Deoxynucleoside salvage enzymes and tissue specific mitochondrial DNA depletion.

PubMed

Wang, L

2010-06-01

Adequate mitochondrial DNA (mtDNA) copies are required for normal mitochondria function and reductions in mtDNA copy number due to genetic alterations cause tissue-specific mtDNA depletion syndrome (MDS). There are eight nuclear genes, directly or indirectly involved in mtDNA replication and mtDNA precursor synthesis, which have been identified as the cause of MDS. However, the tissue specific pathology of these nuclear gene mutations is not well understood. Here, mtDNA synthesis, mtDNA copy number control, and mtDNA turnover, as well as the synthesis of mtDNA precursors in relation to the levels of salvage enzymes are discussed. The question why MDS caused by TK2 and p53R2 mutations are predominantly muscle specific while dGK deficiency affected mainly liver will be addressed.

Enzyme-specific sensors via aggregation of charged p-phenylene ethynylenes.

PubMed

Hill, Eric H; Zhang, Yue; Evans, Deborah G; Whitten, David G

2015-03-11

Chemical and biological sensors are sought for their ability to detect enzymes as biomarkers for symptoms of various disorders, or the presence of chemical pollutants or poisons. p-Phenylene ethynylene oligomers with pendant charged groups have been recently shown to have ideal photophysical properties for sensing. In this study, one anionic and one cationic oligomer are combined with substrates that are susceptible to enzymatic degradation by phospholipases or acetylcholinesterases. The photophysical properties of the J-aggregated oligomers with the substrate are ideal for sensing, with fluorescence quantum yields of the sensors enhanced between 30 and 66 times compared to the oligomers without substrate. The phospholipase sensor was used to monitor the activity of phospholipase A1 and A2 and obtain kinetic information, though phospholipase C did not degrade the sensor. The acetylcholinesterase sensor was used to monitor enzyme activity and was also used to detect the inhibition of acetylcholinesterase by three different inhibitors. Phospholipase A2 is a biomarker for heart and circulatory disease, and acetylcholinesterase is a biomarker for Alzheimer's, and indicative of exposure to certain pesticides and nerve agents. This work shows that phenylene ethynylene oligomers can be tailored to enzyme-specific sensors by careful selection of substrates that induce formation of a molecular aggregate, and that the sensing of enzymes can be extended to enzyme kinetics and detection of inhibition. Furthermore, the aggregates were studied through all-atom molecular dynamics, providing a molecular-level view of the formation of the molecular aggregates and their structure.

KAS IV: a 3-ketoacyl-ACP synthase from Cuphea sp. is a medium chain specific condensing enzyme.

PubMed

Dehesh, K; Edwards, P; Fillatti, J; Slabaugh, M; Byrne, J

1998-08-01

cDNA clones encoding a novel 3-ketoacyl-ACP synthase (KAS) have been isolated from Cuphea. The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic Brassica plants over-expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic Brassica seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co-expression of the Cuphea KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co-expression of the Cuphea KAS along with an 18:0/18.1-ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the Cuphea KAS reported here has a different acyl-chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.

Efficient, environmentally-friendly and specific valorization of lignin: promising role of non-radical lignolytic enzymes.

PubMed

Wang, Wenya; Zhang, Chao; Sun, Xinxiao; Su, Sisi; Li, Qiang; Linhardt, Robert J

2017-06-01

Lignin is the second most abundant bio-resource in nature. It is increasingly important to convert lignin into high value-added chemicals to accelerate the development of the lignocellulose biorefinery. Over the past several decades, physical and chemical methods have been widely explored to degrade lignin and convert it into valuable chemicals. Unfortunately, these developments have lagged because of several difficulties, of which high energy consumption and non-specific cleavage of chemical bonds in lignin remain the greatest challenges. A large number of enzymes have been discovered for lignin degradation and these are classified as radical lignolytic enzymes and non-radical lignolytic enzymes. Radical lignolytic enzymes, including laccases, lignin peroxidases, manganese peroxidases and versatile peroxidases, are radical-based bio-catalysts, which degrade lignins through non-specific cleavage of chemical bonds but can also catalyze the radical-based re-polymerization of lignin fragments. In contrast, non-radical lignolytic enzymes selectively cleave chemical bonds in lignin and lignin model compounds and, thus, show promise for use in the preparation of high value-added chemicals. In this mini-review, recent developments on non-radical lignolytic enzymes are discussed. These include recently discovered non-radical lignolytic enzymes, their metabolic pathways for lignin conversion, their recent application in the lignin biorefinery, and the combination of bio-catalysts with physical/chemical methods for industrial development of the lignin refinery.

Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation.

PubMed

Savary, B J

2001-08-01

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.

A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major

PubMed Central

Catharina, Larissa; Lima, Carlyle Ribeiro; Franca, Alexander; Guimarães, Ana Carolina Ramos; Alves-Ferreira, Marcelo; Tuffery, Pierre; Derreumaux, Philippe; Carels, Nicolas

2017-01-01

We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens, only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens. Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development. PMID:28638238

A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major.

PubMed

Catharina, Larissa; Lima, Carlyle Ribeiro; Franca, Alexander; Guimarães, Ana Carolina Ramos; Alves-Ferreira, Marcelo; Tuffery, Pierre; Derreumaux, Philippe; Carels, Nicolas

2017-01-01

We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens , only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens . Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development.

Enzyme

MedlinePlus

Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

SigrafW: An easy-to-use program for fitting enzyme kinetic data.

PubMed

Leone, Francisco Assis; Baranauskas, José Augusto; Furriel, Rosa Prazeres Melo; Borin, Ivana Aparecida

2005-11-01

SigrafW is Windows-compatible software developed using the Microsoft® Visual Basic Studio program that uses the simplified Hill equation for fitting kinetic data from allosteric and Michaelian enzymes. SigrafW uses a modified Fibonacci search to calculate maximal velocity (V), the Hill coefficient (n), and the enzyme-substrate apparent dissociation constant (K). The estimation of V, K, and the sum of the squares of residuals is performed using a Wilkinson nonlinear regression at any Hill coefficient (n). In contrast to many currently available kinetic analysis programs, SigrafW shows several advantages for the determination of kinetic parameters of both hyperbolic and nonhyperbolic saturation curves. No initial estimates of the kinetic parameters are required, a measure of the goodness-of-the-fit for each calculation performed is provided, the nonlinear regression used for calculations eliminates the statistical bias inherent in linear transformations, and the software can be used for enzyme kinetic simulations either for educational or research purposes. Persons interested in receiving a free copy of the software should contact Dr. F. A. Leone. Copyright © 2005 International Union of Biochemistry and Molecular Biology, Inc.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

PubMed

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Evolutionary dynamics of enzymes.

PubMed

Demetrius, L

1995-08-01

This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion-controlled rates and absolute

Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

USDA-ARS?s Scientific Manuscript database

Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

α-Amylase: an enzyme specificity found in various families of glycoside hydrolases.

PubMed

Janeček, Štefan; Svensson, Birte; MacGregor, E Ann

2014-04-01

α-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4-7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α)8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α)7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.

Phospholipid substrate-specificity of the L-serine base-exchange enzyme in rat liver microsomal fraction.

PubMed Central

Bjerve, K S

1984-01-01

The specificity of the L-serine base-exchange enzyme towards the fatty acid composition of the phospholipid substrate was investigated with a rat liver microsomal fraction. The relative rates of L-serine incorporation into saturated-hexaenoic, saturated-pentaenoic, saturated-tetraenoic, saturated-trienoic, dienoic-dienoic, monoenoic-dienoic, saturated-dienoic and saturated-monoenoic + saturated-saturated phosphatidylserine molecular species were 42, 5, 23, 4, 5, 4, 5 and 11% respectively. This is similar to, but not identical with, the relative mass abundance of these molecular species in total liver cell phosphatidylserines. The results indicate that the substrate-specificity of the L-serine base-exchange enzyme can at least in part explain the observed fatty acid composition of rat liver phosphatidylserines. PMID:6430274

Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

PubMed

Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

2009-12-11

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

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

Moving college students to a better understanding of substrate specificity of enzymes through utilizing multimedia pre-training and an interactive enzyme model

NASA Astrophysics Data System (ADS)

Saleh, Mounir R.

Scientists' progress in understanding enzyme specificity uncovered a complex natural phenomenon. However, not all of the currently available biology textbooks seem to be up to date on this progress. Students' understanding of how enzymes work is a core requirement in biochemistry and biology tertiary education. Nevertheless, current pre-college science education does not provide students with enough biochemical background to enable them to understand complex material such as this. To bridge this gap, a multimedia pre-training presentation was prepared to fuel the learner's prior knowledge with discrete facts necessary to understand the presented concept. This treatment is also known to manage intrinsic cognitive load during the learning process. An interactive instructional enzyme model was also built to motivate students to learn about substrate specificity of enzymes. Upon testing the effect of this combined treatment on 111 college students, desirable learning outcomes were found in terms of cognitive load, motivation, and achievement. The multimedia pre-training group reported significantly less intrinsic cognitive load, higher motivation, and demonstrated higher transfer performance than the control and post-training groups. In this study, a statistical mediation model is also proposed to explain how cognitive load and motivation work in concert to foster learning from multimedia pre-training. This type of research goes beyond simple forms of "what works" to a deeper understanding of "how it works", thus enabling informed decisions for multimedia instructional design. Multimedia learning plays multiple roles in science education. Therefore, science learners would be some of the first to benefit from improving multimedia instructional design. Accordingly, complex scientific phenomena can be introduced to college students in a motivating, informative, and cognitively efficient learning environment.

Vacuolar processing enzyme in plant programmed cell death

PubMed Central

Hatsugai, Noriyuki; Yamada, Kenji; Goto-Yamada, Shino; Hara-Nishimura, Ikuko

2015-01-01

Vacuolar processing enzyme (VPE) is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an ortholog of animal asparaginyl endopeptidase (AEP/VPE/legumain). VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD) pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1. PMID:25914711

Reliability of programs specified with equational specifications

NASA Astrophysics Data System (ADS)

Nikolik, Borislav

Ultrareliability is desirable (and sometimes a demand of regulatory authorities) for safety-critical applications, such as commercial flight-control programs, medical applications, nuclear reactor control programs, etc. A method is proposed, called the Term Redundancy Method (TRM), for obtaining ultrareliable programs through specification-based testing. Current specification-based testing schemes need a prohibitively large number of testcases for estimating ultrareliability. They assume availability of an accurate program-usage distribution prior to testing, and they assume the availability of a test oracle. It is shown how to obtain ultrareliable programs (probability of failure near zero) with a practical number of testcases, without accurate usage distribution, and without a test oracle. TRM applies to the class of decision Abstract Data Type (ADT) programs specified with unconditional equational specifications. TRM is restricted to programs that do not exceed certain efficiency constraints in generating testcases. The effectiveness of TRM in failure detection and recovery is demonstrated on formulas from the aircraft collision avoidance system TCAS.

Development of a genetically programed vanillin-sensing bacterium for high-throughput screening of lignin-degrading enzyme libraries.

PubMed

Sana, Barindra; Chia, Kuan Hui Burton; Raghavan, Sarada S; Ramalingam, Balamurugan; Nagarajan, Niranjan; Seayad, Jayasree; Ghadessy, Farid J

2017-01-01

Lignin is a potential biorefinery feedstock for the production of value-added chemicals including vanillin. A huge amount of lignin is produced as a by-product of the paper industry, while cellulosic components of plant biomass are utilized for the production of paper pulp. In spite of vast potential, lignin remains the least exploited component of plant biomass due to its extremely complex and heterogenous structure. Several enzymes have been reported to have lignin-degrading properties and could be potentially used in lignin biorefining if their catalytic properties could be improved by enzyme engineering. The much needed improvement of lignin-degrading enzymes by high-throughput selection techniques such as directed evolution is currently limited, as robust methods for detecting the conversion of lignin to desired small molecules are not available. We identified a vanillin-inducible promoter by RNAseq analysis of Escherichia coli cells treated with a sublethal dose of vanillin and developed a genetically programmed vanillin-sensing cell by placing the 'very green fluorescent protein' gene under the control of this promoter. Fluorescence of the biosensing cell is enhanced significantly when grown in the presence of vanillin and is readily visualized by fluorescence microscopy. The use of fluorescence-activated cell sorting analysis further enhances the sensitivity, enabling dose-dependent detection of as low as 200 µM vanillin. The biosensor is highly specific to vanillin and no major response is elicited by the presence of lignin, lignin model compound, DMSO, vanillin analogues or non-specific toxic chemicals. We developed an engineered E. coli cell that can detect vanillin at a concentration as low as 200 µM. The vanillin-sensing cell did not show cross-reactivity towards lignin or major lignin degradation products including vanillin analogues. This engineered E. coli cell could potentially be used as a host cell for screening lignin-degrading enzymes that

Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

PubMed

Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

2015-01-01

The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs.

Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme.

PubMed

Cerminati, Sebastián; Eberhardt, Florencia; Elena, Claudia E; Peirú, Salvador; Castelli, María E; Menzella, Hugo G

2017-06-01

Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14 g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

DARPA Internet Program. Internet and Transmission Control Specifications,

DTIC Science & Technology

1981-09-01

Internet Program Protocol Specification", RFC 791, USC/ Information Sciences Institute, September 1981. [34] Postel, J., ed., "Transmission Control Protocol...DARPA Internet Program Protocol Specification", RFC 793, USC/ Information Sciences Institute, September 1981. [35] Postel, J., "Echo Process", RFC 347...Newman, March 1981. [53] Postel, J., " Internet Control Message Protocol - DARPA Internet Program Protocol Specification", RFC 792, USC/ Information

Specifications for Developing Secondary Program Proposals.

ERIC Educational Resources Information Center

Maryland State Dept. of Education, Baltimore. Div. of Career Technology and Adult Learning.

This document, which is designed for individuals developing local career and technology education (CTE) program proposals and/or designing new CTE facilities in Maryland, presents the CTE program specifications that were developed to ensure the quality of CTE programs in public schools in Maryland. The document is divided into four sections. The…

Evolution of substrate specificity in a retained enzyme driven by gene loss

DOE PAGES

Juárez-Vázquez, Ana Lilia; Edirisinghe, Janaka N.; Verduzco-Castro, Ernesto A.; ...

2017-03-31

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence oftrpandhisgenes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficientmore » form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Finally, our results show how gene loss can drive the evolution of substrate specificity from retained enzymes.« less

Evolution of Substrate Specificity in A Retained Enzyme Driven by Gene Loss

DOE PAGES

Juarez-Vazquez, Ana L.; Edirisinghe, Janaka N.; Verduzco-Castro, Ernesto A.; ...

2017-03-31

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. Here, we apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We also observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to amore » monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. These results show how gene loss can drive the evolution of substrate specificity from retained enzymes.« less

Evolution of substrate specificity in a retained enzyme driven by gene loss

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

Juárez-Vázquez, Ana Lilia; Edirisinghe, Janaka N.; Verduzco-Castro, Ernesto A.

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence oftrpandhisgenes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficientmore » form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Finally, our results show how gene loss can drive the evolution of substrate specificity from retained enzymes.« less

Evolution of Substrate Specificity in A Retained Enzyme Driven by Gene Loss

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

Juarez-Vazquez, Ana L.; Edirisinghe, Janaka N.; Verduzco-Castro, Ernesto A.

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. Here, we apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We also observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to amore » monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. These results show how gene loss can drive the evolution of substrate specificity from retained enzymes.« less

Enzyme cascades activated on topologically programmed DNA scaffolds

NASA Astrophysics Data System (ADS)

Wilner, Ofer I.; Weizmann, Yossi; Gill, Ron; Lioubashevski, Oleg; Freeman, Ronit; Willner, Itamar

2009-04-01

The ability of DNA to self-assemble into one-, two- and three-dimensional nanostructures, combined with the precision that is now possible when positioning nanoparticles or proteins on DNA scaffolds, provide a promising approach for the self-organization of composite nanostructures. Predicting and controlling the functions that emerge in self-organized biomolecular nanostructures is a major challenge in systems biology, and although a number of innovative examples have been reported, the emergent properties of systems in which enzymes are coupled together have not been fully explored. Here, we report the self-assembly of a DNA scaffold made of DNA strips that include `hinges' to which biomolecules can be tethered. We attach either two enzymes or a cofactor-enzyme pair to the scaffold, and show that enzyme cascades or cofactor-mediated biocatalysis can proceed effectively; similar processes are not observed in diffusion-controlled homogeneous mixtures of the same components. Furthermore, because the relative position of the two enzymes or the cofactor-enzyme pair is determined by the topology of the DNA scaffold, it is possible to control the reactivity of the system through the design of the individual DNA strips. This method could lead to the self-organization of complex multi-enzyme cascades.

Amplification of the enzyme-linked immunosorbent assay (ELISA) in the detection of class-specific antibodies.

PubMed

Butler, J E; McGivern, P L; Swanson, P

1978-01-01

A modification of the standard enzyme-linked immunosorbent assay (ELISA) is described which circumvents the requirement for specifically purified antibodies from which antibody-enzyme complexes are made. The assay utilizes the principle of a soluble anti-alkaline phosphatase immune complex (AP-A-AP) and has been called the amplified ELISA. Methods for preparing and evidence for the specificity of rabbit anti-rat gamma-FC, IgM (mu) and IgA (alpha) are presented. These reagents are used to measure anti-DNP antibodies belonging to classes IgG, IgM and IgA in rat serum. Using antiglobulin and anti-enzyme reagents prepared in guinea pigs, anti-ovalbumin antibodies are measured in rabbit serum. Titration curves are similar when the amplified ELISA is compared to the standard ELISA. A change in slope suggesting an effect of saturation of antigen sites, occurs at the same input antibody concentration for both assays. Determination of the anti-DNP concentration of unknown sera by extrapopulation from titration graphs of a known serum suggests that the value is overestimated, i.e., amplified when the amplified ELISA is used. In addition, the amplified ELISA has an improved ability to detect low levels of antibody. Evidence is presented which illustrates how the use of optimally conjugated DNP-proteins, age of conjugates, and optimal dilutions of secondary antiglobulins and the AP-A-AP reduce non-specific binding in the amplified ELISA. The amplified ELISA is capable of detecting 2.4 ng of antibody to ovalbumin in a one: one million dilution of rabbit serum with high reproducibility and low background.

Enzyme Informatics

PubMed Central

Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

2012-01-01

Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

PubMed Central

Zhao, Hua

2015-01-01

There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule. PMID:26949281

Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

PubMed

Haraya, Shiomi; Karasawa, Koji; Sano, Yoshihiro; Ozawa, Kimiko; Kato, Nobumasa; Arakawa, Hidetoshi

2017-01-01

Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

General and program-specific moderators of two eating disorder prevention programs.

PubMed

Stice, Eric; Marti, Nathan; Shaw, Heather; O'Neil, Kelly

2008-11-01

To investigate general and program-specific factors hypothesized to moderate the effects of two eating disorder prevention programs. High-risk adolescent girls (N = 481; M age = 17) were randomized to a dissonance-based thin-ideal internalization reduction program, a healthy weight management program, an expressive-writing control condition, or an assessment-only control condition. Participants completed diagnostic interviews and surveys at pretest, post-test, 6-month follow-up, and 12-month follow-up. Dissonance program effects on bulimic symptoms were stronger for participants with initial elevations in body image distress, bulimic symptoms, and thin-ideal internalization. Healthy weight program effects on bulimic symptoms were stronger for adolescents with initial elevations in body image distress, bulimic symptoms, readiness to change, body mass, and emotional eating. Overall, intervention effects tended to be amplified for high-risk versus low-risk adolescents. However, certain moderator effects appeared to be specific to the two different prevention programs.

A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1.

PubMed

Stewart, James J P

2016-11-01

A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol -1 to ±0.1 kcal mol -1 . This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates.

Enzyme Prodrug Therapy Achieves Site-Specific, Personalized Physiological Responses to the Locally Produced Nitric Oxide

PubMed Central

2018-01-01

Nitric oxide (NO) is a highly potent but short-lived endogenous radical with a wide spectrum of physiological activities. In this work, we developed an enzymatic approach to the site-specific synthesis of NO mediated by biocatalytic surface coatings. Multilayered polyelectrolyte films were optimized as host compartments for the immobilized β-galactosidase (β-Gal) enzyme through a screen of eight polycations and eight polyanions. The lead composition was used to achieve localized production of NO through the addition of β-Gal–NONOate, a prodrug that releases NO following enzymatic bioconversion. The resulting coatings afforded physiologically relevant flux of NO matching that of the healthy human endothelium. The antiproliferative effect due to the synthesized NO in cell culture was site-specific: within a multiwell dish with freely shared media and nutrients, a 10-fold inhibition of cell growth was achieved on top of the biocatalytic coatings compared to the immediately adjacent enzyme-free microwells. The physiological effect of NO produced via the enzyme prodrug therapy was validated ex vivo in isolated arteries through the measurement of vasodilation. Biocatalytic coatings were deposited on wires produced using alloys used in clinical practice and successfully mediated a NONOate concentration-dependent vasodilation in the small arteries of rats. The results of this study present an exciting opportunity to manufacture implantable biomaterials with physiological responses controlled to the desired level for personalized treatment. PMID:29570264

Mannitol and the Mannitol-Specific Enzyme IIB Subunit Activate Vibrio cholerae Biofilm Formation

PubMed Central

Ymele-Leki, Patrick; Houot, Laetitia

2013-01-01

Vibrio cholerae is a halophilic, Gram-negative rod found in marine environments. Strains that produce cholera toxin cause the diarrheal disease cholera. V. cholerae use a highly conserved, multicomponent signal transduction cascade known as the phosphoenolpyruvate phosphotransferase system (PTS) to regulate carbohydrate uptake and biofilm formation. Regulation of biofilm formation by the PTS is complex, involving many different regulatory pathways that incorporate distinct PTS components. The PTS consists of the general components enzyme I (EI) and histidine protein (HPr) and carbohydrate-specific enzymes II. Mannitol transport by V. cholerae requires the mannitol-specific EII (EIIMtl), which is expressed only in the presence of mannitol. Here we show that mannitol activates V. cholerae biofilm formation and transcription of the vps biofilm matrix exopolysaccharide synthesis genes. This regulation is dependent on mannitol transport. However, we show that, in the absence of mannitol, ectopic expression of the B subunit of EIIMtl is sufficient to activate biofilm accumulation. Mannitol, a common compatible solute and osmoprotectant of marine organisms, is a main photosynthetic product of many algae and is secreted by algal mats. We propose that the ability of V. cholerae to respond to environmental mannitol by forming a biofilm may play an important role in habitat selection. PMID:23728818

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

NASA Astrophysics Data System (ADS)

Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

2016-05-01

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

Evolution of substrate specificity in a retained enzyme driven by gene loss

PubMed Central

Juárez-Vázquez, Ana Lilia; Edirisinghe, Janaka N; Verduzco-Castro, Ernesto A; Michalska, Karolina; Wu, Chenggang; Noda-García, Lianet; Babnigg, Gyorgy; Endres, Michael; Medina-Ruíz, Sofía; Santoyo-Flores, Julián; Carrillo-Tripp, Mauricio; Ton-That, Hung; Joachimiak, Andrzej; Henry, Christopher S; Barona-Gómez, Francisco

2017-01-01

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Our results show how gene loss can drive the evolution of substrate specificity from retained enzymes. DOI: http://dx.doi.org/10.7554/eLife.22679.001 PMID:28362260

Toward reducing immunogenicity of enzyme replacement therapy: altering the specificity of human β-glucuronidase to compensate for α-iduronidase deficiency.

PubMed

Chuang, Huai-Yao; Suen, Ching-Shu; Hwang, Ming-Jing; Roffler, Steve R

2015-11-01

Enzyme replacement therapy (ERT) is an effective treatment for many patients with lysosomal storage disorders caused by deficiency in enzymes involved in cell metabolism. However, immune responses that develop against the administered enzyme in some patients can hinder therapeutic efficacy and cause serious side effects. Here we investigated the feasibility of a general approach to decrease ERT immunogenicity by altering the specificity of a normal endogenous enzyme to compensate for a defective enzyme. We sought to identify human β-glucuronidase variants that display α-iduronidase activity, which is defective in mucopolysaccharidosis (MPS) type I patients. A human β-glucuronidase library was screened by the Enzyme Cleavable Surface-Tethered All-purpose Screen sYstem to isolate variants that exhibited 100-290-fold greater activity against the α-iduronidase substrate 4-methylumbelliferyl α-l-iduronide and 7900-24 500-fold enzymatic specificity shift when compared with wild-type β-glucuronidase. In vitro treatment of MPS I cells with the β-glucuronidase variants significantly restored lysosome appearance similar to treatment with α-iduronidase. Our study suggests that β-glucuronidase variants can be isolated to display α-iduronidase activity and produce significant phenotype improvement of MPS I cells. This strategy may represent a possible approach to produce enzymes that display therapeutic benefits with potentially less immunogenicity. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

14 CFR 91.1017 - Amending program manager's management specifications.

Code of Federal Regulations, 2014 CFR

2014-01-01

... proposed amendment. (2) The Flight Standards District Office that issued the program manager's management... presented, the Flight Standards District Office that issued the program manager's management specifications... Standards District Office that issued the program manager's management specifications issues an amendment of...

14 CFR 91.1017 - Amending program manager's management specifications.

Code of Federal Regulations, 2012 CFR

2012-01-01

... proposed amendment. (2) The Flight Standards District Office that issued the program manager's management... presented, the Flight Standards District Office that issued the program manager's management specifications... Standards District Office that issued the program manager's management specifications issues an amendment of...

Enzyme Sorption onto Soil and Biocarbon Amendments Alters Catalytic Capacity and Depends on the Specific Protein and pH

NASA Astrophysics Data System (ADS)

Foster, E.; Fogle, E. J.; Cotrufo, M. F.

2017-12-01

Enzymes catalyze biogeochemical reactions in soils and play a key role in nutrient cycling in agricultural systems. Often, to increase soil nutrients, agricultural managers add organic amendments and have recently experimented with charcoal-like biocarbon products. These amendments can enhance soil water and nutrient holding capacity through increasing porosity. However, the large surface area of the biocarbon has the potential to sorb nutrients and other organic molecules. Does the biocarbon decrease nutrient cycling through sorption of enzymes? In a laboratory setting, we compared the interaction of two purified enzymes β-glucosidase and acid phosphatase with a sandy clay loam and two biocarbons. We quantified the sorbed enzymes at three different pHs using a Bradford protein assay and then measured the activity of the sorbed enzyme via high-throughput fluorometric analysis. Both sorption and activity depended upon the solid phase, pH, and specific enzyme. Overall the high surface area biocarbon impacted the catalytic capacity of the enzymes more than the loam soil, which may have implications for soil nutrient management with these organic amendments.

[Human drug metabolizing enzymes. II. Conjugation enzymes].

PubMed

Vereczkey, L; Jemnitz, K; Gregus, Z

1998-09-01

In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

Targeted enzyme prodrug therapies.

PubMed

Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

2010-09-01

The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

A Measure of the Broad Substrate Specificity of Enzymes Based on ‘Duplicate’ Catalytic Residues

PubMed Central

Chakraborty, Sandeep; Ásgeirsson, Bjarni; Rao, Basuthkar J.

2012-01-01

The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing ‘duplicate’ residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. ‘Duplicate’ residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins. PMID:23166637

Space Research and Technology Program: Program and specific objectives, document approval

NASA Technical Reports Server (NTRS)

1982-01-01

A detailed view of the Space Research and Technology program work breakdown structure is provided down to the specific objective level. Goals or objectives at each of these levels are set forth. The specific objective narratives are structured into several parts. First, a short paragraph statement of the specific objective is given. This is followed by a list of subobjectives. A list of targets is then provided for those areas of the specific objective that are amenable to a quantitative description of technical accomplishment and schedule. Fluid and thermal physics, materials and structures, computer science and electronics, space energy conversion, multidisciplinary research, controls and human factors, chemical propulsion, spacecraft systems, transportation systems, platform systems, and spacecraft systems technology comprise the principal research programs.

A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. Enzymes catalyzing aldehyde oxidation in an adult moth

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

Tasayco, M.L.; Prestwich, G.D.

1990-02-25

Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor ofmore » this enzyme. This labeled vinyl ketone, (3H)(Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes.« less

Enzyme-linked immunosorbent assays for determination of plasma aldosterone using highly specific polyclonal antibodies.

PubMed

Schwartz, F; Hadas, E; Harnik, M; Solomon, B

1990-01-01

Two enzyme-linked immunosorbent assays were established and compared for the estimation of plasma aldosterone. In the first method immobilized aldosterone-protein complexes on the ELISA plates compete with aldosterone to be determined for the binding of certain amount of anti-aldosterone antibodies. The sensitivity of this method depends on the protein carrier used to conjugate with aldosterone. In the second method, anti-aldosterone antibodies adsorbed on ELISA plates compete for binding of known amount of the enzyme-labeled aldosterone and aldosterone to be determined. The highly specific rabbit anti-aldosterone antibodies were obtained by injection of aldosterone-oxime thyroglobulin. The detection limit of aldosterone in both methods ranged between 2-20 pg. The proposed assays are suitable for the determination of aldosterone in biological fluids compared with other reported ELISA assays, as well as with RIA.

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

PubMed

Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

2016-06-07

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

Screening glycosynthase libraries with a fluoride chemosensor assay independently of enzyme specificity: identification of a transitional hydrolase to synthase mutant.

PubMed

Andrés, Eduardo; Aragunde, Hugo; Planas, Antoni

2014-03-01

Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. Enzyme-directed evolution approaches are applied to improve the performance of current glycosynthases and engineer specificity for non-natural substrates. However, simple and general screening methods are required since most of the reported assays are specific for each particular enzyme. In the present paper, we report a general screening assay that is independent of enzyme specificity, and implemented in an HTS (high-throughput screening) format for the screening of cell extracts in directed evolution experiments. Fluoride ion is a general by-product released in all glycosynthase reactions with glycosyl fluoride donors. The new assay is based on the use of a specific chemical sensor (a silyl ether of a fluorogenic methylumbelliferone) to transduce fluoride concentration into a fluorescence signal. As a proof-of-concept, it has been applied to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-β-glucanase. Beyond the expected mutations at the glutamic acid (catalytic) nucleophile, other variants have been shown to acquire glycosynthase activity. Surprisingly, an aspartic acid for glutamic acid replacement renders a highly active glycosynthase, but still retains low hydrolase activity. It appears as an intermediate state between glycosyl hydrolase and glycosynthase.

Recommended Standards: Common to All Programs; Specific to Production Agriculture, Secondary; Specific to Adult Education for Quality Agriculture/Agribusiness Programs in Kansas.

ERIC Educational Resources Information Center

Kansas State Univ., Manhattan. Dept. of Adult and Occupational Education.

This document contains recommended standards for quality vocational programs in agricultural/agribusiness education which are divided into (1) standards common to all programs, (2) standards specific to adult education in agriculture/agribusiness, and (3) standards specific to production agriculture, secondary. The sixty common standards are…

Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis.

PubMed

Tan, Cheng Seng; Hassan, Maizom; Mohamed Hussein, Zeti Azura; Ismail, Ismanizan; Ho, Kok Lian; Ng, Chyan Leong; Zainal, Zamri

2018-02-01

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp 280 , Leu 294 and Ala 303 ). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Program Aids Specification Of Multiple-Block Grids

NASA Technical Reports Server (NTRS)

Sorenson, R. L.; Mccann, K. M.

