare and does not need any complex covalent modification. Second, it has fairly high capture capability and catalytic activity because it is presented as aggregates of abundant antibodies and enzymes. Third, it has enhanced enzymatic stability compared to the free form of enzyme due to the unique hierarchical nanostructure.

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass.

PubMed

Navarro, David; Couturier, Marie; da Silva, Gabriela Ghizzi Damasceno; Berrin, Jean-Guy; Rouau, Xavier; Asther, Marcel; Bignon, Christophe

2010-07-16

To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously process 120 triplicate wheat-straw samples per day. This throughput can be doubled if the incubation time is reduced from 24 h to 4 h (for initial rates measurements, for instance). This method can potentially be used with any insoluble substrate that is micronizable. A video illustrating the method can be seen at the following URL: http://www.youtube.com/watch?v=NFg6TxjuMWU.

Transcriptional switches in the control of macronutrient metabolism.

PubMed

Wise, Alan

2008-06-01

This review shows how some transcription factors respond to alterations in macronutrients. Carbohydrates induce enzymes for their metabolism and fatty acid synthesis. Fatty acids reduce carbohydrate processing, induce enzymes for their metabolism, and increase both gluconeogenesis and storage of fat. Fat stores help control carbohydrate uptake by other cells. The following main transcription factors are discussed: carbohydrate response element-binding protein; sterol regulatory element-binding protein-1c, cyclic AMP response element-binding protein, peroxisome proliferator-activated receptor-alpha, and peroxisome proliferator-activated receptor-gamma.

The inhibition of hemicellulosic sugars on cellulose hydrolysis are highly dependant on the cellulase productive binding, processivity, and substrate surface charges.

PubMed

Zhai, Rui; Hu, Jinguang; Saddler, Jack N

2018-06-01

In this study, the influence of major hemicellulosic sugars (mannose and xylose) on cellulose hydrolysis and major enzyme activities were evaluated by using both commercial enzyme cocktail and purified cellulase monocomponents over a "library" of cellulosic substrates. Surprisingly, the results showed that unlike glucose, mannose/xylose did not inhibit individual cellulase activities but significantly decreased their hydrolytic performance on cellulose substrates. When various enzyme-substrate interactions (e.g. adsorption/desorption, productive binding, and processive moving) were evaluated, it appeared that these hemicellulosic sugars significantly reduced the productive binding and processivity of Cel7A, which in turn limited cellulase hydrolytic efficacy. Among a range of major cellulose characteristics (e.g. crystallinity, degree of polymerization, accessibility, and surface charges), the acid group content of the cellulosic substrates seemed to be the main driver that determined the extent of hemicellulosic sugar inhibition. Our results provided new insights for better understanding the sugar inhibition mechanisms of cellulose hydrolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Histochemical location of key enzyme activities involved in receptivity and self-incompatibility in the olive tree (Olea europaea L.).

PubMed

Serrano, Irene; Olmedilla, Adela

2012-12-01

Stigma-surface and style enzymes are important for pollen reception, selection and germination. This report deals with the histochemical location of the activity of four basic types of enzyme involved in these processes in the olive (Olea europaea L.). The detection of peroxidase, esterase and acid-phosphatase activities at the surface of the stigma provided evidence of early receptivity in olive pistils. The stigma maintained its receptivity until the arrival of pollen. Acid-phosphatase activity appeared in the style at the moment of anthesis and continued until the fertilization of the ovule. RNase activity was detected in the extracellular matrix of the styles of flowers just before pollination and became especially evident in pistils after self-pollination. This activity gradually decreased until it practically disappeared in more advanced stages. RNase activity was also detected in pollen tubes growing in pollinated pistils and appeared after in vitro germination in the presence of self-incompatible pistils. These findings suggest that RNases may well be involved in intraspecific pollen rejection in olive flowers. To the best of our knowledge this is the first time that evidence of enzyme activity in stigma receptivity and pollen selection has been described in this species. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Impact of Extraction Parameters on the Recovery of Lipolytic Activity from Fermented Babassu Cake

PubMed Central

Silva, Jaqueline N.; Godoy, Mateus G.; Gutarra, Melissa L. E.; Freire, Denise M. G.

2014-01-01

Enzyme extraction from solid matrix is as important step in solid-state fermentation to obtain soluble enzymes for further immobilization and application in biocatalysis. A method for the recovery of a pool of lipases from Penicillium simplicissimum produced by solid-state fermentation was developed. For lipase recovery different extraction solution was used and phosphate buffer containing Tween 80 and NaCl showed the best results, yielding lipase activity of 85.7 U/g and 65.7 U/g, respectively. The parameters with great impacts on enzyme extraction detected by the Plackett-Burman analysis were studied by Central Composite Rotatable experimental designs where a quadratic model was built showing maximum predicted lipase activity (160 U/g) at 25°C, Tween 80 0.5% (w/v), pH 8.0 and extraction solution 7 mL/g, maintaining constant buffer molarity of 0.1 M and 200 rpm. After the optimization process a 2.5 fold increase in lipase activity in the crude extract was obtained, comparing the intial value (64 U/g) with the experimental design (160 U/g), thus improving the overall productivity of the process. PMID:25090644

Lipase production in solid-state fermentation monitoring biomass growth of aspergillus niger using digital image processing.

PubMed

Dutra, Júlio C V; da C Terzi, Selma; Bevilaqua, Juliana Vaz; Damaso, Mônica C T; Couri, Sônia; Langone, Marta A P; Senna, Lilian F

2008-03-01

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.

Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

NASA Astrophysics Data System (ADS)

Dutra, Julio C. V.; da Terzi, Selma C.; Bevilaqua, Juliana Vaz; Damaso, Mônica C. T.; Couri, Sônia; Langone, Marta A. P.; Senna, Lilian F.

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.

The Role of Oxysterols in a Computational Steroidogenesis Model of Human H295R Cells to Improve Predictability of Biochemical Responses to Endocrine Disruptors

EPA Science Inventory

Steroids, which have an important role in a wide range of physiological processes, are synthesized primarily in the gonads and adrenal glands through a series of enzyme mediated reactions. The activity of steroidogenic enzymes can be altered by a variety of endocrine disruptors (...

A Quantitative Measure of Conformational Changes in Apo, Holo and Ligand-Bound Forms of Enzymes.

PubMed

Singh, Satendra; Singh, Atul Kumar; Wadhwa, Gulshan; Singh, Dev Bukhsh; Dwivedi, Seema; Gautam, Budhayash; Ramteke, Pramod W

2016-06-01

Determination of the native geometry of the enzymes and ligand complexes is a key step in the process of structure-based drug designing. Enzymes and ligands show flexibility in structural behavior as they come in contact with each other. When ligand binds with active site of the enzyme, in the presence of cofactor some structural changes are expected to occur in the active site. Motivation behind this study is to determine the nature of conformational changes as well as regions where such changes are more pronounced. To measure the structural changes due to cofactor and ligand complex, enzyme in apo, holo and ligand-bound forms is selected. Enzyme data set was retrieved from protein data bank. Fifteen triplet groups were selected for the analysis of structural changes based on selection criteria. Structural features for selected enzymes were compared at the global as well as local region. Accessible surface area for the enzymes in entire triplet set was calculated, which describes the change in accessible surface area upon binding of cofactor and ligand with the enzyme. It was observed that some structural changes take place during binding of ligand in the presence of cofactor. This study will helps in understanding the level of flexibility in protein-ligand interaction for computer-aided drug designing.

Preparation of cellulase concoction using differential adsorption phenomenon.

PubMed

Birhade, Sachinkumar; Pednekar, Mukesh; Sagwal, Shilpa; Odaneth, Annamma; Lali, Arvind

2017-05-28

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.

Structural, Thermodynamic, and Functional Mechanisms of Adaptations WrbA and AdoMetDC Proteins in Extremophilic Organisms

DTIC Science & Technology

2007-08-15

thermophilic ) and low (psychrophilic). A model protein used in this study, S- adenosyl-methionine decarboxylase (AdoMetDC), is a key enzyme in the polyamine...experimental characterization of the thermophilic AdoMtDC from Termatoga maritima. The processing of TmAdoMetDC that leads to catalytically active enzyme is... thermophilic organisms. One of the open questions of structural biology is the understanding of the mechanisms by which enzymes adapt to extreme temperatures

Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.

PubMed

East-Seletsky, Alexandra; O'Connell, Mitchell R; Knight, Spencer C; Burstein, David; Cate, Jamie H D; Tjian, Robert; Doudna, Jennifer A

2016-10-13

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

Restricting detergent protease action to surface of protein fibres by chemical modification.

PubMed

Schroeder, M; Lenting, H B M; Kandelbauer, A; Silva, C J S M; Cavaco-Paulo, A; Gübitz, G M

2006-10-01

Due to their excellent properties, such as thermostability, activity over a broad range of pH and efficient stain removal, proteases from Bacillus sp. are commonly used in the textile industry including industrial processes and laundry and represent one of the most important groups of enzymes. However, due to the action of proteases, severe damage on natural protein fibres such as silk and wool result after washing with detergents containing proteases. To include the benefits of proteases in a wool fibre friendly detergent formulation, the soluble polymer polyethylene glycol (PEG) was covalently attached to a protease from Bacillus licheniformis. In contrast to activation of PEG with cyanuric chloride (50%) activation with 1,1'-carbonyldiimidazole (CDI) lead to activity recovery above 90%. With these modified enzymes, hydrolytic attack on wool fibres could be successfully prevented up to 95% compared to the native enzymes. Colour difference (DeltaE) measured in the three dimensional colour space showed good stain removal properties for the modified enzymes. Furthermore, half-life of the modified enzymes in buffers and commercial detergents solutions was nearly twice as high as those of the non-modified enzymes with values of up to 63 min. Out of the different modified proteases especially the B. licheniformis protease with the 2.0-kDa polymer attached both retained stain removal properties and did not hydrolyse/damage wool fibres.

Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications

PubMed Central

Rémy, Benjamin; Plener, Laure; Poirier, Laetitia; Elias, Mikael; Daudé, David; Chabrière, Eric

2016-01-01

Extremozymes have gained considerable interest as they could meet industrial requirements. Among these, SsoPox is a hyperthermostable enzyme isolated from the archaeon Sulfolobus solfataricus. This enzyme is a lactonase catalyzing the hydrolysis of acyl-homoserine lactones; these molecules are involved in Gram-negative bacterial communication referred to as quorum sensing. SsoPox exhibits promiscuous phosphotriesterase activity for the degradation of organophosphorous chemicals including insecticides and chemical warfare agents. Owing to its bi-functional catalytic abilities as well as its intrinsic stability, SsoPox is appealing for many applications, having potential uses in the agriculture, defense, food and health industries. Here we investigate the biotechnological properties of the mutant SsoPox-W263I, a variant with increased lactonase and phosphotriesterase activities. We tested enzyme resistance against diverse process-like and operating conditions such as heat resistance, contact with organic solvents, sterilization, storage and immobilization. Bacterial secreted materials from both Gram-negative and positive bacteria were harmless on SsoPox-W263I activity and could reactivate heat-inactivated enzyme. SsoPox showed resistance to harsh conditions demonstrating that it is an extremely attractive enzyme for many applications. Finally, the potential of SsoPox-W263I to be active at subzero temperature is highlighted and discussed in regards to the common idea that hyperthermophile enzymes are nearly inactive at low temperatures. PMID:27876889

Production optimization of invertase by Lactobacillus brevis Mm-6 and its immobilization on alginate beads.

PubMed

Awad, Ghada E A; Amer, Hassan; El-Gammal, Eman W; Helmy, Wafaa A; Esawy, Mona A; Elnashar, Magdy M M

2013-04-02

A sequential optimization strategy, based on statistical experimental designs, was employed to enhance the production of invertase by Lactobacillus brevis Mm-6 isolated from breast milk. First, a 2-level Plackett-Burman design was applied to screen the bioprocess parameters that significantly influence the invertase production. The second optimization step was performed using fractional factorial design in order to optimize the amounts of variables have the highest positive significant effect on the invertase production. A maximal enzyme activity of 1399U/ml was more than five folds the activity obtained using the basal medium. Invertase was immobilized onto grafted alginate beads to improve the enzyme's stability. Immobilization process increased the operational temperature from 30 to 60°C compared to the free enzyme. The reusability test proved the durability of the grafted alginate beads for 15 cycles with retention of 100% of the immobilized enzyme activity to be more convenient for industrial uses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification and partial characterization of a latent ATP, Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol.

PubMed

Vandenheede, J R; Staquet, S; Merlevede, W

1989-05-04

Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase FA-dependent form upon incubation at 30 degrees C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent Mr in sucrose density-gradient centrifugation (70,000) and in gel filtration (110,000).

Serum angiotensin-1 converting enzyme activity processes a human immunodeficiency virus 1 gp160 peptide for presentation by major histocompatibility complex class I molecules

PubMed Central

1992-01-01

T cell stimulation by the human immunodeficiency virus 1 gp160-derived peptide p18 presented by H-2Dd class I major histocompatibility complex molecules in a cell-free system was found to require proteolytic cleavage. This extracellular processing was mediated by peptidases present in fetal calf serum. In vitro processing of p18 resulted in a distinct reverse phase high performance liquid chromatography profile, from which a biologically active product was isolated and sequenced. This peptide processing can be specifically blocked by the angiotensin- 1 converting enzyme (ACE) inhibitor captopril, and can occur by exposing p18 to purified ACE. The ability of naturally occurring extracellular proteases to convert inactive peptides to T cell antigens has important implications for understanding cytotoxic T lymphocyte responses in vivo, and for rational peptide vaccine design. PMID:1316930

A novel lactone-forming carboxylesterase: molecular identification of a tuliposide A-converting enzyme in tulip.

PubMed

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-06-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.

A Novel Lactone-Forming Carboxylesterase: Molecular Identification of a Tuliposide A-Converting Enzyme in Tulip1[W

PubMed Central

Nomura, Taiji; Ogita, Shinjiro; Kato, Yasuo

2012-01-01

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification. PMID:22474185

Effects of chlorpyrifos on soil carboxylesterase activity at an aggregate-size scale.

PubMed

Sanchez-Hernandez, Juan C; Sandoval, Marco

2017-08-01

The impact of pesticides on extracellular enzyme activity has been mostly studied on the bulk soil scale, and our understanding of the impact on an aggregate-size scale remains limited. Because microbial processes, and their extracellular enzyme production, are dependent on the size of soil aggregates, we hypothesized that the effect of pesticides on enzyme activities is aggregate-size specific. We performed three experiments using an Andisol to test the interaction between carboxylesterase (CbE) activity and the organophosphorus (OP) chlorpyrifos. First, we compared esterase activity among aggregates of different size spiked with chlorpyrifos (10mgkg -1 wet soil). Next, we examined the inhibition of CbE activity by chlorpyrifos and its metabolite chlorpyrifos-oxon in vitro to explore the aggregate size-dependent affinity of the pesticides for the active site of the enzyme. Lastly, we assessed the capability of CbEs to alleviate chlorpyrifos toxicity upon soil microorganisms. Our principal findings were: 1) CbE activity was significantly inhibited (30-67% of controls) in the microaggregates (<0.25mm size) and smallest macroaggregates (<1.0 - 0.25mm), but did not change in the largest macroaggregates (>1.0mm) compared with the corresponding controls (i.e., pesticide-free aggregates), 2) chlorpyrifos-oxon was a more potent CbE inhibitor than chlorpyrifos; however, no significant differences in the CbE inhibition were found between micro- and macroaggregates, and 3) dose-response relationships between CbE activity and chlorpyrifos concentrations revealed the capability of the enzyme to bind chlorpyrifos-oxon, which was dependent on the time of exposure. This chemical interaction resulted in a safeguarding mechanism against chlorpyrifos-oxon toxicity on soil microbial activity, as evidenced by the unchanged activity of dehydrogenase and related extracellular enzymes in the pesticide-treated aggregates. Taken together, these results suggest that environmental risk assessments of OP-polluted soils should consider the fractionation of soil in aggregates of different size to measure the CbE activity, and other potential soil enzyme activities. Copyright © 2017 Elsevier Inc. All rights reserved.

Antioxidant Expression Response to Free Radicals in Active Men and Women Fallowing to a Session Incremental Exercise; Numerical Relationship Between Antioxidants and Free Radicals.

PubMed

Baghaiee, Behrouz; Aliparasti, Mohammad Reza; Almasi, Shohreh; Siahkuhian, Marefat; Baradaran, Behzad

2016-06-01

Energy production is a necessary process to continue physical activities, and exercise is associated with more oxygen consumption and increase of oxidative stress. what seems important is the numerical relationship between antioxidant and free radicals. Although the activity of some enzymes increases with physical activities, but it is possible that gene expression of this enzyme is not changed during exercise. The aim of the present study is to investigate the antioxidant enzymes gene expression and changes in malondialdehyde (MDA) and total antioxidant capacity (TAC) levels in men and women affected by a session of incremental exercise and to carefully and numerically assess the relationship between MDA changes and gene expression and activity of antioxidant enzymes. 12 active men and 12 active women (21 - 24 years old) participated voluntarily in this study. Peripheral blood samples were taken from the subjects in three phases, before and after graduated exercise test (GXT) and 3 hours later (recovery). The gene expression of manganese superoxide dismutase (MnSOD) enzyme increased significantly in women in the recovery phase (P < 0.05). Catalase gene expression significantly increased in men in both phases (immediately & recovery) (P < 0.05). But the changes in active women were only significant immediately after the exercise. TAC levels increased significantly in men in the recovery phase and in active women immediately after the exercise (P < 0.05). MDA activity also increased significantly in men in both phases (P < 0.05). However, in women the increase was significant only in the recovery phase (P < 0.05). There was a reverse relationship between changes in MnSOD and copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) levels and MDA in men (P < 0.05). In active women there was also a significant relationship between changes in MDA and gene expression of Cu/ZnSOD and TAC (P < 0.05). The increase in free radicals during incremental exercises challenges gene expression and activity of antioxidant enzymes. However, despite the negative effects of free radicals, in women, activity and gene expression of antioxidant enzymes respond appropriately to free radicals.

Effects of soybean meal on digestive enzymes activity, expression of inflammation-related genes, and chromatin modifications in marine fish (Sparus aurata L.) larvae.

