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Sample records for enhanced enzymic saccharification

  1. Evaluation of various fungal pretreatment of switchgrass for enhanced saccharification and simultaneous enzyme production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During fungal pretreatment of lignocellulosic biomass for bioethanol production, the treatment effectiveness may vary with different fungal strains in regard to biomass loss, sugar yield, enzyme loading, and co-product yield. In this study, 25 different fungal strains were screened for pretreatment ...

  2. Effects of granule swelling on starch saccharification by granular starch hydrolyzing enzyme.

    PubMed

    Li, Zhaofeng; Cai, Liming; Gu, Zhengbiao; Shi, Yong-Cheng

    2014-08-13

    The effects of granule swelling on enzymatic saccharification of normal corn starch by granular starch hydrolyzing enzyme were investigated. After swelling, Km values for the saccharification of granular starch decreased compared with native granular starch, indicating that granule swelling caused granular starch hydrolyzing enzyme to have higher affinity for starch granules. The partial swelling of starch granules enhanced starch saccharification. Furthermore, the enhancement at an earlier stage of enzymatic reaction was much more significant than that at later stages. For granular starch pretreated at 67.5 °C for 30 min, conversions to glucose after incubation with the enzyme at 32 °C for 4 and 24 h were approximately 3-fold and 26% higher than for native granular starch, respectively. As a result, proper heat pretreatment of granular starch before simultaneous saccharification and fermentation has great potential to facilitate industrial production of ethanol by use of granular starch hydrolyzing enzyme. PMID:25039418

  3. Development of a commercial enzymes system for lignocellulosic biomass saccharification

    SciTech Connect

    Kumar, Manoj

    2012-12-20

    DSM Innovation Inc., in its four year effort was able to evaluate and develop its in-house DSM fungal cellulolytic enzymes system to reach enzyme efficiency mandates set by DoE Biomass program MYPP goals. DSM enzyme cocktail is uniquely active at high temperature and acidic pH, offering many benefits and product differentiation in 2G bioethanol production. Under this project, strain and process development, ratio optimization of enzymes, protein and genetic engineering has led to multitudes of improvement in productivity and efficiency making development of a commercial enzyme system for lignocellulosic biomass saccharification viable. DSM is continuing further improvement by additional biodiversity screening, protein engineering and overexpression of enzymes to continue to further lower the cost of enzymes for saccharification of biomass.

  4. Fungal pretreatment of switchgrass for improved saccharification and simultaneous enzyme production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal pretreatment of switchgrass involving solid state fermentation (SSF) to improve saccharification and simultaneously produce enzymes as co-products was investigated in this study. The results revealed that the fungus Pycnoporus sp. SYBC-L3 can significantly degrade lignin and enhance enzymatic...

  5. Enhanced biological straw saccharification through coculturing of lignocellulose-degrading microorganisms.

    PubMed

    Taha, Mohamed; Shahsavari, Esmaeil; Al-Hothaly, Khalid; Mouradov, Aidyn; Smith, Andrew T; Ball, Andrew S; Adetutu, Eric M

    2015-04-01

    Lignocellulosic waste (LCW) is an abundant, low-cost, and inedible substrate for the induction of lignocellulolytic enzymes for cellulosic bioethanol production using an efficient, environmentally friendly, and economical biological approach. In this study, 30 different lignocellulose-degrading bacterial and 18 fungal isolates were quantitatively screened individually for the saccharification of four different ball-milled straw substrates: wheat, rice, sugarcane, and pea straw. Rice and sugarcane straws which had similar Fourier transform-infrared spectroscopy profiles were more degradable, and resulted in more hydrolytic enzyme production than wheat and pea straws. Crude enzyme produced on native straws performed better than those on artificial substrates (such as cellulose and xylan). Four fungal and five bacterial isolates were selected (based on their high strawase activities) for constructing dual and triple microbial combinations to investigate microbial synergistic effects on saccharification. Combinations such as FUNG16-FUNG17 (Neosartorya fischeri-Myceliophthora thermophila) and RMIT10-RMIT11 (Aeromonas hydrophila-Pseudomonas poae) enhanced saccharification (3- and 6.6-folds, respectively) compared with their monocultures indicating the beneficial effects of synergism between those isolates. Dual isolate combinations were more efficient at straw saccharification than triple combinations in both bacterial and fungal assays. Overall, co-culturing can result in significant increases in saccharification which may offer significant commercial potential for the use of microbial consortia. PMID:25724976

  6. Consequences of antisense down-regulation of a lignification-specific peroxidase on leaf and vascular tissue in tobacco lines demonstrating enhanced enzymic saccharification.

    PubMed

    Kavousi, Bahram; Daudi, Arsalan; Cook, Charis M; Joseleau, Jean-Paul; Ruel, Katia; Devoto, Alessandra; Bolwell, G Paul; Blee, Kristopher A

    2010-04-01

    Tobacco plants expressing an antisense construct for a cationic peroxidase, which down-regulated lignin content at the presumed level of polymerisation, have been further analysed. T(1) plants were derived from a large-scale screen of T(0) mutant lines, previously published, which identified lines demonstrating consistent lignin down-regulation. Of these, line 1074 which had the most robust changes in lignin distribution through several generations was shown to have accompanying down-regulation of transcription of most lignin biosynthesis genes, except cinnamoyl-CoA reductase. The consistent 20% reduction in lignin was not accompanied by significant gross changes in vascular polysaccharide content and composition, despite a modest up-regulation of transcripts of genes involved in cellulose and hemicellulose synthesis. Morphologically, 1074 plants have under-developed xylem with both fibers and vessels having thin cell walls and limited secondary wall thickening with an abnormal S2 layer. However, they were not compromised in overall growth. Nevertheless, these and other lines showed improved potential industrial utility through a threefold increase in enzymic saccharification efficiency compared with wild-type (wt). Therefore, they were profiled for further un-intended effects of transgenesis that might compromise their value for industrial or biofuel processes. Other phenotypic changes included increased leaf thickness and bifurcation at the tip of the leaf. wt-Plants had smaller chloroplasts and higher stomatal numbers than mutants. Transgenic lines also showed a variable leaf pigment distribution with light-green areas that contained measurably less chlorophyll a, b, and carotenoids. Changes in epidermal pavement cells of mutant lines were also observed after exposure to various chemicals, while wt leaves retained their structural integrity. Despite these changes, the mutant plants grew and were viable indicating that lignification patterns can be manipulated

  7. Comparison of raw starch hydrolyzing enzyme with conventional liquefaction and saccharification enzymes in dry-grind corn processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a conventional dry-grind corn process, starch is converted into dextrins using liquefaction enzymes at high temperatures (90–120 deg C) during a liquefaction step. Dextrins are hydrolyzed into sugars using saccharification enzymes during a simultaneous saccharification and fermentation (SSF) step...

  8. Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting

    PubMed Central

    Badhan, Ajay; Wang, Yu-Xi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim A.

    2015-01-01

    Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes. PMID:26180803

  9. Low melting point pyridinium ionic liquid pretreatment for enhancing enzymatic saccharification of cellulosic biomass.

    PubMed

    Uju; Nakamoto, Aya; Shoda, Yasuhiro; Goto, Masahiro; Tokuhara, Wataru; Noritake, Yoshiyuki; Katahira, Satoshi; Ishida, Nobuhiro; Ogino, Chiaki; Kamiya, Noriho

    2013-05-01

    The potential of 1-hexylpyridinium chloride ([Hpy][Cl]), to pretreat cellulosic feedstocks was investigated using microcrystalline cellulose (Avicel) and Bagasse at 80 °C or 100 °C. Short [Hpy][Cl] pretreatments, <30 min, at lower temperature accelerate subsequent enzymatic saccharification of Avicel. Over 95% conversion of pretreated Avicel to glucose was attained after 24h enzymatic saccharification under optimal conditions, whereas regenerated Bagasse showed 1-3-fold higher conversion than untreated biomass. FT-IR analysis of both Avicel and Bagasse samples pretreated with [Hpy][Cl] or 1-ethyl-3-methyimidazolium acetate ([Emim][OAc]) revealed that these ionic liquids behaved differently during pretreatment. [Hpy][Cl] pretreatment for an extended duration (180 min) released mono- and disaccharides without using cellulase enzymes, suggesting [Hpy][Cl] has capability for direct saccharification of cellulosic feedstocks. On the basis of the results obtained, [Hpy][Cl] pretreatment enhanced initial reaction rates in enzymatic saccharification by either crystalline polymorphic alteration of cellulose or partial degradation of the crystalline cellulosic fraction in biomass. PMID:22850175

  10. Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens

    PubMed Central

    Rytioja, Johanna; Hildén, Kristiina; Mäkinen, Susanna; Vehmaanperä, Jari; Hatakka, Annele; Mäkelä, Miia R.

    2015-01-01

    White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification. PMID:26660105

  11. Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens.

    PubMed

    Rytioja, Johanna; Hildén, Kristiina; Mäkinen, Susanna; Vehmaanperä, Jari; Hatakka, Annele; Mäkelä, Miia R

    2015-01-01

    White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification. PMID:26660105

  12. Lignosulfonate and elevated pH can enhance enzymatic saccharification of lignocelluloses

    PubMed Central

    2013-01-01

    Background Nonspecific (nonproductive) binding (adsorption) of cellulase by lignin has been identified as a key barrier to reduce cellulase loading for economical sugar and biofuel production from lignocellulosic biomass. Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) is a relatively new process, but demonstrated robust performance for sugar and biofuel production from woody biomass especially softwoods in terms of yields and energy efficiencies. This study demonstrated the role of lignin sulfonation in enhancing enzymatic saccharification of lignocelluloses – lignosulfonate from SPORL can improve enzymatic hydrolysis of lignocelluloses, contrary to the conventional belief that lignin inhibits enzymatic hydrolysis due to nonspecific binding of cellulase. Results The study found that lignosulfonate from SPORL pretreatment and from a commercial source inhibits enzymatic hydrolysis of pure cellulosic substrates at low concentrations due to nonspecific binding of cellulase. Surprisingly, the reduction in enzymatic saccharification efficiency of a lignocellulosic substrate was fully recovered as the concentrations of these two lignosulfonates increased. We hypothesize that lignosulfonate serves as a surfactant to enhance enzymatic hydrolysis at higher concentrations and that this enhancement offsets its inhibitive effect from nonspecific binding of cellulase, when lignosulfonate is applied to lignocellulosic solid substrates. Lignosulfonate can block nonspecific binding of cellulase by bound lignin on the solid substrates, in the same manner as a nonionic surfactant, to significantly enhance enzymatic saccharification. This enhancement is linearly proportional to the amount of lignosulfonate applied which is very important to practical applications. For a SPORL-pretreated lodgepole pine solid, 90% cellulose saccharification was achieved at cellulase loading of 13 FPU/g glucan with the application of its corresponding pretreatment hydrolysate

  13. Saccharification of orange peel wastes with crude enzymes from new isolated Aspergillus japonicus PJ01.

    PubMed

    Li, Pei-Jun; Xia, Jin-Lan; Nie, Zhen-Yuan; Shan, Yang

    2016-03-01

    This study investigated the saccharification of orange peel wastes with crude enzymes from Aspergillus japonicus PJ01. Pretreated orange peel powder was hydrolyzed by submerged fermentation (SmF) and solid-state fermentation (SSF) crude enzymes, the results showed that 4 % (w/v) of solid loading, undiluted crude enzymes, and 45 °C were suitable saccharification conditions. The hydrolysis kinetics showed that the apparent Michaelis-Menten constant [Formula: see text] and maximal reaction rate [Formula: see text] were 73.32 g/L and 0.118 g/(L min) for SmF enzyme, and 41.45 g/L and 0.116 g/(L min) for SSF enzyme, respectively. After 48 h of hydrolysis, the saccharification yields were 58.5 and 78.7 %, the reducing sugar concentrations were 14.9 and 20.1 mg/mL by SmF and SSF enzymes. Material balance showed that the SmF enzymatic hydrolysate was enriched galacturonic acid > arabinose > galactose > xylose, and the SSF enzymatic hydrolysate was enriched galacturonic acid > xylose > galactose > arabinose. PMID:26718204

  14. Modeling fixed and fluidized reactors for cassava starch Saccharification with immobilized enzyme

    SciTech Connect

    Zanin, G.M.; De Moraes, F.F.

    1997-12-31

    Cassava starch saccharification in fixed-and fluidized-bed reactors using immobilized enzyme was modeled in a previous paper using a simple model in which all dextrins were grouped in a single substrate. In that case, although good fit of the model to experimental data was obtained, physical inconsistency appeared as negative kinetic constants. In this work, a multisubstrate model, developed earlier for saccharification with free enzyme, is adapted for immobilized enzyme. This latter model takes into account the formation of intermediate substrates, which are dextrins competing for the catalytic site of the enzyme, reversibility of some reactions, inhibition by substrate and product, and the formation of isomaltose. Kinetic parameters to be used with this model were obtained from initial velocity saccharification tests using the immobilized enzyme and different liquefied starch concentrations. The new model was found to be valid for modeling both fixed- and fluidized-bed reactors. It did not present inconsistencies as the earlier one had and has shown that apparent glucose inhibition is about seven times higher in the fixed-bed than in fluidized-bed reactor. 13 refs., 5 figs., 1 tab.

  15. Enzymic saccharification of sugarcane bagasse pretreated by autohydrolysis-steam explosion

    SciTech Connect

    Dekker, R.F.H.; Wallis, A.F.A.

    1983-12-01

    Pretreatment of bagasse by autohydrolysis at 200 degrees C for 4 min and explosive defibration resulted in the solubilization of 90% of the hemicellulose (a heteroxylan) and in the production of a pulp that was highly susceptible to hydrolysis by cellulases from Trichoderma reesei C-30 and QM 9414, and by a commercial preparation, Meicelase. Saccharification yields of 50% resulted after 24 h at 50 degrees C (pH 5.0) in enzymic digests containing 10% (w/v) bagasse pulps and 20 filter paper cellulase units (FPU). Saccharifications could be increased to more than 80% at 24 h by the addition of exogeneous ..beta..-glucosidase from Aspergillus niger. The crystallinity of cellulose in bagasse remained unchanged following autohydrolysis-explosion and did not appear to hinder the rate or extent of hydrolysis of cellulose. Autohydrolysis-exploded pulps extracted with alkali or ethanol to remove lignin resulted in lower conversions of cellulose (28-36% after 25 h) than unextracted pulps. Alkali extracted pulps arising from autohydrolysis times of more than 10 min at 200 degrees C were less susceptible to enzymic hydrolysis than unextracted pulps and alkali-extracted pulps arising from short autohydrolysis times (e.g., 2 min at 200 degrees C). Autohydrolysis-explosion was as effective a pretreatment method as 0.25M NaOH (70 degrees C/2 h); both yielded pulps that resulted in high cellulose conversions with T. reesei cellulase preparations and Meicelase. Supplementation of T. reesei C-30 cellulase preparations with A. niger ..beta..-glucosidases was effective in promoting the conversion of cellulose into glucose. A ratio of FPU to ..beta..-glucosidase of 1:1.25 was the minimum requirement to achieve more than 80% conversion of cellulose into glucose within 24 h. Other factors which influenced the extent of saccharification were the enzyme-substrate ratio, the substrate concentration, and the saccharification mode. (Refs. 30).

  16. Solid fermentation of wheat bran for hydrolytic enzymes production and saccharification content by a local isolate Bacillus megatherium

    PubMed Central

    2014-01-01

    Back ground For enzyme production, the costs of solid state fermentation (SSF) techniques were lower and the production higher than submerged cultures. A large number of fungal species was known to grow well on moist substrates, whereas many bacteria were unable to grow under this condition. Therefore, the aim of this study was to isolate a highly efficient strain of Bacillus sp utilizing wheat bran in SSF and optimizing the enzyme production and soluble carbohydrates. Results A local strain Bacillus megatherium was isolated from dung sheep. The maximum production of pectinase, xylanase and α-amylase, and saccharification content (total soluble carbohydrates and reducing sugars) were obtained by application of the B. megatherium in SSF using wheat bran as compared to grasses, palm leaves and date seeds. All enzymes and saccharification content exhibited their maximum production during 12–24 h, at the range of 40–80% moisture content of wheat bran, temperature 37-45°C and pH 5–8. An ascending repression of pectinase production was observed by carbon supplements of lactose, glucose, maltose, sucrose and starch, respectively. All carbon supplements improved the production of xylanase and α-amylase, except of lactose decreased α-amylase production. A little increase in the yield of total reducing sugars was detected for all carbon supplements. Among the nitrogen sources, yeast extract induced a significant repression to all enzyme productivity. Sodium nitrate, urea and ammonium chloride enhanced the production of xylanase, α-amylase and pectinase, respectively. Yeast extract, urea, ammonium sulphate and ammonium chloride enhanced the productivity of reducing sugars. Conclusions The optimization of enzyme production and sccharification content by B. megatherium in SSF required only adjustment of incubation period and temperature, moisture content and initial pH. Wheat bran supplied enough nutrients without any need for addition of supplements of carbon and

  17. Use of new endophytic fungi as pretreatment to enhance enzymatic saccharification of Eucalyptus globulus.

    PubMed

    Martín-Sampedro, Raquel; Fillat, Úrsula; Ibarra, David; Eugenio, María E

    2015-11-01

    New endophytic fungi are assessed for the first time as pretreatment to enhance saccharification of Eucalyptus globulus wood. The fungi are all laccase-producing ascomycetes and were isolated from eucalyptus trees in Spain. After five endophytes had been assayed alone or in combination with white-rot fungus Trametes sp. I-62, three were pre-selected. To improve sugar production, an autohydrolysis pretreatment was performed before or after fungal treatment. Pretreatment increased sugar production 2.7 times compared to non-pretreated wood. When fungal and autohydrolysis pretreatments were combined, a synergistic increase in saccharification was observed in all cases. Endophytic fungi Ulocladium sp. and Hormonema sp. produced greater enhancements in saccharification than Trametes sp. I-62 (increase in sugar yields of 8.5, 8.0 and 6.0 times, respectively), demonstrating the high potential of these new endophytic fungi for saccharification enhancement. PMID:26255602

  18. Enhanced bioprocessing of lignocellulose: Wood-rot fungal saccharification and fermentation of corn fiber to ethanol

    NASA Astrophysics Data System (ADS)

    Shrestha, Prachand

    This research aims at developing a biorefinery platform to convert corn-ethanol coproduct, corn fiber, into fermentable sugars at a lower temperature with minimal use of chemicals. White-rot (Phanerochaete chrysosporium), brown-rot (Gloeophyllum trabeum) and soft-rot (Trichoderma reesei) fungi were used in this research to biologically break down cellulosic and hemicellulosic components of corn fiber into fermentable sugars. Laboratory-scale simultaneous saccharification and fermentation (SSF) process proceeded by in-situ cellulolytic enzyme induction enhanced overall enzymatic hydrolysis of hemi/cellulose from corn fiber into simple sugars (mono-, di-, tri-saccharides). The yeast fermentation of hydrolyzate yielded 7.1, 8.6 and 4.1 g ethanol per 100 g corn fiber when saccharified with the white-, brown-, and soft-rot fungi, respectively. The highest corn-to-ethanol yield (8.6 g ethanol/100 g corn fiber) was equivalent to 42 % of the theoretical ethanol yield from starch and cellulose in corn fiber. Cellulase, xylanase and amylase activities of these fungi were also investigated over a week long solid-substrate fermentation of corn fiber. G. trabeum had the highest activities for starch (160 mg glucose/mg protein.min) and on day three of solid-substrate fermentation. P. chrysosporium had the highest activity for xylan (119 mg xylose/mg protein.min) on day five and carboxymethyl cellulose (35 mg glucose/mg protein.min) on day three of solid-substrate fermentation. T. reesei showed the highest activity for Sigma cell 20 (54.8 mg glucose/mg protein.min) on day 5 of solid-substrate fermentation. The effect of different pretreatments on SSF of corn fiber by fungal processes was examined. Corn fiber was treated at 30 °C for 2 h with alkali [2% NaOH (w/w)], alkaline peroxide [2% NaOH (w/w) and 1% H2O 2 (w/w)], and by steaming at 100 °C for 2 h. Mild pretreatment resulted in improved ethanol yields for brown- and soft-rot SSF, while white-rot and Spezyme CP SSFs showed

  19. Development of a Commerical Enzyme System for Lignocellulosic Biomass Saccharification

    SciTech Connect

    Manoj Kumar, PhD

    2011-02-14

    Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  20. Saccharification of corn fiber using enzymes from Aureobasidium sp. strain NRRL Y-2311-1

    SciTech Connect

    Leathers, T.D.; Gupta, S.C.

    1996-06-01

    Crude enzyme preparations from Aureobasidium sp. strain NRRL Y-2311-1 were characterized and tested for the capacity to saccharify corn fiber. Cultures grown on xylan, corn fiber, and alkaline hydrogen peroxide (AHP)-pretreated corn fiber produced specific levels of endoxylanase, amylase, protease, cellulose, and other activities. Using equal units of endoxylanase activity, crude enzymes from AHP-pretreated corn fiber cultures were most effective in saccharification. Multiple enzyme activities were implicated in this process. Pretreatment of corn fiber with AHP nearly doubled the susceptibility of hemicellulose to enzymatic digestion. Up to 138 mg xylose, 125 mg arabinose, and 490 mg glucose were obtained per g pretreated corn fiber under conditions tested. 31 refs., 2 figs., 4 tabs.

  1. Three-stage hydrolysis to enhance enzymatic saccharification of steam-exploded corn stover.

    PubMed

    Yang, Jing; Zhang, Xiaoping; Yong, Qiang; Yu, Shiyuan

    2010-07-01

    The objective of the present research was to explore new approach to reduce the hydrolysis time and to enhance the productivity of enzymatic saccharification. One-stage hydrolysis of steam-exploded corn stover required 72 h to reach a yield of 62.8%, while multi-stage hydrolysis could reduce the time to 24 h. A concept of three-stage hydrolysis was therefore proposed in which cellulosic substrate was hydrolyzed for 6, 6, and 12 h, respectively. High hydrolysis yields, 70.2% with enzyme recycling and 76.1% with the supplement of fresh enzyme to eliminate enzyme recovery procedure, were obtained in 24 h. Analysis indicated that short-time hydrolysis and the removal of end products at each stage improved cellulase activities and benefited the adsorption of cellulase enzyme to the solid substrate. When steam-exploded corn stover was used as the substrate for cellulase synthesis, a hydrolysis yield of 88.6% was achieved in 24 h. PMID:19857959

  2. Saccharification of ozonated sugarcane bagasse using enzymes from Myceliophthora thermophila JCP 1-4 for sugars release and ethanol production.

    PubMed

    Pereira, Josiani de Cassia; Travaini, Rodolfo; Marques, Natalia Paganini; Bolado-Rodríguez, Silvia; Martins, Daniela Alonso Bocchini

    2016-03-01

    The saccharification of ozonated sugarcane bagasse (SCB) by enzymes from Myceliophthora thermophila JCP 1-4 was studied. Fungal enzymes provided slightly higher sugar release than commercial enzymes, working at 50°C. Sugar release increased with temperature increase. Kinetic studies showed remarkable glucose release (4.99 g/L, 3%w/w dry matter) at 60°C, 8 h of hydrolysis, using an enzyme load of 10 FPU (filter paper unit). FPase and β-glucosidase activities increased during saccharification (284% and 270%, respectively). No further significant improvement on glucose release was observed increasing the enzyme load above 7.5 FPU per g of cellulose. Higher dry matter contents increased sugars release, but not yields. The fermentation of hydrolysates by Saccharomyces cerevisiae provided glucose-to-ethanol conversions around to 63%. PMID:26773948

  3. Range of cell-wall alterations enhance saccharification in Brachypodium distachyon mutants.

    PubMed

    Marriott, Poppy E; Sibout, Richard; Lapierre, Catherine; Fangel, Jonatan U; Willats, William G T; Hofte, Herman; Gómez, Leonardo D; McQueen-Mason, Simon J

    2014-10-01

    Lignocellulosic plant biomass is an attractive feedstock for the production of sustainable biofuels, but the commercialization of such products is hampered by the high costs of processing this material into fermentable sugars (saccharification). One approach to lowering these costs is to produce crops with cell walls that are more susceptible to hydrolysis to reduce preprocessing and enzyme inputs. To deepen our understanding of the molecular genetic basis of lignocellulose recalcitrance, we have screened a mutagenized population of the model grass Brachypodium distachyon for improved saccharification with an industrial polysaccharide-degrading enzyme mixture. From an initial screen of 2,400 M2 plants, we selected 12 lines that showed heritable improvements in saccharification, mostly with no significant reduction in plant size or stem strength. Characterization of these putative mutants revealed a variety of alterations in cell-wall components. We have mapped the underlying genetic lesions responsible for increased saccharification using a deep sequencing approach, and here we report the mapping of one of the causal mutations to a narrow region in chromosome 2. The most likely candidate gene in this region encodes a GT61 glycosyltransferase, which has been implicated in arabinoxylan substitution. Our work shows that forward genetic screening provides a powerful route to identify factors that impact on lignocellulose digestibility, with implications for improving feedstock for cellulosic biofuel production. PMID:25246540

  4. Range of cell-wall alterations enhance saccharification in Brachypodium distachyon mutants

    PubMed Central

    Marriott, Poppy E.; Sibout, Richard; Lapierre, Catherine; Fangel, Jonatan U.; Willats, William G. T.; Hofte, Herman; Gómez, Leonardo D.; McQueen-Mason, Simon J.

    2014-01-01

    Lignocellulosic plant biomass is an attractive feedstock for the production of sustainable biofuels, but the commercialization of such products is hampered by the high costs of processing this material into fermentable sugars (saccharification). One approach to lowering these costs is to produce crops with cell walls that are more susceptible to hydrolysis to reduce preprocessing and enzyme inputs. To deepen our understanding of the molecular genetic basis of lignocellulose recalcitrance, we have screened a mutagenized population of the model grass Brachypodium distachyon for improved saccharification with an industrial polysaccharide-degrading enzyme mixture. From an initial screen of 2,400 M2 plants, we selected 12 lines that showed heritable improvements in saccharification, mostly with no significant reduction in plant size or stem strength. Characterization of these putative mutants revealed a variety of alterations in cell-wall components. We have mapped the underlying genetic lesions responsible for increased saccharification using a deep sequencing approach, and here we report the mapping of one of the causal mutations to a narrow region in chromosome 2. The most likely candidate gene in this region encodes a GT61 glycosyltransferase, which has been implicated in arabinoxylan substitution. Our work shows that forward genetic screening provides a powerful route to identify factors that impact on lignocellulose digestibility, with implications for improving feedstock for cellulosic biofuel production. PMID:25246540

  5. Simultaneously saccharification and fermentation approach as a tool for enhanced fossil fuels biodesulfurization.

    PubMed

    Paixão, Susana M; Arez, Bruno F; Roseiro, José C; Alves, Luís

    2016-11-01

    Biodesulfurization can be a complementary technology to the hydrodesulfurization, the commonly physical-chemical process used for sulfur removal from crude oil. The desulfurizing bacterium Gordonia alkanivorans strain 1B as a fructophilic microorganism requires fructose as C-source. In this context, the main goal of this work was the optimization of a simultaneous saccharification and fermentation (SSF) approach using the Zygosaccharomyces bailii strain Talf1 crude enzymes with invertase activity and sucrose as a cheaper fructose-rich commercial C-source (50% fructose) towards dibenzothiophene (DBT) desulfurization by strain 1B. The determination of optimal conditions, for both sucrose hydrolysis and DBT desulfurization was carried out through two sequential experimental uniform designs according to the Doehlert distribution for two factors: pH (5.5-7.5) and temperature (28-38 °C), with the enzyme load of 1.16 U/g/L; and enzyme load (0-4 U/g/L) and temperature (28-38 °C), with pH at 7.5. Based on 2-hydroxybiphenyl production, the analysis of the response surfaces obtained pointed out for pH 7.5, 32 °C and 1.8 U/g/L as optimal conditions. Further optimized SSF of sucrose during the DBT desulfurization process permitted to attain a 4-fold enhanced biodesulfurization. This study opens a new focus of research through the exploitation of sustainable low cost sucrose-rich feedstocks towards a more economical viable bioprocess scale-up. PMID:27505164

  6. Simultaneous pretreatment and saccharification: green technology for enhanced sugar yields from biomass using a fungal consortium.

    PubMed

    Dhiman, Saurabh Sudha; Haw, Jung-Rim; Kalyani, Dayanand; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

    2015-03-01

    Two different biomasses were subjected to simultaneous pretreatment and saccharification (SPS) using a cocktail of hydrolytic and oxidizing enzymes. Application of a novel laccase as a detoxifying agent caused the removal of 49.8% and 32.6% of phenolic contents from the soaked rice straw and willow, respectively. Hydrolysis of soaked substrates using a newly developed fungal consortium resulted in saccharification yield of up to 74.2% and 63.6% for rice straw and willow, respectively. A high saccharification yield was obtained with soaked rice straw and willow without using any hazardous chemicals. The efficiency of each step related to SPS was confirmed by atomic force microscopy. The suitability of the developed SPS process was further confirmed by converting the hydrolysate from the process into bioethanol with 72.4% sugar conversion efficiency. To the best of our knowledge, this is the first report on the development of a less tedious, single-pot, and eco-friendly SPS methodology. PMID:25514402

  7. Assessment of bacterial and fungal (hemi)cellulose-degrading enzymes in saccharification of ammonia fibre expansion-pretreated Arundo donax.

    PubMed

    Giacobbe, Simona; Balan, Venkatesh; Montella, Salvatore; Fagnano, Massimo; Mori, Mauro; Faraco, Vincenza

    2016-03-01

    This study reports enzymatic hydrolysis of the biomass of the giant reed (Arundo donax L.) after ammonia fibre expansion (AFEX) pretreatment. In particular, the capacity of the arabinofuranosidase from the fungus Pleurotus ostreatus recombinantly expressed in Pichia pastoris rPoAbf, its evolved mutant rPoAbf F435Y/Y446F and the endo-cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli to enhance the hydrolysis of AFEX-treated A. donax was investigated, using the corn stover as reference feedstock. The investigated enzymes were assayed using a mixture of purified cellulases (CBHI, CBHII, EGI and βG), endoxylanases (LX3, LX4) and accessory hemicellulases (LarbF and LβX) as reference enzyme mixture and substituting EGI with rCelStrep and LarbF with rPoAbf or rPoAbf F435Y/Y446F. The use of rPoAbf F435Y/Y446F in the substitution of LarbF led to improvements in sugar conversion, giving a glucan, xylan and arabinan conversion after 72 h of around 62, 63 and 80 %, respectively, similar or higher than those (44, 66 and 55 %) achieved by 72 h hydrolysis with commercial enzymes Novozymes Cellic®, Ctec3 and Htec3. The enzymes rPoAbf, rPoAbf F435Y/Y446F and rCelStrep were also investigated for their effect on hydrolysis of AFEX-pretreated A. donax by addition to commercial enzyme mixture Novozymes Cellic®, Ctec3 and Htec3, and it was shown that the addition of rPoAbf and its evolved mutant rPoAbf F435Y/Y446F enhanced both xylan and arabinan conversions, which achieved 80 % after 6 days of saccharification with rPoAbf F435Y/Y446F. PMID:26521250

  8. Addition of alkali to the hydrothermal-mechanochemical treatment of Eucalyptus enhances its enzymatic saccharification.

    PubMed

    Ishiguro, Maki; Endo, Takashi

    2014-02-01

    The effects of alkali on hydrothermal-mechanochemical treatment (hydrothermal treatment combined with wet-milling) were examined with the aim of improving pretreatment of lignocellulosic biomass before enzymatic saccharification. After enzymatic saccharification, the highest glucose yield was obtained by autoclaving at 170°C in the presence of 20% NaOH per substrate weight. The wood fiber was unraveled into finer nanofibers by hydrothermal-mechanochemical treatment, thus increasing the specific surface area of the substrate from 11 to 132m(2)/g. Adding 20% NaOH to the treatment further increased the specific surface area of the already fibrillated substrate by 76% (232m(2)/g) due to lignin removal and ester bond cleavage between lignin and hemicellulose. This increase in specific surface area was closely related to the increase in enzymatic digestibility; therefore, NaOH addition may have enhanced the effect of hydrothermal-mechanochemical treatment. PMID:24378778

  9. Efficient conversion of biomass into lipids by using the simultaneous saccharification and enhanced lipid production process

    PubMed Central

    2013-01-01

    Background Microbial lipid production by using lignocellulosic biomass as the feedstock holds a great promise for biodiesel production and biorefinery. This usually involves hydrolysis of biomass into sugar-rich hydrolysates, which are then used by oleaginous microorganisms as the carbon and energy sources to produce lipids. However, the costs of microbial lipids remain prohibitively high for commercialization. More efficient and integrated processes are pivotal for better techno-economics of microbial lipid technology. Results Here we describe the simultaneous saccharification and enhanced lipid production (SSELP) process that is highly advantageous in terms of converting cellulosic materials into lipids, as it integrates cellulose biomass hydrolysis and lipid biosynthesis. Specifically, Cryptococcus curvatus cells prepared in a nutrient-rich medium were inoculated at high dosage for lipid production in biomass suspension in the presence of hydrolytic enzymes without auxiliary nutrients. When cellulose was loaded at 32.3 g/L, cellulose conversion, cell mass, lipid content and lipid coefficient reached 98.5%, 12.4 g/L, 59.9% and 204 mg/g, respectively. Lipid yields of the SSELP process were higher than those obtained by using the conventional process where cellulose was hydrolyzed separately. When ionic liquid pretreated corn stover was used, both cellulose and hemicellulose were consumed simultaneously. No xylose was accumulated over time, indicating that glucose effect was circumvented. The lipid yield reached 112 mg/g regenerated corn stover. This process could be performed without sterilization because of the absence of auxiliary nutrients for bacterial contamination. Conclusions The SSELP process facilitates direct conversion of both cellulose and hemicellulose of lignocellulosic materials into microbial lipids. It greatly reduces time and capital costs while improves lipid coefficient. Optimization of the SSELP process at different levels should further

  10. Production and characterization of multi-polysaccharide degrading enzymes from Aspergillus aculeatus BCC199 for saccharification of agricultural residues.

    PubMed

    Suwannarangsee, Surisa; Arnthong, Jantima; Eurwilaichitr, Lily; Champreda, Verawat

    2014-10-01

    Enzymatic hydrolysis of lignocellulosic biomass into fermentable sugars is a key step in the conversion of agricultural by-products to biofuels and value-added chemicals. Utilization of a robust microorganism for on-site production of biomass-degrading enzymes has gained increasing interest as an economical approach for supplying enzymes to biorefinery processes. In this study, production of multi-polysaccharide-degrading enzymes from Aspergillus aculeatus BCC199 by solid-state fermentation was improved through the statistical design approach. Among the operational parameters, yeast extract and soybean meal as well as the nonionic surfactant Tween 20 and initial pH were found as key parameters for maximizing production of cellulolytic and hemicellulolytic enzymes. Under the optimized condition, the production of FPase, endoglucanase, β-glucosidase, xylanase, and β-xylosidase was achieved at 23, 663, 88, 1,633, and 90 units/g of dry substrate, respectively. The multi-enzyme extract was highly efficient in the saccharification of alkaline-pretreated rice straw, corn cob, and corn stover. In comparison with commercial cellulase preparations, the BCC199 enzyme mixture was able to produce remarkable yields of glucose and xylose, as it contained higher relative activities of β-glucosidase and core hemicellulases (xylanase and β-xylosidase). These results suggested that the crude enzyme extract from A. aculeatus BCC199 possesses balanced cellulolytic and xylanolytic activities required for the efficient saccharification of lignocellulosic biomass feedstocks, and supplementation of external β-glucosidase or xylanase was dispensable. The work thus demonstrates the high potential of A. aculeatus BCC199 as a promising producer of lignocellulose-degrading enzymes for the biomass conversion industry. PMID:25001556

  11. Ultrasonic pretreatment for enhanced saccharification and fermentation of ethanol production from corn

    NASA Astrophysics Data System (ADS)

    Montalbo-Lomboy, Melissa T.

    The 21st Century human lifestyle has become heavily dependent on hydrocarbon inputs. Energy demand and the global warming effects due to the burning of fossil fuels have continued to increase. Rising awareness of the negative environmental and economic impacts of hydrocarbon dependence has led to a resurgence of interest in renewable energy sources such as ethanol. Fuel ethanol is known to be a cleaner and renewable source of energy relative to gasoline. Many studies have agreed that fuel ethanol has reduced greenhouse gas (GHG) emissions and has larger overall energy benefits compared to gasoline. Currently, the majority of the fuel ethanol in the United States is produced from corn using dry-grind milling process. The typical dry-grind ethanol plant incorporates jet cooking using steam to cook the corn slurry as pretreatment for saccharification; an energy intensive step. In aiming to reduce energy usage, this study evaluated the use of ultrasonics as an alternative to jet cooking. Ultrasonic batch experiments were conducted using a Branson 2000 Series bench-scale ultrasonic unit operating at a frequency of 20 kHz and a maximum output of 2.2 kW. Corn slurry was sonicated at varying amplitudes from 192 to 320 mumpeak-to-peak(p-p) for 0-40 seconds. Enzyme stability was investigated by adding enzyme (STARGEN(TM)001) before and after sonication. Scanning electron micrograph (SEM) images and particle size distribution analysis showed a nearly 20-fold size reduction by disintegration of corn particles due to ultrasonication. The results also showed a 30% improvement in sugar release of sonicated samples relative to the control group (untreated). The efficiency exceeded 100% in terms of relative energy gain from the additional sugar released due to ultrasonication compared to the ultrasonic energy applied. Interestingly, enzymatic activity was enhanced when sonicated at low and medium power. This result suggested that ultrasonic energy did not denature the enzymes

  12. Epigallocatechin gallate incorporation into lignin enhances the alkaline delignification and enzymatic saccharification of cell walls

    PubMed Central

    2012-01-01

    Background Lignin is an integral component of the plant cell wall matrix but impedes the conversion of biomass into biofuels. The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers such as flavonoids into cell wall lignins that are consequently less recalcitrant to biomass processing. In the present study, epigallocatechin gallate (EGCG) was evaluated as a potential lignin bioengineering target for rendering biomass more amenable to processing for biofuel production. Results In vitro peroxidase-catalyzed polymerization experiments revealed that both gallate and pyrogallyl (B-ring) moieties in EGCG underwent radical cross-coupling with monolignols mainly by β–O–4-type cross-coupling, producing benzodioxane units following rearomatization reactions. Biomimetic lignification of maize cell walls with a 3:1 molar ratio of monolignols and EGCG permitted extensive alkaline delignification of cell walls (72 to 92%) that far exceeded that for lignified controls (44 to 62%). Alkali-insoluble residues from EGCG-lignified walls yielded up to 34% more glucose and total sugars following enzymatic saccharification than lignified controls. Conclusions It was found that EGCG readily copolymerized with monolignols to become integrally cross-coupled into cell wall lignins, where it greatly enhanced alkaline delignification and subsequent enzymatic saccharification. Improved delignification may be attributed to internal trapping of quinone-methide intermediates to prevent benzyl ether cross-linking of lignin to structural polysaccharides during lignification, and to the cleavage of ester intra-unit linkages within EGCG during pretreatment. Overall, our results suggest that apoplastic deposition of EGCG for incorporation into lignin would be a promising plant genetic engineering target for improving the delignification and saccharification of biomass crops. PMID:22889353

  13. Enhanced Ethanol Production from De-Ashed Paper Sludge by Simultaneous Saccharification and Fermentation and Simultaneous Saccharification and Co-Fermentation

    SciTech Connect

    Kang, L.; Wang, W.; Pallapolu, V. R.; Lee, Y. Y.

    2011-11-01

    A previous study demonstrated that paper sludges with high ash contents can be converted to ethanol by simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and co-fermentation (SSCF). High ash content in the sludge, however, limited solid loading in the bioreactor, causing low product concentration. To overcome this problem, sludges were de-ashed before SSF and SSCF. Low ash content in sludges also increased the ethanol yield to the extent that the enzyme dosage required to achieve 70% yield in the fermentation process was reduced by 30%. High solid loading in SSF and SSCF decreased the ethanol yield. High agitation and de-ashing of the sludges were able to restore the part of the yield loss caused by high solid loading. Substitution of the laboratory fermentation medium (peptone and yeast extract) with corn steep liquor did not bring about any adverse effects in the fermentation. Fed-batch operation of the SSCF and SSF using low-ash content sludges was effective in raising the ethanol concentration, achieving 47.8 g/L and 60.0 g/L, respectively.

  14. Enhancement of enzymatic saccharification of Eucalyptus globulus: steam explosion versus steam treatment.

    PubMed

    Martin-Sampedro, Raquel; Revilla, Esteban; Villar, Juan C; Eugenio, Maria E

    2014-09-01

    Steam explosion and steam pre-treatment have proved capable of enhancing enzymatic saccharification of lignocellulosic materials. However, until now, these methods had not been compared under the same operational conditions and using the same raw material. Both pre-treatments lead to increased yields in the saccharification of Eucalyptus globulus; but results have been better with steam pre-treatments, despite the more accessible surface of exploded samples. The reason for this finding could be enzymatic inhibition: steam explosion causes a more extensive extraction of hemicelluloses and releases a greater amount of degradation products which can inhibit enzymatic action. Enzymatic inhibition is also dependent on the amount and chemical structure of lignin, which was also a contributing factor to the lower enzymatic yields obtained with the most severe pre-treatment. Thus, the highest yields (46.7% glucose and 73.4% xylose yields) were obtained after two cycle of steam treatment, of 5 and 3 min, at 183°C. PMID:24980031

  15. Lignosulfonate-mediated cellulase adsorption: enhanced enzymatic saccharification of lignocellulose through weakening nonproductive binding to lignin

    PubMed Central

    2013-01-01

    -acknowledged concept in the fields of biofuels and biorefinery that the pretreatment hydrolysate is inhibitory to enzymes. Conclusions The results reported in this study also suggest significant advantages of SPORL pretreatment in terms of water consumption and process integration, that is, it should abolish the steps of solid substrate washing and pretreatment hydrolysate detoxification for direct simultaneous saccharification and combined fermentation (SSCombF) of enzymatic and pretreatment hydrolysate, thereby facilitating bioprocess consolidation. Furthermore, this study not only has practical significance to biorefinery and bioenergy, but it also provides scientific importance to the molecular design of composite enzyme-polyelectrolyte systems, such as immobilized enzymes and enzyme activators, as well as to the design of enzyme separation processes using water-soluble polyelectrolytes. PMID:24188090

  16. Optimization of Arundo donax Saccharification by (Hemi)cellulolytic Enzymes from Pleurotus ostreatus

    PubMed Central

    Liguori, Rossana; Ionata, Elena; Marcolongo, Loredana; Vandenberghe, Luciana Porto de Souza; La Cara, Francesco; Faraco, Vincenza

    2015-01-01

    An enzymatic mixture of cellulases and xylanases was produced by Pleurotus ostreatus using microcrystalline cellulose as inducer, partially characterized and tested in the statistical analysis of Arundo donax bioconversion. The Plackett-Burman screening design was applied to identify the most significant parameters for the enzymatic hydrolysis of pretreated A. donax. As the most significant influence during the enzymatic hydrolysis of A. donax was exercised by the temperature (°C), pH, and time, the combined effect of these factors in the bioconversion by P. ostreatus cellulase and xylanase was analyzed by a 33 factorial experimental design. It is worth noting that the best result of 480.10 mg of sugars/gds, obtained at 45°C, pH 3.5, and 96 hours of incubation, was significant also when compared with the results previously reached by process optimization with commercial enzymes. PMID:26634214

  17. Optimization of Arundo donax Saccharification by (Hemi)cellulolytic Enzymes from Pleurotus ostreatus.

    PubMed

    Liguori, Rossana; Ionata, Elena; Marcolongo, Loredana; Vandenberghe, Luciana Porto de Souza; La Cara, Francesco; Faraco, Vincenza

    2015-01-01

    An enzymatic mixture of cellulases and xylanases was produced by Pleurotus ostreatus using microcrystalline cellulose as inducer, partially characterized and tested in the statistical analysis of Arundo donax bioconversion. The Plackett-Burman screening design was applied to identify the most significant parameters for the enzymatic hydrolysis of pretreated A. donax. As the most significant influence during the enzymatic hydrolysis of A. donax was exercised by the temperature (°C), pH, and time, the combined effect of these factors in the bioconversion by P. ostreatus cellulase and xylanase was analyzed by a 3(3) factorial experimental design. It is worth noting that the best result of 480.10 mg of sugars/gds, obtained at 45 °C, pH 3.5, and 96 hours of incubation, was significant also when compared with the results previously reached by process optimization with commercial enzymes. PMID:26634214

  18. Vertical Integration of Biomass Saccharification of Enzymes for Sustainable Cellulosic Biofuel Production in a Biorefinery

    SciTech Connect

    Manoj Kumar, PhD

    2011-05-09

    Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  19. Simultaneous saccharification and fermentation of dilute alkaline-pretreated corn stover for enhanced butanol production by Clostridium saccharobutylicum DSM 13864.

    PubMed

    Dong, Jin-Jun; Ding, Ji-Cai; Zhang, Yun; Ma, Li; Xu, Guo-Chao; Han, Rui-Zhi; Ni, Ye

    2016-02-01

    Simultaneous saccharification and fermentation (SSF) process was applied for biobutanol production by Clostridium saccharobutylicum DSM 13864 from corn stover (CS). The key influential factors in SSF process, including corn steep liquor concentration, dry biomass and enzyme loading, SSF temperature, inoculation size and pre-hydrolysis time were optimized. In 5-L bioreactor with SSF process, butanol titer and productivity of 12.3 g/L and 0.257 g/L/h were achieved at 48 h, which were 20.6% and 21.2% higher than those in separate hydrolysis and fermentation (SHF), respectively. The butanol yield reached 0.175 g/g pretreated CS in SSF, representing 50.9% increase than that in SHF (0.116 g/g pretreated CS). This study proves the feasibility of efficient and economic production of biobutanol from CS by SSF. PMID:26764423

  20. Enhanced enzymatic saccharification of pretreated biomass using glycerol thermal processing (GTP).

    PubMed

    Zhang, Wei; Sathitsuksanoh, Noppadon; Barone, Justin R; Renneckar, Scott

    2016-01-01

    Biomass was heated (200-240°C) in the presence of glycerol, for 4-12 min, under shear to disrupt the native cell wall architecture. The impact of this method, named glycerol thermal processing (GTP), on saccharification efficiency of the hardwood Liquidambar styraciflua, and a control cellulose sample was studied as a function of treatment severity. Furthermore, the enzymatic conversion of samples with varying compositions was studied after extraction of the structural polymers. Interestingly, the sweet gum processed materials crystallinity index increased by 10% of the initial value. The experiments revealed that the residual lignin was not a barrier to limiting the digestibility of cellulose after pretreatment yielding up to 70% glucose based on the starting wood material. Further xylan removal greatly improved the cellulose hydrolysis rate, converting nearly 70% of the cellulose into glucose within 24h, and reaching 78% of ultimate glucan digestibility after 72 h. PMID:26384086

  1. Thermo-chemical pretreatment and enzymatic hydrolysis for enhancing saccharification of catalpa sawdust.

    PubMed

    Jin, Shuguang; Zhang, Guangming; Zhang, Panyue; Li, Fan; Fan, Shiyang; Li, Juan

    2016-04-01

    To improve the reducing sugar production from catalpa sawdust, thermo-chemical pretreatments were examined and the chemicals used including NaOH, Ca(OH)2, H2SO4, and HCl. The hemicellulose solubilization and cellulose crystallinity index (CrI) were significantly increased after thermo-alkaline pretreatments, and the thermo-Ca(OH)2 pretreatment showed the best improvement for reducing sugar production comparing to other three pretreatments. The conditions of thermo-Ca(OH)2 pretreatment and enzymatic hydrolysis were systematically optimized. Under the optimal conditions, the reducing sugar yield increased by 1185.7% comparing to the control. This study indicates that the thermo-Ca(OH)2 pretreatment is ideal for the saccharification of catalpa sawdust and that catalpa sawdust is a promising raw material for biofuel. PMID:26802185

  2. Simultaneous saccharification and fermentation of solid household waste following mild pretreatment using a mix of hydrolytic enzymes in combination with Saccharomyces cerevisiae.

    PubMed

    Nwobi, A; Cybulska, I; Tesfai, W; Shatilla, Y; Rodríguez, J; Thomsen, M H

    2015-01-01

    Ethanol production from low severity pretreated (85 °C, 1 h) solid household waste was studied using simultaneous saccharification and fermentation (SSF). The aim of the study was to examine typical composition of the organic fraction of municipal solid waste (OFMSW) and to develop a simple method for simultaneous liquefaction and biofuels production. A model waste was prepared based on the composition of the organic waste in Masdar City. Chemical analysis of the OFMSW showed that it contained 37 % total solids with up to 57 g glucan/100 g total solid (TS). Hydrolysis of the wet OFMSW was carried out using a mix of hydrolytic enzymes: amylase, cellulase, protease, lipase, hemicellulase, and pectate lyase. The enzymatic hydrolysis using this enzyme mix was studied using different dilutions of the OFMSW at different enzyme loadings. This study has demonstrated that SSF of low severity pretreated OFMSW can be carried out using Saccharomyces cerevisiae without dilution (addition of water), and liquefaction of the undiluted OFMSW can be achieved in less than 24 h of hydrolysis. Also, SSF of the pretreated waste can be carried out with very low enzyme loading (10 % of the company recommended dosage)-0.1 % cellulase, 0.1 % amylase, 0.02 % protease, 0.02 % hemicellulase, 0.02 % lipase, and 0.02 % pectate lyase (w/w per TS) following mild heat pretreatment conditions of 85 °C for 1 h. PMID:25176443

  3. Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification

    PubMed Central

    Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino

    2015-01-01

    Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry. PMID:26137472

  4. Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification.

    PubMed

    Longoni, Paolo; Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino

    2015-01-01

    Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry. PMID:26137472

  5. Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-L-arabinofuranosidase.

    PubMed

    Delabona, Priscila da Silva; Cota, Júnio; Hoffmam, Zaira Bruna; Paixão, Douglas Antonio Alvaredo; Farinas, Cristiane Sanchez; Cairo, João Paulo Lourenço Franco; Lima, Deise Juliana; Squina, Fábio Marcio; Ruller, Roberto; Pradella, José Geraldo da Cruz

    2013-03-01

    Supplementation of cellulase cocktails with accessory enzymes can contribute to a higher hydrolytic capacity in releasing fermentable sugars from plant biomass. This study investigated which enzymes were complementary to the enzyme set of Trichoderma harzianum in the degradation of sugarcane bagasse. Specific activities of T. harzianum extract on different substrates were compared with the extracts of Penicillium echinulatum and Trichoderma reesei, and two commercial cellulase preparations. Complementary analysis of the secretome of T. harzianum was also used to identify which enzymes were produced during growth on pretreated sugarcane bagasse. These analyses enabled the selection of the enzymes pectinase and α-L-arabinofuranosidase (AF) to be further investigated as supplements to the T. harzianum extract. The effect of enzyme supplementation on the efficiency of sugarcane bagasse saccharification was evaluated using response surface methodology. The supplementation of T. harzianum enzymatic extract with pectinase and AF increased the efficiency of hydrolysis by up to 116%. PMID:23391738

  6. Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock

    PubMed Central

    2014-01-01

    Background Biofuel production from lignocellulosic material is hampered by biomass recalcitrance towards enzymatic hydrolysis due to the compact architecture of the plant cell wall and the presence of lignin. The purpose of this work is to study the ability of an industrially available laccase-mediator system to modify and remove lignin during pretreatment of wood (Eucalyptus globulus) feedstock, thus improving saccharification, and to analyze the chemical modifications produced in the whole material and especially in the recalcitrant lignin moiety. Results Up to 50% lignin removal from ground eucalypt wood was attained by pretreatment with recombinant Myceliophthora thermophila laccase and methyl syringate as mediator, followed by alkaline peroxide extraction in a multistage sequence. The lignin removal directly correlated with increases (approximately 40%) in glucose and xylose yields after enzymatic hydrolysis. The pretreatment using laccase alone (without mediator) removed up to 20% of lignin from eucalypt wood. Pyrolysis-gas chromatography/mass spectrometry of the pretreated wood revealed modifications of the lignin polymer, as shown by lignin markers with shortened side chains and increased syringyl-to-guaiacyl ratio. Additional information on the chemical modifications produced was obtained by two-dimensional nuclear magnetic resonance of the whole wood swollen in dimethylsulfoxide-d6. The spectra obtained revealed the removal of guaiacyl and syringyl lignin units, although with a preferential removal of the former, and the lower number of aliphatic side-chains per phenylpropane unit (involved in main β-O-4ʹ and β-βʹ inter-unit linkages), in agreement with the pyrolysis-gas chromatography/mass spectrometry results, without a substantial change in the wood polysaccharide signals. However, the most noticeable modification observed in the spectra was the formation of Cα-oxidized syringyl lignin units during the enzymatic treatment. Further insight into

  7. Incorporation of flavonoid derivatives or pentagalloyl glucose into lignin enhances cell wall saccharification following mild alkaline or acidic pretreatments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Partial substitution of normal monolignols with phenolic precursors from other metabolic pathways may improve the susceptibility of lignified biomass to chemical pretreatment and enzymatic saccharification for biofuel production. Flavonoids and gallate esters readily undergo oxidative coupling react...