1993-01-01

3DPREP computer program aids specification of multiple-block computational grids. Highly interactive graphical preprocessing program designed for use on powerful graphical scientific computer workstation. Divided into three main parts, each corresponding to principal graphical-and-alphanumerical display. Relieves user of some burden of collecting and formatting many data needed to specify blocks and grids, and prepares input data for NASA's 3DGRAPE grid-generating computer program.

Direct Electron Transfer of Enzymes in a Biologically Assembled Conductive Nanomesh Enzyme Platform.

PubMed

Lee, Seung-Woo; Lee, Ki-Young; Song, Yong-Won; Choi, Won Kook; Chang, Joonyeon; Yi, Hyunjung

2016-02-24

Nondestructive assembly of a nanostructured enzyme platform is developed in combination of the specific biomolecular attraction and electrostatic coupling for highly efficient direct electron transfer (DET) of enzymes with unprecedented applicability and versatility. The biologically assembled conductive nanomesh enzyme platform enables DET-based flexible integrated biosensors and DET of eight different enzyme with various catalytic activities. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

13 CFR 130.350 - Specific program responsibilities.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 13 Business Credit and Assistance 1 2011-01-01 2011-01-01 false Specific program responsibilities. 130.350 Section 130.350 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION SMALL BUSINESS... Program policies and procedures to improve the delivery of services by SBDCs to the small business...

13 CFR 130.350 - Specific program responsibilities.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Specific program responsibilities. 130.350 Section 130.350 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION SMALL BUSINESS... Program policies and procedures to improve the delivery of services by SBDCs to the small business...

Detection of anti-Yta antibodies using a sensitive and specific enzyme-linked immunosorbent assay.

PubMed

Geen, J; Hullin, D A; Hogg, S I

1999-01-01

A specific, sensitive and semi-quantitative enzyme-linked immunosorbent assay (ELISA) is described to detect anti-Yta antibodies in human serum. Recombinant acetylcholinesterase (AChE E.C.3.1.1.7) was employed as the coating antigen in the microtitre plate and horseradish peroxidase (HRP)-conjugated specific antibody (IgG) was used as the secondary antibody. The method developed showed excellent sensitivity, detecting a titre > 1 in 600,000 (3.5 ng/mL mouse IgG protein) for mouse monoclonal (mMAb) anti-AChE antibody. No cross-reaction was seen with other common blood group antibodies, confirming the specificity of the method. The recombinant antigen's AChE phenotype was confirmed as Yta, as no reaction was detected with anti-Ytb-positive sera. The ELISA method correlated closely with the established serological grading system used routinely in blood transfusion laboratories.

Induction Specificity and Catabolite Repression of the Early Enzymes in Camphor Degradation by Pseudomonas putida

PubMed Central

Hartline, Richard A.; Gunsalus, I. C.

1971-01-01

The ability of bornane and substituted bornanes to induce the early enzymes for d(+)-camphor degradation and control of these enzymes by catabolite repression were studied in a strain of a Pseudomonas putida. Bornane and 20 substituted bornane compounds showed induction. Of these 21 compounds, bornane and 8 of the substituted bornanes provided induction without supporting growth. Oxygen, but not nitrogen, enhanced the inductive potency of the unsubstituted bornane ring. All bornanedione isomers caused induction, and those with substituents on each of the three consecutive carbon atoms, including the methyl group at the bridgehead carbon, showed induction without supporting growth. Although it was not possible to obtain experimental data for a case of absolute gratuitous induction by compounds not supporting growth, indirect evidence in support of gratuitous induction is presented. It is proposed that the ability of P. putida to tolerate the unusually high degree of possible gratuitous induction observed for camphor catabolism may be related to the infrequent occurrence of bicyclic ring structures in nature. Survival of an organism with a broad specificity for gratuitous induction is discussed. Glucose and succinate, but not glutamate, produced catabolite repression of the early camphor-degrading enzymes. Pathway enzymes differ in their degree of sensitivity to succinate-provoked catabolite repression. The ability of a compound to produce catabolite repression is not, however, directly related to the duration of the lag period (diauxic lag) between growth on camphor and growth on the repressing compound. PMID:5573731

Early evolution of efficient enzymes and genome organization

PubMed Central

2012-01-01

Background Cellular life with complex metabolism probably evolved during the reign of RNA, when it served as both information carrier and enzyme. Jensen proposed that enzymes of primordial cells possessed broad specificities: they were generalist. When and under what conditions could primordial metabolism run by generalist enzymes evolve to contemporary-type metabolism run by specific enzymes? Results Here we show by numerical simulation of an enzyme-catalyzed reaction chain that specialist enzymes spread after the invention of the chromosome because protocells harbouring unlinked genes maintain largely non-specific enzymes to reduce their assortment load. When genes are linked on chromosomes, high enzyme specificity evolves because it increases biomass production, also by reducing taxation by side reactions. Conclusion The constitution of the genetic system has a profound influence on the limits of metabolic efficiency. The major evolutionary transition to chromosomes is thus proven to be a prerequisite for a complex metabolism. Furthermore, the appearance of specific enzymes opens the door for the evolution of their regulation. Reviewers This article was reviewed by Sándor Pongor, Gáspár Jékely, and Rob Knight. PMID:23114029

[The specific enzyme inhibitors for potential therapeutic use].

PubMed

Bretner, Maria

2015-01-01

Therapy for hepatitis C virus (HCV) initially consisted on administering ribavirin - having a broad spectrum of action - and pegylated interferon, and was only effective in 40-50% of patients. Appropriate was to find effective inhibitors of viral replication e.g. by inhibition of a viral enzyme, NTPase/helicase required in the process of translation and RNA replication of the HCV. We developed methods of synthesis of many compounds belonging to different groups - derivatives of nucleosides, benzotriazole, benzimidazole, tropolone and epirubicine. Some of the derivatives inhibit HCV helicase activity at low concentrations and reduces replication of the viral RNA in subgenomic replicon system. In the process of HCV replication casein kinase CK2 plays an important role. It regulates the level of phosphorylation of HCV protein NS5A, which affects the production of infectious virions of HCV. Effective and selective inhibitors of kinase CK2 could be of use in the treatment of HCV in combination with other drugs. CK2 kinase phosphorylates approximately 300 proteins that affect the growth, differentiation, proliferation or apoptosis. Elevated CK2 kinase activity has been observed in several types of cancer and other diseases, therefore, inhibitors of this enzyme are potential therapeutic importance, particularly for anti-cancer treatment. Research carried out in collaboration with prof. Shugar led to the synthesis of one of the most selective inhibitors of this enzyme which is 4,5,6,7-tetrabromo-1H-benzotriazole, used for the study of the role of kinase CK2 in a number of metabolic processes in tumor cells.

Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

PubMed

Bate, Paul; Warwicker, Jim

2004-07-02

Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

Molecular diversity of tuliposide B-converting enzyme in tulip (Tulipa gesneriana): identification of the root-specific isozyme.

PubMed

Nomura, Taiji; Ueno, Ayaka; Ogita, Shinjiro; Kato, Yasuo

2017-06-01

6-Tuliposide B (PosB) is a glucose ester accumulated in tulip (Tulipa gesneriana) as a major secondary metabolite. PosB serves as the precursor of the antimicrobial lactone tulipalin B (PaB), which is formed by PosB-converting enzyme (TCEB). The gene TgTCEB1, encoding a TCEB, is transcribed in tulip pollen but scarcely transcribed in other tissues (e.g. roots) even though those tissues show high TCEB activity. This led to the prediction of the presence of a TCEB isozyme with distinct tissue specificity. Herein, we describe the identification of the TgTCEB-R gene from roots via native enzyme purification; this gene is a paralog of TgTCEB1. Recombinant enzyme characterization verified that TgTCEB-R encodes a TCEB. Moreover, TgTCEB-R was localized in tulip plastids, as found for pollen TgTCEB1. TgTCEB-R is transcribed almost exclusively in roots, indicating a tissue preference for the transcription of TCEB isozyme genes.

ENZVU--An Enzyme Kinetics Computer Simulation Based upon a Conceptual Model of Enzyme Action.

ERIC Educational Resources Information Center

Graham, Ian

1985-01-01

Discusses a simulation on enzyme kinetics based upon the ability of computers to generate random numbers. The program includes: (1) enzyme catalysis in a restricted two-dimensional grid; (2) visual representation of catalysis; and (3) storage and manipulation of data. Suggested applications and conclusions are also discussed. (DH)

Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors.

PubMed

Jian, Hui; Wang, Yingwu; Bai, Yan; Li, Rong; Gao, Renjun

2016-07-11

Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

Enzymes from Extreme Environments and Their Industrial Applications

PubMed Central

Littlechild, Jennifer A.

2015-01-01

This article will discuss the importance of specific extremophilic enzymes for applications in industrial biotechnology. It will specifically address those enzymes that have applications in the area of biocatalysis. Such enzymes now play an important role in catalyzing a variety of chemical conversions that were previously carried out by traditional chemistry. The biocatalytic process is carried out under mild conditions and with greater specificity. The enzyme process does not result in the toxic waste that is usually produced in a chemical process that would require careful disposal. In this sense, the biocatalytic process is referred to as carrying out “green chemistry” which is considered to be environmentally friendly. Some of the extremophilic enzymes to be discussed have already been developed for industrial processes such as an l-aminoacylase and a γ-lactamase. The industrial applications of other extremophilic enzymes, including transaminases, carbonic anhydrases, dehalogenases, specific esterases, and epoxide hydrolases, are currently being assessed. Specific examples of these industrially important enzymes that have been studied in the authors group will be presented in this review. PMID:26528475

29 CFR 99.235 - Program-specific audits.

Code of Federal Regulations, 2012 CFR

2012-07-01

... auditee shall submit to the Federal clearinghouse designated by the OMB, the data collection form prepared...-specific audit guide. (3) When a program-specific audit guide is not available, the reporting package for a... section, and the auditor's report(s) described in paragraph (b)(4) of this section. The data collection...

29 CFR 99.235 - Program-specific audits.

Code of Federal Regulations, 2010 CFR

2010-07-01

... auditee shall submit to the Federal clearinghouse designated by the OMB, the data collection form prepared...-specific audit guide. (3) When a program-specific audit guide is not available, the reporting package for a... section, and the auditor's report(s) described in paragraph (b)(4) of this section. The data collection...

7 CFR 3052.235 - Program-specific audits.

Code of Federal Regulations, 2014 CFR

2014-01-01

... is available, the auditee shall submit to the Federal clearinghouse designated by OMB the data...-specific audit guide. (3) When a program-specific audit guide is not available, the reporting package for a... section, and the auditor's report(s) described in paragraph (b)(4) of this section. The data collection...

29 CFR 99.235 - Program-specific audits.

Code of Federal Regulations, 2013 CFR

2013-07-01

... auditee shall submit to the Federal clearinghouse designated by the OMB, the data collection form prepared...-specific audit guide. (3) When a program-specific audit guide is not available, the reporting package for a... section, and the auditor's report(s) described in paragraph (b)(4) of this section. The data collection...

7 CFR 3052.235 - Program-specific audits.

Code of Federal Regulations, 2011 CFR

2011-01-01

... is available, the auditee shall submit to the Federal clearinghouse designated by OMB the data...-specific audit guide. (3) When a program-specific audit guide is not available, the reporting package for a... section, and the auditor's report(s) described in paragraph (b)(4) of this section. The data collection...

29 CFR 99.235 - Program-specific audits.

Code of Federal Regulations, 2011 CFR

2011-07-01

... auditee shall submit to the Federal clearinghouse designated by the OMB, the data collection form prepared...-specific audit guide. (3) When a program-specific audit guide is not available, the reporting package for a... section, and the auditor's report(s) described in paragraph (b)(4) of this section. The data collection...

7 CFR 3052.235 - Program-specific audits.

Code of Federal Regulations, 2012 CFR

2012-01-01

... is available, the auditee shall submit to the Federal clearinghouse designated by OMB the data...-specific audit guide. (3) When a program-specific audit guide is not available, the reporting package for a... section, and the auditor's report(s) described in paragraph (b)(4) of this section. The data collection...

Enzyme specificity under dynamic control

NASA Astrophysics Data System (ADS)

Ota, Nobuyuki; Agard, David A.

2002-03-01

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of alpha-lytic protease. The wild-type alpha-lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190Ala binding pocket mutant has a much broader specificity, actively hydrolyzing substrates ranging from Ala to Phe. We performed a principal component analysis using 1-nanosecond molecular dynamics simulations using solvent boundary condition. We found that the walls of the wild-type substrate binding pocket move in tandem with one another, causing the pocket size to remain fixed so that only small substrates are recognized. In contrast, the M190A mutant shows uncoupled movement of the binding pocket walls, allowing the pocket to sample both smaller and larger sizes, which appears to be the cause of the observed broad specificity. The results suggest that the protein dynamics of alpha-lytic protease may play a significant role in defining the patterns of substrate specificity.

Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

PubMed

Azad, Kimi; Banerjee, Manidipa; Johnson, John E

2017-09-29

Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

Lung function, atopy, specific hypersensitivity, and smoking of workers in the enzyme detergent industry over 11 years.

PubMed

Flood, D F; Blofeld, R E; Bruce, C F; Hewitt, J I; Juniper, C P; Roberts, D M

1985-01-01

A study of 2800 workers employed in three factories of the two major manufacturers of enzymatic products in the United Kingdom covering 11 years of operation from 1969 to 1980 showed that 2344 workers had sufficient lung function data to meet the operational criteria and these were analysed in three separate groups by factory locations. Spirometry and prick tests for specific skin reactions to standardised enzyme were performed at six monthly intervals for the first six years of the study and then annually. Factory enzyme dust and total dust measurements were made to determine the degree of dust exposure of the subjects. The lung function of the factory groups was analysed for the effects of working in the detergent industry, the degree of exposure to enzymes, skin prick test positivity to enzymes, atopicity, and smoking. The 4.5% of workers who had experienced respiratory effects from enzymes were analysed separately. Exposure to the enzyme allergen has had no significant long term effect on the lung function of the detergent workers. A higher proportion of atopics than non-atopics became skin test positive to the allergen and more smokers than non-smokers were sensitised. The overall lung function of detergent workers showed 39 ml/year loss in FEV1 on the 11 year longitudinal study and 51 ml/year loss on the lateral (cross sectional) analysis with better lung function in the south east than the north west of England. In the development of the methodology for the study several potential problems were discovered that could remain unrecognised in a cross sectional analysis performed in isolation.

Development of immunoglobulin class-specific enzyme-linked immunosorbent assays for measuring antibodies against avian rotavirus.

PubMed

Myers, T J; Schat, K A; Mockett, A P

1989-01-01

Immunoglobulin class-specific enzyme-linked immunosorbent assays were developed for detecting antibodies against avian rotavirus in serum, intestinal contents, and bile from experimentally infected specific-pathogen-free (SPF) chickens. Both indirect and antibody-capture (AbC) assays were developed based on monoclonal antibodies specific for chicken IgG, IgM, and IgA. Treatment of purified rotavirus with sodium thiocyanate before coating the plate improved the rotavirus-specific reading in the indirect assay. Use of Immunolon 2 plates facilitated attachment of monoclonal antibodies to the plate in the AbC assay. Addition of 5% powdered skim milk to the diluent buffer reduced nonspecific background readings. The indirect assay was superior for detecting rotavirus-specific IgG, whereas the AbC assay was better for detecting rotavirus-specific IgM and IgA. The presence of intestinal contents in the assay wells did not reduce the measurable titers of IgG, IgM, or IgA. These assays showed that SPF chickens produced systemic and mucosal antibodies against avian rotavirus.

Structure and function of α-glucan debranching enzymes.

PubMed

Møller, Marie Sofie; Henriksen, Anette; Svensson, Birte

2016-07-01

α-Glucan debranching enzymes hydrolyse α-1,6-linkages in starch/glycogen, thereby, playing a central role in energy metabolism in all living organisms. They belong to glycoside hydrolase families GH13 and GH57 and several of these enzymes are industrially important. Nine GH13 subfamilies include α-glucan debranching enzymes; isoamylase and glycogen debranching enzymes (GH13_11); pullulanase type I/limit dextrinase (GH13_12-14); pullulan hydrolase (GH13_20); bifunctional glycogen debranching enzyme (GH13_25); oligo-1 and glucan-1,6-α-glucosidases (GH13_31); pullulanase type II (GH13_39); and α-amylase domains (GH13_41) in two-domain amylase-pullulanases. GH57 harbours type II pullulanases. Specificity differences, domain organisation, carbohydrate binding modules, sequence motifs, three-dimensional structures and specificity determinants are discussed. The phylogenetic analysis indicated that GH13_39 enzymes could represent a "missing link" between the strictly α-1,6-specific debranching enzymes and the enzymes with dual specificity and α-1,4-linkage preference.

Logic programming and metadata specifications

NASA Technical Reports Server (NTRS)

Lopez, Antonio M., Jr.; Saacks, Marguerite E.

1992-01-01

Artificial intelligence (AI) ideas and techniques are critical to the development of intelligent information systems that will be used to collect, manipulate, and retrieve the vast amounts of space data produced by 'Missions to Planet Earth.' Natural language processing, inference, and expert systems are at the core of this space application of AI. This paper presents logic programming as an AI tool that can support inference (the ability to draw conclusions from a set of complicated and interrelated facts). It reports on the use of logic programming in the study of metadata specifications for a small problem domain of airborne sensors, and the dataset characteristics and pointers that are needed for data access.

38 CFR 41.235 - Program-specific audits.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Federal clearinghouse designated by OMB the data collection form prepared in accordance with § 41.320(b...-specific audit guide is not available, the reporting package for a program-specific audit shall consist of...) described in paragraph (b)(4) of this section. The data collection form prepared in accordance with § 41.320...

38 CFR 41.235 - Program-specific audits.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Federal clearinghouse designated by OMB the data collection form prepared in accordance with § 41.320(b...-specific audit guide is not available, the reporting package for a program-specific audit shall consist of...) described in paragraph (b)(4) of this section. The data collection form prepared in accordance with § 41.320...

38 CFR 41.235 - Program-specific audits.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Federal clearinghouse designated by OMB the data collection form prepared in accordance with § 41.320(b...-specific audit guide is not available, the reporting package for a program-specific audit shall consist of...) described in paragraph (b)(4) of this section. The data collection form prepared in accordance with § 41.320...

38 CFR 41.235 - Program-specific audits.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Federal clearinghouse designated by OMB the data collection form prepared in accordance with § 41.320(b...-specific audit guide is not available, the reporting package for a program-specific audit shall consist of...) described in paragraph (b)(4) of this section. The data collection form prepared in accordance with § 41.320...

Enzymes approved for human therapy: indications, mechanisms and adverse effects.

PubMed

Baldo, Brian A

2015-02-01

Research and drug developments fostered under orphan drug product development programs have greatly assisted the introduction of efficient and safe enzyme-based therapies for a range of rare disorders. The introduction and regulatory approval of 20 different recombinant enzymes has enabled, often for the first time, effective enzyme-replacement therapy for some lysosomal storage disorders, including Gaucher (imiglucerase, taliglucerase, and velaglucerase), Fabry (agalsidase alfa and beta), and Pompe (alglucosidase alfa) diseases and mucopolysaccharidoses I (laronidase), II (idursulfase), IVA (elosulfase), and VI (galsulfase). Approved recombinant enzymes are also now used as therapy for myocardial infarction (alteplase, reteplase, and tenecteplase), cystic fibrosis (dornase alfa), chronic gout (pegloticase), tumor lysis syndrome (rasburicase), leukemia (L-asparaginase), some collagen-based disorders such as Dupuytren's contracture (collagenase), severe combined immunodeficiency disease (pegademase bovine), detoxification of methotrexate (glucarpidase), and vitreomacular adhesion (ocriplasmin). The development of these efficacious and safe enzyme-based therapies has occurred hand in hand with some remarkable advances in the preparation of the often specifically designed recombinant enzymes; the manufacturing expertise necessary for commercial production; our understanding of underlying mechanisms operative in the different diseases; and the mechanisms of action of the relevant recombinant enzymes. Together with information on these mechanisms, safety findings recorded so far on the various adverse events and problems of immunogenicity of the recombinant enzymes used for therapy are presented.

47 CFR 76.75 - Specific EEO program requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO... necessary. Nothing in this section shall be interpreted to require a multichannel video programming...) In addition to using such recruitment sources, a multichannel video programming distributor...

47 CFR 76.75 - Specific EEO program requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO... necessary. Nothing in this section shall be interpreted to require a multichannel video programming...) In addition to using such recruitment sources, a multichannel video programming distributor...

47 CFR 76.75 - Specific EEO program requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO... necessary. Nothing in this section shall be interpreted to require a multichannel video programming...) In addition to using such recruitment sources, a multichannel video programming distributor...

47 CFR 76.75 - Specific EEO program requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO... necessary. Nothing in this section shall be interpreted to require a multichannel video programming...) In addition to using such recruitment sources, a multichannel video programming distributor...

Closing the Gap Between Specification and Programming: VDM++ and SCALA

NASA Technical Reports Server (NTRS)

Havelund, Klaus

2011-01-01

We argue that a modern programming language such as Scala offers a level of succinctness, which makes it suitable for program and systems specification as well as for high-level programming. We illustrate this by comparing the language with the Vdm++ specification language. The comparison also identifies areas where Scala perhaps could be improved, inspired by Vdm++. We furthermore illustrate Scala's potential as a specification language by augmenting it with a combination of parameterized state machines and temporal logic, defined as a library, thereby forming an expressive but simple runtime verification framework.

A Program for Iron Economy during Deficiency Targets Specific Fe Proteins.

PubMed

Hantzis, Laura J; Kroh, Gretchen E; Jahn, Courtney E; Cantrell, Michael; Peers, Graham; Pilon, Marinus; Ravet, Karl

2018-01-01

Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis ( Arabidopsis thaliana ) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome- b 6 f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency. © 2018 American Society of Plant Biologists. All Rights Reserved.

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.

Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase 1

PubMed Central

Hack, Ethan; Kemp, John D.

1980-01-01

A single enzyme catalyzes the synthesis of all four N2-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N2-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (Km) differ, the ratio V/Km is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:16661312

Tissue Specificity of Human Angiotensin I-Converting Enzyme

PubMed Central

Kryukova, Olga V.; Tikhomirova, Victoria E.; Golukhova, Elena Z.; Evdokimov, Valery V.; Kalantarov, Gavreel F.; Trakht, Ilya N.; Schwartz, David E.; Dull, Randal O.; Gusakov, Alexander V.; Uporov, Igor V.; Kost, Olga A.; Danilov, Sergei M.

2015-01-01

Background Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. Methods/Principal Findings We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. Conclusions Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs. PMID:26600189

Tissue specific distribution of pyrimidine deoxynucleoside salvage enzymes shed light on the mechanism of mitochondrial DNA depletion.

PubMed

Wang, L; Eriksson, S

2010-06-01

Deficiency in thymidine kinase 2 (TK2) activity due to genetic alterations caused tissue specific mitochondrial DNA (mtDNA) depletion syndrome with symptoms resembling these of AIDS patients treated with nucleoside analogues. Mechanisms behind this mitochondrial effects is still not well understood. With rat as a model we isolated mitochondrial and cytosolic fractions from major organs and studied enzymes involved in thymidine (dT) and deoxycytidine (dC) phosphorylation by using ionic exchange column chromatography. A cytosolic form of TK2 was identified in all tested tissues in addition to mitochondrial TK2. TK1 was detected in liver and spleen cytosolic extracts while dCK was found in liver, spleen and lung cytosolic extracts. Thus, the nature of dT and dC salvage enzymes in each tissue type was determined. In most tissues TK2 is the only salvage enzyme present except liver and spleen. These results may help to explain the mechanisms of mitochondrial toxicity of antiviral nucleoside analogues and mtDNA depletion caused by TK2 deficiency.

Northwest Energy Efficient Manufactured Housing Program Specification Development

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

Hewes, Tom; Peeks, Brady

2013-02-01

The DOE research team Building America Partnership for Improved Residential Construction (BA-PIRC), Bonneville Power Administration (BPA), and Northwest Energy Works (NEW), the current Northwest Energy Efficient Manufactured Home Program (NEEM) program administrator, collaborated to research a new specification that would reduce the energy requirements of a NEEM home.This research identified and developed combinations of cost-effective high performance building assemblies and mechanical systems that can readily can be deployed in the manufacturing setting that reduce energy used for space conditioning, water heating and lighting by 50% over the present NEEM specifications.

21 CFR 184.1287 - Enzyme-modified fats.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...

21 CFR 184.1287 - Enzyme-modified fats.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...

21 CFR 184.1287 - Enzyme-modified fats.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...

21 CFR 184.1287 - Enzyme-modified fats.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...

KINEXP: Computer Simulation in Enzyme Kinetics.

ERIC Educational Resources Information Center

Gelpi, Josep Lluis; Domenech, Carlos

1988-01-01

Describes a program which allows students to identify and characterize several kinetic inhibitory mechanisms. Uses the generic model of reversible inhibition of a monosubstrate enzyme but can be easily modified to run other models such as bisubstrate enzymes. Uses MS-DOS BASIC. (MVL)

Highly broad-specific and sensitive enzyme-linked immunosorbent assay for screening sulfonamides: Assay optimization and application to milk samples

USDA-ARS?s Scientific Manuscript database

A broad-specific and sensitive immunoassay for the detection of sulfonamides was developed by optimizing the conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, de...

Muscle enzyme and fiber type-specific sarcomere protein increases in serum after inertial concentric-eccentric exercise.

PubMed

Carmona, G; Guerrero, M; Cussó, R; Padullés, J M; Moras, G; Lloret, M; Bedini, J L; Cadefau, J A

2015-12-01

Muscle damage induced by inertial exercise performed on a flywheel device was assessed through the serum evolution of muscle enzymes, interleukin 6, and fiber type-specific sarcomere proteins such as fast myosin (FM) and slow myosin (SM). We hypothesized that a model of muscle damage could be constructed by measuring the evolution of serum concentration of muscle proteins following inertial exercise, according to their molecular weight and the fiber compartment in which they are located. Moreover, by measuring FM and SM, the type of fibers that are affected could be assessed. Serum profiles were registered before and 24, 48, and 144 h after exercise in 10 healthy and recreationally active young men. Creatine kinase (CK) and CK-myocardial band isoenzyme increased in serum early (24 h) and returned to baseline values after 48 h. FM increased in serum late (48 h) and remained elevated 144 h post-exercise. The increase in serum muscle enzymes suggests increased membrane permeability of both fast and slow fibers, and the increase in FM reveals sarcomere disruption as well as increased membrane permeability of fast fibers. Consequently, FM could be adopted as a fiber type-specific biomarker of muscle damage. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants.

PubMed

Pati, Sarah G; Kohler, Hans-Peter E; Pabis, Anna; Paneth, Piotr; Parales, Rebecca E; Hofstetter, Thomas B

2016-07-05

Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent (13)C- and (2)H-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O2 activation and aromatic C oxygenation. The contribution of enzymatic O2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants.

Self-powered enzyme micropumps

NASA Astrophysics Data System (ADS)

Sengupta, Samudra; Patra, Debabrata; Ortiz-Rivera, Isamar; Agrawal, Arjun; Shklyaev, Sergey; Dey, Krishna K.; Córdova-Figueroa, Ubaldo; Mallouk, Thomas E.; Sen, Ayusman

2014-05-01

Non-mechanical nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of adenosine triphosphate function as self-powered micropumps in the presence of their respective substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid density generated by the enzymatic reaction. The pumping velocity increases with increasing substrate concentration and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small molecules and proteins in response to specific chemical stimuli, including the release of insulin in response to glucose.

Dissipative Dynamics of Enzymes

NASA Astrophysics Data System (ADS)

Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

2014-11-01

We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C ; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

Dissipative Dynamics of Enzymes

NASA Astrophysics Data System (ADS)

Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni; Zocchi LabMolecular Biophysics Team

2015-03-01

We explore enzyme conformational dynamics at sub - Å resolution, specifically temperature effects. The ensemble averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor 2 as the temperature is raised from 10 C to 45 C; the elastic parameter K shows a similar decrease. Thus when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

Dissipative dynamics of enzymes.

PubMed

Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

2014-11-07

We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

An assessment of catalytic residue 3D ensembles for the prediction of enzyme function.

PubMed

Žváček, Clemens; Friedrichs, Gerald; Heizinger, Leonhard; Merkl, Rainer

2015-11-04

The central element of each enzyme is the catalytic site, which commonly catalyzes a single biochemical reaction with high specificity. It was unclear to us how often sites that catalyze the same or highly similar reactions evolved on different, i. e. non-homologous protein folds and how similar their 3D poses are. Both similarities are key criteria for assessing the usability of pose comparison for function prediction. We have analyzed the SCOP database on the superfamily level in order to estimate the number of non-homologous enzymes possessing the same function according to their EC number. 89% of the 873 substrate-specific functions (four digit EC number) assigned to mono-functional, single-domain enzymes were only found in one superfamily. For a reaction-specific grouping (three digit EC number), this value dropped to 35%, indicating that in approximately 65% of all enzymes the same function evolved in two or more non-homologous proteins. For these isofunctional enzymes, structural similarity of the catalytic sites may help to predict function, because neither high sequence similarity nor identical folds are required for a comparison. To assess the specificity of catalytic 3D poses, we compiled the redundancy-free set ENZ_SITES, which comprises 695 sites, whose composition and function are well-defined. We compared their poses with the help of the program Superpose3D and determined classification performance. If the sites were from different superfamilies, the number of true and false positive predictions was similarly high, both for a coarse and a detailed grouping of enzyme function. Moreover, classification performance did not improve drastically, if we additionally used homologous sites to predict function. For a large number of enzymatic functions, dissimilar sites evolved that catalyze the same reaction and it is the individual substrate that determines the arrangement of the catalytic site and its local environment. These substrate-specific requirements

Stabilization of enzymes in ionic liquids via modification of enzyme charge.

PubMed

Nordwald, Erik M; Kaar, Joel L

2013-09-01

Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.

Heat Stable Enzymes from Thermophiles

DTIC Science & Technology

1998-02-01

final product and is somewhat messy to work with. Therefore, alternatives were tested. However, no combination of corn syrup , alternative sugars and...INTRODUCTION 9 CLONING OF ALKALINE PHOSPHATASE GENE AND PRODUCTION OF HIGH SPECIFIC ACTIVITY ENZYME 9 Cloning into E. coil and expression of high activity...JKR209, into an alternative, better producing organism. CLONING OF ALKALINE PHOSPHATASE GENE AND PRODUCTION OF HIGH SPECIFIC ACTIVITY ENZYME Cloning into

Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation.

PubMed

Yu, Feifan; Alesand, Veronica; Nygren, Per-Åke

2018-02-27

Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

2 CFR 200.507 - Program-specific audits.

Code of Federal Regulations, 2014 CFR

2014-01-01

... auditee must electronically submit to the FAC the data collection form prepared in accordance with § 200... submitted to the FAC. (d) Other sections of this part may apply. Program-specific audits are subject to: (1...

Access channels to the buried active site control substrate specificity in CYP1A P450 enzymes.

PubMed

Urban, Philippe; Truan, Gilles; Pompon, Denis

2015-04-01

A cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity. Time course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps. The global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not. Size discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition. Catalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active site. Copyright © 2014 Elsevier B.V. All rights reserved.

21 CFR 184.1063 - Enzyme-modified lecithin.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Enzyme-modified lecithin. 184.1063 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1063 Enzyme-modified lecithin. (a) Enzyme-modified... Lysolecithin Content of Enzyme-Modified Lecithin: Method I,” dated 1985, which is incorporated by reference in...