PubMed

Perera, Erick; Yúfera, Manuel

2017-04-01

The effects of soybean meal (SBM) in early diet of Sparus aurata larvae at two developmental windows were assessed. Prolonged (beyond 14 days post-hatch, dph) feeding with SBM decreased the activity of pancreatic enzymes of larvae. In the absence of SBM these larvae later resumed enzyme activities, but exhibited a significant delay in development. Larvae response to SBM involved up-regulation of extracellular matrix remodeling enzymes and pro-inflammatory cytokines, coupled with a drop in putative intestinal enzymes. Larvae receiving SBM at first feeding appear later to have lower expression of inflammation-related genes, especially those fed SBM until 14 dph. Multivariate analysis confirmed that the duration of the SBM early feeding period drives the physiology of larvae in different directions. Feeding larvae with SBM increased global histone H3 acetylation, whereas upon removal of SBM the process was reverted. A more in deep analysis revealed a dynamic interplay among several reversible histone modifications such as H3K14ac and H3K27m3. Finally, we showed that SBM feeding of larvae results in global hypomethylation that persist after SBM removal. This study is the first demonstrating an effect of diet on marine fish epigenetics. It is concluded that there are limitations for extending SBM feeding of S. aurata larvae beyond 14 dph even under co-feeding with live feed, affecting key physiological processes and normal growth. However, up to 14 dph, SBM does not affect normal development, and produces apparently lasting effects on some key enzymes, genes, and chromatin modifications.

Activity Based Profiling of Deubiquitylating Enzymes and Inhibitors in Animal Tissues.

PubMed

McLellan, Lauren; Forder, Cassie; Cranston, Aaron; Harrigan, Jeanine; Jacq, Xavier

2016-01-01

The attachment of ubiquitin or ubiquitin-like modifiers to proteins is an important signal for the regulation of a variety of biological processes including the targeting of substrates for degradation, receptor internalization, regulation of gene expression, and DNA repair. Posttranslational modification of proteins by ubiquitin controls many cellular processes, and aberrant ubiquitylation can contribute to cancer, immunopathologies, and neurodegeneration. Thus, deubiquitylating enzymes (DUBs) that remove ubiquitin from proteins have become attractive therapeutic targets. Monitoring the activity of DUBs in cells or in tissues is critical for understanding the biological function of DUBs in particular pathways and is essential for determining the physiological specificity and potency of small-molecule DUB inhibitors. Here, we describe a method for the homogenization of animal tissues and incubation of tissue lysates with ubiquitin-based activity probes to monitor DUB activity in mouse tissues and target engagement following treatment of animals with small-molecule DUB inhibitors.

[Oxygen-dependent processes in the blood cells from children with arterial hypertension concomitant with biliary dyskinesia].

PubMed

Mikashinovich, Z I; Nagornaia, G Iu; Kovalenko, T D; Zvereva, E A

2011-02-01

Age individuality is characterized by an imbalance of the molecular mechanisms of antioxidant defense in adolescents with arterial hypertension and biliary dyskinesia, as documented by an enzyme imbalance of the first line of antioxidant defense and H2O, accumulation, by a substantial increase in glutathione peroxidase activity, and by inhibition of the activity of glutathione-dependent enzymes. The considerable rise of 2,3-diphosphoglycerate suggests tissue hypoxia. With this, enhanced neutrophil elastase activity causes damage to the structural components of vascular wall connective tissue, resulting in the development of endothelial dysfunction.

Stoichiometry constrains microbial response to root exudation - insights from a model and a field experiment in a temperate forest

NASA Astrophysics Data System (ADS)

Drake, J. E.; Darby, B. A.; Giasson, M.-A.; Kramer, M. A.; Phillips, R. P.; Finzi, A. C.

2012-06-01

Healthy plant roots release a wide range of chemicals into soils. This process, termed root exudation, is thought to increase the activity of microbes and the exo-enzymes they synthesize, leading to accelerated rates of carbon (C) mineralization and nutrient cycling in rhizosphere soils relative to bulk soils. The causal role of exudation, however, is difficult to isolate with in-situ observations, given the complex nature of the rhizosphere environment. We investigated the potential effects of root exudation on microbial and exo-enzyme activity using a theoretical model of decomposition and a field experiment, with a specific focus on the stoichiometric constraint of nitrogen (N) availability. The field experiment isolated the effect of exudation by pumping solutions of exudate mimics through microlysimeter "root simulators" into intact forest soils over two 50-day periods. Using a combined model-experiment approach, we tested two hypotheses: (1) exudation alone is sufficient to stimulate microbial and exo-enzyme activity in rhizosphere soils, and (2) microbial response to C-exudates (carbohydrates and organic acids) is constrained by N-limitation. Experimental delivery of exudate mimics containing C and N significantly increased microbial respiration, microbial biomass, and the activity of exo-enzymes that decompose labile components of soil organic matter (SOM, e.g., cellulose, amino sugars), while decreasing the activity of exo-enzymes that degrade recalcitrant SOM (e.g., polyphenols, lignin). However, delivery of C-only exudates had no effect on microbial biomass or overall exo-enzyme activity, and only increased microbial respiration. The theoretical decomposition model produced complementary results; the modeled microbial response to C-only exudates was constrained by limited N supply to support the synthesis of N-rich microbial biomass and exo-enzymes, while exuding C and N together elicited an increase in modeled microbial biomass, exo-enzyme activity, and decomposition. Thus, hypothesis (2) was supported, while hypothesis (1) was only supported when C and N compounds were exuded together. This study supports a cause-and-effect relationship between root exudation and enhanced microbial activity, and suggests that exudate stoichiometry is an important and underappreciated driver of microbial activity in rhizosphere soils.

S-Adenosylmethionine decarboxylase from human prostate. Activation by putrescine

PubMed Central

Zappia, Vincenzo; Cartenì-Farina, Maria; Pietra, Gennaro Della

1972-01-01

1. The presence of S-adenosylmethionine decarboxylase in human prostate gland is reported. A satisfactory radiochemical enzymic assay was developed and the enzyme was partially characterized. 2. Putrescine stimulates the reaction rate by up to 6-fold at pH7.5: the apparent activation constant was estimated to be 0.13mm. The stimulation is pH-dependent and a maximal effect is observed at acid pH values. 3. Putrescine activation is rather specific: other polyamines, such as spermidine and spermine, did not show any appreciable effect. 4. The apparent Km for the substrate is 4×10−5m. The calculated S-adenosylmethionine content of human prostate (0.18μmol/g wet wt. of tissue) demonstrates that the cellular amounts of sulphonium compound are saturating with respect to the enzyme. 5. The enzyme is moderately stable at 0°C and is rapidly inactivated at 40°C. The optimum pH is about 7.5, with one-half of the maximal activity occurring at pH6.6. 6. Several carboxy-14C-labelled analogues and derivatives of S-adenosylmethionine were tested as substrates. The enzyme appears to be highly specific: the replacement of the 6′-amino group of the sulphonium compound alone results in a complete loss of activity. 7. Inhibition of the enzyme activity by several carbonyl reagents suggests an involvement of either pyridoxal phosphate or pyruvate in the catalytic process. 8. The inhibitory effect of thiol reagents indicates the presence of `essential' thiol groups. PMID:4658995

Who is who in litter decomposition? Metaproteomics reveals major microbial players and their biogeochemical functions

PubMed Central

Schneider, Thomas; Keiblinger, Katharina M; Schmid, Emanuel; Sterflinger-Gleixner, Katja; Ellersdorfer, Günther; Roschitzki, Bernd; Richter, Andreas; Eberl, Leo; Zechmeister-Boltenstern, Sophie; Riedel, Kathrin

2012-01-01

Leaf-litter decomposition is a central process in carbon cycling; however, our knowledge about the microbial regulation of this process is still scarce. Metaproteomics allows us to link the abundance and activity of enzymes during nutrient cycling to their phylogenetic origin based on proteins, the ‘active building blocks' in the system. Moreover, we employed metaproteomics to investigate the influence of environmental factors and nutrients on the decomposer structure and function during beech litter decomposition. Litter was collected at forest sites in Austria with different litter nutrient content. Proteins were analyzed by 1-D-SDS-PAGE followed by liquid-chromatography and tandem mass-spectrometry. Mass spectra were assigned to phylogenetic and functional groups by a newly developed bioinformatics workflow, assignments being validated by complementary approaches. We provide evidence that the litter nutrient content and the stoichiometry of C:N:P affect the decomposer community structure and activity. Fungi were found to be the main producers of extracellular hydrolytic enzymes, with no bacterial hydrolases being detected by our metaproteomics approach. Detailed investigation of microbial succession suggests that it is influenced by litter nutrient content. Microbial activity was stimulated at higher litter nutrient contents via a higher abundance and activity of extracellular enzymes. PMID:22402400

A review on lipase-catalyzed reactions in ultrasound-assisted systems.

PubMed

Lerin, Lindomar A; Loss, Raquel A; Remonatto, Daniela; Zenevicz, Mara Cristina; Balen, Manuela; Netto, Vendelino Oenning; Ninow, Jorge L; Trentin, Cláudia M; Oliveira, J Vladimir; de Oliveira, Débora

2014-12-01

The named "green chemistry" has been receiving increasing prominence due to its environmentally friendly characteristics. The use of enzymes as catalysts in processes of synthesis to replace the traditional use of chemical catalysts present as main advantage the fact of following the principles of the green chemistry. However, processes of enzymatic nature generally provide lower yields when compared to the conventional chemical processes. Therefore, in the last years, the ultrasound has been extensively used in enzymatic processes, such as the production of esters with desirable characteristics for the pharmaceutical, cosmetics, and food industry, for the hydrolysis and glycerolysis of vegetable oils, production of biodiesel, etc. Several works found in the open literature suggest that the energy released by the ultrasound during the cavitation phenomena can be used to enhance mass transfer (substrate/enzyme), hence increasing the rate of products formation, and also contributing to enhance the enzyme catalytic activity. Furthermore, the ultrasound is considered a "green" technology due to its high efficiency, low instrumental requirement and significant reduction of the processing time in comparison to other techniques. The main goal of this review was to summarize studies available to date regarding the application of ultrasound in enzyme-catalyzed esterification, hydrolysis, glycerolysis and transesterification reactions.

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

PubMed Central

Geiser, Elena; Reindl, Michèle; Blank, Lars M.; Feldbrügge, Michael

2016-01-01

ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. PMID:27316952

Biomass-C specific temperature responses of microbial C transformations reveal consistency regardless of microbial community structure across diverse timescales of inquiry

NASA Astrophysics Data System (ADS)

Min, K.; Buckeridge, K. M.; Ziegler, S. E.; Edwards, K. A.; Bagchi, S.; Billings, S. A.

2016-12-01

The responses of heterotrophic microbial process rates to temperature in soils are often investigated in the short-term (hours to months), making it difficult to predict longer-term temperature responses. Here, we integrate the temperature sensitivity obtained from the Arrhenius model with the concepts of microbial resistance, resilience, and susceptibility to assess temporal dynamics of microbial temperature responses. We collected soils along a boreal forest climate gradient (long-term effect), and quantified exo-enzyme activities and CO2 respiration at 5, 15, and 25°C for 84 days (relatively short-term effect). Microbial process rates were examined at two levels (per g microbial biomass-C; and per g dry soil) along with community structure, to characterize driving mechanisms for temporal patterns (e.g., size of biomass, physiological plasticity, community composition). Although temperature sensitivity of exo-enzyme activities on a per g dry soil basis showed both resistance and resilience depending on the types of exo-enzyme, biomass -C-specific responses always exhibited resistance regardless of distinct community composition. Temperature sensitivity of CO2 respiration was constant across time and different communities at both units. This study advances our knowledge in two ways. First, resistant temperature sensitivity of exo-enzymes and respiration at biomass-C specific level across distinct communities and diverse timescales indicates a common relationship between microbial physiology and temperature at a fundamental level, a useful feature allowing microbial process models to be reasonably simplified. Second, different temporal responses of exo-enzymes depending on the unit selected provide a cautionary tale for those projecting future microbial behaviors, because interpretation of ecosystem process rates may vary with the unit of observation.

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

PubMed

Honda, Yuki; Zang, Qian; Shimizu, Yasuhiro; Dadashipour, Mohammad; Zhang, Zilian; Kawarabayasi, Yutaka

2017-02-01

The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k cat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k cat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity. Copyright © 2017 American Society for Microbiology.

Mercury behaviour and C, N, and P biogeochemical cycles during ecological restoration processes of old mining sites in French Guiana.

PubMed

Couic, Ewan; Grimaldi, Michel; Alphonse, Vanessa; Balland-Bolou-Bi, Clarisse; Livet, Alexandre; Giusti-Miller, Stéphanie; Sarrazin, Max; Bousserrhine, Noureddine

2018-04-25

Several decades of gold mining extraction activities in the Amazonian rainforest have caused deforestation and pollution. While ecological rehabilitation is essential for restoring biodiversity and decreasing erosion on deforested lands, few studies note the behaviour or toxicity of trace elements during the rehabilitation process. Our original study focused on the potential use of microbial activity and Hg speciation and compared them with As, Cu, Zn and Cr speciation in assessing the chemical and biological quality of ecological restoration efforts. We sampled two sites in French Guyana 17 years after rehabilitation efforts began. The former site was actively regenerated (R) with the leguminous species Clitoria racemosa and Acacia mangium, and the second site was passively regenerated with spontaneous vegetation (Sv). We also sampled soil from a control site without a history of gold mining (F). We performed microcosm soil experiments for 30 days, where trace element speciation and enzyme activities (i.e., FDA, dehydrogenase, β-glucosidase, urease, alkaline and acid phosphatase) were estimated to characterise the behaviour of trace elements and the soil microbial activity. As bioindicators, the use of soil microbial carbon biomass and soil enzyme activities related to the carbon and phosphorus cycles seems to be relevant for assessing soil quality in rehabilitated and regenerated old mining sites. Our results showed that restoration with leguminous species had a positive effect on soil chemical quality and on soil microbial bioindicators, with activities that tended toward natural non-degraded soil (F). Active restoration processes also had a positive effect on Hg speciation by reducing its mobility. While in Sv we found more exchangeable and soluble mercury, in regenerated sites, Hg was mostly bound to organic matter. These results also suggested that enzyme activities and mercury cycles are sensitive to land restoration and must be considered when evaluating the efficiency of restoration processes.

Engineering Novel and Improved Biocatalysts by Cell Surface Display

PubMed Central

Smith, Mason R.; Khera, Eshita; Wen, Fei

2017-01-01

Biocatalysts, especially enzymes, have the ability to catalyze reactions with high product selectivity, utilize a broad range of substrates, and maintain activity at low temperature and pressure. Therefore, they represent a renewable, environmentally friendly alternative to conventional catalysts. Most current industrial-scale chemical production processes using biocatalysts employ soluble enzymes or whole cells expressing intracellular enzymes. Cell surface display systems differ by presenting heterologous enzymes extracellularly, overcoming some of the limitations associated with enzyme purification and substrate transport. Additionally, coupled with directed evolution, cell surface display is a powerful platform for engineering enzymes with enhanced properties. In this review, we will introduce the molecular and cellular principles of cell surface display and discuss how it has been applied to engineer enzymes with improved properties as well as to develop surface-engineered microbes as whole-cell biocatalysts. PMID:29056821

Microbial enzymes: industrial progress in 21st century.

PubMed

Singh, Rajendra; Kumar, Manoj; Mittal, Anshumali; Mehta, Praveen Kumar

2016-12-01

Biocatalytic potential of microorganisms have been employed for centuries to produce bread, wine, vinegar and other common products without understanding the biochemical basis of their ingredients. Microbial enzymes have gained interest for their widespread uses in industries and medicine owing to their stability, catalytic activity, and ease of production and optimization than plant and animal enzymes. The use of enzymes in various industries (e.g., food, agriculture, chemicals, and pharmaceuticals) is increasing rapidly due to reduced processing time, low energy input, cost effectiveness, nontoxic and eco-friendly characteristics. Microbial enzymes are capable of degrading toxic chemical compounds of industrial and domestic wastes (phenolic compounds, nitriles, amines etc.) either via degradation or conversion. Here in this review, we highlight and discuss current technical and scientific involvement of microorganisms in enzyme production and their present status in worldwide enzyme market.

Biomimetic/Bioinspired Design of Enzyme@capsule Nano/Microsystems.

PubMed

Shi, J; Jiang, Y; Zhang, S; Yang, D; Jiang, Z

2016-01-01

Enzyme@capsule nano/microsystems, which refer to the enzyme-immobilized capsules, have received tremendous interest owing to the combination of the high catalytic activities of encapsulated enzymes and the hierarchical structure of the capsule. The preparation of capsules and simultaneous encapsulation of enzymes is recognized as the core process for the rational design and construction of enzyme@capsule nano/microsystems. The strategy used has three major steps: (a) generation of the templates, (b) surface coating on the templates, and (c) removal of the templates, and it has been proven to be effective and versatile for the construction of enzyme@capsule nano/microsystems. Several conventional methods, including layer-by-layer assembly of polyelectrolytes, liquid crystalline templating method, etc., were used to design and construct enzyme@capsule nano/microsystems, but these have two major drawbacks. One is the low mechanical stability of the systems and the second is the harsh conditions used in the construction process. Learning from nature, several biomimetic/bioinspired methods such as biomineralization, biomimetic/bioinspired adhesion, and their combination have been exploited for the construction of enzyme@capsule nano/microsystems. In this chapter, we will present a general protocol for the construction of enzyme@capsule nano/microsystems using the latter approach. Some suggestions for improved design, construction, and characterization will also be presented with detailed procedures for specific examples. © 2016 Elsevier Inc. All rights reserved.

Expression and Regulation of Drug Transporters and Metabolizing Enzymes in the Human Gastrointestinal Tract.

PubMed

Drozdzik, M; Oswald, S

2016-01-01

Orally administered drugs must pass through the intestinal wall and then through the liver before reaching systemic circulation. During this process drugs are subjected to different processes that may determine the therapeutic value. The intestinal barrier with active drug metabolizing enzymes and drug transporters in enterocytes plays an important role in the determination of drug bioavailability. Accumulating information demonstrates variable distribution of drug metabolizing enzymes and transporters along the human gastrointestinal tract (GI), that creates specific barrier characteristics in different segments of the GI. In this review, expression of drug metabolizing enzymes and transporters in the healthy and diseased human GI as well as their regulatory aspects: genetic, miRNA, DNA methylation are outlined. The knowledge of unique interplay between drug metabolizing enzymes and transporters in specific segments of the GI tract allows more precise definition of drug release sites within the GI in order to assure more complete bioavailability and prediction of drug interactions.