  8. Mechanistic insight into ultrasound induced enhancement of simultaneous saccharification and fermentation of Parthenium hysterophorus for ethanol production.

    PubMed

    Singh, Shuchi; Agarwal, Mayank; Sarma, Shyamali; Goyal, Arun; Moholkar, Vijayanand S

    2015-09-01

    This paper presents investigations into mechanism of ultrasound assisted bioethanol synthesis using Parthenium hysterophorus biomass through simultaneous saccharification and fermentation (SSF) mode. Approach of coupling experimental results to mathematical model for SSF using Genetic Algorithm based optimization has been adopted. Comparison of model parameters for experiments with mechanical shaking and sonication (10% duty cycle) give an interesting mechanistic account of influence of ultrasound on SSF system. A 4-fold rise in ethanol and cell mass productivity is seen with ultrasound. The analysis reveals following facets of influence of ultrasound on SSF: increase in Monod constant for glucose for cell growth, maximal specific growth rate and inhibition constant of cell growth by glucose and reduction in specific cell death rate. Values of inhibition constant of cell growth by ethanol (K3E), and constants for growth associated (a) and non-growth associated (b) ethanol production remained unaltered with sonication. Beneficial effects of ultrasound are attributed to enhanced cellulose hydrolysis, enhanced trans-membrane transport of substrate and products as well as dilution of the toxic substances due to micro-convection induced by ultrasound. Intrinsic physiological functioning of cells remained unaffected by ultrasound as indicated by unaltered values of K3E, a and b. PMID:25813894

  9. Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass

    PubMed Central

    2014-01-01

    Background Previous research focused on pretreatment of biomass, production of fermentable sugars and their consumption to produce ethanol. The main goal of the work was to economise the production process cost of fermentable sugars. Therefore, the objective of the present work was to investigate enzyme hydrolysis of microcrystalline cellulose and hemp hurds (natural cellulosic substrate) using free and immobilised enzymes. Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability. Results Up to 94% protein binding was achieved during immobilisation of cellulase on nanoparticles. Successful binding was confirmed using Fourier transform infrared spectroscopy (FTIR). The free and immobilised enzymes exhibited identical pH optima (pH 4.0) and differing temperature optima at 50°C and 60°C, respectively. The K M values obtained for the free and immobilised enzymes were 0.87 mg/mL and 2.6 mg/mL respectively. The immobilised enzyme retained 50% enzyme activity up to five cycles, with thermostability at 80°C superior to that of the free enzyme. Optimum hydrolysis of carboxymethyl cellulose (CMC) with free and immobilised enzymes was 88% and 81%, respectively. With pretreated hemp hurd biomass (HHB), the free and immobilised enzymes resulted in maximum hydrolysis in 48 h of 89% and 93%, respectively. Conclusion The current work demonstrated the advantages delivered by immobilised enzymes by minimising the consumption of cellulase during substrate hydrolysis and making the production process of fermentable sugars economical and feasible. The activity of cellulase improved as a result of the immobilisation, which provided a better stability at higher temperatures. The immobilised enzyme provided an advantage over the free enzyme through the reusability and longer

  10. The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails: A comparative study.

    PubMed

    Maitan-Alfenas, Gabriela Piccolo; Visser, Evan Michael; Alfenas, Rafael Ferreira; Nogueira, Bráulio Ris G; de Campos, Guilherme Galvão; Milagres, Adriane Ferreira; de Vries, Ronald P; Guimarães, Valéria Monteze

    2015-09-01

    Biomass enzymatic hydrolysis depends on the pretreatment methods employed, the composition of initial feedstock and the enzyme cocktail used to release sugars for subsequent fermentation into ethanol. In this study, sugarcane bagasse was pretreated with 1% H2SO4 and 1% NaOH and the biomass saccharification was performed with 8% solids loading using 10 FPase units/g of bagasse of the enzymatic extract from Chrysoporthe cubensis and three commercial cocktails for a comparative study. Overall, the best glucose and xylose release was obtained from alkaline pretreated sugarcane bagasse. The C. cubensis extract promoted higher release of glucose (5.32 g/L) and xylose (9.00 g/L) than the commercial mixtures. Moreover, the C. cubensis extract presented high specific enzyme activities when compared to commercial cocktails mainly concerning to endoglucanase (331.84 U/mg of protein), β-glucosidase (29.48 U/mg of protein), β-xylosidase (2.95 U/mg of protein), pectinase (127.46 U/mg of protein) and laccase (2.49 U/mg of protein). PMID:26094192

  11. Enhancement of enzymatic saccharification of corn stover with sequential Fenton pretreatment and dilute NaOH extraction.

    PubMed

    He, Yu-Cai; Ding, Yun; Xue, Yu-Feng; Yang, Bin; Liu, Feng; Wang, Cheng; Zhu, Zheng-Zhong; Qing, Qing; Wu, Hao; Zhu, Cheng; Tao, Zhi-Cheng; Zhang, Dan-Ping

    2015-10-01

    In this study, an effective method by the sequential Fenton pretreatment and dilute NaOH extraction (FT-AE) was chosen for pretreating corn stover. Before dilute NaOH (0.75 wt%) extraction at 90 °C for 1h, Fenton reagent (0.95 g/L of FeSO4 and 29.8 g/L of H2O2) was employed to pretreat CS at a solid/liquid ratio of 1/20 (w/w) at 35 °C for 30 min. The changes in the cellulose structural characteristics (porosity, morphology, and crystallinity) of the pretreated solid residue were correlated with the enhancement of enzymatic saccharification. After being enzymatically hydrolyzed for 72 h, the reducing sugars and glucose from the hydrolysis of 60 g/L FT-AE-CS pretreated could be obtained at 40.96 and 23.61 g/L, respectively. Finally, the recovered hydrolyzates containing glucose had no inhibitory effects on the ethanol fermenting microorganism. In conclusion, the sequential Fenton pretreatment and dilute NaOH extraction has high potential application in future. PMID:26142999

  12. Integration of mild acid hydrolysis in γ-valerolactone/water system for enhancement of enzymatic saccharification from cotton stalk.

    PubMed

    Wu, Miao; Yan, Zhong Ya; Zhang, Xue Ming; Xu, Feng; Sun, Run Cang

    2016-01-01

    In this study, mild acid hydrolysis using γ-valerolactone (GVL)/water system integrated with enzymatic hydrolysis was carried out for the enhancement of enzymatic saccharification efficiency. The quantitative analysis of soluble carbohydrates and structural characterizations of solid residues were conducted. Results showed that the soluble carbohydrates in the water-phase were mainly composed of monomers and oligomers from xylose and glucose, while the contents of which were depended on the ratio of GVL to water. Moreover, the inhibitors were hardly detected due to the moderate pretreatment severity. Compared with the untreated feedstock, the yields of enzymatic hydrolysis from pretreated samples increased by two-fold with the mixture of 80/20 GVL/H2O. Combined with the amount of glucose (14.6%) dissolved in the water-phase, over 92.6% of glucose in cotton stalk was released and recovered. Based on the comprehensive analysis, treatment with GVL/H2O system provided us a more effective approach for sugar production from biomass. PMID:26476160

  13. Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process

    PubMed Central

    2014-01-01

    Background Cassava starch is considered as a potential source for the commercial production of bioethanol because of its availability and low market price. It can be used as a basic source to support large-scale biological production of bioethanol using microbial amylases. With the progression and advancement in enzymology, starch liquefying and saccharifying enzymes are preferred for the conversion of complex starch polymer into various valuable metabolites. These hydrolytic enzymes can selectively cleave the internal linkages of starch molecule to produce free glucose which can be utilized to produce bioethanol by microbial fermentation. Results In the present study, several filamentous fungi were screened for production of amylases and among them Aspergillus fumigatus KIBGE-IB33 was selected based on maximum enzyme yield. Maximum α-amylase, amyloglucosidase and glucose formation was achieved after 03 days of fermentation using cassava starch. After salt precipitation, fold purification of α-amylase and amyloglucosidase increased up to 4.1 and 4.2 times with specific activity of 9.2 kUmg-1 and 393 kUmg-1, respectively. Concentrated amylolytic enzyme mixture was incorporated in cassava starch slurry to give maximum glucose formation (40.0 gL-1), which was further fermented using Saccharomyces cerevisiae into bioethanol with 84.0% yield. The distillate originated after recovery of bioethanol gave 53.0% yield. Conclusion An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch. The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production. PMID:24885587

  14. Optimization of Enzymatic Saccharification of Alkali Pretreated Parthenium sp. Using Response Surface Methodology

    PubMed Central

    Pandiyan, K.; Tiwari, Rameshwar; Singh, Surender; Nain, Pawan K. S.; Rana, Sarika; Arora, Anju; Singh, Shashi B.; Nain, Lata

    2014-01-01

    Parthenium sp. is a noxious weed which threatens the environment and biodiversity due to its rapid invasion. This lignocellulosic weed was investigated for its potential in biofuel production by subjecting it to mild alkali pretreatment followed by enzymatic saccharification which resulted in significant amount of fermentable sugar yield (76.6%). Optimization of enzymatic hydrolysis variables such as temperature, pH, enzyme, and substrate loading was carried out using central composite design (CCD) in response to surface methodology (RSM) to achieve the maximum saccharification yield. Data obtained from RSM was validated using ANOVA. After the optimization process, a model was proposed with predicted value of 80.08% saccharification yield under optimum conditions which was confirmed by the experimental value of 85.80%. This illustrated a good agreement between predicted and experimental response (saccharification yield). The saccharification yield was enhanced by enzyme loading and reduced by temperature and substrate loading. This study reveals that under optimized condition, sugar yield was significantly increased which was higher than earlier reports and promises the use of Parthenium sp. biomass as a feedstock for bioethanol production. PMID:24900917

  15. Liquid chromatography-mass spectrometry investigation of enzyme-resistant xylooligosaccharide structures of switchgrass associated with ammonia pretreatment, enzymatic saccharification, and fermentation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Switchgrass is a potential source of renewable biomass for conversion to liquid biofuels. Efficient conversion requires effective strategies for pretreatment and enzymatic saccharification to produce fermentable sugars. Standard analysis of fermentation liquids includes detection of monosaccharides ...

  16. Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification.

    PubMed

    Eudes, Aymerick; George, Anthe; Mukerjee, Purba; Kim, Jin S; Pollet, Brigitte; Benke, Peter I; Yang, Fan; Mitra, Prajakta; Sun, Lan; Cetinkol, Ozgül P; Chabout, Salem; Mouille, Grégory; Soubigou-Taconnat, Ludivine; Balzergue, Sandrine; Singh, Seema; Holmes, Bradley M; Mukhopadhyay, Aindrila; Keasling, Jay D; Simmons, Blake A; Lapierre, Catherine; Ralph, John; Loqué, Dominique

    2012-06-01

    Lignocellulosic biomass is utilized as a renewable feedstock in various agro-industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these 'DP reducers' is achieved by expressing a bacterial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side-chain of hydroxycinnamoyl-CoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild-type plants. However, cell wall characterization of extract-free stems by thioacidolysis and by 2D-NMR revealed an increased amount of unusual C₆C₁ lignin monomers most likely linked with lignin as end-groups. Moreover the analysis of lignin isolated from these plants using size-exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C₆C₁ monomers ('DP reducers') into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis. PMID:22458713

  17. Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

    PubMed Central

    2012-01-01

    Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases

  18. Enhanced production of bioethanol from waste of beer fermentation broth at high temperature through consecutive batch strategy by simultaneous saccharification and fermentation.

    PubMed

    Khattak, Waleed Ahmad; Khan, Taous; Ha, Jung Hwan; Ul-Islam, Mazhar; Kang, Min-Kyung; Park, Joong Kon

    2013-10-10

    Malt hydrolyzing enzymes and yeast glycolytic and fermentation enzymes in the waste from beer fermentation broth (WBFB) were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A new 'one-pot consecutive batch strategy' was developed for efficient bio-ethanol production by simultaneous saccharification and fermentation (SSF) using WBFB without additional enzymes, microbial cells, or carbohydrates. Bio-ethanol production was conducted in batches using WBFB supernatant in the first phase at 25-67°C and 50rpm, followed by the addition of 3% WBFB solid residue to the existing culture broth in the second phase at 67°C. The ethanol production increased from 50 to 102.5g/L when bare supernatant was used in the first phase, and then to 219g ethanol/L in the second phase. The amount of ethanol obtained using this strategy was almost equal to that obtained using the original WBFB containing 25% solid residue at 33°C, and more than double that obtained when bare supernatant was used. Microscopic and gel electrophoresis studies revealed yeast cell wall degradation and secretion of cellular material into the surrounding medium. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) supported the existence of enzymes in WBFB involved in bioethanol production at elevated temperatures. The results of this study will provide insight for the development of new strategies for biofuel production. PMID:24034431

  19. Integrated biorefinery concept for grass silage using a combination of adapted pulping methods for advanced saccharification and extraction of lignin.

    PubMed

    Schwarz, Dominik; Dörrstein, Jörg; Kugler, Sabine; Schieder, Doris; Zollfrank, Cordt; Sieber, Volker

    2016-09-01

    An integrated refining and pulping process for ensiled biomass from permanent grassland was established on laboratory scale. The liquid phase, containing the majority of water-soluble components, including 24% of the initial dry matter (DM), was first separated by mechanical pressing. The fiber fraction was subjected to high solid load saccharification (25% DM) to enhance the lignin content in the feed for subsequent organosolvation. The saccharification enzymes were pre-selected applying experimental design approaches. Cellulose convertibility was improved by a secondary pressing step during liquefaction. Combined saccharification and organosolvation showed high degree of saccharide solubilization with recovery of 98% of the glucan and 73% of the xylan from the fiber fraction in the hydrolysates, and enabled the recovery of 41% of the grass silage lignin. The effects of the treatment were confirmed by XRD and SEM tracking of cellulose crystallinity and fiber morphology throughout the pulping procedure. PMID:27262721

  20. Fast Enzymatic Saccharification of Switchgrass After Pretreatment with Ionic Liquids

    SciTech Connect

    Zhou, Hua; Baker, Gary A; Cowins, Janet V.

    2010-01-01

    The pretreatment of cellulose using ionic liquids (ILs) has been shown to be an effective method for improving the enzymatic hydrolysis of cellulose; this technique affords a fast and complete saccharification of cellulose into reducing sugars (Dadi et al., Biotechnol Bioeng. 2006; 95:904 910; Liu and Chen, Chinese Sci Bull. 2006; 51:2432 2436; Zhao et al., J Biotechnol. 2009; 139:47 54). Motivated by these advances, this study examines the effect of IL-pretreatment on the enzymatic hydrolysis of purified xylan (as a model system of hemicellulose) and switchgrass (as a real lignocellulose). The IL-pretreatment resulted in no improvement in the hydrolysis of xylan. The likely reason is that pure xylan has a low degree of polymerization (DP), and is readily biodegraded even without any pretreatment. However, in real cellulosic materials (such as switchgrass), xylan is entrapped within the cellulosic matrix, and cannot be conveniently accessed by enzymes. Our data demonstrate that the IL-pretreatment of switchgrass significantly improved the enzymatic saccharification of both cellulose (96% D-glucose yield in 24 h) and xylan (63% D-xylose yield in 24 h). The compositional analysis of switchgrass suggests a lower lignin content after IL-pretreatment. In addition, the infrared spectrum of regenerated switchgrass indicates a lower substrate crystallinity, whereas the enzyme adsorption isotherm further implies that the regenerated substrate is more accessible to enzymes. This study has further confirmed that ILpretreatment is an effective tool in enhancing the enzymatic hydrolysis of cellulosic biomass, and allowing a more complete saccharification.

  1. Enzymatic saccharification of pretreated rice straw and biomass production

    SciTech Connect

    Araujo, A.; D'Souza, J.

    1986-10-01

    A comparative study on the saccharification of pretreated rice straw was brought about by using cellulase enzyme produced by Aspergillus terreus ATCC 52430 and its mutant strain UNGI-40. The effect of enzyme and substrate concentrations on the saccharification rate at 24 and 48 were studied. A syrup with 7% sugar concentration was obtained with a 10% substrate concentration for the mutant case, whereas a syrup with 6.8% sugar concentration was obtained with 3.5 times concentrated enzyme from the wild strain. A high saccharification value was obtained with low substrate concentration; the higher the substrate concentration used, the lower the percent saccharification. The glucose content in the hydrolysate comprised 80-82% of total reducing sugars; the remainder was cellobiose and xylose together. The hydrolysate supported the growth of yeasts Candida utilis and Saccharomyces cerevisiae ATCC 52431. A biomass with a 48% protein content was obtained. The essential amino acid composition of yeast biomass was determined.

  2. Sapropterin Hydrochloride: Enzyme Enhancement Therapy for Phenylketonuria

    PubMed Central

    Lachmann, Robin

    2011-01-01

    Phenylketonuria (PKU) is an inherited disorder of amino acid metabolism caused by deficiency of the enzyme phenylalanine hydroxylase (PAH). Historically PKU was a common genetic cause of severe learning difficulties and developmental delay, but with the introduction of newborn screening and early dietary management, it has become a treatable disease and people born with PKU should now have IQs and achievements similar to their peers. Dietary treatment, however, involves lifestyle changes that pervade most aspects of daily life for an individual and their family. A simple pharmacological treatment for PKU would have a great appeal. Sapropterin hydrochloride is a synthetic form of tetrahydrobiopterin, the cofactor for PAH. A proportion of mutant PAH enzymes show enhanced activity in the presence of pharmacological doses of sapropterin and, for some patients with milder forms of PKU, sapropterin can effectively lower plasma phenylalanine levels. This article discusses the potential place for sapropterin in the management of PKU and how this expensive orphan drug is being integrated into patient care in different healthcare systems. PMID:23148178

  3. Enzymes Enhance Biofilm Removal Efficiency of Cleaners

    PubMed Central

    Stiefel, Philipp; Mauerhofer, Stefan; Schneider, Jana; Maniura-Weber, Katharina; Rosenberg, Urs

    2016-01-01

    Efficient removal of biofilms from medical devices is a big challenge in health care to avoid hospital-acquired infections, especially from delicate devices like flexible endoscopes, which cannot be reprocessed using harsh chemicals or high temperatures. Therefore, milder solutions such as enzymatic cleaners have to be used, which need to be carefully developed to ensure efficacious performance. In vitro biofilm in a 96-well-plate system was used to select and optimize the formulation of novel enzymatic cleaners. Removal of the biofilm was quantified by crystal violet staining, while the disinfecting properties were evaluated by a BacTiter-Glo assay. The biofilm removal efficacy of the selected cleaner was further tested by using European standard (EN) for endoscope cleaning EN ISO 15883, and removal of artificial blood soil was investigated by treating TOSI (Test Object Surgical Instrument) cleaning indicators. Using the process described here, a novel enzymatic endoscope cleaner was developed, which removed 95% of Staphylococcus aureus and 90% of Pseudomonas aeruginosa biofilms in the 96-well plate system. With a >99% reduction of CFU and a >90% reduction of extracellular polymeric substances, this cleaner enabled subsequent complete disinfection and fulfilled acceptance criteria of EN ISO 15883. Furthermore, it efficiently removed blood soil and significantly outperformed comparable commercial products. The cleaning performance was stable even after storage of the cleaner for 6 months. It was demonstrated that incorporation of appropriate enzymes into the cleaner enhanced performance significantly. PMID:27044552

  4. Enzymes Enhance Biofilm Removal Efficiency of Cleaners.

    PubMed

    Stiefel, Philipp; Mauerhofer, Stefan; Schneider, Jana; Maniura-Weber, Katharina; Rosenberg, Urs; Ren, Qun

    2016-06-01

    Efficient removal of biofilms from medical devices is a big challenge in health care to avoid hospital-acquired infections, especially from delicate devices like flexible endoscopes, which cannot be reprocessed using harsh chemicals or high temperatures. Therefore, milder solutions such as enzymatic cleaners have to be used, which need to be carefully developed to ensure efficacious performance. In vitro biofilm in a 96-well-plate system was used to select and optimize the formulation of novel enzymatic cleaners. Removal of the biofilm was quantified by crystal violet staining, while the disinfecting properties were evaluated by a BacTiter-Glo assay. The biofilm removal efficacy of the selected cleaner was further tested by using European standard (EN) for endoscope cleaning EN ISO 15883, and removal of artificial blood soil was investigated by treating TOSI (Test Object Surgical Instrument) cleaning indicators. Using the process described here, a novel enzymatic endoscope cleaner was developed, which removed 95% of Staphylococcus aureus and 90% of Pseudomonas aeruginosa biofilms in the 96-well plate system. With a >99% reduction of CFU and a >90% reduction of extracellular polymeric substances, this cleaner enabled subsequent complete disinfection and fulfilled acceptance criteria of EN ISO 15883. Furthermore, it efficiently removed blood soil and significantly outperformed comparable commercial products. The cleaning performance was stable even after storage of the cleaner for 6 months. It was demonstrated that incorporation of appropriate enzymes into the cleaner enhanced performance significantly. PMID:27044552

  5. Methods of saccharification of polysaccharides in plants

    DOEpatents

    Howard, John; Fake, Gina

    2014-04-29

    Saccharification of polysaccharides of plants is provided, where release of fermentable sugars from cellulose is obtained by adding plant tissue composition. Production of glucose is obtained without the need to add additional .beta.-glucosidase. Adding plant tissue composition to a process using a cellulose degrading composition to degrade cellulose results in an increase in the production of fermentable sugars compared to a process in which plant tissue composition is not added. Using plant tissue composition in a process using a cellulose degrading enzyme composition to degrade cellulose results in decrease in the amount of cellulose degrading enzyme composition or exogenously applied cellulase required to produce fermentable sugars.

  6. Enzymatic saccharification of high pressure assist-alkali pretreated cotton stalk and structural characterization.

    PubMed

    Du, Shuang-kui; Su, Xia; Yang, Weihua; Wang, Yanqin; Kuang, Meng; Ma, Lei; Fang, Dan; Zhou, Dayun

    2016-04-20

    Cotton stalk is a potential biomass for bioethanol production, while the conversion of direct saccharification or biotransformation of cotton stalk is extremely low due to the recalcitrant nature of lignocellulose. To enhance the enzymatic conversion of cotton stalks, the enzymatic saccharification parameters of high pressure assist-alkali pretreatment (HPAP) cotton stalk were optimized in the present study. Results indicated that a maximum reducing sugar yield of 54.7g/100g dry biomass cellulose was achieved at a substrate concentration of 2%, 100rpm agitation, 0.6g/g enzyme loading, 40°C hydrolysis temperature, 50h saccharification time, and pH 5.0. Scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy were used to identify structural changes in native, pretreated biomass and hydrolyzed residues. Structural analysis revealed large part of amorphous cellulose and partial crystalline cellulose in the HPAP cotton stalk were hydrolyzed during enzymatic treatment. HPAP cotton stalk can be used as a potential feed stock for bioethanol production. PMID:26876855

  7. Process for concentrated biomass saccharification

    DOEpatents

    Hennessey, Susan M.; Seapan, Mayis; Elander, Richard T.; Tucker, Melvin P.

    2010-10-05

    Processes for saccharification of pretreated biomass to obtain high concentrations of fermentable sugars are provided. Specifically, a process was developed that uses a fed batch approach with particle size reduction to provide a high dry weight of biomass content enzymatic saccharification reaction, which produces a high sugars concentration hydrolysate, using a low cost reactor system.

  8. Mapping and candidate genes associated with saccharification yield in sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum [Sorghum bicolor (L.) Moench] is a potentially high-yielding hardy energy crop to produce lignocellulosic biofuels. Saccharification is a process by which hydrolytic enzymes break down lignocellulosic materials to fermentable sugars for biofuel production. Mapping and identifying genes und...

  9. Enhancing pulp and paper mill biosludge dewaterability using enzymes.

    PubMed

    Bonilla, Sofia; Tran, Honghi; Allen, D Grant

    2015-01-01

    There have been limited studies on the potential use of enzymes for enhancing the dewaterability of biosludge. The mechanisms for such enhancement have not been investigated despite the environmental advantages of using enzymes over synthetic polymers for biosludge conditioning. In order to find enzymes with this potential, a screening of commercially available enzymes was carried out using capillary suction time to assess biosludge dewaterability. The only enzyme that showed dewatering improvements in the screening tests was a lysozyme which reduced the capillary suction time by 36% and increased the cake solids content from 5.6 to 8.9 DS%. Lysozyme aided in the flocculation of particles reducing the polymer demand from 11% to 6%. Active and inactive lysozyme exhibited a similar ability for enhancing sludge dewatering, indicating that the conditioning mechanism of lysozyme is similar to that of a flocculant. PMID:25462773

  10. Silica distinctively affects cell wall features and lignocellulosic saccharification with large enhancement on biomass production in rice.

    PubMed

    Zhang, Jing; Zou, Weihua; Li, Ying; Feng, Yongqing; Zhang, Hui; Wu, Zhiliang; Tu, Yuanyuan; Wang, Yanting; Cai, Xiwen; Peng, Liangcai

    2015-10-01

    Rice is a typical silicon-accumulating crop with enormous biomass residues for biofuels. Silica is a cell wall component, but its effect on the plant cell wall and biomass production remains largely unknown. In this study, a systems biology approach was performed using 42 distinct rice cell wall mutants. We found that silica levels are significantly positively correlated with three major wall polymers, indicating that silica is associated with the cell wall network. Silicon-supplied hydroculture analysis demonstrated that silica distinctively affects cell wall composition and major wall polymer features, including cellulose crystallinity (CrI), arabinose substitution degree (reverse Xyl/Ara) of xylans, and sinapyl alcohol (S) proportion in three typical rice mutants. Notably, the silicon supplement exhibited dual effects on biomass enzymatic digestibility in the mutant and wild type (NPB) after pre-treatments with 1% NaOH and 1% H2SO4. In addition, silicon supply largely enhanced plant height, mechanical strength and straw biomass production, suggesting that silica rescues mutant growth defects. Hence, this study provides potential approaches for silicon applications in biomass process and bioenergy rice breeding. PMID:26398793

  11. Micromorphological changes and mechanism associated with wet ball milling of Pinus radiata substrate and consequences for saccharification at low enzyme loading.

    PubMed

    Vaidya, Alankar A; Donaldson, Lloyd A; Newman, Roger H; Suckling, Ian D; Campion, Sylke H; Lloyd, John A; Murton, Karl D

    2016-08-01

    In this work, substrates prepared from thermo-mechanical treatment of Pinus radiata chips were vibratory ball milled for different times. In subsequent enzymatic hydrolysis, percent glucan conversion passed through a maximum value at a milling time of around 120min and then declined. Scanning electron microscopy revealed breakage of fibers to porous fragments in which lamellae and fibrils were exposed during ball milling. Over-milling caused compression of the porous fragments to compact globular particles with a granular texture, decreasing accessibility to enzymes. Carbon-13 NMR spectroscopy showed partial loss of interior cellulose in crystallites, leveling off once fiber breakage was complete. A mathematical model based on observed micromorphological changes supports ball milling mechanism. At a low enzyme loading of 2FPU/g of substrate and milling time of 120min gave a total monomeric sugar yield of 306g/kg of pulp which is higher than conventional pretreatment method such as steam exploded wood. PMID:27131293

  12. Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes

    PubMed Central

    2011-01-01

    Background Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. Results First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i) the production of a large amount of gluconic acid, (ii) increased hemicellulose degradation, and (iii) increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH) expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM). Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. Conclusions We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material. PMID:22204630

  13. The optimization of saccharification of desulfurated red seaweed-derived polysaccharides and analysis of their composition.

    PubMed

    Li, Xiaolin; Li, Yinping; Mou, Haijin; Gao, Yan; Hwang, Hueymin; Wang, Peng

    2014-01-01

    Currently processes of ethanol production from desulfurated red seaweed derived polysaccharides (De-RSDP) are well established. However, the optimization of the enzymatic saccharification process has not been reported. In this study, De-RSDP from Kappaphycus alvarezii was subjected to saccharification by different enzymes, including pectinase, cellobiase, cellulase, and hemicellulase. The best saccharification enzyme was determined as pectinase. In order to obtain more reducing sugar (RS), the single-factor experiment followed by central composite rotatable design (CCD) was used to optimize the parameters for enzymatic hydrolysis. The optimal saccharification parameters of De-RSDP were 4400 U/g pectinase dosage, 96 hr, and 55.5°C, respectively, and the yield of RS reached 81.3%. Subsequently, the saccharification liquids of De-RSDP were isolated and purified with gel permeation chromatography (GPC) to separate saccharides with different molecular weights. Simultaneously, the composition of saccharification liquids was analyzed by 1-phenyl-3-methyl-5-pyrazolone high-pressure liquid chromatography (PMP-HPLC) and mass spectroscopy (MS). The result showed that the content is mainly composed of galactose (65.2%) with the average molecular mass in the fourth peak 181.1 Da. The saccharification liquids of De-RSDP inoculated with Saccharomyces cerevisiae L-4 for ethanol fermentation produced 0.3 g ethanol/g De-RSDP, which corresponded to 71.8% of the theoretical yield (0.38 g ethanol/g). PMID:24117151

  14. Rapid saccharification for production of cellulosic biofuels.

    PubMed

    Lee, Dae-Seok; Wi, Seung Gon; Lee, Soo Jung; Lee, Yoon-Gyo; Kim, Yeong-Suk; Bae, Hyeun-Jong

    2014-04-01

    The economical production of biofuels is hindered by the recalcitrance of lignocellulose to processing, causing high consumption of processing enzymes and impeding hydrolysis of pretreated lignocellulosic biomass. We determined the major rate-limiting factor in the hydrolysis of popping pre-treated rice straw (PPRS) by examining cellulase adsorption to lignin and cellulose, amorphogenesis of PPRS, and re-hydrolysis. Based on the results, equivalence between enzyme loading and the open structural area of cellulose was required to significantly increase productive adsorption of cellulase and to accelerate enzymatic saccharification of PPRS. Amorphogenesis of PPRS by phosphoric acid treatment to expand open structural area of the cellulose fibers resulted in twofold higher cellulase adsorption and increased the yield of the first re-hydrolysis step from 13% to 46%. The total yield from PPRS was increased to 84% after 3h. These results provide evidence that cellulose structure is one of major effects on the enzymatic hydrolysis. PMID:24607460

  15. Potential halophilic cellulases for in situ enzymatic saccharification of ionic liquids pretreated lignocelluloses.

    PubMed

    Gunny, Ahmad Anas Nagoor; Arbain, Dachyar; Edwin Gumba, Rizo; Jong, Bor Chyan; Jamal, Parveen

    2014-03-01

    Ionic liquids (ILs) have been used as an alternative green solvent for lignocelluloses pretreatment. However, being a salt, ILs exhibit an inhibitory effect on cellulases activity, thus making the subsequent saccharification inefficient. The aim of the present study is to produce salt-tolerant cellulases, with the rationale that the enzyme also tolerant to the presence of ILs. The enzyme was produced from a locally isolated halophilic strain and was characterized and assessed for its tolerance to different types of ionic liquids. The results showed that halophilic cellulases produced from Aspergillus terreus UniMAP AA-6 exhibited higher tolerance to ILs and enhanced thermo stability in the presence of high saline conditions. PMID:24457303

  16. An investigation into keratinolytic enzymes to enhance ungual drug delivery.

    PubMed

    Mohorcic, M; Torkar, A; Friedrich, J; Kristl, J; Murdan, S

    2007-03-01

    The topical therapy of nail diseases is limited by the low permeability of drugs through the nail plate. To increase drug penetration, the integrity of the nail plate must be compromised to a certain extent. We hypothesised that keratinolytic enzymes might decrease the barrier properties of the nail plate by hydrolysing the nail keratins, and thereby enhance ungual drug permeation. To determine enzyme action on nail plates, nail clippings were incubated at 35 degrees C, in the presence of keratinase at optimal pH for 48h, after which the nail plates were examined using scanning electron microscopy. It was found that the enzyme acted on the intercellular matrix which holds nail cells together, such that corneocytes on the dorsal surface separated from one another and 'lifted off' the nail plate. In addition, the surface of the corneocytes was corroded. Permeation studies using modified Franz diffusion cells and bovine hoof membranes as a model for the nail plate showed that the enzyme enhanced drug permeation through the hoof membrane. The permeability and partition coefficients, and the drug flux were found to be significantly increased in the presence of the enzyme. We can conclude that the enzyme, via its hydrolytic action on nail plate proteins, could increase ungual drug delivery. PMID:17097244

  17. Enzyme Enhancers for the Treatment of Fabry and Pompe Disease

    PubMed Central

    Lukas, Jan; Pockrandt, Anne-Marie; Seemann, Susanne; Sharif, Muhammad; Runge, Franziska; Pohlers, Susann; Zheng, Chaonan; Gläser, Anne; Beller, Matthias; Rolfs, Arndt; Giese, Anne-Katrin

    2015-01-01

    Lysosomal storage disorders (LSD) are a group of heterogeneous diseases caused by compromised enzyme function leading to multiple organ failure. Therapeutic approaches involve enzyme replacement (ERT), which is effective for a substantial fraction of patients. However, there are still concerns about a number of issues including tissue penetrance, generation of host antibodies against the therapeutic enzyme, and financial aspects, which render this therapy suboptimal for many cases. Treatment with pharmacological chaperones (PC) was recognized as a possible alternative to ERT, because a great number of mutations do not completely abolish enzyme function, but rather trigger degradation in the endoplasmic reticulum. The theory behind PC is that they can stabilize enzymes with remaining function, avoid degradation and thereby ameliorate disease symptoms. We tested several compounds in order to identify novel small molecules that prevent premature degradation of the mutant lysosomal enzymes α-galactosidase A (for Fabry disease (FD)) and acid α-glucosidase (GAA) (for Pompe disease (PD)). We discovered that the expectorant Ambroxol when used in conjunction with known PC resulted in a significant enhancement of mutant α-galactosidase A and GAA activities. Rosiglitazone was effective on α-galactosidase A either as a monotherapy or when administered in combination with the PC 1-deoxygalactonojirimycin. We therefore propose both drugs as potential enhancers of pharmacological chaperones in FD and PD to improve current treatment strategies. PMID:25409744

  18. Enzyme enhancers for the treatment of Fabry and Pompe disease.

    PubMed

    Lukas, Jan; Pockrandt, Anne-Marie; Seemann, Susanne; Sharif, Muhammad; Runge, Franziska; Pohlers, Susann; Zheng, Chaonan; Gläser, Anne; Beller, Matthias; Rolfs, Arndt; Giese, Anne-Katrin

    2015-03-01

    Lysosomal storage disorders (LSD) are a group of heterogeneous diseases caused by compromised enzyme function leading to multiple organ failure. Therapeutic approaches involve enzyme replacement (ERT), which is effective for a substantial fraction of patients. However, there are still concerns about a number of issues including tissue penetrance, generation of host antibodies against the therapeutic enzyme, and financial aspects, which render this therapy suboptimal for many cases. Treatment with pharmacological chaperones (PC) was recognized as a possible alternative to ERT, because a great number of mutations do not completely abolish enzyme function, but rather trigger degradation in the endoplasmic reticulum. The theory behind PC is that they can stabilize enzymes with remaining function, avoid degradation and thereby ameliorate disease symptoms. We tested several compounds in order to identify novel small molecules that prevent premature degradation of the mutant lysosomal enzymes α-galactosidase A (for Fabry disease (FD)) and acid α-glucosidase (GAA) (for Pompe disease (PD)). We discovered that the expectorant Ambroxol when used in conjunction with known PC resulted in a significant enhancement of mutant α-galactosidase A and GAA activities. Rosiglitazone was effective on α-galactosidase A either as a monotherapy or when administered in combination with the PC 1-deoxygalactonojirimycin. We therefore propose both drugs as potential enhancers of pharmacological chaperones in FD and PD to improve current treatment strategies. PMID:25409744

  19. Enzymatic saccharification and fermentation of cellulosic date palm wastes to glucose and lactic acid

    PubMed Central

    Alrumman, Sulaiman A.

    2016-01-01

    The bioconversion of cellulosic wastes into high-value bio-products by saccharification and fermentation processes is an important step that can reduce the environmental pollution caused by agricultural wastes. In this study, enzymatic saccharification of treated and untreated date palm cellulosic wastes by the cellulases from Geobacillus stearothermophilus was optimized. The alkaline pre-treatment of the date palm wastes was found to be effective in increasing the saccharification percentage. The maximum rate of saccharification was found at a substrate concentration of 4% and enzyme concentration of 30 FPU/g of substrate. The optimum pH and temperature for the bioconversions were 5.0 and 50 °C, respectively, after 24 h of incubation, with a yield of 31.56 mg/mL of glucose at a saccharification degree of 71.03%. The saccharification was increased to 94.88% by removal of the hydrolysate after 24 h by using a two-step hydrolysis. Significant lactic acid production (27.8 mg/mL) was obtained by separate saccharification and fermentation after 72 h of incubation. The results indicate that production of fermentable sugar and lactic acid is feasible and may reduce environmental pollution by using date palm wastes as a cheap substrate. PMID:26887233

  20. Enzymatic saccharification and fermentation of cellulosic date palm wastes to glucose and lactic acid.

    PubMed

    Alrumman, Sulaiman A

    2016-01-01

    The bioconversion of cellulosic wastes into high-value bio-products by saccharification and fermentation processes is an important step that can reduce the environmental pollution caused by agricultural wastes. In this study, enzymatic saccharification of treated and untreated date palm cellulosic wastes by the cellulases from Geobacillus stearothermophilus was optimized. The alkaline pre-treatment of the date palm wastes was found to be effective in increasing the saccharification percentage. The maximum rate of saccharification was found at a substrate concentration of 4% and enzyme concentration of 30 FPU/g of substrate. The optimum pH and temperature for the bioconversions were 5.0 and 50°C, respectively, after 24h of incubation, with a yield of 31.56mg/mL of glucose at a saccharification degree of 71.03%. The saccharification was increased to 94.88% by removal of the hydrolysate after 24h by using a two-step hydrolysis. Significant lactic acid production (27.8mg/mL) was obtained by separate saccharification and fermentation after 72h of incubation. The results indicate that production of fermentable sugar and lactic acid is feasible and may reduce environmental pollution by using date palm wastes as a cheap substrate. PMID:26887233

  1. Enzymatic saccharification of brown seaweed for production of fermentable sugars.

    PubMed

    Sharma, Sandeep; Horn, Svein Jarle

    2016-08-01

    This study shows that high drying temperatures negatively affect the enzymatic saccharification yield of the brown seaweed Saccharina latissima. The optimal drying temperature of the seaweed in terms of enzymatic sugar release was found to be 30°C. The enzymatic saccharification process was optimized by investigating factors such as kinetics of sugar release, enzyme dose, solid loading and different blend ratios of cellulases and an alginate lyase. It was found that the seaweed biomass could be efficiently hydrolysed to fermentable sugars using a commercial cellulase cocktail. The inclusion of a mono-component alginate lyase was shown to improve the performance of the enzyme blend, in particular at high solid loadings. At 25% dry matter loading a combined glucose and mannitol concentration of 74g/L was achieved. PMID:26961713

  2. Saccharification of sunflower stalks using lignocellulases from a fungal consortium comprising Pholiota adiposa and Armillaria gemina.

    PubMed

    Ramachandran, Priyadharshini; Kim, Tae-Su; Dhiman, Saurabh Sudha; Li, Jinglin; Park, Ji-Hyun; Choi, Joon-Ho; Kim, Jae Young; Kim, Dongwook; Lee, Jung-Kul

    2015-09-01

    Lignocellulases from Armillaria gemina and Pholiota adiposa are efficient in hydrolyzing aspen and poplar biomass, respectively. In the present study, lignocellulosic enzymes obtained from a fungal consortium comprising P. adiposa and A. gemina were used for the saccharification of sunflower stalks. Sunflower stalks were thermochemically pretreated using 2 % NaOH at 50 °C for 24 h. The saccharification process parameters including substrate concentration, enzyme loading, pH, and temperature were optimized using response surface methodology to improve the saccharification yield. The highest enzymatic hydrolysis (84.3 %) was obtained using the following conditions: enzyme loading 10 FPU/g-substrate, substrate 5.5 %, temperature 50 °C, and pH 4.5. The hydrolysis yield obtained using the enzymes from the fungal consortium was equivalent to that obtained using a mixture of commercial enzymes Celluclast and Novozyme β-glucosidase. Addition of up to 500 ppm of heavy metal ions (As, Cu, Fe, Mn, Ni, Pb, and Zn) during saccharification did not significantly affect the saccharification yield. Thus, the biomass grown for phytoremediation of heavy metals can be used for the production of reducing sugars followed by ethanol fermentation. PMID:25924967

  3. Enhanced Production of Ligninolytic Enzymes by a Mushroom Stereum ostrea

    PubMed Central

    Usha, K. Y.; Praveen, K.; Reddy, B. Rajasekhar

    2014-01-01

    The white rot fungi Stereum ostrea displayed a wide diversity in their response to supplemented inducers, surfactants, and copper sulphate in solid state fermentation. Among the inducers tested, 0.02% veratryl alcohol increased the ligninolytic enzyme production to a significant extent. The addition of copper sulphate at 300 μM concentration has a positive effect on laccase production increasing its activity by 2 times compared to control. Among the surfactants, Tween 20, Tween 80, and Triton X 100, tested in the studies, Tween 80 stimulated the production of ligninolytic enzymes. Biosorption of dyes was carried out by using two lignocellulosic wastes, rice bran and wheat bran, in 50 ppm of remazol brilliant blue and remazol brilliant violet 5R dyes. These dye adsorbed lignocelluloses were then utilized for the production of ligninolytic enzymes in solid state mode. The two dye adsorbed lignocelluloses enhanced the production of laccase and manganese peroxidase but not lignin peroxidase. PMID:25610656

  4. Enhanced Production of Ligninolytic Enzymes by a Mushroom Stereum ostrea.

    PubMed

    Usha, K Y; Praveen, K; Reddy, B Rajasekhar

    2014-01-01

    The white rot fungi Stereum ostrea displayed a wide diversity in their response to supplemented inducers, surfactants, and copper sulphate in solid state fermentation. Among the inducers tested, 0.02% veratryl alcohol increased the ligninolytic enzyme production to a significant extent. The addition of copper sulphate at 300 μM concentration has a positive effect on laccase production increasing its activity by 2 times compared to control. Among the surfactants, Tween 20, Tween 80, and Triton X 100, tested in the studies, Tween 80 stimulated the production of ligninolytic enzymes. Biosorption of dyes was carried out by using two lignocellulosic wastes, rice bran and wheat bran, in 50 ppm of remazol brilliant blue and remazol brilliant violet 5R dyes. These dye adsorbed lignocelluloses were then utilized for the production of ligninolytic enzymes in solid state mode. The two dye adsorbed lignocelluloses enhanced the production of laccase and manganese peroxidase but not lignin peroxidase. PMID:25610656

  5. Enhancing enzyme stability by construction of polymer-enzyme conjugate micelles for decontamination of organophosphate agents.

    PubMed

    Suthiwangcharoen, Nisaraporn; Nagarajan, Ramanathan

    2014-04-14

    Enhancing the stability of enzymes under different working environments is essential if the potential of enzyme-based applications is to be realized for nanomedicine, sensing and molecular diagnostics, and chemical and biological decontamination. In this study, we focus on the enzyme, organophosphorus hydrolase (OPH), which has shown great promise for the nontoxic and noncorrosive decontamination of organophosphate agents (OPs) as well as for therapeutics as a catalytic bioscavanger against nerve gas poisoning. We describe a facile approach to stabilize OPH using covalent conjugation with the amphiphilic block copolymer, Pluronic F127, leading to the formation of F127-OPH conjugate micelles, with the OPH on the micelle corona. SDS-PAGE and MALDI-TOF confirmed the successful conjugation, and transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed ∼100 nm size micelles. The conjugates showed significantly enhanced stability and higher activity compared to the unconjugated OPH when tested (i) in aqueous solutions at room temperature, (ii) in aqueous solutions at higher temperatures, (iii) after multiple freeze/thaw treatments, (iv) after lyophilization, and (v) in the presence of organic solvents. The F127-OPH conjugates also decontaminated paraoxon (introduced as a chemical agent simulant) on a polystyrene film surface and on a CARC (Chemical Agent Resistant Coating) test panel more rapidly and to a larger extent compared to free OPH. We speculate that, in the F127-OPH conjugates (both in the micellar form as well as in the unaggregated conjugate), the polypropylene oxide block of the copolymer interacts with the surface of the OPH and this confinement of the OPH reduces the potential for enzyme denaturation and provides robustness to OPH at different working environments. The use of such polymer-enzyme conjugate micelles with improved enzyme stability opens up new opportunities for numerous civilian and Warfighter applications. PMID

  6. Alkali-based pretreatments distinctively extract lignin and pectin for enhancing biomass saccharification by altering cellulose features in sugar-rich Jerusalem artichoke stem.

    PubMed

    Li, Meng; Wang, Jun; Yang, Yuezhou; Xie, Guanghui

    2016-05-01

    Jerusalem artichoke (JA) has been known as a potential nonfood feedstock for biofuels. Based on systems analysis of total 59 accessions, both soluble sugar and ash could positively affect biomass digestibility after dilute sodium hydroxide pretreatment (A). In this study, one representative accession (HEN-3) was used to illustrate its enzymatic digestibility with pretreatments of ultrasonic-assisted dilute sodium hydroxide (B), alkaline peroxide (C), and ultrasonic-assisted alkaline peroxide (D). Pretreatment D exhibited the highest hexose release rate (79.4%) and total sugar yield (10.4 g/L), which were 2.4 and 2.6 times higher, respectively, than those of the control. The analysis of cellulose crystalline index (CrI), cellulose degree of polymerization (DP), thermal behavior and SEM suggested that alkali-based pretreatments could distinctively extract lignin and pectin polymers, leading to significant alterations of cellulose CrI and DP for high biomass saccharification. Additionally, hydrogen peroxide (H2O2) could significant reduce the generation of fermentation inhibitors during alkali-based pretreatments. PMID:26918836

  7. Enhancement of Penicillium echinulatum glycoside hydrolase enzyme complex.

    PubMed

    dos Santos Costa, Patrícia; Büchli, Fernanda; Robl, Diogo; Delabona, Priscila da Silva; Rabelo, Sarita Candida; Pradella, José Geraldo da Cruz

    2016-05-01

    The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and β-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and β-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance β-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and β-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance β-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed β-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-β-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with β-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the

  8. Chemical characteristics and enzymatic saccharification of lignocellulosic biomass treated using high-temperature saturated steam: comparison of softwood and hardwood.

    PubMed

    Asada, Chikako; Sasaki, Chizuru; Hirano, Takeshi; Nakamura, Yoshitoshi

    2015-04-01

    This study investigated the effect of high-temperature saturated steam treatments on the chemical characteristics and enzymatic saccharification of softwood and hardwood. The weight loss and chemical modification of cedar and beech wood pieces treated at 25, 35, and 45 atm for 5 min were determined. Fourier transform infrared and X-ray diffraction analyses indicated that solubilization and removal of hemicellulose and lignin occurred by the steam treatment. The milling treatment of steam-treated wood enhanced its enzymatic saccharification. Maximum enzymatic saccharification (i.e., 94% saccharification rate of cellulose) was obtained using steam-treated beech at 35 atm for 5 min followed by milling treatment for 1 min. However, the necessity of the milling treatment for efficient enzymatic saccharification is dependent on the wood species. PMID:25704097

  9. Laboratory-scale method for enzymatic saccharification of lignocellulosic biomass at high-solids loadings

    PubMed Central

    2009-01-01

    Background Screening new lignocellulosic biomass pretreatments and advanced enzyme systems at process relevant conditions is a key factor in the development of economically viable lignocellulosic ethanol. Shake flasks, the reaction vessel commonly used for screening enzymatic saccharifications of cellulosic biomass, do not provide adequate mixing at high-solids concentrations when shaking is not supplemented with hand mixing. Results We identified roller bottle reactors (RBRs) as laboratory-scale reaction vessels that can provide adequate mixing for enzymatic saccharifications at high-solids biomass loadings without any additional hand mixing. Using the RBRs, we developed a method for screening both pretreated biomass and enzyme systems at process-relevant conditions. RBRs were shown to be scalable between 125 mL and 2 L. Results from enzymatic saccharifications of five biomass pretreatments of different severities and two enzyme preparations suggest that this system will work well for a variety of biomass substrates and enzyme systems. A study of intermittent mixing regimes suggests that mass transfer limitations of enzymatic saccharifications at high-solids loadings are significant but can be mitigated with a relatively low amount of mixing input. Conclusion Effective initial mixing to promote good enzyme distribution and continued, but not necessarily continuous, mixing is necessary in order to facilitate high biomass conversion rates. The simplicity and robustness of the bench-scale RBR system, combined with its ability to accommodate numerous reaction vessels, will be useful in screening new biomass pretreatments and advanced enzyme systems at high-solids loadings. PMID:19889202

  10. Enzymes and other agents that enhance cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

  11. Enhanced Diffusion of Enzymes that Catalyze Exothermic Reactions

    NASA Astrophysics Data System (ADS)

    Golestanian, Ramin

    2015-09-01

    Enzymes have been recently found to exhibit enhanced diffusion due to their catalytic activities. A recent experiment [C. Riedel et al., Nature (London) 517, 227 (2015)] has found evidence that suggests this phenomenon might be controlled by the degree of exothermicity of the catalytic reaction involved. Four mechanisms that can lead to this effect, namely, self-thermophoresis, boost in kinetic energy, stochastic swimming, and collective heating are critically discussed, and it is shown that only the last two can be strong enough to account for the observations. The resulting quantitative description is used to examine the biological significance of the effect.

  12. A Weibull statistics-based lignocellulose saccharification model and a built-in parameter accurately predict lignocellulose hydrolysis performance.

    PubMed

    Wang, Mingyu; Han, Lijuan; Liu, Shasha; Zhao, Xuebing; Yang, Jinghua; Loh, Soh Kheang; Sun, Xiaomin; Zhang, Chenxi; Fang, Xu

    2015-09-01

    Renewable energy from lignocellulosic biomass has been deemed an alternative to depleting fossil fuels. In order to improve this technology, we aim to develop robust mathematical models for the enzymatic lignocellulose degradation process. By analyzing 96 groups of previously published and newly obtained lignocellulose saccharification results and fitting them to Weibull distribution, we discovered Weibull statistics can accurately predict lignocellulose saccharification data, regardless of the type of substrates, enzymes and saccharification conditions. A mathematical model for enzymatic lignocellulose degradation was subsequently constructed based on Weibull statistics. Further analysis of the mathematical structure of the model and experimental saccharification data showed the significance of the two parameters in this model. In particular, the λ value, defined the characteristic time, represents the overall performance of the saccharification system. This suggestion was further supported by statistical analysis of experimental saccharification data and analysis of the glucose production levels when λ and n values change. In conclusion, the constructed Weibull statistics-based model can accurately predict lignocellulose hydrolysis behavior and we can use the λ parameter to assess the overall performance of enzymatic lignocellulose degradation. Advantages and potential applications of the model and the λ value in saccharification performance assessment were discussed. PMID:26121186

  13. Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of limonene concentration, enzyme loading, and pH on ethanol production from simultaneous saccharification and fermentation (SSF) of citrus peel waste by Saccharomyces cerevisiae were studied at 37 C. Prior to SSF, citrus peel waste underwent a steam explosion process combined with fla...

  14. Process for whole cell saccharification of lignocelluloses to sugars using a dual bioreactor system

    DOEpatents

    Lu, Jue; Okeke, Benedict

    2012-03-27

    The present invention describes a process for saccharification of lignocelluloses to sugars using whole microbial cells, which are enriched from cultures inoculated with paper mill waste water, wood processing waste and soil. A three-member bacterial consortium is selected as a potent microbial inocula and immobilized on inedible plant fibers for biomass saccharification. The present invention further relates the design of a dual bioreactor system, with various biocarriers for enzyme immobilization and repeated use. Sugars are continuously removed eliminating end-product inhibition and consumption by cell.

  15. High-Solids Enzymatic Saccharification Screening Method for Lignocellulosic Biomass (Poster)

    SciTech Connect

    Roche, C. M.; Stickel, J. J.