21 CFR 184.1063 - Enzyme-modified lecithin.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Enzyme-modified lecithin. 184.1063 Section 184.1063... Listing of Specific Substances Affirmed as GRAS § 184.1063 Enzyme-modified lecithin. (a) Enzyme-modified... Lysolecithin Content of Enzyme-Modified Lecithin: Method I,” dated 1985, which is incorporated by reference in...

Aeronautics Research and Technology Program and specific objectives, fiscal year 1982

NASA Technical Reports Server (NTRS)

Olstad, W. B.

1981-01-01

The Aeronautics Research and Technology program is broken down into two program areas (research and technology base, and systems technology programs) which are further broken down into succeedingly more detailed activities to form a work breakdown structure for the aeronautics program: program area, program/discipline objective, specific objective, and research and technology objective and plan (RTOP). A detailed view of this work breakdown structure down to the specific objective level is provided, and goals or objectives at each of these levels are set forth. What is to be accomplished and why are addressed, but not how. The letter falls within the domain of the RTOP.

Targeted metabolomics connects thioredoxin-interacting protein (TXNIP) to mitochondrial fuel selection and regulation of specific oxidoreductase enzymes in skeletal muscle.

PubMed

DeBalsi, Karen L; Wong, Kari E; Koves, Timothy R; Slentz, Dorothy H; Seiler, Sarah E; Wittmann, April H; Ilkayeva, Olga R; Stevens, Robert D; Perry, Christopher G R; Lark, Daniel S; Hui, Simon T; Szweda, Luke; Neufer, P Darrell; Muoio, Deborah M

2014-03-21

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIP(SKM-/-)) Txnip deficiency. Compared with littermate controls, both TKO and TXNIP(SKM-/-) mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability.

Targeted Metabolomics Connects Thioredoxin-interacting Protein (TXNIP) to Mitochondrial Fuel Selection and Regulation of Specific Oxidoreductase Enzymes in Skeletal Muscle*

PubMed Central

DeBalsi, Karen L.; Wong, Kari E.; Koves, Timothy R.; Slentz, Dorothy H.; Seiler, Sarah E.; Wittmann, April H.; Ilkayeva, Olga R.; Stevens, Robert D.; Perry, Christopher G. R.; Lark, Daniel S.; Hui, Simon T.; Szweda, Luke; Neufer, P. Darrell; Muoio, Deborah M.

2014-01-01

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIPSKM−/−) Txnip deficiency. Compared with littermate controls, both TKO and TXNIPSKM−/− mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability. PMID:24482226

A dermal HOX transcriptional program regulates site-specific epidermal fate

PubMed Central

Rinn, John L.; Wang, Jordon K.; Allen, Nancy; Brugmann, Samantha A.; Mikels, Amanda J.; Liu, Helen; Ridky, Todd W.; Stadler, H. Scott; Nusse, Roel; Helms, Jill A.; Chang, Howard Y.

2008-01-01

Reciprocal epithelial–mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration. PMID:18245445

A dermal HOX transcriptional program regulates site-specific epidermal fate.

PubMed

Rinn, John L; Wang, Jordon K; Allen, Nancy; Brugmann, Samantha A; Mikels, Amanda J; Liu, Helen; Ridky, Todd W; Stadler, H Scott; Nusse, Roel; Helms, Jill A; Chang, Howard Y

2008-02-01

Reciprocal epithelial-mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration.

The enzymic hydrolysis of amygdalin

PubMed Central

Haisman, D. R.; Knight, D. J.

1967-01-01

Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond β-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the β-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components. PMID:4291788

42 CFR 457.1180 - Program specific review process: Notice.

Code of Federal Regulations, 2012 CFR

2012-10-01

... SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO STATES State Plan Requirements: Applicant and Enrollee Protections § 457.1180 Program specific review process... explanation of applicable rights to review of that determination, the standard and expedited time frames for...

42 CFR 457.1180 - Program specific review process: Notice.

Code of Federal Regulations, 2013 CFR

2013-10-01

... SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO STATES State Plan Requirements: Applicant and Enrollee Protections § 457.1180 Program specific review process... explanation of applicable rights to review of that determination, the standard and expedited time frames for...

42 CFR 457.1180 - Program specific review process: Notice.

Code of Federal Regulations, 2011 CFR

2011-10-01

... SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO STATES State Plan Requirements: Applicant and Enrollee Protections § 457.1180 Program specific review process... explanation of applicable rights to review of that determination, the standard and expedited time frames for...

42 CFR 457.1180 - Program specific review process: Notice.

Code of Federal Regulations, 2014 CFR

2014-10-01

... SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO STATES State Plan Requirements: Applicant and Enrollee Protections § 457.1180 Program specific review process... explanation of applicable rights to review of that determination, the standard and expedited time frames for...

42 CFR 457.1180 - Program specific review process: Notice.

Code of Federal Regulations, 2010 CFR

2010-10-01

... SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS TO STATES State Plan Requirements: Applicant and Enrollee Protections § 457.1180 Program specific review process... explanation of applicable rights to review of that determination, the standard and expedited time frames for...

A novel pH-enzyme-dependent mesalamine colon-specific delivery system.

PubMed

Jin, Lei; Ding, Yi-Cun; Zhang, Yu; Xu, Xiao-Qing; Cao, Qin

2016-01-01

The aim of the present study was to design a new pH-enzyme double-dependent mesalamine colon-specific delivery system. The drug release behaviors in vitro and pharmacokinetics and biodistribution in vivo were further evaluated. The mean particle diameters of mesalamine-coated microparticles were 312.2 µm. In vitro, a small amount of mesalamine was released in HCl at a pH of 1.2 and PBS medium at a pH of 7.4 for 5 hours, and 71% of the entrapped mesalamine was further released during the subsequent 20 hours of incubation. A greater area under the plasma concentration-time curve (AUC)0-t was obtained for the coated microparticles (1.9-fold) compared to the suspensions group, which indicated that the encapsulated mesalamine had mostly been absorbed in rats over the period of 12 hours. The AUC0-t of the coated microparticles in colon was 2.63-fold higher compared to the suspensions (P<0.05). Hence, mesalamine-coated microparticles are considered to maintain the drug concentration within target ranges for a long period of time.

Enzymes in Fermented Fish.

PubMed

Giyatmi; Irianto, H E

Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

SPECIFIC HEAT DATA ANALYSIS PROGRAM FOR THE IBM 704 DIGITAL COMPUTER

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

Roach, P.R.

1962-01-01

A computer program was developed to calculate the specific heat of a substance in the temperature range from 0.3 to 4.2 deg K, given temperature calibration data for a carbon resistance thermometer, experimental temperature drift, and heating period data. The speciftc heats calculated from these data are then fitted by a curve by the methods of least squares and the specific heats are corrected for the effect of the curvature of the data. The method, operation, program details, and program stops are discussed. A program listing is included. (M.C.G.)

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

PubMed

Schallmey, Marcus; Koopmeiners, Julia; Wells, Elizabeth; Wardenga, Rainer; Schallmey, Anett

2014-12-01

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Structural aspects of denitrifying enzymes.

PubMed

Moura, I; Moura, J J

2001-04-01

The reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide is the metabolic pathway usually known as denitrification, a key step in the nitrogen cycle. As observed for other elemental cycles, a battery of enzymes are utilized, namely the reductases for nitrate, nitrite, nitric oxide and nitrous oxide, as well as multiple electron donors that interact with these enzymes, in order to carry out the stepwise reactions that involve key intermediates. Because of the importance of this pathway (of parallel importance to the nitrogen-fixation pathway), efforts are underway to understand the structures of the participating enzymes and to uncover mechanistic aspects. Three-dimensional structures have been solved for the majority of these enzymes in the past few years, revealing the architecture of the active metal sites as well as global structural aspects, and possible mechanistic aspects. In addition, the recognition of specific electron-transfer partners raises important questions regarding specific electron-transfer pathways, partner recognition and control of metabolism.

Roles of germination-specific lytic enzymes CwlJ and SleB in Bacillus anthracis.

PubMed

Heffron, Jared D; Orsburn, Benjamin; Popham, David L

2009-04-01

The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJ1 or SleB. Compared to the wild type, germinating spores without CwlJ1 suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CwlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.

A Study in Enzyme Kinetics Using an Ion-Specific Electrode.

ERIC Educational Resources Information Center

Turchi, Sandra; And Others

1989-01-01

Describes an undergraduate biochemistry laboratory experiment on enzyme kinetics using the D-amino acid oxidase system and an ammonia electrode. Preparation of an ammonia standard curve, a sample preparation, and inhibition studies are discussed. (YP)

Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae.

PubMed

Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W; Gruber, Karl; Macheroux, Peter

2016-01-01

Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.

Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

PubMed

Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

2014-03-04

Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

Structure, function, and engineering of enzymes in isoflavonoid biosynthesis.

PubMed

Wang, Xiaoqiang

2011-03-01

Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.

A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome.

PubMed

Chung, Yo Kyung; Sohn, Young Bae; Sohn, Jong Mun; Lee, Jieun; Chang, Mi Sun; Kwun, Younghee; Kim, Chi Hwa; Lee, Jin Young; Yook, Yeon Joo; Ko, Ah-Ra; Jin, Dong-Kyu

2014-05-01

Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase(®), Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase(®), Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes.The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4 ± 0.9 vs. 68.1 ± 2.2 %, P < 0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6 ± 1.1 vs. 27.8 ± 0.9 nmol/min/μg protein, P < 0.001). The levels of M6P and sialic acid were not significantly different (2.4 ± 0.1 vs 2.4 ± 0.3 mol/mol protein for M6P and 12.3 ± 0.7 vs. 12.4 ± 0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09 ± 0.96 vs. 6.50 ± 1.28 nM protein, P = 0.017).In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.

Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin-converting enzyme

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

Muzykantov, V.R.; Puchnina, E.A.; Atochina, E.N.

The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats. Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema. The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion. In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v. injection. Endotoxin injection induces reduction of specific pulmonary uptake of this antibody. Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without anymore » change in accumulation of control nonspecific IgG. The antibody distribution in other organs and its blood level were almost the same as in the control. In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.« less

Specific starch digestion of maize alpha-limit dextrins by recombinant mucosal glucosidase enzymes

USDA-ARS?s Scientific Manuscript database

Starch digestion requires two luminal enzymes, salivary and pancreatic alpha-amylase (AMY), and four small intestinal mucosal enzyme activities from the N- and C-terminals of maltase-glucoamylase (MGAM) and sucrose-isomaltase (SI) complexes. AMY is not a requirement for starch digestion to glucose b...

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

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

Khadempour, Lily; Burnum-Johnson, Kristin E.; Baker, Erin S.

Herbivores use symbiotic microbes to help gain access to energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, having tremendous impact on their ecosystems as dominant generalist herbivores through cultivation of a fungus, Leucoagaricus gongylophorous. Here we examine how this mutualism could facilitate the flexible substrate incorporation of the ants by providing leaf-cutter ant subcolonies four substrate types: leaves, flowers, oats, and a mixture of all three. Through metaproteomic analysis of the fungus gardens, we were able to identify and quantify 1766 different fungal proteins, including 161 biomass-degrading enzymes. This analysis revealed that fungal protein profiles weremore » significantly different between subcolonies fed different substrates with the highest abundance of cellulolytic enzymes observed in the leaf and flower treatments. When the fungus garden is provided with leaves and flowers, which contain the majority of their energy in recalcitrant material, it increases its production of proteins that break down cellulose: endoglucanases, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, the mixed treatment closely resembled the treatment with oats alone. This suggests that when provided a mixture of substrates, the fungus garden preferentially produces enzymes necessary for breakdown of simpler, more digestible substrates. This flexible, substrate-specific response of the fungal cultivar allows the leaf-cutter ants to derive energy from a wide range of substrates, which may contribute to their ability to be dominant generalist herbivores.« less

A highly specific competitive direct enzyme immunoassay for sterigmatocystin as a tool for rapid immunochemotaxonomic differentiation of mycotoxigenic Aspergillus species.

PubMed

Wegner, S; Bauer, J I; Dietrich, R; Märtlbauer, E; Usleber, E; Gottschalk, C; Gross, M

2017-02-01

A simplified method to produce specific polyclonal rabbit antibodies against sterigmatocystin (STC) was established, using a STC-glycolic acid-ether derivative (STC-GE) conjugated to keyhole limpet haemocyanin (immunogen). The competitive direct enzyme immunoassay (EIA) established for STC had a detection limit (20% binding inhibition) of 130 pg ml -1 . The test was highly specific for STC, with minor cross-reactivity with O-methylsterigmatocystin (OMSTC, 0·87%) and negligible reactivity with aflatoxins (<0·02%). STC-EIA was used in combination with a previously developed specific EIA for aflatoxins (<0·1% cross-reactivity with STC and OMSTC), to study the STC/aflatoxin production profiles of reference strains of Aspergillus species. This immunochemotaxonomic procedure was found to be a convenient tool to identify STC- or aflatoxin-producing strains. The carcinogenic mycotoxin sterigmatocystin (STC) is produced by several Aspergillus species, either alone or together with aflatoxins. Here, we report a very simple and straightforward procedure to obtain highly sensitive and specific anti-STC antibodies, and their use in the first ever real STC-specific competitive direct enzyme immunoassay (EIA). In combination with a previous EIA for aflatoxins, this study for the first time demonstrates the potential of a STC/aflatoxin EIA pair for what is branded as 'immunochemotaxonomic' identification of mycotoxigenic Aspergillus species. This new analytical tool enhances analytical possibilities for differential analysis of STC and aflatoxins. © 2016 The Society for Applied Microbiology.

Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

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

Maltz, Lauren

By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure ofmore » the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.« less

Digestive Enzyme Supplementation in Gastrointestinal Diseases.

PubMed

Ianiro, Gianluca; Pecere, Silvia; Giorgio, Valentina; Gasbarrini, Antonio; Cammarota, Giovanni

2016-01-01

Digestive enzymes are able to break down proteins and carbohydrates and lipids, and their supplementation may play a role in the management of digestive disorders, from lactose intolerance to cystic fibrosis. To date, several formulations of digestive enzymes are available on the market, being different each other in terms of enzyme type, source and origin, and dosage. This review, performed through a non-systematic search of the available literature, will provide an overview of the current knowledge of digestive enzyme supplementation in gastrointestinal disorders, discussion of the use of pancreatic enzymes, lactase (β-galactosidase) and conjugated bile acids, and also exploring the future perspective of digestive enzyme supplementation. Currently, the animal-derived enzymes represent an established standard of care, however the growing study of plant-based and microbe-derived enzymes offers great promise in the advancement of digestive enzyme therapy. New frontiers of enzyme replacement are being evaluated also in the treatment of diseases not specifically related to enzyme deficiency, whereas the combination of different enzymes might constitute an intriguing therapeutic option in the future.

Advanced development of immobilized enzyme reactors

NASA Technical Reports Server (NTRS)

Jolly, Clifford D.; Schussel, Leonard J.; Carter, Layne

1991-01-01

Fixed-bed reactors have been used at NASA-Marshall to purify wastewater generated by an end-use equipment facility, on the basis of a combination of multifiltration unibeds and enzyme unibeds. The enzyme beds were found to effectively remove such targeted organics as urea, alcohols, and aldehydes, down to levels lying below detection limits. The enzyme beds were also found to remove organic contaminants not specifically targeted.

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.

Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes.

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

Zhang, R.; Evans, G.; Rotella, F.

1999-06-01

IMP dehydrogenase (IMPDH) is an essential enzyme of de novo guanine nucleotide synthesis. IMPDH inhibitors have clinical utility as antiviral, anticancer or immunosuppressive agents. The essential nature of this enzyme suggests its therapeutic applications may be extended to the development of antimicrobial agents. Bacterial IMPDH enzymes show bio- chemical and kinetic characteristics that are different than the mammalian IMPDH enzymes, suggesting IMPDH may be an attractive target for the development of antimicrobial agents. We suggest that the biochemical and kinetic differences between bacterial and mammalian enzymes are a consequence of the variance of specific, identifiable amino acid residues. Identification ofmore » these residues or combination of residues that impart this mammalian or bacterial enzyme signature is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. We used sequence alignments of IMPDH proteins to identify sequence signatures associated with bacterial or eukaryotic IMPDH enzymes. These selections were further refined to discern those likely to have a role in catalysis using information derived from the bacterial and mammalian IMPDH crystal structures and site-specific mutagenesis. Candidate bacterial sequence signatures identified by this process include regions involved in subunit interactions, the active site flap and the NAD binding region. Analysis of sequence alignments in these regions indicates a pattern of catalytic residues conserved in all enzymes and a secondary pattern of amino acid conservation associated with the major phylogenetic groups. Elucidation of the basis for this mammalian/bacterial IMPDH signature will provide insight into the catalytic mechanism of this enzyme and the foundation for the development of highly specific inhibitors.« less

Structural analysis of enzymes used for bioindustry and bioremediation.

PubMed

Tanokura, Masaru; Miyakawa, Takuya; Guan, Lijun; Hou, Feng

2015-01-01

Microbial enzymes have been widely applied in the large-scale, bioindustrial manufacture of food products and pharmaceuticals due to their high substrate specificity and stereoselectivity, and their effectiveness under mild conditions with low environmental burden. At the same time, bioremedial techniques using microbial enzymes have been developed to solve the problem of industrial waste, particularly with respect to persistent chemicals and toxic substances. And finally, structural studies of these enzymes have revealed the mechanistic basis of enzymatic reactions, including the stereoselectivity and binding specificity of substrates and cofactors. The obtained structural insights are useful not only to deepen our understanding of enzymes with potential bioindustrial and/or bioremedial application, but also for the functional improvement of enzymes through rational protein engineering. This review shows the structural bases for various types of enzymatic reactions, including the substrate specificity accompanying cofactor-controlled and kinetic mechanisms.

Biomimicry enhances sequential reactions of tethered glycolytic enzymes, TPI and GAPDHS.

PubMed

Mukai, Chinatsu; Gao, Lizeng; Bergkvist, Magnus; Nelson, Jacquelyn L; Hinchman, Meleana M; Travis, Alexander J

2013-01-01

Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.

Biomimicry Enhances Sequential Reactions of Tethered Glycolytic Enzymes, TPI and GAPDHS

PubMed Central

Mukai, Chinatsu; Gao, Lizeng; Bergkvist, Magnus; Nelson, Jacquelyn L.; Hinchman, Meleana M.; Travis, Alexander J.

2013-01-01

Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices. PMID:23626684

A novel pH–enzyme-dependent mesalamine colon-specific delivery system

PubMed Central

Jin, Lei; Ding, Yi-cun; Zhang, Yu; Xu, Xiao-qing; Cao, Qin

2016-01-01

The aim of the present study was to design a new pH–enzyme double-dependent mesalamine colon-specific delivery system. The drug release behaviors in vitro and pharmacokinetics and biodistribution in vivo were further evaluated. The mean particle diameters of mesalamine-coated microparticles were 312.2 µm. In vitro, a small amount of mesalamine was released in HCl at a pH of 1.2 and PBS medium at a pH of 7.4 for 5 hours, and 71% of the entrapped mesalamine was further released during the subsequent 20 hours of incubation. A greater area under the plasma concentration–time curve (AUC)0–t was obtained for the coated microparticles (1.9-fold) compared to the suspensions group, which indicated that the encapsulated mesalamine had mostly been absorbed in rats over the period of 12 hours. The AUC0–t of the coated microparticles in colon was 2.63-fold higher compared to the suspensions (P<0.05). Hence, mesalamine-coated microparticles are considered to maintain the drug concentration within target ranges for a long period of time. PMID:27382255

Changes in digestive enzyme activities during larval development of Chinese loach Paramisgurnus dabryanus (Dabry de Thiersant, 1872).

PubMed

Zhang, Yun-Long; Wu, Qiao-Wan; Hu, Wei-Hua; Wang, Fan; Zhao, Zhong-Bo; He, Hui; Shao, Wei-Han; Fan, Qi-Xue

2015-12-01

The digestive physiology of Chinese loach (Paramisgurnus dabryanus) was studied by assessing the specific and total activities of different pancreatic (trypsin, chymotrypsin, amylase and lipase), gastric (pepsin) and intestinal (alkaline phosphatase and leucine-aminopeptidase) enzymes from hatching to 40 days after hatching (DAH). Larvae were reared at 24.4 ± 0.4 °C and fed with rotifers from mouth opening (4 DAH) to 15 DAH, from 10 to 35 DAH with Cladocera and from 30 to 40 DAH with compound diet. Enzyme activities for trypsin, chymotrypsin, amylase and lipase were detected before the onset of exogenous feeding, indicating that these enzymes were genetically pre-programmed. Most of the pancreatic enzyme specific activities increased until 20 DAH and decreased thereafter. The pepsin activity of Chinese loach was firstly detected at 30 DAH, indicating the appearance of functional gastric gland. Alkaline phosphatase specific activity was detected from hatching onward, showed marked increase and reached the second peak at 20 DAH, while a gradual increase in specific leucine-aminopeptidase activity was observed until the end of the experiment. Accordingly, the larvae of Chinese loach possess a functional digestive system before the onset of exogenous feeding and the digestive capacity gradually increases as development progresses. The abrupt increase in intestinal enzyme activities between 10 and 20 DAH demonstrates onset of juvenile-like digestive mode in Chinese loach larvae. The increase in pepsin activity after 30 DAH indicates the shift from alkaline to acidic digestion in Chinese loach larvae, which may be considered as the onset of weaning.

Broad Specification Fuels Combustion Technology Program, Phase 2

NASA Technical Reports Server (NTRS)

Lohmann, R. P.; Jeroszko, R. A.; Kennedy, J. B.

1990-01-01

An experimental evaluation of two advanced technology combustor concepts was conducted to evolve and assess their capability for operation on broadened properties fuels. The concepts were based on the results of Phase 1 of the Broad Specification Fuel Combustor Technology Program which indicated that combustors with variable geometry or staged combustion zones had a flexibility of operation that could facilitate operation on these fuels. Emphasis in defining these concepts included the use of single pipe as opposed to duplex or staged fuels systems to avoid the risk of coking associated with the reduction in thermal stability expected in broadened properties fuels. The first concept was a variable geometry combustor in which the airflow into the primary zone could be altered through valves on the front while the second was an outgrowth of the staged Vorbix combustor, evolved under the NASA/P&W ECCP and EEE programs incorporating simplified fuel and air introduction. The results of the investigation, which involved the use of Experimental Referee Broad Specification (ERBS) fuel, indicated that in the form initially conceived, both of these combustor concepts were deficient in performance relative to many of the program goals for performance emissions. However, variations of both combustors were evaluated that incorporated features to simulate conceptual enhancement to demonstrate the long range potential of the combustor. In both cases, significant improvements relative to the program goals were observed.

Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

PubMed

Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

2014-01-01

During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

Dihydroflavonol 4-Reductase Genes Encode Enzymes with Contrasting Substrate Specificity and Show Divergent Gene Expression Profiles in Fragaria Species

PubMed Central

Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

2014-01-01

During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (K cat/K m values) of DFR1 combined with the loss of F3’H activity late in fruit development of F.×ananassa. PMID

Lessons Learned about Workplace Literacy from Military Job-Specific Reading Programs.

ERIC Educational Resources Information Center

Philippi, Jorie W.

For almost half a century the United States military services have incorporated formal literacy programs into job training for those enlistees who are less than fully qualified. Over the years, several successful job-specific reading programs have evolved. In 1975, the Army began the Functional Literacy (FLIT) Program, a program based on a…

Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura.

PubMed

Boswell, H D; Dräger, B; McLauchlan, W R; Portsteffen, A; Robins, D J; Robins, R J; Walton, N J

1999-11-01

The enzymes N-methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida x aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.

Effect of formal specifications on program complexity and reliability: An experimental study

NASA Technical Reports Server (NTRS)

Goel, Amrit L.; Sahoo, Swarupa N.

1990-01-01

The results are presented of an experimental study undertaken to assess the improvement in program quality by using formal specifications. Specifications in the Z notation were developed for a simple but realistic antimissile system. These specifications were then used to develop 2 versions in C by 2 programmers. Another set of 3 versions in Ada were independently developed from informal specifications in English. A comparison of the reliability and complexity of the resulting programs suggests the advantages of using formal specifications in terms of number of errors detected and fault avoidance.

Rational approaches for engineering novel functionalities in carbon-carbon bond forming enzymes

PubMed Central

Baker, Perrin; Seah, Stephen Y. K.

2012-01-01

Enzymes that catalyze carbon-carbon bond formation can be exploited as biocatalyst for synthetic organic chemistry. However, natural enzymes frequently do not possess the required properties or specificities to catalyze industrially useful transformations. This mini-review describes recent work using knowledge-guided site-specific mutagenesis of key active site residues to alter substrate specificity, stereospecificity and reaction specificity of these enzymes. In addition, examples of de novo designed enzymes that catalyze C-C bond reactions not found in nature will be discussed. PMID:24688644

Caenorhabditis elegans glutamylating enzymes function redundantly in male mating.

PubMed

Chawla, Daniel G; Shah, Ruchi V; Barth, Zachary K; Lee, Jessica D; Badecker, Katherine E; Naik, Anar; Brewster, Megan M; Salmon, Timothy P; Peel, Nina

2016-09-15

Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons. © 2016. Published by The Company of Biologists Ltd.

Antibody-capture enzyme-linked immunosorbent assays that use enzyme-labelled antigen for detection of virus-specific immunoglobulin M, A and G in patients with varicella or herpes zoster.

PubMed Central

van Loon, A. M.; van der Logt, J. T.; Heessen, F. W.; Heeren, M. C.; Zoll, J.

1992-01-01

Antibody-capture enzyme-linked immunosorbent assays (AC-ELISA) which use enzyme-labelled antigen were developed for detection of varicella-zoster virus-(VZV) specific IgM, IgA and IgG antibody in patients with varicella or herpes zoster and in sera from healthy individuals. All 18 patients with varicella developed a VZV-IgM and a VZV-IgG response, 17 also a VZV-IgA response. In contrast, all 19 patients with herpes zoster were shown to be positive for VZV-IgA whereas only 13 of these reacted positively for VZV-IgM. A VZV-IgM response was detected in only two sera from 100 healthy individuals and an IgA response in only one. The presence of virus-specific IgA and IgG in the cerebrospinal fluid as determined by AC-ELISA was a useful indicator of VZV infection of the central nervous system. By AC-ELISA, VZV-IgG was detected predominantly in sera from patients with acute or recent VZV infection. Only 14 sera from 100 healthy individuals were positive for VZV-IgG by AC-ELISA, whereas all were positive by an indirect ELISA. These results indicate that AC-ELISA's may be useful assays for determination for acute or recurrent VZV infection, but are not suitable for determination of past infection with this virus. PMID:1312479

Pericyclic reactions catalyzed by chorismate-utilizing enzymes

PubMed Central

Lamb, Audrey L.

2011-01-01

One of the fundamental questions of enzymology is how catalytic power is derived. This review focuses on recent developments in the structure-function relationships of chorismate-utilizing enzymes involved in siderophore biosynthesis to provide insight into the biocatalysis of pericyclic reactions. Specifically, salicylate synthesis by the two-enzyme pathway in Pseudomonas aeruginosa is examined. The isochorismate-pyruvate lyase is discussed in the context of its homologues, the chorismate mutases, and the isochorismate synthase is compared to its homologues in the MST-family (menaquinone, siderophore or tryptophan biosynthesis) of enzymes. The tentative conclusion is that the activities observed cannot be reconciled by inspection of the active site participants alone. Instead, individual activities must arise from unique dynamic properties of each enzyme that are tuned to promote specific chemistries. PMID:21823653

A phylogenetic analysis of normal modes evolution in enzymes and its relationship to enzyme function

PubMed Central

Lai, Jason; Jin, Jing; Kubelka, Jan; Liberles, David A.

2012-01-01

Since the dynamic nature of protein structures is essential for enzymatic function, it is expected that the functional evolution can be inferred from the changes in the protein dynamics. However, dynamics can also diverge neutrally with sequence substitution between enzymes without changes of function. In this study, a phylogenetic approach is implemented to explore the relationship between enzyme dynamics and function through evolutionary history. Protein dynamics are described by normal mode analysis based on a simplified harmonic potential force field applied to the reduced Cα representation of the protein structure while enzymatic function is described by Enzyme Commission (EC) numbers. Similarity of the binding pocket dynamics at each branch of the protein family’s phylogeny was analyzed in two ways: 1) explicitly by quantifying the normal mode overlap calculated for the reconstructed ancestral proteins at each end and 2) implicitly using a diffusion model to obtain the reconstructed lineage-specific changes in the normal modes. Both explicit and implicit ancestral reconstruction identified generally faster rates of change in dynamics compared with the expected change from neutral evolution at the branches of potential functional divergences for the alpha-amylase, D-isomer specific 2-hydroxyacid dehydrogenase, and copper-containing amine oxidase protein families. Normal modes analysis added additional information over just comparing the RMSD of static structures. However, the branch-specific changes were not statistically significant compared to background function-independent neutral rates of change of dynamic properties and blind application of the analysis would not enable prediction of changes in enzyme specificity. PMID:22651983

A phylogenetic analysis of normal modes evolution in enzymes and its relationship to enzyme function.

PubMed

Lai, Jason; Jin, Jing; Kubelka, Jan; Liberles, David A

2012-09-21

Since the dynamic nature of protein structures is essential for enzymatic function, it is expected that functional evolution can be inferred from the changes in protein dynamics. However, dynamics can also diverge neutrally with sequence substitution between enzymes without changes of function. In this study, a phylogenetic approach is implemented to explore the relationship between enzyme dynamics and function through evolutionary history. Protein dynamics are described by normal mode analysis based on a simplified harmonic potential force field applied to the reduced C(α) representation of the protein structure while enzymatic function is described by Enzyme Commission numbers. Similarity of the binding pocket dynamics at each branch of the protein family's phylogeny was analyzed in two ways: (1) explicitly by quantifying the normal mode overlap calculated for the reconstructed ancestral proteins at each end and (2) implicitly using a diffusion model to obtain the reconstructed lineage-specific changes in the normal modes. Both explicit and implicit ancestral reconstruction identified generally faster rates of change in dynamics compared with the expected change from neutral evolution at the branches of potential functional divergences for the α-amylase, D-isomer-specific 2-hydroxyacid dehydrogenase, and copper-containing amine oxidase protein families. Normal mode analysis added additional information over just comparing the RMSD of static structures. However, the branch-specific changes were not statistically significant compared to background function-independent neutral rates of change of dynamic properties and blind application of the analysis would not enable prediction of changes in enzyme specificity. Copyright © 2012 Elsevier Ltd. All rights reserved.

A chemogenomic analysis of the human proteome: application to enzyme families.