Dimerization of Matrix Metalloproteinase-2 (MMP-2)

PubMed Central

Koo, Bon-Hun; Kim, Yeon Hyang; Han, Jung Ho; Kim, Doo-Sik

2012-01-01

Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca2+ ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys102 and the neighboring Cys102. Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2. PMID:22577146

Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS

PubMed Central

2014-01-01

Background Environmental screening programs are used to find new enzymes that may be utilized in large-scale industrial processes. Among microbial sources of new enzymes, the rationale for screening fungal endophytes as a potential source of such enzymes relates to the hypothesised mutualistic relationship between the endophyte and its host plant. There is a need for new microbial amylases that are active at low temperature and alkaline conditions as these would find industrial applications as detergents. Results An α-amylase produced by Preussia minima, isolated from the Australian native plant, Eremophilia longifolia, was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion exchange chromatography. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L-asparagine as the nitrogen source. Bioreactor studies showed that enzyme activity was comparable to that obtained in shaker cultures, which encourages scale-up fermentation for enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9-mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae. Conclusions The findings of the present study indicate that the purified α-amylase exhibits a number of promising properties that make it a strong candidate for application in the detergent industry. To our knowledge, this is the first amylase isolated from a Preussia minima strain of endophytic origin. PMID:24602289

Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS.

PubMed

Zaferanloo, Bita; Bhattacharjee, Shatabdi; Ghorbani, Mahmood M; Mahon, Peter J; Palombo, Enzo A

2014-03-07

Environmental screening programs are used to find new enzymes that may be utilized in large-scale industrial processes. Among microbial sources of new enzymes, the rationale for screening fungal endophytes as a potential source of such enzymes relates to the hypothesised mutualistic relationship between the endophyte and its host plant. There is a need for new microbial amylases that are active at low temperature and alkaline conditions as these would find industrial applications as detergents. An α-amylase produced by Preussia minima, isolated from the Australian native plant, Eremophilia longifolia, was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion exchange chromatography. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L-asparagine as the nitrogen source. Bioreactor studies showed that enzyme activity was comparable to that obtained in shaker cultures, which encourages scale-up fermentation for enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9-mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae. The findings of the present study indicate that the purified α-amylase exhibits a number of promising properties that make it a strong candidate for application in the detergent industry. To our knowledge, this is the first amylase isolated from a Preussia minima strain of endophytic origin.

Metal active site elasticity linked to activation of homocysteine in methionine synthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.

2008-04-02

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry uponmore » binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.« less

Effects of Lead Exposure and Genetic Polymorphisms on ALAD and GPx Activities in Brazilian Battery Workers.

PubMed

da Cunha Martins, Airton; Mazzaron Barcelos, Gustavo Rafael; Jacob Ferreira, Anna Laura Bechara; de Souza, Marilesia Ferreira; de Syllos Cólus, Ilce Mara; Antunes, Lusânia Maria Greggi; Bastos Paoliello, Monica Maria; Adeyemi, Joseph A; Barbosa, Fernando

2015-01-01

Lead (Pb) is a toxic metal that is widely used by metallurgical industries such as car battery recycling. Exposure to the metal may modify the redox status of the cells and consequently result in changes in activities of important enzymes such as delta-aminolevulinic acid dehydratase (ALAD) and glutathione peroxidase (GPx). Similarly, genetic polymorphisms may modulate the activities of enzymes related to detoxification processes of the metal and may modify Pb body burden. Therefore, the aims of the present study were (i) to evaluate the correlation between blood lead levels (BLL) and activities of the enzymes ALAD and GPx, and (ii) to determine whether activities of these enzymes may be influenced by polymorphisms in ALAD and GPx genes in Brazilian automotive battery workers chronically exposed to Pb, as well as the effects of these polymorphisms on BLL. Our study included 257 participants; BLL were determined by inductively couple plasma-mass spectrometry (ICP-MS), and the activities of the enzymes ALAD and GPx were quantified spectrophotometrically; and genotyping of ALAD (rs1800435) and GPx-1 (rs1800668) polymorphisms was performed by TaqMan assays (real-time polymerase chain reaction, RT-PCR). Significant negative correlations were found between BLL and ALAD activity. Subjects who carried at least one polymorphic allele for ALAD gene displayed markedly lower ALAD activities, while no significant effect was observed regarding GPx-1 polymorphism and activity of the same enzyme. Further, ALAD and GPx-1 polymorphisms exerted no marked influence on BLL. Taken together, our results showed that BLL affected ALAD but not GPx activities, and these were not modulated by polymorphisms in ALAD and GPx gene. Further, the rs1800435 SNP showed a tendency to modulate ALAD activity, while the rs1800668 SNP did not modulate GPx activity in Brazilian automotive battery workers exposed to Pb.

The evolution of new enzyme function: lessons from xenobiotic metabolizing bacteria versus insecticide-resistant insects

PubMed Central

Russell, Robyn J; Scott, Colin; Jackson, Colin J; Pandey, Rinku; Pandey, Gunjan; Taylor, Matthew C; Coppin, Christopher W; Liu, Jian-Wei; Oakeshott, John G

2011-01-01

Here, we compare the evolutionary routes by which bacteria and insects have evolved enzymatic processes for the degradation of four classes of synthetic chemical insecticide. For insects, the selective advantage of such degradative activities is survival on exposure to the insecticide, whereas for the bacteria the advantage is simply a matter of access to additional sources of nutrients. Nevertheless, bacteria have evolved highly efficient enzymes from a wide variety of enzyme families, whereas insects have relied upon generalist esterase-, cytochrome P450- and glutathione-S-transferase-dependent detoxification systems. Moreover, the mutant insect enzymes are less efficient kinetically and less diverged in sequence from their putative ancestors than their bacterial counterparts. This presumably reflects several advantages that bacteria have over insects in the acquisition of new enzymatic functions, such as a broad biochemical repertoire from which new functions can be evolved, large population sizes, high effective mutation rates, very short generation times and access to genetic diversity through horizontal gene transfer. Both the insect and bacterial systems support recent theory proposing that new biochemical functions often evolve from ‘promiscuous’ activities in existing enzymes, with subsequent mutations then enhancing those activities. Study of the insect enzymes will help in resistance management, while the bacterial enzymes are potential bioremediants of insecticide residues in a range of contaminated environments. PMID:25567970

Endothelial Targeting of Semi-permeable Polymer Nanocarriers for Enzyme Therapies

PubMed Central

Dziubla, Thomas D; Shuvaev, Vladimir V.; Hong, Nan Kang; Hawkins, Brian; Muniswamy, Madesh; Takano, Hajime; Simone, Eric; Nakada, Marian T.; Fisher, Aron; Albelda, Steven M.; Muzykantov, Vladimir R.

2007-01-01

The medical utility of proteins, e.g. therapeutic enzymes, is greatly restricted by their liable nature and inadequate delivery. Most therapeutic enzymes do not accumulate in their targets and are inactivated by proteases. Targeting of enzymes encapsulated into substrate-permeable Polymeric Nano-Carriers (PNC) impermeable for proteases might overcome these limitations. To test this hypothesis, we designed endothelial targeted PNC loaded with catalase, the H2O2-detoxifying enzyme, and tested if this approach protects against vascular oxidative stress, a pathological process implicated in ischemia-reperfusion and other disease conditions. Encapsulation of catalase (MW 240KD), peroxidase (MW 42kD) and xanthine oxidase (XO, MW 300 kD) into ~300nm diameter PNC composed of co-polymers of PEG-PLGA (polyethylene glycol and poly-lactic/poly-glycolic acid) was in the range ~10% for all enzymes. PNC/catalase and PNC/peroxidase were protected from external proteolysis and exerted the enzymatic activity on their PNC diffusible substrates, H2O2 and ortho-phenylendiamine, whereas activity of encapsulated XO was negligible due to polymer impermeability to the substrate. PNC targeted to platelet-endothelial cell adhesion molecule-1 delivered active encapsulated catalase to endothelial cells and protected the endothelium against oxidative stress in cell culture and animal studies. Vascular targeting of PNC-loaded detoxifying enzymes may find wide medical applications including management of oxidative stress and other toxicities. PMID:17950837

Biochemical and Thermodynamical Characterization of Glucose Oxidase, Invertase, and Alkaline Phosphatase Secreted by Antarctic Yeasts.

PubMed

Yuivar, Yassef; Barahona, Salvador; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

2017-01-01

The use of enzymes in diverse industries has increased substantially over past decades, creating a well-established and growing global market. Currently, the use of enzymes that work better at ambient or lower temperatures in order to decrease the temperatures of production processes is desirable. There is thus a continuous search for enzymes in cold environments, especially from microbial sources, with amylases, proteases, lipases and, cellulases being the most studied. Other enzymes, such as glucose oxidase (GOD), invertase (Inv), and alkaline phosphatase (ALP), also have a high potential for application, but have been much less studied in microorganisms living in cold-environments. In this work, secretion of these three enzymes by Antarctic yeast species was analyzed, and five, three, and five species were found to produce extracellular GOD, Inv, and ALP, respectively. The major producers of GOD, Inv, and ALP were Goffeauzyma gastrica, Wickerhamomyces anomalus , and Dioszegia sp., respectively, from which the enzymes were purified and characterized. Contrary to what was expected, the highest GOD and Inv activities were found at 64°C and 60°C, respectively, and at 47°C for ALP. However, the three enzymes maintained a significant percentage of activity at lower temperatures, especially ALP that kept a 67 and 43% of activity at 10°C and 4°C, respectively.

Biochemical and Thermodynamical Characterization of Glucose Oxidase, Invertase, and Alkaline Phosphatase Secreted by Antarctic Yeasts

PubMed Central

Yuivar, Yassef; Barahona, Salvador; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

2017-01-01

The use of enzymes in diverse industries has increased substantially over past decades, creating a well-established and growing global market. Currently, the use of enzymes that work better at ambient or lower temperatures in order to decrease the temperatures of production processes is desirable. There is thus a continuous search for enzymes in cold environments, especially from microbial sources, with amylases, proteases, lipases and, cellulases being the most studied. Other enzymes, such as glucose oxidase (GOD), invertase (Inv), and alkaline phosphatase (ALP), also have a high potential for application, but have been much less studied in microorganisms living in cold-environments. In this work, secretion of these three enzymes by Antarctic yeast species was analyzed, and five, three, and five species were found to produce extracellular GOD, Inv, and ALP, respectively. The major producers of GOD, Inv, and ALP were Goffeauzyma gastrica, Wickerhamomyces anomalus, and Dioszegia sp., respectively, from which the enzymes were purified and characterized. Contrary to what was expected, the highest GOD and Inv activities were found at 64°C and 60°C, respectively, and at 47°C for ALP. However, the three enzymes maintained a significant percentage of activity at lower temperatures, especially ALP that kept a 67 and 43% of activity at 10°C and 4°C, respectively. PMID:29312954

Glutaminase free l-asparaginase from Vibrio cholerae: Heterologous expression, purification and biochemical characterization.

PubMed

Radha, Remya; Arumugam, Nagarajan; Gummadi, Sathyanarayana N

2018-05-01

l-asparaginase is used as a pivotal agent in treatment of lympho proliferative disorders and holds an excessive demand for food processing aids. Most of the l-asparaginases possess glutaminase activity which might lead to side effects during the treatment. The search for new therapeutic enzymes is of great interest in both medical and food applications. In this study, Vibrio cholerael-asparaginase was recombinantly overexpressed in E. coli, purified to homogeneity and molecular size was estimated to be 36.6kDa. Recombinant enzyme showed an optimum pH and temperature of 7.0 and 37°C respectively. The K m and V max of the enzyme for l-asparagine was 1.1mM and 1006μM/min respectively. The enzyme is highly specific for l-asparagine and did not show glutaminase activity. The enzyme possessed high half life time and good stability over a wide range of physiological conditions. T m of the enzyme was found to be 81°C by circular dichroism and differential scanning calorimetric techniques. This enzyme possesses novel properties such as (i) glutaminase free l-asparaginase activity, (ii) very high V max value with high affinity and (iii) high stability at 30°C (half-life time of 2.9days). Copyright © 2018 Elsevier B.V. All rights reserved.

Microgravity

NASA Image and Video Library

2004-04-15

The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Pharmaceutical companies have been developing drugs that can inhibit the function of neuraminidase hoping to create an effective weapon against the flu. Researchers from the pharmaceutical industry and from the Center for Macromolecular Crystallography have grown crystals of neuraminidase in space. These improved, space-grown crystals have provided information that have helped design drugs which form a stronger interaction with the enzyme. These drugs inhibit neuraminidase by attaching themselves to the enzyme. Since the drugs are less likely to detach from the enzyme, they are more effective, require smaller dosages, and have fewer side effects. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

Neuraminidase

NASA Technical Reports Server (NTRS)

2004-01-01

The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Pharmaceutical companies have been developing drugs that can inhibit the function of neuraminidase hoping to create an effective weapon against the flu. Researchers from the pharmaceutical industry and from the Center for Macromolecular Crystallography have grown crystals of neuraminidase in space. These improved, space-grown crystals have provided information that have helped design drugs which form a stronger interaction with the enzyme. These drugs inhibit neuraminidase by attaching themselves to the enzyme. Since the drugs are less likely to detach from the enzyme, they are more effective, require smaller dosages, and have fewer side effects. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

Eicosapentaenoic acid and docosahexaenoic acid increase the degradation of amyloid-β by affecting insulin-degrading enzyme.

PubMed

Grimm, Marcus O W; Mett, Janine; Stahlmann, Christoph P; Haupenthal, Viola J; Blümel, Tamara; Stötzel, Hannah; Grimm, Heike S; Hartmann, Tobias

2016-12-01

Omega-3 polyunsaturated fatty acids (PUFAs) have been proposed to be highly beneficial in Alzheimer's disease (AD). AD pathology is closely linked to an overproduction and accumulation of amyloid-β (Aβ) peptides as extracellular senile plaques in the brain. Total Aβ levels are not only dependent on its production by proteolytic processing of the amyloid precursor protein (APP), but also on Aβ-clearance mechanisms, including Aβ-degrading enzymes. Here we show that the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increase Aβ-degradation by affecting insulin-degrading enzyme (IDE), the major Aβ-degrading enzyme secreted into the extracellular space of neuronal and microglial cells. The identification of the molecular mechanisms revealed that EPA directly increases IDE enzyme activity and elevates gene expression of IDE. DHA also directly stimulates IDE enzyme activity and affects IDE sorting by increasing exosome release of IDE, resulting in enhanced Aβ-degradation in the extracellular milieu. Apart from the known positive effect of DHA in reducing Aβ production, EPA and DHA might ameliorate AD pathology by increasing Aβ turnover.

Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling

PubMed Central

2013-01-01

Background It is necessary to develop efficient methods to produce renewable fuels from lignocellulosic biomass. One of the main challenges to the industrialization of lignocellulose conversion processes is the large amount of cellulase enzymes used for the hydrolysis of cellulose. One method for decreasing the amount of enzyme used is to recycle the enzymes. In this study, the recycle of enzymes associated with the insoluble solid fraction after the enzymatic hydrolysis of cellulose was investigated for pretreated corn stover under a variety of recycling conditions. Results It was found that a significant amount of cellulase activity could be recovered by recycling the insoluble biomass fraction, and the enzyme dosage could be decreased by 30% to achieve the same glucose yields under the most favorable conditions. Enzyme productivity (g glucose produced/g enzyme applied) increased between 30 and 50% by the recycling, depending on the reaction conditions. While increasing the amount of solids recycled increased process performance, the methods applicability was limited by its positive correlation with increasing total solids concentrations, reaction volumes, and lignin content of the insoluble residue. However, increasing amounts of lignin rich residue during the recycle did not negatively impact glucose yields. Conclusions To take advantage of this effect, the amount of solids recycled should be maximized, based on a given processes ability to deal with higher solids concentrations and volumes. Recycling of enzymes by recycling the insoluble solids fraction was thus shown to be an effective method to decrease enzyme usage, and research should be continued for its industrial application. PMID:23336604

Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion.

PubMed

Condori, Jose; Acosta, Walter; Ayala, Jorge; Katta, Varun; Flory, Ashley; Martin, Reid; Radin, Jonathan; Cramer, Carole L; Radin, David N

2016-02-01

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called "hard-to-treat" tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid β-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid β-galactosidase enzyme were produced using a plant-based bioproduction platform. β-gal:RTB and RTB:β-gal fusion products retained both lectin activity and β-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived β-galactosidase. In contrast, plant-derived β-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native β-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1-4h, both β-gal:RTB and RTB:β-gal were processed to the ~64kDa "activated" β-gal form; the RTB lectin was cleaved and rapidly degraded. The activated β-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of β-gal:RTB supported significantly greater accumulation of β-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived β-gal. These data demonstrate that plant-made β-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis - delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

Inhibitory activities of Moringa oleifera leaf extract against α-glucosidase enzyme in vitro

NASA Astrophysics Data System (ADS)

Natsir, H.; Wahab, A. W.; Laga, A.; Arif, A. R.

2018-03-01

Alpha-glucosidase is a key enzyme in the final process of breaking carbohydrates into glucose. Inhibition of α-glucosidase affected more absorption of glucose, so it can reduce hyperglycemia condition. The aims of this study is to determine the effectiveness of inhibition wet and dried Moringa oleifera leaf extract through α-glucosidase activity in vitro. The effectiveness study of inhibition on the activity of α-glucosidase enzyme obtained from white glutinous rice (Oryza sativa glutinosa) was carried out using wet and dried kelor leaf extract of 13% (w/v) with 10 mM α-D-glucopyranoside (PNPG) substrate. A positive control used 1% acarbose and substrate without addition of extract was a negative control. Inhibitory activity was measured using spectrophotometers at a wavelength of 400 nm. The result showed that the inhibition activity against α-glucosidase enzyme of dried leaf extract, wet leaf extract and acarbose was 81,39%, 83,94%, and 95,4%, respectively on pH 7,0. The effectiveness inhibition of the wet Moringa leaf extract was greater than the dried leaf extract. The findings suggest that M. oleifera leaf has the potential to be developed as an alternative food therapy for diabetics.

Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.

PubMed

Renata, Hans; Wang, Z Jane; Arnold, Frances H

2015-03-09

High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature's known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzymatic Conversion of CO2 to Bicarbonate in Functionalized Mesoporous Silica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yuehua; Chen, Baowei; Qi, Wen N.

2012-05-01

We report here that carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in functionalized mesoporous silica (FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) due to the dominant electrostatic interaction. The binding of CA to HOOC-FMS can result in the protein’s conformational change comparing to the enzyme free in solution, but can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency. The electrostatically bound CA can bemore » released by changing pH. The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. This work opens up a new approach converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.« less

Human tyrosinase produced in insect cells: a landmark for the screening of new drugs addressing its activity.