    2009-05-01

    The ability to screen new biomass pretreatments and advanced enzyme systems at process-relevant conditions is key to developing economically viable lignocellulosic ethanol. While much research is being invested in developing pretreatment technologies and enzyme systems that will more efficiently convert cellulosic biomass to sugars, the current standard reactor vessel, a shake flask, that is used for screening enzymatic saccharification of cellulosic biomass is inadequate at high-solids conditions. Shake flasks do not provide adequate mixing at high solids conditions. In this work, a roller bottle reactor was identified as a small-scale high-solids saccharification reaction vessel, and a method was developed for use in screening both pretreated biomass and enzyme systems at process-relevant conditions. This new method addresses mixing issues observed in high-solids saccharifications. In addition, yield calculations from sugar concentrations on a mass basis were used to account for the two-phase nature of the saccharification slurry, which eliminates discontinuities in comparing high-solids to low-solids saccharifications that occur when using concentrations on a volume basis. The roller bottle reactors out-performed the shake flasks by 5% for an initial insoluble solids loading of 15% and 140% for an initial soluble solids loading of 30%. The reactor system and method was compared at bench and floor scales and determined to be scalable for initial insoluble solids loading in the range of 15% to 30%. Pretreatment and enzyme screening results indicate that mid severity pretreated biomass is more digestible than the low and high severity biomass and GC220 is a superior enzyme to Spezyme CP.

  16. Synergistic effect and application of xylanases as accessory enzymes to enhance the hydrolysis of pretreated bagasse.

    PubMed

    Gonçalves, Geisa A L; Takasugi, Yusaku; Jia, Lili; Mori, Yutaro; Noda, Shuhei; Tanaka, Tsutomu; Ichinose, Hirofumi; Kamiya, Noriho

    2015-05-01

    Recently, the new trend in the second-generation ethanol industry is to use mild pretreatments, in order to reduce costs and to keep higher content of hemicellulose in the biomass. Nevertheless, a high enzyme dosage is still required in the conversion of (hemi)cellulose. The interaction between cellulases and xylanases seems to be an effective alternative to reduce enzyme loading in the saccharification process. At first, to evaluate the synergism of xylanases on bagasse degradation, we have produced two xylanases from glycoside hydrolase family 10 (GH10) and three xylanases from glycoside hydrolase family 11 (GH11), from two thermophilic organisms, Thermobifida fusca and Clostridium thermocellum, and one mesophilic organism, Streptomyces lividans. Peracetic acid (PAA) pretreated bagasse was used as substrate. The combination of XynZ-C (GH10, from C. thermocellum), and XlnB (GH11, from S. lividans) presented the highest degree of synergy after 6h (3.62). However, the combination of XynZ-C and Xyn11A (GH11, from T. fusca) resulted in the highest total yield of reducing sugars. To evaluate the synergism between xylanases and cellulases, commercial cellulase preparation from Trichoderma reesei was combined with the selected xylanases, XynZ-C and Xyn11A. About 2-fold increase was observed in the concentration of reducing sugars, when both xylanases, XynZ-C and Xyn11A, were added together with T. reesei cellulases in the reaction mixture. PMID:25837503

  17. 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 ...

  18. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    NASA Astrophysics Data System (ADS)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-02-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  19. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    PubMed Central

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-01-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

  20. Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

    NASA Astrophysics Data System (ADS)

    Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2015-03-01

    Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

  1. Enhanced plasma persistence of therapeutic enzymes by coupling to soluble dextran.

    PubMed Central

    Sherwood, R F; Baird, J K; Atkinson, T; Wiblin, C N; Rutter, D A; Ellwood, D C

    1977-01-01

    Conjugation of carboxypeptidase G and arginase, two enzymes of therapeutic interest, to a soluble dextran significantly enhanced plasma persistence in normal and tumour-bearing mice. A prolonged decrease in arginine concentrations in plasma of tumour-bearing mice was demonstrated by using the dextran-linked arginase. Gel filtration of dextran-enzyme conjugate showed that enzyme activity co-chromatographed as a single peak with carbohydrate, and enzyme was shown to be covalently linked to the dextran. PMID:880251

  2. Enhancing a Pathway-Genome Database (PGDB) to Capture Subcellular Localization of Metabolites and Enzymes: The Nucleotide-Sugar Biosynthetic Pathways of Populus trichocarpa

    SciTech Connect

    Nag, A.; Karpinets, T. V.; Chang, C. H.; Bar-Peled, M.

    2012-01-01

    Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s).

  3. Saccharification of bamboo carbohydrates for the production of ethanol

    SciTech Connect

    De Menezes, T.J.B.; Azzini, A.; Dos Santos, C.L.M.

    1983-04-01

    Bamboo carbohydrates were hydrolyzed with commercial amylases and a mixture of fungal culture broths containing cellulolytic and hemicellulolytic enzymes. The effects of cooking temperature and the size of fiber particles were also investigated. It was found that the higher the cooking temperature, the higher the rate of sugar formation and the lower the viscosity of the slurry. Additions of cellulose and hemicellulose digesting enzymes increased the sugar yield and decreased the viscosity of both the cooked and noncooked slurries. A smaller size of particle appeared to favor the average saccharification rate. Although glucose, xylose, and cellobiose were present in the hydrolysates, only 50% of the total carbohydrate was digested, and 78.9% of this was converted to reducing sugars. The alcohol efficiency for the fermentation of cooked and noncooked mashes by Saccharomyces was about 85%.

  4. Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

    PubMed Central

    Liang, Youyun; Si, Tong

    2014-01-01

    Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast. PMID:25149522

  5. The addition of accessory enzymes enhances the hydrolytic performance of cellulase enzymes at high solid loadings.

    PubMed

    Hu, Jinguang; Chandra, Richard; Arantes, Valdeir; Gourlay, Keith; van Dyk, J Susan; Saddler, Jack N

    2015-06-01

    The pretreatment process used and the nature of the biomass feedstock will influence the role that accessory enzymes can play in synergistically interacting with cellulases to effectively deconstruct the substrate. The work reported here assessed the possible boosting effects of the xylanase and lytic polysaccharide monooxygenase (AA9, formerly known as GH61) on the hydrolytic potential of cellulase enzyme mixtures during hydrolysis of steam pretreated poplar and corn stover at high (10-20% w/v) substrate concentrations. A higher proportion of xylanase was required when the substrate had a relatively high xylan content and at high substrate concentrations. In contrast, a relatively small amount of AA9 (about 2 mg/g cellulose) was enough, regardless of the nature or concentration of the substrate. The overall protein loading required to achieve effective hydrolysis of high concentrations of pretreated biomass substrates could be substantially reduced by optimizing the ratio of enzymes in the "cellulase" mixture. PMID:25812819

  6. Enhanced bioremediation of subsurface contamination: Enzyme recruitment and redesign

    SciTech Connect

    Brockman, F.J.; Ornstein, R.L.

    1991-12-01

    Subsurface systems containing radionuclide, heavy metal, and organic wastes must be carefully attended to avoid further impacts to the environment or exposures to human populations. It is appropriate, therefore, to invest in basic research to develop the requisite tools and methods for addressing complex cleanup problems. The rational modification of subsurface microoganisms by enzyme recruitment and enzyme design, in concert with engineered systems for delivery of microorganisms and nutrients to the contaminated zone, are potentially useful tools in the spectrum of approaches that will be required for successful remediation of deep subsurface contamination.

  7. Enzyme Assay: An Investigative Approach to Enhance Science Process Skills

    ERIC Educational Resources Information Center

    Vartak, Rekha; Ronad, Anupama; Ghanekar, Vikrant

    2013-01-01

    Scientific investigations play a vital role in teaching and learning the process of science. An investigative task that was developed for pre-university students is described here. The task involves extraction of an enzyme from a vegetable source and its detection by biochemical method. At the beginning of the experiment, a hypothesis is presented…

  8. Poly(ethylene glycol) conjugated enzyme with enhanced hydrophobic compatibility for self-cleaning coatings.

    PubMed

    Zhang, Liting; Wu, Songtao; Buthe, Andreas; Zhao, Xueyan; Jia, Hongfei; Zhang, Songping; Wang, Ping

    2012-11-01

    Enzyme-based smart materials constitute a rapidly growing group of functional materials. Often the natively evolved enzymes are not compatible with hydrophobic synthetic materials, thus significantly limiting the performance of enzymes. This work investigates the use of a polyethylene glycol (PEG)-conjugated detergent enzyme for self-cleaning coatings. As a result, PEG conjugated α-amylase demonstrated a much more homogeneous distribution in polyurethane coatings than the parent native enzyme as detected by both fluorescent microscopy and scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (SEM-EDX). Additionally, the conjugated enzyme showed enhanced retention in the coating and much improved thermal stability with a halflife of 20 days detected at 80 °C and over 350 days under room temperature. Such coating-incorporated enzyme afforded interesting self-cleaning functionality against starch-based stains as examined through a slipping drop test. PMID:23067105

  9. Open-mouthed hybrid microcapsules with elevated enzyme loading and enhanced catalytic activity.

    PubMed

    Shi, Jiafu; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi

    2014-10-25

    Open-mouthed hybrid microcapsules (HMCs) are synthesized through a hard-templating method. When utilized for enzyme immobilization and enzymatic catalysis, the open-mouthed HMCs show high enzyme loading capability, enhanced catalytic activity and desirable recycling stability, due to their fully exposed outer and inner surfaces. PMID:25189769

  10. A study of overproduction and enhanced secretion of enzymes. Quarterly report

    SciTech Connect

    Dashek, W.V.

    1993-09-01

    Wood decay within forests, a significant renewable photosynthetic energy resource, is caused primarily by Basidiomycetous fungi, e.g., white rot fungi. These organisms possess the ability to degrade lignin, cellulose and hemicellulose, the main organic polymers of wood. In the case of the white rot fungi, e.g., Coriolus versicolor, the capacity results from the fungus` ability to elaborate extracellular cellulolytic and ligninolytic enzymes. With regard to the latter, at least one of the enzymes, polyphenol oxidase (PPO) appears within a defined growth medium. This proposal focuses on the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. There are two major sections to the proposal: (1) overproduction of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electro microscopical techniques and (2) the biochemical/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO enzymes.

  11. Addition of metal ions to a (hemi)cellulolytic enzymatic cocktail produced in-house improves its activity, thermostability, and efficiency in the saccharification of pretreated sugarcane bagasse.

    PubMed

    Vasconcellos, V M; Tardioli, P W; Giordano, R L C; Farinas, C S

    2016-05-25

    High activity and stability are essential for (hemi)cellulolytic enzymes used in biomass conversion, while non-productive binding of cellulases to lignin reduces saccharification efficiency and needs to be avoided. One potential strategy is the addition of inexpensive metal ions. This paper describes the influence of divalent metal ions on the activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes produced in-house by Aspergillus niger under solid-state fermentation (SSF). The use of Mn(2+) provided the best (hemi)cellulolytic activity and stability, with an increase in endoglucanase activity of up to 57%. The use of Mn(2+) was then investigated in the saccharification of sugarcane bagasse submitted to acid, steam-explosion, and hydrothermal pretreatments. The addition of Mn(2+) ions at 10mM in the saccharification of acid-pretreated bagasse resulted in a 34% increase in glucose release. These positive effects appeared to be due to a reduction in non-productive enzyme adsorption. The findings suggest that the addition of inexpensive metal ions can help to improve activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes. PMID:26709004

  12. Acetone-butanol-ethanol production from Kraft paper mill sludge by simultaneous saccharification and fermentation.

    PubMed

    Guan, Wenjian; Shi, Suan; Tu, Maobing; Lee, Yoon Y

    2016-01-01

    Paper mill sludge (PS), a solid waste from pulp and paper industry, was investigated as a feedstock for acetone-butanol-ethanol (ABE) production by simultaneous saccharification and fermentation (SSF). ABE fermentation of paper sludge by Clostridium acetobutylicum required partial removal of ash in PS to enhance its enzymatic digestibility. Enzymatic hydrolysis was found to be a rate-limiting step in the SSF. A total of 16.4-18.0g/L of ABE solvents were produced in the SSF of de-ashed PS with solid loading of 6.3-7.4% and enzyme loading of 10-15FPU/g-glucan, and the final solvent yield reached 0.27g/g sugars. No pretreatment and pH control were needed in ABE fermentation of paper sludge, which makes it an attractive feedstock for butanol production. The results suggested utilization of paper sludge should not only consider the benefits of buffering effect of CaCO3 in fermentation, but also take into account its inhibitory effect on enzymatic hydrolysis. PMID:26562687

  13. Enzyme immunoassay by dynamic enhanced vibrational spectroscopy of the enzyme reaction product

    NASA Astrophysics Data System (ADS)

    Zhao, Haiying; Dou, Xiaoming

    2005-01-01

    This paper reports a kind of application of surface-enhanced Raman scattering (SERS) to immunology. In the proposed system, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibody labeled with peroxidase. If this immunocomplex is subjected to reaction with o-phenylenediamine and hydrogenperoxide at 37°C, azoaniline is generated. This azo compound is adsorbed on a silver colloid and only the azo compound gives a strong surface-enhanced resonance Raman (SERRS) spectrum. A linear relationship was observed between the peak intensity of the N=N stretching band and the concentration of antigen, revealing that one can determine the concentration of antigen by the SERRS measurement of the reaction product.

  14. Enzymatic Saccharification of Lignocellulosic Residues by Cellulases Obtained from Solid State Fermentation Using Trichoderma viride

    PubMed Central

    Sartori, Tanara; Tibolla, Heloisa; Prigol, Elenizi; Colla, Luciane Maria; Costa, Jorge Alberto Vieira; Bertolin, Telma Elita

    2015-01-01

    The aim of this study was to verify the viability of lignocellulosic substrates to obtain renewable energy source, through characterization of the cellulolytic complex, which was obtained by solid state fermentation using Trichoderma viride. Enzymatic activity of the cellulosic complex was measured during saccharification of substrates filter paper, eucalyptus sawdust, and corncob, and compared with the activity of commercial cellulase. The characterization of the enzymes was performed by a 22 Full Factorial Design, where the pH and temperature were the variables of study. Enzymatic saccharification of different substrates appearedviable until 12 to be viable until 12 h; after this period the activity decreased for both enzymatic forms (cellulolytic complex and commercial cellulase). The enzymatic activity of the commercial cellulase was favored with the use of corncob as substrate, while the cellulolytic complex does not show any difference in its specificity by the substrates studied. The largest activities of both enzymes were obtained in the temperature and pH range between 40°C and 50°C and 4.8 and 5.2, respectively. The cellulolytic complex obtained appeared to be viable for the saccharification of lignocellulosic residues compared with the commercial cellulase. PMID:26137476

  15. High-throughput Saccharification assay for lignocellulosic materials.

    PubMed

    Gomez, Leonardo D; Whitehead, Caragh; Roberts, Philip; McQueen-Mason, Simon J

    2011-01-01

    Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest (1). In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification (2). These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system. This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2

  16. Enzyme sensitive, surface engineered nanoparticles for enhanced delivery of camptothecin.

    PubMed

    Yu, Hongliang; Chen, Jiao; Liu, Sen; Lu, Qian; He, Jian; Zhou, Zhengyang; Hu, Yong

    2015-10-28

    To achieve a drug delivery system combining the programmable long circulation and targeting ability, surface engineering nanoparticles (NPs), having a sandwich structure consisting of a long circulating outmost layer, a targeting middle layer and a hydrophobic innermost core were constructed by mixing a matrix metalloproteinase MMP2 and MMP9-sensitive copolymers (mPEG-Pep-PCL) and folate receptor targeted copolymers (FA-PEG-PCL). Their physiochemical traits including morphology, particle size, drug loading content, and in vitro release profiles were studied. In vitro studies validated that the inhibition efficiency of tumor cells was effectively correlated with NP concentrations. Furthermore, The PEG layer would detach from the NPs due to the up-regulated extracellular MMP2 and MMP9 in tumors, resulting in the exposure of folate to enhance the cellular internalization via folate receptor mediated endocytosis, which accelerated the release rate of CPT in vivo. The antitumor efficacy, tumor targeting ability and bio-distribution of the NPs were examined in a B16 melanoma cells xenograft mouse model. These NPs showed improved tumor target ability and enhanced aggregation of camptothecin (CPT) in tumor site and prominent suppression of tumor growth. Thus this mPEG-Pep-PCL@FA-PEG-PCL core-shell structure NP could be a better candidate for the tumor specific delivery of hydrophobic drug. PMID:26282096

  17. Dilute acid saccharification of lignocellulosic biomass

    SciTech Connect

    Penner, M.H.; Hashimoto, A.G.

    1995-12-01

    Aqueous dilute sulfuric acid solutions have been evaluated in terms of their effectiveness for the saccharification of the insoluble xylan fraction of poplar and switchgrass feedstocks. Acid concentrations ranging from .6 to 1.2% have been tested at temperatures ranging from 120 to 160{degrees}C. Treatments at optimum time, temperature, and acid combinations provided xylose yields of approximately 90% theoretical. Rate constants associated with xylan hydrolysis and xylose degradation for each of the feed-stocks have been evaluated. In general, optimum yields were associated with high temperature treatments for relatively short reaction times. Results from our laboratory will be presented with reference to previously published studies on hemicellulose saccharification and in the general context of converting lignocellulosic biomass to useful products.

  18. Progress on implantable biofuel cell: Nano-carbon functionalization for enzyme immobilization enhancement.

    PubMed

    Babadi, Arman Amani; Bagheri, Samira; Hamid, Sharifah Bee Abdul

    2016-05-15

    Biofuel cells are bio-electrochemical devices, which are suitable for the environmentally friendly generation of energy. Enzymatic biofuel cell (EBFC) operates at ambient temperature and pH. Biofuel cells utilize vegetable and animal fluids (e.g. glucose) as a biofuel to produce energy. Fundamental part of each Glucose biofuel cell (GBFC) is two bioelectrodes which their surface utilizes as an enzyme immobilized site. Glucose oxidase (GOx) or glucose dehydrogenase (GDH) were immobilized on bioanode and oxidize glucose while oxygen reduced in biocathode using immobilized laccase or bilirubin oxidase in order to generate sufficient power. Glucose biofuel cells are capable to generate sufficient power for implanted devices. The key step of manufacturing a bioelectrode is the effective enzyme immobilization on the electrode surface. Due to the thin diameter of carbon nanomaterials, which make them accessible to the enzyme active sites, they are applicable materials to establish electronic communication with redox enzymes. Carbon nanomaterials regenerate the biocatalysts either by direct electron transfer or redox mediators which serve as intermediated for the electron transfer. Nano-carbon functionalization is perfectly compatible with other chemical or biological approaches to enhance the enzyme functions in implantable biofuel cells. Efficient immobilization of enzyme using the functionalized nano-carbon materials is the key point that greatly increases the possibilities of success. Current review highlights the progress on implantable biofuel cell, with focus on the nano-carbon functionalization for enzyme immobilization enhancement in glucose/O2 biofuel cells. PMID:26785309

  19. Deep eutectic solvent pretreatment and subsequent saccharification of corncob.

    PubMed

    Procentese, Alessandra; Johnson, Erin; Orr, Valerie; Garruto Campanile, Anna; Wood, Jeffery A; Marzocchella, Antonio; Rehmann, Lars

    2015-09-01

    Ionic liquid (ILs) pretreatment of lignocellulosic biomass has attracted broad scientific interest, despite high costs, possible toxicity and energy intensive recycling. An alternative group of ionic solvents with similar physicochemical properties are deep eutectic solvents (DESs). Corncob residues were pretreated with three different DES systems: choline chloride and glycerol, choline chloride and imidazole, choline chloride and urea. The pretreated biomass was characterised in terms of lignin content, sugars concentration, enzymatic digestibility and crystallinity index. A reduction of lignin and hemicellulose content resulted in increased crystallinity of the pretreated biomass while the crystallinity of the cellulose fraction could be reduced, depending on DES system and operating conditions. The subsequent enzymatic saccharification was enhanced in terms of rate and extent. A total of 41 g fermentable sugars (27 g glucose and 14 g xylose) could be recovered from 100g corncob, representing 76% (86% and 63%) of the initially available carbohydrates. PMID:26005926

  20. The rumen: a unique source of enzymes for enhancing livestock production.

    PubMed

    Selinger, L B; Forsberg, C W; Cheng, K J

    1996-10-01

    Increasing competition in the livestock industry has forced producers to cut costs by adopting new technologies aimed at increasing production efficiency. One particularly promising technology is feeding enzymes as supplements for animal diets. Supplementation of diets for non-ruminants (e.g., swine and poultry) with fibrolytic enzymes, such as cellulases, xylanases and beta-glucanases, increases the feed conversion efficiency and growth rate of the animals. Enzymatic hydrolysis of plant cell wall polymers (e.g., cellulose, xylan, beta-glucans) releases glucose and xylose and eliminates the antinutritional effects of beta-glucans and arabinoxylans. Enzyme supplementation of diets for ruminants has also been shown to improve growth performance, even though the rumen itself represents the most potent fibrolytic fermentation system known. Implementation of this technology in the livestock industry has been limited largely because of the cost of development and production of enzymes. Over the last decade, however, developments in recombinant DNA technology have increased the efficiency of existing microbial production systems and facilitated exploitation of alternative sources of industrial enzymes. The ruminal ecosystem is among the novel enzyme sources currently being explored. Understanding the role of enzymes in feed digestion through characterization of the enzymology and genetics involved in digestion of feedstuffs by ruminants will provide insight required to improve the products currently available to producers. Characterization of genes encoding a variety of hydrolytic enzymes, such as cellulases, xylanases, beta-glucanases, amylases, pectinases, proteases, phytases and tannases, will foster the development of more efficacious enzyme supplements and enzyme expression systems for enhancing nutrient utilization by domestic animals. Characteristics of the original source organism need no longer restrict the production of a useful enzyme. Recent reports of

  1. Evaluation of nanoparticle-immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions

    SciTech Connect

    Lupoi, Jason; Smith, Emily

    2011-12-01

    Ethanol yields were 2.1 (P = 0.06) to 2.3 (P = 0.01) times higher in simultaneous saccharification and fermentation (SSF) reactions of microcrystalline cellulose when cellulase was physisorbed on silica nanoparticles compared to enzyme in solution. In SSF reactions, cellulose is hydrolyzed to glucose by cellulase while yeast simultaneously ferments glucose to ethanol. The 35 C temperature and the presence of ethanol in SSF reactions are not optimal conditions for cellulase. Immobilization onto solid supports can stabilize the enzyme and promote activity at non-optimum reaction conditions. Mock SSF reactions that did not contain yeast were used to measure saccharification products and identify the mechanism for the improved ethanol yield using immobilized cellulase. Cellulase adsorbed to 40 nm silica nanoparticles produced 1.6 times (P = 0.01) more glucose than cellulase in solution in 96 h at pH 4.8 and 35 C. There was no significant accumulation (<250 {mu}g) of soluble cellooligomers in either the solution or immobilized enzyme reactions. This suggests that the mechanism for the immobilized enzyme's improved glucose yield compared to solution enzyme is the increased conversion of insoluble cellulose hydrolysis products to soluble cellooligomers at 35 C and in the presence of ethanol. The results show that silica-immobilized cellulase can be used to produce increased ethanol yields in the conversion of lignocellulosic materials by SSF.

  2. Immobilization of Enzymes to Silver Island Films for Enhanced Enzymatic Activity

    PubMed Central

    Abel, Biebele; Aslan, Kadir

    2013-01-01

    Hypothesis The performance of the enzyme-based biosensors depends on the enzymatic activity and the use of an appropriate technique for immobilization of enzymes. The incorporation of silver island films (SIFs) into the enzyme-based biosensors is expected to enhance the enzymatic activity and to increase the detectability of analytes of interest. Experiments Two enzymes, β-galactosidase (β-Gal) and alkaline phosphatase (AP) were immobilized onto SIFs using the interactions of avidin-modified enzymes with (i) a monolayer of biotinylated bovine serum albumin (b-BSA) and/or (ii) a monolayer of biotinylated poly(ethylene-glycol)-amine (BEA molecular weight: 550 to 10000 Da). To confirm the effect of SIFs on enzymatic activity, two control surfaces (no silver) were also employed. Findings No enhancement in enzymatic activity for β-Gal on all SIFs was observed, which was attributed to the inhibition of β-Gal activity due to direct interactions of β-Gal with SIFs. The AP activity on SIFs with BEA was significantly larger than that observed on SIFs with b-BSA, where a 300% increase in AP activity was observed as compared to control surfaces. These observations suggest that SIFs can significantly enhance AP activity, which could help improve the detection limits of ELISAs and immunoassays that employ AP. PMID:24267340

  3. Surface enhanced Raman scattering as a probe of the cholesterol oxidase enzyme

    NASA Astrophysics Data System (ADS)

    Wojnarowska, R.; Polit, J.; Broda, D.; Gonchar, M.; Sheregii, E. M.

    2015-03-01

    In this work, we present a sensitive method for the determination of the enzyme concentration of cholesterol oxidase, which is one of the most important analytical enzymes. Although the method is affected by sensitivity limitations, recently the Raman scattering experimental data carried out on cholesterol oxidase conjugated via a 16-mercaptohexadecanoic acid organic linker with gold nanoparticles due to the surface plasmon resonance confirmed the observation of surface enhanced Raman scattering, which enables us to detect the vibrational lines belonging to PO and C=C bonds assigned to the flavin prosthetic group. This means there is a stable binding of the enzyme with nanoparticles as well as the enzyme remaining active and substantiates the possibility that prepared bio-nanosystems can be used for analytical purposes as a sensing element.

  4. An internal electron reservoir enhances catalytic CO2 reduction by a semisynthetic enzyme.

    PubMed

    Schneider, Camille R; Shafaat, Hannah S

    2016-08-01

    The development of an artificial metalloenzyme for CO2 reduction is described. The small-molecule catalyst [Ni(II)(cyclam)](2+) has been incorporated within azurin. Selectivity for CO generation over H(+) reduction is enhanced within the protein environment, while the azurin active site metal impacts the electrochemical overpotential and photocatalytic activity. The enhanced catalysis observed for copper azurin suggests an important role for intramolecular electron transfer, analogous to native CO2 reducing enzymes. PMID:27406946

  5. NanoCluster Beacons as Reporter Probes in Rolling Circle Enhanced Enzyme Activity Detection†

    PubMed Central

    Juul, Sissel; Obliosca, Judy M.; Liu, Cong; Liu, Yen-Liang; Chen, Yu-An; Imphean, Darren M.; Knudsen, Birgitta R.; Ho, Yi-Ping; Leong, Kam W.; Yeh, Hsin-Chih

    2015-01-01

    As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp.. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics. PMID:25901841

  6. The ultrasound-enhanced bioscouring performance of four polygalacturonase enzymes obtained from rhizopus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An analytical and statistical method has been developed to measure the ultrasound-enhanced bioscouring performance of milligram quantities of endo- and exo-polygalacturonase enzymes obtained from Rhizopus oryzae fungi. UV-Vis spectrophotometric data and a general linear mixed models procedure indic...

  7. Optimization of cellulase production by Enhydrobacter sp. ACCA2 and its application in biomass saccharification

    PubMed Central

    Premalatha, Nagaiah; Gopal, Nellaiappan O.; Jose, Polpass Arul; Anandham, Rangasamy; Kwon, Soon-Wo

    2015-01-01

    Cellulase finds use in saccharification of lignocellulosic agroresidues to fermentable sugars which can be used for production of commercially important metabolites. This study reports endoglucanase (CMCase) production by Enhydrobacter sp. ACCA2. The CMCase activity of the strain ACCA2 was successively improved by optimization of range of physical and nutritional parameter in a set of non-statistical and statistical experiments. Initial non-statistical selection of carbon source, incubation time, temperature and pH resulted in 1.07 fold increase of CMCase activity. In a subsequent statistical method, response surface methodology, optimization of medium components such as carboxymethylcellulose, peptone, NaCl, MgSO4, K2HPO4, and (NH4)2SO4 yielded further increase up to 2.39 fold CMCase activity. The cellulolytic potential was evaluated in biomass saccharification with different plant materials and the results revealed that the enzyme produced by strain may have significant commercial values for industrial saccharification process. Moreover, this is the first report of cellulase production by an Enhydrobacter spp. PMID:26500615

  8. Compositions for enhancing hydroysis of cellulosic material by cellulolytic enzyme compositions

    DOEpatents

    Quinlan, Jason; Xu, Feng; Sweeney, Matthew; Johansen, Katja Salomon

    2014-09-30

    The present invention relates to compositions comprising a GH61 polypeptide having cellulolytic enhancing activity and an organic compound comprising a carboxylic acid moiety, a lactone moiety, a phenolic moiety, a flavonoid moiety, or a combination thereof, wherein the combination of the GH61 polypeptide having cellulolytic enhancing activity and the organic compound enhances hydrolysis of a cellulosic material by a cellulolytic enzyme compared to the GH61 polypeptide alone or the organic compound alone. The present invention also relates to methods of using the compositions.

  9. The cellulase-mediated saccharification on wood derived from transgenic low-lignin lines of black cottonwood (Populus trichocarpa).

    PubMed

    Min, Douyong; Li, Quanzi; Jameel, Hasan; Chiang, Vincent; Chang, Hou-min

    2012-10-01

    Downregulated lignin transgenic black cottonwood (Populus trichocarpa) was used to elucidate the effect of lignin and xylan content on enzymatic saccharification. The lignin contents of three transgenic samples (4CL1-1, 4CL1-4, and CH8-1-4) were 19.3, 16.7, and 15.0 %, respectively, as compared with the wild type (21.3 %). The four pretreatments were dilute acid (0.1 % sulfuric acid, 185 °C, 30 min), green liquor (6 % total titratable alkali, 25 % sulfidity based on TTA, 185 °C, and 15 min.), autohydrolysis (185 °C, 30 min), and ozone delignification (25 °C, 30 min). Following the pretreatment, enzymatic saccharification was carried out using an enzyme charge of 5 FPU/g of substrates. The removal of lignin and hemicellulose varies with both the types of pretreatments and the lignin content of the transgenic trees. Due to the greatest removal of lignin, green liquor induced the highest sugar production and saccharification efficiency, followed by acid, ozone, and autohydrolysis in descending order. The results indicated that lignin is the main recalcitrance of biomass degradation. At a given lignin content, pretreatment with ozone delignification had lower saccharification efficiency than the other pretreatment methods due to higher xylan content. PMID:22903324

  10. The heat released during catalytic turnover enhances the diffusion of an enzyme.

    PubMed

    Riedel, Clement; Gabizon, Ronen; Wilson, Christian A M; Hamadani, Kambiz; Tsekouras, Konstantinos; Marqusee, Susan; Pressé, Steve; Bustamante, Carlos

    2015-01-01

    Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis. Although this observation has been reported and characterized for several different systems, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein-solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). This novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme. PMID:25487146

  11. The heat released during catalytic turnover enhances the diffusion of an enzyme

    SciTech Connect

    Riedel, Clement; Gabizon, Ronen; Wilson, Christian A. M.; Hamadani, Kambiz; Tsekouras, Konstantinos; Marqusee, Susan; Pressé, Steve; Bustamante, Carlos

    2014-12-10

    Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis. Although this observation has been reported and characterized for several different systems, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein-solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). We find this novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme.

  12. The heat released during catalytic turnover enhances the diffusion of an enzyme

    DOE PAGESBeta

    Riedel, Clement; Gabizon, Ronen; Wilson, Christian A. M.; Hamadani, Kambiz; Tsekouras, Konstantinos; Marqusee, Susan; Pressé, Steve; Bustamante, Carlos

    2014-12-10

    Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis. Although this observation has been reported and characterized for several different systems, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theorymore » to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein-solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). We find this novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme.« less

  13. The heat released during catalytic turnover enhances the diffusion of an enzyme

    PubMed Central

    Riedel, Clement; Gabizon, Ronen; Wilson, Christian A. M.; Hamadani, Kambiz; Tsekouras, Konstantinos; Marqusee, Susan; Pressé, Steve; Bustamante, Carlos

    2015-01-01

    Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis1,2. Although this observation has been reported and characterized for several different systems3–10, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms11,12. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein–solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). This novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme. PMID:25487146

  14. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

  15. Enzymatic saccharification of sugar cane bagasse by continuous xylanase and cellulase production from cellulomonas flavigena PR-22.

    PubMed

    Rojas-Rejón, Óscar A; Poggi-Varaldo, Héctor M; Ramos-Valdivia, Ana C; Ponce-Noyola, Teresa; Cristiani-Urbina, Eliseo; Martínez, Alfredo; de la Torre, Mayra

    2016-03-01

    Cellulase (CMCase) and xylanase enzyme production and saccharification of sugar cane bagasse were coupled into two stages and named enzyme production and sugar cane bagasse saccharification. The performance of Cellulomonas flavigena (Cf) PR-22 cultured in a bubble column reactor (BCR) was compared to that in a stirred tank reactor (STR). Cells cultured in the BCR presented higher yields and productivity of both CMCase and xylanase activities than those grown in the STR configuration. A continuous culture with Cf PR-22 was run in the BCR using 1% alkali-pretreated sugar cane bagasse and mineral media, at dilution rates ranging from 0.04 to 0.22 1/h. The highest enzymatic productivity values were found at 0.08 1/h with 1846.4 ± 126.4 and 101.6 ± 5.6 U/L·h for xylanase and CMCase, respectively. Effluent from the BCR in steady state was transferred to an enzymatic reactor operated in fed-batch mode with an initial load of 75 g of pretreated sugar cane bagasse; saccharification was then performed in an STR at 55°C and 300 rpm for 90 h. The constant addition of fresh enzyme as well as the increase in time of contact with the substrate increased the total soluble sugar concentration 83% compared to the value obtained in a batch enzymatic reactor. This advantageous strategy may be used for industrial enzyme pretreatment and saccharification of lignocellulosic wastes to be used in bioethanol and chemicals production from lignocellulose. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:321-326, 2016. PMID:26701152

  16. Enhanced removal of PAHs by Peniophora incarnata and ascertainment of its novel ligninolytic enzyme genes.

    PubMed

    Lee, Hwanhwi; Jang, Yeongseon; Lee, Young Min; Lee, Hanbyul; Kim, Gyu-Hyeok; Kim, Jae-Jin

    2015-12-01

    The hazardous effects of the PAHs should be managed by removal using white rot fungal ligninolytic enzymes. The white rot fungus Peniophora incarnataKUC8836 was stimulated to produce ligninolytic enzymes in a liquid medium by the addition of four substances: 0.5 g L(-1) Tween 80, 70 mg L(-1) CuSO4·5H2O, 10 mg L(-1) MnSO4·H2O, and 0.3 g L(-1) veratryl alcohol. The experiments were carried out in two different media: basal salt and 2% malt extract (ME) liquid medium. Under the experimental conditions, both laccase and manganese-dependent peroxidase (MnP) demonstrated with the highest activities in 2% ME liquid medium following the addition of Tween 80. The biodegradation of anthracene and pyrene was significantly enhanced by the induced ligninolytic enzymes when Tween 80 was added. Tween 80 is a viable co-substrate for P. incarnata, as it enhances the ability of P. incarnata to manage effective biodegradation of PAHs. Most of all, the novel laccase and MnP genes ascertained in this study, showed that the genes were involved in the production of ligninolytic enzymes from P. incarnataKUC8836. PMID:26342262

  17. A study of over production and enhanced secretion of enzymes. Quarterly report 1

    SciTech Connect

    Dashek, W.V.

    1992-12-28

    The current project is concerned with the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. The project is divided into two segments: over-production of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electron microscopical techniques. The former approach employs recombinant DNA procedures, ligation of appropriate nuclease generated DNA fragments into a vector and the subsequent transformation of Escherichia coli to yield E. coli harboring a C. versicolor DNA insert. The biochemistry/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO inhibitors to elevate C.versicolor`s ability to synthesize and secrete lignocellulosic enzymes. In this connection, cell fractionation/kinetic analysis, TEM immunoelectron microscopic localization and TEM substrate localization of PPO are being employed to assess the route of secretion. Both approaches will culminate in the batch culture of either E. coli or C. versicolor, in a fermentor with the subsequent development of rapid isolation and purification procedures to yield elevated quantities of pure lignocellulosic enzymes. During the past year, research effort were directed toward determining the route of polyphenol oxidase (PPO) secretion by the wood-decay fungus, Coriolus versicolor. In addition, research activities were continued to over-produce and to purify PPO as well as define the time-dependent intra- and extra-cellular appearances of C. versicolor ligninases and cellulases.

  18. Enzymes for enhancing bioremediation of petroleum-contaminated soils: a brief review.

    PubMed

    Fan, C Y; Krishnamurthy, S

    1995-06-01

    During the 1950s and 1960s, hundreds of thousands of underground storage tanks (and above-ground storage tanks) containing petroleum products and hazardous chemicals were installed. Many of these tanks either have been abandoned or have exceeded their useful lives and are leaking, thereby posing a serious threat to the nation's surface and groundwater supplies, as well as to public health. Cleaning up releases of petroleum hydrocarbons or other organic chemicals in the subsurface environment is a real-world problem. Biological treatment of hydrocarbon-contaminated soil is considered to be a relatively low-cost and safe technology; however, its potential for effectively treating recalcitrant wastes has not been fully explored. For millions of years, microorganisms such as bacteria, fungi, actinomycete, protozoa, and others have performed the function of recycling organic matter from which new plant life can grow. This paper examines the biological treatment technology for cleaning up petroleum product-contaminated soils, with special emphasis on microbial enzyme systems for enhancing the rate of biodegradation of petroleum hydrocarbons. Classifications and functions of enzymes, as well as the microbes, in degrading the organic contaminants are discussed. In addition, the weathering effect on biodegradation, types of hydrocarbon degraders, advantages associated with enzyme use, methods of enzyme extraction, and future research needs for development and evaluation of enzyme-assisted bioremediation are examined. PMID:7788508

  19. Steam pretreatment of agricultural residues facilitates hemicellulose recovery while enhancing enzyme accessibility to cellulose.

    PubMed

    Chandra, Richard P; Arantes, Valdeir; Saddler, Jack

    2015-06-01

    The origins of lignocellulosic biomass and the pretreatment used to enhance enzyme accessibility to the cellulosic component are known to be strongly influenced by various substrate characteristics. To assess the impact that fibre properties might have on enzymatic hydrolysis, seven agricultural residues were characterised before and after steam pretreatment using a single pretreatment condition (190°C, 5min, 3% SO2) previously shown to enhance fractionation and hydrolysis of the cellulosic component of corn stover. When the fibre length, width and coarseness, viscosity, water retention value and cellulose crystallinity were monitored, no clear correlation was observed between any single substrate characteristic and the substrate's ease of enzymatic hydrolysis. However, the amount of hemicellulose that was solubilised during pretreatment correlated (r(2)=0.98) with the effectiveness of enzyme hydrolysis of each pretreated substrate. Simons's staining, to measure the cellulose accessibility, showed good correlation (r(2)=0.83) with hemicellulose removal and the extent of enzymatic hydrolysis. PMID:25780906

  20. Enzyme catalysis enhanced dark-field imaging as a novel immunohistochemical method.

    PubMed

    Fan, Lin; Tian, Yanyan; Yin, Rong; Lou, Doudou; Zhang, Xizhi; Wang, Meng; Ma, Ming; Luo, Shouhua; Li, Suyi; Gu, Ning; Zhang, Yu

    2016-04-28

    Conventional immunohistochemistry is limited to subjective judgment based on human experience and thus it is clinically required to develop a quantitative immunohistochemical detection. 3,3'-Diaminobenzidin (DAB) aggregates, a type of staining product formed by conventional immunohistochemistry, were found to have a special optical property of dark-field imaging for the first time, and the mechanism was explored. On this basis, a novel immunohistochemical method based on dark-field imaging for detecting HER2 overexpressed in breast cancer was established, and the quantitative analysis standard and relevant software for measuring the scattering intensity was developed. In order to achieve a more sensitive detection, the HRP (horseradish peroxidase)-labeled secondary antibodies conjugated gold nanoparticles were constructed as nanoprobes to load more HRP enzymes, resulting in an enhanced DAB deposition as a dark-field label. Simultaneously, gold nanoparticles also act as a synergistically enhanced agent due to their mimicry of enzyme catalysis and dark-field scattering properties. PMID:26786242

  1. The simultaneous saccharification and fermentation of pretreated woody crops to ethanol

    SciTech Connect

    Spindler, D.D.; Wyman, C.E.; Grohmann, K.

    1991-12-31

    Four promising woody crops (Populus maximowiczii x nigra (NE388), P. trichocarpa x deltoides (N11), P. tremuloides, and Sweetgum Liquidambar styraciflua) were pretreated by dilute sulfuric acid and evaluated in the simultaneous saccharification and fermentation (SSF) process for ethanol production. The yeast Saccharomyces cerevisiae was used in the fermentations alone, and in mixed cultures with {beta}-glucosidase producing Brettanomyces clausenii. Commercial Genencor 150L cellulose enyme was either employed alone or supplemented with {beta}-glucosidase. All SSFs were run at 37{degrees}C for 8 d and compared to saccharifications at 45{degrees}C under the same enzyme loadings. S. cerevisiae alone achieved the highest ethanol yields and rates of hydrolysis at the higher enzyme loadings, whereas the mixed culture performed better at the lower enzyme loadings without {beta}-glucosidase supplementation. The best overall rates of fermentation (3 d) and final theoretical ethanol yields (86-90%) were achieved with P. maximowiczii x nigra (NE388) and Sweetgum Liquidambar styraciflua, followed by P. tremuloides and P. trichocarpa x deltoides (N11) with slightly slower rates and lower yields. Although there were some differences in SSF performance, all these pretreated woody crops show promise as substrates for ethanol production.

  2. High levels of β-xylosidase in Thermomyces lanuginosus: potential use for saccharification.

    PubMed

    Corrêa, Juliana Moço; Christi, Divair; Torre, Carla Lieko Della; Henn, Caroline; da Conceição-Silva, José Luis; Kadowaki, Marina Kimiko; Simão, Rita de Cássia Garcia

    2016-01-01

    A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its β-xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of β-xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high β-xylosidases activities 1003U/mL. According our known, this study is the first to show so high levels of β-xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce β-xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60°C showing better thermostability at 55°C. The saccharification ability of β-xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50°C. Our data strongly indicated that the β-xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production. PMID:27256169

  3. NanoCluster Beacons as reporter probes in rolling circle enhanced enzyme activity detection

    NASA Astrophysics Data System (ADS)

    Juul, Sissel; Obliosca, Judy M.; Liu, Cong; Liu, Yen-Liang; Chen, Yu-An; Imphean, Darren M.; Knudsen, Birgitta R.; Ho, Yi-Ping; Leong, Kam W.; Yeh, Hsin-Chih

    2015-04-01

    As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics.As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics. Electronic

  4. Apoferritin Nanoparticle: A Novel and Biocompatible Carrier for Enzyme Immobilization with Enhanced Activity and Stability

    SciTech Connect

    Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong J.; Lin, Chiann Tso; Lin, Yuehe

    2011-11-01

    Apoferritin is a nanostructured material with a uniform size and spherical structure, and it has excellent bio-compatibility. In this work, we report the use of apoferritin as a novel and biocompatible carrier for stabilizing enzymes and their activities. We used glucose oxidase (GOx) as a model enzyme. GOx was immobilized on the surface of the apoferritin through a green synthetic approach taking advantage of bioaffinity binding between streptavidin and biotin. As a result, a glucose oxidase-biotin/streptavidin/biotin-apoferritin conjugate (Apo-GOx) was prepared using streptavidin as a bridge. The synthesized Apo-GOx was characterized with transmission electron microscopy, ultraviolet, and fluorescence spectroscopy. The activity and stability of GOx on the surface of the apoferritin were studied in different environments, such as temperature, chemicals, and pH, in comparison with the biotinylated GOx (B-GOx). The results showed that the activity of GOx on the apoferritin surface was significantly enhanced. The thermal and chemical stability of the GOx on the apoferritin was also greatly improved compared to free B-GOx in a solution. It was found that the activity of the GOx on the apoferritin only lost 30% in comparison to a 70% loss of free B-GOx after a 2 h incubation at 50oC. There was almost no decrease in activity for the GOx on the apoferritin as compared to an 80% activity decrease for free B-GOx after 30 min incubation in a 5 M urea solution. Glucose detection was used as a model application for the enzyme immobilization method developed in this work. The GOx immobilized apoferritin nanoparticles exhibited high sensitivity for glucose detection with a detection limit of 3 nM glucose. This work offers a novel approach for immobilizing enzymes with enhanced stability and activity, and this method may find a number of applications, such as in enzyme catalysis, DNA assays and immunoassays.

  5. Fundamental Challenges in Mechanistic Enzymology: Progress toward Understanding the Rate Enhancements of Enzymes

    PubMed Central

    Herschlag, Daniel; Natarajan, Aditya

    2013-01-01

    Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multi-faceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis. PMID:23488725

  6. Barrier height enhancement of metal/semiconductor contact by an enzyme biofilm interlayer

    NASA Astrophysics Data System (ADS)

    Ocak, Yusuf Selim; Gul Guven, Reyhan; Tombak, Ahmet; Kilicoglu, Tahsin; Guven, Kemal; Dogru, Mehmet

    2013-06-01

    A metal/interlayer/semiconductor (Al/enzyme/p-Si) MIS device was fabricated using α-amylase enzyme as a thin biofilm interlayer. It was observed that the device showed an excellent rectifying behavior and the barrier height value of 0.78 eV for Al/α-amylase/p-Si was meaningfully larger than the one of 0.58 eV for conventional Al/p-Si metal/semiconductor (MS) contact. Enhancement of the interfacial potential barrier of Al/p-Si MS diode was realized using enzyme interlayer by influencing the space charge region of Si semiconductor. The electrical properties of the structure were executed by the help of current-voltage and capacitance-voltage measurements. The photovoltaic properties of the structure were executed under a solar simulator with AM1.5 global filter between 40 and 100 mW/cm2 illumination conditions. It was also reported that the α-amylase enzyme produced from Bacillus licheniformis had a 3.65 eV band gap value obtained from optical method.

  7. Spatiotemporal patterns enhanced by intra- and inter-molecular fluctuations in arrays of allosterically regulated enzymes

    NASA Astrophysics Data System (ADS)

    Togashi, Yuichi; Casagrande, Vanessa

    2015-03-01

    Enzymatic reactions often involve slow conformational changes, with reaction cycles that sometimes require milliseconds or seconds to complete. The dynamics are strongly affected by fluctuations at the nanoscale. However, such enzymes often occur in small numbers in a cell; hence, the fluctuations caused by finite numbers of molecules should also be substantial. Because of these factors, the behavior of the system is likely to deviate from that of classical reaction-diffusion equations, in which immediate reaction events are assumed to occur without memory and between a huge number of molecules. In this work, we model each enzyme as a unit represented by a phase variable to investigate the effects of fluctuations in arrays of enzymes. Using an analysis based on partial differential equations and stochastic simulations, we show that fluctuations arising from internal states of enzymes (intramolecular fluctuations) and those arising from the stochastic nature of interactions between molecules (intermolecular fluctuations) have distinctive effects on spatiotemporal patterns; the former generally disturb synchronization at high frequencies, whereas the latter can enhance synchronization. The balance of the two types of fluctuations may determine the spatiotemporal behavior of biochemical processes.

  8. Enhancing enzyme stability against TiO2-UV induced inactivation.

    PubMed

    Lele, Bhalchandra S; Russell, Alan J

    2005-01-01

    The use of enzymes in conjunction with inorganic photocatalysts requires stability against photooxidation. In this paper, we describe enhanced stabilization of a model enzyme, chymotrypsin, to photooxidation driven by titanium dioxide exposed to ultraviolet light (TiO(2)-UV). Stabilization is achieved conjugating the enzyme with an oligomeric adduct of UV-absorbing (2-[3-(2H-benzotriazol-2-yl)-4-hydroxyphenyl]ethyl methacrylate) (HBMA) and free radical-absorbing 2-methacryloyloxyethyl-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (Trolox-HEMA). Juxtaposition of the antioxidant Trolox with the UV absorber HBMA within a single chain reduced the rate of deactivation of the former by TiO(2)-UV. This enables modified enzyme, which is adsorbed on TiO(2), to absorb both UV-light and free radicals and locally reduce the rate of photooxidation. Interestingly, Trolox was more readily deactivated by TiO(2)-UV when it was conjugated separately to chymotrypsin that had been pre-modified with HBMA moieties. PMID:15638555

  9. Application of a new xylanase activity from Bacillus amyloliquefaciens XR44A in brewer's spent grain saccharification

    PubMed Central

    Amore, Antonella; Parameswaran, Binod; Kumar, Ramesh; Birolo, Leila; Vinciguerra, Roberto; Marcolongo, Loredana; Ionata, Elena; La Cara, Francesco; Pandey, Ashok; Faraco, Vincenza

    2015-01-01

    Background Cellulases and xylanases are the key enzymes involved in the conversion of lignocelluloses into fermentable sugars. Western Ghat region (India) has been recognized as an active hot spot for the isolation of new microorganisms. The aim of this work was to isolate new microorganisms producing cellulases and xylanases to be applied in brewer's spent grain saccharification. Results 93 microorganisms were isolated from Western Ghat and screened for the production of cellulase and xylanase activities. Fourteen cellulolytic and seven xylanolytic microorganisms were further screened in liquid culture. Particular attention was focused on the new isolate Bacillus amyloliquefaciens XR44A, producing xylanase activity up to 10.5 U mL−1. A novel endo-1,4-beta xylanase was identified combining zymography and proteomics and recognized as the main enzyme responsible for B. amyloliquefaciens XR44A xylanase activity. The new xylanase activity was partially characterized and its application in saccharification of brewer's spent grain, pretreated by aqueous ammonia soaking, was investigated. Conclusion The culture supernatant of B. amyloliquefaciens XR44A with xylanase activity allowed a recovery of around 43% xylose during brewer's spent grain saccharification, similar to the value obtained with a commercial xylanase from Trichoderma viride, and a maximum arabinose yield of 92%, around 2-fold higher than that achieved with the commercial xylanase. © 2014 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:25866429

  10. In vivo cytochrome P450 drug metabolizing enzyme characterization using surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Yanfang; Bachmann, Kenneth A.; Cameron, Brent D.

    2003-07-01

    The development of a rapid, inexpensive, and accurate in vivo phenotyping methodology for characterizing drug-metabolizing phenotypes with reference to the cytochrome P450 (CYP450) enzymes would be very beneficial. In terms of application, in the wake of the human genome project, considerable interest is focused on the development of new drugs whose uses will be tailored to specific genetic polymorphisms, and on the individualization of dosing regimens that are also tailored to meet individual patient needs depending upon genotype. In this investigation, chemical probes for CYP450 enzymes were characterized and identified with Raman spectroscopy. Furthermore, gold-based metal colloid clusters were utilized to generate surface enhanced Raman spectra for each of the chemical probes. Results will be presented demonstrating the ability of SERS to identify minute quantities of these probes on the order needed for in vivo application.

  11. Plant Growth-Promoting Rhizobacteria Enhance Salinity Stress Tolerance in Okra through ROS-Scavenging Enzymes.

    PubMed

    Habib, Sheikh Hasna; Kausar, Hossain; Saud, Halimi Mohd

    2016-01-01

    Salinity is a major environmental stress that limits crop production worldwide. In this study, we characterized plant growth-promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and examined their effect on salinity stress tolerance in okra through the induction of ROS-scavenging enzyme activity. PGPR inoculated okra plants exhibited higher germination percentage, growth parameters, and chlorophyll content than control plants. Increased antioxidant enzyme activities (SOD, APX, and CAT) and upregulation of ROS pathway genes (CAT, APX, GR, and DHAR) were observed in PGPR inoculated okra plants under salinity stress. With some exceptions, inoculation with Enterobacter sp. UPMR18 had a significant influence on all tested parameters under salt stress, as compared to other treatments. Thus, the ACC deaminase-containing PGPR isolate Enterobacter sp. UPMR18 could be an effective bioresource for enhancing salt tolerance and growth of okra plants under salinity stress. PMID:26951880

  12. Plant Growth-Promoting Rhizobacteria Enhance Salinity Stress Tolerance in Okra through ROS-Scavenging Enzymes

    PubMed Central

    Habib, Sheikh Hasna; Kausar, Hossain; Saud, Halimi Mohd

    2016-01-01

    Salinity is a major environmental stress that limits crop production worldwide. In this study, we characterized plant growth-promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and examined their effect on salinity stress tolerance in okra through the induction of ROS-scavenging enzyme activity. PGPR inoculated okra plants exhibited higher germination percentage, growth parameters, and chlorophyll content than control plants. Increased antioxidant enzyme activities (SOD, APX, and CAT) and upregulation of ROS pathway genes (CAT, APX, GR, and DHAR) were observed in PGPR inoculated okra plants under salinity stress. With some exceptions, inoculation with Enterobacter sp. UPMR18 had a significant influence on all tested parameters under salt stress, as compared to other treatments. Thus, the ACC deaminase-containing PGPR isolate Enterobacter sp. UPMR18 could be an effective bioresource for enhancing salt tolerance and growth of okra plants under salinity stress. PMID:26951880

  13. Application safety enhancement model using self-checking with software enzymes

    NASA Astrophysics Data System (ADS)

    Subramaniam, Chandrasekaran; Ravishankar, Arthi; Gopal, Deepthi; Subramanian, Dhaarini

    2011-12-01

    The objective of the paper is to propose a safety enhancement model for application software in accelerating the respective self checking strategies similar to bio enzymatic actions. The application software components which are safety critical may have to be assessed periodically or on demand to achieve not only the functional correctness but also the safety specifications or features while getting executed. The design and deployment of such software modules can be formally verified for possible safety flaws using self checking capabilities and software enzymatic actions. The self checks must sense the safety holes in the software and decide to activate the built-in software components called enzymes to do the safe guard operations in a timely manner to mitigate the safety faults using the proposed enzyme calculus. The various application hazards due to the boolean faults in the functional and behavioral model that lead to software safety issues are considered in this approach.