PubMed

Bernasconi, Paul; Chen, Min; Galasinski, Scott; Popa-Burke, Ioana; Bobasheva, Anna; Coudurier, Louis; Birkos, Steve; Hallam, Rhonda; Janzen, William P

2007-10-01

Sequence-based phylogenies (SBP) are well-established tools for describing relationships between proteins. They have been used extensively to predict the behavior and sensitivity toward inhibitors of enzymes within a family. The utility of this approach diminishes when comparing proteins with little sequence homology. Even within an enzyme family, SBPs must be complemented by an orthogonal method that is independent of sequence to better predict enzymatic behavior. A chemogenomic approach is demonstrated here that uses the inhibition profile of a 130,000 diverse molecule library to uncover relationships within a set of enzymes. The profile is used to construct a semimetric additive distance matrix. This matrix, in turn, defines a sequence-independent phylogeny (SIP). The method was applied to 97 enzymes (kinases, proteases, and phosphatases). SIP does not use structural information from the molecules used for establishing the profile, thus providing a more heuristic method than the current approaches, which require knowledge of the specific inhibitor's structure. Within enzyme families, SIP shows a good overall correlation with SBP. More interestingly, SIP uncovers distances within families that are not recognizable by sequence-based methods. In addition, SIP allows the determination of distance between enzymes with no sequence homology, thus uncovering novel relationships not predicted by SBP. This chemogenomic approach, used in conjunction with SBP, should prove to be a powerful tool for choosing target combinations for drug discovery programs as well as for guiding the selection of profiling and liability targets.

Conformational diversity and computational enzyme design

PubMed Central

Lassila, Jonathan K.

2010-01-01

The application of computational protein design methods to the design of enzyme active sites offers potential routes to new catalysts and new reaction specificities. Computational design methods have typically treated the protein backbone as a rigid structure for the sake of computational tractability. However, this fixed-backbone approximation introduces its own special challenges for enzyme design and it contrasts with an emerging picture of natural enzymes as dynamic ensembles with multiple conformations and motions throughout a reaction cycle. This review considers the impact of conformational variation and dynamics on computational enzyme design and it highlights new approaches to addressing protein conformational diversity in enzyme design including recent advances in multistate design, backbone flexibility, and computational library design. PMID:20829099

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

PubMed

Coitinho, Juliana B; Pereira, Mozart S; Costa, Débora M A; Guimarães, Samuel L; Araújo, Simara S; Hengge, Alvan C; Brandão, Tiago A S; Nagem, Ronaldo A P

2016-09-27

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis.

PubMed

Jones, Hannah B L; Wells, Stephen A; Prentice, Erica J; Kwok, Anthony; Liang, Liyin L; Arcus, Vickery L; Pudney, Christopher R

2017-09-01

Our understanding of how enzymes work is coloured by static structure depictions where the enzyme scaffold is presented as either immobile, or in equilibrium between well-defined static conformations. Proteins, however, exhibit a large degree of motion over a broad range of timescales and magnitudes and this is defined thermodynamically by the enzyme free energy landscape (FEL). The role and importance of enzyme motion is extremely contentious. Much of the challenge is in the experimental detection of so called 'conformational sampling' involved in enzyme turnover. Herein we apply combined pressure and temperature kinetics studies to elucidate the full suite of thermodynamic parameters defining an enzyme FEL as it relates to enzyme turnover. We find that the key thermodynamic parameters governing vibrational modes related to enzyme turnover are the isobaric expansivity term and the change in heat capacity for enzyme catalysis. Variation in the enzyme FEL affects these terms. Our analysis is supported by a range of biophysical and computational approaches that specifically capture information on protein vibrational modes and the FEL (all atom flexibility calculations, red edge excitation shift spectroscopy and viscosity studies) that provide independent evidence for our findings. Our data suggest that restricting the enzyme FEL may be a powerful strategy when attempting to rationally engineer enzymes, particularly to alter thermal activity. Moreover, we demonstrate how rational predictions can be made with a rapid computational approach. © 2017 Federation of European Biochemical Societies.

AMPHION: Specification-based programming for scientific subroutine libraries

NASA Technical Reports Server (NTRS)

Lowry, Michael; Philpot, Andrew; Pressburger, Thomas; Underwood, Ian; Waldinger, Richard; Stickel, Mark

1994-01-01

AMPHION is a knowledge-based software engineering (KBSE) system that guides a user in developing a diagram representing a formal problem specification. It then automatically implements a solution to this specification as a program consisting of calls to subroutines from a library. The diagram provides an intuitive domain oriented notation for creating a specification that also facilitates reuse and modification. AMPHION'S architecture is domain independent. AMPHION is specialized to an application domain by developing a declarative domain theory. Creating a domain theory is an iterative process that currently requires the joint expertise of domain experts and experts in automated formal methods for software development.

Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. II. Metabolic characteristics of the enzyme

NASA Technical Reports Server (NTRS)

Leznicki, A. J.; Bandurski, R. S.

1988-01-01

The synthesis of indole-3-acetyl-1-O-beta-D-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as rho-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed.

The unique functional role of the C-HS hydrogen bond in the substrate specificity and enzyme catalysis of type 1 methionine aminopeptidase.

PubMed

Reddi, Ravikumar; Singarapu, Kiran Kumar; Pal, Debnath; Addlagatta, Anthony

2016-07-19

It is intriguing how nature attains recognition specificity between molecular interfaces where there is no apparent scope for classical hydrogen bonding or polar interactions. Methionine aminopeptidase (MetAP) is one such enzyme where this fascinating conundrum is at play. In this study, we demonstrate that a unique C-HS hydrogen bond exists between the enzyme methionine aminopeptidase (MetAP) and its N-terminal-methionine polypeptide substrate, which allows specific interaction between apparent apolar interfaces, imposing a strict substrate recognition specificity and efficient catalysis, a feature replicated in Type I MetAPs across all kingdoms of life. We evidence this evolutionarily conserved C-HS hydrogen bond through enzyme assays on wild-type and mutant MetAP proteins from Mycobacterium tuberculosis that show a drastic difference in catalytic efficiency. The X-ray crystallographic structure of the methionine bound protein revealed a conserved water bridge and short contacts involving the Met side-chain, a feature also observed in MetAPs from other organisms. Thermal shift assays showed a remarkable 3.3 °C increase in melting temperature for methionine bound protein compared to its norleucine homolog, where C-HS interaction is absent. The presence of C-HS hydrogen bonding was also corroborated by nuclear magnetic resonance spectroscopy through a change in chemical shift. Computational chemistry studies revealed the unique role of the electrostatic environment in facilitating the C-HS interaction. The significance of this atypical hydrogen bond is underscored by the fact that the function of MetAP is essential for any living cell.

47 CFR 76.75 - Specific EEO program requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO... or sex is prohibited and that they may notify the Equal Employment Opportunity Commission, the... necessary. Nothing in this section shall be interpreted to require a multichannel video programming...

Strategic enzyme patterning for microfluidic biofuel cells

NASA Astrophysics Data System (ADS)

Kjeang, E.; Sinton, D.; Harrington, D. A.

The specific character of biological enzyme catalysts enables combined fuel and oxidant channels and simplified non-compartmentalized fuel cell assemblies. In this work, a microstructured enzymatic biofuel cell architecture is proposed, and species transport phenomena combined with consecutive chemical reactions are studied computationally in order to provide guidelines for optimization. This is the first computational study of this technology, and a 2D CFD model for species transport coupled with laminar fluid flow and Michaelis-Menten enzyme kinetics is established. It is shown that the system is reaction rate limited, indicating that enzyme specific turnover numbers are key parameters for biofuel cell performance. Separated and mixed enzyme patterns in different proportions are analyzed for various Peclet numbers. High fuel utilization is achieved in the diffusion dominated and mixed species transport regimes with separated enzymes arranged in relation to individual turnover rates. However, the Peclet number has to be above a certain threshold value to obtain satisfying current densities. The mixed transport regime is particularly attractive while current densities are maintained close to maximum levels. Optimum performance is achieved by mixed enzyme patterning tailored with respect to individual turnover rates, enabling high current densities combined with nearly complete fuel utilization.

Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

PubMed

Bi, Xiaodong; Liu, Zhen

2014-12-16

Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

Crystal Structures of the Helicobacter pylori MTAN Enzyme Reveal Specific Interactions between S-Adenosylhomocysteine and the 5'-Alkylthio Binding Subsite

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

Mishra, Vidhi; Ronning, Donald R.

2012-11-13

The bacterial 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzyme is a multifunctional enzyme that catalyzes the hydrolysis of the N-ribosidic bond of at least four different adenosine-based metabolites: S-adenosylhomocysteine (SAH), 5'-methylthioadenosine (MTA), 5'-deoxyadenosine (5'-DOA), and 6-amino-6-deoxyfutalosine. These activities place the enzyme at the hub of seven fundamental bacterial metabolic pathways: S-adenosylmethionine (SAM) utilization, polyamine biosynthesis, the purine salvage pathway, the methionine salvage pathway, the SAM radical pathways, autoinducer-2 biosynthesis, and menaquinone biosynthesis. The last pathway makes MTAN essential for Helicobacter pylori viability. Although structures of various bacterial and plant MTANs have been described, the interactions between the homocysteine moiety of SAH and themore » 5'-alkylthiol binding site of MTAN have never been resolved. We have determined crystal structures of an inactive mutant form of H. pylori MTAN bound to MTA and SAH to 1.63 and 1.20 Å, respectively. The active form of MTAN was also crystallized in the presence of SAH, allowing the determination of the structure of a ternary enzyme–product complex resolved at 1.50 Å. These structures identify interactions between the homocysteine moiety and the 5'-alkylthiol binding site of the enzyme. This information can be leveraged for the development of species-specific MTAN inhibitors that prevent the growth of H. pylori.« less

Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-.

PubMed

Tobe, Seiichi; Shimogaki, Hisao; Ohdera, Motoyasu; Asai, Yoshio; Oba, Kenkichi; Iwama, Masanori; Irie, Masachika

2006-01-01

An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA.

Multi-Enzyme Complexes in the Thermophilic Archaea: The Effects of Temperature on Stability, Catalysis and Enzyme Interactions in a Multi-Component System

DTIC Science & Technology

2012-01-01

COVERED (From - To) 4. TITLE AND SUBTITLE Multi- enzyme complexes in the thermophilic archaea: The effects of temperature on stability, catalysis and... enzyme interactions in a multi- component system 5a. CONTRACT NUMBER 5b. GRANT NUMBER FA9550-07-1-0058 5c. PROGRAM ELEMENT NUMBER 61102F 6...involves cloning of the genes for the relevant lipoylation enzymes , and characterisation of the protein products 15. SUBJECT TERMS 16. SECURITY

Alterations in Circulatory and Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 in Fetal Programmed Hypertension

PubMed Central

Shaltout, Hossam A.; Figueroa, Jorge P.; Rose, James C.; Diz, Debra I.; Chappell, Mark C.

2009-01-01

Antenatal betamethasone treatment is a widely accepted therapy to accelerate lung development and improve survival in preterm infants. However, there are reports that infants who receive antenatal glucocorticoids exhibit higher systolic blood pressure in their early adolescent years. We have developed an experimental model of programming whereby the offspring of pregnant sheep administered clinically relevant doses of betamethasone exhibit elevated blood pressure. We tested the hypothesis as to whether alterations in angiotensin-converting enzyme (ACE), ACE2, and neprilysin in serum, urine, and proximal tubules are associated with this increase in mean arterial pressure. Male sheep were administered betamethasone (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle at the 80th day of gestation and delivered at term. Sheep were instrumented at adulthood (1.8 years) for direct conscious recording of mean arterial pressure. Serum and urine were collected and proximal tubules isolated from the renal cortex. Betamethasone-treated animals had elevated mean arterial pressure (97±3 versus 83±2 mm Hg; P<0.05) and a 25% increase in serum ACE activity (48.4±7.0 versus 36.0±2.7 fmol/mL per minute) but a 40% reduction in serum ACE2 activity (18.8±1.2 versus 31.4±4.4 fmol/mL per minute). In isolated proximal tubules, ACE2 activity and expression were 50% lower in the treated sheep with no significant change in ACE or neprilysin activities. We conclude that antenatal steroid treatment results in the chronic alteration of ACE and ACE2 in the circulatory and tubular compartments, which may contribute to the higher blood pressure in this model of fetal programming-induced hypertension. PMID:19047579

The nodulation factor hydrolase of Medicago truncatula: characterization of an enzyme specifically cleaving rhizobial nodulation signals.

PubMed

Tian, Ye; Liu, Wei; Cai, Jie; Zhang, Lan-Yue; Wong, Kam-Bo; Feddermann, Nadja; Boller, Thomas; Xie, Zhi-Ping; Staehelin, Christian

2013-11-01

Nodule formation induced by nitrogen-fixing rhizobia depends on bacterial nodulation factors (NFs), modified chitin oligosaccharides with a fatty acid moiety. Certain NFs can be cleaved and inactivated by plant chitinases. However, the most abundant NF of Sinorhizobium meliloti, an O-acetylated and sulfated tetramer, is resistant to hydrolysis by all plant chitinases tested so far. Nevertheless, this NF is rapidly degraded in the host rhizosphere. Here, we identify and characterize MtNFH1 (for Medicago truncatula Nod factor hydrolase 1), a legume enzyme structurally related to defense-related class V chitinases (glycoside hydrolase family 18). MtNFH1 lacks chitinase activity but efficiently hydrolyzes all tested NFs of S. meliloti. The enzyme shows a high cleavage preference, releasing exclusively lipodisaccharides from NFs. Substrate specificity and kinetic properties of MtNFH1 were compared with those of class V chitinases from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), which cannot hydrolyze tetrameric NFs of S. meliloti. The Michaelis-Menten constants of MtNFH1 for NFs are in the micromolar concentration range, whereas nonmodified chitin oligosaccharides represent neither substrates nor inhibitors for MtNFH1. The three-dimensional structure of MtNFH1 was modeled on the basis of the known structure of class V chitinases. Docking simulation of NFs to MtNFH1 predicted a distinct binding cleft for the fatty acid moiety, which is absent in the class V chitinases. Point mutation analysis confirmed the modeled NF-MtNFH1 interaction. Silencing of MtNFH1 by RNA interference resulted in reduced NF degradation in the rhizosphere of M. truncatula. In conclusion, we have found a novel legume hydrolase that specifically inactivates NFs.

Differences in the Glucuronidation of Resveratrol and Pterostilbene: Altered Enzyme Specificity and Potential Gender Differences

PubMed Central

Dellinger, Ryan W.; Gomez Garcia, Angela M.; Meyskens, Frank L.

2015-01-01

Summary Resveratrol, a natural polyphenol found in grapes, berries and other plants, has been proposed as an ideal chemopreventative agent due to its plethora of health promoting activities. However, despite its lofty promise as a cancer prevention agent its success in human clinical trials has been limited due to its poor bioavailability. Thus, interest in other natural polyphenols is intensifying including the naturally occurring dimethylated analog of resveratrol, pterostilbene. The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the metabolism of both resveratrol and pterostilbene. The current study sought to elucidate the UGT family members responsible for the metabolism of pterostilbene and to examine gender differences in the glucuronidation of resveratrol and pterostilbene. We demonstrate that UGT1A1 and UGT1A3 are mainly responsible for pterostilbene glucuronidation although UGT1A8, UGT1A9 and UGT1A10 also had detectable activity. Intriguingly, UGT1A1 exhibits the highest activity against both resveratrol and pterostilbene despite altered hydroxyl group specificity. Using pooled human liver microsomes, enzyme kinetics were determined for pterostilbene and resveratrol glucuronides. In all cases females were more efficient than males, indicating potential gender differences in stilbene metabolism. Importantly, the glucuronidation of pterostilbene is much less efficient than that of resveratrol, indicating that pterostilbene will have dramatically decreased metabolism in humans. PMID:23965644

Applications of Microbial Enzymes in Food Industry.

PubMed

Raveendran, Sindhu; Parameswaran, Binod; Ummalyma, Sabeela Beevi; Abraham, Amith; Mathew, Anil Kuruvilla; Madhavan, Aravind; Rebello, Sharrel; Pandey, Ashok

2018-03-01

The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed.

Applications of Microbial Enzymes in Food Industry

PubMed Central

2018-01-01

Summary The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed. PMID:29795993

Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes.

PubMed

Merz, Michael; Eisele, Thomas; Berends, Pieter; Appel, Daniel; Rabe, Swen; Blank, Imre; Stressler, Timo; Fischer, Lutz

2015-06-17

Flavourzyme is sold as a peptidase preparation from Aspergillus oryzae. The enzyme preparation is widely and diversely used for protein hydrolysis in industrial and research applications. However, detailed information about the composition of this mixture is still missing due to the complexity. The present study identified eight key enzymes by mass spectrometry and partially by activity staining on native polyacrylamide gels or gel zymography. The eight enzymes identified were two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one α-amylase from the A. oryzae strain ATCC 42149/RIB 40 (yellow koji mold). Various specific marker substrates for these Flavourzyme enzymes were ascertained. An automated, time-saving nine-step protocol for the purification of all eight enzymes within 7 h was designed. Finally, the purified Flavourzyme enzymes were biochemically characterized with regard to pH and temperature profiles and molecular sizes.

The programming language HAL: A specification

NASA Technical Reports Server (NTRS)

1971-01-01

HAL accomplishes three significant objectives: (1) increased readability, through the use of a natural two-dimensional mathematical format; (2) increased reliability, by providing for selective recognition of common data and subroutines, and by incorporating specific data-protect features; (3) real-time control facility, by including a comprehensive set of real-time control commands and signal conditions. Although HAL is designed primarily for programming on-board computers, it is general enough to meet nearly all the needs in the production, verification and support of aerospace, and other real-time applications.

MAIL LOG, program summary and specifications

NASA Technical Reports Server (NTRS)

Harris, D. K.

1979-01-01

The summary and specifications to obtain the software package, MAIL LOG, developed for the Scout Project Automatic Data System, SPADS are provided. The MAIL LOG program has four modes of operation: (1) input - putting new records into the data base; (2) revise - changing or modifying existing records in the data base; (3) search - finding special records existing in the data base; and (4) archive - store or put away existing records in the data base. The output includes special printouts of records in the data base and results from the input and search modes.

Quantitative measurement of human thyroglobulin-specific antibodies by use of a sensitive enzyme-linked immunoassay.

PubMed

Kuppers, R C; Outschoorn, I M; Hamilton, R G; Burek, C L; Rose, N R

1993-04-01

A quantitative enzyme-linked immunoassay that measures in absolute terms the subclass concentration of human thyroglobulin (huTg)-specific IgG autoantibody was developed. Unique to this study was the use of an affinity-purified anti-huTg standard with a known concentration of the four IgG subclasses. The sensitivity of the ELISA assay was 1-5 ng/ml depending on the IgG subclass being measured. We examined 22 sera of patients with autoimmune thyroid disease. The total huTg-specific antibody concentrations in serum ranged from 0 to nearly 3000 micrograms/ml of IgG. The IgG subclass distribution in individuals with low huTg-specific IgG (< 10 micrograms/ml) was primarily IgG1 and IgG3 Ab. Patients with intermediate levels of huTg IgG (10-600 micrograms/ml) expressed all four subclasses; however, no particular subclass was dominant. Individuals with > 1000 micrograms/ml also showed huTg-Ab in all four subclasses, however, IgG1 and IgG2 were dominant. All four IgG subclasses were used in the response to huTg, although the pattern of usage varied between individuals. There was no dominant subclass usage seen in this patient population.

A Functional-Notional Approach for English for Specific Purposes (ESP) Programs.

ERIC Educational Resources Information Center

Kim, Young-Min

English for Specific Purposes (ESP) programs, characterized by the special needs of the language learners, are described and a review of the literature on a functional-notional approach to the syllabus design of ESP programs is presented. It is suggested that effective ESP programs should teach the language skills necessary to function and perform…

Phage lytic enzymes: a history.

PubMed

Trudil, David

2015-02-01

There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

A DNA enzyme with N-glycosylase activity

NASA Technical Reports Server (NTRS)

Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

2000-01-01

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

Modified kinetics of enzymes interacting with nanoparticles

NASA Astrophysics Data System (ADS)

Díaz, Sebastián. A.; Breger, Joyce C.; Malanoski, Anthony; Claussen, Jonathan C.; Walper, Scott A.; Ancona, Mario G.; Brown, Carl W.; Stewart, Michael H.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

2015-08-01

Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.

Detergents as selective inhibitors and inactivators of enzymes.

PubMed

Vincenzini, M T; Favilli, F; Stio, M; Vanni, P; Treves, C

1985-01-01

In order to study the detergent-enzyme interaction and to clarify whether such an interaction produces specific or non-specific effects, we investigated the action of natural and synthetic detergents on enzymatic systems of different levels of complexity (crystalline enzymes, crude homogenates, organ preparations, organisms in toto i.e. rats and germinating seeds). The enzyme-detergent interaction was examined both as a time-independent phenomenon (inhibition) and as a time-dependent phenomenon (inactivation). In in vitro experiments a clear inhibition of pyridine-dependent dehydrogenases by long-chain anionic detergents was found. Cationic detergents have their greatest effect on lipase, LDH, MDH and ICDH from rat liver homogenates. At low concentrations SDS inactivates all the dehydrogenase enzymes studied. With high concentrations (10 mM) of SDS and dodecyltrimethylammonium bromide (C12), there was a sharp and non-specific decrease of enzymatic activities. In the in vivo studies, rats were given detergents to drink; the cationic detergent (C12) was far more effective than SDS with enzymes from both intestine and liver homogenates. SDS and C12 do not seem to interfere with enzyme activities at the beginning of the germination of Pinus pinea and Triticum durum seeds. However a marked reduction of activities does occur at the respective maximum germination times of these seeds. The nonionic detergent is ineffective both as inhibitor and as inactivator.

Micellar Polymer Encapsulation of Enzymes.

PubMed

Besic, Sabina; Minteer, Shelley D

2017-01-01

Although enzymes are highly efficient and selective catalysts, there have been problems incorporating them into fuel cells. Early enzyme-based fuel cells contained enzymes in solution rather than immobilized on the electrode surface. One problem utilizing an enzyme in solution is an issue of transport associated with long diffusion lengths between the site of bioelectrocatalysis and the electrode. This issue drastically decreases the theoretical overall power output due to the poor electron conductivity. On the other hand, enzymes immobilized at the electrode surface have eliminated the issue of poor electron conduction due to close proximity of electron transfer between electrode and the biocatalyst. Another problem is inefficient and short term stability of catalytic activity within the enzyme that is suspended in free flowing solution. Enzymes in solutions are only stable for hours to days, whereas immobilized enzymes can be stable for weeks to months and now even years. Over the last decade, there has been substantial research on immobilizing enzymes at electrode surfaces for biofuel cell and sensor applications. The most commonly used techniques are sandwich or wired. Sandwich techniques are powerful and successful for enzyme immobilization; however, the enzymes optimal activity is not retained due to the physical distress applied by the polymer limiting its applications as well as the non-uniform distribution of the enzyme and the diffusion of analyte through the polymer is slowed significantly. Wired techniques have shown to extend the lifetime of an enzyme at the electrode surface; however, this technique is very hard to master due to specific covalent bonding of enzyme and polymer which changes the three-dimensional configuration of enzyme and with that decreases the optimal catalytic activity. This chapter details encapsulation techniques where an enzyme will be immobilized within the pores/pockets of the hydrophobically modified micellar polymers such as

Evaluation of an enzyme immunoassay system for measuring herpes simplex virus (HSV) type 1-specific and HSV type 2-specific IgG antibodies.

PubMed

Prince, H E; Ernst, C E; Hogrefe, W R

2000-01-01

MRL Diagnostics has developed a dual enzyme immunoassay (EIA) system that employs the recombinant Herpes Simplex Virus (HSV) type-specific glycoproteins G1 (HSV1) and G2 (HSV2) to detect HSV type-specific IgG antibodies. This system was evaluated using 155 consecutive sera previously tested in a conventional dual EIA system (Zeus) that employs multiple HSV1 and HSV2 proteins to detect type-common as well as type-specific antibodies. Sera were also analyzed by Western blot to determine the true HSV type-specific IgG reactivity pattern. Of 110 sera giving concordant reactivity patterns in the MRL and Zeus EIA systems, 108 (98%) also displayed concordant Western blot patterns; two sera gave false positive HSV2 reactivity in both EIA systems. Of 45 sera giving discordant MRL and Zeus EIA reactivity patterns, 41 (91%) displayed a Western blot reactivity pattern that matched the MRL reactivity pattern. Both the HSV1 IgG component and the HSV2 IgG component of the MRL EIA system were 100% sensitive and > 95% specific. In contrast, the Zeus HSV1 IgG EIA was 98% sensitive and 79% specific, and the Zeus HSV2 IgG EIA was 85% sensitive and 79% specific. An analysis of the distribution of index values in the MRL EIA system showed that low-positive values (1.0-3.0) were rare, but, when detected, often represented false positive results; only 11 MRL low-positive results were observed, but all 6 MRL false positive results were found within this low-positive subgroup. These findings show that the MRL dual EIA system effectively detects HSV type-specific IgG antibodies. Copyright 2000 Wiley-Liss, Inc.

Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization

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

Kim, Moon Il; Kim, Jungbae; Lee, Jinwoo

2007-02-01

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shakingmore » condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.« less

Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression.

PubMed

Ziesack, Marika; Rollins, Nathan; Shah, Aashna; Dusel, Brendon; Webster, Gordon; Silver, Pamela A; Way, Jeffrey C

2018-05-15

Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 ( Cp FatB1) (C 8 specificity, lower activity) and Cp FatB2 (C 14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Notably, chimera 4 exhibited an increased C 8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify Cp FatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a "positive patch" within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from Cp FatB1 into Cp FatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Thus, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control. IMPORTANCE Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other

Non-homologous isofunctional enzymes: a systematic analysis of alternative solutions in enzyme evolution.

PubMed

Omelchenko, Marina V; Galperin, Michael Y; Wolf, Yuri I; Koonin, Eugene V

2010-04-30

Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins. We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE) that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. These results indicate that at least some of the non-homologous isofunctional enzymes were recruited relatively recently from enzyme families that are active against related substrates and are sufficiently flexible to accommodate changes in substrate specificity.

SLS-SPEC-159 Cross-Program Design Specification for Natural Environments (DSNE) Revision D

NASA Technical Reports Server (NTRS)

Roberts, Barry C.

2015-01-01

This document is derived from the former National Aeronautics and Space Administration (NASA) Constellation Program (CxP) document CxP 70023, titled "The Design Specification for Natural Environments (DSNE), Revision C." The original document has been modified to represent updated Design Reference Missions (DRMs) for the NASA Exploration Systems Development (ESD) Programs. The DSNE completes environment-related specifications for architecture, system-level, and lower-tier documents by specifying the ranges of environmental conditions that must be accounted for by NASA ESD Programs. To assure clarity and consistency, and to prevent requirements documents from becoming cluttered with extensive amounts of technical material, natural environment specifications have been compiled into this document. The intent is to keep a unified specification for natural environments that each Program calls out for appropriate application. This document defines the natural environments parameter limits (maximum and minimum values, energy spectra, or precise model inputs, assumptions, model options, etc.), for all ESD Programs. These environments are developed by the NASA Marshall Space Flight Center (MSFC) Natural Environments Branch (MSFC organization code: EV44). Many of the parameter limits are based on experience with previous programs, such as the Space Shuttle Program. The parameter limits contain no margin and are meant to be evaluated individually to ensure they are reasonable (i.e., do not apply unrealistic extreme-on-extreme conditions). The natural environments specifications in this document should be accounted for by robust design of the flight vehicle and support systems. However, it is understood that in some cases the Programs will find it more effective to account for portions of the environment ranges by operational mitigation or acceptance of risk in accordance with an appropriate program risk management plan and/or hazard analysis process. The DSNE is not intended

Screening of white-rot fungi manganese peroxidases: a comparison between the specific activities of the enzyme from different native producers

PubMed Central

2012-01-01

In this study manganese peroxidase (MnP) enzymes from selected white-rot fungi were isolated and compared for potential future recombinant production. White-rot fungi were cultivated in small-scale in liquid media and a simplified process was established for the purification of extracellular enzymes. Five lignin degrading organisms were selected (Bjerkandera sp., Phanerochaete (P.) chrysosporium, Physisporinus (P.) rivulosus, Phlebia (P.) radiata and Phlebia sp. Nf b19) and studied for MnP production in small-scale. Extracellular MnP activity was followed and cultivations were harvested at proximity of the peak activity. The production of MnPs varied in different organisms but was clearly regulated by inducing liquid media components (Mn2+, veratryl alcohol and malonate). In total 8 different MnP isoforms were purified. Results of this study reinforce the conception that MnPs from distinct organisms differ substantially in their properties. Production of the extracellular enzyme in general did not reach a substantial level. This further suggests that these native producers are not suitable for industrial scale production of the enzyme. The highest specific activities were observed with MnPs from P. chrysosporium (200 U mg-1), Phlebia sp. Nf b19 (55 U mg-1) and P. rivulosus (89 U mg-1) and these MnPs are considered as the most potential candidates for further studies. The molecular weight of the purified MnPs was estimated to be between 45–50 kDa. PMID:23190610

Employer Specific Training Program for Program Year 1988-89. Annual Report to the Governor and Legislature.

ERIC Educational Resources Information Center

New York State Education Dept., Albany.

The Employer Specific Skills Training Program helps build the superior work force called for by the National Alliance of Business and other significant employer, union, government, and educational groups. Through a combination of state and federal funds, the New York State Department of Education has crafted a flexible and responsible program.…

Measurement of Enzyme Isotope Effects.

PubMed

Kholodar, Svetlana A; Ghosh, Ananda K; Kohen, Amnon

2017-01-01

Enzyme isotope effects, or the kinetic effects of "heavy" enzymes, refer to the effect of isotopically labeled protein residues on the enzyme's activity or physical properties. These effects are increasingly employed in the examination of the possible contributions of protein dynamics to enzyme catalysis. One hypothesis assumed that isotopic substitution of all 12 C, 14 N, and nonexchangeable 1 H by 13 C, 15 N, and 2 H, would slow down protein picosecond to femtosecond dynamics without any effect on the system's electrostatics following the Born-Oppenheimer approximation. It was suggested that reduced reaction rates reported for several "heavy" enzymes accords with that hypothesis. However, numerous deviations from the predictions of that hypothesis were also reported. Current studies also attempt to test the role of individual residues by site-specific labeling or by labeling a pattern of residues on activity. It appears that in several systems the protein's fast dynamics are indeed reduced in "heavy" enzymes in a way that reduces the probability of barrier crossing of its chemical step. Other observations, however, indicated that slower protein dynamics are electrostatically altered in isotopically labeled enzymes. Interestingly, these effects appear to be system dependent, thus it might be premature to suggest a general role of "heavy" enzymes' effect on catalysis. © 2017 Elsevier Inc. All rights reserved.

Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

PubMed

Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H

2011-02-17

Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.

Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

PubMed

Tóth, Eszter; Huszár, Krisztina; Bencsura, Petra; Kulcsár, Péter István; Vodicska, Barbara; Nyeste, Antal; Welker, Zsombor; Tóth, Szilvia; Welker, Ervin

2014-01-01

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease--a Body Double of the Type IIP enzyme--is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.

Recent advances in rational approaches for enzyme engineering

PubMed Central

Steiner, Kerstin; Schwab, Helmut

2012-01-01

Enzymes are an attractive alternative in the asymmetric syntheses of chiral building blocks. To meet the requirements of industrial biotechnology and to introduce new functionalities, the enzymes need to be optimized by protein engineering. This article specifically reviews rational approaches for enzyme engineering and de novo enzyme design involving structure-based approaches developed in recent years for improvement of the enzymes’ performance, broadened substrate range, and creation of novel functionalities to obtain products with high added value for industrial applications. PMID:24688651

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity.

PubMed

Zhou, Bangjun; Mural, Ravi V; Chen, Xuanyang; Oates, Matt E; Connor, Richard A; Martin, Gregory B; Gough, Julian; Zeng, Lirong

2017-02-01

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart. © 2017 American Society of Plant Biologists. All Rights Reserved.