PubMed

Fogal, Stefano; Carotti, Marcello; Giaretta, Laura; Lanciai, Federico; Nogara, Leonardo; Bubacco, Luigi; Bergantino, Elisabetta

2015-01-01

Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, L-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.

Ascorbic acid metabolism during bilberry (Vaccinium myrtillus L.) fruit development.

PubMed

Cocetta, Giacomo; Karppinen, Katja; Suokas, Marko; Hohtola, Anja; Häggman, Hely; Spinardi, Anna; Mignani, Ilaria; Jaakola, Laura

2012-07-15

Bilberry (Vaccinium myrtillus L.) possesses a high antioxidant capacity in berries due to the presence of anthocyanins and ascorbic acid (AsA). Accumulation of AsA and the expression of the genes encoding the enzymes of the main AsA biosynthetic route and of the ascorbate-glutathione cycle, as well as the activities of the enzymes involved in AsA oxidation and recycling were investigated for the first time during the development and ripening of bilberry fruit. The results showed that the AsA level remained relatively stable during fruit maturation. The expression of the genes encoding the key enzymes in the AsA main biosynthetic route showed consistent trends with each other as well as with AsA levels, especially during the first stages of fruit ripening. The expression of genes and activities of the enzyme involved in the AsA oxidation and recycling route showed more prominent developmental stage-dependent changes during the ripening process. Different patterns of activity were found among the studied enzymes and the results were, for some enzymes, in accordance with AsA levels. In fully ripe berries, both AsA content and gene expression were significantly higher in skin than in pulp. Copyright © 2012 Elsevier GmbH. All rights reserved.

Determination of some significant batch culture conditions affecting acetyl-xylan esterase production by Penicillium notatum NRRL-1249

PubMed Central

2011-01-01

Background Acetyl-xylan esterase (AXE, EC 3.1.1.72) hydrolyses acetate group from the linear chain of xylopyranose residues bound by β-1,4-linkage. The enzyme finds commercial applications in bio-bleaching of wood pulp, treating animal feed to increase digestibility, processing food to increase clarification and converting lignocellulosics to feedstock and fuel. In the present study, we report on the production of an extracellular AXE from Penicillium notatum NRRL-1249 by solid state fermentation (SSF). Results Wheat bran at a level of 10 g (with 4 cm bed height) was optimized as the basal substrate for AXE production. An increase in enzyme activity was observed when 7.5 ml of mineral salt solution (MSS) containing 0.1% KH2PO4, 0.05% KCl, 0.05% MgSO4.7H2O, 0.3% NaNO3, 0.001% FeSO4.2H2O and 0.1% (v/w) Tween-80 as an initial moisture content was used. Various nitrogen sources including ammonium sulphate, urea, peptone and yeast extract were compared for enzyme production. Maximal enzyme activity of 760 U/g was accomplished which was found to be highly significant (p ≤ 0.05). A noticeable enhancement in enzyme activity was observed when the process parameters including incubation period (48 h), initial pH (5), 0.2% (w/w) urea as nitrogen source and 0.5% (v/w) Tween-80 as a stimulator were further optimized using a 2-factorial Plackett-Burman design. Conclusion From the results it is clear that an overall improvement of more than 35% in terms of net enzyme activity was achieved compared to previously reported studies. This is perhaps the first report dealing with the use of P. notatum for AXE production under batch culture SSF. The Plackett-Burman model terms were found highly significant (HS), suggesting the potential commercial utility of the culture used (df = 3, LSD = 0.126). PMID:21575210

Trypsin from the processing waste of the lane snapper (Lutjanus synagris) and its compatibility with oxidants, surfactants and commercial detergents.

PubMed

Espósito, Talita S; Marcuschi, Marina; Amaral, Ian P G; Carvalho, Luiz B; Bezerra, Ranilson S

2010-05-26

A trypsin from the viscera of the lane snapper (Lutjanus synagris) was purified by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-p-nitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulfonyl fluoride (PMSF), which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and 45 degrees C and retained 100% of the activity after incubation at the optimal temperature for 30 min. At a concentration of 10 mM, activity was slightly activated by Ca(2+) and inhibited by the following ions in decreasing order: Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+). The effects of Ba(2+), K(1+) and Li(1+) proved to be less intensive. Using 1% (w/v) azocasein as substrate, the enzyme revealed high resistance (60% residual activity) when incubated with 10% H(2)O(2) for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi, Surf and Ala, retaining more than 50% of initial activity after 60 min at 25 degrees C and 30 min at 40 degrees C. The thermostability and compatibility of this enzyme with commercial detergents suggest a good potentiality for application in the detergent industry.

The role of apelin in the modulation of gastric and pancreatic enzymes activity in adult rats.

PubMed

Antuschevich, H; Kapica, M; Krawczynska, A; Herman, A; Kato, I; Kuwahara, A; Zabielski, R

2016-06-01

Apelin is considered as important gut regulatory peptide ligand of APJ receptor with a potential physiological role in gastrointestinal cytoprotection, regulation of food intake and drinking behavior. Circulating apelin inhibits secretion of pancreatic juice through vagal- cholecystokinin-dependent mechanism and reduces local blood flow. Our study was aimed to determine the effect of fundectomy and intraperitoneal or intragastric administration of apelin-13 on pancreatic and gastric enzymes activities in adult rats. Fundectomy is a surgical removal of stomach fundus - maine site apelin synthesis. Three independent experiments were carried out on Wistar rats. In the first and second experiment apelin-13 was given by intragastric or intraperitoneal way twice a day for 10 days (100 nmol/kg b.w.). Control groups received the physiological saline respectively. In the third experiment the group of rats after fundectomy were used. Fundectomized rats did not receive apelin and the rats from control group were 'sham operated'. At the end of experiment rats were sacrificed and blood from rats was withdrawn for apelin and CCK (cholecystokinin) radioimmunoassay analysis and pancreas and stomach tissues were collected for enzyme activity analyses. Intragastric and intraperitoneal administrations of apelin-13 increased basal plasma CCK level and stimulated gastric and pancreatic enzymes activity in rats. In animals after fundectomy decreased activity of studied enzymes was observed, as well as basal plasma apelin and CCK levels. In conclusion, apelin can effects on CCK release and stimulates some gastric and pancreatic enzymes activity in adult rats while fudectomy suppresses those processes. Changes in the level of pancreatic lipase activity point out that apelin may occurs as a regulator of lipase secretion.

Effects of Recurring Droughts on Extracellular Enzyme Activity in Mountain Grassland

NASA Astrophysics Data System (ADS)

Fuchslueger, L.; Bahn, M.; Kienzl, S.; Hofhansl, F.; Schnecker, J.; Richter, A.

2015-12-01

Water availability is a key factor for biogeochemical processes and determines microbial activity and functioning, and thereby organic matter decomposition in soils by affecting the osmotic potential, soil pore connectivity, substrate diffusion and nutrient availability. Low water availability during drought periods therefore directly affects microbial activity. Recurring drought periods likely induce shifts in microbial structure that might be reflected in altered responses of microbial turnover of organic matter by extracellular enzymes. To study this we measured a set of potential extracellular enzyme activity rates (cellobiohydrolase CBH; leucine-amino-peptidase LAP; phosphatase PHOS; phenoloxidase POX), in grassland soils that were exposed to extreme experimental droughts during the growing seasons of up to five subsequent years. During the first drought period after eight weeks of rain exclusion all measured potential enzyme activities were significantly decreased. In parallel, soil extractable organic carbon and nitrogen concentrations increased and microbial community structure, determined by phospholipid fatty acid analysis, changed. In soils that were exposed to two and three drought periods only PHOS decreased. After four years of drought again CBH, PHOS and POX decreased, while LAP was unaffected; after five years of drought PHOS and POX decreased and CBH and LAP remained stable. Thus, our results suggest that recurring extreme drought events can cause different responses of extracellular enzyme activities and that the responses change over time. We will discuss whether and to what degree these changes were related to shifts in microbial community composition. However, independent of whether a solitary or a recurrent drought was imposed, in cases when enzyme activity rates were altered during drought, they quickly recovered after rewetting. Overall, our data suggest that microbial functioning in mountain grassland is sensitive to drought, but highly resilient even after five years of drought.

The Secretion and Action of Brush Border Enzymes in the Mammalian Small Intestine.

PubMed

Hooton, Diane; Lentle, Roger; Monro, John; Wickham, Martin; Simpson, Robert

2015-01-01

Microvilli are conventionally regarded as an extension of the small intestinal absorptive surface, but they are also, as latterly discovered, a launching pad for brush border digestive enzymes. Recent work has demonstrated that motor elements of the microvillus cytoskeleton operate to displace the apical membrane toward the apex of the microvillus, where it vesiculates and is shed into the periapical space. Catalytically active brush border digestive enzymes remain incorporated within the membranes of these vesicles, which shifts the site of BB digestion from the surface of the enterocyte to the periapical space. This process enables nutrient hydrolysis to occur adjacent to the membrane in a pre-absorptive step. The characterization of BB digestive enzymes is influenced by the way in which these enzymes are anchored to the apical membranes of microvilli, their subsequent shedding in membrane vesicles, and their differing susceptibilities to cleavage from the component membranes. In addition, the presence of active intracellular components of these enzymes complicates their quantitative assay and the elucidation of their dynamics. This review summarizes the ontogeny and regulation of BB digestive enzymes and what is known of their kinetics and their action in the peripheral and axial regions of the small intestinal lumen.

Substrate-dependent temperature sensitivity of soil organic matter decomposition

NASA Astrophysics Data System (ADS)

Myachina, Olga; Blagodatskaya, Evgenia

2015-04-01

Activity of extracellular enzymes responsible for decomposition of organics is substrate dependent. Quantity of the substrate is the main limiting factor for enzymatic or microbial heterotrophic activity in soils. Different mechanisms of enzymes response to temperature suggested for low and high substrate availability were never proved for real soil conditions. We compared the temperature responses of enzymes-catalyzed reactions in soils. Basing on Michaelis-Menten kinetics we determined the enzymes affinity to substrate (Km) and mineralization potential of heterotrophic microorganisms (Vmax) 1) for three hydrolytic enzymes: β-1,4-glucosidase, N-acetyl- β -D-glucosaminidase and phosphatase by the application of fluorogenically labeled substrates and 2) for mineralization of 14C-labeled glucose by substrate-dependent respiratory response. Here we show that the amount of available substrate is responsible for temperature sensitivity of hydrolysis of polymers in soil, whereas monomers oxidation to CO2 does not depend on substrate amount and is mainly temperature governed. We also found that substrate affinity of enzymes (which is usually decreases with the temperature) differently responded to warming for the process of depolymerisation versus monomers oxidation. We suggest the mechanism to temperature acclimation based on different temperature sensitivity of enzymes kinetics for hydrolysis of polymers and for monomers oxidation.

Enhanced cellulose degradation using cellulase-nanosphere complexes.

PubMed

Blanchette, Craig; Lacayo, Catherine I; Fischer, Nicholas O; Hwang, Mona; Thelen, Michael P

2012-01-01

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.

Enhanced Cellulose Degradation Using Cellulase-Nanosphere Complexes

PubMed Central

Blanchette, Craig; Lacayo, Catherine I.; Fischer, Nicholas O.; Hwang, Mona; Thelen, Michael P.

2012-01-01

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production. PMID:22870287

Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure.

PubMed

Colombo, Lívia Tavares; de Oliveira, Marcelo Nagem Valério; Carneiro, Deisy Guimarães; de Souza, Robson Assis; Alvim, Mariana Caroline Tocantins; Dos Santos, Josenilda Carlos; da Silva, Cynthia Canêdo; Vidigal, Pedro Marcus Pereira; da Silveira, Wendel Batista; Passos, Flávia Maria Lopes

2016-09-01

Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes β-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three β-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.

The mouse liver displays daily rhythms in the metabolism of phospholipids and in the activity of lipid synthesizing enzymes.

PubMed

Gorné, Lucas D; Acosta-Rodríguez, Victoria A; Pasquaré, Susana J; Salvador, Gabriela A; Giusto, Norma M; Guido, Mario Eduardo

2015-02-01

The circadian system involves central and peripheral oscillators regulating temporally biochemical processes including lipid metabolism; their disruption leads to severe metabolic diseases (obesity, diabetes, etc). Here, we investigated the temporal regulation of glycerophospholipid (GPL) synthesis in mouse liver, a well-known peripheral oscillator. Mice were synchronized to a 12:12 h light-dark (LD) cycle and then released to constant darkness with food ad libitum. Livers collected at different times exhibited a daily rhythmicity in some individual GPL content with highest levels during the subjective day. The activity of GPL-synthesizing/remodeling enzymes: phosphatidate phosphohydrolase 1 (PAP-1/lipin) and lysophospholipid acyltransferases (LPLATs) also displayed significant variations, with higher levels during the subjective day and at dusk. We evaluated the temporal regulation of expression and activity of phosphatidylcholine (PC) synthesizing enzymes. PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) as a key regulatory enzyme or through the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. The PC/PE content ratio exhibited a daily variation with lowest levels at night, while ChoKα and PEMT mRNA expression displayed maximal levels at nocturnal phases. Our results demonstrate that mouse liver GPL metabolism oscillates rhythmically with a precise temporal control in the expression and/or activity of specific enzymes.

Trace elements and antioxidant enzymes in Behçet's disease.

PubMed

Saglam, K; Serce, A F; Yilmaz, M I; Bulucu, F; Aydin, A; Akay, C; Sayal, A

2002-07-01

Free oxygen radicals and insufficiency of antioxidant enzymes have been implicated in the pathogenesis of Behçet's disease (BD). Trace elements function as cofactors to antioxidant enzymes. The antioxidant system and trace elements were investigated in many different studies, including BD, but these subjects have not been investigated as a whole in these patients. The aim of the present study was to investigate the antioxidative system and trace elements in BD to contribute to the knowledge of pathogenesis and treatment of this disease. We examined glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities together with selenium (Se), copper (Cu), zinc (Zn), manganese (Mn), and iron (Fe) levels in plasma and erythrocytes of 50 patients with BD and 30 healthy controls. It was found that in patients with BD, erythrocyte GSH-Px and SOD activities and erythrocyte Se, plasma Fe, Mn, and Zn levels were significantly lower than those of controls and that plasma Cu, erythrocyte Zn, and Mn levels were significantly higher in patients with BD. Insufficient antioxidant enzyme activities were observed in patients with BD. The mechanism(s) of this phenomenon is not clear. Therefore, supplementation with trace elements involved in the antioxidative processes may increase scavenger enzyme activities, and consequently, an improvement in clinical symptoms may be expected.

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase.

PubMed

Miginiac-Maslow, M; Jacquot, J P; Droux, M

1985-09-01

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxinm and f (Tdm, Tdf) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m(-2) under nitrogen and 50 W·m(-2) under air, while NADP photoreduction was saturated at 240 W·m(-2). Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N2 by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O2 was located at the level of Fd.

Of enzyme use in cost-effective high solid simultaneous saccharification and fermentation processes.

PubMed

Sóti, Valentin; Lenaerts, Silvia; Cornet, Iris

2018-03-20

Enzyme cost is considered to be one of the most significant factors defining the final product price in lignocellulose hydrolysis and fermentation. Enzyme immobilization and recycling can be a tool to decrease costs. However, high solid loading is a key factor towards high product titers, and recovery of immobilized enzymes from this thick liquid is often overlooked. This paper aims to evaluate the economic feasibility of immobilized enzymes in simultaneous saccharification and fermentation (SSF) of lignocellulose biomass in general, as well as the recuperation of magnetic immobilized enzymes (m-CLEAs) during high solid loading in simultaneous saccharification, detoxification and fermentation processes (SSDF) of lignocellulose biomass. Enzyme prices were obtained from general cost estimations by Klein-Marcuschamer et al. [Klein-Marcuschamer et al. (2012) Biotechnol. Bioeng. 109, 1083-1087]. During enzyme cost analysis, the influence of inoculum recirculation as well as a shortened fermentation time was explored. Both resulted in 15% decrease of final enzyme product price. Enzyme recuperation was investigated experimentally and 99.5 m/m% of m-CLEAs was recovered from liquid medium in one step, while 88 m/m% could still be recycled from a thick liquid with high solid concentrations (SSF fermentation broth). A mathematical model was constructed to calculate the cost of immobilized and free enzyme utilization and showed that, with current process efficiencies and commercial enzyme prices, the cost reduction obtained by enzyme immobilization can reach around 60% compared to free enzyme utilization, while lower enzyme prices will result in a lower percentage of immobilization related savings, but overall enzyme costs will decrease significantly. These results are applied in a case study, estimating the viability of shifting from sugar to lignocellulose substrate for a 100 t lactic acid fermentation batch. It was concluded that it will only be economically feasible if the enzymes are produced at the most optimistic variable cost and either the activity of the immobilized catalyst or the recovery efficiency is further increased. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular dynamics simulation of the last step of a catalytic cycle: product release from the active site of the enzyme chorismate mutase from Mycobacterium tuberculosis.

PubMed

Choutko, Alexandra; van Gunsteren, Wilfred F

2012-11-01

The protein chorismate mutase MtCM from Mycobacterium tuberculosis catalyzes one of the few pericyclic reactions known in biology: the transformation of chorismate to prephenate. Chorismate mutases have been widely studied experimentally and computationally to elucidate the transition state of the enzyme catalyzed reaction and the origin of the high catalytic rate. However, studies about substrate entry and product exit to and from the highly occluded active site of the enzyme have to our knowledge not been performed on this enzyme. Crystallographic data suggest a possible substrate entry gate, that involves a slight opening of the enzyme for the substrate to access the active site. Using multiple molecular dynamics simulations, we investigate the natural dynamic process of the product exiting from the binding pocket of MtCM. We identify a dominant exit pathway, which is in agreement with the gate proposed from the available crystallographic data. Helices H2 and H4 move apart from each other which enables the product to exit from the active site. Interestingly, in almost all exit trajectories, two residues arginine 72 and arginine 134, which participate in the burying of the active site, are accompanying the product on its exit journey from the catalytic site. Copyright © 2012 The Protein Society.

Iminosugars Inhibit Dengue Virus Production via Inhibition of ER Alpha-Glucosidases--Not Glycolipid Processing Enzymes.