  14. SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Resilience.

    PubMed

    Schoene, C; Bennett, S P; Howarth, M

    2016-01-01

    Enzymes often have marginal stability, with unfolding typically leading to irreversible denaturation. This sensitivity is a major barrier, both for de novo enzyme development and for expanding enzyme impact beyond the laboratory. Seeking an approach to enhance resilience to denaturation that could be applied to a range of different enzymes, we developed SpyRing cyclization. SpyRings contain genetically encoded SpyTag (13 amino acids) on the N-terminus and SpyCatcher (12kDa) on the C-terminus of the enzyme, so that the Spy partners spontaneously react together through an irreversible isopeptide bond. SpyRing cyclization gave major increases in thermal resilience, including on a model for enzyme evolution, β-lactamase, and an industrially important enzyme in agriculture and nutrition, phytase. We outline the SpyRing rationale, including comparison of SpyRing cyclization to other cyclization strategies. The cloning strategy is presented for the simple insertion of enzyme genes for recombinant expression. We discuss structure-based approaches to select suitable enzyme cyclization targets. Approaches to evaluate the cyclization reaction and its effect on enzyme resilience are described. We also highlight the use of differential scanning calorimetry to understand how SpyRing cyclization promotes enzyme refolding. Efficiently searching sequence space will continue to be important for enzyme improvement, but the SpyRing platform may be a valuable rational adjunct for conferring resilience. PMID:27586332

  15. Optimization of pretreatment and saccharification for the production of bioethanol from water hyacinth by Saccharomyces cerevisiae.

    PubMed

    Ahn, Deuk Joo; Kim, Se Kyung; Yun, Hyun Shik

    2012-01-01

    Alkaline-oxidative (A/O) pretreatment and enzymatic saccharification were optimized for bioethanol fermentation from water hyacinth by Saccharomyces cerevisiae. Water hyacinth was subjected to A/O pretreatment at various NaOH and H(2)O(2) concentrations and reaction temperatures for the optimization of bioethanol fermentation by S. cerevisiae. The most effective condition for A/O pretreatment was 7% (w/v) NaOH at 100 °C and 2% (w/v) H(2)O(2). The carbohydrate content was analyzed after reaction at various enzyme concentrations and enzyme ratios using Celluclast 1.5 L and Viscozyme L to determine the effective conditions for enzymatic saccharification. After ethanol fermentation using S. cerevisiae KCTC 7928, the concentration of glucose, ethanol and glycerol was analyzed by HPLC using a RI detector. The yield of ethanol in batch fermentation was 0.35 g ethanol/g biomass. Continuous fermentation was carried out at a dilution rate of 0.11 (per h) and the ethanol productivity was 0.77 [g/(l h)]. PMID:21909939

  16. Integrated delignification and simultaneous saccharification and fermentation of hard wood by a white-rot fungus, Phlebia sp. MG-60.

    PubMed

    Kamei, Ichiro; Hirota, Yoshiyuki; Meguro, Sadatoshi

    2012-12-01

    We propose a new process of unified aerobic delignification and anaerobic saccharification and fermentation of wood by a single microorganism, the white-rot fungus Phlebia sp. MG-60. This fungus is able to selectively degrade lignin under aerobic solid state fermentation conditions, and to produce ethanol directly from delignified oak wood under semi-aerobic liquid culture conditions. After 56 d aerobic incubation, 40.7% of initial lignin and negligible glucan were degraded. Then under semi-aerobic conditions without the addition of cellulase, 43.9% of theoretical maximum ethanol was produced after 20 d. Changing from aerobic conditions (biological delignification pretreatment) to semi-aerobic conditions (saccharification and fermentation) enabled the fermentation of wood by solely biological processes. This is the first report of ethanol production from woody biomass using a single microorganism without addition of chemicals or enzymes. PMID:23073100

  17. β2 Agonists enhance the efficacy of simultaneous enzyme replacement therapy in murine Pompe disease.

    PubMed

    Koeberl, Dwight D; Li, Songtao; Dai, Jian; Thurberg, Beth L; Bali, Deeksha; Kishnani, Priya S

    2012-02-01

    Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has improved clinical outcomes in patients with Pompe disease; however, the response of skeletal muscle and the central nervous system to ERT has been attenuated. The poor response of skeletal muscle to ERT has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR), which mediates receptor-mediated uptake of rhGAA. Hence the ability of adjunctive therapy with β2-agonists to increase CI-MPR expression in skeletal muscle was evaluated during ERT in murine Pompe disease with regard to reversal of neuromuscular involvement. Mice with Pompe disease were treated with weekly rhGAA injections (20 mg/kg) and a selective β2-agonist, either albuterol (30 mg/l in drinking water) or low-dose clenbuterol (6 mg/l in drinking water). Biochemical correction was enhanced by β2-agonist treatment in both muscle and the cerebellum, indicating that adjunctive therapy could enhance efficacy from ERT in Pompe disease with regard to neuromuscular involvement. Intriguingly, clenbuterol slightly reduced muscle glycogen content independent of CI-MPR expression, as demonstrated in CI-MPR knockout/GAA knockout mice that were otherwise resistant to ERT. Thus, adjunctive therapy with β2 agonists might improve the efficacy of ERT in Pompe disease and possibly other lysosomal storage disorders through enhancing receptor-mediated uptake of recombinant lysosomal enzymes. PMID:22154081

  18. β2 Agonists Enhance the Efficacy of Simultaneous Enzyme Replacement Therapy in Murine Pompe Disease

    PubMed Central

    Koeberl, Dwight D.; Li, Songtao; Dai, Jian; Thurberg, Beth L.; Bali, Deeksha; Kishnani, Priya S.

    2011-01-01

    Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has improved clinical outcomes in patients with Pompe disease; however, the response of skeletal muscle and the central nervous system to ERT has been attenuated. The poor response of skeletal muscle to ERT has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR), which mediates receptor-mediated uptake of rhGAA. Hence the ability of adjunctive therapy with β2-agonists to increase CI-MPR expression in skeletal muscle was evaluated during ERT in murine Pompe disease with regard to reversal of neuromuscular involvement. Mice with Pompe disease were treated with weekly rhGAA injections (20 mg/kg) and a selective β2-agonist, either albuterol (30 mg/l in drinking water) or low-dose clenbuterol (6 mg/l in drinking water). Biochemical correction was enhanced by β2-agonist treatment in both muscle and the cerebellum, indicating that adjunctive therapy could enhance efficacy from ERT in Pompe disease with regard to neuromuscular involvement. Intriguingly, clenbuterol slightly reduced muscle glycogen content independent of CI-MPR expression, as demonstrated in CI-MPR knockout/GAA knockout mice that were otherwise resistant to ERT. Thus, adjunctive therapy with β2 agonists might improve the efficacy of ERT in Pompe disease and possibly other lysosomal storage disorders through enhancing receptor-mediated uptake of recombinant lysosomal enzymes. PMID:22154081

  19. Identification and characterization of ambroxol as an enzyme enhancement agent for Gaucher disease.

    PubMed

    Maegawa, Gustavo H B; Tropak, Michael B; Buttner, Justin D; Rigat, Brigitte A; Fuller, Maria; Pandit, Deepangi; Tang, Liangiie; Kornhaber, Gregory J; Hamuro, Yoshitomo; Clarke, Joe T R; Mahuran, Don J

    2009-08-28

    Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243-249, 310-312, and 386-400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes. PMID:19578116

  20. Identification and Characterization of Ambroxol as an Enzyme Enhancement Agent for Gaucher Disease*

    PubMed Central

    Maegawa, Gustavo H. B.; Tropak, Michael B.; Buttner, Justin D.; Rigat, Brigitte A.; Fuller, Maria; Pandit, Deepangi; Tang, Liangiie; Kornhaber, Gregory J.; Hamuro, Yoshitomo; Clarke, Joe T. R.; Mahuran, Don J.

    2009-01-01

    Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243–249, 310–312, and 386–400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes. PMID:19578116

  1. Overexpression of angiotensin-converting enzyme in myelomonocytic cells enhances the immune response

    PubMed Central

    Bernstein, Kenneth E.; Khan, Zakir; Giani, Jorge F.; Zhao, Tuantuan; Eriguchi, Masahiro; Bernstein, Ellen A.; Gonzalez-Villalobos, Romer A.; Shen, Xiao Z.

    2016-01-01

    Angiotensin-converting enzyme (ACE) converts angiotensin I to the vasoconstrictor angiotensin II and thereby plays an important role in blood pressure control. However, ACE is relatively non-specific in its substrate specificity and cleaves many other peptides. Recent analysis of mice overexpressing ACE in monocytes, macrophages, and other myelomonocytic cells shows that these animals have a marked increase in resistance to experimental melanoma and to infection by Listeria monocytogenes or methicillin-resistant Staphylococcus aureus (MRSA). Several other measures of immune responsiveness, including antibody production, are enhanced in these animals. These studies complement a variety of studies indicating an important role of ACE in the immune response. PMID:27018193

  2. Enzyme catalysis enhanced dark-field imaging as a novel immunohistochemical method

    NASA Astrophysics Data System (ADS)

    Fan, Lin; Tian, Yanyan; Yin, Rong; Lou, Doudou; Zhang, Xizhi; Wang, Meng; Ma, Ming; Luo, Shouhua; Li, Suyi; Gu, Ning; Zhang, Yu

    2016-04-01

    Conventional immunohistochemistry is limited to subjective judgment based on human experience and thus it is clinically required to develop a quantitative immunohistochemical detection. 3,3'-Diaminobenzidin (DAB) aggregates, a type of staining product formed by conventional immunohistochemistry, were found to have a special optical property of dark-field imaging for the first time, and the mechanism was explored. On this basis, a novel immunohistochemical method based on dark-field imaging for detecting HER2 overexpressed in breast cancer was established, and the quantitative analysis standard and relevant software for measuring the scattering intensity was developed. In order to achieve a more sensitive detection, the HRP (horseradish peroxidase)-labeled secondary antibodies conjugated gold nanoparticles were constructed as nanoprobes to load more HRP enzymes, resulting in an enhanced DAB deposition as a dark-field label. Simultaneously, gold nanoparticles also act as a synergistically enhanced agent due to their mimicry of enzyme catalysis and dark-field scattering properties.Conventional immunohistochemistry is limited to subjective judgment based on human experience and thus it is clinically required to develop a quantitative immunohistochemical detection. 3,3'-Diaminobenzidin (DAB) aggregates, a type of staining product formed by conventional immunohistochemistry, were found to have a special optical property of dark-field imaging for the first time, and the mechanism was explored. On this basis, a novel immunohistochemical method based on dark-field imaging for detecting HER2 overexpressed in breast cancer was established, and the quantitative analysis standard and relevant software for measuring the scattering intensity was developed. In order to achieve a more sensitive detection, the HRP (horseradish peroxidase)-labeled secondary antibodies conjugated gold nanoparticles were constructed as nanoprobes to load more HRP enzymes, resulting in an enhanced DAB

  3. Strategies for enhancing resveratrol production and the expression of pathway enzymes.

    PubMed

    Lu, Yao; Shao, Dongyan; Shi, Junling; Huang, Qingsheng; Yang, Hui; Jin, Mingliang

    2016-09-01

    Trans-resveratrol (trans-3,5,4'-trihydroxystilbene) is one of the most promising stilbenes, a type of natural phenol that is produced naturally by some plant species in response to stress. Resveratrol exhibits multiple bioactivities and is used in the agriculture, medical, food, and cosmetic industries due to its antitumor, anti-inflammatory, cardioprotective, and antioxidant properties. Due to the increasing demand, an active area of investigation is the use of plant cell culture and metabolic engineering techniques to produce large quantities of active resveratrol. However, most recent studies have focused on the efficiency of synthesizing resveratrol in vitro, but have not investigated the contributions of the transcriptional activities of the genes encoding the related enzymes in the biosynthesis pathway. This article reviews recently developed methods for the biosynthesis of resveratrol and comprehensively reviews the current state of knowledge of the function of the key pathway enzymes in resveratrol synthesis. Approaches for enhancing resveratrol production, such as introducing non-pathway genes and co-localizing enzymes are described in detail. PMID:27405437

  4. [Dual promoters enhance heterologous enzyme production from bacterial phage based recombinant Bacillus subtilis].

    PubMed

    Liu, Gang; Zhang, Yan; Xing, Miao

    2006-03-01

    The effect of dual promoters on recombinant protein production from bacterial phage based Bacillus subtilis expression system was investigated. Alpha amylase (from Bacillus amyloliquefaciens) and penicillin acylase (from Bacillus megaterium) were selected as the indicating enzymes. Both the promoterless genes and the promoter-bearing genes were isolated through PCR amplification with properly designed primers, and were inserted into plasmid pSG703 that contains the lacZ-cat expression cartridge. The lysogenic B. subtilis (phi105 MU331) was transformed with the resultant recombinant plasmids, and the heterologous genes were thereby integrated into the chromosommal DNA of B. subtilis via homologous recombination. The transformants were designated as B. subtilis AMY1, B. subtilis AMY2, B. subtilis PA1, and B. subtilis PA2, respectively. In the recombinant B. subtilis strains, the inserted sequences were located down stream of a strong phage promoter that could be activated by thermal induction. In B. subtilis AMY1 and B. subtilis PA1, transcription of the heterologous genes was only initiated by the phage promoter after heat shock, whereas in B. subtilis AMY2 and B. subtilis PA2, transcription of the heterologous genes was initiated by dual promoters, the phage promoter and the native promoter. The application of dual promoters increased the productivity of both enzymes, with 133% enhancement for alpha-amylase production and 113% enhancement for penicillin acylase production. PMID:16607942

  5. Enhanced dewatering of waste-activated sludge by composite hydrolysis enzymes.

    PubMed

    Wu, Boran; Chai, Xiaoli; Zhao, Youcai

    2016-04-01

    The feasibility of composite hydrolysis enzymes in enhanced dewatering of waste-activated sludge (WAS) was verified in this study. A Pearson correlation analysis was conducted to explore the roles of different extracellular polymeric substance (EPS) fractions on WAS dewaterability. The results indicated that tightly bound EPS (TB-EPS) was released into the liquid phase consistently during enzymatic hydrolysis to form soluble EPS (S-EPS) and loosely bound EPS and that the TB-EPS content was positively correlated with the capillary suction time of WAS. A kinetic analysis was carried out to gain further insights into the kinetic variation in TB-EPS removal. It was found that TB-EPS reduction fit a first-order kinetic model and that mild temperature (25-30 °C) and a slightly acidic condition were favorable for the improvement of enzyme activity. Solid phase extraction combined with UV-Vis spectroscopy analysis was used to characterize the processes of migration and transformation of the hydrophobic (HPO), transphilic and hydrophilic (HPI) fractions in EPS during the enzymatic process. The results revealed that HPO and HPI were mainly composed of PN and PS, respectively, and that the enzymatic hydrolysis could enhance the transformation of HPI from TB-EPS to S-EPS, which was the dominant mechanism of improving WAS dewaterability. PMID:26815556

  6. Hematopoietic Stem Cell Regeneration Enhanced by Ectopic Expression of ROS-detoxifying Enzymes in Transplant Mice

    PubMed Central

    Miao, Weimin; XuFeng, Richard; Park, Moo-Rim; Gu, Haihui; Hu, Linping; Kang, Jin Wook; Ma, Shihui; Liang, Paulina H; Li, Yanxin; Cheng, Haizi; Yu, Hui; Epperly, Michael; Greenberger, Joel; Cheng, Tao

    2013-01-01

    High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo. PMID:23295952

  7. Hematopoietic stem cell regeneration enhanced by ectopic expression of ROS-detoxifying enzymes in transplant mice.

    PubMed

    Miao, Weimin; Xufeng, Richard; Park, Moo-Rim; Gu, Haihui; Hu, Linping; Kang, Jin Wook; Ma, Shihui; Liang, Paulina H; Li, Yanxin; Cheng, Haizi; Yu, Hui; Epperly, Michael; Greenberger, Joel; Cheng, Tao

    2013-02-01

    High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo. PMID:23295952

  8. Reporter-encapsulated liposomes on graphene field effect transistors for signal enhanced detection of physiological enzymes.

    PubMed

    Chen, Hu; Lim, Seng Koon; Chen, Peng; Huang, Jingfeng; Wang, Yi; Palaniappan, Alagappan; Platt, Mark; Liedberg, Bo; Tok, Alfred Iing Yoong

    2015-02-01

    A novel approach for enzymatic assay using reporter-encapsulated liposomes on graphene field effect transistors (FET) is proposed. This approach involves real time monitoring of drain current (Id) of reduced graphene oxide (rGO) upon rupture of reporter-encapsulated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes triggered by enzymes. For validation of the proposed approach, 2,4,6-trinitrophenol (TNP) is used as the reporter for specific detection of phospholipase A2 (PLA2), a key enzyme in various membrane related physiological processes. Experimental results revealed that Id increased with PLA2 concentration, which is attributed to the interaction between released TNP and rGO. The limit of detection (LOD) achieved by the proposed approach was 80 pM, which is superior to most assays reported previously and much lower than the cut-off level of circulating secretory PLA2 (2.07 nM). Besides the high accuracy of the electronic detection methodology, the signal enhancement effect realized by the excess concentration of TNP (approximately 1 mM) in liposomes is believed to be the main reason for the significantly enhanced sensitivity of the proposed assay, indicating great potential for further improvement in the sensitivity by increasing the concentration of TNP. In addition, the proposed approach is rapid (incubation time ≤ 10 min) and label-free, thus showing great potential for practical applications in the future. PMID:25531209

  9. A GHF7 cellulase from the protist symbiont community of Reticulitermes flavipes enables more efficient lignocellulose processing by host enzymes.

    PubMed

    Sethi, Amit; Kovaleva, Elena S; Slack, Jeffrey M; Brown, Susan; Buchman, George W; Scharf, Michael E

    2013-12-01

    Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7-3, GHF7-5, and GHF7-6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full-length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus-insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7-3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and β-glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell-1), beta-glucosidase (β-glu), and laccase (LacA). GHF7-3 was the only GHF7 to enhance glucose release by Cell-1 and β-glu. Finally, GHF7-3, Cell-1, and β-glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only β-glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7-3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases. PMID:24186432

  10. Comparative study on the conventional and non thermal simultaneous saccharification and fermentation of Manihot glaziovii root starch

    NASA Astrophysics Data System (ADS)

    Hargono, Kumoro, Andri Cahyo; Jos, Bakti

    2015-12-01

    Inconventional ethanol production process, starch is converted into dextrins via liquefaction using α-amylase enzyme at high temperature (90-120°C). Then, dextrins are saccharified by glucoamylase to obtain to monomeric sugars (glucose). Recently, a granular starch hydrolyzing enzymes (GSHE), Stargen 002, was developed to convert starch into dextrins at low temperature (< 32°C) and hydrolyzes dextrins into glucose. The subject of this research was to compare ethanol production using a granular starch hydrolyzing enzymes and conventional enzymatic liquefaction and saccharification in cassava starch processing. Starch slurry concentrations were 20% w/v, and dosage of enzymes 0.50, 1.0 and 2%, respectively, were studied. After 48 hr process the final ethanol concentration for the respective enzyme concentration for conventional process were 34.90, 36.16 and 42.10 g/L, whereas for the non-thermal treatment, final ethanol concentration were 46.4, 57.62 and 59.65 g/L, respectively. By implementation of this non thermal process, the use of energy can be saved by carrying out saccharification step at lower temperature (30°C) could be realized.

  11. Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance.

    PubMed

    Sun, Baodong; Bird, Andrew; Young, Sarah P; Kishnani, Priya S; Chen, Y-T; Koeberl, Dwight D

    2007-11-01

    Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid alpha -glucosidase (GAA) (also called "acid maltase"), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects' overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund's adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector-mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases. PMID:17924344

  12. Enhanced Response to Enzyme Replacement Therapy in Pompe Disease after the Induction of Immune Tolerance

    PubMed Central

    Sun, Baodong ; Bird, Andrew ; Young, Sarah P. ; Kishnani, Priya S. ; Chen, Y.-T. ; Koeberl, Dwight D. 

    2007-01-01

    Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid α-glucosidase (GAA) (also called “acid maltase”), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects’ overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund’s adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector–mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases. PMID:17924344

  13. Enhancement of fruit shelf life by suppressing N-glycan processing enzymes

    PubMed Central

    Meli, Vijaykumar S.; Ghosh, Sumit; Prabha, T. N.; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2010-01-01

    In a globalized economy, the control of fruit ripening is of strategic importance because excessive softening limits shelf life. Efforts have been made to reduce fruit softening in transgenic tomato through the suppression of genes encoding cell wall–degrading proteins. However, these have met with very limited success. N-glycans are reported to play an important role during fruit ripening, although the role of any particular enzyme is yet unknown. We have identified and targeted two ripening-specific N-glycoprotein modifying enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex). We show that their suppression enhances fruit shelf life, owing to the reduced rate of softening. Analysis of transgenic tomatoes revealed ≈2.5- and ≈2-fold firmer fruits in the α-Man and β-Hex RNAi lines, respectively, and ≈30 days of enhanced shelf life. Overexpression of α-Man or β-Hex resulted in excessive fruit softening. Expression of α-Man and β-Hex is induced by the ripening hormone ethylene and is modulated by a regulator of ripening, rin (ripening inhibitor). Furthermore, transcriptomic comparative studies demonstrate the down-regulation of cell wall degradation- and ripening-related genes in RNAi fruits. It is evident from these results that N-glycan processing is involved in ripening-associated fruit softening. Genetic manipulation of N-glycan processing can be of strategic importance to enhance fruit shelf life, without any negative effect on phenotype, including yield. PMID:20133661

  14. Correlation analysis of enzyme activities and deconstruction of ammonia-pretreated switchgrass by bacterial-fungal communities.

    PubMed

    Jain, Abhiney; Bediako, Sandra H; Henson, J Michael

    2016-10-01

    The mixed microbial communities that occur naturally on lignocellulosic feedstocks can provide feedstock-specific enzyme mixtures to saccharify lignocelluloses. Bacterial-fungal communities were enriched from switchgrass bales to deconstruct ammonia-pretreated switchgrass (DSG). Correlation analysis was carried out to elucidate the relationship between microbial decomposition of DSG by these communities, enzymatic activities produced and enzymatic saccharification of DSG using these enzyme mixtures. Results of the analysis showed that β-glucosidase and xylosidase activities limited the extent of microbial deconstruction and enzymatic saccharification of DSG. The results also underlined the importance of ligninase activity for the enzymatic saccharification of pretreated lignocellulosic feedstock. The bacterial-fungal communities developed in this research can be used to produce enzyme mixtures to deconstruct DSG, and the results from the correlation analysis can be used to optimize these enzyme mixtures for efficient saccharification of DSG to produce second-generation biofuels. PMID:27469088

  15. Enhanced competitive chemiluminescent enzyme immunoassay for the trace detection of insecticide triazophos.

    PubMed

    Jin, Maojun; Shao, Hua; Jin, Fen; Gui, Wenjun; Shi, Xiaomei; Wang, Jing; Zhu, Guonian

    2012-05-01

    A direct competitive chemiluminescent enzyme immunoassay (CLEIA) for triazophos was developed, which was based on the anti-THHe IgG monoclonal antibody and a heterogeneous enzyme tracer (THHu-HRP). Several components of chemiluminescent enhanced solution (CES) were optimized. The results showed that 1 mM of p-iodo-phenol, 0.625 mM of luminol, and 4 mM of H(2)O(2) had the best performance. Based on the study of CES, the influence of several factors (assay buffer, blocking substance, and solvent) on the immunoassay was investigated. The sensitivity for detection, IC(50) value was 0.87 ng/mL at a practical working concentration range between 0.04 ng/mL and 5 ng/mL and the limit of detection for triazophos was 0.063 ng/mL. The average recovery of triazophos added to lettuce, carrot, apple, water, and soil were 78.71%, 67.52%, 118.3%, 117.2%, and 122.0%, respectively. Finally, comparison between the methods of CLEIA and high-performance liquid chromatography-tandem mass spectrum (HPLC-MS/MS) was performed. The results obtained from CLEIA were in agreement with those of HPLC-MS/MS. PMID:22490114

  16. A sensitive surface-enhanced Raman scattering enzyme-catalyzed immunoassay of respiratory syncytial virus.

    PubMed

    Zhan, Lei; Zhen, Shu Jun; Wan, Xiao Yan; Gao, Peng Fei; Huang, Cheng Zhi

    2016-02-01

    Respiratory viruses have become a major global health challenge which would benefit from advances in screening methods for early diagnosis. Respiratory syncytial virus (RSV) is one of the most important pathogen causing severe lower respiratory tract infections. Here we present a novel surface-enhanced Raman scattering (SERS) enzyme-catalyzed immunoassay of RSV by employing peroxidase substrate 3, 3'-5, 5'-tetramethylbenzidine (TMB) as Raman molecule. Horseradish peroxidase (HRP) attached to the detection antibody in a novel sandwich immunoassay catalyzes the oxidation of TMB by H2O2 to give a radical cation (TMB(+)), which could be easily adsorbed on the negatively charged surface of silver nanoparticles (AgNPs) through electrostatic interaction, inducing the aggregation of AgNPs and thus giving a strong SERS signal. A linear relationship was obtained between the Raman intensity and the amount of RSV in the range from 0.5 to 20 pg/mL, and the minimum detectable concentration of this SERS-based enzyme immunoassay was 0.05 pg/mL, which was 20 times lower than that found in the colorimetric method. PMID:26653454

  17. Potential chemoprevention activity of pterostilbene by enhancing the detoxifying enzymes in the HT-29 cell line.

    PubMed

    Harun, Zaliha; Ghazali, Ahmad Rohi

    2012-01-01

    Detoxifying enzymes are present in most epithelial cells of the human gastrointestinal tract where they protect against xenobiotics which may cause cancer. Induction of examples such as glutathione S-transferase (GST) and its thiol conjugate, glutathione (GSH) as well as NAD(P)H: quinoneoxidoreductase (NQO1) facilitate the excretion of carcinogens and thus preventing colon carcinogenesis. Pterostilbene, an analogue of resveratrol, has demonstrated numerous pharmacological activities linked with chemoprevention. This study was conducted to investigate the potential of pterostilbene as a chemopreventive agent using the HT-29 colon cancer cell line to study the modulation of GST and NQO1 activities as well as the GSH level. Initially, our group, established the optimum dose of 24 hours pterostilbene treatment using MTT assays. Then, effects of pterostilbene (0-50 μM) on GST and NQO1 activity and GSH levels were determined using GST, NQO1 and Ellman assays, respectively. MTT assay of pterostilbene (0-100 μM) showed no cytotoxicity toward the HT-29 cell line. Treatment increased GST activity in the cell line significantly (p<0.05) at 12.5 and 25.0 μM. In addition, treatment at 50 μM increased the GSH level significantly (p<0.05). Pterostilbene also enhanced NQO1 activity significantly (p<0.05) at 12.5 μM and 50 μM. Hence, pterostilbene is a potential chemopreventive agent capable of modulation of detoxifiying enzyme levels in HT-29 cells. PMID:23464466

  18. Rational enhancement of enzyme performance in organic solvents. Final technical report, 1992--1996

    SciTech Connect

    Klibanov, A.M.

    1996-12-31

    This research focused on the following: the dependence of enzymatic activity of several model hydrolases in nonaqueous solvents; control of substrate selectivity of the protease subtilisin Carlsberg by the solvent; control of catalytic activity and enantioselectivity of this enzyme in organic solvents by immobilization support; lipase-catalyzed acylation of sugars in anhydrous hydrophobic media; the possibility of accelerating enzymatic processes in organic solvents by certain cosolvents; whether lipase catalysis in organic solvents can be enhanced by introducing interfaces in the in the reaction medium; the structure of proteins suspended in organic solvents; improving enzymatic enantioselectivity in organic solvents; analyzing the plunge in enzymatic activity upon replacing water with organic solvents; and the structural basis for the phenomenon of molecular memory of imprinted proteins in organic solvents.

  19. Enhanced Transformation of TNT by Arabidopsis Plants Expressing an Old Yellow Enzyme

    PubMed Central

    Zhu, Bo; Peng, Ri-He; Fu, Xiao-Yan; Jin, Xiao-Fen; Zhao, Wei; Xu, Jing; Han, Hong-Juan; Gao, Jian-Jie; Xu, Zhi-Sheng; Bian, Lin; Yao, Quan-Hong

    2012-01-01

    2,4,6-Trinitrotoluene (TNT) is released in nature from manufacturing or demilitarization facilities, as well as after the firing or detonation of munitions or leakage from explosive remnants of war. Environmental contamination by TNT is associated with human health risks, necessitating the development of cost-effective remediation techniques. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. In this study, we present a system for TNT phytoremediation by overexpressing the old yellow enzyme (OYE3) gene from Saccharomyces cerevisiae. The resulting transgenic Arabidopsis plants demonstrated significantly enhanced TNT tolerances and a strikingly higher capacity to remove TNT from their media. The current work indicates that S. cerevisiae OYE3 overexpression in Arabidopsis is an efficient method for the phytoremoval and degradation of TNT. Our findings have the potential to provide a suitable remediation strategy for sites contaminated by TNT. PMID:22808068

  20. Catecholamines enhance dihydrolipoamide dehydrogenase inactivation by the copper Fenton system. Enzyme protection by copper chelators.

    PubMed

    Correa, J G; Stoppani, A O

    1996-04-01

    Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) and o-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II)/H2O2 (Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H2O2 and CA concentrations. Similar inhibitions were obtained with the assayed CAs and o-diphenols. CAs enhanced HO. radical production by Cu(II)/H2O2, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipoamide, dihydrolipoic acid, DL-dithiothreitol, GSSG, trypanothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H2O2/CAs systems. Denatured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH increased and Captopril inhibited epinephrine oxidation by Cu(II)/H2O2 and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO. from H2O2 by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the enzyme active site by site-specifically generated HO. radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation. PMID:8731015

  1. Saccharification and fermentation of sugar cane bagasse by Klebsiella oxytoca P2 containing chromosomally integrated genes encoding the Zymomonas mobilis ethanol pathway

    SciTech Connect

    Doran, J.B.; Aldrich, H.C.; Ingram, L.O. . Dept. of Microbiology and Cell Science)

    1994-06-20

    Pretreatment of sugar cane bagasse is essential for a simultaneous saccharification and fermentation (SSF) process which uses recombinant Klebsiella oxytoca strain P2 and Genencor Spezyme CE. Strain P2 has been genetically engineered to express Zymomonas mobilis genes encoding the ethanol pathway and retains the native ability to transport and metabolize cellobiose (minimizing the need for extracellular cellobiase). In SSF studies with this organism, both the rate of ethanol production and ethanol yield were limited by saccharification at 10 and 20 filter paper units (FPU) g[sup [minus]1] acid-treated bagasse. Dilute slurries of biomass were converted to ethanol more efficiently (over 72% of theoretical yield) in simple batch fermentations than slurries containing high solids, albeit with the production of lower levels of ethanol. With high solids (i.e., 160 g acid-treated bagasse L[sup [minus]1]), a combination of 20 FPU cellulase g[sup [minus]1] bagasse, preincubation under saccharification conditions, and additional grinding (to reduce particle size) were required to produce ca. 40 g ethanol L[sup [minus]1]. Alternatively, almost 40 g ethanol L[sup [minus]1] was produced with 10 FPU cellulase g[sup [minus]1] bagasse by incorporating a second saccharification step (no further enzyme addition) followed by a second inoculation and short fermentation. In this way, a theoretical ethanol yield of over 70% was achieved with the production of 20 g ethanol 800 FPU[sup [minus]1] of commercial cellulase.

  2. Podospora anserina Hemicellulases Potentiate the Trichoderma reesei Secretome for Saccharification of Lignocellulosic Biomass▿

    PubMed Central

    Couturier, Marie; Haon, Mireille; Coutinho, Pedro M.; Henrissat, Bernard; Lesage-Meessen, Laurence; Berrin, Jean-Guy

    2011-01-01

    To improve the enzymatic hydrolysis (saccharification) of lignocellulosic biomass by Trichoderma reesei, a set of genes encoding putative polysaccharide-degrading enzymes were selected from the coprophilic fungus Podospora anserina using comparative genomics. Five hemicellulase-encoding genes were successfully cloned and expressed as secreted functional proteins in the yeast Pichia pastoris. These novel fungal CAZymes belonging to different glycoside hydrolase families (PaMan5A and PaMan26A mannanases, PaXyn11A xylanase, and PaAbf51A and PaAbf62A arabinofuranosidases) were able to break down their predicted cognate substrates. Although PaMan5A and PaMan26A displayed similar specificities toward a range of mannan substrates, they differed in their end products, suggesting differences in substrate binding. The N-terminal CBM35 module of PaMan26A displayed dual binding specificity toward xylan and mannan. PaXyn11A harboring a C-terminal CBM1 module efficiently degraded wheat arabinoxylan, releasing mainly xylobiose as end product. PaAbf51A and PaAbf62A arabinose-debranching enzymes exhibited differences in activity toward arabinose-containing substrates. Further investigation of the contribution made by each P. anserina auxiliary enzyme to the saccharification of wheat straw and spruce demonstrated that the endo-acting hemicellulases (PaXyn11A, PaMan5A, and PaMan26A) individually supplemented the secretome of the industrial T. reesei CL847 strain. The most striking effect was obtained with PaMan5A that improved the release of total sugars by 28% and of glucose by 18%, using spruce as lignocellulosic substrate. PMID:21037302

  3. Continuous enzymatic liquefaction of starch for saccharification

    SciTech Connect

    Carr, M.E.; Black, L.T.; Bagby, M.O.

    1982-01-01

    A process was explored for continuous enzymatic liquefaction of corn starch at high concentration and subsequent saccharification to glucose. The process appears to be quite efficient for conversion of starch to glucose and enzymatic liquefaction and should be readily adaptable to industrial fermentation processes. Preliminary work indicated that milled corn or other cereal grains also can be suitably converted by such a process. Essentially, the process involved incorporation of a thermostable, bacterial alpha-amylase for liquefaction and, subsequently, of a glucoamylase into the continuous mixer under conditions conductive to rapid enzymatic hydrolyses. Also studied was the effect on substrate liquefaction of variables such as starch concentration (40-70%), level of alpha-amylase (0.14-0.4%, dry starch basis), temperature (70-100 degrees C), pH (5.8-7.1), and residence time (6 and 12 minutes). The degree of liquefaction was assessed by determining 1) the Brookfield viscosity, 2) the amount of reducing groups, and 3) the rate and extent of glucose formed after glucoamylase treatment. Best liquefaction processing conditions were achieved by using 50-60% starch concentration, at 95 degrees C, with 0.4% alpha-amylase, and a 6 minute residence period in the mixer. Under these conditions, rates and extents of glucose obtained after glucoamylase treatment approached those obtained in longer laboratory batch liquefactions. The amount of glucose formed in 24 hours with the use of 0.4% glucoamylase was 86% of theory after a 6-min continuous liquefaction, compared to 90% for a 30-min laboratory batch liquefaction (95 degrees C, 0.4% alpha-amylase). (Refs. 15).

  4. Self-assembly of amphiphilic janus particles into monolayer capsules for enhanced enzyme catalysis in organic media.

    PubMed

    Cao, Wei; Huang, Renliang; Qi, Wei; Su, Rongxin; He, Zhimin

    2015-01-14

    Encapsulation of enzymes during the creation of an emulsion is a simple and efficient route for enhancing enzyme catalysis in organic media. Herein, we report a capsule with a shell comprising a monolayer of silica Janus particles (JPs) (referred to as a monolayer capsule) and a Pickering emulsion for the encapsulation of enzyme molecules for catalysis purposes in organic media using amphiphilic silica JPs as building blocks. We demonstrate that the JP capsules had a monolayer shell consisting of closely packed silica JPs (270 nm). The capsules were on average 5-50 μm in diameter. The stability of the JP capsules (Pickering emulsion) was investigated with the use of homogeneous silica nanoparticles as a control. The results show that the emulsion stabilized via amphiphilic silica JPs presented no obvious changes in physical appearance after 15 days, indicating the high stability of the emulsions and JP capsules. Furthermore, the lipase from Candida sp. was chosen as a model enzyme for encapsulation within the JP capsules during their formation. The catalytic performance of lipase was evaluated according to the esterification of 1-hexanol with hexanoic acid. It was found that the specific activity of the encapsulated enzymes (28.7 U mL(-1)) was more than 5.6 times higher than that of free enzymes in a biphasic system (5.1 U mL(-1)). The enzyme activity was further increased by varying the volume ratio of water to oil and the JPs loadings. The enzyme-loaded capsule also exhibited high stability during the reaction process and good recyclability. In particular, the jellification of agarose in the JP capsules further enhanced their operating stability. We believe that the monolayer structure of the JP capsules, together with their high stability, rendered the capsules to be ideal enzyme carriers and microreactors for enzyme catalysis in organic media because they created a large interfacial area and had low mass transfer resistance through the monolayer shell. PMID

  5. Fungal delignification of lignocellulosic biomass improves the saccharification of cellulosics.

    PubMed

    Gupta, Rishi; Mehta, Girija; Khasa, Yogender Pal; Kuhad, Ramesh Chander

    2011-07-01

    The biological delignification of lignocellulosic feedstocks, Prosopis juliflora and Lantana camara was carried out with Pycnoporus cinnabarinus, a white rot fungus, at different scales under solid-state fermentation (SSF) and the fungal treated substrates were evaluated for their acid and enzymatic saccharification. The fungal fermentation at 10.0 g substrate level optimally delignified the P. juliflora by 11.89% and L. camara by 8.36%, and enriched their holocellulose content by 3.32 and 4.87%, respectively, after 15 days. The fungal delignification when scaled up from 10.0 g to 75.0, 200.0 and 500.0 g substrate level, the fungus degraded about 7.69-10.08% lignin in P. juliflora and 6.89-7.31% in L. camara, and eventually enhanced the holocellulose content by 2.90-3.97 and 4.25-4.61%, respectively. Furthermore, when the fungal fermented L. camara and P. juliflora was hydrolysed with dilute sulphuric acid, the sugar release was increased by 21.4-42.4% and the phenolics content in hydrolysate was decreased by 18.46 and 19.88%, as compared to the unfermented substrate acid hydrolysis, respectively. The reduction of phenolics in acid hydrolysates of fungal treated substrates decreased the amount of detoxifying material (activated charcoal) by 25.0-33.0% as compared to the amount required to reduce almost the same level of phenolics from unfermented substrate hydrolysates. Moreover, an increment of 21.1-25.1% sugar release was obtained when fungal treated substrates were enzymatically hydrolysed as compared to the hydrolysis of unfermented substrates. This study clearly shows that fungal delignification holds potential in utilizing plant residues for the production of sugars and biofuels. PMID:20711746

  6. Carbon dioxide enhances the development of the ethylene forming enzyme in tobacco leaf discs

    SciTech Connect

    Philosoph-Hadas, S.; Aharoni, N.; Yang, S.F.

    1986-01-01

    Since CO/sub 2/ is known to stimulate ethylene production by promoting the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, the effect of CO/sub 2/ on the activity and the development of the ethylene forming enzyme (EFE) was studied in tobacco (Nicotiana tabacum L. cv Havana 425 and Xanthi) leaf discs. In addition to previous observations that EFE activity is dependent on CO/sub 2/ concentration and is saturable with 2% CO/sub 2/, present data show two saturation curves at 2% and 10% CO/sub 2/. Promotion of EFE development was dependent also on CO/sub 2/ concentration (saturated at 2% CO/sub 2/) and duration (maximum at 24 in the dark), and was abolished by 20 micromolar cycloheximide. Application of exogenous ethylene (20 microliters per liter) or light treatment further increased the CO/sub 2/-enhanced development of EFE, implying that these two factors can also affect EFE development via interaction with CO/sub 2/. The results suggest that CO/sub 2/ exerts its stimulatory effect on the conversion of ACC to ethylene by enhancing not only the activity but also the synthesis of EFE in leaf discs.

  7. How cell wall complexity influences saccharification efficiency in Miscanthus sinensis.

    PubMed

    De Souza, Amanda P; Alvim Kamei, Claire L; Torres, Andres F; Pattathil, Sivakumar; Hahn, Michael G; Trindade, Luisa M; Buckeridge, Marcos S

    2015-07-01

    The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis. PMID:25908240

  8. Methods and compositions for simultaneous saccharification and fermentation

    DOEpatents

    Ingram, Lonnie O'Neal; Zhou, Shengde

    2006-04-11

    The invention provides compositions and methods for the synergistic degradation of oligosaccharides by endoglucanases. The invention further provides recombinant host cells containing one or more genes encoding endoglucanses which are capable of the synergistic degradation of oligosaccharides. Preferred host cells of the invention are ethanologenic and capable of carrying out simultaneous saccharification and fermentation resulting in the production of ethanol from complex cellulose substrates.

  9. Linkage Mapping of Stem Saccharification Digestibility in Rice

    PubMed Central

    Hua, Cangmei; Sun, Lili; Ali, Imran; Huang, Linli; Yu, Chunyan; Simister, Rachael; Steele-King, Clare; Gan, Yinbo; McQueen-Mason, Simon J.

    2016-01-01

    Rice is the staple food of almost half of the world population, and in excess 90% of it is grown and consumed in Asia, but the disposal of rice straw poses a problem for farmers, who often burn it in the fields, causing health and environmental problems. However, with increased focus on the development of sustainable biofuel production, rice straw has been recognized as a potential feedstock for non-food derived biofuel production. Currently, the commercial realization of rice as a biofuel feedstock is constrained by the high cost of industrial saccharification processes needed to release sugar for fermentation. This study is focused on the alteration of lignin content, and cell wall chemotypes and structures, and their effects on the saccharification potential of rice lignocellulosic biomass. A recombinant inbred lines (RILs) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 271 molecular markers for quantitative trait SNP (QTS) analyses was used. After association analysis of 271 markers for saccharification potential, 1 locus and 4 pairs of epistatic loci were found to contribute to the enzymatic digestibility phenotype, and an inverse relationship between reducing sugar and lignin content in these recombinant inbred lines was identified. As a result of QTS analyses, several cell-wall associated candidate genes are proposed that may be useful for marker-assisted breeding and may aid breeders to produce potential high saccharification rice varieties. PMID:27415441

  10. How cell wall complexity influences saccharification efficiency in Miscanthus sinensis

    PubMed Central

    De Souza, Amanda P.; Kamei, Claire L. Alvim; Torres, Andres F.; Pattathil, Sivakumar; Hahn, Michael G.; Trindade, Luisa M.; Buckeridge, Marcos S.

    2015-01-01

    The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis. PMID:25908240

  11. Linkage Mapping of Stem Saccharification Digestibility in Rice.

    PubMed

    Liu, Bohan; Gómez, Leonardo D; Hua, Cangmei; Sun, Lili; Ali, Imran; Huang, Linli; Yu, Chunyan; Simister, Rachael; Steele-King, Clare; Gan, Yinbo; McQueen-Mason, Simon J

    2016-01-01

    Rice is the staple food of almost half of the world population, and in excess 90% of it is grown and consumed in Asia, but the disposal of rice straw poses a problem for farmers, who often burn it in the fields, causing health and environmental problems. However, with increased focus on the development of sustainable biofuel production, rice straw has been recognized as a potential feedstock for non-food derived biofuel production. Currently, the commercial realization of rice as a biofuel feedstock is constrained by the high cost of industrial saccharification processes needed to release sugar for fermentation. This study is focused on the alteration of lignin content, and cell wall chemotypes and structures, and their effects on the saccharification potential of rice lignocellulosic biomass. A recombinant inbred lines (RILs) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 271 molecular markers for quantitative trait SNP (QTS) analyses was used. After association analysis of 271 markers for saccharification potential, 1 locus and 4 pairs of epistatic loci were found to contribute to the enzymatic digestibility phenotype, and an inverse relationship between reducing sugar and lignin content in these recombinant inbred lines was identified. As a result of QTS analyses, several cell-wall associated candidate genes are proposed that may be useful for marker-assisted breeding and may aid breeders to produce potential high saccharification rice varieties. PMID:27415441

  12. Genetically engineered immunomodulatory Streptococcus thermophilus strains producing antioxidant enzymes exhibit enhanced anti-inflammatory activities.

    PubMed

    Del Carmen, Silvina; de Moreno de LeBlanc, Alejandra; Martin, Rebeca; Chain, Florian; Langella, Philippe; Bermúdez-Humarán, Luis G; LeBlanc, Jean Guy

    2014-02-01

    The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti

  13. Dual Inhibition of Endocannabinoid Catabolic Enzymes Produces Enhanced Antiwithdrawal Effects in Morphine-Dependent Mice

    PubMed Central

    Ramesh, Divya; Gamage, Thomas F; Vanuytsel, Tim; Owens, Robert A; Abdullah, Rehab A; Niphakis, Micah J; Shea-Donohue, Terez; Cravatt, Benjamin F; Lichtman, Aron H

    2013-01-01

    Inhibition of the endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH) attenuates naloxone-precipitated opioid withdrawal signs in mice via activation of CB1 receptors. Complete FAAH inhibition blocks only a subset of withdrawal signs, whereas complete MAGL inhibition elicits enhanced antiwithdrawal efficacy, but is accompanied with some cannabimimetic side effects. Thus, the primary objective of the present study was to determine whether combined, full FAAH inhibition and partial MAGL represents an optimal strategy to reduce opioid withdrawal. To test this hypothesis, we examined whether combined administration of high-dose of the FAAH inhibitor PF-3845 and low-dose of the MAGL inhibitor JZL184, as well as the novel dual FAAH-MAGL inhibitor SA-57, which is 100-fold more potent in inhibiting FAAH than MAGL, would prevent spontaneous withdrawal in morphine-dependent mice, a model with greater face validity than precipitating withdrawal with μ-opioid receptor antagonists. Strikingly, a combination of low-dose JZL184 and high-dose PF-3845 as well as the dual inhibitor SA-57 reduced all abrupt withdrawal signs (ie, platform jumping, paw flutters, head shakes, diarrhea, and total body weight loss), but did not elicit any cannabimimetic side effects. In addition, JZL184 or PF-3845 blocked naloxone-precipitated hypersecretion in morphine-dependent small intestinal tissue. Collectively, these results are the first to show that endocannabinoid catabolic enzyme inhibitors reduce abrupt withdrawal in morpine-dependent mice and are effective in a novel in vitro model of opioid withdrawal. More generally, these findings support the idea that joint MAGL and FAAH inhibition represents a promising approach for the treatment of opioid dependence. PMID:23303065

  14. Use of exogenous fibrolytic enzymes to enhance in vitro fermentation of alfalfa hay and corn silage.

    PubMed

    Eun, J-S; Beauchemin, K A; Schulze, H

    2007-03-01

    Two in vitro experiments were performed to identify promising exogenous fibrolytic enzyme products (EFE) and optimum dose rates (DR) for improving the degradation of alfalfa hay and corn silage. The relationship between enzymatic activity and fermentation responses was examined to identify optimum formulations. In experiment 1, 5 EFE containing mainly endoglucanase and xylanase activities, with different ratios between the 2 activities, were assessed at a DR of 0.7, 1.4, and 2.1 mg/g of DM forage. Milled alfalfa hay or corn silage was incubated in an in vitro batch culture with buffer, ruminal fluid, and EFE. Gas production (GP) was measured during 24 h of incubation, and degradabilities of DM and fiber were measured after terminating the incubation at 24 h. Two (E1 and E3) EFE substantially improved GP and degradation of alfalfa hay and corn silage fiber. The optimum DR of these EFE was 1.4 mg/g of DM for both forages with improvements in NDF degradability up to 20.6% for alfalfa hay and up to 60.3% for corn silage. Whereas added activities of endoglucanase and exoglucanase were positively correlated with improvement in NDF degradability for alfalfa hay and corn silage, there was no relationship between added xylanase activity and NDF degradability. The 2 most promising EFE from experiment 1 were reevaluated in experiment 2, alone and in combination with a high xylanase EFE, to determine whether their effectiveness could be enhanced by decreasing the endoglucanase to xylanase ratio. The 2 EFE improved GP and fiber degradation in a manner similar to that observed in experiment 1, but the combination treatments resulted in no further beneficial effects. Exogenous fibrolytic enzyme products can greatly improve forage utilization, but DR and the activities supplied are critical for achieving this response. Products used with alfalfa hay and corn silage should contain high endoglucanase activity, with an ideal ratio of endoglucanse to xylanase. PMID:17297117

  15. Genetically Engineered Immunomodulatory Streptococcus thermophilus Strains Producing Antioxidant Enzymes Exhibit Enhanced Anti-Inflammatory Activities

    PubMed Central

    del Carmen, Silvina; de Moreno de LeBlanc, Alejandra; Martin, Rebeca; Chain, Florian; Langella, Philippe; Bermúdez-Humarán, Luis G.

    2014-01-01

    The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti

  16. Co-solvent pretreatment reduces costly enzyme requirements for high sugar and ethanol yields from lignocellulosic biomass.

    PubMed

    Nguyen, Thanh Yen; Cai, Charles M; Kumar, Rajeev; Wyman, Charles E

    2015-05-22

    We introduce a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) to reduce enzyme costs dramatically for high sugar yields from hemicellulose and cellulose, which is essential for the low-cost conversion of biomass to fuels. CELF employs THF miscible with aqueous dilute acid to obtain up to 95 % theoretical yield of glucose, xylose, and arabinose from corn stover even if coupled with enzymatic hydrolysis at only 2 mgenzyme  gglucan (-1) . The unusually high saccharification with such low enzyme loadings can be attributed to a very high lignin removal, which is supported by compositional analysis, fractal kinetic modeling, and SEM imaging. Subsequently, nearly pure lignin product can be precipitated by the evaporation of volatile THF for recovery and recycling. Simultaneous saccharification and fermentation of CELF-pretreated solids with low enzyme loadings and Saccharomyces cerevisiae produced twice as much ethanol as that from dilute-acid-pretreated solids if both were optimized for corn stover. PMID:25677100

  17. Electron beam irradiation pretreatment and enzymatic saccharification of used newsprint and paper mill wastes

    NASA Astrophysics Data System (ADS)

    Waheed Khan, A.; Labrie, Jean-Pierre; McKeown, Joseph

    Electron beam pretreatment of used newsprint, pulp, as well as pulp recovered from clarifier sludge and paper mill sludge, caused the dissociation of cellulose from lignin, and rendered them suitable for enzymatic hydrolysis. A maximum dose of 1 MGy for newsprint and 1.5—2.0 MGy for pulp and paper mill sludge was required to render cellulose present in them in a form which, could be enzymatically saccharified to 90% of completion. Saccharification approaching the theoretical yield was obtained in 2 days with a cellulolytic enzyme system obtained from Trichoderma reesei. As a result of irradiation, water soluble lignin breakdown products, NaOH- soluble lignin, free cellobiose, glucose, mannose, xylose and their polymers, and acetic acid were produced from these materials.

  18. Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in-vitro blood clot lysis potential.

    PubMed

    Narasimhan, Manoj Kumar; Chandrasekaran, Muthukumaran; Rajesh, Mathur

    2015-01-01

    The discovery of plasmin-like microbial fibrinolytic enzymes having high specificity and negligible side effects is crucial for thrombolytic therapy. Herein, we report one such extra-cellular fibrinolytic enzyme producing Bacillus cereus SRM-001 isolated from the blood-laden soil of a chicken dump yard. The potency of the enzyme was established with fibrin plate assay and in-vitro blood clot lysis assay. The shake-flask operating parameters and media composition were optimized for maximizing the productivity of the enzyme. The operating parameters, pH 7, 37°C, 1% inoculum volume and 24 h inoculum age, were found to be the optimum. The levels of media components, corn flour (0.3% w/v), soyabean powder (1.9% w/v) and MnSO4 (11.5 mM) were optimized by statistical analysis using Box-Behnken design derived RSM. This resulted in an almost 1.8 fold increase in fibrinolytic enzyme productivity. The 3D response surface plots showed soyabean powder and MnSO4 to be the key ingredients for enhancing the enzyme productivity, whereas corn flour had a marginal effect. The in-vitro blood clot lysis assay conducted at near physiological pH 7 at 37°C showed the enzyme to be a potential therapeutic thrombolytic agent. PMID:26582284

  19. Enhancement of enzymatic hydrolysis of cellulose by surfactant

    SciTech Connect

    Ooshima, H.; Sakata, M.; Harano, Y.