(Re-)programming of subtype specific cardiomyocytes.

PubMed

Hausburg, Frauke; Jung, Julia Jeannine; Hoch, Matti; Wolfien, Markus; Yavari, Arash; Rimmbach, Christian; David, Robert

2017-10-01

Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel use of a N2-specific enzyme-linked immunosorbent assay for differentiation of infected from vaccinated animals (DIVA)-based identification of avian influenza.

PubMed

Kwon, Ji-Sun; Kim, Min-Chul; Jeong, Ok-Mi; Kang, Hyun-Mi; Song, Chang-Seon; Kwon, Jun-Hun; Lee, Youn-Jeong

2009-05-21

Proper vaccination with validated companion differentiation of infected from vaccinated animals (DIVA) tests using a vaccine containing a heterologous neuraminidase to the field virus can be effective to control avian influenza (AI). However, indirect immunofluorescent assay, the only field validated DIVA test, has limitations to be set up as high throughput screening test and the assay requires subjective interpretation of the results. To apply the DIVA strategy to the Korean H9N2 low pathogenic AI (LPAI) vaccine program and overcome these limitations, we generated a reassortant H9N8 virus (rgH9N8) vaccine using plasmid-based reverse genetics and developed a novel N2-specific enzyme-linked immunosorbent assay (N2-ELISA). The rgH9N8 vaccine showed adequate immunogenicity and protection, and the optimized N2-ELISA showed that the sensitivity was 97.0% and specificity was 96.4% compared with a hemagglutination inhibition test. In vaccination-challenge experiments in specific pathogen-free chickens, the sera of chickens vaccinated with rgH9N8 vaccine and uninfected were negative by the N2-ELISA (S/P< or =0.4), whereas infected sera with H9N2 were positive (S/P>0.4). These results suggest that the rgH9N8 vaccine and the companion DIVA test, N2-ELISA, allow the utilization of the DIVA strategy for the control of H9N2 LPAI infections in Korea.

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme

PubMed Central

Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P. C.; Ovaa, Huib; Drag, Marcin; Lima, Christopher D.; Huang, Tony T.

2015-01-01

Ubiquitin (Ub) and the ubiquitin-like modifier interferon stimulated gene 15 (ISG15) participate in the host defense of viral infections. Viruses, including the Severe Acute Respiratory Syndrome human coronavirus (SARS hCoV), have co-opted Ub/ISG15-conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub/ISG15-conjugated host proteins. Here, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle Eastern Respiratory Syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that similar to SARS PLpro, MERS PLpro is both a deubiquitinating and a deISGylating enzyme. Further analysis of the intrinsic deubiquitinating enzyme (DUB) activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, while SARS PLpro prefers to cleave Lys48-linked polyUb chains. Second, MERS PLpro cleaves polyUb chains in a “mono-distributive” manner (one Ub at a time), and SARS PLpro prefers to cleave K48-linked poly-Ub chains by sensing a di-Ub moiety as a minimal recognition element using a “di-distributive” cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses. PMID:25764917

Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

PubMed Central

Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

2011-01-01

Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high

The roles of ubiquitin modifying enzymes in neoplastic disease.

PubMed

Kumari, Nishi; Jaynes, Patrick William; Saei, Azad; Iyengar, Prasanna Vasudevan; Richard, John Lalith Charles; Eichhorn, Pieter Johan Adam

2017-12-01

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFβ, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

Lignocellulose-degrading enzymes from termites and their symbiotic microbiota.

PubMed

Ni, Jinfeng; Tokuda, Gaku

2013-11-01

Lignocellulose-the dry matter of plants, or "plant biomass"-digestion is of increasing interest in organismal metabolism research, specifically the conversion of biomass into biofuels. Termites efficiently decompose lignocelluloses, and studies on lignocellulolytic systems may elucidate mechanisms of efficient lignocellulose degradation in termites as well as offer novel enzyme sources, findings which have significant potential industrial applications. Recent progress in metagenomic and metatranscriptomic research has illuminated the diversity of lignocellulolytic enzymes within the termite gut. Here, we review state-of-the-art research on lignocellulose-degrading systems in termites, specifically cellulases, xylanases, and lignin modification enzymes produced by termites and their symbiotic microbiota. We also discuss recent investigations into heterologous overexpression of lignocellulolytic enzymes from termites and their symbionts. Copyright © 2013 Elsevier Inc. All rights reserved.

Enzymes in bast fibrous plant processing.

PubMed

Kozlowski, Ryszard; Batog, Jolanta; Konczewicz, Wanda; Mackiewicz-Talarczyk, Maria; Muzyczek, Malgorzata; Sedelnik, Natalia; Tanska, Bogumila

2006-05-01

The program COST Action 847 Textile Quality and Biotechnology (2000-2005) has given an excellent chance to review the possibilities of the research, aiming at development of the industrial application of enzymes for bast fibrous plant degumming and primary processing. The recent advancements in enzymatic processing of bast fibrous plants (flax, hemp, jute, ramie and alike plants) and related textiles are given. The performance of enzymes in degumming, modification of bast fibres, roving, yarn, related fabrics as well as enzymatic bonding of lignocellulosic composites is provided.

Enzyme-Mediated Individual Nanoparticle Release Assay

PubMed Central

Glass, James R.; Dickerson, Janet C.; Schultz, David A.

2007-01-01

Numerous methods have been developed to measure the presence of macromolecular species in a sample, however methods that detect functional activity, or modulators of that activity are more limited. To address this limitation, an approach was developed that utilizes the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here we describe their use in a release assay format where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time is described. This approach was used to detect and discriminate between non-methylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzyme activity. PMID:16620746

Micromotors Powered by Enzyme Catalysis.

PubMed

Dey, Krishna K; Zhao, Xi; Tansi, Benjamin M; Méndez-Ortiz, Wilfredo J; Córdova-Figueroa, Ubaldo M; Golestanian, Ramin; Sen, Ayusman

2015-12-09

Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.

Elucidating sequence and solvent specific design targets to protect and stabilize enzymes for biocatalysis in ionic liquids.

PubMed

Sprenger, K G; Plaks, J G; Kaar, J L; Pfaendtner, J

2017-07-05

For many different frameworks, the structure, function, and dynamics of an enzyme is largely determined by the nature of its interactions with the surrounding host environment, thus a molecular level understanding of enzyme/host interactions is essential to the design of new processes and applications. Ionic liquid (IL) solvents are a popular class of solvents in which to study enzyme behavior, yet it is still not possible to predict how a given enzyme will behave in a given IL solvent. Furthermore, a dearth of experimental data with which to evaluate simulation force fields has prevented the full integration of experimental and computational techniques to gain a complete picture of enzyme/IL interactions. Utilizing recently published crystallographic data of an enzyme in complex with an IL, this study aims to validate the use of current molecular force fields for studying enzyme/IL interactions, and to provide new mechanistic insight into enzyme stabilization in IL solvents. Classical molecular dynamics (MD) simulations have been performed on both the folded and unfolded state of Bacillus subtilis lipase A and a quadruple-mutant version of lipase A, in solutions of aqueous 1-butyl-3-methylimidazolium chloride. Results show classical MD simulations can predict the preferred surface binding locations of IL cations as well as reductions in IL anion binding to mutated surface residues with high accuracy. The results also point to a mechanistic difference between IL binding to the folded and unfolded state of an enzyme, which we call the "counter-ion effect". These findings could have important implications for future rational design efforts to stabilize enzymes in non-conventional media.

Rennin--a Neglected Enzyme?

ERIC Educational Resources Information Center

Gill, John; Saunders, Terry

1987-01-01

Presents investigations to explore the substrate specificity, pH, concentration, and temperature relations of an enzyme with only inexpensive commercial rennet and basic laboratory equipment. Describes how the activities were carried out with a group of 15-year-old students. (CW)

21 CFR 184.1387 - Lactase enzyme preparation from Candida pseudotropicalis.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Lactase enzyme preparation from Candida... Specific Substances Affirmed as GRAS § 184.1387 Lactase enzyme preparation from Candida pseudotropicalis. (a) This enzyme preparation is derived from the nonpathogenic, nontoxicogenic yeast C...

21 CFR 184.1924 - Urease enzyme preparation from Lactobacillus fermentum.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Urease enzyme preparation from Lactobacillus... Specific Substances Affirmed as GRAS § 184.1924 Urease enzyme preparation from Lactobacillus fermentum. (a) This enzyme preparation is derived from the nonpathogenic, nontoxicogenic bacterium Lactobacillus...

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No...

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No...

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No...

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No...

Functional characterization and expression of GASCL1 and GASCL2, two anther-specific chalcone synthase like enzymes from Gerbera hybrida.

PubMed

Kontturi, Juha; Osama, Raisa; Deng, Xianbao; Bashandy, Hany; Albert, Victor A; Teeri, Teemu H

2017-02-01

The chalcone synthase superfamily consists of type III polyketidesynthases (PKSs), enzymes responsible for producing plant secondary metabolites with various biological and pharmacological activities. Anther-specific chalcone synthase-like enzymes (ASCLs) represent an ancient group of type III PKSs involved in the biosynthesis of sporopollenin, the main component of the exine layer of moss spores and mature pollen grains of seed plants. In the latter, ASCL proteins are localized in the tapetal cells of the anther where they participate in sporopollenin biosynthesis and exine formation within the locule. It is thought that the enzymes responsible for sporopollenin biosynthesis are highly conserved, and thus far, each angiosperm species with a genome sequenced has possessed two ASCL genes, which in Arabidopsis thaliana are PKSA and PKSB. The Gerbera hybrida (gerbera) PKS protein family consists of three chalcone synthases (GCHS1, GCHS3 and GCHS4) and three 2-pyrone synthases (G2PS1, G2PS2 and G2PS3). In previous studies we have demonstrated the functions of chalcone synthases in flavonoid biosynthesis, and the involvement of 2-pyrone synthases in the biosynthesis of antimicrobial compounds found in gerbera. In this study we expanded the gerbera PKS-family by functionally characterizing two gerbera ASCL proteins. In vitro enzymatic studies using purified recombinant proteins showed that both GASCL1 and GASCL2 were able to use medium and long-chain acyl-CoA starters and perform two to three condensation reactions of malonyl-CoA to produce tri- and tetraketide 2-pyrones, usually referred to as alpha-pyrones in sporopollenin literature. Both GASCL1 and GASCL2 genes were expressed only in floral organs, with most expression observed in anthers. In the anthers, transcripts of both genes showed strict tapetum-specific localization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Using the SCR Specification Technique in a High School Programming Course.

ERIC Educational Resources Information Center

Rosen, Edward; McKim, James C., Jr.

1992-01-01

Presents the underlying ideas of the Software Cost Reduction (SCR) approach to requirements specifications. Results of applying this approach to the teaching of programing to high school students indicate that students perform better in writing programs. An appendix provides two examples of how the method is applied to problem solving. (MDH)

NASIS data base management system: IBM 360 TSS implementation. Volume 4: Program design specifications

NASA Technical Reports Server (NTRS)

1973-01-01

The design specifications for the programs and modules within the NASA Aerospace Safety Information System (NASIS) are presented. The purpose of the design specifications is to standardize the preparation of the specifications and to guide the program design. Each major functional module within the system is a separate entity for documentation purposes. The design specifications contain a description of, and specifications for, all detail processing which occurs in the module. Sub-models, reference tables, and data sets which are common to several modules are documented separately.

Solvent effects on enzymes - Implications for extraterrestrial life.

NASA Technical Reports Server (NTRS)

Heinrich, M. R.

1972-01-01

Review of several studies on the alterations taking place in the structure, catalytic activity, specificity, and stability of an enzyme when some or all of the water in the medium is replaced by another solvent. These studies show the utility of solvents as a tool for probing enzyme function. They also suggest that solvents other than water should be investigated as media for controlling and directing enzyme reactions.

Can the national surgical quality improvement program provide surgeon-specific outcomes?

PubMed

Kuhnen, Angela H; Marcello, Peter W; Roberts, Patricia L; Read, Thomas E; Schoetz, David J; Rusin, Lawrence C; Hall, Jason F; Ricciardi, Rocco

2015-02-01

Efforts to improve the quality of surgical care and reduce morbidity and mortality have resulted in outcomes reporting at the service and institutional level. Surgeon-specific outcomes are not readily available. The aim of this study is to compare surgeon-specific outcomes from the National Surgical Quality Improvement Program and 100% capture institutional quality data. We conducted a cohort study evaluating institutional and surgeon-specific outcomes following colorectal surgery procedures at 1 institution over 5 years. All patients who underwent an operation by a colorectal surgeon at Lahey Hospital & Medical Center from January 1, 2008 through December 31, 2012 were identified. Thirty-day mortality, reoperation, urinary tract infection, deep vein thrombosis, pneumonia, superficial surgical site infection, and organ space infection were the primary outcomes measured. We compared annual and 5-year institutional and surgeon-specific adverse event rates between the data sets. In addition, we categorized individual surgeons as low-outlier, average, or high-outlier in relation to aggregate averages and determined the concordance between the data sets in identifying outliers. Concordance was designated if the 2 databases classified outlier status similarly for the same adverse event category. In the 100% capture institutional data, 6459 operative encounters were identified in comparison with 1786 National Surgical Quality Improvement Program encounters (28% sampled). Annual aggregate adverse event rates were similar between the institutional data and the National Surgical Quality Improvement Program. For annual surgeon-specific comparisons, concordance in identifying outliers between the 2 data sets was 51.4%, and gross discordance between outlier status was in 8.2%. Five-year surgeon-specific comparisons demonstrated 59% concordance in identifying outlier status with 8.2% gross discordance for the group. The inclusion of data from only 1 academic referral center is a

Substrate-driven chemotactic assembly in an enzyme cascade.

PubMed

Zhao, Xi; Palacci, Henri; Yadav, Vinita; Spiering, Michelle M; Gilson, Michael K; Butler, Peter J; Hess, Henry; Benkovic, Stephen J; Sen, Ayusman

2018-03-01

Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

Substrate-driven chemotactic assembly in an enzyme cascade

NASA Astrophysics Data System (ADS)

Zhao, Xi; Palacci, Henri; Yadav, Vinita; Spiering, Michelle M.; Gilson, Michael K.; Butler, Peter J.; Hess, Henry; Benkovic, Stephen J.; Sen, Ayusman

2018-03-01

Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

Compounds from Silicones Alter Enzyme Activity in Curing Barnacle Glue and Model Enzymes

PubMed Central

Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H.

2011-01-01

Background Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. Methodology/Principal Findings GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Conclusions/Significance Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management. PMID:21379573

21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Lipase enzyme preparation derived from Rhizopus... Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No. 9001-62-1), which is obtained from the...

Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity

PubMed Central

Mazzoni, Esteban O; Mahony, Shaun; Closser, Michael; Morrison, Carolyn A; Nedelec, Stephane; Williams, Damian J; An, Disi; Gifford, David K; Wichterle, Hynek

2013-01-01

Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation–sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type–specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types. PMID:23872598

21 CFR 184.1924 - Urease enzyme preparation from Lactobacillus fermentum.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Urease enzyme preparation from Lactobacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1924 Urease enzyme preparation from Lactobacillus fermentum. (a) This enzyme preparation is derived from the nonpathogenic...

21 CFR 184.1924 - Urease enzyme preparation from Lactobacillus fermentum.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Urease enzyme preparation from Lactobacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1924 Urease enzyme preparation from Lactobacillus fermentum. (a) This enzyme preparation is derived from the nonpathogenic...

21 CFR 184.1924 - Urease enzyme preparation from Lactobacillus fermentum.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Urease enzyme preparation from Lactobacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1924 Urease enzyme preparation from Lactobacillus fermentum. (a) This enzyme preparation is derived from the nonpathogenic...

Efficient elimination of nonstoichiometric enzyme inhibitors from HTS hit lists.

PubMed

Habig, Michael; Blechschmidt, Anke; Dressler, Sigmar; Hess, Barbara; Patel, Viral; Billich, Andreas; Ostermeier, Christian; Beer, David; Klumpp, Martin

2009-07-01

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.

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

Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection.

PubMed

Marden, James H

2013-12-01

Metabolic enzyme loci were some of the first genes accessible for molecular evolution and ecology research. New technologies now make the whole genome, transcriptome or proteome readily accessible, allowing unbiased scans for loci exhibiting significant differences in allele frequency or expression level and associated with phenotypes and/or responses to natural selection. With surprising frequency and in many cases in proportions greater than chance relative to other genes, glycolysis and TCA cycle enzyme loci appear among the genes with significant associations in these studies. Hence, there is an ongoing need to understand the basis for fitness effects of metabolic enzyme polymorphisms. Allele-specific effects on the binding affinity and catalytic rate of individual enzymes are well known, but often of uncertain significance because metabolic control theory and in vivo studies indicate that many individual metabolic enzymes do not affect pathway flux rate. I review research, so far little used in evolutionary biology, showing that metabolic enzyme substrates affect signalling pathways that regulate cell and organismal biology, and that these enzymes have moonlighting functions. To date there is little knowledge of how alleles in natural populations affect these phenotypes. I discuss an example in which alleles of a TCA enzyme locus associate with differences in a signalling pathway and development, organismal performance, and ecological dynamics. Ultimately, understanding how metabolic enzyme polymorphisms map to phenotypes and fitness remains a compelling and ongoing need for gaining robust knowledge of ecological and evolutionary processes. © 2013 John Wiley & Sons Ltd.

Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection

NASA Astrophysics Data System (ADS)

Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

2016-03-01

Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn2+ and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn2+ and adenosine monophosphate (AMP) with a simple mixing step. Most

Beyond Vmax and Km: How details of enzyme function influence geochemical cycles

NASA Astrophysics Data System (ADS)

Steen, A. D.

2015-12-01

Enzymes catalyze the vast majority of chemical reactions relevant to geomicrobiology. Studies of the activities of enzymes in environmental systems often report Vmax (the maximum possible rate of reaction; often proportional to the concentration of enzymes in the system) and sometimes Km (a measure of the affinity between enzymes and their substrates). However, enzyme studies - particularly those related to enzymes involved in organic carbon oxidation - are often limited to only those parameters, and a relatively limited and mixed set of enzymes. Here I will discuss some novel methods to assay and characterize the specific sets of enzymes that may be important to the carbon cycle in aquatic environments. First, kinetic experiments revealed the collective properties of the complex mixtures of extracellular peptidases that occur where microbial communities are diverse. Crystal structures combined with biochemical characterization of specific enzymes can yield more detailed information about key steps in organic carbon transformations. These new techniques have the potential to provide mechanistic grounding to geomicrobiological models.

Enhanced enzyme stability through site-directed covalent immobilization.

PubMed

Wu, Jeffrey Chun Yu; Hutchings, Christopher Hayden; Lindsay, Mark Jeffrey; Werner, Christopher James; Bundy, Bradley Charles

2015-01-10

Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To determine the impact of orientation on covalently immobilized enzyme activity and stability, we apply our PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddition "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. Our results demonstrate that orientation control of covalently immobilized enzymes can provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chemical denaturant treatments. Copyright © 2014 Elsevier B.V. All rights reserved.

Sequential Reactions of Surface-Tethered Glycolytic Enzymes

PubMed Central

Mukai, Chinatsu; Bergkvist, Magnus; Nelson, Jacquelyn L.; Travis, Alexander J.

2014-01-01

SUMMARY The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the flagellar principal piece by means of glycolytic enzymes, several of which are tethered to a cytoskeletal support via germ cell-specific targeting domains. Inspired by this design, we have produced recombinant hexokinase type 1 and glucose-6-phosphate isomerase capable of oriented immobilization on a nickel-nitrilotriacetic acid modified surface. Specific activities of enzymes tethered via this strategy were substantially higher than when randomly adsorbed. Furthermore, these enzymes showed sequential activities when tethered onto the same surface. This is the first demonstration of surface-tethered pathway components showing sequential enzymatic activities, and it provides a first step toward reconstitution of glycolysis on engineered hybrid devices. PMID:19778729

21 CFR 184.1924 - Urease enzyme preparation from Lactobacillus fermentum.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Urease enzyme preparation from Lactobacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1924 Urease enzyme..., nontoxicogenic bacterium Lactobacillus fermentum. It contains the enzyme urease (CAS Reg. No. 9002-13-5), which...

Pressure effects on enzyme-catalyzed quantum tunneling events arise from protein-specific structural and dynamic changes.

PubMed

Hay, Sam; Johannissen, Linus O; Hothi, Parvinder; Sutcliffe, Michael J; Scrutton, Nigel S

2012-06-13

The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions.

Non-antigenic and low allergic gelatin produced by specific digestion with an enzyme-coupled matrix.

PubMed

Sakai, Y; Yamato, R; Onuma, M; Kikuta, T; Watanabe, M; Nakayama, T

1998-04-01

Porcine gelatin (heat-denatured collagen) was digested with a bioreactor using an enzyme-coupled matrix (ECM) with purified collagenase. The digested gelatin, FreAlagin type R (M.W. range 200-10000 Da), was further purified by an HPLC system depending upon molecular size. The molecular weight range of the purified fractions, FreAlagin type P and type AD, were 200-500 and 2000-10000 Da, respectively, and glycine was the N-terminal amino acid of both types (> or =93%). ECM has the capability of digesting gelatin at a specific point in the sequence before glycine, and it was determined that FreAlagin type P consists of a tri-peptide fraction with the amino acid sequence Gly-X-Y. No types of FreAlagin exhibited any reactivity with gelatin-specific IgG antibody raised in guinea pigs, and they also possessed an extremely low reactivity with gelatin-specific IgE antibody from the sera of patients who had experienced an anaphylactic reaction against gelatin after vaccination or after eating gelatin-containing foods. From these results, it was determined that FreAlagin types R and AD were non-antigenic, low-allergic gelatins. FreAlagin type R, and especially type AD, had strong adsorption-blocking activity comparable to the level of bovine serum albumin, whereas type P and glycine had virtually no adsorption-blocking activity. Therefore, the new types of gelatin, FreAlagin types R and AD, are suitable for pharmaceutical use to avoid gelatin allergy.

Carboxylesterases: General detoxifying enzymes

PubMed Central

Hatfield, M. Jason; Umans, Robyn A.; Hyatt, Janice L.; Edwards, Carol C; Wierdl, Monika; Tsurkan, Lyudmila; Taylor, Michael R.; Potter, Philip M.

2016-01-01

Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents). PMID:26892220

Microbial responses to multi-factor climate change: effects on soil enzymes.

PubMed

Steinweg, J Megan; Dukes, Jeffrey S; Paul, Eldor A; Wallenstein, Matthew D

2013-01-01

The activities of extracellular enzymes, the proximate agents of decomposition in soils, are known to depend strongly on temperature, but less is known about how they respond to changes in precipitation patterns, and the interaction of these two components of climate change. Both enzyme production and turnover can be affected by changes in temperature and soil moisture, thus it is difficult to predict how enzyme pool size may respond to altered climate. Soils from the Boston-Area Climate Experiment (BACE), which is located in an old field (on abandoned farmland), were used to examine how climate variables affect enzyme activities and microbial biomass carbon (MBC) in different seasons and in soils exposed to a combination of three levels of precipitation treatments (ambient, 150% of ambient during growing season, and 50% of ambient year-round) and four levels of warming treatments (unwarmed to ~4°C above ambient) over the course of a year. Warming, precipitation and season had very little effect on potential enzyme activity. Most models assume that enzyme dynamics follow microbial biomass, because enzyme production should be directly controlled by the size and activity of microbial biomass. We observed differences among seasons and treatments in mass-specific potential enzyme activity, suggesting that this assumption is invalid. In June 2009, mass-specific potential enzyme activity, using chloroform fumigation-extraction MBC, increased with temperature, peaking under medium warming and then declining under the highest warming. This finding suggests that either enzyme production increased with temperature or turnover rates decreased. Increased maintenance costs associated with warming may have resulted in increased mass-specific enzyme activities due to increased nutrient demand. Our research suggests that allocation of resources to enzyme production could be affected by climate-induced changes in microbial efficiency and maintenance costs.

Engineered control of enzyme structural dynamics and function.

PubMed

Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F

2018-04-01

Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine. © 2018 The Protein Society.

Biochemistry students' ideas about how an enzyme interacts with a substrate.

PubMed

Linenberger, Kimberly J; Bretz, Stacey Lowery

2015-01-01

Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an enzyme and be able to reason from simplistic lock and key or induced fit models to the more complex energetics model of transition state theory. Learning to understand these many facets of enzyme-substrate interactions and reasoning from multiple models present challenges where students incorrectly make connections between concepts or make no connection at all. This study investigated biochemistry students' understanding of enzyme-substrate interactions through the use of clinical interviews and a national administration (N = 707) of the Enzyme-Substrate Interactions Concept Inventory. Findings include misconceptions regarding the nature of enzyme-substrate interactions, naïve ideas about the active site, a lack of energetically driven interactions, and an incomplete understanding of the specificity pocket. © 2015 by the International Union of Biochemistry and Molecular Biology.

Regulation of Ubiquitin Enzymes in the TGF-β Pathway.

PubMed

Iyengar, Prasanna Vasudevan

2017-04-20

The transforming growth factor-β (TGF-β) pathway has a tumor suppressor role in normal and premalignant cells but promotes oncogenesis in advanced cancer cells. Components of the pathway are tightly controlled by ubiquitin modifying enzymes and aberrations in these enzymes are frequently observed to dysregulate the pathway causing diseases such as bone disorders, cancer and metastasis. These enzymes and their counterparts are increasingly being tested as druggable targets, and thus a deeper understanding of the enzymes is required. This review summarizes the roles of specific ubiquitin modifying enzymes in the TGF-β pathway and how they are regulated.

Microbial extracellular enzymes in biogeochemical cycling of ecosystems.

PubMed

Luo, Ling; Meng, Han; Gu, Ji-Dong

2017-07-15

Extracellular enzymes, primarily produced by microorganisms, affect ecosystem processes because of their essential roles in degradation, transformation and mineralization of organic matter. Extracellular enzymes involved in the cycling of carbon (C), nitrogen (N) and phosphorus (P) have been widely investigated in many different ecosystems, and several enzymes have been recognized as key components in regulating C storage and nutrient cycling. In this review, it was the first time to summarize the specific extracellular enzymes related to C storage and nutrient cycling for better understanding the important role of microbial extracellular enzymes in biogeochemical cycling of ecosystems. Subsequently, ecoenzymatic stoichiometry - the relative ratio of extracellular enzyme, has been reviewed and further provided a new perspective for understanding biogeochemical cycling of ecosystems. Finally, the new insights of using microbial extracellular enzyme in indicating biogeochemical cycling and then protecting ecosystems have been suggested. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Application of enzymes in pulp and paper industry].

PubMed

Lin, Ying

2014-01-01

The application of enzymes has a high potential in the pulp and paper industry to improve the economics of the paper production process and to achieve, at the same time, a reduced environmental burden. Specific enzymes contribute to reduce the amount of chemicals, water and energy in various processes. This review is aimed at presenting the latest progresses of applying enzymes in bio-pulping, bio-bleaching, bio-deinking, enzymatic control of pitch and enzymatic modification of fibers.

41 CFR 101-29.221 - Federal Specifications, Standards and Commercial Item Description Program (Federal...

Code of Federal Regulations, 2010 CFR

2010-07-01

... standardization program developed under authority of the Federal Property and Administrative Services Act of 1949..., Standards and Commercial Item Description Program (Federal Standardization Program). 101-29.221 Section 101...-Definitions § 101-29.221 Federal Specifications, Standards and Commercial Item Description Program (Federal...

41 CFR 101-29.221 - Federal Specifications, Standards and Commercial Item Description Program (Federal...

Code of Federal Regulations, 2011 CFR

2011-07-01

... standardization program developed under authority of the Federal Property and Administrative Services Act of 1949..., Standards and Commercial Item Description Program (Federal Standardization Program). 101-29.221 Section 101...-Definitions § 101-29.221 Federal Specifications, Standards and Commercial Item Description Program (Federal...

Prevalence of sensitization to 'improver' enzymes in UK supermarket bakers.

PubMed

Jones, M; Welch, J; Turvey, J; Cannon, J; Clark, P; Szram, J; Cullinan, P

2016-07-01

Supermarket bakers are exposed not only to flour and alpha-amylase but also to other 'improver' enzymes, the nature of which is usually shrouded by commercial sensitivity. We aimed to determine the prevalence of sensitization to 'improver' enzymes in UK supermarket bakers. We examined the prevalence of sensitization to enzymes in 300 bakers, employed by one of two large supermarket bakeries, who had declared work-related respiratory symptoms during routine health surveillance. Sensitization was determined using radioallergosorbent assay to eight individual enzymes contained in the specific 'improver' mix used by each supermarket. The prevalence of sensitization to 'improver' enzymes ranged from 5% to 15%. Sensitization was far more likely if the baker was sensitized also to either flour or alpha-amylase. The prevalence of sensitization to an 'improver' enzyme did not appear to be related to the concentration of that enzyme in the mix. We report substantial rates of sensitization to enzymes other than alpha-amylase in UK supermarket bakers; in only a small proportion of bakers was there evidence of sensitization to 'improver mix' enzymes without sensitization to either alpha-amylase or flour. The clinical significance of these findings needs further investigation, but our findings indicate that specific sensitization in symptomatic bakers may not be identified without consideration of a wide range of workplace antigens. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity.

PubMed Central

Batista, F R; Hernández, L; Fernández, J R; Arrieta, J; Menéndez, C; Gómez, R; Támbara, Y; Pons, T

1999-01-01

beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases. PMID:9895294

21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Cellulase enzyme preparation derived from... SAFE Listing of Specific Substances Affirmed as GRAS § 184.1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase enzyme preparation is derived from a nonpathogenic...

Archaeal Enzymes and Applications in Industrial Biocatalysts.

PubMed

Littlechild, Jennifer A

2015-01-01

Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in "extreme" conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

Ionizable Side Chains at Catalytic Active Sites of Enzymes

PubMed Central

Jimenez-Morales, David; Liang, Jie

2012-01-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

Linking protein motion to enzyme catalysis.

PubMed

Singh, Priyanka; Abeysinghe, Thelma; Kohen, Amnon

2015-01-13

Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate binding, catalysis of the chemical conversion, and product release. The relationship between protein motions and catalytic activity is of contemporary interest in enzymology. To understand the factors influencing the rates of enzyme-catalyzed reactions, the dynamics of the protein-solvent-ligand complex must be considered. The current review presents two case studies of enzymes-dihydrofolate reductase (DHFR) and thymidylate synthase (TSase)-and discusses the role of protein motions in their catalyzed reactions. Specifically, we will discuss the utility of kinetic isotope effects (KIEs) and their temperature dependence as tools in probing such phenomena.

Structures of ribonucleoprotein particle modification enzymes

PubMed Central

Liang, Bo; Li, Hong

2016-01-01

Small nucleolar and Cajal body ribonucleoprotein particles (RNPs) are required for the maturation of ribosomes and spliceosomes. They consist of small nucleolar RNA or Cajal body RNA combined with partner proteins and represent the most complex RNA modification enzymes. Recent advances in structure and function studies have revealed detailed information regarding ribonucleoprotein assembly and substrate binding. These enzymes form intertwined RNA–protein assemblies that facilitate reversible binding of the large ribosomal RNA or small nuclear RNA. These revelations explain the specificity among the components in enzyme assembly and substrate modification. The multiple conformations of individual components and those of complete RNPs suggest a dynamic assembly process and justify the requirement of many assembly factors in vivo. PMID:21108865

Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection.

PubMed

Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

2016-03-21

Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn(2+) and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.