PubMed

Sayce, Andrew C; Alonzi, Dominic S; Killingbeck, Sarah S; Tyrrell, Beatrice E; Hill, Michelle L; Caputo, Alessandro T; Iwaki, Ren; Kinami, Kyoko; Ide, Daisuke; Kiappes, J L; Beatty, P Robert; Kato, Atsushi; Harris, Eva; Dwek, Raymond A; Miller, Joanna L; Zitzmann, Nicole

2016-03-01

It has long been thought that iminosugar antiviral activity is a function of inhibition of endoplasmic reticulum-resident α-glucosidases, and on this basis, many iminosugars have been investigated as therapeutic agents for treatment of infection by a diverse spectrum of viruses, including dengue virus (DENV). However, iminosugars are glycomimetics possessing a nitrogen atom in place of the endocyclic oxygen atom, and the ubiquity of glycans in host metabolism suggests that multiple pathways can be targeted via iminosugar treatment. Successful treatment of patients with glycolipid processing defects using iminosugars highlights the clinical exploitation of iminosugar inhibition of enzymes other than ER α-glucosidases. Evidence correlating antiviral activity with successful inhibition of ER glucosidases together with the exclusion of alternative mechanisms of action of iminosugars in the context of DENV infection is limited. Celgosivir, a bicyclic iminosugar evaluated in phase Ib clinical trials as a therapeutic for the treatment of DENV infection, was confirmed to be antiviral in a lethal mouse model of antibody-enhanced DENV infection. In this study we provide the first evidence of the antiviral activity of celgosivir in primary human macrophages in vitro, in which it inhibits DENV secretion with an EC50 of 5 μM. We further demonstrate that monocyclic glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. By comparison to bicyclic glucose-mimicking iminosugars which inhibit glycoprotein processing but do not inhibit glycolipid processing and galactose-mimicking iminosugars which do not inhibit glycoprotein processing but do inhibit glycolipid processing, we demonstrate that inhibition of endoplasmic reticulum-resident α-glucosidases, not glycolipid processing, is responsible for iminosugar antiviral activity against DENV. Our data suggest that inhibition of ER α-glucosidases prevents release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.

Reconciling Apparent Variability in Effects of Biochar Amendment on Soil Enzyme Activities by Assay Optimization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bailey, Vanessa L.; Fansler, Sarah J.; Smith, Jeffery L.

2011-02-01

Applying biochar to soils as an ameliorative substance and mechanism for C sequestration has received a great deal of interest in light of the sustained fertility observed in the Terra Preta soils of Brazil. The effects of synthetic biochars on biochemical processes needs to be better understood in order to determine if this is a reasonable practice in managed systems. The biochar studied was formed from the fast-pyrolysis of a switchgrass feedstock. Four soil enzymes were studied: β-glucosidase, β-N-acetylglucosaminidase, lipase, and leucine aminopeptidase. Both colorimetric and fluorescent assays were used for β-glucosidase and β-N-acetylglucosaminidase. Seven days after biochar was addedmore » to microcosms of a Palouse silt loam, the fluorescence-based assays indicated increased activities of the four enzymes, compared to non-amended soil. To clarify the mechanisms of the observed effects,in the absence of soil, purified enzymes or substrates were briefly exposed to biochar and then assayed. Except for β-N-acetylglucosaminidase, the exposure of substrate to biochar reduced the apparent activity of the remaining three enzymes in vitro, suggesting that sorption reactions between the substrate and biochar either removed the substrate from the assays or impeded the enzyme binding. The activity of purified β-N-acetylglucosaminidase increased significantly following biochar exposure, suggesting a chemical stimulation of enzyme functioning. We conclude that biochar added to soil acts as a substrate that can stimulate the soil microbial biomass and its activity. Our in vitro study suggests that biochar is not biochemically inert. Biochar amendments are likely to have effects that are currently difficult to predict, and that could impact overall soil function.« less

Characterization of thermo- and detergent stable antigenic glycosylated cysteine protease of Euphorbia nivulia Buch.-Ham. and evaluation of its ecofriendly applications.

PubMed

Badgujar, Shamkant B; Mahajan, Raghunath T

2013-01-01

An antigenic glycosylated cysteine protease has been purified from the latex of Euphorbia nivulia Buch.-Ham. It exhibits remarkable protease activity in the presence of metal ions, oxidizing agents, organic solvents, and detergents. This enzyme showed potential role in leather processing industry due to its dehairing activity for animal hide without hydrolyzing fibrous proteins, producing, by this way, a better quality product. The enzyme can also be used for silver recovering from X-ray plates. In addition, the stability (temperature and surfactants) and hydrolysis of blood stain data also revealed its application in detergent industries. Agriculturally, this protease finds application in biocontrol process against the infectious management of root knot nematode, Meloidogyne incognita. Biologically, it shows noticeable wound healing, haemostatic and antibacterial activity.

Characterization of Thermo- and Detergent Stable Antigenic Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham. and Evaluation of Its Ecofriendly Applications

PubMed Central

Badgujar, Shamkant B.; Mahajan, Raghunath T.

2013-01-01

An antigenic glycosylated cysteine protease has been purified from the latex of Euphorbia nivulia Buch.-Ham. It exhibits remarkable protease activity in the presence of metal ions, oxidizing agents, organic solvents, and detergents. This enzyme showed potential role in leather processing industry due to its dehairing activity for animal hide without hydrolyzing fibrous proteins, producing, by this way, a better quality product. The enzyme can also be used for silver recovering from X-ray plates. In addition, the stability (temperature and surfactants) and hydrolysis of blood stain data also revealed its application in detergent industries. Agriculturally, this protease finds application in biocontrol process against the infectious management of root knot nematode, Meloidogyne incognita. Biologically, it shows noticeable wound healing, haemostatic and antibacterial activity. PMID:24348183

Excited-State Charge Separation in the Photochemical Mechanism of the Light-Driven Enzyme Protochlorophyllide Oxidoreductase**

PubMed Central

Heyes, Derren J; Hardman, Samantha J O; Hedison, Tobias M; Hoeven, Robin; Greetham, Greg M; Towrie, Michael; Scrutton, Nigel S

2015-01-01

The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride- and proton-transfer chemistry, have so far proven difficult to detect. We have used a combination of time-resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond–microsecond time range, to propose a new mechanism for the photochemistry. Excited-state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized and highly reactive double bond. This so-called “reactive” intramolecular charge-transfer state creates an electron-deficient site across the double bond to trigger the subsequent nucleophilic attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited-state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light-activated chemical and biological catalysts. PMID:25488797

Excited-state charge separation in the photochemical mechanism of the light-driven enzyme protochlorophyllide oxidoreductase.

PubMed

Heyes, Derren J; Hardman, Samantha J O; Hedison, Tobias M; Hoeven, Robin; Greetham, Greg M; Towrie, Michael; Scrutton, Nigel S

2015-01-26

The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride- and proton-transfer chemistry, have so far proven difficult to detect. We have used a combination of time-resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond-microsecond time range, to propose a new mechanism for the photochemistry. Excited-state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized and highly reactive double bond. This so-called "reactive" intramolecular charge-transfer state creates an electron-deficient site across the double bond to trigger the subsequent nucleophilic attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited-state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light-activated chemical and biological catalysts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Concerted In Vitro Trimming of Viral HLA-B27-Restricted Ligands by Human ERAP1 and ERAP2 Aminopeptidases

PubMed Central

Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen; Jiménez, Mercedes; López, Daniel

2013-01-01

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2. PMID:24223975

Concerted in vitro trimming of viral HLA-B27-restricted ligands by human ERAP1 and ERAP2 aminopeptidases.

PubMed

Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen; Jiménez, Mercedes; López, Daniel

2013-01-01

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling

DOE PAGES

Liu, Yun; Fredrickson, James K.; Sadler, Natalie C.; ...

2015-09-25

Here, the development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosicmore » bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.« less

Xylose induces cellulase production in Thermoascus aurantiacus.

PubMed

Schuerg, Timo; Prahl, Jan-Philip; Gabriel, Raphael; Harth, Simon; Tachea, Firehiwot; Chen, Chyi-Shin; Miller, Matthew; Masson, Fabrice; He, Qian; Brown, Sarah; Mirshiaghi, Mona; Liang, Ling; Tom, Lauren M; Tanjore, Deepti; Sun, Ning; Pray, Todd R; Singer, Steven W

2017-01-01

Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus . Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted to produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. Xylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.

Continuous enzymatic hydrolysis of lignocellulosic biomass with simultaneous detoxification and enzyme recovery.

PubMed

Gurram, Raghu N; Menkhaus, Todd J

2014-07-01

Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical conversion of lignocellulosic materials into renewable biofuels and biochemicals. Here, we show that enzymatic hydrolysis of acid-pretreated pine wood with continuous and/or fed-batch enzyme addition improved sugar conversion efficiencies by over sixfold. In addition, specific activity of the hydrolysis enzymes (cellulases, hemicellulases, etc.) increased as a result of continuously washing the residual solids with removal of glucose (avoiding the end product inhibition) and other enzymatic inhibitory compounds (e.g., furfural, hydroxymethyl furfural, organic acids, and phenolics). As part of the continuous hydrolysis, anion exchange resin was tested for its dual application of simultaneous enzyme recovery and removal of potential enzymatic and fermentation inhibitors. Amberlite IRA-96 showed favorable adsorption profiles of inhibitors, especially furfural, hydroxymethyl furfural, and acetic acid with low affinity toward sugars. Affinity of hydrolysis enzymes to adsorb onto the resin allowed for up to 92 % of the enzymatic activity to be recovered using a relatively low-molar NaCl wash solution. Integration of an ion exchange column with enzyme recovery into the proposed fed-batch hydrolysis process can improve the overall biorefinery efficiency and can greatly reduce the production costs of lignocellulosic biorenewable products.

Furin is a chemokine-modifying enzyme: in vitro and in vivo processing of CXCL10 generates a C-terminally truncated chemokine retaining full activity.

PubMed

Hensbergen, Paul J; Verzijl, Dennis; Balog, Crina I A; Dijkman, Remco; van der Schors, Roel C; van der Raaij-Helmer, Elizabeth M H; van der Plas, Mariena J A; Leurs, Rob; Deelder, André M; Smit, Martine J; Tensen, Cornelis P

2004-04-02

Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the total spectrum of chemokine variants. We have recently shown that the native chemokine CXCL10 is processed at the C terminus, thereby shedding the last four amino acids. The present study was performed to elucidate the mechanism in vivo and in vitro and to study the biological activity of this novel isoform of CXCL10. Using a combination of protein purification and mass spectrometric techniques, we show that the production of C-terminally truncated CXCL10 by primary keratinocytes is inhibited in vivo by a specific inhibitor of pro-protein convertases (e.g. furin) but not by inhibition of matrix metalloproteinases. Moreover, CXCL10 is processed by furin in vitro, which is abrogated by a mutation in the furin recognition site. Using GTPgammaS binding, Ca(2+) mobilization, and chemotaxis assays, we demonstrate that the C-terminally truncated CXCL10 variant is a potent ligand for CXCR3. Moreover, the inverse agonist activity on the virally encoded receptor ORF74 and the direct antibacterial activity of CXCL10 are fully retained. Hence, we have identified furin as a novel chemokine-modifying enzyme in vitro and most probably also in vivo, generating a C-terminally truncated CXCL10, which fully retains its (inverse) agonistic properties.

The 53-kDa proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity.

PubMed

Ravi-Kumar, K; Venkatesh, K S; Umesh-Kumar, S

2007-04-01

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent alpha-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of alpha-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.

Rhizobium cellulase CelC2 is essential for primary symbiotic infection of legume host roots

PubMed Central

Robledo, M.; Jiménez-Zurdo, J. I.; Velázquez, E.; Trujillo, M. E.; Zurdo-Piñeiro, J. L.; Ramírez-Bahena, M. H.; Ramos, B.; Díaz-Mínguez, J. M.; Dazzo, F.; Martínez-Molina, E.; Mateos, P. F.

2008-01-01

The rhizobia–legume, root-nodule symbiosis provides the most efficient source of biologically fixed ammonia fertilizer for agricultural crops. Its development involves pathways of specificity, infectivity, and effectivity resulting from expressed traits of the bacterium and host plant. A key event of the infection process required for development of this root-nodule symbiosis is a highly localized, complete erosion of the plant cell wall through which the bacterial symbiont penetrates to establish a nitrogen-fixing, intracellular endosymbiotic state within the host. This process of wall degradation must be delicately balanced to avoid lysis and destruction of the host cell. Here, we describe the purification, biochemical characterization, molecular genetic analysis, biological activity, and symbiotic function of a cell-bound bacterial cellulase (CelC2) enzyme from Rhizobium leguminosarum bv. trifolii, the clover-nodulating endosymbiont. The purified enzyme can erode the noncrystalline tip of the white clover host root hair wall, making a localized hole of sufficient size to allow wild-type microsymbiont penetration. This CelC2 enzyme is not active on root hairs of the nonhost legume alfalfa. Microscopy analysis of the symbiotic phenotypes of the ANU843 wild type and CelC2 knockout mutant derivative revealed that this enzyme fulfils an essential role in the primary infection process required for development of the canonical nitrogen-fixing R. leguminosarum bv. trifolii-white clover symbiosis. PMID:18458328

Purification and Characterization of a Novel Thermo-Alkali-Stable Catalase from Thermus brockianus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, Vicki Sue; Schaller, Kastli Dianne; Apel, William Arnold

2003-10-01

A novel thermo-alkali-stable catalase from Thermus brockianus was purified and characterized. The protein was purified from a T. brockianus cell extract in a three-step procedure that resulted in 65-fold purification to a specific activity of 5300 U/mg. The enzyme consisted of four identical subunits of 42.5 kDa as determined by SDS-PAGE and a total molecular mass measured by gel filtration of 178 kDa. The catalase was active over a temperature range from 30 to 94 C and a pH range from 6 to 10, with optimum activity occurring at 90 C and pH 8. At pH 8, the enzyme wasmore » extremely stable at elevated temperatures with half-lives of 330 h at 80 C and 3 h at 90 C. The enzyme also demonstrated excellent stability at 70 C and alkaline pH with measured half-lives of 510 h and 360 h at pHs of 9 and 10, respectively. The enzyme had an unusual pyridine hemochrome spectrum and appears to utilize eight molecules of heme c per tetramer rather than protoheme IX present in the majority of catalases studied to date. The absorption spectrum suggested that the heme iron of the catalase was in a 6-coordinate low spin state rather than the typical 5-coordinate high spin state. A Km of 35.5 mM and a Vmax of 20.3 mM/min·mg protein for hydrogen peroxide was measured, and the enzyme was not inhibited by hydrogen peroxide at concentrations up to 450 mM. The enzyme was strongly inhibited by cyanide and the traditional catalase inhibitor 3-amino-1,2,4-triazole. The enzyme also showed no peroxidase activity to peroxidase substrates o-dianisidine and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), a trait of typical monofunctional catalases. However, unlike traditional monofunctional catalases, the T. brockianus catalase was easily reduced by dithionite, a characteristic of catalase-peroxidases. The above properties indicate that this catalase has potential for applications in industrial bleaching processes to remove residual hydrogen peroxide from process streams.« less

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis.

PubMed

Burschowsky, Daniel; van Eerde, André; Ökvist, Mats; Kienhöfer, Alexander; Kast, Peter; Hilvert, Donald; Krengel, Ute

2014-12-09

For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.

Tannase Production by Solid State Fermentation of Cashew Apple Bagasse

NASA Astrophysics Data System (ADS)

Podrigues, Tigressa H. S.; Dantas, Maria Alcilene A.; Pinto, Gustavo A. S.; Gonçalves, Luciana R. B.

The ability of Aspergillus oryzae for the production of tannase by solid state fermentation was investigated using cashew apple bagasse (CAB) as substrate. The effect of initial water content was studied and maximum enzyme production was obtained when 60 mL of water was added to 100.0 g of CAB. The fungal strain was able to grow on CAB without any supplementation but a low enzyme activity was obtained, 0.576 U/g of dry substrate (gds). Optimization of process parameters such as supplementation with tannic acid, phosphorous, and different organic and inorganic nitrogen sources was studied. The addition of tannic acid affected the enzyme production and maximum tannase activity (2.40 U/gds) was obtained with 2.5% (w/w) supplementation. Supplementation with ammonium nitrate, peptone, and yeast extract exerted no influence on tannase production. Ammonium sulphate improved the enzyme production in 3.75-fold compared with control. Based on the experimental results, CAB is a promising substrate for solid state fermentation, enabling A. oryzae growth and the production of tannase, with a maximum activity of 3.42 U/gds and enzyme productivity of 128.5×10-3 U·gds -1·h-1.

Potency of Amylase-producing Bacteria and Optimization Amylase Activities

NASA Astrophysics Data System (ADS)

Indriati, G.; Megahati, R. R. P.; Rosba, E.

2018-04-01

Enzymes are capable to act as biocatalyst for a wide variety of chemical reactions. Amylase have potential biotechnological applications in a wide range of industrial processes and account for nearly 30% of the world’s enzyme market. Amylase are extracellular enzymes that catalyze the hydrolysis of internal α-1,4-glycosidic linkages in starch to dextrin, and other small carbohydrate molecules constituted of glucose units. Although enzymes are produced from animal and plant sources, the microbial sources are generally the most suitable for commercial applications. Bacteria from hot springs is widely used as a source of various enzymes, such as amylase. But the amount of amylase-producing bacteria is still very limited. Therefore it is necessary to search sources of amylase-producing bacteria new, such as from hot springs Pariangan. The purpose of this study was to isolation of amylase-producing bacteria from Pariangan hot spring, West Sumatera and amylase activity optimization. The results were obtained 12 isolates of thermophilic bacteria and 5 isolates of amyalse-producing bacteria with the largest amylolytic index of 3.38 mm. The highest amylase activity was obtained at 50°C and pH 7.5.

Preparation and activity of bubbling-immobilized cellobiase within chitosan-alginate composite.

PubMed

Wang, Fang; Su, Rong-Xin; Qi, Wei; Zhang, Ming-Jia; He, Zhi-Min

2010-01-01

Cellobiase can hydrolyze cellobiose into glucose; it plays a key role in the process of cellulose hydrolysis by reducing the product inhibition. To reuse the enzyme and improve the economic value of cellulosic ethanol, cellobiase was immobilized using sodium alginate and chitosan as carriers by the bubbling method. The immobilization conditions were optimized as follows: enzyme loading of 100 U cellobiase/g carrier, 30 min immobilization, 3.5 wt% sodium alginate, 0.25 wt% chitosan, and 2 wt% calcium chloride. Compared to free enzyme, the immobilized cellobiase had a decreased apparent K(m) and the maximum activity at a lower pH, indicating its higher acidic and thermal stability. The immobilized cellobiase was further tested in the hydrolysis of cellobiose and various cellulosic substrates (microcrystalline cellulose, filter paper, and ammonia-pretreated corn cobs). Together with cellulases, the immobilized cellobiase converted the cellulosic substrates into glucose with the rate and extent similar to the free enzyme.