    1986-01-01

    Effects of surfactants on enzymatic saccharification of cellulose have been studied. Nonionic, amphoteric, and cationic surfactants enhanced the saccharification, while anionic surfactant did not. Cationic and anionic surfactants denatured cellulase in their relatively low concentrations, namely, more than 0.008 and 0.001%, respectively. Using nonionic surfactant Tween 20, which is most effective to the enhancement (e.g., the fractional conversion attained by 72 h saccharification of 5 wt % Avicel in the presence of 0.05 wt % Tween 20 is increased by 35%), actions of surfactant have been examined. As the results, it was suggested that Tween 20 plays an important role in the hydrolysis of crystalline cellulose and that Tween 20 disturbs the adsorption of endoglucanase on cellulose, i.e., varies the adsorption balance of endo- and exoglucanase, resulting in enhancing the reaction. The influence of Tween 20 to the saccharification was found to remain in simultaneous saccharification and fermentation of Avicel.

  20. Modification of PEGylated enzyme with glutaraldehyde can enhance stability while avoiding intermolecular crosslinking†

    PubMed Central

    McShane, M. J.

    2015-01-01

    We demonstrate an enzyme stabilization approach whereby a model enzyme is PEGylated, followed by controlled chemical modification with glutaraldehyde. Using this stabilization strategy, size increases and aggregation due to intermolecular crosslinking are avoided. Immediately following synthesis, the PEGylated enzyme with and without glutaraldehyde modification possessed specific activities of 372.9 ± 20.68 U/mg and 373.9 ± 15.14 U/mg, respectively (vs. 317.7 ± 19.31 U/mg for the native enzyme). The glutaraldehyde-modified PEGylated enzyme retains 73% original activity after 4 weeks at 37 °C (vs. 2% retention for control). PMID:26052433

  1. Enhancing Biosynthesis of a Ginsenoside Precursor by Self-Assembly of Two Key Enzymes in Pichia pastoris.

    PubMed

    Zhao, Chengcheng; Gao, Xin; Liu, Xinbin; Wang, Yong; Yang, Shengli; Wang, Fengqing; Ren, Yuhong

    2016-05-01

    Ginsenosides from the edible and medicinal plant ginseng have demonstrated various pharmacological activities. However, producing ginsenoside efficiently remains a challenge. Engineering metabolic pathways through protein assembly in yeast is a promising way for ginsenoside production. In the biosynthetic pathway of ginsenosides, dammarenediol-II synthase and squalene epoxidase are two key enzymes that determine the production rate of the dammarane-type ginsenoside precursor dammarenediol-II. In this work, a strategy to enhance the biosynthesis of dammarenediol-II in Pichia pastoris was developed by the self-assembly of the two key enzymes via protein-protein interaction. After being modified by interacting proteins, the two enzymes were successfully co-localized, resulting in a 2.1-fold enhancement in dammarenediol-II yields. PMID:27074597

  2. Malbranchea cinnamomea: A thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes.

    PubMed

    Mahajan, Chhavi; Basotra, Neha; Singh, Surender; Di Falco, Marcos; Tsang, Adrian; Chadha, B S

    2016-01-01

    This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. PMID:26476165

  3. Activity, life time and effect of hydrolytic enzymes for enhanced biogas production from sludge anaerobic digestion.

    PubMed

    Odnell, Anna; Recktenwald, Michael; Stensén, Katarina; Jonsson, Bengt-Harald; Karlsson, Martin

    2016-10-15

    As an alternative to energy intensive physical methods, enzymatic treatment of sludge produced at wastewater treatment plants for increased hydrolysis and biogas production was investigated. Several hydrolytic enzymes were assessed with a focus on how enzyme activity and life time was influenced by sludge environments. It could be concluded that the activity life time of added enzymes was limited (<24 h) in both waste activated sludge and anaerobic digester sludge environments and that this was, for the majority of enzymes, due to endogenous protease activity. In biogas in situ experiments, subtilisin at a 1% mixture on basis of volatile solids, was the only enzyme providing a significantly increased biomethane production of 37%. However, even at this high concentration, subtilisin could not hydrolyze all available substrate within the life time of the enzyme. Thus, for large scale implementation, enzymes better suited to the sludge environments are needed. PMID:27498254

  4. Beijerinckia indica var. penicillanicum penicillin V acylase: enhanced enzyme production by catabolite repression-resistant mutant and effect of solvents on enzyme activity.

    PubMed

    Ambedkar, S S; Deshpande, B S; Sudhakaran, V K; Shewale, J G

    1991-04-01

    Beijerinckia indica var. penicillanicum mutant UREMS-5, producing 168% more penicillin V acylase, was obtained by successive treatment with UV, gamma-irradiation and ethylmethane sulfonate. Penicillin V acylase production by the mutant strain was resistant to catabolite repression by glucose. Incorporation of glucose, sodium glutamate and vegetable oils in the medium enhanced enzyme production. The maximum specific production of penicillin V acylase was 244 IU/g dry weight of cells. Effect of solvents on hydrolysis of penicillin V by soluble penicillin V acylase and whole cells was studied. Methylene chloride, chloroform and carbon tetrachloride significantly stimulated the rate of penicillin V hydrolysis by whole cells. PMID:1367509

  5. Improvement of alpha-L: -arabinofuranosidase production by Talaromyces thermophilus and agro-industrial residues saccharification.

    PubMed

    Guerfali, Mohamed; Chaabouni, Moncef; Gargouri, Ali; Belghith, Hafedh

    2010-02-01

    This study is an application of an experimental design methodology for the optimization of the culture conditions of alpha-L: -arabinofuranosidase production by Talaromyces thermophilus. Wheat bran and yeast extract were first selected as the best carbon and nitrogen sources, respectively, for enzyme production. A Plackett-Burman design was then used to evaluate the effects of eight variables. Statistical analyses showed that while pH had a negative effect on alpha-L: -arabinofuranosidase production, wheat bran and MgSO(4) had a significantly positive effect. The values of the latter three parameters were further optimised using a central composite design and a response surface methodology. The experimental results were fitted to a second-order polynomial model that yielded a determination coefficient of R(2) = 0.91. The statistical output showed that the linear and quadric terms of the three variables had significant effects. Using optimal conditions, the experimental value of alpha-L: -arabinofuranosidase activity produced was very close to the model-predicted value. The optimal temperature and pH of enzyme activity were 55 degrees C and 7.0, respectively. This enzyme was very stable over a considerable pH range from 4 to 9. The crude enzyme of T. thermophilus rich in alpha-L: -arabinofuranosidase was also used for saccharification of lignocellulosic materials and arabinose production. PMID:19697020

  6. Gene Amplification-Associated Overexpression of the RNA Editing Enzyme ADAR1 Enhances Human Lung Tumorigenesis

    PubMed Central

    Anadón, Carmen; Guil, Sonia; Simó-Riudalbas, Laia; Moutinho, Catia; Setien, Fernando; Martínez-Cardús, Anna; Moran, Sebastian; Villanueva, Alberto; Calaf, Monica; Vidal, August; Lazo, Pedro A.; Zondervan, Ilse; Savola, Suvi; Kohno, Takashi; Yokota, Jun; Ribas de Pouplana, Lluís; Esteller, Manel

    2015-01-01

    The introduction of new therapies against particular genetic mutations in non-small cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis. PMID:26640150

  7. Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

    PubMed

    Anadón, C; Guil, S; Simó-Riudalbas, L; Moutinho, C; Setien, F; Martínez-Cardús, A; Moran, S; Villanueva, A; Calaf, M; Vidal, A; Lazo, P A; Zondervan, I; Savola, S; Kohno, T; Yokota, J; de Pouplana, L R; Esteller, M

    2016-08-18

    The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis. PMID:26640150

  8. Enhancing of sugar cane bagasse hydrolysis by Annulohypoxylon stygium glycohydrolases.

    PubMed

    Robl, Diogo; Costa, Patrícia dos Santos; Büchli, Fernanda; Lima, Deise Juliana da Silva; Delabona, Priscila da Silva; Squina, Fabio Marcio; Pimentel, Ida Chapaval; Padilla, Gabriel; Pradella, José Geraldo da Cruz

    2015-02-01

    The aim of this study was to develop a bioprocess for the production of β-glucosidase and pectinase from the fungus Annulohypoxylon stygium DR47. Media optimization and bioreactor cultivation using citrus bagasse and soybean bran were explored and revealed a maximum production of 6.26 U/mL of pectinase at pH 4.0 and 10.13 U/mL of β-glucosidase at pH 5.0. In addition, the enzymes extracts were able to replace partially Celluclast 1.5L in sugar cane bagasse hydrolysis. Proteomic analysis from A. stygium cultures revealed accessory enzymes, mainly belong to the families GH3 and GH54, that would support enhancement of commercial cocktail saccharification yields. This is the first report describing bioreactor optimization for enzyme production from A. stygium with a view for more efficient degradation of sugar cane bagasse. PMID:25496945

  9. Enzyme Characterization of Cellulase and Hemicellulases Component Enzymes and Saccharification of Ionic Liquid Pretreated Lignocellulosic Biomass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignocellulosic biomass is comprised of cellulose and hemicellulose, sources of polysaccharides, and lignin, a macromolecule with extensive aromaticity. Terrestrial biomass can provide a renewable carbon based feedstock for fuel and chemical production. However, recalcitrance of biomass to deconstru...

  10. 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. PMID:23626684

  11. Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity.

    PubMed

    Cunha, Eva S; Hatem, Christine L; Barrick, Doug

    2016-08-01

    Biomass deconstruction to small simple sugars is a potential approach to biofuels production; however, the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyperstable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation. Proteins 2016; 84:1043-1054. © 2016 Wiley Periodicals, Inc. PMID:27071357

  12. The identification of and relief from Fe3+ inhibition for both cellulose and cellulase in cellulose saccharification catalyzed by cellulases from Penicillium decumbens.

    PubMed

    Wang, Mingyu; Mu, Ziming; Wang, Junli; Hou, Shaoli; Han, Lijuan; Dong, Yanmei; Xiao, Lin; Xia, Ruirui; Fang, Xu

    2013-04-01

    Lignocellulosic biomass is an underutilized, renewable resource that can be converted to biofuels. The key step in this conversion is cellulose saccharification catalyzed by cellulase. In this work, the effect of metal ions on cellulose hydrolysis by cellulases from Penicillium decumbens was reported for the first time. Fe(3+) and Cu(2+) were shown to be inhibitory. Further studies on Fe(3+) inhibition showed the inhibition takes place on both enzyme and substrate levels. Fe(3+) treatment damages cellulases' capability to degrade cellulose and inhibits all major cellulase activities. Fe(3+) treatment also reduces the digestibility of cellulose, due to its oxidation. Treatment of Fe(3+)-treated cellulose with DTT and supplementation of EDTA to saccharification systems partially relieved Fe(3+) inhibition. It was concluded that Fe(3+) inhibition in cellulose degradation is a complicated process in which multiple inhibition events occur, and that relief from Fe(3+) inhibition can be achieved by the supplementation of reducing or chelating agents. PMID:23455222

  13. Enhancing water removal from whole stillage by enzyme addition during fermentation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The removal of water from coproducts in the fuel ethanol process requires a significant energy input. In this study, the addition of cell-wall-degrading enzymes was investigated to determine whether or not the enzymes could reduce the amount of water bound within the wet grains. This would have the ...

  14. Comparative study of sulfite pretreatments for robust enzymatic saccharification of corn cob residue

    PubMed Central

    2012-01-01

    Background Corn cob residue (CCR) is a kind of waste lignocellulosic material with enormous potential for bioethanol production. The moderated sulphite processes were used to enhance the hydrophily of the material by sulfonation and hydrolysis. The composition, FT-IR spectra, and conductometric titrations of the pretreated materials were measured to characterize variations of the CCR in different sulfite pretreated environments. And the objective of this study is to compare the saccharification rate and yield of the samples caused by these variations. Results It was found that the lignin in the CCR (43.2%) had reduced to 37.8%, 38.0%, 35.9%, and 35.5% after the sulfite pretreatment in neutral, acidic, alkaline, and ethanol environments, respectively. The sulfite pretreatments enhanced the glucose yield of the CCR. Moreover, the ethanol sulfite sample had the highest glucose yield (81.2%, based on the cellulose in the treated sample) among the saccharification samples, which was over 10% higher than that of the raw material (70.6%). More sulfonic groups and weak acid groups were produced during the sulfite pretreatments. Meanwhile, the ethanol sulfite treated sample had the highest sulfonic group (0.103 mmol/g) and weak acid groups (1.85 mmol/g) in all sulfite treated samples. In FT-IR spectra, the variation of bands at 1168 and 1190 cm-1 confirmed lignin sulfonation during sulfite pretreatment. The disappearance of the band at 1458 cm-1 implied the methoxyl on lignin had been removed during the sulfite pretreatments. Conclusions It can be concluded that the lignin in the CCR can be degraded and sulfonated during the sulfite pretreatments. The pretreatments improve the hydrophility of the samples because of the increase in sulfonic group and weak acid groups, which enhances the glucose yield of the material. The ethanol sulfite pretreatment is the best method for lignin removal and with the highest glucose yield. PMID:23206858

  15. Enhanced efficacy of enzyme replacement therapy in Pompe disease through mannose-6-phosphate receptor expression in skeletal muscle.

    PubMed

    Koeberl, Dwight D; Luo, Xiaoyan; Sun, Baodong; McVie-Wylie, Alison; Dai, Jian; Li, Songtao; Banugaria, Suhrad G; Chen, Y-T; Bali, Deeksha S

    2011-06-01

    Enzyme replacement therapy (ERT) with acid α-glucosidase has become available for Pompe disease; however, the response of skeletal muscle, as opposed to the heart, has been attenuated. The poor response of skeletal muscle has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle compared to heart. To further understand the role of CI-MPR in Pompe disease, muscle-specific CI-MPR conditional knockout (KO) mice were crossed with GAA-KO (Pompe disease) mice. We evaluated the impact of CI-MPR-mediated uptake of GAA by evaluating ERT in CI-MPR-KO/GAA-KO (double KO) mice. The essential role of CI-MPR was emphasized by the lack of efficacy of ERT as demonstrated by markedly reduced biochemical correction of GAA deficiency and of glycogen accumulations in double KO mice, in comparison with the administration of the same therapeutic doses in GAA-KO mice. Clenbuterol, a selective β(2)-agonist, enhanced the CI-MPR expression in skeletal tissue and also increased efficacy from GAA therapy, thereby confirming the key role of CI-MPR with regard to enzyme replacement therapy in Pompe disease. Biochemical correction improved in both muscle and non-muscle tissues, indicating that therapy could be similarly enhanced in other lysosomal storage disorders. In summary, enhanced CI-MPR expression might improve the efficacy of enzyme replacement therapy in Pompe disease through enhancing receptor-mediated uptake of GAA. PMID:21397538

  16. Enhanced Efficacy of Enzyme Replacement Therapy in Pompe Disease Through Mannose-6-Phosphate Receptor Expression in Skeletal Muscle

    PubMed Central

    Koeberl, Dwight D.; Luo, Xiaoyan; Sun, Baodong; McVie-Wylie, Alison; Dai, Jian; Li, Songtao; Banugaria, Suhrad G.; Chen, Y-T; Bali, Deeksha S.

    2011-01-01

    Enzyme replacement therapy (ERT) with acid α-glucosidase has become available for Pompe disease; however, the response of skeletal muscle, as opposed to the heart, has been attenuated. The poor response of skeletal muscle has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle compared to heart. To further understand the role of CI-MPR in Pompe disease, muscle-specific CI-MPR conditional knockout (KO) mice were crossed with GAA-KO (Pompe disease) mice. We evaluated the impact of CI-MPR-mediated uptake of GAA by evaluating ERT in CI-MPR-KO/GAA-KO (double KO) mice. The essential role of CI-MPR was emphasized by the lack of efficacy of ERT as demonstrated by markedly reduced biochemical correction of GAA deficiency and of glycogen accumulations in double KO mice, in comparison with administration of the same therapeutic doses in GAA-KO mice. Clenbuterol, a selective β2-agonist, enhanced CI-MPR expression in skeletal tissue and also increased efficacy from GAA therapy, thereby confirming the key role of CI-MPR with regard to enzyme replacement therapy in Pompe disease. Biochemical correction improved in both muscle and non-muscle tissues, indicating that therapy could be similarly enhanced in other lysosomal storage disorders. In summary, enhanced CI-MPR expression might improve the efficacy of enzyme replacement therapy in Pompe disease through enhancing receptor-mediated uptake of GAA. PMID:21397538

  17. Recombinant hosts suitable for simultaneous saccharification and fermentation

    DOEpatents

    Ingram, Lonnie O'Neal; Zhou, Shengde

    2007-06-05

    The invention provides recombinant host cells containing at least one heterologous polynucleotide encoding a polysaccharase under the transcriptional control of a surrogate promoter capable of increasing the expression of the polysaccharase. In addition, the invention further provides such hosts with genes encoding secretory protein/s to facilitate the secretion of the expressed polysaccharase. Preferred hosts of the invention are ethanologenic and capable of carrying out simultaneous saccharification fermentation resulting in the production of ethanol from complex cellulose substrates.

  18. Enhanced saccharification of biologically pretreated wheat straw for ethanol production.

    PubMed

    López-Abelairas, M; Lu-Chau, T A; Lema, J M

    2013-02-01

    The biological pretreatment of lignocellulosic biomass with white-rot fungi for the production of bioethanol is an alternative to the most used physico-chemical processes. After biological treatment, a solid composed of cellulose, hemicellulose, and lignin-this latter is with a composition lower than that found in the initial substrate-is obtained. On the contrary, after applying physico-chemical methods, most of the hemicellulose fraction is solubilized, while cellulose and lignin fractions remain in the solid. The optimization of the combination of cellulases and hemicellulases required to saccharify wheat straw pretreated with the white-rot fungus Irpex lacteus was carried out in this work. The application of the optimal dosage made possible the increase of the sugar yield from 33 to 54 %, and at the same time the reduction of the quantity of enzymatic mixture in 40 %, with respect to the initial dosage. The application of a pre-hydrolysis step with xylanases was also studied. PMID:23306886

  19. Organic acids associated with saccharification of cellulosic wastes during solid-state fermentation.

    PubMed

    El-Naggar, Noura El-Ahmady; El-Hersh, Mohammed Saad

    2011-02-01

    Saccharification of five cellulosic wastes, i.e. rice husks, wheat bran, corn cobs, wheat straw and rice straw by three cellulytic fungi, i.e. Aspergillus glaums MN1, Aspergillus oryzae MN2 and Penicillium purpurogenum MN3, during solid-state fermentation (SSF) was laboratory studied. Rice husks, wheat bran, and corn cobs were selected as inducers of glucose production in the tested fungi. An incubation interval of 10 days was optimal for glucose production. Maximal activities of the cellulases FP-ase, CMC-ase, and p-glucosidase were detected during SSF of rice husks by P. purpurogenum; however, a-amylase activity (7.2 U/g) was comparatively reduced. Meanwhile, the productivities of FP-ase, CMC-ase, and β-glucosidase were high during SSF of rice husks by A glaucus; however, they decreased during SSF of corn cobs by P. purpurogenum. Addition of rock phosphate (RP) (75 mg P(2)O(5)) decreased the pH of SSF media. (NH(4))(2)SO(4) was found to be less inducer of cellulytic enzymes, during SSF of rice husks by A. glaucus or A. oryzae; it also induced phytase production and solubilization of RP. The organic acids associated with saccharification of the wastes studied have also been investigated. The highest concentration of levulinic acid was detected (46.15 mg/g) during SSF of corn cobs by P. purpurogenum. Likewise, oxalic acid concentration was 43.20 mg/g during SSF of rice husks by P. purpurogenum. PMID:21369980

  20. Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities.

    PubMed

    Alvarez, Maricel; Huygens, Dries; Olivares, Erick; Saavedra, Isabel; Alberdi, Miren; Valenzuela, Eduardo

    2009-08-01

    Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs. PMID:19470091

  1. Elucidation of Factors Effecting Enzymatic Saccharification using Transgenic Hardwoods

    NASA Astrophysics Data System (ADS)

    Min, Douyong

    Three groups of transgenic wood samples were used as starting materials to elucidate the recalcitrance of enzymatic saccharification with/without pretreatments. The first group of transgenic wood samples is low lignin P. trichocarpa. The second group is low xylan P. trichocarpa. The third one is 12 hybrid poplars which have different levels of S/V ratio and lignin content. Four pretreatments were carried out in this research including dilute sulfuric acid, green liquor, auto hydrolysis and ozone delignification. The behavior among pretreatments as a function of removal of lignin appears to be different. Lignin is the major factor of recalcitrance of the lignocellulosic material to ethanol conversion process. Xylan also plays key role in this process. In addition, the crude milled wood lignin was isolated from these three groups of transgenic samples. Lignin carbohydrate complexes was characterized by 1H-13C HMQC and 13C NMR. Thus the effect of LCCs on enzymatic saccharification was elucidated. High S/V ratio propels the lignin removal during pretreatments however; high S/V ratio retards the enzymatic saccharification on the lignocellulosic material without pretreatments. The level of LCCs linkages accounts for additional recalcitrance of the lignocellulosic material to ethanol conversion process. The amount of LCCs linkages is affected by xylan content, lignin content and S/V ratio.

  2. Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes.

    PubMed

    Cahn, J K B; Baumschlager, A; Brinkmann-Chen, S; Arnold, F H

    2016-01-01

    NAD(P)H-dependent enzymes are ubiquitous in metabolism and cellular processes and are also of great interest for pharmaceutical and industrial applications. Here, we present a structure-guided enzyme engineering strategy for improving catalytic properties of NAD(P)H-dependent enzymes toward native or native-like reactions using mutations to the enzyme's adenine-binding pocket, distal to the site of catalysis. Screening single-site saturation mutagenesis libraries identified mutations that increased catalytic efficiency up to 10-fold in 7 out of 10 enzymes. The enzymes improved in this study represent three different cofactor-binding folds (Rossmann, DHQS-like, and FAD/NAD binding) and utilize both NADH and NADPH. Structural and biochemical analyses show that the improved activities are accompanied by minimal changes in other properties (cooperativity, thermostability, pH optimum, uncoupling), and initial tests on two enzymes (ScADH6 and EcFucO) show improved functionality in Escherichia coli. PMID:26512129

  3. N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin.

    PubMed

    Smith, A Ian; Rajapakse, Niwanthi W; Kleifeld, Oded; Lomonte, Bruno; Sikanyika, Nkumbu L; Spicer, Alexander J; Hodgson, Wayne C; Conroy, Paul J; Small, David H; Kaye, David M; Parkington, Helena C; Whisstock, James C; Kuruppu, Sanjaya

    2016-01-01

    Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer's disease. PMID:26931059

  4. N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin

    PubMed Central

    Smith, A. Ian; Rajapakse, Niwanthi W.; Kleifeld, Oded; Lomonte, Bruno; Sikanyika, Nkumbu L.; Spicer, Alexander J.; Hodgson, Wayne C.; Conroy, Paul J.; Small, David H.; Kaye, David M.; Parkington, Helena C.; Whisstock, James C.; Kuruppu, Sanjaya

    2016-01-01

    Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer’s disease. PMID:26931059

  5. Simultaneous Saccharification and Fermentation and Partial Saccharification and Co-Fermentation of Lignocellulosic Biomass for Ethanol Production

    NASA Astrophysics Data System (ADS)

    Doran-Peterson, Joy; Jangid, Amruta; Brandon, Sarah K.; Decrescenzo-Henriksen, Emily; Dien, Bruce; Ingram, Lonnie O.

    Ethanol production by fermentation of lignocellulosic biomass-derived sugars involves a fairly ancient art and an ever-evolving science. Production of ethanol from lignocellulosic biomass is not avant-garde, and wood ethanol plants have been in existence since at least 1915. Most current ethanol production relies on starch- and sugar-based crops as the substrate; however, limitations of these materials and competing value for human and animal feeds is renewing interest in lignocellulose conversion. Herein, we describe methods for both simultaneous saccharification and fermentation (SSF) and a similar but separate process for partial saccharification and cofermentation (PSCF) of lignocellulosic biomass for ethanol production using yeasts or pentose-fermenting engineered bacteria. These methods are applicable for small-scale preliminary evaluations of ethanol production from a variety of biomass sources.

  6. Impact of pretreatments on morphology and enzymatic saccharification of shedding bark of Melaleuca leucadendron.

    PubMed

    Ahmed, Ibrahim Nasser; Santoso, Shella Permatasari; Tran-Nguyen, Phuong Lan; Huynh, Lien Huong; Ismadji, Suryadi; Ju, Yi-Hsu

    2013-07-01

    The effects of subcritical water (SCW) and dilute acid pretreatments on the shedding bark of Melaleuca leucadendron (paper bark tree, PBT) biomass morphology, crystallinity index (CrI) and enzymatic saccharification were studied. The morphology of PBT bark was characterized by X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy. SCW pretreatment mainly extracted amorphous parts of the biomass hence its CrI increased, partial decrystallization of cellulose and exposing of intact nanofibers of cellulose were observed for SCW pretreatment at 180°C. On the other hand, dilute acid pretreatment at 160°C exhibited a large decrease in CrI, an increase in surface area, a decrease in lignin content and decrystallization of cellulose as well as the peel-off and degradation of some nanofiber bundles. Dilute acid and SCW pretreatments of PBT biomass resulted in about 4.5 fold enhancement in glucose release relative to the untreated one. PMID:23697662

  7. Enhanced lipid recovery from Nannochloropsis microalgae by treatment with optimized cell wall degrading enzyme mixtures.

    PubMed

    Zuorro, Antonio; Miglietta, Selenia; Familiari, Giuseppe; Lavecchia, Roberto

    2016-07-01

    A statistical mixture design approach was used to investigate the effects of cell wall degrading enzymes on the recovery of lipids from Nannochloropsis sp. A preliminary screening of potentially suitable enzyme preparations, including lysozyme, cellulase and different types of hemicellulases, was carried out. The most effective preparations were then taken as basic components for the formulation of enzyme mixtures. Optimized ternary mixtures consisting of cellulase and two hemicellulases were obtained which allowed the recovery of up to 37.2g of lipids per 100g of dry biomass. SEM and TEM images of the enzymatically treated microalga revealed extensive cell damage, with degradation of the cell wall and release of intracellular material. Overall, the results obtained demonstrate that the mixture design method can be used to prepare cell wall degrading enzyme cocktails that can significantly improve the recovery of lipids or other valuable components from microalgae. PMID:27078205

  8. Cognitive enhancing effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers on learning and memory

    PubMed Central

    Nade, V. S.; Kawale, L. A.; Valte, K. D.; Shendye, N. V.

    2015-01-01

    Objective: The present study was designed to investigate cognitive enhancing property of angiotensin-converting enzymes inhibitors (ACEI) and angiotensin receptor blockers (ARBs) in rats. Materials and Methods: The elevated plus maze (EPM), passive avoidance test (PAT), and water maze test (WMT) were used to assess cognitive enhancing activity in young and aged rats. Ramipril (10 mg/kg, p.o.), perindopril (10 mg/kg, i.p), losartan (20 mg/kg, i.p), and valsartan (20 mg/kg, p.o) were administered to assess their effect on learning and memory. Scopolamine (1 mg/kg, i.p) was used to impair cognitive function. Piracetam (200 mg/kg, i.p) was used as reference drug. Results: All the treatments significantly attenuated amnesia induced by aging and scopolamine. In EPM, aged and scopolamine-treated rats showed an increase in transfer latency (TL) whereas, ACEI and ARBs showed a significant decrease in TL. Treatment with ACEI and ARBs significantly increased step down latencies and decreased latency to reach the platform in target quadrant in young, aged and scopolamine-treated animals in PAT and WMT, respectively. The treatments inhibited acetylcholinesterase (AChE) enzyme in the brain. Similarly, all the treatments attenuated scopolamine-induced lipid peroxidation and normalize antioxidant enzymes. Conclusion: The results suggest that the cognitive enhancing effect of ACEI and ARBs may be due to inhibition of AChE or by regulation of antioxidant system or increase in formation of angiotensin IV. PMID:26069362

  9. Cost-effective production of biotechnologically important hydrolytic enzymes by Sporotrichum thermophile.

    PubMed

    Bala, Anju; Singh, Bijender

    2016-01-01

    Economical production of xylanase and three cellulases, endo-β-1,4-glucanase (CMCase), exo-β-1,4-glucanase (FPase), β-glucosidase (BGL) was studied in submerged fermentation using cane molasses medium. A statistical optimization approach involving Plackett-Burman design and response surface methodology (RSM) resulted in the production of 72,410, 36,420, 32,420 and 5180 U/l of xylanase, CMCase, FPase and β-glucosidase, respectively. Optimization resulted in more than fourfold improvements in production of xylanolytic and cellulolytic enzymes. Scale up of enzymes production in shake flasks of varied volumes was sustainable, suggesting a good scope for large scale enzyme production. Addition of microparticles engineered fungal morphology and enhanced enzymes production. Xylanase of S. thermophile is a neutral xylanase displaying its optimal activity at 60 °C while all the cellulases are optimally active at pH 5.0 and 60 °C. The efficacy of enzyme cocktail in waste tea cup paper and rice straw hydrolysis showed that maximum sugar yield of 578.12 and 421.79 mg/g substrate for waste tea cup and rice straw, respectively, were achieved after 24 h. Therefore, concomitant production of cellulolytic and xylanolytic enzymes will be beneficial for the saccharification of lignocellulosics in generating both monomeric and oligomeric sugars for biofuels and other biotechnological applications. PMID:26581490

  10. Enhancing stabilities of lipase by enzyme aggregate coating immobilized onto ionic liquid modified mesoporous materials

    NASA Astrophysics Data System (ADS)

    Zou, Bin; Song, Chunyan; Xu, Xiaping; Xia, Jiaojiao; Huo, Shuhao; Cui, Fengjie

    2014-08-01

    Mesoporous material SBA-15 as the matrix and hydrophilic methyl imidazolium ionic liquids [MSiIM]+BF4- as modifier were involved in preparing ionic liquid modified materials as enzyme carriers through after-grafting silane coupling reaction. The method of enzyme aggregates coating was firstly used to immobilize porcine pancreatic lipase (PPL) onto ionic liquid modified SBA-15. Characterization before and after modification and immobilization were conducted using infrared spectroscopy (FT-IR), differential thermal-thermal analysis (DTA-TG) and N2 adsorption-desorption method (BET). The results indicated that the ordering degree of SBA-15 declined after ionic liquid modification, but mesoporous structure remained. After enzyme immobilization, pore size and specific surface area of carrier became smaller. The cross-linking agent amount, reaction temperature and pH were optimized in this paper. The result demonstrated that the initial activity of enzyme was raised from 35% to 53% after five times recycle by enzyme aggregate coating. 74% of the original activity remained after 25 days storage.

  11. Enzyme-assisted supercritical carbon dioxide extraction of black pepper oleoresin for enhanced yield of piperine-rich extract.

    PubMed

    Dutta, Sayantani; Bhattacharjee, Paramita

    2015-07-01

    Black pepper (Piper nigrum L.), the King of Spices is the most popular spice globally and its active ingredient, piperine, is reportedly known for its therapeutic potency. In this work, enzyme-assisted supercritical carbon dioxide (SC-CO2) extraction of black pepper oleoresin was investigated using α-amylase (from Bacillus licheniformis) for enhanced yield of piperine-rich extract possessing good combination of phytochemical properties. Optimization of the extraction parameters (without enzyme), mainly temperature and pressure, was conducted in both batch and continuous modes and the optimized conditions that provided the maximum yield of piperine was in the batch mode, with a sample size of 20 g of black pepper powder (particle diameter 0.42 ± 0.02 mm) at 60 °C and 300 bar at 2 L/min of CO2 flow. Studies on activity of α-amylase were conducted under these optimized conditions in both batch and continuous modes, with varying amounts of lyophilized enzyme (2 mg, 5 mg and 10 mg) and time of exposure of the enzyme to SC-CO2 (2.25 h and 4.25 h). The specific activity of the enzyme increased by 2.13 times when treated in the continuous mode than in the batch mode (1.25 times increase). The structural changes of the treated enzymes were studied by (1)H NMR analyses. In case of α-amylase assisted extractions of black pepper, both batch and continuous modes significantly increased the yields and phytochemical properties of piperine-rich extracts; with higher increase in batch mode than in continuous. PMID:25617183

  12. Aqueous ammonia soaking of switchgrass followed by simultaneous saccharification and fermentation.

    PubMed

    Isci, Asli; Himmelsbach, Jennifer N; Pometto, Anthony L; Raman, D Raj; Anex, Robert P

    2008-01-01

    Simultaneous saccharification and fermentation (SSF) of switchgrass was performed following aqueous ammonia pretreatment. Switchgrass was soaked in aqueous ammonium hydroxide (30%) with different liquid-solid ratios (5 and 10 ml/g) for either 5 or 10 days. The pretreatment was carried out at atmospheric conditions without agitation. A 40-50% delignification (Klason lignin basis) was achieved, whereas cellulose content remained unchanged and hemicellulose content decreased by approximately 50%. The Sacccharomyces cerevisiae (D5A)-mediated SSF of ammonia-treated switchgrass was investigated at two glucan loadings (3 and 6%) and three enzyme loadings (26, 38.5, and 77 FPU/g cellulose), using Spezyme CP. The percentage of maximum theoretical ethanol yield achieved was 72. Liquid-solid ratio and steeping time affected lignin removal slightly, but did not cause a significant change in overall ethanol conversion yields at sufficiently high enzyme loadings. These results suggest that ammonia steeping may be an effective method of pretreatment for lignocellulosic feedstocks. PMID:18415988

  13. Complete saccharification of β-glucan using hyperthermophilic endocellulase and β-glucosidase from Pyrococcus furiosus.

    PubMed

    Kataoka, Misumi; Ishikawa, Kazuhiko

    2014-01-01

    Hyperthermophilic cellulase is an industrially important enzyme for biomass saccharification at high temperature. Two hyperthermophilic cellulases from the hyperthermophile Pyrococcus furiosus, endocellulase (EGPf) and β-glucosidase (BGLPf), exhibit optimal activity at 90-105 °C and a combination of two enzymes can hydrolyze a wide range of β-linked substrates. EGPf cleaves the β(1→4) bond of various substrates containing either only the β(1→4) linkage or β(1→3),(1→4) mixed-linkages. In contrast, BGLPf preferentially hydrolyzes the β(1→3) linkage over the β(1→4) linkage of disaccharides. β-Glucans are polysaccharides of D-glucose monomers formed by β(1→3),(1→4) mixed-linkage bonds. They occur most commonly as cellulose in plants, in the bran of cereal grains, the cell wall of baker's yeast, and in certain fungi, mushrooms, and bacteria. We reveal that β-glucan can be completely degraded to glucose at high temperature with a combination of EGPf and BGLPf. PMID:25209501

  14. Parameter Estimation for Simultaneous Saccharification and Fermentation of Food Waste Into Ethanol Using Matlab Simulink

    NASA Astrophysics Data System (ADS)

    Davis, Rebecca Anne

    The increase in waste disposal and energy costs has provided an incentive to convert carbohydrate-rich food waste streams into fuel. For example, dining halls and restaurants discard foods that require tipping fees for removal. An effective use of food waste may be the enzymatic hydrolysis of the waste to simple sugars and fermentation of the sugars to ethanol. As these wastes have complex compositions which may change day-to-day, experiments were carried out to test fermentability of two different types of food waste at 27° C using Saccharomyces cerevisiae yeast (ATCC4124) and Genencor's STARGEN™ enzyme in batch simultaneous saccharification and fermentation (SSF) experiments. A mathematical model of SSF based on experimentally matched rate equations for enzyme hydrolysis and yeast fermentation was developed in Matlab Simulink®. Using Simulink® parameter estimation 1.1.3, parameters for hydrolysis and fermentation were estimated through modified Michaelis-Menten and Monod-type equations with the aim of predicting changes in the levels of ethanol and glycerol from different initial concentrations of glucose, fructose, maltose, and starch. The model predictions and experimental observations agree reasonably well for the two food waste streams and a third validation dataset. The approach of using Simulink® as a dynamic visual model for SSF represents a simple method which can be applied to a variety of biological pathways and may be very useful for systems approaches in metabolic engineering in the future.

  15. Enzymatic liquefaction and saccharification of pretreated corn stover at high-solids concentrations in a horizontal rotating bioreactor.

    PubMed

    Du, Jian; Zhang, Fazhan; Li, Yuanyuan; Zhang, Hongman; Liang, Jingrui; Zheng, Hongbo; Huang, He

    2014-02-01

    A self-designed horizontal rotating bioreactor (HRR) was applied for enzymatic hydrolysis of pretreated corn stover to improve the process economics of ethanol production. The mixing principle was based on gravity and free fall employed with tank-rotating. The liquefaction performances using the HRR and the vertical stirred-tank reactor (VSTR) with a helical impeller were compared and analyzed by measuring rheological properties of the slurry. During the enzymatic hydrolysis, viscosity decreased dramatically in the initial phase for both bioreactors and more pronouncedly for the HRR. Rheological parameters fitted to the power law showed that shear thinning properties of the slurry weakened during the reaction. The glucose concentration was used to define the efficiency of the saccharification reaction. The HRR also proved to be more efficient for glucose release with both the constant and fed-batch substrate addition modes. Liquefaction and saccharification at 25% w/w dry matter (DM) and enzyme loading of 7 FPU/g DM resulted in the optimal glucose concentration of 86 g/kg. Results revealed a decrease in cellulose conversion at increasing initial DM, which was slighter in the HRR compared with that in the VSTR. PMID:23771162

  16. Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase

    PubMed Central

    Van Acker, Rebecca; Leplé, Jean-Charles; Aerts, Dirk; Storme, Véronique; Goeminne, Geert; Ivens, Bart; Légée, Frédéric; Lapierre, Catherine; Piens, Kathleen; Van Montagu, Marc C. E.; Santoro, Nicholas; Foster, Clifton E.; Ralph, John; Soetaert, Wim; Pilate, Gilles; Boerjan, Wout

    2014-01-01

    Lignin is one of the main factors determining recalcitrance to enzymatic processing of lignocellulosic biomass. Poplars (Populus tremula x Populus alba) down-regulated for cinnamoyl-CoA reductase (CCR), the enzyme catalyzing the first step in the monolignol-specific branch of the lignin biosynthetic pathway, were grown in field trials in Belgium and France under short-rotation coppice culture. Wood samples were classified according to the intensity of the red xylem coloration typically associated with CCR down-regulation. Saccharification assays under different pretreatment conditions (none, two alkaline, and one acid pretreatment) and simultaneous saccharification and fermentation assays showed that wood from the most affected transgenic trees had up to 161% increased ethanol yield. Fermentations of combined material from the complete set of 20-mo-old CCR–down-regulated trees, including bark and less efficiently down-regulated trees, still yielded ∼20% more ethanol on a weight basis. However, strong down-regulation of CCR also affected biomass yield. We conclude that CCR down-regulation may become a successful strategy to improve biomass processing if the variability in down-regulation and the yield penalty can be overcome. PMID:24379366

  17. Facilitating the enzymatic saccharification of pulped bamboo residues by degrading the remained xylan and lignin-carbohydrates complexes.

    PubMed

    Huang, Caoxing; He, Juan; Li, Xin; Min, Douyong; Yong, Qiang

    2015-09-01

    Kraft pulping was performed on bamboo residues and its impact on the chemical compositions and the enzymatic digestibility of the samples were investigated. To improve the digestibility of sample by degrading the xylan and lignin-carbohydrates complexes (LCCs), xylanase and α-L-arabinofuranosidase (AF) were supplemented with cellulase. The results showed more carbohydrates were remained in the samples pulped with low effective alkali (EA) charge, compared to conventional kraft pulping. When 120 IU/g xylanase and 15 IU/g AF were supplemented with 20 FPU/g cellulase, the xylan degradation yield of the sample pulped with 12% EA charge increased from 68.20% to 88.35%, resulting in an increased enzymatic saccharification efficiency from 58.98% to 83.23%. The amount of LCCs in this sample decreased from 8.63/100C9 to 2.99/100C9 after saccharification with these enzymes. The results indicated that degrading the remained xylan and LCCs in the pulp could improve its enzymatic digestibility. PMID:26080104

  18. Site-saturation mutagenesis of Glomerella cingulata cutinase gene for enhanced enzyme thermostability

    NASA Astrophysics Data System (ADS)

    Hanapi, Wan Nurhidayah Wan; Iuan-Sheau, Chin; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu

    2015-09-01

    Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82_V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.

  19. New isolate of Trichoderma viride strain for enhanced cellulolytic enzyme complex production.

    PubMed

    Jiang, Xinde; Geng, Anli; He, Ning; Li, Qingbiao

    2011-02-01

    A new Trichoderma viride stain was isolated from Singapore soil samples. Its mutants were developed by using ethyl methyl sulfonate (EMS) treatment and UV-irradiation followed by a semi-quantitative plate clearing assay on phosphoric-acid-swollen cellulose plates. Mutant EU2-77 proved to be the most promising extracellular cellulase producer among 20 mutants in a screening program performed in shake flask fermentation after plate screening. Soluble protein content, filter paper cellulase (FPase) activity, β-glucosidase activity and endoglucanase (CMCase) activity of the fermentation broths of the mutant strain were increased to 1.67, 2.49, 2.16, and 2.61 folds, respectively, compared with the wild strain. This enzyme complex produced by mutant EU2-77 contained FPase (2.19 IU/ml), CMCase (16.46 IU/ml), β-glucosidase (4.04 IU/ml), xylanase (42.37 IU/ml), and β-xylosidase (0.12 IU/ml). The soluble protein concentration in the enzyme complex was 1.69 mg/ml. The hydrolytic capacities of fermentation supernatants of T. reesei Rut-C30, the wild strain T. viride NP13a and mutant T. viride EU2-77 were compared with the commercial enzymes on the hydrolysis of waste newspaper. The crude enzymes prepared by T. viride EU2-77 showed much higher hydrolysis performance than that from the commercial strain Rut-C30 and demonstrated much comparable hydrolytic performances with the commercial enzyme mixtures. T. viride mutant EU2-77 produced high levels of extracellular cellulases as well as β-glucosidase, rendering the supplementation of β-glucosidase unnecessary in waste newspaper hydrolysis. PMID:21071269

  20. Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution.

    PubMed

    Sun, Zhoutong; Lonsdale, Richard; Kong, Xu-Dong; Xu, Jian-He; Zhou, Jiahai; Reetz, Manfred T

    2015-10-12

    Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme. PMID:25891639

  1. The analysis of saccharification in biomass using an automated high-throughput method.

    PubMed

    Whitehead, Caragh; Gomez, Leonardo D; McQueen-Mason, Simon J

    2012-01-01

    The recalcitrance of the cell wall to enzymatic hydrolysis represents one of the greatest challenges for using biomass to replace the petroleum as a feedstock for fuels and chemicals. Cell walls are complex in architecture and composition, posing a biochemical challenge for the development of efficient enzymes to release the sugars from the polysaccharide components. The complex composition of the polymers that constitute the cell wall requires a mixture of enzymes to hydrolyze the different glycosidic bonds present in biomass. The improvement of the properties of biomass, in turn, requires the screening of large populations of plants in order to identify markers associated with saccharification potential or pinpoint the genes that regulate recalcitrance. The improvement of both, enzymes and biomass together, requires the capacity to deal with large numbers of variables in a combinatorial approach. We have developed a high-throughput system that allows the determination of cellulolytic activity in a 96-well plate format by automatically handling biomass materials, carrying out hydrolytic reactions, and determining the release of reducing sugars. This platform consists of a purpose-made robot that grinds, formats, and dispenses precise amounts of solids into 96-well plates, and a liquid-handling station specifically designed to carry out pretreatments, hydrolysis, and the determination of released reducing sugar equivalents using a colorimetric assay. These modules can be used individually or in combination according to the function needed. Here we show some examples of the capabilities of the platforms in terms of enzyme and biomass evaluation, as well as combining the robot with off-line analytical tools. PMID:22608720

  2. Improved in situ saccharification of cellulose pretreated by dimethyl sulfoxide/ionic liquid using cellulase from a newly isolated Paenibacillus sp. LLZ1.

    PubMed

    Hu, Dongxue; Ju, Xin; Li, Liangzhi; Hu, Cuiying; Yan, Lishi; Wu, Tianyun; Fu, Jiaolong; Qin, Ming

    2016-02-01

    A cellulase producing strain was newly isolated from soil samples and identified as Paenibacillus sp. LLZ1. A novel aqueous-dimethyl sulfoxide (DMSO)/1-ethyl-3-methylimidazolium diethyl phosphate ([Emin]DEP)-cellulase system was designed and optimized. In the pretreatment, DMSO was found to be a low-cost substitute of up to 70% ionic liquid to enhance the cellulose dissolution. In the enzymatic saccharification, the optimum pH and temperature of the Paenibacillus sp. LLZ1 cellulase were identified as 6.0 and 40°C, respectively. Under the optimized reaction condition, the conversion of microcrystalline cellulose and bagasse cellulose increased by 39.3% and 37.6%, compared with unpretreated cellulose. Compared to current methods of saccharification, this new approach has several advantages including lower operating temperature, milder pH, and less usage of ionic liquid, indicating a marked progress in environmental friendly hydrolysis of biomass-based materials. PMID:26618784

  3. Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels.

    PubMed

    Greene, Eric R; Himmel, Michael E; Beckham, Gregg T; Tan, Zhongping

    2015-01-01

    Cellulose in plant cell walls is the largest reservoir of renewable carbon on Earth. The saccharification of cellulose from plant biomass into soluble sugars can be achieved using fungal and bacterial cellulolytic enzymes, cellulases, and further converted into fuels and chemicals. Most fungal cellulases are both N- and O-glycosylated in their native form, yet the consequences of glycosylation on activity and structure are not fully understood. Studying protein glycosylation is challenging as glycans are extremely heterogeneous, stereochemically complex, and glycosylation is not under direct genetic control. Despite these limitations, many studies have begun to unveil the role of cellulase glycosylation, especially in the industrially relevant cellobiohydrolase from Trichoderma reesei, Cel7A. Glycosylation confers many beneficial properties to cellulases including enhanced activity, thermal and proteolytic stability, and structural stabilization. However, glycosylation must be controlled carefully as such positive effects can be dampened or reversed. Encouragingly, methods for the manipulation of glycan structures have been recently reported that employ genetic tuning of glycan-active enzymes expressed from homogeneous and heterologous fungal hosts. Taken together, these studies have enabled new strategies for the exploitation of protein glycosylation for the production of enhanced cellulases for biofuel production. PMID:26613815

  4. Model analysis for enhancement of enzyme-catalyzed alcohol oxidation by solvent extraction of product

    SciTech Connect

    Hidaka, Nobuyuki; Matsumoto, Toshitatsu; Morooka, Shigeharu

    1995-07-01

    Enzymatic oxidation of ethanol and butanol was conducted with alcohol oxidase in a one-phase system of water and a two-phase system of water and toluene. The conversion of the reaction in the two-phase system was higher than that in the one-phase system. The inhibition of the enzyme was relieved in the two-phase system by extracting the aldehyde product into the organic phase. The dissolved oxygen concentration in the water phase also affected the reaction rate. A kinetic model of the reaction was developed by considering both the inhibition of enzyme activity and the dissolved oxygen concentration. Parameters used in the model were evaluated experimentally. The reaction rate calculated using the model was in good agreement with the data.

  5. Strategies for enhancing the effectiveness of metagenomic-based enzyme discovery in lignocellulytic microbial communities

    SciTech Connect

    DeAngelis, K.M.; Gladden, J.G.; Allgaier, M.; D'haeseleer, P.; Fortney, J.L.; Reddy, A.; Hugenholtz, P.; Singer, S.W.; Vander Gheynst, J.; Silver, W.L.; Simmons, B.; Hazen, T.C.

    2010-03-01

    Producing cellulosic biofuels from plant material has recently emerged as a key U.S. Department of Energy goal. For this technology to be commercially viable on a large scale, it is critical to make production cost efficient by streamlining both the deconstruction of lignocellulosic biomass and fuel production. Many natural ecosystems efficiently degrade lignocellulosic biomass and harbor enzymes that, when identified, could be used to increase the efficiency of commercial biomass deconstruction. However, ecosystems most likely to yield relevant enzymes, such as tropical rain forest soil in Puerto Rico, are often too complex for enzyme discovery using current metagenomic sequencing technologies. One potential strategy to overcome this problem is to selectively cultivate the microbial communities from these complex ecosystems on biomass under defined conditions, generating less complex biomass-degrading microbial populations. To test this premise, we cultivated microbes from Puerto Rican soil or green waste compost under precisely defined conditions in the presence dried ground switchgrass (Panicum virgatum L.) or lignin, respectively, as the sole carbon source. Phylogenetic profiling of the two feedstock-adapted communities using SSU rRNA gene amplicon pyrosequencing or phylogenetic microarray analysis revealed that the adapted communities were significantly simplified compared to the natural communities from which they were derived. Several members of the lignin-adapted and switchgrass-adapted consortia are related to organisms previously characterized as biomass degraders, while others were from less well-characterized phyla. The decrease in complexity of these communities make them good candidates for metagenomic sequencing and will likely enable the reconstruction of a greater number of full length genes, leading to the discovery of novel lignocellulose-degrading enzymes adapted to feedstocks and conditions of interest.

  6. A study of over-production and enhanced secretion of enzymes. Quarterly report 2

    SciTech Connect

    Dashek, W.V.

    1993-04-08

    This project is concerned with the over-production of ligno-cellulolytic enzymes which are relevant to the paper-pulp industry and agricultural community. Since ligno-cellulosics are components of wood, the project involves the forest, a renewable energy resource. Attention is focused on the following: over-production of polyphenol oxidase; establishment of the route of polyphenol oxidase secretion; regulation of polyphenol oxidase secretion; purification of extracellular oxidase.

  7. Enhancing in vitro degradation of alfalfa hay and corn silage using feed enzymes.

    PubMed

    Eun, J-S; Beauchemin, K A

    2007-06-01

    A series of in vitro fermentation experiments was performed to assess the effects of 4 feed enzyme products (FE) that varied in enzymatic activities on the degradation of alfalfa hay and corn silage. The FE contained a range of endoglucanase, exoglucanase, xylanase, and protease activities, and a range of dose rates (DR) was used. The objective of the study was to identify effective formulations and optimum DR, and to establish if combining FE would further improve fiber degradation. For alfalfa hay, quadratic increases in gas production and degradation of dry matter (DM) and fiber were observed for all FE, with maximum responses at low to medium DR. For corn silage, none of the FE increased gas production or DM degradation, but all FE increased NDF degradation, with optimum DR in the low to medium range. The proteolytic enzyme papain improved fiber degradation of alfalfa hay and corn silage in a manner similar to that observed for polysaccharidase FE. Among the polysaccharidase FE, added activities of endoglucanase and exoglucanase were positively correlated with improvement in neutral detergent fiber (NDF) degradability of corn silage, whereas only added endoglucanase activity tended to be correlated with improvement in NDF degradability of alfalfa hay. Combining effective polysaccharidase FE further improved fiber degradation of both forages, with greater improvements for corn silage. Combining polysaccharidase and proteolytic FE further improved NDF degradation of corn silage, but not alfalfa hay. Combination treatments generally resulted in additive effects with increases in fiber degradation equal to the sum of the improvements for the individual enzyme components. Improved fiber degradation of corn silage was associated with decreased acetate to propionate ratios. Enzyme products that improve in vitro degradation of forages may have the potential to improve lactational performance of dairy cows. PMID:17517724

  8. Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced substrate and stereo specificities.

    PubMed

    Fesko, Kateryna

    2016-03-01

    Threonine aldolases have emerged as a powerful tool for asymmetric carbon-carbon bond formation. These enzymes catalyse the unnatural aldol condensation of different aldehydes and glycine to produce highly valuable β-hydroxy-α-amino acids with complete stereocontrol at the α-carbon and moderate specificity at the β-carbon. A range of microbial threonine aldolases has been recently recombinantly produced by several groups and their biochemical properties were characterized. Numerous studies have been conducted to improve the reaction protocols to enable higher conversions and investigate the substrate scope of enzymes. However, the application of threonine aldolases in organic synthesis is still limited due to often moderate yields and low diastereoselectivities obtained in the aldol reaction. This review briefly summarizes the screening techniques recently applied to discover novel threonine aldolases as well as enzyme engineering and mutagenesis studies which were accomplished to improve the catalytic activity and substrate specificity. Additionally, the results from new investigations on threonine aldolases including crystal structure determinations and structural-functional characterization are reviewed. PMID:26810201

  9. Zinc enhancement of cytidine deaminase activity highlights a potential allosteric role of loop-3 in regulating APOBEC3 enzymes

    PubMed Central

    Marx, Ailie; Galilee, Meytal; Alian, Akram

    2015-01-01

    The strong association of APOBEC3 cytidine deaminases with somatic mutations leading to cancers accentuates the importance of their tight intracellular regulation to minimize cellular transformations. We reveal a novel allosteric regulatory mechanism of APOBEC3 enzymes showing that APOBEC3G and APOBEC3A coordination of a secondary zinc ion, reminiscent to ancestral deoxycytidylate deaminases, enhances deamination activity. Zinc binding is pinpointed to loop-3 which whilst highly variable harbors a catalytically essential and spatially conserved asparagine at its N-terminus. We suggest that loop-3 may play a general role in allosterically tuning the activity of zinc-dependent cytidine deaminase family members. PMID:26678087

  10. Zinc enhancement of cytidine deaminase activity highlights a potential allosteric role of loop-3 in regulating APOBEC3 enzymes.