Biochemistry Students' Ideas about How an Enzyme Interacts with a Substrate

ERIC Educational Resources Information Center

Linenberger, Kimberly J.; Bretz, Stacey Lowery

2015-01-01

Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an…

NASIS data base management system - IBM 360/370 OS MVT implementation. 4: Program design specifications

NASA Technical Reports Server (NTRS)

1973-01-01

The design specifications for the programs and modules within the NASA Aerospace Safety Information System (NASIS) are presented. The purpose of the design specifications is to standardize the preparation of the specifications and to guide the program design. Each major functional module within the system is a separate entity for documentation purposes. The design specifications contain a description of, and specifications for, all detail processing which occurs in the module. Sub-modules, reference tables, and data sets which are common to several modules are documented separately.

Preliminary characterization of digestive enzymes in freshwater mussels

USGS Publications Warehouse

Sauey, Blake W.; Amberg, Jon J.; Cooper, Scott T.; Grunwald, Sandra K.; Newton, Teresa J.; Haro, Roger J.

2015-01-01

Resource managers lack an effective chemical tool to control the invasive zebra mussel Dreissena polymorpha. Zebra mussels clog water intakes for hydroelectric companies, harm unionid mussel species, and are believed to be a reservoir of avian botulism. Little is known about the digestive physiology of zebra mussels and unionid mussels. The enzymatic profile of the digestive glands of zebra mussels and native threeridge (Amblema plicata) and plain pocketbook mussels (Lampsilis cardium) are characterized using a commercial enzyme kit, api ZYM, and validated the kit with reagent-grade enzymes. A linear correlation was shown for only one of nineteen enzymes, tested between the api ZYM kit and a specific enzyme kit. Thus, the api ZYM kit should only be used to make general comparisons of enzyme presence and to observe trends in enzyme activities. Enzymatic trends were seen in the unionid mussel species, but not in zebra mussels sampled 32 days apart from the same location. Enzymatic classes, based on substrate, showed different trends, with proteolytic and phospholytic enzymes having the most change in relative enzyme activity.

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

Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes.

PubMed

Tencer, Adam H; Liang, Qin; Zhuang, Zhihao

2016-08-23

Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and polyubiquitination of proteins and play essential roles in numerous cellular processes. Close to 100 human DUBs have been identified and are classified into five families, with the ubiquitin-specific protease (USP) family being the largest (>50 members). The binding of ubiquitin (Ub) to USP is strikingly different from that observed for the DUBs in the ubiquitin C-terminal hydrolase (UCH) and ovarian tumor domain protease (OTU) families. We generated a panel of mutant ubiquitins and used them to probe the ubiquitin's interaction with a number of USPs. Our results revealed a remarkable divergence of USP-Ub interactions among the USP catalytic domains. Our double-mutant cycle analysis targeting the ubiquitin residues located in the tip, the central body, and the tail of ubiquitin also demonstrated different crosstalk among the USP-Ub interactions. This work uncovered intriguing divergence in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hot spots on USPs for selective inhibition of USPs by small molecule antagonists.

Integrative genomic mining for enzyme function to enable engineering of a non-natural biosynthetic pathway.

PubMed

Mak, Wai Shun; Tran, Stephen; Marcheschi, Ryan; Bertolani, Steve; Thompson, James; Baker, David; Liao, James C; Siegel, Justin B

2015-11-24

The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function. Here we utilize two approaches to discover enzymes that enable specific production of longer-chain (C5-C8) alcohols from sugar. The first approach combines bioinformatics and molecular modelling to mine sequence databases, resulting in a diverse panel of enzymes capable of catalysing the targeted reaction. The median catalytic efficiency of the computationally selected enzymes is 75-fold greater than a panel of naively selected homologues. This integrative genomic mining approach establishes a unique avenue for enzyme function discovery in the rapidly expanding sequence databases. The second approach uses computational enzyme design to reprogramme specificity. Both approaches result in enzymes with >100-fold increase in specificity for the targeted reaction. When enzymes from either approach are integrated in vivo, longer-chain alcohol production increases over 10-fold and represents >95% of the total alcohol products.

alpha-1,4-Glucan lyase, a new class of starch/glycogen degrading enzyme. III. Substrate specificity, mode of action, and cleavage mechanism.

PubMed

Yu, S; Ahmad, T; Kenne, L; Pedersén, M

1995-05-11

The alpha-1,4-glucan lyase (EC 4.2.2.-), purified from the red alga Gracilariopsis lemaneiformis, is a single polypeptide with a molecular mass of 116,654 Da as determined by matrix-assisted laser-desorption mass spectrometry. It degraded maltose, maltosaccharides, amylose, amylopectin and glycogen, forming 1,5-anhydro-D-fructose from the non-reducing end groups. The substrate specificity, mode of action, and cleavage mechanism of the enzyme were studied by using various naturally occurring and synthesized substrates. This enzyme was highly specific for the alpha-1,4-D-glucosidic bond. When a linear alpha-1,4-glucan was used as substrate, the enzyme split the substrate from the non-reducing end and released 1,5-anhydro-D-fructose successively until only one glucose unit was left. When a branched pentasaccharide of 6(2)-alpha-maltosylmaltotriose, obtained from glycogen by alpha-amylase limitation, was used as substrate, the glucose group in the 4-position of the 4,6-branched residue was not cleaved off. Using maltoheptaose as substrate and following the reaction with HPLC and 1H-NMR spectroscopy, it was found that the action mode of the lyase followed a multichain attack mechanism. 1H- and 13C-NMR spectroscopic studies on unlabelled and labelled amylose (1-2H, 2-2H, 1-13C) as substrates indicated that the lyase cleaved the C-(1')-O(4) bond forming a double bond between C-1' and C-2', thus forming the enol form of 1,5-anhydro-D-fructose. It also indicated that the catalytic process of the lyase involved proton exchanges among C-1, C-2, C-3 and the solvent.

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity1[OPEN

PubMed Central

Connor, Richard A.

2017-01-01

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart. PMID:27909045

Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

PubMed Central

Valero, E; Varón, R; García-Carmona, F

1995-01-01

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

Competitive Enzyme-Linked Immunosorbent Assay Based on a Rhoptry-Associated Protein 1 Epitope Specifically Identifies Babesia bovis-Infected Cattle

PubMed Central

Goff, Will L.; McElwain, Terry F.; Suarez, Carlos E.; Johnson, Wendell C.; Brown, Wendy C.; Norimine, Junzo; Knowles, Donald P.

2003-01-01

The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool. PMID:12522037

Subcellular localization of pituitary enzymes

NASA Technical Reports Server (NTRS)

Smith, R. E.

1970-01-01

A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

Efficacy of function specific 3D-motifs in enzyme classification according to their EC-numbers.

PubMed

Rahimi, Amir; Madadkar-Sobhani, Armin; Touserkani, Rouzbeh; Goliaei, Bahram

2013-11-07

Due to the increasing number of protein structures with unknown function originated from structural genomics projects, protein function prediction has become an important subject in bioinformatics. Among diverse function prediction methods, exploring known 3D-motifs, which are associated with functional elements in unknown protein structures is one of the most biologically meaningful methods. Homologous enzymes inherit such motifs in their active sites from common ancestors. However, slight differences in the properties of these motifs, results in variation in the reactions and substrates of the enzymes. In this study, we examined the possibility of discriminating highly related active site patterns according to their EC-numbers by 3D-motifs. For each EC-number, the spatial arrangement of an active site, which has minimum average distance to other active sites with the same function, was selected as a representative 3D-motif. In order to characterize the motifs, various points in active site elements were tested. The results demonstrated the possibility of predicting full EC-number of enzymes by 3D-motifs. However, the discriminating power of 3D-motifs varies among different enzyme families and depends on selecting the appropriate points and features. © 2013 Elsevier Ltd. All rights reserved.

[Cytochemical localization and properties of selected nucleolytic enzymes].

PubMed

Sierakowska, Halina

2015-01-01

In the article there are shortly outlined studies on cytochemical localization of selected nucleolytic enzymes carried out between 1957-1986 by David Shugar and his coworkers. The histochemical localization of several nucleolytic enzymes in animal and plant tissues was determined by synthesis of specific substrates, alpha-naphthyl esters of 5'- and 3'-nucleotides and their derivatives. In rat tissues phosphodiesterase I was localized in the plasma membrane whereas phosphodiesterase II in the lizosomes, reflecting their physiological roles. The localization of pancreatic type ribonuclease in animal tissues was determined, indicating its role in extracellular digestion. Plant nucleotide pyrophosphatase was localized in several tissues, purified to near homogeneity from potato tubers and its properties and substrate specificity were determined. Application of this enzyme for removal of m7GMP from the "cap" of eukaryotic mRNA allowed to elucidate the role of "cap" in mRNA binding to ribosomes in the process of translation. Furthermore, cyclic nucleotide phosphodiesterase was isolated from potato tubers and its physicochemical properties, oligomeric structure and substrate specificity were elucidated.

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false α-Amylase enzyme preparation from Bacillus... Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture filtrate that results from a pure...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

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

Boernke, W. E.; Millard, C. S.; Stevens, P. W.

1995-09-10

Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the nativemore » enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the

A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

PubMed

Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

2014-03-03

Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

Improving colorimetric assays through protein enzyme-assisted gold nanoparticle amplification.

PubMed

Xie, Xiaoji; Xu, Wei; Liu, Xiaogang

2012-09-18

The discovery of the DNA-mediated assembly of gold nanoparticles was a great moment in the history of science; this understanding and chemical control enabled the rational design of functional nanomaterials as novel probes in biodetection. In contrast with conventional probes such as organic dyes, gold nanoparticles exhibit high photostability and unique size-dependent optical properties. Because of their high extinction coefficients and strong distance dependent optical properties, these nanoparticles have emerged over the past decade as a promising platform for rapid, highly sensitive colorimetric assays that allow for the visual detection of low concentrations of metal ions, small molecules, and biomacromolecules. These discoveries have deepened our knowledge of biological phenomena and facilitated the development of many new diagnostic and therapeutic tools. Despite these many advances and continued research efforts, current nanoparticle-based colorimetric detection systems still suffer from several drawbacks, such as limited sensitivity and selectivity. This Account describes the recent development of colorimetric assays based on protein enzyme-assisted gold nanoparticle amplification. The benefits of such detection systems include significantly improved detection sensitivity and selectivity. First, we discuss the general design of enzyme-modified nanoparticle systems in colorimetric assays. We show that a quantitative understanding of the unique properties of different enzymes is paramount for effective biological assays. We then examine the assays for nucleic acid detection based on different types of enzymes, including endonucleases, ligases, and polymerases. For each of these assays, we identify the underlying principles that contribute to the enhanced detection capability of nanoparticle systems and illustrate them with selected examples. Furthermore, we demonstrate that the combination of gold nanoparticles and specific enzymes can probe enzyme dynamics

ORENZA: a web resource for studying ORphan ENZyme activities

PubMed Central

Lespinet, Olivier; Labedan, Bernard

2006-01-01

Background Despite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers) defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap. Description We designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities. Complete lists of all EC numbers and of orphan EC numbers are available and will be periodically updated. ORENZA allows one to browse the complete list of EC numbers or the subset associated with orphan enzymes or to query a specific EC number, an enzyme name or a species name for those interested in particular organisms. It is possible to search ORENZA for the different biochemical properties of the defined enzymes, the metabolic pathways in which they participate, the taxonomic data of the organisms whose genomes encode them, and many other features. The association of an enzyme activity with an amino acid sequence is clearly underlined, making it easy to identify at once the orphan enzyme activities. Interactive publishing of suggestions by the community would provide expert evidence for re-annotation of orphan EC numbers in public databases. Conclusion ORENZA is a Web resource designed to progressively bridge the unwanted gap between function (enzyme activities) and sequence (dataset present in public databases). ORENZA should increase interactions between communities of biochemists and of genomicists. This is expected to reduce the number of orphan enzyme activities by allocating gene

Archaeal Enzymes and Applications in Industrial Biocatalysts

PubMed Central

Littlechild, Jennifer A.

2015-01-01

Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches. PMID:26494981

SLS-SPEC-159 Cross-Program Design Specification for Natural Environments (DSNE) Revision E

NASA Technical Reports Server (NTRS)

Roberts, Barry C.

2017-01-01

The DSNE completes environment-related specifications for architecture, system-level, and lower-tier documents by specifying the ranges of environmental conditions that must be accounted for by NASA ESD Programs. To assure clarity and consistency, and to prevent requirements documents from becoming cluttered with extensive amounts of technical material, natural environment specifications have been compiled into this document. The intent is to keep a unified specification for natural environments that each Program calls out for appropriate application. This document defines the natural environments parameter limits (maximum and minimum values, energy spectra, or precise model inputs, assumptions, model options, etc.), for all ESD Programs. These environments are developed by the NASA Marshall Space Flight Center (MSFC) Natural Environments Branch (MSFC organization code: EV44). Many of the parameter limits are based on experience with previous programs, such as the Space Shuttle Program. The parameter limits contain no margin and are meant to be evaluated individually to ensure they are reasonable (i.e., do not apply unrealistic extreme-on-extreme conditions). The natural environments specifications in this document should be accounted for by robust design of the flight vehicle and support systems. However, it is understood that in some cases the Programs will find it more effective to account for portions of the environment ranges by operational mitigation or acceptance of risk in accordance with an appropriate program risk management plan and/or hazard analysis process. The DSNE is not intended as a definition of operational models or operational constraints, nor is it adequate, alone, for ground facilities which may have additional requirements (for example, building codes and local environmental constraints). "Natural environments," as the term is used here, refers to the environments that are not the result of intended human activity or intervention. It

Spatial distribution of enzyme driven reactions at micro-scales

NASA Astrophysics Data System (ADS)

Kandeler, Ellen; Boeddinghaus, Runa; Nassal, Dinah; Preusser, Sebastian; Marhan, Sven; Poll, Christian

2017-04-01

Studies of microbial biogeography can often provide key insights into the physiologies, environmental tolerances, and ecological strategies of soil microorganisms that dominate in natural environments. In comparison with aquatic systems, soils are particularly heterogeneous. Soil heterogeneity results from the interaction of a hierarchical series of interrelated variables that fluctuate at many different spatial and temporal scales. Whereas spatial dependence of chemical and physical soil properties is well known at scales ranging from decimetres to several hundred metres, the spatial structure of soil enzymes is less clear. Previous work has primarily focused on spatial heterogeneity at a single analytical scale using the distribution of individual cells, specific types of organisms or collective parameters such as bacterial abundance or total microbial biomass. There are fewer studies that have considered variations in community function and soil enzyme activities. This presentation will give an overview about recent studies focusing on spatial pattern of different soil enzymes in the terrestrial environment. Whereas zymography allows the visualization of enzyme pattern in the close vicinity of roots, micro-sampling strategies followed by MUF analyses clarify micro-scale pattern of enzymes associated to specific microhabitats (micro-aggregates, organo-mineral complexes, subsoil compartments).

Peer counseling in a culturally specific adolescent pregnancy prevention program.

PubMed

Ferguson, S L

1998-08-01

This study evaluated the effects of peer counseling in a culturally specific adolescent pregnancy prevention program for African American females. A random pretest and multiple posttest experimental and comparison group design was used to obtain data on a sample of 63 female African American adolescents, ages 12 to 16, who lived in four public housing developments. Descriptive data and tests of significance revealed that none of the participants who received peer counseling became pregnant within three months of the intervention. Findings revealed a statistically significant increase in reproductive and other self-related knowledge topics among the experimental group when comparing pretest and eight-week posttest scores. Most participants had not had sexual intercourse; the average age of sexual onset was 12 years in the experimental group and 11 years in the controls. Designing and implementing culturally specific adolescent pregnancy prevention programs for adolescents younger than age 11 and/or before sexually active seems appropriate.

Ionizable side chains at catalytic active sites of enzymes.

PubMed

Jimenez-Morales, David; Liang, Jie; Eisenberg, Bob

2012-05-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å(3). The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

Substrate specificity of low-molecular mass bacterial DD-peptidases.

PubMed

Nemmara, Venkatesh V; Dzhekieva, Liudmila; Sarkar, Kumar Subarno; Adediran, S A; Duez, Colette; Nicholas, Robert A; Pratt, R F

2011-11-22

The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and low-molecular mass (LMM) enzymes. The latter group, which is subdivided into classes A-C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 dd-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD

[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.

Clustering and optimal arrangement of enzymes in reaction-diffusion systems.

PubMed

Buchner, Alexander; Tostevin, Filipe; Gerland, Ulrich

2013-05-17

Enzymes within biochemical pathways are often colocalized, yet the consequences of specific spatial enzyme arrangements remain poorly understood. We study the impact of enzyme arrangement on reaction efficiency within a reaction-diffusion model. The optimal arrangement transitions from a cluster to a distributed profile as a single parameter, which controls the probability of reaction versus diffusive loss of pathway intermediates, is varied. We introduce the concept of enzyme exposure to explain how this transition arises from the stochastic nature of molecular reactions and diffusion.

Sensitising effects of genetically modified enzymes used in flavour, fragrance, detergence and pharmaceutical production: cross-sectional study.

PubMed

Budnik, Lygia T; Scheer, Edwin; Burge, P Sherwood; Baur, Xaver

2017-01-01

The use of genetically engineered enzymes in the synthesis of flavourings, fragrances and other applications has increased tremendously. There is, however, a paucity of data on sensitisation and/or allergy to the finished products. We aimed to review the use of genetically modified enzymes and the enormous challenges in human biomonitoring studies with suitable assays of specific IgE to a variety of modified enzyme proteins in occupational settings and measure specific IgE to modified enzymes in exposed workers. Specific IgE antibodies against workplace-specific individual enzymes were measured by the specific fluorescence enzyme-labelled immunoassay in 813 exposed workers seen in cross-sectional surveys. Twenty-three per cent of all exposed workers showed type I sensitisation with IgE antibodies directed against respective workplace-specific enzymes. The highest sensitisation frequencies observed were for workers exposed enzymes derived from α-amylase (44%), followed by stainzyme (41%), pancreatinin (35%), savinase (31%), papain (31%), ovozyme (28%), phytase (16%), trypsin (15%) and lipase (4%). The highest individual antibody levels (up to 110 kU/L) were detected in workers exposed to phytase, xylanase and glucanase. In a subgroup comprising 134 workers, detailed clinical diagnostics confirmed work-related symptoms. There was a strong correlation (r=0.75, p<0.0001) between the symptoms and antibody levels. Workers with work-related respiratory symptoms showed a higher prevalence for the presence of specific IgE antibodies against workplace-specific enzymes than asymptomatic exposed workers (likelihood ratio 2.32, sensitivity 0.92, specificity 0.6). Our data confirm the previous findings showing that genetically engineered enzymes are potent allergens eliciting immediate-type sensitisation. Owing to lack of commercial diagnostic tests, few of those exposed receive regular surveillance including biomonitoring with relevant specific IgE assays. Published by the

Tissue enzyme studies in Macaca nemestrina monkeys.

NASA Technical Reports Server (NTRS)

Hubbard, R. W.; Hoffman, R. A.; Jenkins, D.

1971-01-01

Total enzyme activities in fresh tissue specimens from major organs of Macaca nemestrina were analyzed for lactic dehydrogenase (LDH), creatine phosphokinase (CPK), and aldolase. The concentration of these enzymes varied among the different tissue with skeletal muscle, heart, and brain having the highest activities. LDH isozymes determinations for the various tissues were also made. The spectrum of LDH isozyme distribution appears to be quite specific and characteristic for at least some of the tissues analyzed.

Ubiquitin enzymes in the regulation of immune responses.

PubMed

Ebner, Petra; Versteeg, Gijs A; Ikeda, Fumiyo

2017-08-01

Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.

Synthetic, switchable enzymes

PubMed Central

Norris, Vic; Krylov, Sergey N.; Agarwal, Pratul K.; White, Glenn J.

2017-01-01

The construction of switchable, radiation-controlled, aptameric enzymes alias swenzymes is, in principle, feasible. We propose a strategy to make such catalysts from two (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a two-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker so bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low intensity, non-ionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate, product-capturing, aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis. PMID:28448969

Synthetic, Switchable Enzymes.

PubMed

Norris, Vic; Krylov, Sergey N; Agarwal, Pratul K; White, Glenn J

2017-01-01

The construction of switchable, radiation-controlled, aptameric enzymes - "swenzymes" - is, in principle, feasible. We propose a strategy to make such catalysts from 2 (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a 2-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker, thus bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low-intensity, nonionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate product-capturing aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis. © 2017 S. Karger AG, Basel.

Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endo-epsilon (gamma-Glu)-Lys isopeptide bonds and help to dissolve blood clots.

PubMed

Zavalova, L; Lukyanov, S; Baskova, I; Snezhkov, E; Akopov, S; Berezhnoy, S; Bogdanova, E; Barsova, E; Sverdlov, E D

1996-11-27

We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-epsilon(gamma-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine gamma-carboxamide and the epsilon-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.

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.

The Relationship between Institutional, Departmental and Program-Specific Variables and the Academic Performance of Division I FBS Football Programs

ERIC Educational Resources Information Center

Eigenbrot, Steven C.

2012-01-01

This study investigated the connection between the academic evaluation of Division I FBS football programs and the various social settings that influenced these student-athletes. These social settings were classified as: institutional, departmental and program-specific. The experience of the student-athlete is thought to be impacted by all three…

Specificity of the trypanothione-dependent Leishmania major glyoxalase I: structure and biochemical comparison with the human enzyme.

PubMed

Ariza, Antonio; Vickers, Tim J; Greig, Neil; Armour, Kirsten A; Dixon, Mark J; Eggleston, Ian M; Fairlamb, Alan H; Bond, Charles S

2006-02-01

Trypanothione replaces glutathione in defence against cellular damage caused by oxidants, xenobiotics and methylglyoxal in the trypanosomatid parasites, which cause trypanosomiasis and leishmaniasis. In Leishmania major, the first step in methylglyoxal detoxification is performed by a trypanothione-dependent glyoxalase I (GLO1) containing a nickel cofactor; all other characterized eukaryotic glyoxalases use zinc. In kinetic studies L. major and human enzymes were active with methylglyoxal derivatives of several thiols, but showed opposite substrate selectivities: N1-glutathionylspermidine hemithioacetal is 40-fold better with L. major GLO1, whereas glutathione hemithioacetal is 300-fold better with human GLO1. Similarly, S-4-bromobenzylglutathionylspermidine is a 24-fold more potent linear competitive inhibitor of L. major than human GLO1 (Kis of 0.54 microM and 12.6 microM, respectively), whereas S-4-bromobenzylglutathione is >4000-fold more active against human than L. major GLO1 (Kis of 0.13 microM and >500 microM respectively). The crystal structure of L. major GLO1 reveals differences in active site architecture to both human GLO1 and the nickel-dependent Escherichia coli GLO1, including increased negative charge and hydrophobic character and truncation of a loop that may regulate catalysis in the human enzyme. These differences correlate with the differential binding of glutathione and trypanothione-based substrates, and thus offer a route to the rational design of L. major-specific GLO1 inhibitors.

Enzyme-Powered Pumps: From Fundamentals to Applications

NASA Astrophysics Data System (ADS)

Ortiz-Rivera, Isamar

Non-mechanical nano and microfluidic devices that function without the aid of an external power source, and can be tailored to meet specific needs, represent the next generation of smart devices. Recently, we have shown that surface-bound enzymes can act as pumps driving large-scale fluid flows in the presence of any substance that triggers the enzymatic reaction (e.g. substrate, co-factor, or biomarker). The fluid velocities attained in such systems depend directly on the enzymatic reaction rate and the concentration of the substance that initiates enzymatic catalysis. The use of biochemical reactions to power a micropump offers the advantages of specificity, sensitivity, and selectively, eliminating at the same time the need of an external power source, while providing biocompatibility. More importantly, these self-powered pumps overcome a significant obstacle in nano- and micro-fluidics: the need to use external pressure-driven pumps to push fluids through devices. Certainly, the development of enzyme-powered devices opens up new venues in biochemical engineering, particularly in the biomedical field. The work highlighted in this dissertation covers all the studies performed with enzyme-powered pumps, from the development of the micropump design, to the efforts invested in understanding the enzyme pump concept as a whole. The data collected to date, aims to expand our knowledge about enzyme-powered micropumps from the inside out: not only by exploring the different applications of these devices at the macroscale, but also by investigating in depth the mechanism of pump activation behind these systems. Specifically, we have focused on: (1) The general features that characterize the pumping behavior observed in enzyme-powered pumps, as well as the optimization of the device, (2) the possible mechanisms behind fluid motion, including the role of enzyme coverage and/or activity on the transduction of chemical energy into mechanical fluid flow in these devices

The Nodulation Factor Hydrolase of Medicago truncatula: Characterization of an Enzyme Specifically Cleaving Rhizobial Nodulation Signals1[W][OPEN

PubMed Central

Tian, Ye; Liu, Wei; Cai, Jie; Zhang, Lan-Yue; Wong, Kam-Bo; Feddermann, Nadja; Boller, Thomas; Xie, Zhi-Ping; Staehelin, Christian

2013-01-01

Nodule formation induced by nitrogen-fixing rhizobia depends on bacterial nodulation factors (NFs), modified chitin oligosaccharides with a fatty acid moiety. Certain NFs can be cleaved and inactivated by plant chitinases. However, the most abundant NF of Sinorhizobium meliloti, an O-acetylated and sulfated tetramer, is resistant to hydrolysis by all plant chitinases tested so far. Nevertheless, this NF is rapidly degraded in the host rhizosphere. Here, we identify and characterize MtNFH1 (for Medicago truncatula Nod factor hydrolase 1), a legume enzyme structurally related to defense-related class V chitinases (glycoside hydrolase family 18). MtNFH1 lacks chitinase activity but efficiently hydrolyzes all tested NFs of S. meliloti. The enzyme shows a high cleavage preference, releasing exclusively lipodisaccharides from NFs. Substrate specificity and kinetic properties of MtNFH1 were compared with those of class V chitinases from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), which cannot hydrolyze tetrameric NFs of S. meliloti. The Michaelis-Menten constants of MtNFH1 for NFs are in the micromolar concentration range, whereas nonmodified chitin oligosaccharides represent neither substrates nor inhibitors for MtNFH1. The three-dimensional structure of MtNFH1 was modeled on the basis of the known structure of class V chitinases. Docking simulation of NFs to MtNFH1 predicted a distinct binding cleft for the fatty acid moiety, which is absent in the class V chitinases. Point mutation analysis confirmed the modeled NF-MtNFH1 interaction. Silencing of MtNFH1 by RNA interference resulted in reduced NF degradation in the rhizosphere of M. truncatula. In conclusion, we have found a novel legume hydrolase that specifically inactivates NFs. PMID:24082029

Two-fold Bioorthogonal Derivatization by Different Formylglycine-Generating Enzymes.

PubMed

Krüger, Tobias; Weiland, Stefanie; Falck, Georg; Gerlach, Marcus; Boschanski, Mareile; Alam, Sarfaraz; Müller, Kristian M; Dierks, Thomas; Sewald, Norbert

2018-03-26

Formylglycine-generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site-specific oxidation of a cysteine residue to the aldehyde-containing amino acid C α -formylglycine (FGly). This non-canonical residue can be generated within any desired target protein and can subsequently be used for bioorthogonal conjugation reactions. The prototypic formylglycine-generating enzyme (FGE) and the iron-sulfur protein AtsB display slight variations in their recognition sequences. We designed specific tags in peptides and proteins that were selectively converted by the different enzymes. Combination of the different tag motifs within a single peptide or recombinant protein enabled the independent and consecutive introduction of two formylglycine residues and the generation of heterobifunctionalized protein conjugates. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microbial expression of alkaloid biosynthetic enzymes for characterization of their properties.

PubMed

Minami, Hiromichi; Ikezawa, Nobuhiro; Sato, Fumihiko

2010-01-01

A wide variety of secondary metabolites are produced in higher plants. These metabolites are synthesized in specific organs/cells at certain developmental stages and/or under specific environmental conditions. Since these biosynthetic activities are rather restricted and difficult to detect, the biochemical characterization of biosynthetic enzymes involved in secondary metabolism has been limited compared to those involved in primary metabolism. Recently, however, progress in tissue culture and molecular biology has made it easier to study biosynthetic enzymes. Here we describe protocols for expressing some biosynthetic enzymes in Escherichia coli expression systems, since this system is both efficient and cost-effective. First, we describe a standard system for expressing biosynthetic enzymes as a soluble protein under the T7 promoter of the pET expression system in E. coli. In addition, the successful expression of cytochrome P450 in E. coli in an active soluble form with N-terminal modification is discussed, since P450 is the critical enzyme in secondary metabolite biosynthesis.

The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors.

PubMed

Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

2016-11-15

Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Draft Genome of the Invasive Walking Stick, Medauroidea extradendata, Reveals Extensive Lineage-Specific Gene Family Expansions of Cell Wall Degrading Enzymes in Phasmatodea

PubMed Central

Brand, Philipp; Lin, Wei; Johnson, Brian R.

2018-01-01

Plant cell wall components are the most abundant macromolecules on Earth. The study of the breakdown of these molecules is thus a central question in biology. Surprisingly, plant cell wall breakdown by herbivores is relatively poorly understood, as nearly all early work focused on the mechanisms used by symbiotic microbes to breakdown plant cell walls in insects such as termites. Recently, however, it has been shown that many organisms make endogenous cellulases. Insects, and other arthropods, in particular have been shown to express a variety of plant cell wall degrading enzymes in many gene families with the ability to break down all the major components of the plant cell wall. Here we report the genome of a walking stick, Medauroidea extradentata, an obligate herbivore that makes uses of endogenously produced plant cell wall degrading enzymes. We present a draft of the 3.3Gbp genome along with an official gene set that contains a diversity of plant cell wall degrading enzymes. We show that at least one of the major families of plant cell wall degrading enzymes, the pectinases, have undergone a striking lineage-specific gene family expansion in the Phasmatodea. This genome will be a useful resource for comparative evolutionary studies with herbivores in many other clades and will help elucidate the mechanisms by which metazoans breakdown plant cell wall components. PMID:29588379

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.

PubMed

Lu, Haibin; Chandrasekar, Balakumaran; Oeljeklaus, Julian; Misas-Villamil, Johana C; Wang, Zheming; Shindo, Takayuki; Bogyo, Matthew; Kaiser, Markus; van der Hoorn, Renier A L

2015-08-01

Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins. © 2015 American Society of Plant Biologists. All Rights Reserved.

Enzyme-modified electrolyte-gated organic field-effect transistors

NASA Astrophysics Data System (ADS)

Buth, Felix; Donner, Andreas; Stutzmann, Martin; Garrido, Jose A.

2012-10-01

Organic solution-gated field-effect transistors (SGFETs) can be operated at low voltages in aqueous environments, paving the way to the use of organic semiconductors in bio-sensing applications. However, it has been shown that these devices exhibit only a rather weak sensitivity to standard electrolyte parameters such as pH and ionic strength. In order to increase the sensitivity and to add specificity towards a given analyte, the covalent attachment of functional groups and enzymes to the device surface would be desirable. In this contribution we demonstrate that enzyme modified organic SGFETs can be used for the in-situ detection of penicillin in the low μM regime. In a first step, silane molecules with amine terminal groups are grafted to α-sexithiophene-based thin film transistors. Surface characterization techniques like X-ray photoemission confirm the modification of the surface with these functional groups, which are stable in standard aqueous electrolytes. We show that the presence of surface-bound amphoteric groups (e.g. amino or carboxylic moieties) increases the pH-sensitivity of the organic SGFETs. In addition, these groups serve as anchoring sites for the attachment of the enzyme penicillinase. The resulting enzyme-FETs are used for the detection of penicillin, enabling the study of the influence of the buffer strength and the pH of the electrolyte on the enzyme kinetics. The functionalization of the organic FETs shown here can be extended to a large variety of enzymes, allowing the specific detection of different chemical and biochemical analytes.