Mutations that Allow SIR2 Orthologs to Function in a NAD+-Depleted Environment.

PubMed

Ondracek, Caitlin R; Frappier, Vincent; Ringel, Alison E; Wolberger, Cynthia; Guarente, Leonard

2017-03-07

Sirtuin enzymes depend on NAD + to catalyze protein deacetylation. Therefore, the lowering of NAD + during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD + -depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog, SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD + -limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Kinetics of acrylodan-labelled cAMP-dependent protein kinase catalytic subunit denaturation.

PubMed

Kivi, Rait; Loog, Mart; Jemth, Per; Järv, Jaak

2013-10-01

Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components.

PubMed

Lu, Qiaosheng; Wierzbicki, Sara; Krasilnikov, Andrey S; Schmitt, Mark E

2010-03-01

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

Design of experiment (DOE) based screening of factors affecting municipal solid waste (MSW) composting.

PubMed

Kazemi, Khoshrooz; Zhang, Baiyu; Lye, Leonard M; Cai, Qinghong; Cao, Tong

2016-12-01

A design of experiment (DOE) based methodology was adopted in this study to investigate the effects of multiple factors and their interactions on the performance of a municipal solid waste (MSW) composting process. The impact of four factors, carbon/nitrogen ratio (C/N), moisture content (MC), type of bulking agent (BA) and aeration rate (AR) on the maturity, stability and toxicity of compost product was investigated. The statistically significant factors were identified using final C/N, germination index (GI) and especially the enzyme activities as responses. Experimental results validated the use of enzyme activities as proper indices during the course of composting. Maximum enzyme activities occurred during the active phase of decomposition. MC has a significant effect on dehydrogenase activity (DGH), β-glucosidase activity (BGH), phosphodiesterase activity (PDE) and the final moisture content of the compost. C/N is statistically significant for final C/N, DGH, BGH, and GI. The results provided guidance to optimize a MSW composting system that will lead to increased decomposition rate and the production of more stable and mature compost. Copyright © 2016 Elsevier Ltd. All rights reserved.

Study of mango endogenous pectinases as a tool to engineer mango purée consistency.

PubMed

Jamsazzadeh Kermani, Zahra; Shpigelman, Avi; Houben, Ken; ten Geuzendam, Belinda; Van Loey, Ann M; Hendrickx, Marc E

2015-04-01

The objective of this work was to evaluate the possibility of using mango endogenous pectinases to change the viscosity of mango purée. Hereto, the structure of pectic polysaccharide and the presence of sufficiently active endogenous enzymes of ripe mango were determined. Pectin of mango flesh had a high molecular weight and was highly methoxylated. Pectin methylesterase showed a negligible activity which is related to the confirmed presence of a pectin methylesterase inhibitor. Pectin contained relatively high amounts of galactose and considerable β-galactosidase (β-Gal) activity was observed. The possibility of stimulating β-Gal activity during processing (temperature/pressure, time) was investigated. β-Gal of mango was rather temperature labile but pressure stable relatively to the temperature and pressure levels used to inactivate destructive enzymes in industry. Creating processing conditions allowing endogenous β-Gal activity did not substantially change the consistency of mango purée. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Litter decomposition and lignocellulose enzyme activities of Actinothuidium hookeri and Cys- topteris montana in alpine timberline ecotone of western Sichuan, China].

PubMed

Chen, Ya-mei; He, Run-lian; Deng, Chang-chun; Yang, Wan-qin; Zhang, Jian; Yang, Lin; Liu, Yang

2015-11-01

The mass loss and lignocellulose enzyme activities of Actinothuidium hookeri residues and Cystopteris montana leaf litter in coniferous forest and timberline of western Sichuan, China were investigated. The results showed that both the mass loss rates of A. hookeri and C. Montana in timberline were higher than those in coniferous forest, while enzyme activities in timberline were lower than those in coniferous forest which was contrast with the hypothesis. The mass loss of two ground covers had significant differences in different seasons. The mass loss rate of A. hookeri in snow-covered season accounted for 69.8% and 83.0% of the whole year' s in timberline and coniferous forest, while that of C. montana in the growing season accounted for 82.6% and 83.4% of the whole year' s in timberline and coniferous forest, respectively. C. montana leaf litter decayed faster in the growing season, which was consistent with its higher cellulase activity in the growing season. The result illustrated that the enzymatic hydrolysis of cellulose and hemicellulose might be the main driving force for the early stage of litter decomposition. Multiple linear regression analysis showed that environmental factors and initial litter quality could explain 45.8%-85.1% variation of enzyme activity. The enzyme activities of A. hookeri and C. montana in the process of decomposition were mainly affected by the freeze-thaw cycle in snow-covered season.

Signal transduction and amplification through enzyme-triggered ligand release and accelerated catalysis.

PubMed

Goggins, Sean; Marsh, Barrie J; Lubben, Anneke T; Frost, Christopher G

2015-08-01

Signal transduction and signal amplification are both important mechanisms used within biological signalling pathways. Inspired by this process, we have developed a signal amplification methodology that utilises the selectivity and high activity of enzymes in combination with the robustness and generality of an organometallic catalyst, achieving a hybrid biological and synthetic catalyst cascade. A proligand enzyme substrate was designed to selectively self-immolate in the presence of the enzyme to release a ligand that can bind to a metal pre-catalyst and accelerate the rate of a transfer hydrogenation reaction. Enzyme-triggered catalytic signal amplification was then applied to a range of catalyst substrates demonstrating that signal amplification and signal transduction can both be achieved through this methodology.

Bacterial quorum sensing and nitrogen cycling in rhizosphere soil

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeAngelis, K.M.; Lindow, S.E.; Firestone, M.K.

2008-10-01

Plant photosynthate fuels carbon-limited microbial growth and activity, resulting in increased rhizosphere nitrogen (N)-mineralization. Most soil organic N is macromolecular (chitin, protein, nucleotides); enzymatic depolymerization is likely rate-limiting for plant N accumulation. Analyzing Avena (wild oat) planted in microcosms containing sieved field soil, we observed increased rhizosphere chitinase and protease specific activities, bacterial cell densities, and dissolved organic nitrogen (DON) compared to bulk soil. Low-molecular weight DON (<3000 Da) was undetectable in bulk soil but comprised 15% of rhizosphere DON. Extracellular enzyme production in many bacteria requires quorum sensing (QS), cell-density dependent group behavior. Because proteobacteria are considered major rhizospheremore » colonizers, we assayed the proteobacterial QS signals acyl-homoserine lactones (AHLs), which were significantly increased in the rhizosphere. To investigate the linkage between soil signaling and N cycling, we characterized 533 bacterial isolates from Avena rhizosphere: 24% had chitinase or protease activity and AHL production; disruption of QS in 7 of 8 eight isolates disrupted enzyme activity. Many {alpha}-Proteobacteria were newly found with QS-controlled extracellular enzyme activity. Enhanced specific activities of N-cycling enzymes accompanied by bacterial density-dependent behaviors in rhizosphere soil gives rise to the hypothesis that QS could be a control point in the complex process of rhizosphere N-mineralization.« less

Long-term fertilization, but not warming, shifts rates of ectomycorrhizal nutrient cycling in Arctic tussock tundra.

NASA Astrophysics Data System (ADS)

Dunleavy, H.; Mack, M. C.

2017-12-01

The role of ectomycorrhizae (ECM) in Arctic nutrient cycling may be changing as temperature, nutrient availability, and ECM shrub abundance and size increase. A shift in ECM function has been proposed as a possible mechanism for shrub expansion. While several studies demonstrate a higher abundance of ECM as well as community compositional shifts in response to long-term experimental warming and fertilization, direct measurements of functional responses are missing. To understand the potential role of ECM in soil biogeochemical processes of the changing Arctic, we investigated the functional response of ECM to 30 years of summer warming and increased nutrient availability by measuring potential activities of extracellular enzymes associated with nitrogen (N) and phosphorous (P) acquisition on ECM root tips. We hypothesize ECM enzyme activities will be higher with warmer temperatures. Conversely, fertilization will lower ECM enzyme activities as N and P become less limiting to host plants. Preliminary results strongly support our latter hypothesis, but not the first. Warming decreased hydrolytic P-associated and labile N-associated enzyme activities on individual root tips (pmol/min/mm2 root tip) by 30% and 83%, respectively. However, warming increased ECM abundance and did not alter community-level activities (pmol/min/cm3 soil). Fertilization decreased hydrolytic and oxidative enzymatic activities on individual root tips by 34 to 80% as well as on a community level by 67 to 93%, even though ECM shrubs were almost monodominant. The combined effect of warming and fertilization decreased labile N-associated enzyme activity by 82%, but had little effect on oxidative and other hydrolytic enzyme activities. Although both warming and fertilization decreased root tip activities, reflecting a potential reduction in plant allocation to mycorrhizal nutrient acquisition, only fertilization lowered rates of ECM nutrient cycling. The indirect relationship between ECM abundance and individual root tip activity highlights the importance of measuring ECM function to assess the role of this symbiosis in nutrient cycling.

Process relevant screening of cellulolytic organisms for consolidated bioprocessing.

PubMed

Antonov, Elena; Schlembach, Ivan; Regestein, Lars; Rosenbaum, Miriam A; Büchs, Jochen

2017-01-01

Although the biocatalytic conversion of cellulosic biomass could replace fossil oil for the production of various compounds, it is often not economically viable due to the high costs of cellulolytic enzymes. One possibility to reduce costs is consolidated bioprocessing (CBP), integrating cellulase production, hydrolysis of cellulose, and the fermentation of the released sugars to the desired product into one process step. To establish such a process, the most suitable cellulase-producing organism has to be identified. Thereby, it is crucial to evaluate the candidates under target process conditions. In this work, the chosen model process was the conversion of cellulose to the platform chemical itaconic acid by a mixed culture of a cellulolytic fungus with Aspergillus terreus as itaconic acid producer. Various cellulase producers were analyzed by the introduced freeze assay that measures the initial carbon release rate, quantifying initial cellulase activity under target process conditions. Promising candidates were then characterized online by monitoring their respiration activity metabolizing cellulose to assess the growth and enzyme production dynamics. The screening of five different cellulase producers with the freeze assay identified Trichoderma   reesei and Penicillium   verruculosum as most promising. The measurement of the respiration activity revealed a retarded induction of cellulase production for P.   verruculosum but a similar cellulase production rate afterwards, compared to T.   reesei . The freeze assay measurement depicted that P.   verruculosum reaches the highest initial carbon release rate among all investigated cellulase producers. After a modification of the cultivation procedure, these results were confirmed by the respiration activity measurement. To compare both methods, a correlation between the measured respiration activity and the initial carbon release rate of the freeze assay was introduced. The analysis revealed that the different initial enzyme/cellulose ratios as well as a discrepancy in cellulose digestibility are the main differences between the two approaches. With two complementary methods to quantify cellulase activity and the dynamics of cellulase production for CBP applications, T.   reesei and P.   verruculosum were identified as compatible candidates for the chosen model process. The presented methods can easily be adapted to screen for suitable cellulose degrading organisms for various other applications.

Strategies to improve fiber utilization in swine

PubMed Central

2013-01-01

Application of feed processing methods and use of exogenous feed additives in an effort to improve nutrient digestibility of plant-based feed ingredients for swine has been studied for decades. The following review will discuss several of these topics, including: fiber characterization, impact of dietary fiber on gastrointestinal physiology, energy, and nutrient digestibility, mechanical processing of feed on fiber and energy digestibility, and the use of exogenous enzymes in diets fed to growing pigs. Taken together, the diversity and concentration of chemical characteristics that exists among plant-based feed ingredients, as well as interactions among constituents within feed ingredients and diets, suggests that improvements in nutrient digestibility and pig performance from mechanical processing or adding exogenous enzymes to diets fed to swine depends on a better understanding of these characteristics, but also relating enzyme activity to targeted substrates. It may be that an enzyme must not only match a target substrate(s), but there may also need to be a ′cocktail′ of enzymes to effectively breakdown the complex matrixes of fibrous carbohydrates, such that the negative impact of these compounds on nutrient digestibility or voluntary feed intake are alleviated. With the inverse relationship between fiber content and energy digestibility being well described for several feed ingredients, it is only logical that development of processing techniques or enzymes that degrade fiber, and thereby improve energy digestibility or voluntary feed intake, will be both metabolically and economically beneficial to pork production. PMID:23497595

Enzyme Engineering for In Situ Immobilization.

PubMed

Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

2016-10-14

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

Modelling in situ enzyme potential of soils: a tool to predict soil respiration from agricultural fields

NASA Astrophysics Data System (ADS)

Shahbaz Ali, Rana; Poll, Christian; Demyan, Scott; Nkwain Funkuin, Yvonne; Ingwersen, Joachim; Wizemann, Hans-Dieter; Kandeler, Ellen

2014-05-01

The fate of soil organic carbon (SOC) is one of the largest uncertainties in predicting future climate and terrestrial ecosystem functions. Extra-cellular enzymes, produced by microorganisms, perform the very first step in SOC degradation and serve as key components in global carbon cycling. Very little information is available about the seasonal variation in the temperature sensitivity of soil enzymes. Here we aim to model in situ enzyme potentials involved in the degradation of either labile or recalcitrant organic compounds to understand the temporal variability of degradation processes. To identify the similarities in seasonal patterns of soil respiration and in situ enzyme potentials, we compared the modelled in situ enzyme activities with weekly measured soil CO2 emissions. Arable soil samples from two different treatments (4 years fallow and currently vegetated plots; treatments represent range of carbon input into soil) were collected every month from April, 2012 to April, 2013, from two different study regions (Kraichgau and Swabian Alb) in Southwest Germany. The vegetation plots were under crop rotation in both study areas. We measured activities of three enzymes including β-glucosidase, xylanase and phenoloxidase at five different temperatures. We also measured soil microbial biomass in form of microbial carbon (Cmic). Land-use and area had significant effects (P < 0.001) on the microbial biomass; fallow plots having less Cmic than vegetation plots. Potential activities of β-glucosidase (P < 0.001) and xylanase (P < 0.01) were significantly higher in the vegetation plots of the Swabian Alb region than in the Kraichgau region. In both study areas, enzyme activities were higher during vegetation period and lower during winter which points to the importance of carbon input and/or temperature and soil moisture. We calculated the temperature sensitivity (Q10) of enzyme activities based on laboratory measurements of enzyme activities at a range of incubation temperatures. Q10 of β-glucosidase activity changed significantly across the year (Q10 values ranges from 1.5 to 2.0 in Kraichgau and 1.6 to 2.1 in Swabian Alb), while for xylanase activity, no significant effects were found (Q10 values ranges from 1.2 to 3.0 in Kraichgau and 1.3 to 3.3 in Swabian Alb) in both study regions. By using laboratory based enzyme activities, calculated Q10 values, and daily soil temperature data, we modelled in situ enzyme potentials in soils for labile and recalcitrant carbon pools for both study regions. We observed an increase in modelled in situ enzyme activities during the summer period and a substantial decrease during winter indicating temperature as a strong controlling factor. A significant higher positive correlation of soil surface CO2 flux with modelled in situ β-glucosidase activity was found in both study regions compared to modelled in situ xylanase activity. These results demonstrate that (1) Q10 values are site and season specific and should be added into carbon models and (2) the indication of the relevance of greater contribution of labile carbon pool to soil CO2 emissions.

Bioinspired construction of multi-enzyme catalytic systems.

PubMed

Shi, Jiafu; Wu, Yizhou; Zhang, Shaohua; Tian, Yu; Yang, Dong; Jiang, Zhongyi

2018-06-18

Enzyme catalysis, as a green, efficient process, displays exceptional functionality, adaptivity and sustainability. Multi-enzyme catalysis, which can accomplish the tandem synthesis of valuable materials/chemicals from renewable feedstocks, establishes a bridge between single-enzyme catalysis and whole-cell catalysis. Multi-enzyme catalysis occupies a unique and indispensable position in the realm of biological reactions for energy and environmental applications. Two complementary strategies, i.e., compartmentalization and substrate channeling, have been evolved by living organisms for implementing the complex in vivo multi-enzyme reactions (MERs), which have been applied to construct multi-enzyme catalytic systems (MECSs) with superior catalytic activity and stabilities in practical biocatalysis. This tutorial review aims to present the recent advances and future prospects in this burgeoning research area, stressing the features and applications of the two strategies for constructing MECSs and implementing in vitro MERs. The concluding remarks are presented with a perspective on the construction of MECSs through rational combination of compartmentalization and substrate channeling.

Biocatalysts: application and engineering for industrial purposes.

PubMed

Jemli, Sonia; Ayadi-Zouari, Dorra; Hlima, Hajer Ben; Bejar, Samir

2016-01-01

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.

Model-based analysis of N-glycosylation in Chinese hamster ovary cells

PubMed Central

Krambeck, Frederick J.; Bennun, Sandra V.; Betenbaugh, Michael J.

2017-01-01

The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower likelihood of causing immunogenic responses. Because glycan structures are the product of the concerted action of intracellular enzymes, it is difficult to predict a priori how the effects of genetic manipulations alter glycan structures of cells and therapeutic properties. For that reason, quantitative models able to predict glycosylation have emerged as promising tools to deal with the complexity of glycosylation processing. For example, an earlier version of the same model used in this study was used by others to successfully predict changes in enzyme activities that could produce a desired change in glycan structure. In this study we utilize an updated version of this model to provide a comprehensive analysis of N-glycosylation in ten Chinese hamster ovary (CHO) cell lines that include a wild type parent and nine mutants of CHO, through interpretation of previously published mass spectrometry data. The updated N-glycosylation mathematical model contains up to 50,605 glycan structures. Adjusting the enzyme activities in this model to match N-glycan mass spectra produces detailed predictions of the glycosylation process, enzyme activity profiles and complete glycosylation profiles of each of the cell lines. These profiles are consistent with biochemical and genetic data reported previously. The model-based results also predict glycosylation features of the cell lines not previously published, indicating more complex changes in glycosylation enzyme activities than just those resulting directly from gene mutations. The model predicts that the CHO cell lines possess regulatory mechanisms that allow them to adjust glycosylation enzyme activities to mitigate side effects of the primary loss or gain of glycosylation function known to exist in these mutant cell lines. Quantitative models of CHO cell glycosylation have the potential for predicting how glycoengineering manipulations might affect glycoform distributions to improve the therapeutic performance of glycoprotein products. PMID:28486471

Structural similarities and functional differences clarify evolutionary relationships between tRNA healing enzymes and the myelin enzyme CNPase.