    PubMed

    Marx, Ailie; Galilee, Meytal; Alian, Akram

    2015-01-01

    The strong association of APOBEC3 cytidine deaminases with somatic mutations leading to cancers accentuates the importance of their tight intracellular regulation to minimize cellular transformations. We reveal a novel allosteric regulatory mechanism of APOBEC3 enzymes showing that APOBEC3G and APOBEC3A coordination of a secondary zinc ion, reminiscent to ancestral deoxycytidylate deaminases, enhances deamination activity. Zinc binding is pinpointed to loop-3 which whilst highly variable harbors a catalytically essential and spatially conserved asparagine at its N-terminus. We suggest that loop-3 may play a general role in allosterically tuning the activity of zinc-dependent cytidine deaminase family members. PMID:26678087

  11. Lignocellulosic Fermentation of Wild Grass Employing Recombinant Hydrolytic Enzymes and Fermentative Microbes with Effective Bioethanol Recovery

    PubMed Central

    Das, Saprativ P.; Ghosh, Arabinda; Gupta, Ashutosh; Das, Debasish

    2013-01-01

    Simultaneous saccharification and fermentation (SSF) studies of steam exploded and alkali pretreated different leafy biomass were accomplished by recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for bioethanol production. The recombinant C. thermocellum GH5 cellulase and GH43 hemicellulase genes expressed in Escherichia coli cells were grown in repetitive batch mode, with the aim of enhancing the cell biomass production and enzyme activity. In batch mode, the cell biomass (A600 nm) of E. coli cells and enzyme activities of GH5 cellulase and GH43 hemicellulase were 1.4 and 1.6 with 2.8 and 2.2 U·mg−1, which were augmented to 2.8 and 2.9 with 5.6 and 3.8 U·mg−1 in repetitive batch mode, respectively. Steam exploded wild grass (Achnatherum hymenoides) provided the best ethanol titres as compared to other biomasses. Mixed enzyme (GH5 cellulase, GH43 hemicellulase) mixed culture (Saccharomyces cerevisiae, Candida shehatae) system gave 2-fold higher ethanol titre than single enzyme (GH5 cellulase) single culture (Saccharomyces cerevisiae) system employing 1% (w/v) pretreated substrate. 5% (w/v) substrate gave 11.2 g·L−1 of ethanol at shake flask level which on scaling up to 2 L bioreactor resulted in 23 g·L−1 ethanol. 91.6% (v/v) ethanol was recovered by rotary evaporator with 21.2% purification efficiency. PMID:24089676

  12. Effects of moisture enhancement, enzyme treatment, and blade tenderization on the processing characteristics and tenderness of beef semimembranosus steaks.

    PubMed

    Pietrasik, Z; Shand, P J

    2011-05-01

    The individual and combined effects of moisture enhancement with a salt/phosphate solution (ME), blade tenderization (BT), and enzyme injection with proteinases derived from Aspergillus oryzae or Bacillus subtilis on cooking properties, Warner-Bratzler shear force (WBSF), and sensory characteristics of beef semimembranosus were investigated. ME significantly (P < 0.01) reduced WBSF and increased (P < 0.05) sensory scores for juiciness and tenderness. BT increased (P < 0.05) initial and overall tenderness scores and made connective tissue less perceptible. BT combined with ME resulted in the highest initial and overall tenderness scores, however, combining ME with either proteinase was as effective for reducing WBSF and increasing tenderness, particularly at 20 (vs. 10) ppm enzyme inclusion. Tenderness of enzyme-injected steaks was increased without compromising other palatability attributes. All treatments increased the frequency of steaks rated slightly tender or higher, with the ME+BT combination, or ME with inclusion of 20 ppm of either proteinase, being most effective. PMID:21194850

  13. An Electrochemical Impedance Spectroscopy System for Monitoring Pineapple Waste Saccharification

    PubMed Central

    Conesa, Claudia; Ibáñez Civera, Javier; Seguí, Lucía; Fito, Pedro; Laguarda-Miró, Nicolás

    2016-01-01

    Electrochemical impedance spectroscopy (EIS) has been used for monitoring the enzymatic pineapple waste hydrolysis process. The system employed consists of a device called Advanced Voltammetry, Impedance Spectroscopy & Potentiometry Analyzer (AVISPA) equipped with a specific software application and a stainless steel double needle electrode. EIS measurements were conducted at different saccharification time intervals: 0, 0.75, 1.5, 6, 12 and 24 h. Partial least squares (PLS) were used to model the relationship between the EIS measurements and the sugar determination by HPAEC-PAD. On the other hand, artificial neural networks: (multilayer feed forward architecture with quick propagation training algorithm and logistic-type transfer functions) gave the best results as predictive models for glucose, fructose, sucrose and total sugars. Coefficients of determination (R2) and root mean square errors of prediction (RMSEP) were determined as R2 > 0.944 and RMSEP < 1.782 for PLS and R2 > 0.973 and RMSEP < 0.486 for artificial neural networks (ANNs), respectively. Therefore, a combination of both an EIS-based technique and ANN models is suggested as a promising alternative to the traditional laboratory techniques for monitoring the pineapple waste saccharification step. PMID:26861317

  14. An Electrochemical Impedance Spectroscopy System for Monitoring Pineapple Waste Saccharification.

    PubMed

    Conesa, Claudia; Ibáñez Civera, Javier; Seguí, Lucía; Fito, Pedro; Laguarda-Miró, Nicolás

    2016-01-01

    Electrochemical impedance spectroscopy (EIS) has been used for monitoring the enzymatic pineapple waste hydrolysis process. The system employed consists of a device called Advanced Voltammetry, Impedance Spectroscopy & Potentiometry Analyzer (AVISPA) equipped with a specific software application and a stainless steel double needle electrode. EIS measurements were conducted at different saccharification time intervals: 0, 0.75, 1.5, 6, 12 and 24 h. Partial least squares (PLS) were used to model the relationship between the EIS measurements and the sugar determination by HPAEC-PAD. On the other hand, artificial neural networks: (multilayer feed forward architecture with quick propagation training algorithm and logistic-type transfer functions) gave the best results as predictive models for glucose, fructose, sucrose and total sugars. Coefficients of determination (R²) and root mean square errors of prediction (RMSEP) were determined as R² > 0.944 and RMSEP < 1.782 for PLS and R² > 0.973 and RMSEP < 0.486 for artificial neural networks (ANNs), respectively. Therefore, a combination of both an EIS-based technique and ANN models is suggested as a promising alternative to the traditional laboratory techniques for monitoring the pineapple waste saccharification step. PMID:26861317

  15. Enhanced enzyme production with the pelleted form of D. squalens in laboratory bioreactors using added natural lignin inducer.

    PubMed

    Babič, Janja; Pavko, Aleksander

    2012-03-01

    White-rot fungi are extensively used in various submerged biotechnology processes to produce ligninolytic enzymes. Transfer of the process from the laboratory to the industrial level requires optimization of the cultivation conditions on the laboratory scale. An interesting area of optimization is pellet growth since this morphological form solves problems such as the decreased oxygen concentration, limited heat, and nutrient transport, which usually occur in dispersed mycelium cultures. Many submerged fermentations with basidiomycetes in pellet form were done with Phanerochaete, Trametes, and Bjerkandera species, among others. In our study, another promising basidiomycete, D. squalens, was used for ligninolytic enzyme production. With the addition of wood particles (sawdust) as a natural inducer and optimization of mixing and aeration conditions in laboratory stirred tank (STR) and bubble column (BCR) reactors on pellet growth and morphology, the secretion of laccase and the manganese-dependent peroxidase into the medium was substantially enhanced. The maximum mean pellet radius was achieved after 10 days in the BCR (5.1 mm) where pellets were fluffy and 5 days in the STR (3.5 mm) where they were round and smooth. The maximum Lac activity (1,882 U l(-1)) was obtained after 12 days in the STR, while maximum MnP activity (449.8 U l(-1)) occurred after 18 days in the BCR. The pellet size and morphology depended on the agitation and aeration conditions and consequently influenced a particular enzyme synthesis. The enzyme activities were high and comparable with the activities found for other investigations in reactors with basidiomycetes in the form of pellets. PMID:21922328

  16. Ganoderma lucidum (Fr.) P. Karst enhances activities of heart mitochondrial enzymes and respiratory chain complexes in the aged rat.

    PubMed

    Sudheesh, N P; Ajith, T A; Janardhanan, K K

    2009-10-01

    Aging is associated with increased oxidative damage at multiple cellular levels, decline in cellular energy production and enhanced free radical status. The effect of the medicinal mushroom, Ganoderma lucidum on the activities of tricarboxylic acid (Krebs) cycle enzymes and mitochondrial complexes I-IV of the electron transport chain in aged rats were investigated. The activity of Krebs cycle enzymes, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV were determined in heart of aged male Wistar rats orally administrated with 70% ethanolic extract (50 and 250 mg/kg) of G. lucidum. DL-alpha-lipoic acid (100 mg/kg) was taken as the positive control. Administration of the G. lucidum, once daily for 15 days, was significantly (P < 0.05) effective to enhance the Krebs cycle dehydrogenases, and mitochondrial electron transport chain complex IV activities in aged rats. The profound activity of the extract can be correlated to the significant antioxidant property of G. lucidum. The results of the study revealed that G. lucidum is effective to ameliorate the age associated decline of cellular energy status. PMID:19123066

  17. Immobilization of peroxidase enzyme onto the porous silicon structure for enhancing its activity and stability

    NASA Astrophysics Data System (ADS)

    Sahare, Padmavati; Ayala, Marcela; Vazquez-Duhalt, Rafael; Agrawal, Vivechana

    2014-08-01

    In this work, a commercial peroxidase was immobilized onto porous silicon (PS) support functionalized with 3-aminopropyldiethoxysilane (APDES) and the performance of the obtained catalytic microreactor was studied. The immobilization steps were monitored and the activity of the immobilized enzyme in the PS pores was spectrophotometrically determined. The enzyme immobilization in porous silicon has demonstrated its potential as highly efficient enzymatic reactor. The effect of a polar organic solvent (acetonitrile) and the temperature (up to 50°C) on the activity and stability of the biocatalytic microreactor were studied. After 2-h incubation in organic solvent, the microreactor retained 80% of its initial activity in contrast to the system with free soluble peroxidase that lost 95% of its activity in the same period of time. Peroxidase immobilized into the spaces of the porous silicon support would be perspective for applications in treatments for environmental security such as removal of leached dye in textile industry or in treatment of different industrial effluents. The system can be also applied in the field of biomedicine.

  18. Amplified electrochemiluminescent aptasensor using mimicking bi-enzyme nanocomplexes as signal enhancement.

    PubMed

    Zhuo, Ying; Ma, Meng-nan; Chai, Ya-qin; Zhao, Min; Yuan, Ruo

    2014-01-27

    In this work, a sandwich-type electrochemiluminescence (ECL) aptasensor for ultrasensitive detection of thrombin (TB) was designed based on mimicking bi-enzyme cascade catalysis to in situ generate coreactant of dissolved oxygen (O2) for signal amplification. We utilized hollow Au nanoparticles (HAuNPs) as carriers to immobilize glucose oxidase nanoparticles (GOxNPs) and Pt nanoparticles (PtNPs) by electrostatic adsorption. Then, the detection aptamer of thrombin (TBA 2) was immobilized on the PtNPs/GOxNPs/HAuNPs nanocomplexes. Finally, hemin was intercalated into the TBA 2 to obtain the hemin/G-quadruplex structure. The hemin/G-quadruplex was an interesting DNAzyme that commonly mimiced horseradish peroxidase (HRP). Herein, GOxNPs, hemin/G-quadruplex and PtNPs could form mimicking bi-enzyme cascade catalysis system to in situ generate dissolved O2 as coreactant in peroxydisulfate solution when the testing buffer contained proper amounts of glucose. This method had successfully overcome the disadvantage of difficulty to label the dissolved O2 and realized the ECL signal amplification. The experiment proved that the aptasensor had good linear relationship on low concentration of TB. The linear range was 1×10(-6)-10 nM, with a detection limit of 0.3 fM. PMID:24418132

  19. Immobilization of peroxidase enzyme onto the porous silicon structure for enhancing its activity and stability

    PubMed Central

    2014-01-01

    In this work, a commercial peroxidase was immobilized onto porous silicon (PS) support functionalized with 3-aminopropyldiethoxysilane (APDES) and the performance of the obtained catalytic microreactor was studied. The immobilization steps were monitored and the activity of the immobilized enzyme in the PS pores was spectrophotometrically determined. The enzyme immobilization in porous silicon has demonstrated its potential as highly efficient enzymatic reactor. The effect of a polar organic solvent (acetonitrile) and the temperature (up to 50°C) on the activity and stability of the biocatalytic microreactor were studied. After 2-h incubation in organic solvent, the microreactor retained 80% of its initial activity in contrast to the system with free soluble peroxidase that lost 95% of its activity in the same period of time. Peroxidase immobilized into the spaces of the porous silicon support would be perspective for applications in treatments for environmental security such as removal of leached dye in textile industry or in treatment of different industrial effluents. The system can be also applied in the field of biomedicine. PMID:25221454

  20. High bioethanol titre from Manihot glaziovii through fed-batch simultaneous saccharification and fermentation in Automatic Gas Potential Test System.

    PubMed

    Moshi, Anselm P; Crespo, Carla F; Badshah, Malik; Hosea, Kenneth M M; Mshandete, Anthony Manoni; Mattiasson, Bo

    2014-03-01

    A process for the production of high bioethanol titre was established through fed-batch and simultaneous saccharification and fermentation (FB-SSF) of wild, non-edible cassava Manihot glaziovii. FB-SSF allowed fermentation of up to 390g/L of starch-derived glucose achieving high bioethanol concentration of up to 190g/L (24% v/v) with yields of around 94% of the theoretical value. The wild cassava M. glaziovii starch is hydrolysable with a low dosage of amylolytic enzymes (0.1-0.15% v/w, Termamyl® and AMG®). The Automatic Gas Potential Test System (AMPTS) was adapted to yeast ethanol fermentation and demonstrated to be an accurate, reliable and flexible device for studying the kinetics of yeast in SSF and FB-SSF. The bioethanol derived stoichiometrically from the CO2 registered in the AMPTS software correlated positively with samples analysed by HPLC (R(2)=0.99). PMID:24534761

  1. High consistency enzymatic saccharification of sweet sorghum bagasse pretreated with liquid hot water.

    PubMed

    Wang, Wen; Zhuang, Xinshu; Yuan, Zhenhong; Yu, Qiang; Qi, Wei; Wang, Qiong; Tan, Xuesong

    2012-03-01

    A laboratory set-up was designed to carry out high consistency enzymatic saccharification of sweet sorghum bagasse (SSB) which was pretreated by liquid hot water (LHW). The effects of two impellers on enzymatic hydrolysis of SSB were investigated. Compared with the double-curved-blade impeller (DCBI), the plate-and-frame impeller (PFI) could improve glucose production by 10%. Tween80 and fed-batch hydrolysis method adopted in this study produced total sugar of 17.06 g/L more than batch hydrolysis and raised the substrate consistency to 30%. At the final substrate loading of 30%, the concentrations of cellobiose, glucose and xylose reached to 15.01 g/L, 88.95 g/L and 9.80 g/L, respectively, and the ethanol concentration reached to 43.36 g/L in the case of cellobiose and xylose were not fermented by Saccharomyces cerevisiae Y2034. This study is an attempt at improvement of enzyme hydrolyzing LHW-pretreated material at high consistency. PMID:22281144

  2. Conversion of Aqueous Ammonia-Treated Corn Stover to Lactic Acid by Simultaneous Saccharification and Cofermentation

    NASA Astrophysics Data System (ADS)

    Zhu, Yongming; Lee, Y. Y.; Elander, Richard T.

    Treatment of corn stover with aqueous ammonia removes most of the structural lignin, whereas retaining the majority of the carbohydrates in the solids. After treatment, both the cellulose and hemicellulose in corn stover become highly susceptible to enzymatic digestion. In this study, corn stover treated by aqueous ammonia was investigated as the substrate for lactic acid production by simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Spezyme-CP) and Lactobacillus pentosus American Type Culture Collection (ATCC) 8041 (Spanish Type Culture Collection [CECT]-4023) were used for hydrolysis and fermentation, respectively. In batch SSCF operation, the carbohydrates in the treated corn stover were converted to lactic acid with high yields, the maximum lactic acid yield reaching 92% of the stoichiometric maximum based on total fermentable carbohydrates (glucose, xylose, and arabinose). A small amount of acetic acid was also produced from pentoses through the phosphoketolase pathway. Among the major process variables for batch SSCF, enzyme loading and the amount of yeast extract were found to be the key factors affecting lactic acid production. Further tests on nutrients indicated that corn steep liquor could be substituted for yeast extract as a nitrogen source to achieve the same lactic acid yield. Fed-batch operation of the SSCF was beneficial in raising the concentration of lactic acid to a maximum value of 75.0 g/L.

  3. Simultaneous saccharification and fermentation of hemicellulose to butanol by a non-sporulating Clostridium species.

    PubMed

    Li, Tinggang; He, Jianzhong

    2016-11-01

    Production of lignocellulosic butanol has drawn increasing attention. However, currently few microorganisms can produce biofuels, particularly butanol, from lignocellulosic biomass via simultaneous saccharification and fermentation. Here we report discovery of a wild-type, mesophilic Clostridium sp. strain MF28 that ferments xylan to produce butanol (up to 3.2g/L) without the addition of saccharolytic enzymes and without any chemical pretreatments. Application of selective pressure from 2-deoxy-d-glucose facilitated isolation of strain MF28, which exhibits inactivation of genes (gid and ccp genes) responsible for carbon catabolite repression, thus allowing strain MF28 to simultaneously ferment a combination of glucose (30g/L), xylose (15g/L), and arabinose (15g/L) to produce 11.9g/L of butanol. Strain MF28 possesses several unique features: (i) non-sporulating, (ii) no acetone/ethanol, (iii) complete hemicellulose-binding enzymatic domain, and (iv) absence of carbon catabolite repression. These unique characteristics demonstrate the industrial potential of strain MF28 for cost-effective biofuel generation from lignocellulosic biomass. PMID:27513648

  4. Aqueous ammonia pretreatment, saccharification, and fermentation evaluation of oil palm fronds for ethanol production.

    PubMed

    Jung, Young Hoon; Kim, Sooah; Yang, Taek Ho; Lee, Hee Jong; Seung, Doyoung; Park, Yong-Cheol; Seo, Jin-Ho; Choi, In-Geol; Kim, Kyoung Heon

    2012-11-01

    Oil palm fronds are the most abundant lignocellulosic biomass in Malaysia. In this study, fronds were tested as the potential renewable biomass for ethanol production. The soaking in aqueous ammonia pretreatment was applied, and the fermentability of pretreated fronds was evaluated using simultaneous saccharification and fermentation. The optimal pretreatment conditions were 7 % (w/w) ammonia, 80 °C, 20 h of pretreatment, and 1:12 S/L ratio, where the enzymatic digestibility was 41.4 % with cellulase of 60 FPU/g-glucan. When increasing the cellulase loading in the hydrolysis of pretreated fronds, the enzymatic digestibility increased until the enzyme loading reached 60 FPU/g-glucan. With 3 % glucan loading in the SSF of pretreated fronds, the ethanol concentration and yield based on the theoretical maximum after 12 and 48 h of the SSF were 7.5 and 9.7 g/L and 43.8 and 56.8 %, respectively. The ethanol productivities found at 12 and 24 h from pretreated fronds were 0.62 and 0.36 g/L/h, respectively. PMID:22644062

  5. Phenobarbital mechanistic data and risk assessment: enzyme induction, enhanced cell proliferation, and tumor promotion.

    PubMed

    Whysner, J; Ross, P M; Williams, G M

    1996-01-01

    Chronic exposure to high doses of phenobarbital (PB) causes hepatocellular adenomas in both mice and rats and hepatocellular carcinomas in some strains of mice. Long-term PB therapy has not been found to cause human tumors. PB is not DNA reactive, and most genotoxicity tests have yielded negative results. PB has been extensively studied as an epigenetic, rodent liver tumor promoter. At exposures causing rodent liver tumors, PB has measurable effects on hepatocytes: PB inhibits cell-to-cell communication; PB induces enzymes, including P450 cytochromes; PB stimulates proliferation and inhibits apoptosis of hepatocytes in neoplastic foci. Threshold exposures for some of these endpoints coincide with the threshold exposure for tumorigenesis. PMID:8910954

  6. An ultrasensitive and universal photoelectrochemical immunoassay based on enzyme mimetics enhanced signal amplification.

    PubMed

    Wang, Guang-Li; Shu, Jun-Xian; Dong, Yu-Ming; Wu, Xiu-Ming; Li, Zai-Jun

    2015-04-15

    An ultrasensitive photoelectrochemical (PEC) immunoassay based on signal amplification by enzyme mimetics was fabricated for the detection of mouse IgG (as a model protein). The PEC immunosensor was constructed by a layer-by-layer assembly of poly (diallyldimethylammonium chloride) (PDDA), CdS quantum dots (QDs), primary antibody (Ab1, polyclonal goat antimouse IgG), and the antigen (Ag, mouse IgG) on an indium-tin oxide (ITO) electrode. Then, the secondary antibody (Ab2, polyclonal goat antimouse IgG) combined to a bio-bar-coded Pt nanoparticle(NP)-G-quadruplex/hemin probe was used for signal amplification. The bio-bar-coded Pt NP-G-quadruplex/hemin probe could catalyze the oxidation of hydroquinone (HQ) using H2O2 as an oxidant, demonstrating its intrinsic enzyme-like activity. High sensitivity for the target Ag was achieved by using the bio-bar-coded probe as signal amplifier due to its high catalytic activity, a competitive nonproductive absorption of hemin and the steric hindrance caused by the polymeric oxidation products of HQ. For most important, the oxidation product of HQ acted as an efficient electron acceptor of the illuminated CdS QDs. The target Ag could be detected from 0.01pg/mL to 1.0ng/mL with a low detection limit of 6.0fg/mL. The as-obtained immunosensor exhibited high sensitivity, good stability and acceptable reproducibility. This method might be attractive for clinical and biomedical applications. PMID:25437365

  7. Simultaneous saccharification and fermentation of cellulose in ionic liquid for efficient production of α-ketoglutaric acid by Yarrowia lipolytica.

    PubMed

    Ryu, Seunghyun; Labbé, Nicole; Trinh, Cong T

    2015-05-01

    Ionic liquids (ILs) are benign solvents that are highly effective for biomass pretreatment. However, their applications for scale-up biorefinery are limited due to multiple expensive IL recovery and separation steps that are required. To overcome this limitation, it is very critical to develop a compatible enzymatic and microbial biocatalyst system to carry the simultaneous saccharification and fermentation in IL environments (SSF-IL). While enzymatic biocatalysts have been demonstrated to be compatible with various IL environments, it is challenging to develop microbial biocatalysts that can thrive and perform efficient biotransformation under the same conditions (pH and temperature). In this study, we harnessed the robust metabolism of Yarrowia lipolytica as a microbial platform highly compatible with the IL environments such as 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]). We optimized the enzymatic and microbial biocatalyst system using commercial cellulases and demonstrated the capability of Y. lipolytica to convert cellulose into high-value organics such as α-ketoglutaric acid (KGA) in the SSF-IL process at relatively low temperature 28 °C and high pH 6.3. We showed that SSF-IL not only enhanced the enzymatic saccharification but also produced KGA up to 92% of the maximum theoretical yield. PMID:25783627

  8. The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect?

    PubMed Central

    2011-01-01

    Background We and other workers have shown that accessory enzymes, such as β-glucosidase, xylanase, and cellulase cofactors, such as GH61, can considerably enhance the hydrolysis effectiveness of cellulase cocktails when added to pretreated lignocellulosic substrates. It is generally acknowledged that, among the several factors that hamper our current ability to attain efficient lignocellulosic biomass conversion yields at low enzyme loadings, a major problem lies in our incomplete understanding of the cooperative action of the different enzymes acting on pretreated lignocellulosic substrates. Results The reported work assessed the interaction between cellulase and xylanase enzymes and their potential to improve the hydrolysis efficiency of various pretreated lignocellulosic substrates when added at low protein loadings. When xylanases were added to the minimum amount of cellulase enzymes required to achieve 70% cellulose hydrolysis of steam pretreated corn stover (SPCS), or used to partially replace the equivalent cellulase dose, both approaches resulted in enhanced enzymatic hydrolysis. However, the xylanase supplementation approach increased the total protein loading required to achieve significant improvements in hydrolysis (an additive effect), whereas the partial replacement of cellulases with xylanase resulted in similar improvements in hydrolysis without increasing enzyme loading (a synergistic effect). The enhancement resulting from xylanase-aided synergism was higher when enzymes were added simultaneously at the beginning of hydrolysis. This co-hydrolysis of the xylan also influenced the gross fiber characteristics (for example, fiber swelling) resulting in increased accessibility of the cellulose to the cellulase enzymes. These apparent increases in accessibility enhanced the steam pretreated corn stover digestibility, resulting in three times faster cellulose and xylan hydrolysis, a seven-fold decrease in cellulase loading and a significant increase in

  9. Nutrient value of spray field forages fed to pigs and the use of feed enzymes to enhance nutrient digestibility.

    PubMed

    Passos, A A; Andrade, C; Phillips, C E; Coffey, M T; Kim, S W

    2015-04-01

    forages was poorly utilized. In conclusion, spray field forages including Bermuda grass, forage sorghum, and sweet sorghum can partly be utilized in pig feed to provide energy, although N is rather poorly digested. Feed enzymes could enhance both energy and N utilization in Bermuda grass but not sorghum. PMID:26020194

  10. Fuel ethanol production from pretreated agricultural residues by simultaneous saccharification and fermentation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The production of ethanol from any lignocellulosic biomass generally involves four process steps: feedstock pretreatment, enzymatic saccharification, fermentation, and ethanol recovery. Integration of these process steps is essential in order to reduce the overall production cost. Moreover, the enz...

  11. Switchgrass alkaline pretreatment, enzymatic saccharification, and fermentation with residual oligosaccharide product analysis by mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Switchgrass (SG) is a potential renewable biomass source for conversion to liquid biofuels. Efficient conversion requires effective strategies for pretreatment and enzymatic saccharification to fermentable sugars. Standard analysis of fermentation broth includes detection of monosaccharides and etha...

  12. Biological energy from the igneous rock enhances cell growth and enzyme activity.

    PubMed

    Lin, Y; Kuo, H; Chen, C; Kuo, S

    2000-08-01

    Some effects from natural resources might be ignored and unused by humans. Environmental hormesis could be a phenomena necessary to bio-organism existence on earth. Since 1919, radiation and some heavy metal hormesis from the environment were proved in various reports. In this study, igneous rock with very low radioactivity and high ferrous activity was measured by multichannel analyzer and inductively coupled plasma analyzer. The water treated by igneous rock, both directly soaked or indirectly in contact, induced increased activities of glucose oxidase, catalase, peroxidase, and superoxide dismutase. It also increased cell growth of SC-M1, HCT-15, Raji, and fibroblast cell lines. The water after treatment of igneous rock had no change in pH values, but displayed decreased conductivity values. We assume that the igneous rock could transfer energy to water to change the molecular structure or conformation of water cluster, or by radiation hormesis effect could then induce increased enzyme activity and cell growth. It is also possible that the energy from rock may combine radiation hormesis with other transferable biological energy forms to change water cluster conformation. PMID:11056378

  13. Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

    PubMed Central

    Yui, Daishi; Nishida, Yoichiro; Nishina, Tomoko; Mogushi, Kaoru; Tajiri, Mio; Ishibashi, Satoru; Ajioka, Itsuki; Ishikawa, Kinya; Mizusawa, Hidehiro; Murayama, Shigeo; Yokota, Takanori

    2015-01-01

    Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD. PMID:26637123

  14. Influence of blade tenderization, moisture enhancement and pancreatin enzyme treatment on the processing characteristics and tenderness of beef semitendinosus muscle.

    PubMed

    Pietrasik, Z; Aalhus, J L; Gibson, L L; Shand, P J

    2010-03-01

    The combined effect of blade tenderization (BT), moisture enhancement and enzymatic tenderization on drip loss, cook loss, Warner-Bratzler shear force (WBSF) and sensory characteristics of beef semitendinosus (ST) steaks from cattle under 30 months of age was investigated. Injection with phosphate/chloride solution improved tenderness and juiciness of ST muscles (P<0.01). No additional improvement in tenderness was observed with incorporation of a pancreatin enzyme preparation into the moisture enhancement solution (P>0.1). Injection of pancreatin alone tended to improve overall tenderness (P=0.09) and did not adversely affect other palatability attributes. Blade tenderization of ST muscles improved tenderness, as indicated by lower WBSF and increased sensory tenderness scores than for control samples, without decreasing flavour and juiciness. The results suggest that moisture enhancement and blade tenderization can be effectively utilized to reduce the variability in and improve both tenderness and palatability of ST muscles. Pancreatin was not particularly effective at the 0.02% level used. PMID:20374818

  15. Ultrasensitive enhanced chemiluminescence enzyme immunoassay for the determination of alpha-fetoprotein amplified by double-codified gold nanoparticles labels.

    PubMed

    Yang, Xiao-Yan; Guo, Ying-Shu; Bi, Sai; Zhang, Shu-Sheng

    2009-04-15

    A novel enhanced chemiluminescent (CL) immunoassay for ultrasensitive determination of alpha-fetoprotein (AFP) was reported. The method made full use of 4-(4'-iodo)phenylphenol (IPP) as a new potential signal enhancer and double-codified gold nanoparticles (DC-AuNPs) labels modified with horseradish peroxidase (HRP)-conjugated anti-AFP used for further signal amplification. This protocol involved a sandwich format, in which the antigen in the sample was first captured by the immobilized primary antibody on the surface of magnetic beads, and then recognized by the second antibody labeled with DC-AuNPs. The combination of the remarkable sensitivity of the enhanced CL method and the use of AuNPs as an anti-AFP-HRP carrier for the enzymatic signal amplification, provided a linear response range of AFP from 0.008 to 0.3 ng mL(-1) with an extremely low detection limit of 5 pg mL(-1), much lower than those achieved by the classical enzyme-linked immunosorbent assay (ELISA). This new system can be easily extended to a variety of immunodetection as well as DNA analysis. PMID:19152783

  16. L-malate enhances the gene expression of carried proteins and antioxidant enzymes in liver of aged rats.

    PubMed

    Zeng, X; Wu, J; Wu, Q; Zhang, J

    2015-01-01

    Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. To investigate the antioxidant mechanism of L-malate in the mitochondria, we analyzed the change in gene expression of two malate-aspartate shuttle (MAS)-related carried proteins (AGC, aspartate/glutamate carrier and OMC, oxoglutarate/malate carrier) in the inner mitochondrial membrane, and three antioxidant enzymes (CAT, SOD, and GSH-Px) in the mitochondria. The changes in gene expression of these proteins and enzymes were examined by real-time RT-PCR in the heart and liver of aged rats treated with L-malate. L-malate was orally administered in rats continuously for 30 days using a feeding atraumatic needle. We found that the gene expression of OMC and GSH-Px mRNA in the liver increased by 39 % and 38 %, respectively, in the 0.630 g/kg L-malate treatment group than that in the control group. The expression levels of SOD mRNA in the liver increased by 39 %, 56 %, and 78 % in the 0.105, 0.210, and 0.630 g/kg L-malate treatment groups, respectively. No difference were observed in the expression levels of AGC, OMC, CAT, SOD, and GSH-Px mRNAs in the heart of rats between the L-malate treatment and control groups. These results predicted that L-malate may increase the antioxidant capacity of mitochondria by enhancing the expression of mRNAs involved in the MAS and the antioxidant enzymes. PMID:25194133

  17. Impact of Cell Wall Acetylation on Corn Stover Hydrolysis by Cellulolytic and Xylanolytic Enzymes

    SciTech Connect

    Selig, M. J.; Adney, W. S.; Himmel, M. E.; Decker, S. R.

    2009-01-01

    Analysis of variously pretreated corn stover samples showed neutral to mildly acidic pretreatments were more effective at removing xylan from corn stover and more likely to maintain the acetyl to xylopyranosyl ratios present in untreated material than were alkaline treatments. Retention of acetyl groups in the residual solids resulted in greater resistance to hydrolysis by endoxylanase alone, although the synergistic combination of endoxylanase and acetyl xylan esterase enzymes permitted higher xylan conversions to be observed. Acetyl xylan esterase alone did little to improve hydrolysis by cellulolytic enzymes, although a direct relationship was observed between the enzymatic removal of acetyl groups and improvements in the enzymatic conversion of xylan present in substrates. In all cases, effective xylan conversions were found to significantly improve glucan conversions achievable by cellulolytic enzymes. Additionally, acetyl and xylan removal not only enhanced the respective initial rates of xylan and glucan conversion, but also the overall extents of conversion. This work emphasizes the necessity for xylanolytic enzymes during saccharification processes and specifically for the optimization of acetyl esterase and xylanase synergies when biomass processes include milder pretreatments, such as hot water or sulfite steam explosion.

  18. Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

    PubMed Central

    Gao, Zhuangqiang; Hou, Li; Xu, Mingdi; Tang, Dianping

    2014-01-01

    Methods based on enzyme labels have been developed for colorimetric immunoassays, but most involve poor sensitivity and are unsuitable for routine use. Herein, we design an enhanced colorimetric immunoassay for prostate-specific antigen (PSA) coupling with an enzyme-cascade-amplification strategy (ECAS-CIA). In the presence of target PSA, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification. Results indicated that the ECAS-CIA presents good responses toward PSA, and allows detection of PSA at a concentration as low as 0.05 ng mL−1. Intra- and inter-assay coefficients of variation are below 9.5% and 10.7%, respectively. Additionally, the methodology is validated for analysis of clinical serum specimens with consistent results obtained by PSA ELISA kit. Importantly, the ECAS-CIA opens a new horizon for protein diagnostics and biosecurity. PMID:24509941

  19. A highly sensitive immunosensor for calmodulin assay based on enhanced biocatalyzed precipitation adopting a dual-layered enzyme strategy.

    PubMed

    Fu, Ying; Liu, Kai; Sun, Qianqian; Lin, Bin; Lu, Danqin; Xu, Zhiai; Hu, Chen; Fan, Guangjian; Zhang, Shengping; Wang, Chuangui; Zhang, Wen

    2014-06-15

    Calmodulin (CaM) is a ubiquitous protein in eukaryotic cells, and it plays an important role in cancer progression. In this paper, a highly sensitive immunosensor adopting a dual-layered enzyme strategy was proposed for electrochemical detection of CaM. This immunosensor was constructed by introducing honeycomb-like mesoporous carbon (HMPC) as a sensor platform to sequentially immobilize antibody (Ab1), CaM and a multi-functionalized label. The label (HRP-PAupc-Ab1) was synthesized by covalently binding Ab1 and horseradish peroxidase (HRP) to poly(acrylic acid)-functionalized Au popcorn (PAupc) nanoparticles. A novel dual-layered enzyme strategy was employed by incubating HRP-secondary antibody (HRP-Ab2) onto the label surface and the enhanced biocatalyzed precipitation was therefore induced. This immunosensor exhibited satisfactory analytical performances for CaM detection with a linear response ranging from 5.0 pg mL(-1) to 100 ng mL(-1) and a detection limit of 1.5 pg mL(-1). The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF-7) with high sensitivity, which has shown great potency for cancer study. PMID:24508817

  20. Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.

    PubMed Central

    Crouch, C F

    1995-01-01

    AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM. PMID:7560174

  1. Moringa oleifera Enhances Liver Antioxidant Status via Elevation of Antioxidant Enzymes Activity and Counteracts Paracetamol-induced Hepatotoxicity.

    PubMed

    Uma, N; Fakurazi, S; Hairuszah, I

    2010-08-01

    This study investigated the role of antioxidant enzyme system following crude hydroethanolic extract of Moringa oleifera leaves (MO) in acute paracetamol (PCM) induced hepatotoxicity. Hydroethanolic extract (80%) of MO (200 mg/kg and 800 mg/kg; p.o) was pre-administered before a single oral dose of 3 g/kg PCM intoxication to male Sprague-Dawley rats. Pre-treatment of the extract was found to have reduced lipid peroxidation level when compared to the group treated with PCM only. The level of glutathione peroxidase (GPx), glutathione-Stransferase (GST) and glutathione reductase (GR) was restored to near normal in groups that were pre-treated with MO. Histopathological studies have further confirmed the hepatoprotective activity of MO compared to group treated with PCM only. The results obtained were comparable to silymarin (200 mg/kg; p.o). The MO extract was found to have significantly protected the liver against toxicity following PCM intoxication by enhancing the level of antioxidant enzyme activity. PMID:22691934

  2. Uncouplers Stimulate Photosynthesis in Intact Chloroplasts by Enhancing Light-Activation of Enzymes Regulated by the Ferredoxin-Thioredoxin System

    PubMed Central

    Rosa, Luciana; Whatley, F. Robert

    1981-01-01

    Some uncouplers stimulate CO2-dependent O2 evolution by intact spinach chloroplasts at pH 8.6. This effect is not due to alkalinization of the stroma. The stimulation is observed only when photosynthesis has been partly inhibited by the presence of H2O2, generated in a Mehler-type reaction by the broken chloroplasts which always contaminate the intact chloroplast preparations. The addition of methyl viologen increases the Mehler-type reaction and results in greater inhibition of photosynthesis. The addition of excess catalase stimulates photosynthesis by preventing accumulation of H2O2. The uncouplers stimulate photosynthesis primarily by enhancing the light-activation of enzymes that are regulated by the ferredoxin-thioredoxin system, and this effect results from the influence of the uncouplers on the redox poising of the ferredoxin in the intact chloroplasts. PMID:16661918

  3. Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase.

    PubMed

    Aminian, Alieh; Shirzadi, Bahareh; Azizi, Zahra; Maedler, Kathrin; Volkmann, Eike; Hildebrand, Nils; Maas, Michael; Treccani, Laura; Rezwan, Kurosch

    2016-12-01

    Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials. PMID:27612703

  4. Adjunctive albuterol enhances the response to enzyme replacement therapy in late-onset Pompe disease.

    PubMed

    Koeberl, Dwight D; Austin, Stephanie; Case, Laura E; Smith, Edward C; Buckley, Anne F; Young, Sarah P; Bali, Deeksha; Kishnani, Priya S

    2014-05-01

    Effective dosages for enzyme replacement therapy (ERT) in Pompe disease are much higher than for other lysosomal storage disorders, which has been attributed to low cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle. We have previously demonstrated the benefit of increased CI-MPR-mediated uptake of recombinant human acid-α-glucosidase during ERT in mice with Pompe disease following addition of albuterol therapy. Currently we have completed a pilot study of albuterol in patients with late-onset Pompe disease already on ERT for >2 yr, who were not improving further. The 6-min walk test (6MWT) distance increased in all 7 subjects at wk 6 (30±13 m; P=0.002), wk 12 (34±14 m; P=0.004), and wk 24 (42±37 m; P=0.02), in comparison with baseline. Grip strength was improved significantly for both hands at wk 12. Furthermore, individual subjects reported benefits; e.g., a female patient could stand up from sitting on the floor much more easily (time for supine to standing position decreased from 30 to 11 s), and a male patient could readily swing his legs out of his van seat (hip abduction increased from 1 to 2+ on manual muscle testing). Finally, analysis of the quadriceps biopsies suggested increased CI-MPR at wk 12 (P=0.08), compared with baseline. With the exception of 1 patient who succumbed to respiratory complications of Pompe disease in the first week, only mild adverse events have been reported, including tremor, transient difficulty falling asleep, and mild urinary retention (requiring early morning voiding). Therefore, this pilot study revealed initial safety and efficacy in an open label study of adjunctive albuterol therapy in patients with late-onset Pompe disease who had been stable on ERT with no improvements noted over the previous several years. PMID:24443373

  5. Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia

    DOE PAGESBeta

    Montella, Salvatore; Balan, Venkatesh; da Costa Sousa, Leonardo; Gunawan, Christa; Giacobbe, Simona; Pepe, Olimpia; Faraco, Vincenza

    2016-03-02

    Here, the lignocellulosic fractions of municipal solid waste (MSW) can be used as renewable resources due to the widespread availability, predictable and low pricing and suitability for most conversion technologies. In particular, after the typical paper recycling loop, the newspaper waste (NW) could be further valorized as feedstock in biorefinering industry since it still contains up to 70 % polysaccharides. In this study, two different physicochemical methods— ammonia fiber expansion (AFEX) and extractive ammonia (EA) were tested for the pretraetment of NW. Furthermore, based on the previously demonstrated ability of the recombinant enzymes endocellulase rCelStrep, α-larabinofuranosidase rPoAbf and its evolvedmore » variant rPoAbf F435Y/Y446F to improve the saccharification of different lignocellulosic pretreated biomasses (such as corn stover and Arundo donax), in this study these enzymes were tested for the hydrolysis of pretreated NW, with the aim of valorizing the lignocellulosic fractions of the MSW. In particular, a mixture of purified enzymes containing cellulases, xylanases and accessory hemicellulases, was chosen as reference mix and rCelStrep and rPoAbf or its variant were replaced to EGI and Larb. The results showed that these enzymatic mixes are not suitable for the hydrolysis of NW after AFEX or EA pretreatment. On the other hand, when the enzymes rCelStrep, rPoAbf and rPoAbf F435Y/Y446F were tested for their effect in hydrolysis of pretreated NW by addition to a commercial enzyme mixture, it was shown that the total polysaccharides conversion yield reached 37.32 % for AFEX pretreated NW by adding rPoAbf to the mix whilst the maximum sugars conversion yield for EA pretreated NW was achieved 40.80 % by adding rCelStrep. The maximum glucan conversion yield obtained (45.61 % for EA pretreated NW by adding rCelStrep to the commercial mix) is higher than or comparable to those reported in recent manuscripts adopting hydrolysis conditions similar to

  6. Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia.

    PubMed

    Montella, Salvatore; Balan, Venkatesh; da Costa Sousa, Leonardo; Gunawan, Christa; Giacobbe, Simona; Pepe, Olimpia; Faraco, Vincenza

    2016-03-01

    The lignocellulosic fractions of municipal solid waste (MSW) can be used as renewable resources due to the widespread availability, predictable and low pricing and suitability for most conversion technologies. In particular, after the typical paper recycling loop, the newspaper waste (NW) could be further valorized as feedstock in biorefinering industry since it still contains up to 70 % polysaccharides. In this study, two different physicochemical methods-ammonia fiber expansion (AFEX) and extractive ammonia (EA) were tested for the pretraetment of NW. Furthermore, based on the previously demonstrated ability of the recombinant enzymes endocellulase rCelStrep, α-L-arabinofuranosidase rPoAbf and its evolved variant rPoAbf F435Y/Y446F to improve the saccharification of different lignocellulosic pretreated biomasses (such as corn stover and Arundo donax), in this study these enzymes were tested for the hydrolysis of pretreated NW, with the aim of valorizing the lignocellulosic fractions of the MSW. In particular, a mixture of purified enzymes containing cellulases, xylanases and accessory hemicellulases, was chosen as reference mix and rCelStrep and rPoAbf or its variant were replaced to EGI and Larb. The results showed that these enzymatic mixes are not suitable for the hydrolysis of NW after AFEX or EA pretreatment. On the other hand, when the enzymes rCelStrep, rPoAbf and rPoAbf F435Y/Y446F were tested for their effect in hydrolysis of pretreated NW by addition to a commercial enzyme mixture, it was shown that the total polysaccharides conversion yield reached 37.32 % for AFEX pretreated NW by adding rPoAbf to the mix whilst the maximum sugars conversion yield for EA pretreated NW was achieved 40.80 % by adding rCelStrep. The maximum glucan conversion yield obtained (45.61 % for EA pretreated NW by adding rCelStrep to the commercial mix) is higher than or comparable to those reported in recent manuscripts adopting hydrolysis conditions similar to those used

  7. Cloning, Expression, and Purification of Xylanase Gene from Bacillus licheniformis for Use in Saccharification of Plant Biomass.

    PubMed

    Zafar, Asma; Aftab, Muhammad Nauman; Din, Zia Ud; Aftab, Saima; Iqbal, Irfana; Shahid, Anam; Tahir, Arifa; Haq, Ikram Ul

    2016-01-01

    The xylanase gene (xynA) of Bacillus licheniformis 9945A was cloned and expressed in Escherichia coli BL21(DE3) using pET-22b(+) as an expression vector. The recombinant xylanase enzyme was purified by ammonium sulfate precipitation, followed by single-step immobilized metal ion affinity chromatography with a 57.58-fold purification having 138.2 U/mg specific activity and recovery of 70.08 %. Molecular weight of the purified xylanase, 23 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable for up to 70 °C with a broad pH range of 4-9 pH units. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA, indicating that the xylanase was a metalloenzyme. However, an addition of 1-4 % Tween 80, β-mercaptoethanol, and DTT resulted in the increase of enzyme activity by 51, 52, and 5 %, respectively. Organic solvents with a concentration of 10-40 % slightly decreased the enzyme activity. The xylanase enzyme possesses the ability of bioconversion of plant biomasses like wheat straw, rice straw, and sugarcane bagasse. Among the different tested biomasses, the highest saccharification percentage was observed with 1 % sugarcane bagasse after 72 h of incubation at 50 °C with 20 units of enzyme. The results suggest that recombinant xylanase can be used in the bioconversion of natural biomasses into simple sugars which could be further used for the production of biofuel. PMID:26438315

  8. Sorghum mutant RG displays antithetic leaf shoot lignin accumulation resulting in improved stem saccharification properties

    PubMed Central

    2013-01-01

    Background Improving saccharification efficiency in bioenergy crop species remains an important challenge. Here, we report the characterization of a Sorghum (Sorghum bicolor L.) mutant, named REDforGREEN (RG), as a bioenergy feedstock. Results It was found that RG displayed increased accumulation of lignin in leaves and depletion in the stems, antithetic to the trend observed in wild type. Consistent with these measurements, the RG leaf tissue displayed reduced saccharification efficiency whereas the stem saccharification efficiency increased relative to wild type. Reduced lignin was linked to improved saccharification in RG stems, but a chemical shift to greater S:G ratios in RG stem lignin was also observed. Similarities in cellulose content and structure by XRD-analysis support the correlation between increased saccharification properties and reduced lignin instead of changes in the cellulose composition and/or structure. Conclusion Antithetic lignin accumulation was observed in the RG mutant leaf-and stem-tissue, which resulted in greater saccharification efficiency in the RG stem and differential thermochemical product yield in high lignin leaves. Thus, the red leaf coloration of the RG mutant represents a potential marker for improved conversion of stem cellulose to fermentable sugars in the C4 grass Sorghum. PMID:24103129

  9. Optimal production of 4-deoxy-L-erythro-5-hexoseulose uronic acid from alginate for brown macro algae saccharification by combining endo- and exo-type alginate lyases.

    PubMed

    Wang, Da Mao; Kim, Hee Taek; Yun, Eun Ju; Kim, Do Hyoung; Park, Yong-Cheol; Woo, Hee Chul; Kim, Kyoung Heon

    2014-10-01

    Algae are considered as third-generation biomass, and alginate is the main component of brown macroalgae. Alginate can be enzymatically depolymerized by alginate lyases into uronate monomers, such as mannuronic acid and guluronic acid, which are further nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). We have optimized an enzymatic saccharification process using two recombinant alginate lyases, endo-type Alg7D and exo-type Alg17C, for the efficient production of DEH from alginate. When comparing the sequential and simultaneous additions of Alg7D and Alg17C, it was found that the final yield of DEH was significantly higher when the enzymes were added sequentially. The progress of saccharification reactions and production of DEH were verified by thin layer chromatography and gas chromatography-mass spectrometry, respectively. Our results showed that the two recombinant enzymes could be exploited for the efficient production of DEH that is the key substrate for producing biofuels from brown macro algal biomass. PMID:24794171

  10. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    SciTech Connect

    Resch, Michael G.; Donohoe, Byron; Ciesielski, Peter; Nill, Jennifer; McKinney, Kellene; Mittal, Ashutosh; Katahira, Rui; Himmel, Michael; Biddy, Mary; Beckham, Gregg; Decker, Steve

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution.

  11. Formation of disulfide bonds in insect prophenoloxidase enhances immunity through improving enzyme activity and stability.

    PubMed

    Lu, Anrui; Peng, Qin; Ling, Erjun

    2014-06-01

    Type 3 copper proteins, including insect prophenoloxidase (PPO), contain two copper atoms in the active site pocket and can oxidize phenols. Insect PPO plays an important role in immunity. Insects and other invertebrates show limited recovery from pathogen invasion and wounds if phenoloxidase (PO) activity is low. In most insect PPOs, two disulfide bonds are present near the C-terminus. However, in Pimpla hypochondriaca (a parasitoid wasp), each PPO contains one disulfide bond. We thus questioned whether the formation of two sulfide bonds in insect PPOs improved protein stability and/or increased insect innate immunity over time. Using Drosophila melanogaster PPO1 as a model, one or two disulfide bonds were deleted to evaluate the importance of disulfide bonds in insect immunity. rPPO1 and mutants lacking disulfide bonds could be expressed and showed PO activity. However, the PO activities of mutants lacking one or two disulfide bonds significantly decreased. Deletion of disulfide bonds also reduced PPO thermostability. Furthermore, antibacterial activities against Escherichia coli and Bacillus subtilis significantly decreased when disulfide bonds were deleted. Therefore, the formation of two disulfide bond(s) in insect PPO enhances antibacterial activity by increasing PO activity and stability. PMID:24480295

  12. Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy

    PubMed Central

    Rigat, Brigitte A.; Tropak, Michael B.; Buttner, Justin; Crushell, Ellen; Benedict, Daphne; Callahan, John W.; Martin, Douglas R.; Mahuran, Don J.

    2012-01-01

    Deficiencies of lysosomal β-D-galactosidase can result in GM1 gangliosidosis, a severe neurodegenerative disease characterized by massive neuronal storage of GM1 ganglioside in the brain. Currently there are no available therapies that can even slow the progression of this disease. Enzyme enhancement therapy utilizes small molecules that can often cross the blood brain barrier, but are also often competitive inhibitors of their target enzyme. It is a promising new approach for treating diseases, often caused by missense mutations, associated with dramatically reduced levels of functionally folded enzyme. Despite a number of positive reports based on assays performed with patient cells, skepticism persists that an inhibitor-based treatment can increase mutant enzyme activity in vivo. To date no appropriate animal model, i.e., one that recapitulates a responsive human genotype and clinical phenotype, has been reported that could be used to validate enzyme enhancement therapy. In this report, we identify a novel enzyme enhancement-agent, N-nonyl-deoxygalactonojirimycin, that enhances the mutant β-galactosidase activity in the lysosomes of a number of patient cell lines containing a variety of missense mutations. We then demonstrate that treatment of cells from a previously described, naturally occurring feline model (that biochemically, clinically and molecularly closely mimics GM1 gangliosidosis in humans) with this molecule, results in a robust enhancement of their mutant lysosomal β-galactosidase activity. These data indicate that the feline model could be used to validate this therapeutic approach and determine the relationship between the disease stage at which this therapy is initiated and the maximum clinical benefits obtainable. PMID:22784478

  13. White-rot fungal pretreatment of wheat straw with Phanerochaete chrysosporium for biohydrogen production: simultaneous saccharification and fermentation.

    PubMed

    Zhi, Zelun; Wang, Hui

    2014-07-01

    This paper demonstrates biohydrogen production was enhanced by white-rot fungal pretreatment of wheat straw (WS) through simultaneous saccharification and fermentation (SSF). Wheat straw was pretreated by Phanerochaete chrysosporium at 30 °C under solid state fermentation for 12 days, and lignin was removed about 28.5 ± 1.3 %. Microscopic structure observation combined thermal gravity and differential thermal gravity analysis further showed that the lignocellulose structure obviously disrupted after fungal pretreatment. Subsequently, the pretreated WS and crude cellulases prepared from Trichoderma atroviride were applied in SSF for hydrogen production using Clostridium perfringens. The maximum hydrogen yield was obtained to be 78.5 ± 3.4 ml g(-1)-pretreated WS, which was about 1.8-fold than the unpretreated group. Furthermore, the modified Gompertz model was applied study the progress of cumulative H(2) production. This work developed a novel bio-approach to improve fermentative H(2) yield from lignocellulosic biomass. PMID:24429553

  14. Enhancement of trypsin-like enzymes by A23187 ionophore is crucial for sperm penetration through the egg vestment of the giant freshwater prawn.