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.

Network design and analysis for multi-enzyme biocatalysis.

PubMed

Blaß, Lisa Katharina; Weyler, Christian; Heinzle, Elmar

2017-08-10

As more and more biological reaction data become available, the full exploration of the enzymatic potential for the synthesis of valuable products opens up exciting new opportunities but is becoming increasingly complex. The manual design of multi-step biosynthesis routes involving enzymes from different organisms is very challenging. To harness the full enzymatic potential, we developed a computational tool for the directed design of biosynthetic production pathways for multi-step catalysis with in vitro enzyme cascades, cell hydrolysates and permeabilized cells. We present a method which encompasses the reconstruction of a genome-scale pan-organism metabolic network, path-finding and the ranking of the resulting pathway candidates for proposing suitable synthesis pathways. The network is based on reaction and reaction pair data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the thermodynamics calculator eQuilibrator. The pan-organism network is especially useful for finding the most suitable pathway to a target metabolite from a thermodynamic or economic standpoint. However, our method can be used with any network reconstruction, e.g. for a specific organism. We implemented a path-finding algorithm based on a mixed-integer linear program (MILP) which takes into account both topology and stoichiometry of the underlying network. Unlike other methods we do not specify a single starting metabolite, but our algorithm searches for pathways starting from arbitrary start metabolites to a target product of interest. Using a set of biochemical ranking criteria including pathway length, thermodynamics and other biological characteristics such as number of heterologous enzymes or cofactor requirement, it is possible to obtain well-designed meaningful pathway alternatives. In addition, a thermodynamic profile, the overall reactant balance and potential side reactions as well as an SBML file for visualization are generated for each pathway alternative. We present

Affinity labelling enzymes with esters of aromatic sulfonic acids

DOEpatents

Wong, Show-Chu; Shaw, Elliott

1977-01-01

Novel esters of aromatic sulfonic acids are disclosed. The specific esters are nitrophenyl p- and m-amidinophenylmethanesulfonate. Also disclosed is a method for specific inactivation of the enzyme, thrombin, employing nitrophenyl p-amidinophenylmethanesulfonate.

Primary enzyme quantitation

DOEpatents

Saunders, G.C.

1982-03-04

The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

PROTECT YOUR HEART: A CULTURE-SPECIFIC, MULTIMEDIA CARDIOVASCULAR HEALTH EDUCATION PROGRAM

PubMed Central

Shah, Amy; Clayman, Marla L.; Lauderdale, Diane S.; Khurana, Neerja; Glass, Sara; Kandula, Namratha R.

2016-01-01

Objectives South Asians (SAs), the second fastest growing racial/ethnic minority in the United States., have high rates of coronary heart disease (CHD). Few CHD prevention efforts target this population. We developed and tested a culture-specific, multimedia CHD prevention education program in English and Hindi for SAs. Methods Participants were recruited from community organizations in Chicago, IL between June-October 2011. Bilingual interviewers used questionnaires to assess participants’ knowledge and perceptions before and after the patient education program. Change from pre- to post-test score was calculated using a paired t-test. Linear regression was used to determine the association between post-test scores and education and language. Results Participants’ (n=112) average age was 41 years, 67% had more than a high school education, and 50% spoke Hindi. Participants’ mean pre-test score was 15 (Standard Deviation= 4). After the patient education program, post-test scores increased significantly among all participants (post-test score=24, SD=4), including those with limited-English proficiency. Lower education was associated with a lower post-test score (Beta-coefficient= −2.2, 95% CI= −0.68, −3.8) in adjusted regression. Conclusions A culture-specific, multimedia patient education program significantly improved knowledge and perceptions about CHD prevention among SA immigrants. Culturally-salient, multimedia education may be an effective and engaging way to deliver health information to diverse patient populations. PMID:25647363

Novel angiotensin I-converting enzyme inhibitory peptides produced in fermented milk by specific wild Lactococcus lactis strains.

PubMed

Rodríguez-Figueroa, J C; González-Córdova, A F; Torres-Llanez, M J; Garcia, H S; Vallejo-Cordoba, B

2012-10-01

The ability of specific wild Lactococcus lactis strains to hydrolyze milk proteins to release angiotensin I-converting enzyme (ACE) inhibitory peptides was evaluated. The peptide profiles were obtained from the <3 kDa water-soluble extract and subsequently fractionated by reversed-phase HPLC. The fractions with the lowest half-maximal inhibitory concentration estimated values (peptide concentration necessary to inhibit ACE activity by 50%) were Lc. lactis NRRL B-50571 fraction (F)1 (0.034 ± 0.002 μg/mL; mean ± SD) and Lc. lactis NRRL B-50572B F 0005 (0.041 ± 0.003 μg/mL; mean ± SD). All peptide fractions were analyzed by reversed-phase HPLC tandem mass spectrometry. Twenty-one novel peptide sequences associated with ACE inhibitory (ACEI) activity were identified. Several novel ACEI peptides presented peptides encrypted with proven hypotensive activity. In conclusion, specific wild Lc. lactis strains were able to hydrolyze milk proteins to generate potent ACEI peptides. However, further studies are necessary to find out the relationship between Lc. lactis strain proteolytic systems and their ability to biogenerate hypotensive peptides. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Ubiquitin enzymes in the regulation of immune responses

PubMed Central

Ebner, Petra; Versteeg, Gijs A.; Ikeda, Fumiyo

2017-01-01

Abstract Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses. PMID:28524749

Mechanisms, biology and inhibitors of deubiquitinating enzymes.

PubMed

Love, Kerry Routenberg; Catic, André; Schlieker, Christian; Ploegh, Hidde L

2007-11-01

The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes.

Enzyme-Linked Immunospot Assay Detection of Mumps-Specific Antibody-Secreting B Cells as an Alternative Method of Laboratory Diagnosis ▿

PubMed Central

Latner, Donald R.; McGrew, Marcia; Williams, Nobia; Lowe, Luis; Werman, Roniel; Warnock, Eli; Gallagher, Kathleen; Doyle, Peter; Smole, Sandra; Lett, Susan; Cocoros, Noelle; DeMaria, Alfred; Konomi, Raimond; Brown, Cedric J.; Rota, Paul A.; Bellini, William J.; Hickman, Carole J.

2011-01-01

Although high measles, mumps, and rubella (MMR) vaccination coverage has been successful in dramatically reducing mumps disease in the United States, mumps (re)infections occasionally occur in individuals who have been either previously vaccinated or naturally infected. Standard diagnostics that detect virus or virus-specific antibody are dependable for confirming primary mumps infection in immunologically naïve persons, but these methods perform inconsistently for individuals with prior immune exposure. We hypothesized that detection of activated mumps-specific antibody-secreting B cells (ASCs) by enzyme-linked immunospot (ELISPOT) assay could be used as a more reliable diagnostic. To test this, a time course of virus-specific ASC responses was measured by ELISPOT assay following MMR vaccination of 16 previously vaccinated or naturally exposed adult volunteers. Mumps-specific ASCs were detectable in 68% of these individuals at some point during the first 3 weeks following revaccination. In addition, mumps-specific ASCs were detected in 7/7 previously vaccinated individuals who recently had been infected as part of a confirmed mumps outbreak. These data suggest that ELISPOT detection of mumps-specific ASCs has the potential for use as an alternative method of diagnosis when suspect cases cannot be confirmed by detection of IgM or virus. In addition, it was determined that mumps-specific memory B cells are detected at a much lower frequency than measles- or rubella-specific cells, suggesting that mumps infection may not generate robust B-cell memory. PMID:21047998

Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis.

PubMed

Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

2016-03-24

Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the -2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the -1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes.

ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

PubMed Central

Stine, G. J.

1968-01-01

Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged. PMID:4384627

Single-molecule Force Spectroscopy Approach to Enzyme Catalysis*

PubMed Central

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M.

2010-01-01

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed. PMID:20382731

Single-molecule force spectroscopy approach to enzyme catalysis.

PubMed

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M

2010-06-18

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.

Microbial nitrilases: versatile, spiral forming, industrial enzymes.

PubMed

Thuku, R N; Brady, D; Benedik, M J; Sewell, B T

2009-03-01

The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a alpha beta beta alpha-alpha beta beta alpha sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.

Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

PubMed

Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

2005-09-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

Enzyme-Linked Immunosorbent Assays for Detection of Equine Antibodies Specific to Sarcocystis neurona Surface Antigens†

PubMed Central

Hoane, Jessica S.; Morrow, Jennifer K.; Saville, William J.; Dubey, J. P.; Granstrom, David E.; Howe, Daniel K.

2005-01-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM. PMID:16148170

A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

PubMed

Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

2015-04-03

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

PubMed Central

Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

2015-01-01

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

42 CFR 457.1160 - Program specific review process: Time frames.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 4 2010-10-01 2010-10-01 false Program specific review process: Time frames. 457... process: Time frames. (a) Eligibility or enrollment matter. A State must complete the review of a matter described in § 457.1130(a) within a reasonable amount of time. In setting time frames, the State must...

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.

DNA Looping Facilitates Targeting of a Chromatin Remodeling Enzyme

PubMed Central

Yadon, Adam N; Singh, Badri Nath; Hampsey, Michael; Tsukiyama, Toshio

2013-01-01

Summary ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a previously unknown mechanism for the recruitment of a chromatin remodeling enzyme and defines a novel function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes. PMID:23478442

The Enzyme Function Initiative†

PubMed Central

Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

2011-01-01

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID

The Enzyme Function Initiative.

PubMed

Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

2011-11-22

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts. �

Effects of a Specific Numeracy Educational Program in Kindergarten Children: A Pilot Study

ERIC Educational Resources Information Center

Kaufmann, Liane; Delazer, Margarete; Pohl, Renate; Semenza, Carlo; Dowker, Ann

2005-01-01

The present study compared the relative effects of 2 educational programs on kindergarten children. The experimental group took part in a numeracy-specific program, which focused on conceptual knowledge. Children were taught basic numerical skills such as understanding and handling numbers and their relations as well as counting principles. The…

Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis.

PubMed

Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

2006-03-01

Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 A, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 A.

Encapsulation of enzyme via one-step template-free formation of stable organic-inorganic capsules: A simple and efficient method for immobilizing enzyme with high activity and recyclability.

PubMed

Huang, Renliang; Wu, Mengyun; Goldman, Mark J; Li, Zhi

2015-06-01

Enzyme encapsulation is a simple, gentle, and general method for immobilizing enzyme, but it often suffers from one or more problems regarding enzyme loading efficiency, enzyme leakage, mechanical stability, and recyclability. Here we report a novel, simple, and efficient method for enzyme encapsulation to overcome these problems by forming stable organic-inorganic hybrid capsules. A new, facile, one-step, and template-free synthesis of organic-inorganic capsules in aqueous phase were developed based on PEI-induced simultaneous interfacial self-assembly of Fmoc-FF and polycondensation of silicate. Addition of an aqueous solution of Fmoc-FF and sodium silicate into an aqueous solution of PEI gave a new class of organic-inorganic hybrid capsules (FPSi) with multi-layered structure in high yield. The capsules are mechanically stable due to the incorporation of inorganic silica. Direct encapsulation of enzyme such as epoxide hydrolase SpEH and BSA along with the formation of the organic-inorganic capsules gave high yield of enzyme-containing capsules (∼1.2 mm in diameter), >90% enzyme loading efficiency, high specific enzyme loading (158 mg protein g(-1) carrier), and low enzyme leakage (<3% after 48 h incubation). FPSi-SpEH capsules catalyzed the hydrolysis of cyclohexene oxide to give (1R, 2R)-cyclohexane-1,2-diol in high yield and concentration, with high specific activity (6.94 U mg(-1) protein) and the same high enantioselectivity as the free enzyme. The immobilized SpEH demonstrated also excellent operational stability and recyclability: retaining 87% productivity after 20 cycles with a total reaction time of 80 h. The new enzyme encapsulation method is efficient, practical, and also better than other reported encapsulation methods. © 2015 Wiley Periodicals, Inc.

Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway.

PubMed

Geisler, Christoph; Jarvis, Donald L

2012-03-02

Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2) is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing β-N-acetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. N-acetylglucosaminyltransferase I (GNT-I) contributes to branch point intermediate production and can potentially reverse the FDL trimming reaction. However, there has been no concerted effort to evaluate the relationships among these three enzymes in any single insect system. Hence, we extended our previous studies on Spodoptera frugiperda (Sf) FDL to include GNT-I and -II. Sf-GNT-I and -II cDNAs were isolated, the predicted protein sequences were analyzed, and both gene products were expressed and their acceptor substrate specificities and intracellular localizations were determined. Sf-GNT-I transferred N-acetylglucosamine to Man(5)GlcNAc(2), Man(3)GlcNAc(2), and GlcNAc(β1-2)Man(α1-6)[Man(α1-3)]ManGlcNAc(2), demonstrating its role in branch point intermediate production and its ability to reverse FDL trimming. Sf-GNT-II only transferred N-acetylglucosamine to Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2), demonstrating that it initiates complex N-glycan production, but cannot use Man(3)GlcNAc(2) to produce hybrid or complex structures. Fluorescently tagged Sf-GNT-I and -II co-localized with an endogenous Sf Golgi marker and Sf-FDL co-localized with Sf-GNT-I and -II, indicating that all three enzymes are Golgi resident proteins. Unexpectedly, fluorescently tagged Drosophila melanogaster FDL also co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. Thus, the substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the

Extending enzyme molecular recognition with an expanded amino acid alphabet

PubMed Central

Windle, Claire L.; Simmons, Katie J.; Ault, James R.; Trinh, Chi H.; Nelson, Adam

2017-01-01

Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties. PMID:28196894

Independent Evolution of Six Families of Halogenating Enzymes.

PubMed

Xu, Gangming; Wang, Bin-Gui

2016-01-01

Halogenated natural products are widespread in the environment, and the halogen atoms are typically vital to their bioactivities. Thus far, six families of halogenating enzymes have been identified: cofactor-free haloperoxidases (HPO), vanadium-dependent haloperoxidases (V-HPO), heme iron-dependent haloperoxidases (HI-HPO), non-heme iron-dependent halogenases (NI-HG), flavin-dependent halogenases (F-HG), and S-adenosyl-L-methionine (SAM)-dependent halogenases (S-HG). However, these halogenating enzymes with similar biological functions but distinct structures might have evolved independently. Phylogenetic and structural analyses suggest that the HPO, V-HPO, HI-HPO, NI-HG, F-HG, and S-HG enzyme families may have evolutionary relationships to the α/β hydrolases, acid phosphatases, peroxidases, chemotaxis phosphatases, oxidoreductases, and SAM hydroxide adenosyltransferases, respectively. These halogenating enzymes have established sequence homology, structural conservation, and mechanistic features within each family. Understanding the distinct evolutionary history of these halogenating enzymes will provide further insights into the study of their catalytic mechanisms and halogenation specificity.

Independent Evolution of Six Families of Halogenating Enzymes

PubMed Central

Xu, Gangming; Wang, Bin-Gui

2016-01-01

Halogenated natural products are widespread in the environment, and the halogen atoms are typically vital to their bioactivities. Thus far, six families of halogenating enzymes have been identified: cofactor-free haloperoxidases (HPO), vanadium-dependent haloperoxidases (V-HPO), heme iron-dependent haloperoxidases (HI-HPO), non-heme iron-dependent halogenases (NI-HG), flavin-dependent halogenases (F-HG), and S-adenosyl-L-methionine (SAM)-dependent halogenases (S-HG). However, these halogenating enzymes with similar biological functions but distinct structures might have evolved independently. Phylogenetic and structural analyses suggest that the HPO, V-HPO, HI-HPO, NI-HG, F-HG, and S-HG enzyme families may have evolutionary relationships to the α/β hydrolases, acid phosphatases, peroxidases, chemotaxis phosphatases, oxidoreductases, and SAM hydroxide adenosyltransferases, respectively. These halogenating enzymes have established sequence homology, structural conservation, and mechanistic features within each family. Understanding the distinct evolutionary history of these halogenating enzymes will provide further insights into the study of their catalytic mechanisms and halogenation specificity. PMID:27153321

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.

Human-specific bacterial pore-forming toxins induce programmed necrosis in erythrocytes.

PubMed

LaRocca, Timothy J; Stivison, Elizabeth A; Hod, Eldad A; Spitalnik, Steven L; Cowan, Peter J; Randis, Tara M; Ratner, Adam J

2014-08-26

A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC

Thermophilic Enzyme or Mesophilic Enzyme with Enhanced Thermostability: Can We Draw a Line?

PubMed

Jing, Xiaomin; Evangelista Falcon, Wilfredo; Baudry, Jerome; Serpersu, Engin H

2017-07-27

Aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that modifies the C4'-OH site of aminoglycoside antibiotics by nucleotidylation. A few single- and double-residue mutants of this enzyme (T130K, D80Y, and D80Y/T130K) from Bacillus stearothermophilus show increased thermostability. This article investigates how such residue replacements, which are distant from the active site and monomer-monomer interface, result in various changes of the thermostability of the enzyme. In this work, we show that the thermodynamic properties of enzyme-ligand complexes and protein dynamics may be indicators of a thermophilic behavior. Our data suggests that one of the single-site mutants of ANT, D80Y, may be a thermophilic protein and the other thermostable mutant, T130K, is actually a more heat-stable variant of the mesophilic wild type (WT) with a higher T m . Our data also suggest that T130K and D80Y adopt different global dynamics strategies to achieve different levels of thermostability enhancement and that the differences between the properties of the species can be described in terms of global dynamics rather than in terms of specific structural features. Thermophilicity of the D80Y comes at the cost of less favorable thermodynamic parameters for ligand binding relative to WT. On the other hand, the T130K species exhibits the same affinity to ligands and the same thermodynamic parameters of complex formation as the WT enzyme. These observations suggest that a quantitative characterization of ligand binding and protein dynamics can be used to differentiate thermophilic proteins from their simply more heat-stable mesophilic counterparts.

Simultaneous prediction of enzyme orthologs from chemical transformation patterns for de novo metabolic pathway reconstruction

PubMed Central

Tabei, Yasuo; Yamanishi, Yoshihiro; Kotera, Masaaki

2016-01-01

Motivation: Metabolic pathways are an important class of molecular networks consisting of compounds, enzymes and their interactions. The understanding of global metabolic pathways is extremely important for various applications in ecology and pharmacology. However, large parts of metabolic pathways remain unknown, and most organism-specific pathways contain many missing enzymes. Results: In this study we propose a novel method to predict the enzyme orthologs that catalyze the putative reactions to facilitate the de novo reconstruction of metabolic pathways from metabolome-scale compound sets. The algorithm detects the chemical transformation patterns of substrate–product pairs using chemical graph alignments, and constructs a set of enzyme-specific classifiers to simultaneously predict all the enzyme orthologs that could catalyze the putative reactions of the substrate–product pairs in the joint learning framework. The originality of the method lies in its ability to make predictions for thousands of enzyme orthologs simultaneously, as well as its extraction of enzyme-specific chemical transformation patterns of substrate–product pairs. We demonstrate the usefulness of the proposed method by applying it to some ten thousands of metabolic compounds, and analyze the extracted chemical transformation patterns that provide insights into the characteristics and specificities of enzymes. The proposed method will open the door to both primary (central) and secondary metabolism in genomics research, increasing research productivity to tackle a wide variety of environmental and public health matters. Availability and Implementation: Contact: maskot@bio.titech.ac.jp PMID:27307627

Nanostructure enzyme assemblies for biomass conversion

USDA-ARS?s Scientific Manuscript database

Biomass represents a vast resource for production of the world’s fuel and chemical feedstock needs. The use of enzymes to effect these bioconversions offers an alternative that is potentially more specific and environmentally-friendly than harsher chemical methodologies. Some species of anaerobic ...

Protect your heart: a culture-specific multimedia cardiovascular health education program.

PubMed

Shah, Amy; Clayman, Marla L; Glass, Sara; Kandula, Namratha R

2015-04-01

South Asians, the second fastest growing racial/ethnic minority in the United States, have high rates of coronary heart disease. Few coronary heart disease prevention efforts target this population. The authors developed and tested a culture-specific, multimedia coronary heart disease prevention education program in English and Hindi for South Asians. Participants were recruited from community organizations in Chicago, Illinois, between June and October of 2011. Bilingual interviewers used questionnaires to assess participants' knowledge and perceptions before and after the patient education program. The change from pretest score to posttest score was calculated using a paired t test. Linear regression was used to determine the association between posttest scores and education and language. Participants' (N = 112) average age was 41 years, 67% had more than a high school education, and 50% spoke Hindi. Participants' mean pretest score was 15 (SD = 4). After the patient education program, posttest scores increased significantly among all participants (posttest score = 24, SD = 4), including those with limited English proficiency. Lower education was associated with a lower posttest score (β = -2.2, 95% CI [-0.68, -3.83]) in adjusted regression. A culture-specific, multimedia patient education program significantly improved knowledge and perceptions about coronary heart disease prevention among South Asian immigrants. Culturally salient multimedia education may be an effective and engaging way to deliver health information to diverse patient populations.

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

Role of conformational dynamics in the evolution of novel enzyme function.

PubMed

Maria-Solano, Miguel A; Serrano-Hervás, Eila; Romero-Rivera, Adrian; Iglesias-Fernández, Javier; Osuna, Sílvia

2018-05-21

The free energy landscape concept that describes enzymes as an ensemble of differently populated conformational sub-states in dynamic equilibrium is key for evaluating enzyme activity, enantioselectivity, and specificity. Mutations introduced in the enzyme sequence can alter the populations of the pre-existing conformational states, thus strongly modifying the enzyme ability to accommodate alternative substrates, revert its enantiopreferences, and even increase the activity for some residual promiscuous reactions. In this feature article, we present an overview of the current experimental and computational strategies to explore the conformational free energy landscape of enzymes. We provide a series of recent publications that highlight the key role of conformational dynamics for the enzyme evolution towards new functions and substrates, and provide some perspectives on how conformational dynamism should be considered in future computational enzyme design protocols.

Enzyme-coupled nanoparticles-assisted laser desorption ionization mass spectrometry for searching for low-mass inhibitors of enzymes in complex mixtures.

PubMed

Salwiński, Aleksander; Da Silva, David; Delépée, Raphaël; Maunit, Benoît

2014-04-01

In this report, enzyme-coupled magnetic nanoparticles (EMPs) were shown to be an effective affinity-based tool for finding specific interactions between enzymatic targets and the low-mass molecules in complex mixtures using classic MALDI-TOF apparatus. EMPs used in this work act as nonorganic matrix enabling ionization of small molecules without any interference in the low-mass range (enzyme-coupled nanoparticles-assisted laser desorption ionization MS, ENALDI MS) and simultaneously carry the superficial specific binding sites to capture inhibitors present in a studied mixture. We evaluated ENALDI approach in two complementary variations: 'ion fading' (IF-ENALDI), based on superficial adsorption of inhibitors and 'ion hunting' (IH-ENALDI), based on selective pre-concentration of inhibitors. IF-ENALDI was applied for two sets of enzyme-inhibitor pairs: tyrosinase-glabridin and trypsin-leupeptin and for the real plant sample: Sparrmannia discolor leaf and stem methanol extract. The efficacy of IH-ENALDI was shown for the pair of trypsin-leupeptin. Both ENALDI approaches pose an alternative for bioassay-guided fractionation, the common method for finding inhibitors in the complex mixtures.

Identification of PSD-95 Depalmitoylating Enzymes.

PubMed

Yokoi, Norihiko; Fukata, Yuko; Sekiya, Atsushi; Murakami, Tatsuro; Kobayashi, Kenta; Fukata, Masaki

2016-06-15

Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. Protein palmitoylation, the most common lipid modification, dynamically

Identification of PSD-95 Depalmitoylating Enzymes

PubMed Central

Yokoi, Norihiko; Sekiya, Atsushi; Murakami, Tatsuro; Kobayashi, Kenta

2016-01-01

Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. SIGNIFICANCE STATEMENT Protein palmitoylation, the most common lipid

Characterization of Carboxylic Acid Reductases as Enzymes in the Toolbox for Synthetic Chemistry.

PubMed

Finnigan, William; Thomas, Adam; Cromar, Holly; Gough, Ben; Snajdrova, Radka; Adams, Joseph P; Littlechild, Jennifer A; Harmer, Nicholas J

2017-03-20

Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl-CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron-rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α-phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP + , adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.

Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

PubMed

Vieille, C; Zeikus, G J

2001-03-01

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of > 80 degrees C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

Age Specific Programming.

ERIC Educational Resources Information Center

Morse, Neil

Are children going to continue to serve television by providing a trusting audience for its commercial message, or is television going to begin to serve children? Current children's programs are designed for the sole purpose of holding the attention of the broadest age span possible. Today's television fails to enhance the small child's…

Selected soil enzyme activities in an oak-hickory forest following long-term prescribed burning

Treesearch

M. R. Bayan; F. Eivazi

1993-01-01

The biochemical reactions within the soil are mediated by soil flora and fauna, and are catalyzed by enzymes. Therefore, enzymes play a significant role in nutrient cycling. Enzymes are specific for the type of chemical reactions in which they participate. Arylsulfatase is the enzyme that catalyzes the hydrolysis of an arylsulfate anion by fission of the oxygen-sulfur...

Myocardial infarction false alarm: initial electrocardiogram and cardiac enzymes.

PubMed

Gupta, Esha Das; Sakthiswary, Rajalingham

2014-05-01

The objectives of this study were to determine the incidence of a myocardial infarction "false alarm" and evaluate the efficacy of the initial electrocardiogram and cardiac enzymes in diagnosing myocardial infarction in Malaysia. We recruited patients who were admitted with suspected myocardial infarction from June to August 2008. The medical records of these patients were reviewed for the initial electrocardiogram, initial cardiac enzyme levels (creatinine kinase-MB and troponin T), and the final diagnosis upon discharge. The subjects were stratified into 2 groups: true myocardial infarction, and false alarm. 125 patients were enrolled in this study. Following admission and further evaluation, the diagnosis was revised from myocardial infarction to other medical conditions in 48 (38.4%) patients. The sensitivity and specificity of the initial ischemic electrocardiographic changes were 54.5% and 70.8%, respectively. Raised cardiac enzymes had a sensitivity of 44.3% and specificity of 95.8%. A significant proportion of patients in Malaysia are admitted with a false-alarm myocardial infarction. The efficacy of the electrocardiogram in diagnosing myocardial infarction in Malaysia was comparable to the findings of Western studies, but the cardiac enzymes had a much lower sensitivity.

Characteristics of enzyme hydrolyzing natural covalent bond between RNA and protein VPg of encephalomyocarditis virus

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

Drygin, Yu.F.; Siyanova, E.Yu.

1986-08-10

The isolation and a preliminary characterization of the enzyme specifically hydrolyzing the phosphodiester bond between protein VPg and the RNA of encephalomyocarditis virus was the goal of the present investigation. The enzyme was isolated from a salt extract of Krebs II mouse ascites carcinoma cells by ion-exchange and affinity chromatography. It was found that the enzyme actually specifically cleaves the covalent bond between the RNA and protein, however, the isolation procedure does not free the enzyme from impurities which partially inhibit it. The enzyme cleaves the RNA-protein VPg complex of polio virus at a high rate, it is completely inactivatedmore » at 55/sup 0/C, and is partially inhibited by EDTA.« less

Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

PubMed Central

Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

2013-01-01

A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

[Mechanisms of congenital erythrocyte enzyme deficiencies associated with hemolytic anemia].

PubMed

Boivin, P; Kahn, A

1976-01-01

The search for a mechanism for red cell enzyme deficiency associated with congenital hemolytic anemia, requires one to determine the kinetic and thermodynamic properties of the enzyme reaction and study the physico-chemical and immunological characteristics of the protein which supports enzyme activity. The technique of iso-electric focalisation and the use of specific anti-enzyme antibodies, is the reason for recent progress in the understanding of the mechanism of these deficiencies. Examples of application of these techniques are given in relation to glucose-6-dehydrogenase, pyruvate kinase, glucose phosphate isomerase, phosphofructokinase and phosphoglycerate kinase of deficiencies showing the multiplicity of the molecular mechanisms.

Effects of Nanoparticle Size on Multilayer Formation and Kinetics of Tethered Enzymes.

PubMed

Lata, James P; Gao, Lizeng; Mukai, Chinatsu; Cohen, Roy; Nelson, Jacquelyn L; Anguish, Lynne; Coonrod, Scott; Travis, Alexander J

2015-09-16

Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (⌀5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.

Development and evaluation of a Sarcocystis neurona-specific IgM capture enzyme-linked immunosorbent assay.

PubMed

Murphy, J E; Marsh, A E; Reed, S M; Meadows, C; Bolten, K; Saville, W J A

2006-01-01

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses caused primarily by the protozoal parasite Sarcocystis neurona. Currently available antemortem diagnostic testing has low specificity. The hypothesis of this study was that serum and cerebrospinal fluid (CSF) of horses experimentally challenged with S neurona would have an increased S neurona-specific IgM (Sn-IgM) concentration after infection, as determined by an IgM capture enzyme linked immunoassay (ELISA). The ELISA was based on the S neurona low molecular weight protein SNUCD-1 antigen and the monoclonal antibody 2G5 labeled with horseradish peroxidase. The test was evaluated using serum and CSF from 12 horses experimentally infected with 1.5 million S neurona sporocysts and 16 horses experimentally infected with varying doses (100 to 100,000) of S neurona sporocysts, for which results of histopathologic examination of the central nervous system were available. For horses challenged with 1.5 million sporocysts, there was a significant increase in serum Sn-IgM concentrations compared with values before infection at weeks 2-6 after inoculation (P < .0001). For horses inoculated with lower doses of S neurona, there were significant increases in serum Sn-IgM concentration at various points in time after inoculation, depending on the challenge dose (P < .01). In addition, there was a significant increase between the CSF Sn-IgM concentrations before and after inoculation (P < .0001). These results support further evaluation of the assay as a diagnostic test during the acute phase of EPM.