PubMed

Muruganandam, Gopinath; Raasakka, Arne; Myllykoski, Matti; Kursula, Inari; Kursula, Petri

2017-05-16

Eukaryotic tRNA splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5'-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an RNA ligase. The enzymes that catalyze tRNA healing in yeast and higher eukaryotes are homologous to the members of the 2H phosphoesterase superfamily, in particular to the vertebrate myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). We employed different biophysical and biochemical methods to elucidate the overall structural and functional features of the tRNA healing enzymes yeast Trl1 PNK/CPDase and lancelet PNK/CPDase and compared them with vertebrate CNPase. The yeast and the lancelet enzymes have cyclic phosphodiesterase and polynucleotide kinase activity, while vertebrate CNPase lacks PNK activity. In addition, we also show that the healing enzymes are structurally similar to the vertebrate CNPase by applying synchrotron radiation circular dichroism spectroscopy and small-angle X-ray scattering. We provide a structural analysis of the tRNA healing enzyme PNK and CPDase domains together. Our results support evolution of vertebrate CNPase from tRNA healing enzymes with a loss of function at its N-terminal PNK-like domain.

Src kinases play a novel dual role in acute pancreatitis affecting severity but no role in stimulated enzyme secretion.

PubMed

Nuche-Berenguer, Bernardo; Ramos-Álvarez, Irene; Jensen, R T

2016-06-01

In pancreatic acinar cells, the Src family of kinases (SFK) is involved in the activation of several signaling cascades that are implicated in mediating cellular processes (growth, cytoskeletal changes, apoptosis). However, the role of SFKs in various physiological responses such as enzyme secretion or in pathophysiological processes such as acute pancreatitis is either controversial, unknown, or incompletely understood. To address this, in this study, we investigated the role/mechanisms of SFKs in acute pancreatitis and enzyme release. Enzyme secretion was studied in rat dispersed pancreatic acini, in vitro acute-pancreatitis-like changes induced by supramaximal COOH-terminal octapeptide of cholecystokinin (CCK). SFK involvement assessed using the chemical SFK inhibitor (PP2) with its inactive control, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3), under experimental conditions, markedly inhibiting SFK activation. In CCK-stimulated pancreatic acinar cells, activation occurred of trypsinogen, various MAP kinases (p42/44, JNK), transcription factors (signal transducer and activator of transcription-3, nuclear factor-κB, activator protein-1), caspases (3, 8, and 9) inducing apoptosis, LDH release reflective of necrosis, and various chemokines secreted (monocyte chemotactic protein-1, macrophage inflammatory protein-1α, regulated on activation, normal T cell expressed and secreted). All were inhibited by PP2, not by PP3, except caspase activation leading to apoptosis, which was increased, and trypsin activation, which was unaffected, as was CCK-induced amylase release. These results demonstrate SFK activation is playing a dual role in acute pancreatitis, inhibiting apoptosis and promoting necrosis as well as chemokine/cytokine release inducing inflammation, leading to more severe disease, as well as not affecting secretion. Thus, our studies indicate that SFK is a key mediator of inflammation and pancreatic acinar cell death in acute pancreatitis, suggesting it could be a potential therapeutic target in acute pancreatitis. Copyright © 2016 the American Physiological Society.

Beyond the hydrophobic effect: Critical function of water at biological phase boundaries--A hypothesis.

PubMed

Damodaran, Srinivasan

2015-07-01

Many life-sustaining processes in living cells occur at the membrane-water interface. The pertinent questions that need to be asked are what is the evolutionary reason for biology to choose the membrane-water interface as the site for performing and/or controlling crucial biological reactions and what is the key physical principle that is singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this review, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes and receptor activated processes via manipulating the thermodynamic activity of water at the membrane-water interfacial region. In support of this hypothesis, first we establish that the surface pressure of a lipid monolayer is a direct measure of a reduction in the thermodynamic activity of interfacial water. Second, we show that the surface pressure-dependent activation/inactivation of interfacial enzymes is fundamentally related to their dependence on interfacial water activity. We extend this argument to infer that cells might manipulate activities of membrane-associated biological processes via manipulating the activity of interfacial water via localized compression or expansion of the interface. In this paper, we critically analyze literature data on mechano-activation of large pore ion channels in Escherichia coli spheroplasts and G-proteins in reconstituted lipid vesicles, and show that these pressure-induced activation processes are fundamentally and quantitatively related to changes in the thermodynamic state of interfacial water, caused by mechanical stretching of the bilayer. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of endo-β-mannanase from Enterobacter ludwigii MY271 and application in pulp industry.

PubMed

Yang, Miao; Cai, Jun; Wang, Changgao; Du, Xin; Lin, Jianguo

2017-01-01

β-Mannanases are the second most important enzymes for the hydrolysis of hemicelluloses. An endo-β-mannanase from Enterobacter ludwigii MY271 was purified at 11.7 ± 0.2-fold to homogeneity with a final recovery of 15.2 ± 0.2 %. Using purified β-mannanase protein and SDS-PAGE, the molecular mass was found to be 43.16 kDa. The optimal pH and temperature of the enzyme was found to be 7.0 and 55 °C, respectively. The β-mannanase activity was stable over a broad pH range of pH 2.0-10.0. In addition, the purified enzyme was highly activated by several metal ions and chemical reagents, such as Mg 2+ , L-cysteine, glutathione (GSH) and β-mercaptoethanol. Whereas the enzyme was strongly inhibited by Hg 2+ , Cu 2+ , N-bromosuccinimide (NBS), 1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide (EDC), phenylmethanesulfonyl fluoride (PMSF), and sodium dodecyl sulfate (SDS). The β-mannanase was highly active towards glucomannan, and showed endo-activity by producing a mixture of oligosaccharides. Moreover, the enzyme displayed a classical endo-type mode on mannooligosaccharides. The β-mannanase coupled with xylanase significantly improved the brightness of kraft pulp, whereas it has no remarkable effect on the tensile strength of the pulp. Our functional studies of the purified β-mannanase indicate that the enzyme is beneficial to industrial applications, in particular, biotechnological processes, such as food, feed and pulp industry.

Cellulolytic and xylanolytic enzymes from thermophilic Aspergillus terreus RWY.

PubMed

Sharma, Reetika; Kocher, Gurvinder Singh; Bhogal, Ravinder Singh; Oberoi, Harinder Singh

2014-12-01

Thermophilic Aspergillus terreus RWY produced cellulases and xylanases in optimal concentrations at 45 °C in solid state fermentation process, though enzyme production was also observed at 50 and 55 °C. Filter paper cellulase (FP), endoglucanase (EG), β-glucosidase (BGL), cellobiohydrolase (CBH), xylanase, β-xylosidase, α-L-arabinofuranosidase and xylan esterase activities for A. terreus RWY at 45 °C in 72 h were 11.3 ± 0.65, 103 ± 6.4, 122.5 ± 8.7, 10.3 ± 0.66, 872 ± 22.5, 22.1 ± 0.75, 126.4 ± 8.4 and 907 ± 15.5 U (g-ds)(-1) , respectively. Enzyme was optimally active at temperatures and pH ranging between 50-60 °C and 4.0-6.0, respectively. The half life (T1/2 ) of 270 and 240 min at 70 and 75 °C, respectively for the enzyme indicates its stability at higher temperatures. The addition of MnCl2 , CoCl2 , and FeCl3 significantly enhanced cellulase activity. Enzyme demonstrated multiplicity by having seven, one and three isoform(s) for EG, CBH and BGL, respectively. Significant production of functionally active consortium of cellulolytic and xylanolytic enzymes from A. terreus RWY makes it a potential candidate in bioprocessing applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts

PubMed Central

2013-01-01

Background Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Methods Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. Results In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. Conclusion P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs. PMID:23948056

Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts.

PubMed

Makpol, Suzana; Yeoh, Thong Wei; Ruslam, Farah Adilah Che; Arifin, Khaizurin Tajul; Yusof, Yasmin Anum Mohd

2013-08-16

Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs.

Reduction of endogenous nucleic acid in a single-cell protein.

PubMed Central

Yang, H H; Thayer, D W; Yang, S P

1979-01-01

The reduction of nucleic acid by an endogenous polynucleotide phosphorylase and ribonuclease in cells of Brevibacterium JM98A (ATCC 29895) was studied. A simple process was developed for the activation of the endogenous RNA-degrading enzyme(s). RNA degradation was activated by the presence of Pi with 14.2 mumol of ribonucleoside 5'-monophosphate per g of cell mass accumulating extracellularly. The optimum pH for degradation of RNA was 10.5 and the optimum temperature was 55 to 60 degrees C. Enzymatic activity was inhibited by the presence of Ca2+, Zn2+, or Mg2+. Although some of the RNA-degrading enzymatic activity was associated with the ribosomal fraction, most was soluble. Both polynucleotide phosphorylase and ribonuclease activities were identified. PMID:39504

Mechanism of UCH-L5 Activation and Inhibition by DEUBAD Domains in RPN13 and INO80G

PubMed Central

Sahtoe, Danny D.; van Dijk, Willem J.; El Oualid, Farid; Ekkebus, Reggy; Ovaa, Huib; Sixma, Titia K.

2015-01-01

Summary Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in RPN13 activates UCH-L5 by positioning its C-terminal ULD domain and crossover loop to promote substrate binding and catalysis. The related DEUBAD domain in INO80G inhibits UCH-L5 by exploiting similar structural elements in UCH-L5 to promote a radically different conformation, and employs molecular mimicry to block ubiquitin docking. In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates. Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity. These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity. PMID:25702870

A critical switch in the enzymatic properties of the Cid1 protein deciphered from its product-bound crystal structure.

PubMed

Munoz-Tello, Paola; Gabus, Caroline; Thore, Stéphane

2014-03-01

The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1's processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo.

A critical switch in the enzymatic properties of the Cid1 protein deciphered from its product-bound crystal structure

PubMed Central

Munoz-Tello, Paola; Gabus, Caroline; Thore, Stéphane

2014-01-01

The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1's processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo. PMID:24322298

Cold active hydrolytic enzymes production by psychrotrophic Bacilli isolated from three sub-glacial lakes of NW Indian Himalayas.

PubMed

Yadav, Ajar Nath; Sachan, Shashwati Ghosh; Verma, Priyanka; Kaushik, Rajeev; Saxena, Anil Kumar

2016-03-01

The diversity of culturable, cold-active enzymes producing Bacilli was investigated from three sub-glacial lakes of north western Indian Himalayas. Amplified ribosomal DNA restriction analysis (ARDRA) using three restriction enzymes Alu I, Msp I, and Hae III led to the clustering of 136 Bacilli into 26, 23, and 22 clusters at 75% similarity index from Chandratal Lake, Dashair Lake, and Pangong Lake, respectively. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 35 Bacilli that could be grouped in seven families viz.: Bacillaceae (48%), Staphylococcaceae (14%), Bacillales incertae sedis (13%), Planococcaceae (12%), Paenibacillaceae (9%), Sporolactobacillaceae (3%), and Carnobacteriaceae (1%), which included twelve different genera Bacillus, Desemzia, Exiguobacterium, Jeotgalicoccus, Lysinibacillus, Paenibacillus, Planococcus, Pontibacillus, Sinobaca, Sporosarcina, Staphylococcus, and Virgibacillus. Based on their optimal temperature for growth, 35 Bacilli were grouped as psychrophilic (11 strains), psychrotrophic (17 strains), or psychrotolerant (7 strains), respectively. The representative isolates from each cluster were screened for cold-active enzyme activities. Amylase, β-glucosidase, pectinase, and protease activities at 4 °C were detected in more than 80% of the strains while approximately 40, 31, 23, 14, 11, and 9% of strains possessed cellulase, xylanase, β-galactosidase, laccase, chitinase, and lipase activity, respectively. Among 35 Bacilli, Bacillus amyloliquefaciens, Bacillus marisflavi, Exiguobacterium indicum, Paenibacillus terrae, Pontibacillus sp., Sporosarcina globispora, and Sporosarcina psychrophila were efficient producers of different cold-active enzymes. These cold-adapted Bacilli could play an important role in industrial and agricultural processes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biodegradation of composite resin with ester linkages: identifying human salivary enzyme activity with a potential role in the esterolytic process.

PubMed

Cai, Kuihua; Delaviz, Yasaman; Banh, Michael; Guo, Yi; Santerre, J Paul

2014-08-01

The ester linkages contained within dental resin monomers (such as Bisphenol A-glycidylmethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA)) are susceptible to hydrolytic degradation by salivary esterases, however very little is known about the specific esterase activities implicated in this process. The objective of this work was to isolate and identify the dominant proteins from saliva that are associated with the esterase activities shown to be involved in the degradation of BisGMA. Human whole saliva was collected and processed prior to separation in a HiPrep 16/60 Sephacryl S-200 HR column. The fraction with the highest esterase activity was further separated by an anion exchange column (Mono-Q (10/100G)). Isolated fractions were then separated by gel electrophoresis, and compared to a common bench marker esterase, cholesterol esterase (CE), and commercial albumin which has been reported to express esterase activity. Proteins suspected of containing esterase activity were analyzed by Mass Spectroscopy (MS). Commercially available proteins, similar to the salivary esterase proteins identified by MS, were used to replicate the enzymatic complexes and confirm their degradation activity with respect to BisGMA. MS data suggested that the enzyme fraction with the highest esterase activity was contained among a group of proteins consisting of albumin, Zn-α2-glycoprotein, α-amylase, TALDO1 protein, transferrin, lipocalin2, and prolactin-induced protein. Studies concluded that the main esterase bands on the gels in each fraction did not overlap with CE activity, and that albumin activity emerged as a lead candidate with significant esterase activity relative to BisGMA degradation, particularly when it formed a complex with Zn-α2-glycoprotein, under slightly basic conditions. These enzyme complexes can be used as a physiologically relevant formulation to test the biostability of composite resins. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Techno-economic analysis of the industrial production of a low-cost enzyme using E. coli: the case of recombinant β-glucosidase.

PubMed

Ferreira, Rafael da Gama; Azzoni, Adriano Rodrigues; Freitas, Sindelia

2018-01-01

The enzymatic conversion of lignocellulosic biomass into fermentable sugars is a promising approach for producing renewable fuels and chemicals. However, the cost and efficiency of the fungal enzyme cocktails that are normally employed in these processes remain a significant bottleneck. A potential route to increase hydrolysis yields and thereby reduce the hydrolysis costs would be to supplement the fungal enzymes with their lacking enzymatic activities, such as β-glucosidase. In this context, it is not clear from the literature whether recombinant E. coli could be a cost-effective platform for the production of some of these low-value enzymes, especially in the case of on-site production. Here, we present a conceptual design and techno-economic evaluation of the production of a low-cost industrial enzyme using recombinant E. coli . In a simulated baseline scenario for β-glucosidase demand in a hypothetical second-generation ethanol (2G) plant in Brazil, we found that the production cost (316 US$/kg) was higher than what is commonly assumed in the literature for fungal enzymes, owing especially to the facility-dependent costs (45%) and to consumables (23%) and raw materials (25%). Sensitivity analyses of process scale, inoculation volume, and volumetric productivity indicated that optimized conditions may promote a dramatic reduction in enzyme cost and also revealed the most relevant factors affecting production costs. Despite the considerable technical and economic uncertainties that surround 2G ethanol and the large-scale production of low-cost recombinant enzymes, this work sheds light on some relevant questions and supports future studies in this field. In particular, we conclude that process optimization, on many fronts, may strongly reduce the costs of E. coli recombinant enzymes, in the context of tailor-made enzymatic cocktails for 2G ethanol production.

Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions.

PubMed

Libra, Judy A; Borchert, Maren; Banit, Salima

2003-06-20

A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 736-744, 2003.

Evolution of a designed retro-aldolase leads to complete active site remodeling

PubMed Central

Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald

2013-01-01

Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. PMID:23748672

Synthesis and properties of immobilized pectinase onto the macroporous polyacrylamide microspheres.

PubMed

Lei, Zhongli; Jiang, Qin

2011-03-23

Pectinase was covalently immobilized onto the macroporous polyacrylamide (PAM) microspheres synthesized via an inverse suspension polymerization approach, resulting in 81.7% immobilization yield. The stability of the macroporous PAM support, which has a large surface area, is not impeded by the adsorbed proteins despite the fact that up to 296.3 mg of enzyme is immobilized per gram of the carrier particles. The immobilized enzyme retained more than 75% of its initial activity over 30 days, and the optimum temperature/pH also increased to the range of 50-60 °C/3.0-5.0. The immobilized enzyme also exhibited great operational stability, and more than 75% residual activity was observed after 10 batch reactions. The kinetics of a model reaction catalyzed by the immobilized pectinase was finally investigated. Moreover, the immobilized pectinase could be recovered by centrifuging and showed durable activity at the process of recycle.

Water at Biological Phase Boundaries: Its Role in Interfacial Activation of Enzymes and Metabolic Pathways.

PubMed

Damodaran, Srinivasan

2015-01-01

Many life-sustaining activities in living cells occur at the membrane-water interface. The pertinent questions that we need to ask are, what are the evolutionary reasons in biology for choosing the membrane-water interface as the site for performing and/or controlling crucial biological reactions, and what is the key physical principle that is very singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this chapter, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes through manipulation of the thermodynamic activity of water in the lipid-water interfacial region. The hypothesis is based on the fact that the surface pressure of a lipid monolayer is a direct measure of the thermodynamic activity of water at the lipid-water interface. Accordingly, the surface pressure-dependent activation or inactivation of interfacial enzymes is directly related to changes in the thermodynamic activity of interfacial water. Extension of this argument suggests that cells may manipulate conformations (and activities) of membrane-bound enzymes by manipulating the (re)activity of interfacial water at various locations in the membrane by localized compression or expansion of the interface. In this respect, cells may use the membrane-bound hormone receptors, lipid phase transition, and local variations in membrane lipid composition as effectors of local compression and/or expansion of membrane, and thereby local water activity. Several experimental data in the literature will be reexamined in the light of this hypothesis.

Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

NASA Technical Reports Server (NTRS)

Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

2000-01-01

Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

Thrombin-like enzymes from snake venom: Structural characterization and mechanism of action.

PubMed

Ullah, Anwar; Masood, Rehana; Ali, Ijaz; Ullah, Kifayat; Ali, Hamid; Akbar, Haji; Betzel, Christian

2018-07-15

Snake venom thrombin-like enzymes (SVTLEs) constitute the major portion (10-24%) of snake venom and these are the second most abundant enzymes present in the crude venom. During envenomation, these enzymes had shown prominently the various pathological effects, such as disturbance in hemostatic system, fibrinogenolysis, fibrinolysis, platelet aggregation, thrombosis, neurologic disorders, activation of coagulation factors, coagulant, procoagulant etc. These enzymes also been used as a therapeutic agent for the treatment of various diseases such as congestive heart failure, ischemic stroke, thrombotic disorders etc. Although the crystal structures of five SVTLEs are available in the Protein Data Bank (PDB), there is no single article present in the literature that has described all of them. The current work describes the structural aspects, structure-based mechanism of action, processing and inhibition of these enzymes. The sequence analysis indicates that these enzymes show a high sequence identity (57-85%) with each other and low sequence identity with trypsin (36-43%), human alpha-thrombin (29-36%) and other snake venom serine proteinases (57-85%). Three-dimensional structural analysis indicates that the loops surrounding the active site are variable both in amino acids composition and length that may convey variable substrate specificity to these enzymes. The surface charge distributions also vary in these enzymes. Docking analysis with suramin shows that this inhibitor preferably binds to the C-terminal region of these enzymes and causes the destabilization of their three-dimensional structure. Copyright © 2018 Elsevier B.V. All rights reserved.

Bioinspired catalytic materials for energy-relevant conversions

NASA Astrophysics Data System (ADS)

Artero, Vincent

2017-09-01

The structure of active sites of enzymes involved in bioenergetic processes can inspire design of active, stable and cost-effective catalysts for renewable-energy technologies. For these materials to reach maturity, the benefits of bioinspired systems must be combined with practical technological requirements.

Isolation and characterization of marine bacteria from macroalgae Gracilaria salicornia and Gelidium latifolium on agarolitic activity for bioethanol production

NASA Astrophysics Data System (ADS)

Kawaroe, M.; Pratiwi, I.; Sunudin, A.

2017-05-01

Gracilaria salicornia and Gelidium latifolium have high content of agar and potential to be use as raw material for bioethanol. In bioethanol production, one of the processes level is enzyme hydrolysis. Various microorganisms, one of which is bacteria, can carry out the enzyme hydrolysis. Bacteria that degrade the cell walls of macroalgae and produce an agarase enzyme called agarolytic bacteria. The purpose of this study was to isolate bacteria from macroalgae G. salicornia and G. latifolium, which has the highest agarase enzyme activities, and to obtain agarase enzyme characteristic for bioethanol production. There are two isolates bacteria resulted from G. salicornia that are N1 and N3 and there are two isolates from G. latifolium that are BSUC2 and BSUC4. The result of agarase enzyme qualitative test showed that isolates bacteria from G. latifolium were greater than G. salicornia. The highest agarolitic index of bacteria from G. salicornia produced by isolate N3 was 2.32 mm and isolate N3 was 2.27 mm. Bacteria from G. latifolium produced by isolate BSUC4 was 4.28 mm and isolate BSUC2 was 4.18 mm, respectively. Agarase enzyme activities from isolates N1 and N3 were optimum working at pH 7 and temperature 30 °C, while from isolates BSUC4 was optimum at pH 7 and temperature 50 °C. This is indicated that the four bacteria are appropriate to hydrolyze macro alga for bioethanol production.

Enhanced biocatalytic esterification with lipase-immobilized chitosan/graphene oxide beads.

PubMed

Lau, Siaw Cheng; Lim, Hong Ngee; Basri, Mahiran; Fard Masoumi, Hamid Reza; Ahmad Tajudin, Asilah; Huang, Nay Ming; Pandikumar, Alagarsamy; Chia, Chin Hua; Chia, Chi Hua; Andou, Yoshito

2014-01-01

In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the "insoluble" enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60 °C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions.

Enhanced Biocatalytic Esterification with Lipase-Immobilized Chitosan/Graphene Oxide Beads

PubMed Central

Lau, Siaw Cheng; Lim, Hong Ngee; Basri, Mahiran; Fard Masoumi, Hamid Reza; Ahmad Tajudin, Asilah; Huang, Nay Ming; Pandikumar, Alagarsamy; Chia, Chi Hua; Andou, Yoshito

2014-01-01

In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the “insoluble” enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60°C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions. PMID:25127038

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ionsmore » that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.« less

Spatial distribution of total phenolic content, enzymatic activities and browning in white yam (Dioscorea rotundata) tubers.

PubMed

Graham-Acquaah, Seth; Ayernor, George Sodah; Bediako-Amoa, Betty; Saalia, Firibu Kwesi; Afoakwa, Emmanuel Ohene

2014-10-01

Browning in raw and processed yams resulting from enzymes, polyphenol oxidase (PPO) and peroxidase (POD), activities is a major limitation to the industrial utilization of Dioscorea varieties of yams. Two elite cultivars of D. rotundata species were selected to study the spatial distribution of total phenols and enzymes (PPO and POD) activities. The intensities of tissue darkening in fresh yam chips prepared from the tuber sections of cultivars during frozen storage were also studied. Total phenolic content was observed to be highest in the head and mid sections of the cultivars than at the tail end. PPO activity did not have any specific distribution pattern whereas POD activity was found to be more concentrated in the head than in the middle and tail regions. Browning was found to be most intense in the head regions of the two cultivars studied; and was observed to correlate with total phenol and dry matter contents of tubers. Between the two enzymes, POD activity appeared to be more related to browning than PPO.

Super-fine rice-flour production by enzymatic treatment with high hydrostatic pressure processing

NASA Astrophysics Data System (ADS)

Kido, Miyuki; Kobayashi, Kaneto; Chino, Shuji; Nishiwaki, Toshikazu; Homma, Noriyuki; Hayashi, Mayumi; Yamamoto, Kazutaka; Shigematsu, Toru

2013-06-01

In response to the recent expansion of rice-flour use, we established a new rice-flour manufacturing process through the application of high hydrostatic pressure (HP) to the enzyme-treated milling method. HP improved both the activity of pectinase, which is used in the enzyme-treated milling method and the water absorption capacity of rice grains. These results indicate improved damage to the tissue structures of rice grains. In contrast, HP suppressed the increase in glucose, which may have led to less starch damage. The manufacturing process was optimized to HP treatment at 200 MPa (40°C) for 1 h and subsequent wet-pulverization at 11,000 rpm. Using this process, rice flour with an exclusively fine mean particle size less than 20 μm and starch damage less than 5% was obtained from rice grains soaked in an enzyme solution and distilled water. This super-fine rice flour is suitable for bread, pasta, noodles and Western-style sweets.

Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

PubMed Central

East-Seletsky, Alexandra; O’Connell, Mitchell R.; Knight, Spencer C.; Burstein, David; Cate, Jamie H. D.; Tjian, Robert; Doudna, Jennifer A.

2017-01-01

Bacterial adaptive immune systems employ CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage1,2. Although generally targeted to DNA substrates3–5, the Type III and Type VI CRISPR systems direct interference complexes against single-stranded RNA (ssRNA) substrates6–9. In Type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease9,10. How this enzyme acquires mature CRISPR RNAs (crRNAs) essential for immune surveillance and its mechanism of crRNA-mediated RNA cleavage remain unclear. Here we show that C2c2 possesses a unique ribonuclease activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated ssRNA-degradation activity. These dual ribonuclease functions are chemically and mechanistically different from each other and from the crRNA-processing behavior of the evolutionarily unrelated CRISPR enzyme Cpf111. We show that the two ribonuclease activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow for sensitive cellular transcript detection. PMID:27669025

Effects of the microbial secondary metabolite benzothiazole on the nutritional physiology and enzyme activities of Bradysia odoriphaga (Diptera: Sciaridae).

PubMed

Zhao, Yunhe; Xu, Chunmei; Wang, Qiuhong; Wei, Yan; Liu, Feng; Xu, Shuangyu; Zhang, Zhengqun; Mu, Wei

2016-05-01

Bradysia odoriphaga (Diptera: Sciaridae) is the major pest that damages Chinese chive production. As a volatile compound derived from microbial secondary metabolites, benzothiazole has been determined to possess fumigant activity against B. odoriphaga. However, the mechanism of action of benzothiazole is not well understood. In the present study, fourth-instar larvae of B. odoriphaga were exposed to LC10 and LC30 of benzothiazole. Sublethal concentrations (LC10 and LC30) of benzothiazole significantly reduced the food consumption of the larvae on the second day after treatment (2 DAT). However, there were no significant changes in pupal weight among the different treatments. We also measured the protein, lipid, carbohydrate, and trehalose contents and the digestive enzyme activities of the larvae, and the results suggest that benzothiazole reduced the nutrient accumulation and decreased the digestive enzyme activities of B. odoriphaga. In addition, the activity of glutathione S-transferase was significantly decreased at 6h after treatment with benzothiazole, whereas general esterase activities were significantly increased at 6 and 24h after treatment. The results of this study indicate that benzothiazole interferes in the normal food consumption and digestion process by decreasing the activities of digestive enzymes. These results provide valuable information for understanding the toxicity of benzothiazole and for exploring volatile compound for the control of this pest. Copyright © 2015. Published by Elsevier Inc.

[Changes in active cysteine cathepsins in lysosomes from tissues thyroid papillary carcinomas with various biological characteristics].

PubMed

Kalinichenko, O V; Myshunina, T M; Tron'ko, M D

2013-01-01

To clarify possible role of cysteine cathepsin H, B and L in the proteolytic processes that contribute to the progression of tumor growth in the thyroid, we studied their activity in lysosomes isolated from the tissue of papillary carcinomas. It was shown that for these enzymes there is a dependence of the changes in their activity on a number of biological characteristics of the tumors. Thus, the sharp increase in the activity ofcathepsin H observed in lysosomes of tissue carcinomas category T2 and T3, with intra-and ekstrathyroid and lymphatic invasion of tumor cells. An increase in the activity of cathepsin B is set in the lysosomes of tissue heterogeneous follicular structure, especially in the presence of solid areas, in comparison with typical papillary tumors and in the lysosomes of tissue carcinomas in intrathyroid and cathepsin L-at extrathyroid invasion. A common feature of the enzymes is to increase the activity of cathepsins in lysosomes of tissue nonencapsulated papillary carcinomas. These enzymes probably do not take part in the invasion of tumor cells into blood vessels and in the mechanisms of tumor metastasis to regional lymph nodes. The latter shows no changes in the activity of cathepsins in lysosomes of tissue carcinomas category N1. The results indicate the different role of cathepsin H, B and L in thyroid carcinogenesis, where each enzyme has its specific function.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

PubMed Central

2014-01-01

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme. PMID:24934472

Revealing a Novel Otubain-like Enzyme from Leishmania infantum with Deubiquitinating Activity toward K48-linked Substrate

NASA Astrophysics Data System (ADS)

Azevedo, Clênia S.; Guido, Bruna C.; Pereira, Jhonata L.; Nolasco, Diego O.; Corrêa, Rafael; Magalhães, Kelly G.; Motta, Flávia N.; Santana, Jaime M.; Grellier, Philippe; Bastos, Izabela M. D.

2017-03-01

Deubiquitinating enzymes (DUBs) play an important role in regulating a variety of eukaryotic processes. In this context, exploring the role of deubiquitination in Leishmania infantum could be a promising alternative to search new therapeutic targets for leishmaniasis. Here we present the first characterization of a DUB from L. infantum, otubain (OtuLi), and its localization within parasite. The recombinant OtuLi (rOtuLi) showed improved activity on lysine 48 (K48)-linked over K63-linked tetra-ubiquitin (Ub) and site-directed mutations on amino acids close to the catalytic site (F82) or involved in Ub interaction (L265 and F182) caused structural changes as shown by molecular dynamics, resulting in a reduction or loss of enzyme activity, respectively. Furthermore, rOtuLi stimulates lipid droplet biogenesis (an inflammatory marker) in peritoneal macrophages and induces IL-6 and TNF-α secretion in peritoneal macrophages, both proinflammatory cytokines. Our findings suggest that OtuLi is a cytoplasmic enzyme with K48-linked substrate specificity that could play a part in proinflammatory response in stimulated murine macrophages.

Effects of controlled gas environments in microbial enhancement of plant protein recovery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mudgett, R.E.; Bajracharya, R.

Controlled gas environments were maintained by a novel aeration system in solid substrate fermentations for enhanced protein recovery from pressed alfalfa residues. High O/sub 2/ pressures stimulated biomass and enzyme production by an Aspergillus species, isolated from alfalfa, which produced cellulase and pectinase activities in growth-associated metabolism. High CO/sub 2/ pressures also stimulated enzyme production, but had less effect on biomass production, as established from the dissimilation of plant solids. Cellulase and pectinase activities were generally related to protein recoveries. Recoveries were greater than or equal to 50% higher than those obtained by mechanical extraction, with maximum recoveries of greatermore » than or equal to 70% of crude protein contents. Protein not recovered at high cellulase and pectinase activities were believed to be in structurally bound forms not amenable to recovery by nonproteolytic enzymes. Buffering at pH 8 and autoclaving of residues prior to fermentation had little effect on protein recoveries. Controlled gas environments are seen to offer an interesting potential for optimizing industrial fermentation processes for the production of microbial enzymes.« less

Seasonal variations of anti-/apoptotic and antioxidant proteins in the heart and gastrocnemius muscle of the water frog Pelophylax ridibundus.

PubMed

Feidantsis, Konstantinos; Anestis, Andreas; Michaelidis, Basile

2013-10-01

In the present work we investigated the seasonal variations of apoptotic and antioxidant proteins in the heart and gastrocnemius muscle of the amphibian Pelophylax ridibundus. Particularly processes studied included the evaluation of hypoxia through the levels of transcriptional factor Hif-1α, of apoptosis through the determination of Bcl-2 and Bax, ubiquitin conjugates levels and the antioxidant defense through the determination of the activity of enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Due to a general metabolic depression during overwintering, levels of the above mentioned proteins and enzymes are generally retained at low levels of expression and activity in the examined tissues of P. ridibundus. On the other hand recovery from overwintering induces oxidative stress, followed by increased levels of the specific proteins and enzymes. A milder up-regulation of antioxidant enzymes during overwintering probably prepares P. ridibundus for oxidative stress during arousal. The seasonal activation of these mechanisms seems to protect this species from these unfavourable conditions. Copyright © 2013 Elsevier Inc. All rights reserved.

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass

PubMed Central

2010-01-01

Background To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. Results We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Conclusions Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously process 120 triplicate wheat-straw samples per day. This throughput can be doubled if the incubation time is reduced from 24 h to 4 h (for initial rates measurements, for instance). This method can potentially be used with any insoluble substrate that is micronizable. A video illustrating the method can be seen at the following URL: http://www.youtube.com/watch?v=NFg6TxjuMWU PMID:20637080

Enzymes- An Existing and Promising Tool of Food Processing Industry.

PubMed

Ray, Lalitagauri; Pramanik, Sunita; Bera, Debabrata

2016-01-01

The enzyme catalyzed process technology has enormous potential in the food sectors as indicated by the recent patents studies. It is very well realized that the adaptation of the enzyme catalyzed process depends on the availability of enzyme in affordable prices. Enzymes may be used in different food sectors like dairy, fruits & vegetable processing, meat tenderization, fish processing, brewery and wine making, starch processing and many other. Commercially only a small number of enzymes are used because of several factors including instability of enzymes during processing and high cost. More and more enzymes for food technology are now derived from specially selected or genetically modified microorganisms grown in industrial scale fermenters. Enzymes with microbial source have commercial advantages of using microbial fermentation rather than animal and plant extraction to produce food enzymes. At present only a relatively small number of enzymes are used commercially in food processing. But the number is increasing day by day and field of application will be expanded more and more in near future. The purpose of this review is to describe the practical applications of enzymes in the field of food processing.

The Ontogeny of Cytochrome P450 Enzyme Activity and Protein Abundance in Conventional Pigs in Support of Preclinical Pediatric Drug Research.

PubMed

Millecam, Joske; De Clerck, Laura; Govaert, Elisabeth; Devreese, Mathias; Gasthuys, Elke; Schelstraete, Wim; Deforce, Dieter; De Bock, Lies; Van Bocxlaer, Jan; Sys, Stanislas; Croubels, Siska

2018-01-01

Since the implementation of several legislations to improve pediatric drug research, more pediatric clinical trials are being performed. In order to optimize these pediatric trials, adequate preclinical data are necessary, which are usually obtained by juvenile animal models. The growing piglet has been increasingly suggested as a potential animal model due to a high degree of anatomical and physiological similarities with humans. However, physiological data in pigs on the ontogeny of major organs involved in absorption, distribution, metabolism, and excretion of drugs are largely lacking. The aim of this study was to unravel the ontogeny of porcine hepatic drug metabolizing cytochrome P450 enzyme (CYP450) activities as well as protein abundances. Liver microsomes from 16 conventional pigs (8 males and 8 females) per age group: 2 days, 4 weeks, 8 weeks, and 6-7 months were prepared. Activity measurements were performed with substrates of major human CYP450 enzymes: midazolam (CYP3A), tolbutamide (CYP2C), and chlorzoxazone (CYP2E). Next, the hepatic scaling factor, microsomal protein per gram liver (MPPGL), was determined to correct for enzyme losses during the fractionation process. Finally, protein abundance was determined using proteomics and correlated with enzyme activity. No significant sex differences within each age category were observed in enzyme activity or MPPGL. The biotransformation rate of all three substrates increased with age, comparable with human maturation of CYP450 enzymes. The MPPGL decreased from birth till 8 weeks of age followed by an increase till 6-7 months of age. Significant sex differences in protein abundance were observed for CYP1A2, CYP2A19, CYP3A22, CYP4V2, CYP2C36, CYP2E_1, and CYP2E_2. Midazolam and tolbutamide are considered good substrates to evaluate porcine CYP3A/2C enzymes, respectively. However, chlorzoxazone is not advised to evaluate porcine CYP2E enzyme activity. The increase in biotransformation rate with age can be attributed to an increase in absolute amount of CYP450 proteins. Finally, developmental changes were observed regarding the involvement of specific CYP450 enzymes in the biotransformation of the different substrates.