    PubMed

    Watthammawut, Atthaboon; Somrit, Monsicha; Asuvapongpatana, Somluk; Weerachatyanukul, Wattana

    2015-12-01

    We report the presence of trypsin-like enzymes preferring Boc-QAR-MCA substrate in sperm collected from different portions of male reproductive tracts of the giant freshwater prawn, Macrobrachium rosenbergii and compare enzyme activities before and after an A23187 calcium ionophore treatment. Fluorogenic enzyme assays revealed that testicular sperm lysates showed high trypsin-like enzyme activity but the activity was relatively low in vas deferens sperm lysates as well as in the live sperm. Upon sperm treatment with A23187, trypsin-like activity was greatly enhanced in distal vas deferens sperm. Substrate- and inhibitor-based localization studies indicated that the sperm trypsin-like enzymes were not of a soluble type but were rather of a membrane-borne type, localized at the anterior spike and upper part of the main body. Notable structural changes were also evident in A23187-induced sperm including extensive ruffling of the sperm membrane structure at the base of the main body thereby supporting the acrosome reaction response in this species. We further proved by substrate inhibition assays that the enhanced trypsin-like enzyme activity participates in sperm penetration through the vitelline envelope, a novel sperm-egg penetration mechanism that is unique in this species. PMID:26123168

  15. Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

    PubMed

    Eudes, Aymerick; Sathitsuksanoh, Noppadon; Baidoo, Edward E K; George, Anthe; Liang, Yan; Yang, Fan; Singh, Seema; Keasling, Jay D; Simmons, Blake A; Loqué, Dominique

    2015-12-01

    Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass. PMID:25583257

  16. Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

    DOE PAGESBeta

    Eudes, Aymerick; Sathitsuksanoh, Noppadon; Baidoo, Edward E. K.; George, Anthe; Liang, Yan; Yang, Fan; Singh, Seema; Keasling, Jay D.; Simmons, Blake A.; Loqué, Dominique

    2015-01-13

    Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimatemore » dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass.« less

  17. Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

    SciTech Connect

    Eudes, Aymerick; Sathitsuksanoh, Noppadon; Baidoo, Edward E. K.; George, Anthe; Liang, Yan; Yang, Fan; Singh, Seema; Keasling, Jay D.; Simmons, Blake A.; Loqué, Dominique

    2015-01-13

    Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass.

  18. Predicting enzyme adsorption to lignin films by calculating enzyme surface hydrophobicity.

    PubMed

    Sammond, Deanne W; Yarbrough, John M; Mansfield, Elisabeth; Bomble, Yannick J; Hobdey, Sarah E; Decker, Stephen R; Taylor, Larry E; Resch, Michael G; Bozell, Joseph J; Himmel, Michael E; Vinzant, Todd B; Crowley, Michael F

    2014-07-25

    The inhibitory action of lignin on cellulase cocktails is a major challenge to the biological saccharification of plant cell wall polysaccharides. Although the mechanism remains unclear, hydrophobic interactions between enzymes and lignin are hypothesized to drive adsorption. Here we evaluate the role of hydrophobic interactions in enzyme-lignin binding. The hydrophobicity of the enzyme surface was quantified using an estimation of the clustering of nonpolar atoms, identifying potential interaction sites. The adsorption of enzymes to lignin surfaces, measured using the quartz crystal microbalance, correlates to the hydrophobic cluster scores. Further, these results suggest a minimum hydrophobic cluster size for a protein to preferentially adsorb to lignin. The impact of electrostatic contribution was ruled out by comparing the isoelectric point (pI) values to the adsorption of proteins to lignin surfaces. These results demonstrate the ability to predict enzyme-lignin adsorption and could potentially be used to design improved cellulase cocktails, thus lowering the overall cost of biofuel production. PMID:24876380

  19. Predicting Enzyme Adsorption to Lignin Films by Calculating Enzyme Surface Hydrophobicity*

    PubMed Central

    Sammond, Deanne W.; Yarbrough, John M.; Mansfield, Elisabeth; Bomble, Yannick J.; Hobdey, Sarah E.; Decker, Stephen R.; Taylor, Larry E.; Resch, Michael G.; Bozell, Joseph J.; Himmel, Michael E.; Vinzant, Todd B.; Crowley, Michael F.

    2014-01-01

    The inhibitory action of lignin on cellulase cocktails is a major challenge to the biological saccharification of plant cell wall polysaccharides. Although the mechanism remains unclear, hydrophobic interactions between enzymes and lignin are hypothesized to drive adsorption. Here we evaluate the role of hydrophobic interactions in enzyme-lignin binding. The hydrophobicity of the enzyme surface was quantified using an estimation of the clustering of nonpolar atoms, identifying potential interaction sites. The adsorption of enzymes to lignin surfaces, measured using the quartz crystal microbalance, correlates to the hydrophobic cluster scores. Further, these results suggest a minimum hydrophobic cluster size for a protein to preferentially adsorb to lignin. The impact of electrostatic contribution was ruled out by comparing the isoelectric point (pI) values to the adsorption of proteins to lignin surfaces. These results demonstrate the ability to predict enzyme-lignin adsorption and could potentially be used to design improved cellulase cocktails, thus lowering the overall cost of biofuel production. PMID:24876380

  20. Enhanced binding of capsular polysaccharides of Cryptococcus neoformans to polystyrene microtitration plates for enzyme-linked immunosorbent assay.

    PubMed

    Cherniak, R; Cheeseman, M M; Reyes, G H; Reiss, E; Todaro, F

    1988-01-01

    A sensitive enzyme-linked immunosorbent assay (ELISA) to measure antibodies against capsular polysaccharide was developed, based on the enhanced binding of polysaccharide to polystyrene microtitration plates. The wells of the microtitration plate were primed with an adipic acid dihydrazide derivative of bovine serum albumin (AH-BSA) (100 micrograms/mL, 0.01 M NaPO4-0.14 M NaCl, pH 7.2 (PBS]. Capsular polysaccharide, the glucuronoxylomannan of Cryptococcus neoformans serotype A, was oxidized with NaIO4 for 5 min; the reaction was then quenched with ethylene glycol. The partially oxidized polysaccharide was dialyzed vs. PBS, and its concentration was adjusted to 50 micrograms/mL with PBS. This solution (100 microL/well) was covalently bound to the AH-BSA primed microtitration plates through formation of a Schiff base between the hydrazide group on the AH-BSA and the aldehyde groups on the polysaccharide. Antimouse IgG-alkaline phosphatase conjugate was used in an indirect ELISA to measure captured murine monoclonal antibodies directed against glucuronoxylomannan. Mean absorbances, after 15 min, were 0.13 in negative control wells, and greater than 0.7 in test wells. No intermediate steps were required to block nonspecific binding of antibody. PMID:3064947

  1. Comparison of separate hydrolysis and fermentation versus simultaneous saccharification and fermentation of pretreated wheat straw to ethanol by Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethanol production by Saccharomyces cerevisiae NRRL Y- 2034 from wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of glucose from dilute acid pretreated WS (86 g L-1) after enzymatic saccharification was 2...

  2. Chemical characterization of hydrothermally pretreated and enzyme-digested wheat straw: An evaluation of recalcitrance.

    PubMed

    Merali, Zara; Marjamaa, Kaisa; Käsper, Andres; Kruus, Kristiina; Gunning, A Patrick; Morris, Vic J; Waldron, Keith W

    2016-05-01

    There is great interest in understanding changes that occur to cell wall constituents during saccharification of pretreated lignocellulose, particularly in relation to recalcitrance of the residues. This paper reports the effects of hydrothermal pretreatment followed by enzyme hydrolysis on the extractability and properties of recalcitrant wheat straw polymers. The results show that the undigested residue had lost much of its archestructure. Compositional analysis portrayed a considerable loss of cross-linking di-ferulic acid phenolics, hemicellulosic and cellulosic sugars. The remaining cellulosic and non-cellulosic polysaccharides were much more readily extractable in alkali and molecular profiling revealed the presence of low Mw oligomers in the fractions suggesting the partial enzyme hydrolysis of hemicelluloses and cellulose. Simultaneous saccharification and fermentation of the pretreated and enzyme-digested residues surprisingly resulted in ethanol yields of up to 99% of the theoretical. This is discussed in relation to the "recalcitrant" nature of the original pretreated and enzyme digested biomass. PMID:26769515

  3. Bioethanol production from pretreated Melaleuca leucadendron shedding bark--simultaneous saccharification and fermentation at high solid loading.

    PubMed

    Ahmed, Ibrahim Nasser; Nguyen, Phuong Lan Tran; Huynh, Lien Huong; Ismadji, Suryadi; Ju, Yi-Hsu

    2013-05-01

    Bioethanol production from the shedding bark of Melaleuca leucadendron (Paper-bark Tree, PBT) was studied using subcritical water (SCW) pretreatment at various severities (So). High ethanol production was attained by implementing a factorial design on three parameters (So, solid loading and enzyme loading) in simultaneous saccharification and fermentation (SSF) mode. Ethanol concentration of 63.2 g L(-1) corresponding to ethanol yield of 80.9% were achieved from pretreated biomass (So=2.37) at 0.25 g mL(-1) solid and 16 FPU g(-1) glucan enzyme loadings. Similarly at 0.15 g mL(-1) solid loadings both high ethanol concentration (43.7 g L(-1)) and high ethanol yield (91.25%) were achieved. Regression analysis of experimental results shows that all process parameters had significant role on maximum ethanol production, glucose solubility, ethanol yield and ethanol volumetric productivity. SSF of SCW treated PBT biomass is economically feasible for production of bioethanol. PMID:23570711

  4. Aspen pectate lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield

    PubMed Central

    2014-01-01

    Background Wood cell walls are rich in cellulose, hemicellulose and lignin. Hence, they are important sources of renewable biomass for producing energy and green chemicals. However, extracting desired constituents from wood efficiently poses significant challenges because these polymers are highly cross-linked in cell walls and are not easily accessible to enzymes and chemicals. Results We show that aspen pectate lyase PL1-27, which degrades homogalacturonan and is expressed at the onset of secondary wall formation, can increase the solubility of wood matrix polysaccharides. Overexpression of this enzyme in aspen increased solubility of not only pectins but also xylans and other hemicelluloses, indicating that homogalacturonan limits the solubility of major wood cell wall components. Enzymatic saccharification of wood obtained from PL1-27-overexpressing trees gave higher yields of pentoses and hexoses than similar treatment of wood from wild-type trees, even after acid pretreatment. Conclusions Thus, the modification of pectins may constitute an important biotechnological target for improved wood processing despite their low abundance in woody biomass. PMID:24450583

  5. Dieckol enhances the expression of antioxidant and detoxifying enzymes by the activation of Nrf2-MAPK signalling pathway in HepG2 cells.

    PubMed

    Lee, Min-Sup; Lee, Bonggi; Park, Kyoung-Eun; Utsuki, Tadanobu; Shin, Taisun; Oh, Chul Woong; Kim, Hyeung-Rak

    2015-05-01

    Dieckol was previously reported to exhibit antioxidant and anticancer activities in vitro studies. In this study, we characterised the mechanism underlying the dieckol-mediated expression of antioxidant and detoxifying enzymes. Dieckol suppressed the production of intracellular reactive oxygen species in the presence or absence of H2O2 and increased glutathione level in HepG2 cells. Dieckol enhanced the activities of antioxidant enzymes, and the expression of detoxifying enzymes including heme oxygenase-1 (HO-1), NAD(P)H:quinine oxidoreductase 1 (NQO1), and glutathione S-transferase (GST) in HepG2 cells. Enhanced expression of antioxidant and detoxifying enzymes by dieckol was presumed to be the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2) demonstrated by its nuclear translocation and transcriptional activity via activation of mitogen-activated protein kinases in HepG2 cells. Furthermore, we demonstrated dieckol induced the expression of HO-1 in mouse liver. These results demonstrate that the dieckol-mediated cytoprotection in HepG2 cells is mediated through a ROS-independent up-regulation of antioxidant and detoxifying enzymes via Nrf2 activation as well as its intrinsic antioxidant activity, suggesting that dieckol may be used as a natural cytoprotective agent. PMID:25529716

  6. Enzyme Systems for Optimization of Ionic-Liquid Pretreated Biomass Saccharification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biomass, which consists of cellulose, hemicellulose, and lignin, is being developed as a renewable carbon source for fuel and chemical production. The use of biomass as a renewable carbon source requires that it be hydrolyzed to monomeric sugars through efficient processes. The process which shows t...

  7. Improvement of saccharification process for bioethanol production from Undaria sp. by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Yoon, Minchul; Choi, Jong-il; Lee, Ju-Woon; Park, Don-Hee

    2012-08-01

    Recently, many research works have reported on improvements to the saccharification process that increase bioethanol production from cellulosic materials. Gamma irradiation has been studied as an effective method for the depolymerization of complex polysaccharides. In this study, the effect of gamma irradiation on saccharification of Undaria biomass for bioethanol production was investigated. The Undaria biomass was irradiated at doses of 0, 10, 50, 100, 200 and 500 kGy and then hydrolyzed using sulfuric acid. The effects of gamma irradiation were measured through microscopic analysis to determine morphological changes and concentration of the reducing sugar of hydrolysates. Microscopic images show that gamma irradiation causes structure breakage of the Undaria cell wall. The concentration of reducing sugar of hydrolysates significantly increased as a result of gamma irradiation, with or without acid hydrolysis. These results indicate that the combined method of gamma irradiation with acid hydrolysis can significantly improve the saccharification process for bioethanol production from marine algae materials.

  8. Saccharification of Miscanthus x giganteus, incorporation of lignocellulosic by-product in cementitious matrix.

    PubMed

    Le Ngoc Huyen, Tran; Queneudec T'kint, Michèle; Remond, Caroline; Chabbert, Brigitte; Dheilly, Rose-Marie

    2011-11-01

    Given the non competition of miscanthus with food and animal feed, this lignocellulosic species has attracted attention as a possible biofuel resource. However, sustainability of ethanol production from lignocelluloses biomass would imply reduction in the consumption of chemicals and/or energetic means, but also valorization of the lignocellulosic by-product remaining from enzymatic saccharification. Introduction of these by-products into a cementitious matrix could be used in manufacturing a lightweight composite. Miscanthus biomass was submitted to chemical pretreatments followed by saccharification using an enzymatic cocktail. Residues from saccharification were then mixed with a cementitious matrix. Given their mechanical properties and a good adherence between cement and by-product, the hardened materials could be used. However, the delay in the beginning of setting time is too long, which prevents the direct use of by-product into cementitious matrix. Preliminary experiments using a setting accelerator in the cementitious matrix permitted significant reduction in the setting time delay. PMID:22078741

  9. Amphipathic lignin derivatives to accelerate simultaneous saccharification and fermentation of unbleached softwood pulp for bioethanol production.

    PubMed

    Cheng, Ningning; Yamamoto, Yoko; Koda, Keiichi; Tamai, Yutaka; Uraki, Yasumitsu

    2014-12-01

    Amphipathic lignin derivatives (A-LDs) were already demonstrated to improve enzymatic saccharification of lignocellulose. Based on this knowledge, two kinds of A-LDs prepared from black liquor of soda pulping of Japanese cedar were applied to a fed-batch simultaneous saccharification and fermentation (SSF) process for unbleached soda pulp of Japanese cedar to produce bioethanol. Both lignin derivatives slightly accelerated yeast fermentation of glucose but not inhibited it. In addition, ethanol yields based on the theoretical maximum ethanol production in the fed-batch SSF process was increased from 49% without A-LDs to 64% in the presence of A-LDs. PMID:25291627

  10. Programmable Self-Assembly of DNA-Protein Hybrid Hydrogel for Enzyme Encapsulation with Enhanced Biological Stability.

    PubMed

    Wan, Lan; Chen, Qiaoshu; Liu, Jianbo; Yang, Xiaohai; Huang, Jin; Li, Li; Guo, Xi; Zhang, Jue; Wang, Kemin

    2016-04-11

    A DNA-protein hybrid hydrogel was constructed based on a programmable assembly approach, which served as a biomimetic physiologic matrix for efficient enzyme encapsulation. A dsDNA building block tailored with precise biotin residues was fabricated based on supersandwich hybridization, and then the addition of streptavidin triggered the formation of the DNA-protein hybrid hydrogel. The biocompatible hydrogel, which formed a flower-like porous structure that was 6.7 ± 2.1 μm in size, served as a reservoir system for enzyme encapsulation. Alcohol oxidase (AOx), which served as a representative enzyme, was encapsulated in the hybrid hydrogel using a synchronous assembly approach. The enzyme-encapsulated hydrogel was utilized to extend the duration time for ethanol removal in serum plasma and the enzyme retained 78% activity after incubation with human serum for 24 h. The DNA-protein hybrid hydrogel can mediate the intact immobilization on a streptavidin-modified and positively charged substrate, which is very beneficial to solid-phase biosensing applications. The hydrogel-encapsulated enzyme exhibited improved stability in the presence of various denaturants. For example, the encapsulated enzyme retained 60% activity after incubation at 55 °C for 30 min. The encapsulated enzyme also retains its total activity after five freeze-thaw cycles and even suspended in solution containing organic solvents. PMID:27008186

  11. A solid-phase test using antiglobulin and enzyme-enhanced techniques for erythrocyte antibody screening during pregnancy.

    PubMed

    Sallander, S; Shanwell, A; Pegert, S; Jakobsson, L; Wiechel, B

    1995-01-01

    We have modified previously described solid-phase tests for erythrocyte antibody screening to develop a method, suitable for antiglobulin- and enzyme-enhanced techniques (SPH-IAT and SPH-ENZ). In this study we compared the SPH tests with an autoanalyzer (AA) technique. The results were more specific with the SPH tests than with the AA. Of 4,234 unselected samples from pregnant women, screen-positive samples were reduced from 96 (2.27%) by the AA, to 56 (1.32%) by the SPH tests. This difference was due to the reduced number of false-positive reactions with the SPH tests, 0.47% compared to 1.44% with the AA. Antibodies detected by the AA and the SPH-IAT and -ENZ were: 9 Rh prophylaxis, 2 anti-c, 1 anti-K and anti-M, and 1 anti-Jka. Antibodies detected only by the SPH tests were 1 anti-K, 1 anti-Le(a) (SPH-IAT and -ENZ), 1 anti-M (SPH-IAT) and 4 anti-Jka (SPH-ENZ). One anti-C, 2 anti-D, 3 Rh prophylaxis and 1 anti-E were detected by the AA and the SPH-ENZ but failed to react by the SPH-IAT. One anti-Le(a) and 8 Rh prophylaxis antibodies were detected by AA only. All clinically important antibodies were detected by the SPH tests. We conclude that the SPH-IAT and SPH-ENZ are screening methods with high specificity that are readily adaptable to larger series of samples from pregnant women and suitable for automated handling throughout the screening and identification process. PMID:7725668

  12. Plasmon-Enhanced Enzymatic Reactions 2:Optimization of Enzyme Activity by Surface Modification of Silver Island Films with Biotin-Poly (Ethylene-glycol)-Amine.

    PubMed

    Abel, Biebele; Aslan, Kadir

    2012-01-01

    Surface modification of silver island films (SIFs) was carried out with Biotin-Poly (Ethylene-glycol)-Amine (BEA), which acts as a cross-linker between the silver surface and horse radish peroxidase (HRP) enzyme for optimum plasmon-enhanced enzymatic activity. SIFs-deposited blank glass slides and SIFs-deposited 3-Aminopropyltriethoxysilane(APTES)-coated glass slides were used as our plasmonic surfaces.In this regard, three different extent of loading of SIFs were also prepared (low, medium and high) on APTES-coated glass slides. Streptavidin-linked HRP enzyme was attached to SIFs-deposited blank glass slides and SIFs-deposited APTES-coated glass slides through the well-known biotin-streptavidin interactions. The characterization of these surfaces was done using optical absorption spectroscopy. The loading of SIFs on glass slides was observed to have significant effect on the efficiency of plasmon-enhanced enzymatic activity, where an enhancement of 200% in the enzymatic activity was observed when compared to our previously used strategies for enzyme immobilization in our preceding work[1]. In addition, SIFs-deposited on APTES-coated glass slides were found to be re-usable for plasmon-enhanced enzymatic reactions unlike SIFs deposited on to blank glass slides. PMID:22485194

  13. Enzyme-triggered formation of enzyme-tyramine concatamers on nanogold-functionalized dendrimer for impedimetric detection of Hg(II) with sensitivity enhancement.

    PubMed

    Qiu, Zhenli; Tang, Dianyong; Shu, Jian; Chen, Guonan; Tang, Dianping

    2016-01-15

    A new impedimetric sensing strategy based on enzyme-triggered formation of enzyme-tyramine concatamers on the nanogold-functionalized poly(amidoamine) (PAMAM) dendrimer was designed for sensitive detection of mercury(II) (Hg(2+)) ion, coupling with enzymatic biocatalytic precipitation towards 4-choloro-1-naphthol (4-CN) on thymine (T)-rich single-stranded DNA1-modified electrode. Initially, nanogold-decorated PAMAM dendrimer (AuNP-PAMAM) was synthesized by the in-situ reduction method, and then functionalized with horseradish peroxidase (HRP) and another T-rich oligomer (DNA2). Upon target Hg(2+) introduction, probe DNA2 on the AuNP-PAMAM bound to the DNA1 on the electrode owing to the T-Hg(2+)-T coordination chemistry between the two DNA strands. Accompanying the AuNP-PAMAM, the carried HRP could trigger the formation of HRP-tyramine concatamer via the classical tyramine signal amplification strategy in the presence of HRP-tyramine conjugates and hydrogen peroxide. The concatenated HRP molecules in the concatamer catalyzed the 4-CN oxidation to produce an insoluble precipitation on the electrode, thereby resulting in the local alteration in the conductivity. Under optimal conditions, two signal-generation tags including HRP-AuNP-DNA2 and HRP-AuNP-PAMAM-DNA2 with or without tyramine signal amplification strategy (i.e., four schemes) were used for impedimetric detection of target Hg(2+) on the basis of the same assay format. A low detection limit (LOD) of 0.4pM and a wide dynamic working range of 0.001-100nM Hg(2+) by using HRP-AuNP-PAMAM-DNA2 with tyramine signal amplification strategy were obtained in comparison with those of other strategies. The assay had a good repeatability and showed an intermediate precision of down to 9.6%. In addition, the methodology also exhibited high specificity and selectivity towards target Hg(2+) against other metal ions, and was applicable for monitoring Hg(2+) in the spiked drinking water samples. PMID:26301998

  14. Enhanced hydrolysis of lignocellulosic biomass: Bi-functional enzyme complexes expressed in Pichia pastoris improve bioethanol production from Miscanthus sinensis.

    PubMed

    Shin, Sang Kyu; Hyeon, Jeong Eun; Kim, Young In; Kang, Dea Hee; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2015-12-01

    Lignocellulosic biomass is the most abundant utilizable natural resource. In the process of bioethanol production from lignocellulosic biomass, an efficient hydrolysis of cellulose and hemicellulose to release hexose and pentose is essential. We have developed a strain of Pichia pastoris that can produce ethanol via pentose and hexose using an assembly of enzyme complexes. The use of enzyme complexes is one of the strategies for effective lignocellulosic biomass hydrolysis. Xylanase XynB from Clostridium cellulovorans and a chimeric endoglucanase cCelE from Clostridium thermocellum were selected as enzyme subunits, and were bound to a recombinant scaffolding protein mini-CbpA from C. cellulovorans to assemble the enzyme complexes. These complexes efficiently degraded xylan and carboxymethylcellulose (CMC), producing approximately 1.18 and 1.07 g/L ethanol from each substrate, respectively, which is 2.3-fold and 2.7-fold higher than that of the free-enzyme expressing strain. Miscanthus sinensis was investigated as the lignocellulosic biomass for producing bioethanol, and 1.08 g/L ethanol was produced using our recombinant P. pastoris strain, which is approximately 1.9-fold higher than that of the wild-type strain. In future research, construction of enzyme complexes containing various hydrolysis enzymes could be used to develop biocatalysts that can completely degrade lignocellulosic biomass into valuable products such as biofuels. PMID:26479167

  15. Acylated Carrageenan Changes the Physicochemical Properties of Mixed Enzyme-Lipid Ultrathin Films and Enhances the Catalytic Properties of Sucrose Phosphorylase Nanostructured as Smart Surfaces.

    PubMed

    Rocha, Jefferson M; Pavinatto, Adriana; Nobre, Thatyane M; Caseli, Luciano

    2016-06-23

    Control over the catalytic activity of enzymes is important to construct biosensors with a wide range of detectability and higher stability. For this, immobilization of enzymes on solid supports as nanostructured films is a current approach that permits easy control of the molecular architecture as well as tuning of the properties. In this article, we employed acylated carrageenan (AC) mixed with phospholipids at the air-water interface to facilitate the adsorption of the enzyme sucrose phosphorylase (SP). AC stabilized the adsorption of SP at the phospholipid monolayer, as detected by tensiometry, by which thermodynamic parameters could be inferred from the surface pressure-area isotherm. Also, infrared spectroscopy applied in situ over the monolayer showed that the AC-phospholipid system not only permitted the enzyme to be adsorbed but also helped conserve its secondary structure. The mixed monolayers were then transferred onto solid supports as Langmuir-Blodgett (LB) films and investigated with transfer ratio, quartz crystal microbalance, fluorescence spectroscopy, and atomic force microscopy. The enzyme activity of the LB film was then determined, revealing that although there was an expected reduction in activity in relation to the homogeneous environment the activity could be better preserved after 1 month, revealing enhanced stability. PMID:27249064

  16. Kinetic characterization of glucose aerodehydrogenase from Aspergillus niger EMS-150-F after optimizing the dose of mutagen for enhanced production of enzyme

    PubMed Central

    Umbreen, Huma; Zia, Muhammad Anjum; Rasul, Samreen

    2013-01-01

    In the present study enhanced production of glucose aerodehydrogenase from Aspergillus niger has been achieved after optimizing the dose of chemical mutagen ethyl methane sulfonate (EMS) that has not been reported earlier. Different doses of mutagen were applied and a strain was developed basing upon the best production. The selected strain Aspergillus niger EMS-150-F was optimized for nutrient requirements in order to produce enzyme through fermentation and the results showed the best yield at 2% corn steep liquor (CSL), 36 hours fermentation time, pH 5, 30°C temperature, 0.3% KH2PO4, 0.3% urea and 0.06% CaCO3. The enzyme was then purified and resulted in 57.88 fold purification with 52.12% recovery. On kinetic characterization, the enzyme showed optimum activity at pH 6 and temperature 30°C. The Michaelis-Menton constants (Km, Vmax, Kcat and Kcat/Km) were 20 mM, 45.87 U mL−1, 1118.81 s−1 and 55.94 s−1 mM−1, respectively. The enzyme was found to be thermaly stable and the enthalpy and free energy showed an increase with increase in temperature and ΔS* was highly negative proving the enzyme from A. niger EMS-150-F resistant to temperature and showing a very little disorderliness. PMID:24688499

  17. Ultrasound enhanced glucose release from corn in ethanol plants.

    PubMed

    Khanal, Samir Kumar; Montalbo, Melissa; van Leeuwen, J; Srinivasan, Gowrishankar; Grewell, David

    2007-12-01

    This work evaluated the use of high power ultrasonic energy to treat corn slurry in dry corn milling ethanol plants to enhance liquefaction and saccharification for ethanol production. Corn slurry samples obtained before and after jet cooking were subjected to ultrasonic pretreatment for 20 and 40 s at amplitudes of vibration ranging from 180 to 299 microm(pp) (peak to peak amplitude in microm). The resulting samples were then exposed to enzymes (alpha-amylase and glucoamylase) to convert cornstarch into glucose. A comparison of scanning electron micrographs of raw and sonicated samples showed the development of micropores and the disruption of cell walls in corn mash. The corn particle size declined nearly 20-fold following ultrasonic treatment at high power settings. The glucose release rate from sonicated samples increased as much as threefold compared to the control group. The efficiency of ultrasound exceeded 100% in terms of energy gain from the sugar released over the ultrasonic energy supplied. Enzymatic activity was enhanced when the corn slurry was sonicated with simultaneous addition of enzymes. This finding suggests that the ultrasonic energy did not degrade or denature the enzymes during the pretreatment. PMID:17514753

  18. ENZYMATIC SACCHARIFICATION AND FERMENTATION OF ALKALINE PEROXIDE PRETREATED RICE HULLS TO ETHANOL

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rice hulls used in this study contained 35.62 +/- 0.12% cellulose and 11.96 +/- 0.73% hemicellulose. Alkaline H2O2 pretreatment and enzymatic saccharification methods were evaluated for conversion of rice hull cellulose and hemicellulose to simple sugars. The yield of sugars from diluted alkaline ...

  19. Industrial evaluation of a dynamic controller for simultaneous saccharification and fermentation process

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The dry grind corn industry is the largest ethanol producer in the US. Simultaneous saccharification and fermentation (SSF) is one of the most critical process steps that determines the ethanol yields and conversion efficiency of the whole process. Due to its complexity, the SSF process is not compl...

  20. Comparison of Pretreatment Strategies for Enzymatic Saccharification and Fermentation of Barley Straw to Ethanol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley straw used in this study contained 34.1±0.6% cellulose, 22.6±0.4% hemicellulose, and 13.3±0.2% lignin (moisture, 6.5±0.0%). Several pretreatments (dilute acid, lime, and alkaline peroxide) and enzymatic saccharification procedures were evaluated for the conversion of barley straw to monomeri...

  1. Simultaneous detoxification, saccharification, and ethanol fermentation of weak-acid hydrolyzates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignocellulosic feedstocks can be prepared for ethanol fermentation by pre-treatment with a dilute mineral acid catalyst that hydrolyzes the hemicellulose and opens up the plant cell wall fibers for subsequent enzymatic saccharification. The acid catalyzed reaction scheme is sequential whereby rele...

  2. Simultaneous saccharification and fermentation of industrial sweetpotatoes for ethanol production and anthocyanins extraction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A simultaneous saccharification fermentation (SSF) system was studied for ethanol production in flour industrial sweetpotato (ISP) feedstocks (lines: white DM02-180 and purple NC-413) as an integrated cost saving process, and to examine the feasibility of extracting anthocyanins from flour purple IS...

  3. Comparison of Dilute Acid and Ionic Liquid Pretreatment of Switchgrass: Biomass Recalcitrance, Delignification and Enzymatic Saccharification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The efficiency of two biomass pretreatment technologies, dilute acid hydrolysis and dissolution in an ionic liquid, are compared in terms of delignification, saccharification efficiency and saccharide yields with switchgrass serving as a model bioenergy crop. When subject to ionic liquid pretreatme...

  4. Alkaline peroxide pretreatment of corn stover for enzymatic saccharification and ethanol production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alkaline hydrogen peroxide (AHP) pretreatment and enzymatic saccharification were evaluated for conversion of corn stover cellulose and hemicellulose to fermentable sugars. Corn stover used in this study contained 37.0±0.2% cellulose, 26.8±0.2% hemicellulose and 18.0±0.1% lignin on dry basis. Unde...

  5. Utilization of radiation technique on the saccharification and fermentation of biomass

    NASA Astrophysics Data System (ADS)

    Kaetsu, I.; Kumakura, M.; Fujimura, T.; Yoshii, F.; Kojima, T.; Tamada, M.

    The application of irradiation technique to the process of saccharification and subsequent fermentation of cellulosic wastes such as chaff and rice straw to obtain ethanol, was investigated. It was found that when waste raw materials were irradiated by ?-ray or electron beam, they became accessible to the subsequent enzymatic saccharification reaction. Irradiation of 10 7-10 8 Rad was enough for this effect. Some kind of additives reduced necessary dosage for this pretreatment. Cellulase, Trichoderma reesei which produce cellulase, and yeast were immobilized as biocatalysts for biomass conversion by radiation-induced polymerization of glass-forming monomer at low temperature. The immobilized cellulase showed almost same activity of glucose production as the native cellulase. Continuous saccharification reaction was carried out by using the immobilized cellulase. The immobilized Trichoderma reesei and the immobilized yeast showed almost same activity as the intact biocatalysts. It was concluded that the continuous saccharification and subsequent fermentation could be carried out effectively by using the immobilized biocatalysts. Spinach chloroplasts were immobilized by the same method as the first step for the conversion of water into hydrogen gas using solar energy. The immobilized chloroplasts kept the O 2 evolution activity in storage more than 30 days at 4°C. Thermostatility of chloroplasts was also improved greatly by the immobilization.

  6. Evaluation of a recombinant insect-derived amylase performance in simultaneous saccharification and fermentation process with industrial yeasts.

    PubMed

    Celińska, Ewelina; Borkowska, Monika; Białas, Wojciech

    2016-03-01

    Starch is the dominant feedstock consumed for the bioethanol production, accounting for 60 % of its global production. Considering the significant contribution of bioethanol to the global fuel market, any improvement in its major operating technologies is economically very attractive. It was estimated that up to 40 % of the final ethanol unit price is derived from the energy input required for the substrate pre-treatment. Application of raw starch hydrolyzing enzymes (RSHE), combined with operation of the process according to a simultaneous saccharification and fermentation (SSF) strategy, constitutes the most promising solutions to the current technologies limitations. In this study, we expressed the novel RSHE derived from an insect in Saccharomyces cerevisiae strain dedicated for the protein overexpression. Afterwards, the enzyme performance was assessed in SSF process conducted by industrial ethanologenic or thermotolerant yeast species. Comparison of the insect-derived RSHE preparation with commercially available amylolytic RSH preparation was conducted. Our results demonstrate that the recombinant alpha-amylase from rice weevil can be efficiently expressed and secreted with its native signal peptide in S. cerevisiae INVSc-pYES2-Amy1 expression system (accounting for nearly 72 % of the strain's secretome). Application of the recombinant enzyme-based preparation in SSF process secured sufficient amylolytic activity for the yeast cell propagation and ethanol formation from raw starch. (Oligo)saccharide profiles generated by the compared preparations differed with respect to homogeneity of the sugar mixtures. Concomitantly, as demonstrated by a kinetic model developed in this study, the kinetic parameters describing activity of the compared preparations were different. PMID:26545757

  7. Kinetic study of batch and fed-batch enzymatic saccharification of pretreated substrate and subsequent fermentation to ethanol

    PubMed Central

    2012-01-01

    Background Enzymatic hydrolysis, the rate limiting step in the process development for biofuel, is always hampered by its low sugar concentration. High solid enzymatic saccharification could solve this problem but has several other drawbacks such as low rate of reaction. In the present study we have attempted to enhance the concentration of sugars in enzymatic hydrolysate of delignified Prosopis juliflora, using a fed-batch enzymatic hydrolysis approach. Results The enzymatic hydrolysis was carried out at elevated solid loading up to 20% (w/v) and a comparison kinetics of batch and fed-batch enzymatic hydrolysis was carried out using kinetic regimes. Under batch mode, the actual sugar concentration values at 20% initial substrate consistency were found deviated from the predicted values and the maximum sugar concentration obtained was 80.78 g/L. Fed-batch strategy was implemented to enhance the final sugar concentration to 127 g/L. The batch and fed-batch enzymatic hydrolysates were fermented with Saccharomyces cerevisiae and ethanol production of 34.78 g/L and 52.83 g/L, respectively, were achieved. Furthermore, model simulations showed that higher insoluble solids in the feed resulted in both smaller reactor volume and shorter residence time. Conclusion Fed-batch enzymatic hydrolysis is an efficient procedure for enhancing the sugar concentration in the hydrolysate. Restricting the process to suitable kinetic regimes could result in higher conversion rates. PMID:22433563

  8. Effect of non-enzymatic proteins on enzymatic hydrolysis and simultaneous saccharification and fermentation of different lignocellulosic materials.

    PubMed

    Wang, Hui; Kobayashi, Shinichi; Mochidzuki, Kazuhiro

    2015-08-01

    Non-enzymatic proteins were added during hydrolysis of cellulose and simultaneous saccharification and fermentation (SSF) of different biomass materials. Bovine serum albumin (BSA), a model non-enzymatic protein, increased cellulose and xylose conversion efficiency and also enhanced the ethanol yield during SSF of rice straw subjected to varied pretreatments. Corn steep liquor, yeast extract, and peptone also exerted a similar effect as BSA and enhanced the enzymatic hydrolysis of rice straw. Compared to the glucose yields obtained after enzymatic hydrolysis of rice straw in the absence of additives, the glucose yields after 72h of hydrolysis increased by 12.7%, 13.5%, and 13.7% after addition of the corn steep liquor, yeast extract, and peptone, respectively. This study indicated the use of BSA as an alternative to intensive pretreatment of lignocellulosic materials for enhancing enzymatic digestibility. The utilization of non-enzymatic protein additives is promising for application in glucose and ethanol production from lignocellulosic materials. PMID:25974351

  9. Enhancement of Synthetic Trichoderma-Based Enzyme Mixtures for Biomass Conversion with an Alternative Family 5 Glycosyl Hydrolase from Sporotrichum thermophile

    PubMed Central

    Ye, Zhuoliang; Zheng, Yun; Li, Bingyao; Borrusch, Melissa S.; Storms, Reginald; Walton, Jonathan D.

    2014-01-01

    Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4- glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior

  10. Enhancement of synthetic Trichoderma-based enzyme mixtures for biomass conversion with an alternative family 5 glycosyl hydrolase from Sporotrichum thermophile.

    PubMed

    Ye, Zhuoliang; Zheng, Yun; Li, Bingyao; Borrusch, Melissa S; Storms, Reginald; Walton, Jonathan D

    2014-01-01

    Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4-glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance

  11. Efficient chemical and enzymatic saccharification of the lignocellulosic residue from Agave tequilana bagasse to produce ethanol by Pichia caribbica.

    PubMed

    Saucedo-Luna, Jaime; Castro-Montoya, Agustin Jaime; Martinez-Pacheco, Mauro Manuel; Sosa-Aguirre, Carlos Ruben; Campos-Garcia, Jesus

    2011-06-01

    Bagasse of Agave tequilana (BAT) is the residual lignocellulosic waste that remains from tequila production. In this study we characterized the chemical composition of BAT, which was further saccharified and fermented to produce ethanol. BAT was constituted by cellulose (42%), hemicellulose (20%), lignin (15%), and other (23%). Saccharification of BAT was carried out at 147 °C with 2% sulfuric acid for 15 min, yielding 25.8 g/l of fermentable sugars, corresponding to 36.1% of saccharificable material (cellulose and hemicellulose contents, w/w). The remaining lignocellulosic material was further hydrolyzed by commercial enzymes, ~8.2% of BAT load was incubated for 72 h at 40 °C rendering 41 g/l of fermentable sugars corresponding to 73.6% of the saccharificable material (w/w). Mathematic surface response analysis of the acid and enzymatic BAT hydrolysis was used for process optimization. The results showed a satisfactory correlation (R (2) = 0.90) between the obtained and predicted responses. The native yeast Pichia caribbica UM-5 was used to ferment sugar liquors from both acid and enzymatic hydrolysis to ethanol yielding 50 and 87%, respectively. The final optimized process generated 8.99 g ethanol/50 g of BAT, corresponding to an overall 56.75% of theoretical ethanol (w/w). Thus, BAT may be employed as a lignocellulosic raw material for bioethanol production and can contribute to BAT residue elimination from environment. PMID:21072557

  12. High concentrations of cellulosic ethanol achieved by fed batch semi simultaneous saccharification and fermentation of waste-paper

    PubMed Central

    Elliston, Adam; Collins, Samuel R.A.; Wilson, David R.; Roberts, Ian N.; Waldron, Keith W.

    2013-01-01

    A fundamental goal of second generation ethanol production is to increase the ethanol concentration to 10% (v/v) or more to optimise distillation costs. Semi simultaneous saccharification and fermentations (SSSF) were conducted at small pilot scale (5 L) utilising fed-batch additions of solid shredded copier paper substrate. Early addition of Accellerase® 1500 at 16 FPU/g substrate and 30 U/g β-glucosidase followed by substrate only batch addition allowed low final equivalent enzyme concentrations to be achieved (3.7 FPU/g substrate) whilst maintaining digestion. Batch addition resulted in a cumulative substrate concentration equivalent to 65% (w/v). This in turn resulted in the production of high concentrations of ethanol (11.6% v/v). The success of this strategy relied on the capacity of the bioreactor to perform high shear mixing as required. Further research into the timing and number of substrate additions could lead to further improvement in overall yields from the 65.5% attained. PMID:23500568

  13. An ionic liquid tolerant cellulase derived from chemically polluted microhabitats and its application in in situ saccharification of rice straw.

    PubMed

    Xu, Jiaxing; He, Bingfang; Wu, Bin; Wang, Bin; Wang, Chenghua; Hu, Lei

    2014-04-01

    A cellulase-producing fungus was isolated from chemically polluted microhabitats by [Amim][Cl] enrichment and identified as Aspergillus fumigatus. The maximum activity of the cellulase in 30% (v/v) ionic liquids (ILs) was detected in [Emim][DMP], [Amim][Cl] and [Emim][MA] as 127%, 111% and 109%, respectively, of its activity in buffer, suggesting its superior performance in high concentration ILs. Strikingly, although its initial activity varied in each IL, its half-life was longer in most ILs than in buffer, evidence of a high conformational stability of the enzyme that is essential for maintaining the remaining activity in relevant media. It noteworthy that 1-3M NaCl can activate the cellulase somewhat. More gratifyingly, a compatible IL-cellulase system based on the cellulase was developed, and its use significantly improved the saccharification rate of rice straw from 53% to 88% versus the control, demonstrating its potential for efficient transformation of lignocellulose to glucose in a single-step process. PMID:24549238

  14. Thermostable α-amylase immobilization: Enhanced stability and performance for starch biocatalysis.

    PubMed

    Kumar, Gudi Satheesh; Rather, Gulam Mohmad; Gurramkonda, Chandrasekhar; Reddy, Bontha Rajasekhar

    2016-01-01

    The uses of thermostable starch hydrolytic biocatalysts are steadily increasing for the industrial application because of their obvious need for biocatalytic performance at elevated temperatures. The starch liquefaction and saccharification can be carried out simultaneously by the use of thermostable starch hydrolytic biocatalysts, thus minimizing the unit operations, time, and efforts. The cost factor hampers the industrialization of expensive soluble (free) enzymes for biocatalytic applications and the immobilization of enzymes offers promising alternative to the hurdle. The present investigation was aimed for immobilization of thermostable α-amylase using calcium alginate, and statistical optimization studies were carried out for enhanced biocatalytic performance. Initially, one-parameter at a time optimization studies were carried out for identification of significant factors influencing the immobilization. Furthermore, a statistical approach, response surface methodology, was applied for immobilization of α-amylase. The immobilized α-amylase in alginate microbeads showed enhanced stability to temperature and reusable property for up to seven cycles (with the retention of 50% initial activity). Finally, the kinetic behavior of free and immobilized enzyme showed the Km value of 1.2% and 2.6% (w/v) and Vmax of 1,020 and 1,030 U, respectively. Fifty percent reduction in affinity of the immobilized enzyme toward substrate was compensated by its longer stability. PMID:25604037

  15. Simultaneous saccharification and fermentation of Agave tequilana fructans by Kluyveromyces marxianus yeasts for bioethanol and tequila production.

    PubMed

    Flores, Jose-Axel; Gschaedler, Anne; Amaya-Delgado, Lorena; Herrera-López, Enrique J; Arellano, Melchor; Arrizon, Javier

    2013-10-01

    Agave tequilana fructans (ATF) constitute a substrate for bioethanol and tequila industries. As Kluyveromyces marxianus produces specific fructanases for ATF hydrolysis, as well as ethanol, it can perform simultaneous saccharification and fermentation. In this work, fifteen K. marxianus yeasts were evaluated to develop inoculums with fructanase activity on ATF. These inoculums were added to an ATF medium for simultaneous saccharification and fermentation. All the yeasts, showed exo-fructanhydrolase activity with different substrate specificities. The yeast with highest fructanase activity in the inoculums showed the lowest ethanol production level (20 g/l). Five K. marxianus strains were the most suitable for the simultaneous saccharification and fermentation of ATF. The volatile compounds composition was evaluated at the end of fermentation, and a high diversity was observed between yeasts, nevertheless all of them produced high levels of isobutyl alcohol. The simultaneous saccharification and fermentation of ATF with K. marxianus strains has potential for industrial application. PMID:23941710

  16. Enhancement of NNM-induced carcinogenesis in the rat liver by phenobarbital: a combined morphological and enzyme histochemical approach.

    PubMed

    Moore, M A; Hacker, H J; Kunz, H W; Bannasch, P

    1983-01-01

    The influence of sodium phenobarbital (PB) treatment on the sequence of N-nitrosomorpholine (NNM) induced focal preneoplastic lesions in the rat liver was investigated using a combined morphological and enzyme histochemical approach. Quantitative assessment of the different types of foci of altered hepatocytes visible in H&E sections after carcinogen application, namely the clear and acidophilic cell glycogen storage foci and mixed cell foci comprising glycogen storing cells and also more basophilic hepatocytes showing reduction in glycogen reserves, revealed a shift towards mixed cell character and greater size in PB-treated livers in comparison to those receiving NNM alone. Within the three dose levels of PB investigated (0.75, 0.075 or 0.0075 g/l drinking water) a clear dose dependence in appearance of mixed cell foci was apparent. Assessment of alterations in the activities of marker enzymes observed within preneoplastic foci was carried out by comparison of PAS preparations with sections reacted for glucose-6-phosphate dehydrogenase (G6PDH), gamma-glutamyl transpeptidase, glucose-6-phosphatase and adenosine triphosphatase. G6PDH proved the most consistent enzyme marker for small glycogen storage foci whereas larger foci of that type and mixed cell foci were associated with change in activity of all enzymes studied. The results are discussed in relation to the sequence of events occurring during hepatocarcinogenesis and the influence of PB on altered cellular populations. The applicability of enzyme markers is further considered in view of the question of heterogeneity within populations of preneoplastic foci. PMID:6132686

  17. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

  18. A joint analysis of transcriptomic and metabolomic data uncovers enhanced enzyme-metabolite coupling in breast cancer

    NASA Astrophysics Data System (ADS)

    Auslander, Noam; Yizhak, Keren; Weinstock, Adam; Budhu, Anuradha; Tang, Wei; Wang, Xin Wei; Ambs, Stefan; Ruppin, Eytan

    2016-07-01

    Disrupted regulation of cellular processes is considered one of the hallmarks of cancer. We analyze metabolomic and transcriptomic profiles jointly collected from breast cancer and hepatocellular carcinoma patients to explore the associations between the expression of metabolic enzymes and the levels of the metabolites participating in the reactions they catalyze. Surprisingly, both breast cancer and hepatocellular tumors exhibit an increase in their gene-metabolites associations compared to noncancerous adjacent tissues. Following, we build predictors of metabolite levels from the expression of the enzyme genes catalyzing them. Applying these predictors to a large cohort of breast cancer samples we find that depleted levels of key cancer-related metabolites including glucose, glycine, serine and acetate are significantly associated with improved patient survival. Thus, we show that the levels of a wide range of metabolites in breast cancer can be successfully predicted from the transcriptome, going beyond the limited set of those measured.

  19. A joint analysis of transcriptomic and metabolomic data uncovers enhanced enzyme-metabolite coupling in breast cancer

    PubMed Central

    Auslander, Noam; Yizhak, Keren; Weinstock, Adam; Budhu, Anuradha; Tang, Wei; Wang, Xin Wei; Ambs, Stefan; Ruppin, Eytan

    2016-01-01

    Disrupted regulation of cellular processes is considered one of the hallmarks of cancer. We analyze metabolomic and transcriptomic profiles jointly collected from breast cancer and hepatocellular carcinoma patients to explore the associations between the expression of metabolic enzymes and the levels of the metabolites participating in the reactions they catalyze. Surprisingly, both breast cancer and hepatocellular tumors exhibit an increase in their gene-metabolites associations compared to noncancerous adjacent tissues. Following, we build predictors of metabolite levels from the expression of the enzyme genes catalyzing them. Applying these predictors to a large cohort of breast cancer samples we find that depleted levels of key cancer-related metabolites including glucose, glycine, serine and acetate are significantly associated with improved patient survival. Thus, we show that the levels of a wide range of metabolites in breast cancer can be successfully predicted from the transcriptome, going beyond the limited set of those measured. PMID:27406679

  20. Iron-sulfur-based single molecular wires for enhancing charge transport in enzyme-based bioelectronic systems.

    PubMed

    Mahadevan, Aishwarya; Fernando, Teshan; Fernando, Sandun

    2016-04-15

    When redox enzymes are wired to electrodes outside a living cell (ex vivo), their ability to produce a sufficiently powerful electrical current diminishes significantly due to the thermodynamic and kinetic limitations associated with the wiring systems. Therefore, we are yet to harness the full potential of redox enzymes for the development of self-powering bioelectronics devices (such as sensors and fuel cells). Interestingly, nature uses iron-sulfur complexes ([Fe-S]), to circumvent these issues in vivo. Yet, we have not been able to utilize [Fe-S]-based chains ex vivo, primarily due to their instability in aqueous media. Here, a simple technique to attach iron (II) sulfide (FeS) to a gold surface in ethanol media and then complete the attachment of the enzyme in aqueous media is reported. Cyclic voltammetry and spectroscopy techniques confirmed the concatenation of FeS and glycerol-dehydrogenase/nicotinamide-adenine-dinucleotide (GlDH-NAD(+)) apoenzyme-coenzyme molecular wiring system on the base gold electrode. The resultant FeS-based enzyme electrode reached an open circuit voltage closer to its standard potential under a wide range of glycerol concentrations (0.001-1M). When probed under constant potential conditions, the FeS-based electrode was able to amplify current by over 10 fold as compared to electrodes fabricated with the conventional pyrroloquinoline quinone-based composite molecular wiring system. These improvements in current/voltage responses open up a wide range of possibilities for fabricating self-powering, bio-electronic devices. PMID:26657591

  1. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin–cadmium induced diabetic nephrotoxic rats

    SciTech Connect

    Kandasamy, Neelamegam; Ashokkumar, Natarajan

    2014-09-01

    Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)–cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine, blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ–Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ–Cd induced diabetic nephrotoxic rats. - Highlights: • Diabetic rats are more susceptible to cadmium nephrotoxicity. • Cadmium plays as a cumulative

  2. Enhanced therapeutic efficacy of budesonide in experimental colitis with enzyme/pH dual-sensitive polymeric nanoparticles

    PubMed Central

    Naeem, Muhammad; Cao, Jiafu; Choi, Moonjeong; Kim, Woo Seong; Moon, Hyung Ryong; Lee, Bok Luel; Kim, Min-Soo; Jung, Yunjin; Yoo, Jin-Wook

    2015-01-01

    Current colon-targeted drug-delivery approaches for colitis therapy often utilize single pH-triggered systems, which are less reliable due to the variation of gut pH in individuals and in disease conditions. Herein, we prepared budesonide-loaded dual-sensitive nanoparticles using enzyme-sensitive azo-polyurethane and pH-sensitive methacrylate copolymer for the treatment of colitis. The therapeutic potential of the enzyme/pH dual-sensitive nanoparticles was evaluated using a rat colitis model and compared to single pH-triggered nanoparticles. Clinical activity scores, colon/body weight ratios, myeloperoxidase activity, and proinflammatory cytokine levels were markedly decreased by dual-sensitive nanoparticles compared to single pH-triggered nanoparticles and budesonide solution. Moreover, dual-sensitive nanoparticles accumulated selectively in inflamed segments of the colon. In addition, dual-sensitive nanoparticle plasma concentrations were lower than single pH-triggered nanoparticles, and no noticeable in vitro or in vivo toxicity was observed. Our results demonstrate that enzyme/pH dual-sensitive nanoparticles are an effective and safe colon-targeted delivery system for colitis therapy. PMID:26213469

  3. Enhancement of Biodegradable Plastic-degrading Enzyme Production from Paraphoma-like Fungus, Strain B47-9.

    PubMed

    Sameshima-Yamashita, Yuka; Koitabashi, Motoo; Tsuchiya, Wataru; Suzuki, Ken; Watanabe, Takashi; Shinozaki, Yukiko; Yamamoto-Tamura, Kimiko; Yamazaki, Toshimasa; Kitamoto, Hiroko

    2016-03-01

    To improve the productivity of Paraphoma-like fungal strain B47-9 for biodegradable plastic (BP)-degrading enzyme (PCLE), the optimal concentration of emulsified poly(butylene succinate-co-adipate) (PBSA) in the medium was determined. Emulsified PBSA was consumed as a sole carbon source and an inducer of PCLE production by strain B47-9. Among the various concentrations of emulsified PBSA [0.09-0.9% (w/v)] used in flask cultivation, 0.27% yielded the maximum enzyme activity within a short cultivation period. To evaluate the residual concentration of emulsified PBSA in culture, emulsified PBSA in aliquots of culture supernatant was digested in vitro, and the concentration of released monomerised succinic acid was determined. Regardless of the initial concentration of emulsified PBSA in medium, PCLE activity was detected after residual succinic acid decreased below 0.04 mg/mL in culture broth. Jarfermentation was performed at a 0.27% PBSA concentration. Among the various airflow rates tested, 1 LPM resulted in a PCLE production rate of 1.0 U/mL/day. The enzyme activity in the resulting culture filtrate (4.2 U/2 mL) was shown to degrade commercial BP films (1 × 1 cm, 20 µm thickness) within 8 hours. PMID:26876678

  4. The relationship between enhanced enzyme activity and structural dynamics in ionic liquids: a combined computational and experimental study.