Regulation of yeast central metabolism by enzyme phosphorylation

PubMed Central

Oliveira, Ana Paula; Ludwig, Christina; Picotti, Paola; Kogadeeva, Maria; Aebersold, Ruedi; Sauer, Uwe

2012-01-01

As a frequent post-translational modification, protein phosphorylation regulates many cellular processes. Although several hundred phosphorylation sites have been mapped to metabolic enzymes in Saccharomyces cerevisiae, functionality was demonstrated for few of them. Here, we describe a novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics. Focusing on the network of 204 enzymes that constitute the yeast central carbon and amino-acid metabolism, we combined protein and phosphoprotein levels to identify 35 enzymes that change their degree of phosphorylation during growth under five conditions. Correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network, adding to nine known phosphoenzymes. For the pyruvate dehydrogenase complex E1 α subunit Pda1 and the newly identified phosphoregulated glycerol-3-phosphate dehydrogenase Gpd1 and phosphofructose-1-kinase complex β subunit Pfk2, we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry, metabolomics and physiological flux analysis in mutants with genetically removed phosphosites. These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes. PMID:23149688

Intron-loss evolution of hatching enzyme genes in Teleostei

PubMed Central

2010-01-01

Background Hatching enzyme, belonging to the astacin metallo-protease family, digests egg envelope at embryo hatching. Orthologous genes of the enzyme are found in all vertebrate genomes. Recently, we found that exon-intron structures of the genes were conserved among tetrapods, while the genes of teleosts frequently lost their introns. Occurrence of such intron losses in teleostean hatching enzyme genes is an uncommon evolutionary event, as most eukaryotic genes are generally known to be interrupted by introns and the intron insertion sites are conserved from species to species. Here, we report on extensive studies of the exon-intron structures of teleostean hatching enzyme genes for insight into how and why introns were lost during evolution. Results We investigated the evolutionary pathway of intron-losses in hatching enzyme genes of 27 species of Teleostei. Hatching enzyme genes of basal teleosts are of only one type, which conserves the 9-exon-8-intron structure of an assumed ancestor. On the other hand, otocephalans and euteleosts possess two types of hatching enzyme genes, suggesting a gene duplication event in the common ancestor of otocephalans and euteleosts. The duplicated genes were classified into two clades, clades I and II, based on phylogenetic analysis. In otocephalans and euteleosts, clade I genes developed a phylogeny-specific structure, such as an 8-exon-7-intron, 5-exon-4-intron, 4-exon-3-intron or intron-less structure. In contrast to the clade I genes, the structures of clade II genes were relatively stable in their configuration, and were similar to that of the ancestral genes. Expression analyses revealed that hatching enzyme genes were high-expression genes, when compared to that of housekeeping genes. When expression levels were compared between clade I and II genes, clade I genes tends to be expressed more highly than clade II genes. Conclusions Hatching enzyme genes evolved to lose their introns, and the intron-loss events occurred at

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

PubMed Central

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Accelerating the Rate-Limiting Step in Novel Enzymatic Carbohydrate-to-Hydrogen Technology by Enzyme Engineering

DTIC Science & Technology

2011-10-30

stable phosphoglucose isomerase through immobilization of cellulose-binding module-tagged thermophilic enzyme on low- cost high-capacity celiulosic...NOVEL ENZYMATIC CARBOHYDRATE-TO-HYDROGEN TECHNOLOGY BY ENZYME ENGINEERING Grant/Contract Number: FA9550-08-1-0145 Program Manager: Dr. Walt...bbtransformation (SyPaB) is the implementation of complicated biochemical reactions by in vitro assembly of enzyme and coenzymes. Different from in vivo

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins.

PubMed

Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

2015-02-15

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein. Copyright © 2014 Elsevier B.V. All rights reserved.

Resident-Specific Morbidity Reduced Following ACS NSQIP Data-Driven Quality Program.

PubMed

Turrentine, Florence E; Hanks, John B; Tracci, Megan C; Jones, R Scott; Schirmer, Bruce D; Smith, Philip W

2018-04-16

The Accreditation Council for Graduate Medical Education Milestone Project for general surgery provided a more robust method for developing and tracking residents' competence. This framework enhanced systematic and progressive development of residents' competencies in surgical quality improvement. A 22-month interactive, educational program based on resident-specific surgical outcomes data culminated in a quality improvement project for postgraduate year 4 surgery residents. Self- assessment, quality knowledge test, and resident-specific American College of Surgeons National Surgical Quality Improvement Program Quality In-Training Initiative morbidity were compared before and after the intervention. Quality in-training initiative morbidity decreased from 25% (82/325) to 18% (93/517), p = 0.015 despite residents performing more complex cases. All participants achieved level 4 competency (4/4) within the general surgery milestones improvement of care, practice-based learning and improvement competency. Institutional American College of Surgeons National Surgical Quality Improvement Program general surgery morbidity improved from the ninth to the sixth decile. Quality assessment and improvement self-assessment postintervention scores (M = 23.80, SD = 4.97) were not significantly higher than preintervention scores (M = 19.20, SD = 5.26), p = 0.061. Quality Improvement Knowledge Application Tool postintervention test scores (M = 17.4, SD = 4.88), were not significantly higher than pretest scores (M = 13.2, SD = 1.92), p = 0.12. Sharing validated resident-specific clinical data with participants was associated with improved surgical outcomes. Participating fourth year surgical residents achieved the highest score, a level 4, in the practice based learning and improvement competency of the improvement of care practice domain and observed significantly reduced surgical morbidity for cases in which they participated. Copyright © 2018. Published by Elsevier Inc.

Interaction of phospholipase C with liposome: A conformation transition of the enzyme is critical and specific to liposome composition for burst hydrolysis and fusion in concert

NASA Astrophysics Data System (ADS)

Patra, Samir Kumar; Sengupta, Dipta; Deb, Moonmoon; Kar, Swayamsiddha; Kausar, Chahat

2017-02-01

Phospholipase C (PLC)1 is known to help the pathogen B. cereus entry to the host cell and human PLC is over expressed in multiple cancers. Knowledge of dynamic activity of the enzyme PLC while in action on membrane lipids is essential and helpful to drug design and delivery. In view of this, interactions of PLC with liposome of various lipid compositions have been visualized by testing enzyme activity and microenvironments around the intrinsic fluorophores of the enzyme. Overall change of the protein's conformation has been monitored by fluorescence spectroscopy and circular dichroism (CD). Liposome aggregation and fusion were predicted by increase in turbidity and vesicle size. PLC in solution has high fluorescence and exhibit appreciable shift in its emission maxima, upon gradual change in excitation wavelength towards the red edge of the absorption band. REES fluorescence studies indicated that certain Trp fluorophores of inactive PLC are in motionally restricted compact/rigid environments in solution conformation. PLC fluorescence decreased in association with liposome and Trps loosed rigidity where liposome aggregation and fusion occurred. We argue that the structural flexibility is the cause of decrease of fluorescence, mostly to gain optimum conformation for maximum activity of the enzyme PLC. Further studies deciphered that the enzyme PLC undergoes change of conformation when mixed to LUVs prepared with specific lipids. CD data at the far-UV and near-UV regions of PLC in solution are in excellent agreement with the previous reports. CD analyses of PLC with LUVs, showed significant reduction of α-helices, increase of β-sheets; and confirmed dramatic change of orientations of Trps. In case of liposome composed of lipid raft like composition, the enzyme binds very fast, hydrolyze PC with higher rate, exhibit highest structural flexibility and promote vesicle fusion. These data strongly suggest marked differences in conformation transition induced PLC

Nanobio interfaces: charge control of enzyme/inorganic interfaces for advanced biocatalysis.

PubMed

Deshapriya, Inoka K; Kumar, Challa V

2013-11-19

Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding to nanomaterials are vital for engineering advanced, functional nanobiomaterials for biocatalysis, sensing, and biomedical applications. This feature article presents an overview of our recent discoveries on structural, functional, and mechanistic details of how enzymes interact with inorganic nanomaterials and how they can be controlled in a systematic manner using α-Zr(IV)phosphate (α-ZrP) as a model system. The interactions of a number of enzymes having a wide array of surface charges, sizes, and functional groups are investigated. Interactions are carefully controlled to screen unfavorable repulsions and enhance favorable interactions for high affinity, structure retention, and activity preservation. In specific cases, catalytic activities and substrate selectivities are improved over those of the pristine enzymes, and two examples of high activity near the boiling point of water have been demonstrated. Isothermal titration calorimetric studies indicated that enzyme binding is coupled to ion sequestration or release to or from the nanobio interface, and binding is controlled in a rational manner. We learned that (1) bound enzyme stabilities are improved by lowering the entropy of the denatured state; (2) maximal loadings are obtained by matching charge footprints of the enzyme and the nanomaterial surface; (3) binding affinities are improved by ion sequestration at the nanobio interface; and (4) maximal enzyme structure retention is obtained by biophilizing the nanobio interface with protein glues. The chemical and physical manipulations of the nanobio interface are significant not only for understanding the complex behaviors of enzymes at biological interfaces but also for desiging better functional nanobiomaterials for a wide variety of practical applications.

Survey of Microbial Enzymes in Soil, Water, and Plant Microenvironments

PubMed Central

Alves, Priscila Divina Diniz; Siqueira, Flávia de Faria; Facchin, Susanne; Horta, Carolina Campolina Rebello; Victória, Júnia Maria Netto; Kalapothakis, Evanguedes

2014-01-01

Detection of microbial enzymes in natural environments is important to understand biochemical activities and to verify the biotechnological potential of the microorganisms. In the present report, 346 isolates from soil, water, and plants were screened for enzyme production (caseinase, gelatinase, amylase, carboxymethyl cellulase, and esterase). Our results showed that 89.6% of isolates produced at least one tested enzyme. A predominance of amylase in soil samples, carboxymethyl cellulase in plants, as well as esterase and gelatinase in water was observed. Interesting enzymatic profiles were found in some microenvironments, suggesting specificity of available nutrients and/or natural selection. This study revealed the potential of microorganisms present in water, soil, and plant to produce important enzymes for biotechnological exploration. A predominance of certain enzymes was found, depending on the type of environmental sample. The distribution of microbial enzymes in soil, water and plants has been little exploited in previous reports. PMID:24847390

Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

PubMed Central

Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

2006-01-01

Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å. PMID:16511316

Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

USGS Publications Warehouse

Alcorn, S.W.; Pascho, R.J.

2000-01-01

An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

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.

Partial Gene Cloning and Enzyme Structure Modeling of Exolevanase Fragment from Bacillus subtilis

NASA Astrophysics Data System (ADS)

Azhar, M.; Natalia, D.; Syukur, S.; Andriani, N.; Jamsari, J.

2018-04-01

Inulin hydrolysis thermophilic and thermotolerant bacteria are potential sources of inulin hydrolysis enzymes. Partial gene that encodes inulin hydrolysis enzymes had been isolated from Bacillus subtilis using polymerase chain reaction (PCR) method with the DPE.slFandDPE.eR degenerative primers. The partial gene was cloned into pGEM-T Easy vector with E. coli as host cells and analyzed using BLASTx, CrustalW2, and Phyre2 programs. Size of thepartial gene had been found539 bp that encoded 179aminoacid residues of protein fragment. The sequences of protein fragment was more similar to exolevanase than exoinulinase. The protein fragment had conserved motif FSGS, and specific hits GH32 β-fructosidase. It had three residues of active site and five residues of substrate binding. The active site on the protein fragment were D (1-WLNDP-5), D (125-FRDPK-129) and E (177-WEC-179). Substrate binding on the protein fragment were ND (1-WLNDP-5), Q (18-FYQY-21), FS (60-FSGS-63) RD (125-FRDPK-129) and E (177-WEC-179).

Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I

PubMed Central

Dickson, Patricia; Peinovich, Maryn; McEntee, Michael; Lester, Thomas; Le, Steven; Krieger, Aimee; Manuel, Hayden; Jabagat, Catherine; Passage, Merry; Kakkis, Emil D.

2008-01-01

Mucopolysaccharidoses (MPSs) are lysosomal storage diseases caused by a deficit in the enzymes needed for glycosaminoglycan (GAG) degradation. Enzyme replacement therapy with recombinant human α-l-iduronidase successfully reduces lysosomal storage in canines and humans with iduronidase-deficient MPS I, but therapy usually also induces antibodies specific for the recombinant enzyme that could reduce its efficacy. To understand the potential impact of α-l-iduronidase–specific antibodies, we studied whether inducing antigen-specific immune tolerance to iduronidase could improve the effectiveness of recombinant iduronidase treatment in canines. A total of 24 canines with MPS I were either tolerized to iduronidase or left nontolerant. All canines received i.v. recombinant iduronidase at the FDA-approved human dose or a higher dose for 9–44 weeks. Nontolerized canines developed iduronidase-specific antibodies that proportionally reduced in vitro iduronidase uptake. Immune-tolerized canines achieved increased tissue enzyme levels at either dose in most nonreticular tissues and a greater reduction in tissue GAG levels, lysosomal pathology, and urinary GAG excretion. Tolerized MPS I dogs treated with the higher dose received some further benefit in the reduction of GAGs in tissues, urine, and the heart valve. Therefore, immune tolerance to iduronidase improved the efficacy of enzyme replacement therapy with recombinant iduronidase in canine MPS I and could potentially improve outcomes in patients with MPS I and other lysosomal storage diseases. PMID:18654665

Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus.

PubMed

Huang, Yi; Zhu, Youjie; Yang, Mengshi; Zhang, Zhenqing; Song, Donglin; Yuan, Zhiming

2014-12-01

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

Atmospheric, Magnetospheric and Plasmas in space (AMPS) spacelab payload definition study. Volume 4. Part 1, AMPS program specification

NASA Technical Reports Server (NTRS)

Keeley, J. T.

1976-01-01

The AMPS Program Specification delineates the AMPS Program requirements consistent with the resources defined in the AMPS Project Plan. All subsidiary specifications and requirements shall conform to the requirements presented. The requirements hierarchy for the AMPS program is illustrated. A brief description of each of the requirements documents and their intended use is provided.

RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

NASA Technical Reports Server (NTRS)

Ordoukhanian, Phillip; Joyce, Gerald F.

2002-01-01

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction.

PubMed

Siegel, Justin B; Zanghellini, Alexandre; Lovick, Helena M; Kiss, Gert; Lambert, Abigail R; St Clair, Jennifer L; Gallaher, Jasmine L; Hilvert, Donald; Gelb, Michael H; Stoddard, Barry L; Houk, Kendall N; Michael, Forrest E; Baker, David

2010-07-16

The Diels-Alder reaction is a cornerstone in organic synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimolecular Diels-Alder reactions. We describe the de novo computational design and experimental characterization of enzymes catalyzing a bimolecular Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallography confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond-forming reactions should be broadly useful in synthetic chemistry.

25 CFR 39.137 - May schools operate a language development program without a specific appropriation from Congress?

Code of Federal Regulations, 2012 CFR

2012-04-01

... a specific appropriation from Congress? Yes, a school may operate a language development program... 25 Indians 1 2012-04-01 2011-04-01 true May schools operate a language development program without a specific appropriation from Congress? 39.137 Section 39.137 Indians BUREAU OF INDIAN AFFAIRS...

25 CFR 39.137 - May schools operate a language development program without a specific appropriation from Congress?

Code of Federal Regulations, 2013 CFR

2013-04-01

... a specific appropriation from Congress? Yes, a school may operate a language development program... 25 Indians 1 2013-04-01 2013-04-01 false May schools operate a language development program without a specific appropriation from Congress? 39.137 Section 39.137 Indians BUREAU OF INDIAN AFFAIRS...

25 CFR 39.137 - May schools operate a language development program without a specific appropriation from Congress?

Code of Federal Regulations, 2011 CFR

2011-04-01

... a specific appropriation from Congress? Yes, a school may operate a language development program... 25 Indians 1 2011-04-01 2011-04-01 false May schools operate a language development program without a specific appropriation from Congress? 39.137 Section 39.137 Indians BUREAU OF INDIAN AFFAIRS...

25 CFR 39.137 - May schools operate a language development program without a specific appropriation from Congress?

Code of Federal Regulations, 2014 CFR

2014-04-01

... a specific appropriation from Congress? Yes, a school may operate a language development program... 25 Indians 1 2014-04-01 2014-04-01 false May schools operate a language development program without a specific appropriation from Congress? 39.137 Section 39.137 Indians BUREAU OF INDIAN AFFAIRS...

Substance Abuse-Specific Knowledge Transfer or Loss? Treatment Program Turnover versus Professional Turnover among Substance Abuse Clinicians

PubMed Central

Eby, Lillian T.; Curtis, Sara L.

2014-01-01

This longitudinal study investigated the extent to which substance abuse (SA) clinician turnover is associated with SA-specific knowledge loss due to change in professions (professional turnover) versus SA-specific knowledge transfer due to movement from one SA clinical setting to another (treatment program turnover). For this study, clinicians had to voluntarily leave their current treatment program. Eligible clinicians completed a quantitative survey while employed and a qualitative post-employment exit interview 1 year later. Compared to those that exited the SA profession (N = 99), clinicians who changed treatment programs (N = 120) had greater SA-specific formal knowledge and were more likely to be personally in recovery. No differences were found between the two groups in terms of SA-specific practical knowledge. PMID:25115318

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.

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

PubMed Central

Vieille, Claire; Zeikus, Gregory J.

2001-01-01

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described. PMID:11238984

Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism.

PubMed

Pavelka, S

2014-01-01

specific enzyme activity of D1 in WAT and plasma leptin levels was found. The newly developed and adapted radiometric enzyme assays proved to be very useful tools for studies of factors modulating THs metabolism, not only in model animals but also in clinical studies of human obesity.

Empirical evaluation of a virtual laboratory approach to teach lactate dehydrogenase enzyme kinetics.

PubMed

Booth, Christine; Cheluvappa, Rajkumar; Bellinson, Zack; Maguire, Danni; Zimitat, Craig; Abraham, Joyce; Eri, Rajaraman

2016-06-01

Personalised instruction is increasingly recognised as crucial for efficacious learning today. Our seminal work delineates and elaborates on the principles, development and implementation of a specially-designed adaptive, virtual laboratory. We strived to teach laboratory skills associated with lactate dehydrogenase (LDH) enzyme kinetics to 2nd-year biochemistry students using our adaptive learning platform. Pertinent specific aims were to:(1)design/implement a web-based lesson to teach lactate dehydrogenase(LDH) enzyme kinetics to 2nd-year biochemistry students(2)determine its efficacious in improving students' comprehension of enzyme kinetics(3)assess their perception of its usefulness/manageability(vLab versus Conventional Tutorial). Our tools were designed using HTML5 technology. We hosted the program on an adaptive e-learning platform (AeLP). Provisions were made to interactively impart informed laboratory skills associated with measuring LDH enzyme kinetics. A series of e-learning methods were created. Tutorials were generated for interactive teaching and assessment. The learning outcomes herein were on par with that from a conventional classroom tutorial. Student feedback showed that the majority of students found the vLab learning experience "valuable"; and the vLab format/interface "well-designed". However, there were a few technical issues with the 1st roll-out of the platform. Our pioneering effort resulted in productive learning with the vLab, with parity with that from a conventional tutorial. Our contingent discussion emphasises not only the cornerstone advantages, but also the shortcomings of the AeLP method utilised. We conclude with an astute analysis of possible extensions and applications of our methodology.

Quantitation of Lipase Activity from a Bee: An Introductory Enzyme Experiment.

ERIC Educational Resources Information Center

Farley, Kathleen A.; Jones, Marjorie A.

1989-01-01

This four-hour experiment uses a bee as a source of the enzyme which is reacted with a radioactive substrate to determine the specific activity of the enzyme. Uses thin layer chromatography, visible spectrophotometry, and liquid scintillation spectrometry (if not available a Geiger-Muller counter can be substituted). (MVL)

Engineering peptide ligase specificity by proteomic identification of ligation sites.

PubMed

Weeks, Amy M; Wells, James A

2018-01-01

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Type III restriction-modification enzymes: a historical perspective.

PubMed

Rao, Desirazu N; Dryden, David T F; Bheemanaik, Shivakumara

2014-01-01

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

Uronic polysaccharide degrading enzymes.

PubMed

Garron, Marie-Line; Cygler, Miroslaw

2014-10-01

In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polysaccharide lyase families has increased to 23 and more detailed analysis has allowed the identification of more closely related subfamilies, leading to stronger correlation between each subfamily and a unique substrate. The number of as yet unclassified polysaccharide lyases has also increased and we expect that sequencing projects will allow many of these unclassified sequences to emerge as new families. The progress in structural analysis of PLs has led to having at least one representative structure for each of the families and for two unclassified enzymes. The newly determined structures have folds observed previously in other PL families and their catalytic mechanisms follow either metal-assisted or Tyr/His mechanisms characteristic for other PL enzymes. Comparison of PLs with glycoside hydrolases (GHs) shows several folds common to both classes but only for the β-helix fold is there strong indication of divergent evolution from a common ancestor. Analysis of bacterial genomes identified gene clusters containing multiple polysaccharide cleaving enzymes, the Polysaccharides Utilization Loci (PULs), and their gene complement suggests that they are organized to process completely a specific polysaccharide. Copyright © 2014 Elsevier Ltd. All rights reserved.

Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

PubMed

Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

2016-01-01

Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C.

PubMed

Murakami, Chiaki; Mizuno, Satoru; Kado, Sayaka; Sakane, Fumio

2017-06-01

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (≥32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Ballistic Motion of Enzymes that Catalyze Highly Exothermic Reactions

NASA Astrophysics Data System (ADS)

Tsekouras, Konstantinos; Pressé, Steve

Recently we proposed that the experimentally observed enhanced diffusion of enzymes catalyzing highly exothermic reactions is a consequence of their mechanism for dissipating reaction energy. More specifically, we proposed that reaction energy spreads out from the reaction site in the form of an acoustic wave which causes the enzyme to asymmetrically deform into the solvent. The solvent reaction propels the enzyme. However, it has been noted that in water, high viscosity should reduce enzyme momentum to zero within a few ps, so any diffusion increase should not be observable. Here we provide a model explaining how small volumetric expansions of biomolecules inside water may cause fluid compression that in turn creates regions of low fluid density around the biomolecule. We then investigate the dynamics of the biomolecule in the presence of these perturbations.

Isolation of a matrix that binds medial Golgi enzymes

PubMed Central

1994-01-01

Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae. PMID:8106542

Organic Phosphorus Characterisation in Agricultural Soils by Enzyme Addition Assays

NASA Astrophysics Data System (ADS)

Jarosch, Klaus; Frossard, Emmanuel; Bünemann, Else K.

2013-04-01

Phosphorus (P) is a non-renewable resource and it is a building block of many molecules indispensable for life. Up to 80 per cent of total soil P can be in organic form. Hydrolysability and thereby availability to plants and microorganisms differ strongly among the multitude of chemical forms of soil organic P. A recent approach to characterise organic P classes is the addition of specific enzymes which hydrolyse organic P to inorganic orthophosphate, making it detectable by colorimetry. Based on the substrate specificity of the added enzymes, conclusions about the hydrolysed forms of organic P can then be made. The aim of this study was to determine the applicability of enzyme addition assays for the characterisation of organic P species in soil:water suspensions of soils with differing properties. To this end, ten different soil samples originating from four continents, with variable pH (in water) values (4.2-8.0), land management (grassland or cropped land) and P fertilization intensity were analysed. Three different enzymes were used (acid phosphatase, nuclease and phytase). Acid phosphatase alone or in combination with nuclease was applied to determine the content of P in simple monoesters (monoester-like P) and P in DNA (DNA-like P), while P hydrolysed from myo-inositol hexakisphosphate (Ins6P-like P) was calculated from P release after incubation with phytase minus P release by acid phosphatase. To reduce sorption of inorganic P on soil particles of the suspension, especially in highly weathered soils, soil specific EDTA additions were determined in extensive pre-tests. The results of these pre-tests showed that recoveries of at least 30 per cent could be achieved in all soils. Thus, detection of even small organic P pools, such as DNA-like P, was possible in all soils if a suitable EDTA concentration was chosen. The enzyme addition assays provided information about the hydrolysable quantities of the different classes of soil organic P compounds as affected

Homogalacturonan-modifying enzymes: structure, expression, and roles in plants

PubMed Central

Sénéchal, Fabien; Wattier, Christopher; Rustérucci, Christine; Pelloux, Jérôme

2014-01-01

Understanding the changes affecting the plant cell wall is a key element in addressing its functional role in plant growth and in the response to stress. Pectins, which are the main constituents of the primary cell wall in dicot species, play a central role in the control of cellular adhesion and thereby of the rheological properties of the wall. This is likely to be a major determinant of plant growth. How the discrete changes in pectin structure are mediated is thus a key issue in our understanding of plant development and plant responses to changes in the environment. In particular, understanding the remodelling of homogalacturonan (HG), the most abundant pectic polymer, by specific enzymes is a current challenge in addressing its fundamental role. HG, a polymer that can be methylesterified or acetylated, can be modified by HGMEs (HG-modifying enzymes) which all belong to large multigenic families in all species sequenced to date. In particular, both the degrees of substitution (methylesterification and/or acetylation) and polymerization can be controlled by specific enzymes such as pectin methylesterases (PMEs), pectin acetylesterases (PAEs), polygalacturonases (PGs), or pectate lyases-like (PLLs). Major advances in the biochemical and functional characterization of these enzymes have been made over the last 10 years. This review aims to provide a comprehensive, up to date summary of the recent data concerning the structure, regulation, and function of these fascinating enzymes in plant development and in response to biotic stresses. PMID:25056773

Broad specification fuels combustion technology program

NASA Technical Reports Server (NTRS)

Dodds, W. J.; Ekstedt, E. E.

1984-01-01

Design and development efforts to evolve promising aircraft gas turbine combustor configurations for burning broadened-properties fuels were discussed. Design and experimental evaluations of three different combustor concepts in sector combustor rig tests was conducted. The combustor concepts were a state of the art single-annular combustor, a staged double-annular combustor, and a short single-annular combustor with variable geometry to control primary zone stoichiometry. A total of 25 different configurations of the three combustor concepts were evaluated. Testing was conducted over the full range of CF6-80A engine combustor inlet conditions, using four fuels containing between 12% and 14% hydrogen by weight. Good progress was made toward meeting specific program emissions and performance goals with each of the three combustor concepts. The effects of reduced fuel hydrogen content, including increased flame radiation, liner metal temperature, smoke, and NOx emissions were documented. The most significant effect on the baseline combustor was a projected 33% life reduction, for a reduction from 14% to 13% fuel hydrogen content, due to increased liner temperatures.

Sequencing of oligosaccharides using enzyme array digestion with electrochemical and fluorescent detections

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

Sun, M.; Lee, C.S.

1997-12-31

The objective of this study is to develop a rapid and sensitive method for oligosaccharide sequencing. The oligosaccharides are subjected to the enzyme array digestion with exoglycosidases of known and well-defined specificities. The enzyme array method involves the division of oligosaccharide sample into aliquots, and the incubation of each aliquot with a precisely defined mixture of exoglycosidases. In the enzyme array method, the presence of a specific linkage anywhere in the oligosaccharide is determined by the inability of an enzyme mixture lacking a given enzyme to cleave that linkage ( a stop point) and the ability of the other enzymesmore » to cleave the linkage up to that point. The direct quantification of released monosaccharides from the enzyme array can be achieved by using pulsed amperometric detection (PAD) or by fluorescent derivatization with a fluorophoric agent. The measured monosaccharide concentrations in combination with the enzyme array analysis provide detail characterization of oligosaccharides with their sugar composition, configuration, and linkage information, The released monosaccharides are further quantified by anion exchange chromatography and capillary electrophoresis for the comparison with the results obtained from PAD and fluorescence measurements. Our enzyme array-electrochemical (or fluorescent) detection method does not require any separation procedure and any prior labeling of oligosaccharide and have several practical advantages over the current carbohydrate sequencing techniques including simplicity, speed, and the ability to use small amounts of starting material.« less

Exquisite Enzyme-Fenton Biomimetic Catalysts for Hydroxyl Radical Production by Mimicking an Enzyme Cascade.

PubMed

Zhang, Qi; Chen, Shuo; Wang, Hua; Yu, Hongtao

2018-03-14

Hydrogen peroxide (H 2 O 2 ) is a key reactant in the Fenton process. As a byproduct of enzymatic reaction, H 2 O 2 can be obtained via catalytical oxidation of glucose using glucose oxidase in the presence of O 2 . Another oxidation product (gluconic acid) can suitably adjust the microenvironmental pH contributing to the Fe 3+ /Fe 2+ cycle in the Fenton reaction. Enzymes are extremely efficient at catalyzing a variety of reactions with high catalytic activity, substrate specificity, and yields in living organisms. Inspired by the multiple functions of natural multienzyme systems, an exquisite nanozyme-modified α-FeOOH/porous carbon (PC) biomimetic catalyst constructed by in situ growth of glucose oxidase-mimicking Au nanoparticles and crystallization of adsorbed ferric ions within carboxyl into hierarchically PC is developed as an efficient enzyme-Fenton catalyst. The products (H 2 O 2 , ∼4.07 mmol·L -1 ) of the first enzymatic reaction are immediately used as substrates for the second Fenton-like reaction to generate the valuable • OH (∼96.84 μmol·L -1 ), thus mimicking an enzyme cascade pathway. α-FeOOH nanocrystals, attached by C-O-Fe bondings, are encapsulated into the mesoporous PC frameworks, facilitating the electron transfer between α-FeOOH and the PC support and greatly suppressing iron leaching. This study paves a new avenue for designing biomimetic enzyme-based Fenton catalysts mimicking a natural system for • OH production.

Novel fungal FAD glucose dehydrogenase derived from Aspergillus niger for glucose enzyme sensor strips.

PubMed

Sode, Koji; Loew, Noya; Ohnishi, Yosuke; Tsuruta, Hayato; Mori, Kazushige; Kojima, Katsuhiro; Tsugawa, Wakako; LaBelle, Jeffrey T; Klonoff, David C

2017-01-15

In this study, a novel fungus FAD dependent glucose dehydrogenase, derived from Aspergillus niger (AnGDH), was characterized. This enzyme's potential for the use as the enzyme for blood glucose monitor enzyme sensor strips was evaluated, especially by investigating the effect of the presence of xylose during glucose measurements. The substrate specificity of AnGDH towards glucose was investigated, and only xylose was found as a competing substrate. The specific catalytic efficiency for xylose compared to glucose was 1.8%. The specific activity of AnGDH for xylose at 5mM concentration compared to glucose was 3.5%. No other sugars were used as substrate by this enzyme. The superior substrate specificity of AnGDH was also demonstrated in the performance of enzyme sensor strips. The impact of spiking xylose in a sample with physiological glucose concentrations on the sensor signals was investigated, and it was found that enzyme sensor strips using AnGDH were not affected at all by 5mM (75mg/dL) xylose. This is the first report of an enzyme sensor strip using a fungus derived FADGDH, which did not show any positive bias at a therapeutic level xylose concentration on the signal for a glucose sample. This clearly indicates the superiority of AnGDH over other conventionally used fungi derived FADGDHs in the application for SMBG sensor strips. The negligible activity of AnGDH towards xylose was also explained on the basis of a 3D structural model, which was compared to the 3D structures of A. flavus derived FADGDH and of two glucose oxidases. Copyright © 2016 Elsevier B.V. All rights reserved.

Non-metabolic functions of glycolytic enzymes in tumorigenesis.

PubMed

Yu, X; Li, S

2017-05-11

Cancer cells reprogram their metabolism to meet the requirement for survival and rapid growth. One hallmark of cancer metabolism is elevated aerobic glycolysis and reduced oxidative phosphorylation. Emerging evidence showed that most glycolytic enzymes are deregulated in cancer cells and play important roles in tumorigenesis. Recent studies revealed that all essential glycolytic enzymes can be translocated into nucleus where they participate in tumor progression independent of their canonical metabolic roles. These noncanonical functions include anti-apoptosis, regulation of epigenetic modifications, modulation of transcription factors and co-factors, extracellular cytokine, protein kinase activity and mTORC1 signaling pathway, suggesting that these multifaceted glycolytic enzymes not only function in canonical metabolism but also directly link metabolism to epigenetic and transcription programs implicated in tumorigenesis. These findings underscore our understanding about how tumor cells adapt to nutrient and fuel availability in the environment and most importantly, provide insights into development of cancer therapy.

Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains

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

Choi, Ji-Hye; Lee, Heeseob; Kim, Young-Wan

2009-01-09

A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both {alpha}-(1,6)- and {alpha}-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with {sup 14}C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such asmore » maltononaose and maltooctaosyl-{alpha}-(1,6)-{beta}-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.« less