    PubMed

    Kim, Ho Shin; Ha, Sung Ho; Sethaphong, Latsavongsakda; Koo, Yoon-Mo; Yingling, Yaroslava G

    2014-02-21

    Candida antarctica lipase B (CALB) is an efficient biocatalyst for hydrolysis, esterification, and polymerization reactions. In order to understand how to control enzyme activity and stability we performed a combined experimental and molecular dynamics simulation study of CALB in organic solvents and ionic liquids (ILs). Our results demonstrate that the conformational changes of the active site cavity are directly related to enzyme activity and decrease in the following order: [Bmim][TfO] > tert-butanol > [Bmim][Cl]. The entrance to the cavity is modulated by two isoleucines, ILE-189 and ILE-285, one of which is located on the α-10 helix. The α-10 helix can substantially change its conformation due to specific interactions with solvent molecules. This change is acutely evident in [Bmim][Cl] where interactions of LYS-290 with chlorine anions caused a conformational switch between α-helix and turn. Disruption of the α-10 helix structure results in a narrow cavity entrance and, thus, reduced the activity of CALB in [Bmim][Cl]. Finally, our results show that the electrostatic energy between solvents in this study and CALB is correlated with the structural changes leading to differences in enzyme activity. PMID:24424278

  5. Enhanced production of industrial enzymes in Mucoromycotina fungi during solid-state fermentation of agricultural wastes/by-products.

    PubMed

    Takó, Miklós; Kotogán, Alexandra; Krisch, Judit; Vágvölgyi, Csaba; Mondal, Keshab C; Papp, Tamás

    2015-09-01

    Cellulolytic, lipolytic and proteolytic enzyme production of zygomycetes Mucor corticolus, Rhizomucor miehei, Gilbertella persicaria and Rhizopus niveus were investigated using agro-industrial wastes as substrates. Solid-state cultures were carried out on untreated corn residues (stalk and leaf) as single substrate (SSF1) or corn residues and wheat bran in mixed fermentation (SSF2). Rapid production of endoglucanase (CMCase) was observed with maximal activity reaching after about 48-h fermentation, while cellobiohydrolase (CBH) and β-glucosidase enzymes generally had their peak after 72-h incubation. Highest filter paper degrading (FPase), CMCase, CBH and β-glucosidase activities obtained were (U g⁻¹ dss) 17.3, 74.1, 12.2 and 158.3, for R. miehei, G. persicaria, M. corticolus and Rh. niveus, respectively. M. corticolus proved to be the best lipolytic enzyme producer in SSF1 presenting 447.6 U g⁻¹ dss yield, while R. miehei showed 517.7 U g⁻¹ dss activity in SSF2. Rh. niveus exhibited significantly greater protease production than the other strains. Suc-AAPF-pNA hydrolyzing activities of this strain were 1.1 and 1.96 U g⁻¹ dss in SSF1 and SSF2, respectively. We conclude that the used corn stalk and leaf residues could potentially be applicable as strong inducers for cellulase and lipase production by Mucoromycotina fungi. PMID:26344030

  6. Crude soybean hull peroxidase treatment of phenol in synthetic and real wastewater: enzyme economy enhanced by Triton X-100.

    PubMed

    Steevensz, Aaron; Madur, Sneha; Feng, Wei; Taylor, Keith E; Bewtra, Jatinder K; Biswas, Nihar

    2014-02-01

    Soybean peroxidase (SBP)-catalyzed removal of phenol from wastewater has been demonstrated as a feasible wastewater treatment strategy and a non-ionic surfactant, Triton X-100, has the potential for increasing the enzyme economy of the process. Systematic studies on the enzyme-surfactant system have been lacking as well as demonstration of its applicability to industrial wastewater. This paper addresses those two gaps, the latter based on real wastewater from alkyd resin manufacture. The minimum effective Triton X-100 concentrations for crude SBP-catalyzed phenol conversion (≥95%) over 1-10 mM showed a linear trend. To illustrate translation of such lab results to real-world samples, this data were used to optimize crude SBP needed for phenol conversion over that concentration range. Triton X-100 increases enzyme economy by 10- to 13-fold. This treatment protocol was directly applied to tote-scale (700-1000 L) treatment of alkyd resin wastewater, with phenol ranging from 7 to 28 mM and total organic carbon content of >40 g/L, using a crude SBP extract derived from dry soybean hulls by simple aqueous elution. This extract can be used to remove phenol from a complex industrial wastewater and the process is markedly more efficient in the presence of Triton X-100. The water is thus rendered amenable to conventional biological treatment whilst the hulls could still be used in feed, thus adding further value to the crop. PMID:24411447

  7. Enhanced cellulose degradation by nano-complexed enzymes: Synergism between a scaffold-linked exoglucanase and a free endoglucanase.

    PubMed

    Moraïs, Sarah; Heyman, Arnon; Barak, Yoav; Caspi, Jonathan; Wilson, David B; Lamed, Raphael; Shoseyov, Oded; Bayer, Edward A

    2010-06-01

    Protein molecular scaffolds are attracting interest as natural candidates for the presentation of enzymes and acceleration of catalytic reactions. We have previously reported evidence that the stable protein 1 (SP1) from Populustremula can be employed as a molecular scaffold for the presentation of either catalytic or structural binding (cellulosomal cohesin) modules. In the present work, we have displayed a potent exoglucanase (Cel6B) from the aerobic cellulolytic bacterium, Thermobifida fusca, on a cohesin-bearing SP1 scaffold. For this purpose, a chimaeric form of the enzyme, fused to a cellulosomal dockerin module, was prepared. Full incorporation of 12 dockerin-bearing exoglucanase molecules onto the cohesin-bearing scaffold was achieved. Cellulase activity was tested on two cellulosic substrates with different levels of crystallinity, and the activity of the scaffold-linked exoglucanase was significantly reduced, compared to the free dockerin-containing enzyme. However, addition of relatively low concentrations of a free wild-type endoglucanase (T. fusca Cel5A) that bears a cellulose-binding module, in combination with the complexed exoglucanase resulted in a marked rise in activity on both cellulosic substrates. The endoglucanase cleaves internal sites of the cellulose chains, and the new chain ends of the substrate were now readily accessible to the scaffold-borne exoglucanase, thereby resulting in highly effective, synergistic degradation of cellulosic substrates. PMID:20438772

  8. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell-core silica nanospheres based on enzyme-linked immunosorbent assay.

    PubMed

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou

    2015-08-01

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL(-1) with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. PMID:26320802

  9. Enzymatic saccharification and bioethanol production from Cynara cardunculus pretreated by steam explosion.

    PubMed

    Fernandes, Maria C; Ferro, Miguel D; Paulino, Ana F C; Mendes, Joana A S; Gravitis, Janis; Evtuguin, Dmitry V; Xavier, Ana M R B

    2015-06-01

    The correct choice of the specific lignocellulosic biomass pretreatment allows obtaining high biomass conversions for biorefinery implementations and cellulosic bioethanol production from renewable resources. Cynara cardunculus (cardoon) pretreated by steam explosion (SE) was involved in second-generation bioethanol production using separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) processes. Steam explosion pretreatment led to partial solubilisation of hemicelluloses and increased the accessibility of residual polysaccharides towards enzymatic hydrolysis revealing 64% of sugars yield against 11% from untreated plant material. Alkaline extraction after SE pretreatment of cardoon (CSEOH) promoted partial removal of degraded lignin, tannins, extractives and hemicelluloses thus allowing to double glucose concentration upon saccharification step. Bioethanol fermentation in SSF mode was faster than SHF process providing the best results: ethanol concentration 18.7 g L(-1), fermentation efficiency of 66.6% and a yield of 26.6g ethanol/100 g CSEOH or 10.1 g ethanol/100 g untreated cardoon. PMID:25836040

  10. Oral administration recombinant porcine epidermal growth factor enhances the jejunal digestive enzyme genes expression and activity of early-weaned piglets.

    PubMed

    Lee, D N; Chuang, Y S; Chiou, H Y; Wu, F Y; Yen, H T; Weng, C F

    2008-08-01

    This study attempted to determine ingested porcine epidermal growth factor (pEGF) on the gastrointestinal tract development of early-weaned piglets. Thirty-two piglets (14-day weaned) were randomly allotted to supplemented with 0 (control), 0.5, 1.0, or 1.5 mg pEGF/kg diet. Each treatment consisted of four replicates with two pigs per pen for a 14 days experimental period. Piglets were sacrificed and gastrointestinal tract samples were collected to measure mucosa morphology, mRNA expression and activities of digestive enzymes in the gastrointestinal tract of piglets at the end of the experiment. Diets supplemented with pEGF failed to influence growth performance but tended to increase jejunal mucosa weight (p < 0.09) and protein content (p < 0.07). Piglets supplemental pEGF induced incrementally the gastric pepsin activity (p < 0.05) and stimulated jejunal alkaline phosphatase (ALP) and lactase activities accompanied with the increase of jejunal ALP and maltase mRNA expression. No effect of pEGF on the activities of all enzymes in ileum except the stimulation of ileal aminopeptide N mRNA expression. These results reveal that dietary pEGF supplementation might enhance gene expression and activities of digestive enzymes in the stomach and jejunum of piglets. PMID:18662356

  11. Employing bacterial microcompartment technology to engineer a shell-free enzyme-aggregate for enhanced 1,2-propanediol production in Escherichia coli.

    PubMed

    Lee, Matthew J; Brown, Ian R; Juodeikis, Rokas; Frank, Stefanie; Warren, Martin J

    2016-07-01

    Bacterial microcompartments (BMCs) enhance the breakdown of metabolites such as 1,2-propanediol (1,2-PD) to propionic acid. The encapsulation of proteins within the BMC is mediated by the presence of targeting sequences. In an attempt to redesign the Pdu BMC into a 1,2-PD synthesising factory using glycerol as the starting material we added N-terminal targeting peptides to glycerol dehydrogenase, dihydroxyacetone kinase, methylglyoxal synthase and 1,2-propanediol oxidoreductase to allow their inclusion into an empty BMC. 1,2-PD producing strains containing the fused enzymes exhibit a 245% increase in product formation in comparison to un-tagged enzymes, irrespective of the presence of BMCs. Tagging of enzymes with targeting peptides results in the formation of dense protein aggregates within the cell that are shown by immuno-labelling to contain the vast majority of tagged proteins. It can therefore be concluded that these protein inclusions are metabolically active and facilitate the significant increase in product formation. PMID:26969252

  12. Lime pretreatment, enzymatic saccharification, and fermentation of rice hulls to ethanol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rice hulls used in this study contained 35.6±0.1% cellulose and 12.0±0.7% hemicellulose. The maximum yield of monomeric sugars from rice hulls (15.0%, w/v) by lime pretreatment (100 mg g**-1 hulls, 121 deg C, 1 h) and enzymatic saccharification (45 deg C, pH 5.0, 72 h) using a cocktail of three com...

  13. Microwave Pretreatment, Enzymatic Saccharification, and Fermentation of Wheat Straw to Ethanol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat straw used in this study contained 38.8±0.5% cellulose and 31.0±0.3% hemicellulose. The effects of temperature (160-240 deg C, 5 min) and duration (5-20 min at 200 deg C) of microwave pretreatment of wheat straw (8.6%, w/v, in water) on its enzymatic saccharification to fermentable sugars wer...

  14. Increase in cellulose accumulation and improvement of saccharification by overexpression of arabinofuranosidase in rice.

    PubMed

    Sumiyoshi, Minako; Nakamura, Atsuko; Nakamura, Hidemitsu; Hakata, Makoto; Ichikawa, Hiroaki; Hirochika, Hirohiko; Ishii, Tadashi; Satoh, Shinobu; Iwai, Hiroaki

    2013-01-01

    Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have β-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-α-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF) to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX) decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against α-(1,5)-linked l-Ara (LM6) and β-(1,4)-linked d-Xyl (LM10 and LM11) residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol. PMID:24223786

  15. Increase in Cellulose Accumulation and Improvement of Saccharification by Overexpression of Arabinofuranosidase in Rice

    PubMed Central

    Nakamura, Hidemitsu; Hakata, Makoto; Ichikawa, Hiroaki; Hirochika, Hirohiko; Ishii, Tadashi; Satoh, Shinobu; Iwai, Hiroaki

    2013-01-01

    Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have β-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-α-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF) to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX) decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against α-(1,5)-linked l-Ara (LM6) and β-(1,4)-linked d-Xyl (LM10 and LM11) residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol. PMID:24223786

  16. A Novel simultaneous-Saccharification-Fermentation Strategy for Efficient Co-fermentation of C5 and C6 Sugars Using Native, Non-GMO Yeasts

    SciTech Connect

    Varanasi, Sasidhar; Relue, Patricia

    2013-09-30

    Economic bioethanol production is critically dependent upon the ability to convert both the hexose (C6) and pentose (C5) sugars resulting from cellulose and hemicellulose. C5 sugars are not readily fermentable by native Saccharomyces cerevisiae. Genetically Modified Organisms (GMOs) are designed to ferment xylose, but their stability, ethanol yield, environmental impact, and survival under conditions of industrial fermentation are unproven. In this project, we developed a novel approach for efficient fermentation of both C5 and C6 sugars using native S. Cerevisiae by exploiting its ability to produce ethanol from xylulose - the keto-isomer of xylose. While the isomerization of xylose to xylulose can be accomplished via commercially (and cheaply) available Xylose Isomerase (XI) (Sweetzyme™), this conversion has an extremely unfavorable equilibrium (xylose:xylose is about 5:1). To address this, we developed two alternate strategies. In the first, the two enzymes XI and urease are coimmobilized on solid support particles to enable complete isomerization of xylose to xylulose under pH conditions suitable for fermentation, in a simultaneous-isomerization-fermentation (SIF) mode. The ability of our technology to conduct isomerization of xylose under pH conditions suitable for both saccharification and fermentation opens the possibility of SSF with native yeasts for the first time. Herein, we performed specific research tasks for implementation of our technology in several modes of operation, including simultaneous-isomerization-and-fermentation (SIF), simultaneous-saccharification-and-isomerization (SSI) followed by fermentation, and SSF mode with the biomass feedstock poplar. The projected economics of our process are very favorable in comparison to the costs associated with engineering, licensing and propagating GMOs. This novel fermentation technology is readily accessible to rural farming economies for implementation in cellulosic ethanol production facilities.

  17. On-site cellulase production by Trichoderma reesei 3EMS35 mutant and same vessel saccharification and fermentation of acid treated wheat straw for ethanol production

    PubMed Central

    Khokhar, Zia-ullah; Syed, Qurat-ul-Ain; Wu, Jing; Athar, Muhammad Amin

    2014-01-01

    Bioethanol production from lignocellulosic raw materials involves process steps like pre-treatment, enzymatic hydrolysis, fermentation and distillation. In this study, wheat straw was explored as feedstock for on-site cellulase production by T. reesei 3EMS35 mutant, and as a substrate for second generation bioethanol production from baker yeast. Scanning electron microscopy (SEM) and X-ray diffractography (XRD) of untreated wheat straw (UWS) and acid treated wheat straw (TWS) were done to understand the structural organization and changes in the cellulase accessibility and reactivity. The effect of delignification and structural modification for on-site cellulase enzyme production was comparably studied. The efficiency of crude cellulase enzyme for digestion of UWS and TWS and then production of ethanol from TWS was studied using same-vessel saccharification and fermentation (SVSF) technique, both in shaking flasks as well as in fermenters. Two different methods of operation were tested, i.e. the UWSEnz method, where UWS was used for on-site enzyme production, and TWSEnz method where TWS was applied as substrate for cellullase production. Results obtained showed structural modifications in cellulose of TWS due to delignification, removal of wax and change of crystallinity. UWS was better substrate than TWS for cellulase production due to the fact that lignin did not hinder the enzyme production by fungus but acted as a booster. On-site cellulase enzyme produced by T. reesei 3EMS35 mutant hydrolyzed most of cellulose (91 %) in TWS within first 24 hrs. Shake flasks experiments showed that ethanol titers and yields with UWSEnz were 2.9 times higher compared to those obtained with TWSEnz method respectively. Comparatively, titer of ethanol in shake flask experiments was 10 % higher than this obtained in 3 L fermenter with UWSEnz. Outcomes from this investigation clearly demonstrated the potential of on-site cellulase enzyme production and SVSF for ethanol production

  18. Ethanol from a biorefinery waste stream: Saccharification of amylase, protease and xylanase treated wheat bran.

    PubMed

    Wood, Ian P; Cook, Nicola M; Wilson, David R; Ryden, Peter; Robertson, James A; Waldron, Keith W

    2016-05-01

    Biorefining aims to exploit the full value of plant material by sequentially extracting and valorising its components. Many studies focus on the saccharification of virgin biomass sources, but it may be more efficient to pre-extract high-value components before hydrolysis to fermentable sugars. In the current study, a bran residue from de-starched, protein depleted and xylanase treated wheat bran has been subjected to hydrothermal pretreatment, saccharification and fermentation procedures to convert the residue to ethanol. The most effective pretreatment conditions (>190 °C, 10 min) and saccharification conditions were identified following bench-scale liquid hot water pretreatment. Pre-extraction of enzymatically-hydrolysable starch and xylan reduced the release of furfural production, particularly when lower pretreatment severities were used. Pilot-scale steam explosion of the lignocellulosic residue followed by cellulase treatment and conversion to ethanol at a high substrate concentration (19%) gave an ethanol titre of ≈ 25 g/L or a yield of 93% of the theoretical maximum. PMID:26769514

  19. Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine

    PubMed Central

    2014-01-01

    Background Forestry residues consisting of softwood are a major lignocellulosic resource for production of liquid biofuels. Scots pine, a commercially important forest tree, was fractionated into seven fractions of chips: juvenile heartwood, mature heartwood, juvenile sapwood, mature sapwood, bark, top parts, and knotwood. The different fractions were characterized analytically with regard to chemical composition and susceptibility to dilute-acid pretreatment and enzymatic saccharification. Results All fractions were characterized by a high glucan content (38-43%) and a high content of other carbohydrates (11-14% mannan, 2-4% galactan) that generate easily convertible hexose sugars, and by a low content of inorganic material (0.2-0.9% ash). The lignin content was relatively uniform (27-32%) and the syringyl-guaiacyl ratio of the different fractions were within the range 0.021-0.025. The knotwood had a high content of extractives (9%) compared to the other fractions. The effects of pretreatment and enzymatic saccharification were relatively similar, but without pretreatment the bark fraction was considerably more susceptible to enzymatic saccharification. Conclusions Since sawn timber is a main product from softwood species such as Scots pine, it is an important issue whether different parts of the tree are equally suitable for bioconversion processes. The investigation shows that bioconversion of Scots pine is facilitated by that most of the different fractions exhibit relatively similar properties with regard to chemical composition and susceptibility to techniques used for bioconversion of woody biomass. PMID:24641769

  20. Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae.

    PubMed

    Wang, Damao; Yun, Eun Ju; Kim, Sooah; Kim, Do Hyoung; Seo, Nari; An, Hyun Joo; Kim, Jae-Han; Cheong, Nam Yong; Kim, Kyoung Heon

    2016-06-01

    This study was performed to evaluate the effectiveness of acidic pretreatment in increasing the enzymatic digestibility of alginate from brown macroalgae. Pretreatment with 1 % (w/v) sulfuric acid at 120 °C for 30 min produced oligosaccharides, mannuronic acid, and guluronic acid. Enzymatic saccharification of pretreated alginate by alginate lyases produced 52.2 % of the theoretical maximal sugar yield, which was only 7.5 % higher than the sugar yield obtained with unpretreated alginate. Mass spectrometric analyses of products of the two reactions revealed that acidic pretreatment and enzymatic saccharification produced saturated monomers (i.e., mannuronic and guluronic acid) with saturated oligosaccharides and unsaturated monomers (i.e., 4-deoxy-L-erythro-5-hexoseulose uronic acid; DEH), respectively. While DEH is further metabolized by microorganisms, mannuronic acid and guluronic acid are not metabolizable. Because of the poor efficacy in increasing enzymatic digestibility and owing to the formation of non-fermentable saturated monomers, acidic pretreatment cannot be recommended for enzymatic saccharification and fermentation of alginate. PMID:26923145

  1. Whole slurry saccharification and fermentation of maleic acid-pretreated rice straw for ethanol production.

    PubMed

    Jung, Young Hoon; Park, Hyun Min; Kim, Kyoung Heon

    2015-09-01

    We evaluated the feasibility of whole slurry (pretreated lignocellulose) saccharification and fermentation for producing ethanol from maleic acid-pretreated rice straw. The optimized conditions for pretreatment were to treat rice straw at a high temperature (190 °C) with 1 % (w/v) maleic acid for a short duration (3 min ramping to 190 °C and 3 min holding at 190 °C). Enzymatic digestibility (based on theoretical glucose yield) of cellulose in the pretreated rice straw was 91.5 %. Whole slurry saccharification and fermentation of pretreated rice straw resulted in 83.2 % final yield of ethanol based on the initial quantity of glucan in untreated rice straw. These findings indicate that maleic acid pretreatment results in a high yield of ethanol from fermentation of whole slurry even without conditioning or detoxification of the slurry. Additionally, the separation of solids and liquid is not required; therefore, the economics of cellulosic ethanol fuel production are significantly improved. We also demonstrated whole slurry saccharification and fermentation of pretreated lignocellulose, which has rarely been reported. PMID:25930209

  2. Simultaneous saccharification and fermentation of steam exploded duckweed: Improvement of the ethanol yield by increasing yeast titre

    PubMed Central

    Zhao, X.; Moates, G.K.; Elliston, A.; Wilson, D.R.; Coleman, M.J.; Waldron, K.W.

    2015-01-01

    This study investigated the conversion of Lemna minor biomass to bioethanol. The biomass was pre-treated by steam explosion (SE, 210 °C, 10 min) and then subjected to simultaneous saccharification and fermentation (SSF) using Cellic® CTec 2 (20 U or 0.87 FPU g−1 substrate) cellulase plus β-glucosidase (2 U g−1 substrate) and a yeast inoculum of 10% (v/v or 8.0 × 107 cells mL−1). At a substrate concentration of 1% (w/v) an ethanol yield of 80% (w/w, theoretical) was achieved. However at a substrate concentration of 20% (w/v), the ethanol yield was lowered to 18.8% (w/w, theoretical). Yields were considerably improved by increasing the yeast titre in the inoculum or preconditioning the yeast on steam exploded liquor. These approaches enhanced the ethanol yield up to 70% (w/w, theoretical) at a substrate concentration of 20% (w/v) by metabolising fermentation inhibitors. PMID:26210138

  3. Simultaneous saccharification and fermentation of steam exploded duckweed: Improvement of the ethanol yield by increasing yeast titre.

    PubMed

    Zhao, X; Moates, G K; Elliston, A; Wilson, D R; Coleman, M J; Waldron, K W

    2015-10-01

    This study investigated the conversion of Lemna minor biomass to bioethanol. The biomass was pre-treated by steam explosion (SE, 210°C, 10 min) and then subjected to simultaneous saccharification and fermentation (SSF) using Cellic® CTec 2 (20 U or 0.87 FPU g(-1) substrate) cellulase plus β-glucosidase (2 U g(-1) substrate) and a yeast inoculum of 10% (v/v or 8.0×10(7) cells mL(-1)). At a substrate concentration of 1% (w/v) an ethanol yield of 80% (w/w, theoretical) was achieved. However at a substrate concentration of 20% (w/v), the ethanol yield was lowered to 18.8% (w/w, theoretical). Yields were considerably improved by increasing the yeast titre in the inoculum or preconditioning the yeast on steam exploded liquor. These approaches enhanced the ethanol yield up to 70% (w/w, theoretical) at a substrate concentration of 20% (w/v) by metabolising fermentation inhibitors. PMID:26210138

  4. [Simultaneous saccharification and fermentation of Jerusalem artichoke tubers to ethanol with an inulinase-hyperproducing yeast Kluyveromyces cicerisporus].

    PubMed

    Yu, Jing; Jiang, Jiaxi; Zhang, Yongqiang; Lü, Hong; Li, Yuyang; Liu, Jianping

    2010-07-01

    Jerusalem artichoke tubers with inulin as major component are potential feedstock for fuel ethanol production, and Kluyveromyces cicerisporus Y179 expressing high level of inulinase is suitable for ethanol production with this feedstock by simultaneous saccharification and fermentation approach. In this article, the impact of inoculum, aeration and temperature on ethanol production by the yeast was studied. The experimental results illustrated that inoculum with different levels and seed collected at different cultivation times had negligible effect, while anaerobic conditions enhanced ethanol production, and more ethanol was produced by the yeast at 30 degrees C than at 37 degrees C or 42 degrees C. The medium using Jerusalem artichoke tuber meal as sole component with 22% (W/V) total sugars was inoculated with 36 h-precultured seed at 10% (V/V), and the batch fermentation was conducted in a 5 L fermentor at 30 degrees C with a stirring speed of 300 r/min under anaerobic conditions. After 144 h, 12.3% (V/V) ethanol was produced and the yield of ethanol from sugars was 86.9% of its theoretical one, with 93.6% sugars consumed. These results indicate that K. cicerisporus Y179 is a promising candidate for industrial ethanol production using Jerusalem artichoke tuber feedstock. PMID:20954400

  5. Enzyme markers

    MedlinePlus

    ... or defects passed down through families (inherited) can affect how enzymes work. Some enzymes are affected by several genes. Test results are usually reported as a percentage of normal enzyme activity.

  6. Cellulase immobilized on modified nylon for saccharification of cellulose

    SciTech Connect

    Jain, P.; Wilkins, E.S.

    1987-01-01

    The present study deals with the immobilization of cellulase on nylon and nylon incorporated with glass. The immobilized and free enzymes were compared in terms of their yields, using untreated sawdust (yellow pine wood) and carboxymethylcellulose (CMC) as substrates, at a standard pH and temperature. Also, the sawdust was pretreated with 1% alkaline hydrogen peroxide and the yield compared with the untreated sawdust hydrolysis to determine the importance of the substrate pretreatment.

  7. An Enhanced In Vivo Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Model for Quantification of Drug Metabolism Enzymes*

    PubMed Central

    MacLeod, A. Kenneth; Fallon, Padraic G.; Sharp, Sheila; Henderson, Colin J.; Wolf, C. Roland; Huang, Jeffrey T.-J.

    2015-01-01

    Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug–drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of

  8. New FadB homologous enzymes and their use in enhanced biosynthesis of medium-chain-length polyhydroxyalkanoates in FadB mutant Escherichia coli.

    PubMed

    Park, Si Jae; Yup Lee, Sang

    2004-06-20

    Recombinant Escherichia coli harboring the medium-chain-length (MCL) polyhydroxyalkanoate (PHA) synthase gene has been shown to accumulate MCL-PHAs from fatty acids when FadB is inactive. However, the enzymes in fadB mutant E. coli responsible for channeling the beta-oxidation intermediates to PHA biosynthesis have not been fully elucidated. Only recently, two enzymes encoded by yfcX and maoC have been found to be partially responsible for this. In this study, we identified five new FadB homologous enzymes in E. coli: PaaG, PaaF, BhbD, SceH, and YdbU, by protein database search, and examined their roles in the biosynthesis of MCL-PHAs in an fadB mutant E. coli strain. Coexpression of each of these genes along with the Pseudomonas sp. 61-3 phaC2 gene did not allow synthesis of MCL-PHA from fatty acid in recombinant E. coli W3110, which has a fully functional beta-oxidation pathway, but allowed MCL-PHA accumulation in an fadB mutant E. coli WB101. In particular, coexpression of the paaG, paaF, and ydbU genes resulted in a MCL-PHA production up to 0.37, 0.25, and 0.33 g/L, respectively, from 2 g/L of sodium decanoate, which is more than twice higher than that obtained with E. coli WB101 expressing only the phaC2 gene (0.16 g/L). These results suggest that the newly found FadB homologous enzymes, or at least the paaG, paaF, and ydbU genes, are involved in MCL-PHA biosynthesis in an fadB mutant E. coli strain and can be employed for the enhanced production of MCL-PHA. PMID:15137080

  9. Host cell capable of producing enzymes useful for degradation of lignocellulosic material

    SciTech Connect

    Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Damveld, Robbertus Antonius

    2015-08-18

    The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

  10. Dietary ɛ-Polylysine Decreased Serum and Liver Lipid Contents by Enhancing Fecal Lipid Excretion Irrespective of Increased Hepatic Fatty Acid Biosynthesis-Related Enzymes Activities in Rats

    PubMed Central

    Hosomi, Ryota; Yamamoto, Daiki; Otsuka, Ren; Nishiyama, Toshimasa; Yoshida, Munehiro; Fukunaga, Kenji

    2015-01-01

    ɛ-Polylysine (EPL) is used as a natural preservative in food. However, few studies have been conducted to assess the beneficial functions of dietary EPL. The purpose of this study was to elucidate the mechanism underlying the inhibition of neutral and acidic sterol absorption and hepatic enzyme activity-related fatty acid biosynthesis following EPL intake. EPL digest prepared using an in vitro digestion model had lower lipase activity and micellar lipid solubility and higher bile acid binding capacity than casein digest. Male Wistar rats were fed an AIN-93G diet containing 1% (wt/wt) EPL or l-lysine. After 4 weeks of feeding these diets, the marked decrease in serum and liver triacylglycerol contents by the EPL diet was partly attributed to increased fecal fatty acid excretion. The activities of hepatic acetyl-coenzyme A carboxylase and glucose-6-phosphate dehydrogenase, which are key enzymes of fatty acid biosynthesis, were enhanced in rats fed EPL diet. The increased fatty acid biosynthesis activity due to dietary EPL may be prevented by the enhancement of fecal fatty acid excretion. The hypocholesterolemic effect of EPL was mediated by increased fecal neutral and acidic sterol excretions due to the EPL digest suppressing micellar lipid solubility and high bile acid binding capacity. These results show that dietary EPL has beneficial effects that could help prevent lifestyle-related diseases such as hyperlipidemia and atherosclerosis. PMID:25866749

  11. Nanotechnology Enabled Enhancement of Enzyme Activity and Thermostability: Study on Impaired Pectate Lyase from Attenuated Macrophomina phaseolina in Presence of Hydroxyapatite Nanoparticle

    PubMed Central

    Dutta, Nalok; Mukhopadhyay, Arka; Dasgupta, Anjan Kr.; Chakrabarti, Krishanu

    2013-01-01

    In this paper we show that hydroxyapatite nanoparticles (NP) can not only act as a chaperon (by imparting thermostability) but can serve as a synthetic enhancer of activity of an isolated extracellular pectate lyase (APL) with low native state activity. The purified enzyme (an attenuated strain of Macrophomina phaseolina) showed feeble activity at 50°C and pH 5.6. However, on addition of 10.5 µg/ml of hydroxyapatite nanoparticles (NP), APL activity increased 27.7 fold with a 51 fold increase in half-life at a temperature of 90°C as compared to untreated APL. The chaperon like activity of NP was evident from entropy–enthalpy compensation profile of APL. The upper critical temperature for such compensation was elevated from 50°C to 90°C in presence of NP. This dual role of NP in enhancing activity and conferring thermostability to a functionally impaired enzyme is reported for the first time. PMID:23691068

  12. Dietary ɛ-Polylysine Decreased Serum and Liver Lipid Contents by Enhancing Fecal Lipid Excretion Irrespective of Increased Hepatic Fatty Acid Biosynthesis-Related Enzymes Activities in Rats.

    PubMed

    Hosomi, Ryota; Yamamoto, Daiki; Otsuka, Ren; Nishiyama, Toshimasa; Yoshida, Munehiro; Fukunaga, Kenji

    2015-03-01

    ɛ-Polylysine (EPL) is used as a natural preservative in food. However, few studies have been conducted to assess the beneficial functions of dietary EPL. The purpose of this study was to elucidate the mechanism underlying the inhibition of neutral and acidic sterol absorption and hepatic enzyme activity-related fatty acid biosynthesis following EPL intake. EPL digest prepared using an in vitro digestion model had lower lipase activity and micellar lipid solubility and higher bile acid binding capacity than casein digest. Male Wistar rats were fed an AIN-93G diet containing 1% (wt/wt) EPL or l-lysine. After 4 weeks of feeding these diets, the marked decrease in serum and liver triacylglycerol contents by the EPL diet was partly attributed to increased fecal fatty acid excretion. The activities of hepatic acetyl-coenzyme A carboxylase and glucose-6-phosphate dehydrogenase, which are key enzymes of fatty acid biosynthesis, were enhanced in rats fed EPL diet. The increased fatty acid biosynthesis activity due to dietary EPL may be prevented by the enhancement of fecal fatty acid excretion. The hypocholesterolemic effect of EPL was mediated by increased fecal neutral and acidic sterol excretions due to the EPL digest suppressing micellar lipid solubility and high bile acid binding capacity. These results show that dietary EPL has beneficial effects that could help prevent lifestyle-related diseases such as hyperlipidemia and atherosclerosis. PMID:25866749

  13. A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films.

    PubMed

    Barth, Markus; Honak, Annett; Oeser, Thorsten; Wei, Ren; Belisário-Ferrari, Matheus R; Then, Johannes; Schmidt, Juliane; Zimmermann, Wolfgang

    2016-08-01

    TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films. PMID:27214855

  14. Thin-Layer Polymer Wrapped Enzymes Encapsulated in Hierarchically Mesoporous Silica with High Activity and Enhanced Stability

    NASA Astrophysics Data System (ADS)

    Zhang, Fang; Wang, Meitao; Liang, Chao; Jiang, Huangyong; Shen, Jian; Li, Hexing

    2014-03-01

    A novel soft-hard cooperative approach was developed to synthesize bioactive mesoporous composite by pre-wrapping Penicillin G amidase with poly(acrylaimde) nanogel skin and subsequently incorporating such Penicillin G amidase nanocapsules into hierarchically mesoporous silica. The as-received bioactive mesoporous composite exhibited comparable activity and extraordinarily high stability in comparison with native Penicillin G amidase and could be used repetitively in the water-medium hydrolysis of penicillin G potassium salt. Furthermore, this strategy could be extended to the synthesis of multifunctional bioactive mesoporous composite by simultaneously introducing glucose oxidase nanocapsules and horseradish peroxidase nanocapsules into hierarchically mesoporous silica, which demonstrated a synergic effect in one-pot tandem oxidation reaction. Improvements in the catalytic performances were attributed to the combinational unique structure from soft polymer skin and hard inorganic mesoporous silica shell, which cooperatively helped enzyme molecules to retain their appropriate geometry and simultaneously decreased the enzyme-support negative interaction and mass transfer limitation under heterogeneous conditions.

  15. Thin-layer polymer wrapped enzymes encapsulated in hierarchically mesoporous silica with high activity and enhanced stability.

    PubMed

    Zhang, Fang; Wang, Meitao; Liang, Chao; Jiang, Huangyong; Shen, Jian; Li, Hexing

    2014-01-01

    A novel soft-hard cooperative approach was developed to synthesize bioactive mesoporous composite by pre-wrapping Penicillin G amidase with poly(acrylaimde) nanogel skin and subsequently incorporating such Penicillin G amidase nanocapsules into hierarchically mesoporous silica. The as-received bioactive mesoporous composite exhibited comparable activity and extraordinarily high stability in comparison with native Penicillin G amidase and could be used repetitively in the water-medium hydrolysis of penicillin G potassium salt. Furthermore, this strategy could be extended to the synthesis of multifunctional bioactive mesoporous composite by simultaneously introducing glucose oxidase nanocapsules and horseradish peroxidase nanocapsules into hierarchically mesoporous silica, which demonstrated a synergic effect in one-pot tandem oxidation reaction. Improvements in the catalytic performances were attributed to the combinational unique structure from soft polymer skin and hard inorganic mesoporous silica shell, which cooperatively helped enzyme molecules to retain their appropriate geometry and simultaneously decreased the enzyme-support negative interaction and mass transfer limitation under heterogeneous conditions. PMID:24651701

  16. Thin-Layer Polymer Wrapped Enzymes Encapsulated in Hierarchically Mesoporous Silica with High Activity and Enhanced Stability

    PubMed Central

    Zhang, Fang; Wang, Meitao; Liang, Chao; Jiang, Huangyong; Shen, Jian; Li, Hexing

    2014-01-01

    A novel soft-hard cooperative approach was developed to synthesize bioactive mesoporous composite by pre-wrapping Penicillin G amidase with poly(acrylaimde) nanogel skin and subsequently incorporating such Penicillin G amidase nanocapsules into hierarchically mesoporous silica. The as-received bioactive mesoporous composite exhibited comparable activity and extraordinarily high stability in comparison with native Penicillin G amidase and could be used repetitively in the water-medium hydrolysis of penicillin G potassium salt. Furthermore, this strategy could be extended to the synthesis of multifunctional bioactive mesoporous composite by simultaneously introducing glucose oxidase nanocapsules and horseradish peroxidase nanocapsules into hierarchically mesoporous silica, which demonstrated a synergic effect in one-pot tandem oxidation reaction. Improvements in the catalytic performances were attributed to the combinational unique structure from soft polymer skin and hard inorganic mesoporous silica shell, which cooperatively helped enzyme molecules to retain their appropriate geometry and simultaneously decreased the enzyme-support negative interaction and mass transfer limitation under heterogeneous conditions. PMID:24651701

  17. Engineering Cellulase Enzymes for Bioenergy

    NASA Astrophysics Data System (ADS)

    Atreya, Meera Elizabeth

    methods. Protein engineering targets to improve cellulases include reducing enzyme inhibition, improving inter-enzyme synergy, and increasing enzyme thermotolerance. Ameliorating enzyme inhibition could improve catalytic activity and thus the speed of conversion from biomass to fermentable sugars. Improved enzyme synergy could reduce the enzyme loading required to achieve equivalent biomass conversion. Finally, thermostable enzymes could enable more biomass to be processed at a time, due to high temperatures decreasing the viscosity of biomass slurries. A high-temperature enzyme saccharification reaction could also decrease the risk of contamination in the resulting concentrated sugar solution. Throughout my PhD, I have explored research projects broadly across all of these topics, with the most success in addressing the issue of enzyme inhibition. Cellulase enzyme Cel7A is the most abundant cellulase employed by natural systems for cellulose hydrolysis. Cellobiohydrolase enzymes like Cel7A break down cellulose into cellobiose (two glucose molecules). Unfortunately, upon cleavage, this product molecule interferes with continued hydrolysis activity of Cel7A; the strong binding of cellobiose in the active site can obstruct the enzyme from processing down the cellulase chain. This phenomenon, known as product inhibition, is a bottleneck to efficient biomass breakdown. Using insights from computational protein modeling studies, I experimentally generated and tested mutant Cel7A enzymes for improved tolerance to cellobiose. Indeed, this strategy yielded Cel7A enzymes exhibiting reduced product inhibition, including some mutants completely impervious to cellobiose. The improvements in tolerance to cellobiose, however, resulted in an overall reduction of enzyme activity for the mutants tested. Nevertheless, my findings substantiated computational reports with experimental evidence and pinpointed an amino acid residue in the Cel7A product binding site that is of interest for

  18. Enzyme Kinetics in Microgravity

    NASA Astrophysics Data System (ADS)

    Liu, C. C.; Licata, V. J.

    2010-04-01

    The kinetics of some enzymes have been found to be enhanced by the microgravity environment. This is a relatively small effect, but is sufficient to have physiological effects and to impact pharmaceutical therapy in microgravity.

  19. Enzymatic Hydrolysis and Ethanol Fermentation of High Dry Matter Wet-Exploded Wheat Straw at Low Enzyme Loading

    NASA Astrophysics Data System (ADS)

    Georgieva, Tania I.; Hou, Xiaoru; Hilstrøm, Troels; Ahring, Birgitte K.

    Wheat straw was pretreated by wet explosion using three different oxidizing agents (H2O2, O2, and air). The effect of the pretreatment was evaluated based on glucose and xylose liberated during enzymatic hydrolysis. The results showed that pretreatment with the use of O2 as oxidizing agent was the most efficient in enhancing overall convertibility of the raw material to sugars and minimizing generation of furfural as a by-product. For scale-up of the process, high dry matter (DM) concentrations of 15-20% will be necessary. However, high DM hydrolysis and fermentation are limited by high viscosity of the material, higher inhibition of the enzymes, and fermenting microorganism. The wet-explosion pretreatment method enabled relatively high yields from both enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) to be obtained when performed on unwashed slurry with 14% DM and a low enzyme loading of 10 FPU/g cellulose in an industrial acceptable time frame of 96 h. Cellulose and hemicellulose conversion from enzymatic hydrolysis were 70 and 68%, respectively, and an overall ethanol yield from SSF was 68%.

  20. Comparison of simultaneous and separate processes: saccharification and thermophilic L-lactate fermentation of catch crop and aquatic plant biomass.

    PubMed

    Akao, Satoshi; Maeda, Koutaro; Nakatani, Shingo; Hosoi, Yoshihiko; Nagare, Hideaki; Maeda, Morihiro; Fujiwara, Taku

    2012-01-01

    Catch crop candidates (corn, guinea grass) for recovering nutrients from farm soil and aquatic plants (water caltrop, water hyacinth) were utilized to produce L-lactic acid. The efficiencies ofpre-treatment methods for enzymatic saccharification and L-lactate production of two fermentation processes, thermophilic simultaneous saccharification and fermentation (SSF), as well as separate saccharification and fermentation, were compared. Conditions were set at 55 degrees C and pH 5.5 for non-sterile fermentation. Alkaline/peroxide pre-treatment proved the most effective for saccharification in pre-treated corn, guinea grass, water caltrop and water hyacinth with glucose yields of 0.23, 0.20, 0.11 and 0.14 g/g-dry native biomass (24-hour incubation period), respectively. Examination of the two types of thermophilic L-lactate fermentation employed following alkaline/peroxide pre-treatment and saccharification demonstrated that the L-lactate yield obtained using SSF (0.15 g/g in the case of corn) was lower than that obtained using separate saccharification and fermentation (0.28 g/g in the case of corn). The lower yield obtained from SSF is likely to have resulted from the saccharification conditions used in the present study, as the possibility of cellulase deactivation during SSF by thermophilic L-lactate producing bacteria existed. A cellulase that retains high activity levels under non-sterile conditions and a L-lactate producer without cellulose hydrolysis activity would be required in order for SSF to serve as an effective method of L-lactate production. PMID:22988611

  1. Insulin Degrading Enzyme Induces a Conformational Change in Varicella-Zoster Virus gE, and Enhances Virus Infectivity and Stability

    PubMed Central

    Li, Qingxue; Ali, Mir A.; Wang, Kening; Sayre, Dean; Hamel, Frederick G.; Fischer, Elizabeth R.; Bennett, Robert G.; Cohen, Jeffrey I.

    2010-01-01

    Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE), through its unique amino terminal extracellular domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE) enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines. PMID:20593027

  2. Enhanced production of ligninolytic enzymes and decolorization of molasses distillery wastewater by fungi under solid state fermentation.

    PubMed

    Pant, Deepak; Adholeya, Alok

    2007-10-01

    Selected isolates of fungi were grown on wheat straw and corncob in the presence of different moistening agents such as water, molasses, potato dextrose broth and distillery effluent. All the fungal isolates responded differently with respect to growth and ligninolytic enzyme production. Fungal growth on different substrates was checked by calculating ergosterol content, which varied widely within a single species when grown on different substrates. The maximum laccase production was obtained for Aspergillus flavus TERI DB9 grown on wheat straw with molasses. For manganese peroxidase, highest production was in Aspergillus niger TERI DB20 grown on corncob with effluent. Among the two isolates positive for lignin peroxidase, the highest production was in Fusarium verticillioides ITCC 6140. This immobilized fungal biomass was then used for decolorization of effluent from a cane molasses based distillery. Maximum decolorization (86.33%) was achieved in Pleurotus ostreatus (Florida) Eger EM 1303 immobilized on corncob with molasses in a period of 28 days. PMID:17177104

  3. Exogenous IAA treatment enhances phytoremediation of soil contaminated with phenanthrene by promoting soil enzyme activity and increasing microbial biomass.

    PubMed

    Li, Weiming; Wang, Dongsheng; Hu, Feng; Li, Huixin; Ma, Lili; Xu, Li

    2016-06-01

    In this study, we aimed to confirm that indole-3-acetic acid promotes plant uptake of phenanthrene (PHE), stimulates the activity of soil enzymes or microflora, and thereby accelerates the dissipation of PHE in soil. Four treatments were evaluated: PHE-contaminated soil planted with (1) ryegrass (T0), (2) ryegrass and supplemented with 1 mg kg(-1) indole-3-acetic acid (IAA) (T1), (3) ryegrass and supplemented with 5 mg kg(-1) IAA (T5), and (4) ryegrass and supplemented with 10 mg kg(-1) IAA (T10). After 30 days, PHE concentrations were lower for all treatments and the removal rate was 70.19, 89.17, 91.26, and 97.07 % for T0, T1, T5, and T10, respectively. PHE was only detected in the roots and not in the shoots. IAA facilitated the accumulation of PHE in the roots, and plants subjected to the T10 treatment had the highest levels. Exogenous IAA stimulated soil peroxidase activity in a dose-dependent manner, whereas soil polyphenoloxidase activity was not significantly increased, except in T10. Soil microbial biomass also increased in response to IAA treatment, particularly in T10. Furthermore, phospholipid fatty acid analysis showed that IAA treatment increased microbial biomass and alleviated environmental stress. Gram-positive bacteria are largely responsible for polycyclic aromatic hydrocarbon degradation, and we found that the ratio of gram-positive to gram-negative bacteria in the soil significantly increased as the IAA concentrations increased (P < 0.05). Correlation analysis indicated that the increase in soil microbial biomass, enzyme activity, and plant uptake of PHE promotes removal of PHE from the soil. PMID:26884240

  4. An arterial-specific enhancer of the human endothelin converting enzyme 1 (ECE1) gene is synergistically activated by Sox17, FoxC2, and Etv2.

    PubMed

    Robinson, Ashley S; Materna, Stefan C; Barnes, Ralston M; De Val, Sarah; Xu, Shan-Mei; Black, Brian L

    2014-11-15

    Endothelin-converting enzyme-1 (Ece-1), a crucial component of the Endothelin signaling pathway, is required for embryonic development and is an important regulator of vascular tone, yet the transcriptional regulation of the ECE1 gene has remained largely unknown. Here, we define the activity and regulation of an enhancer from the human ECE1 locus in vivo. The enhancer identified here becomes active in endothelial progenitor cells shortly after their initial specification and is dependent on a conserved FOX:ETS motif, a composite binding site for Forkhead transcription factors and the Ets transcription factor Etv2, for activity in vivo. The ECE1 FOX:ETS motif is bound and cooperatively activated by FoxC2 and Etv2, but unlike other described FOX:ETS-dependent enhancers, ECE1 enhancer activity becomes restricted to arterial endothelium and endocardium by embryonic day 9.5 in transgenic mouse embryos. The ECE1 endothelial enhancer also contains an evolutionarily-conserved, consensus SOX binding site, which is required for activity in transgenic mouse embryos. Importantly, the ECE1 SOX site is bound and activated by Sox17, a transcription factor involved in endothelial cell differentiation and an important regulator of arterial identity. Moreover, the ECE1 enhancer is cooperatively activated by the combinatorial action of FoxC2, Etv2, and Sox17. Although Sox17 is required for arterial identity, few direct transcriptional targets have been identified in endothelial cells. Thus, this work has important implications for our understanding of endothelial specification and arterial subspecification. PMID:25179465

  5. Methyl jasmonate effectively enhanced some defense enzymes activity and Total Antioxidant content in harvested "Sabrosa" strawberry fruit.

    PubMed

    Asghari, Mohammadreza; Hasanlooe, Ali Rashid

    2016-05-01

    The use of chemicals in postharvest technology of horticultural crops is highly restricted and it is necessary to introduce safe food preserving methods. Strawberry is very susceptible to postharvest losses and more than 50% of harvested fruit is lost in Iran. Effect of postharvest treatment with methyl jasmonate (at 0, 8, and 16 μmol L(-1)) on some quality attributes of Sabrosa strawberry fruit during storage at 1 ± 0.5°C with 90-95% RH for 14 days followed by 24 h at 20°C was studied. Methyl jasmonate, at both concentrations, decreased weight loss and retained marketability of fruits. Catalase activity of treated fruits was decreased during the first days, but showed a substantial increase during the second week. Methyl jasmonate, in a concentration-dependent manner, enhanced peroxidase activity. Fruit total antioxidant capacity was enhanced by methyl jasmonate treatment. The results indicated that methyl jasmonate plays a key role in establishing resistance against stresses, enhancing fruit defense systems, antioxidant capacity, and storage life leading to decreased postharvest losses. This phytochemical has a good potential to be used in postharvest technology of Sabrosa strawberry fruit and enhance the fruit postharvest life. PMID:27247768

  6. Understanding of alkaline pretreatment parameters for corn stover enzymatic saccharification

    PubMed Central

    2013-01-01

    Background Previous research on alkaline pretreatment has mainly focused on optimization of the process parameters to improve substrate digestibility. To achieve satisfactory sugar yield, extremely high chemical loading and enzyme dosages were typically used. Relatively little attention has been paid to reduction of chemical consumption and process waste management, which has proven to be an indispensable component of the bio-refineries. To indicate alkali strength, both alkali concentration in pretreatment solution (g alkali/g pretreatment liquor or g alkali/L pretreatment liquor) and alkali loading based on biomass solids (g alkali/g dry biomass) have been widely used. The dual approaches make it difficult to compare the chemical consumption in different process scenarios while evaluating the cost effectiveness of this pretreatment technology. The current work addresses these issues through pretreatment of corn stover at various combinations of pretreatment conditions. Enzymatic hydrolysis with different enzyme blends was subsequently performed to identify the effects of pretreatment parameters on substrate digestibility as well as process operational and capital costs. Results The results showed that sodium hydroxide loading is the most dominant variable for enzymatic digestibility. To reach 70% glucan conversion while avoiding extensive degradation of hemicellulose, approximately 0.08 g NaOH/g corn stover was required. It was also concluded that alkali loading based on total solids (g NaOH/g dry biomass) governs the pretreatment efficiency. Supplementing cellulase with accessory enzymes such as α-arabinofuranosidase and β-xylosidase significantly improved the conversion of the hemicellulose by 6–17%. Conclusions The current work presents the impact of alkaline pretreatment parameters on the enzymatic hydrolysis of corn stover as well as the process operational and capital investment costs. The high chemical consumption for alkaline pretreatment technology

  7. Selecting β-glucosidases to support cellulases in cellulose saccharification

    PubMed Central

    2013-01-01

    Background Enzyme end-product inhibition is a major challenge in the hydrolysis of lignocellulose at a high dry matter consistency. β-glucosidases (BGs) hydrolyze cellobiose into two molecules of glucose, thereby relieving the product inhibition of cellobiohydrolases (CBHs). However, BG inhibition by glucose will eventually lead to the accumulation of cellobiose and the inhibition of CBHs. Therefore, the kinetic properties of candidate BGs must meet the requirements determined by both the kinetic properties of CBHs and the set-up of the hydrolysis process. Results The kinetics of cellobiose hydrolysis and glucose inhibition of thermostable BGs from Acremonium thermophilum (AtBG3) and Thermoascus aurantiacus (TaBG3) was studied and compared to Aspergillus sp. BG purified from Novozyme®188 (N188BG). The most efficient cellobiose hydrolysis was achieved with TaBG3, followed by AtBG3 and N188BG, whereas the enzyme most sensitive to glucose inhibition was AtBG3, followed by TaBG3 and N188BG. The use of higher temperatures had an advantage in both increasing the catalytic efficiency and relieving the product inhibition of the enzymes. Our data, together with data from a literature survey, revealed a trade-off between the strength of glucose inhibition and the affinity for cellobiose; therefore, glucose-tolerant BGs tend to have low specificity constants for cellobiose hydrolysis. However, although a high specificity constant is always an advantage, in separate hydrolysis and fermentation, the priority may be given to a higher tolerance to glucose inhibition. Conclusions The specificity constant for cellobiose hydrolysis and the inhibition constant for glucose are the most important kinetic parameters in selecting BGs to support cellulases in cellulose hydrolysis. PMID:23883540

  8. Simultaneous saccharification and co-fermentation for bioethanol production using corncobs at lab, PDU and demo scales

    PubMed Central

    2013-01-01

    Background While simultaneous saccharification and co-fermentation (SSCF) is considered to be a promising process for bioconversion of lignocellulosic materials to ethanol, there are still relatively little demo-plant data and operating experiences reported in the literature. In the current work, we designed a SSCF process and scaled up from lab to demo scale reaching 4% (w/v) ethanol using xylose rich corncobs. Results Seven different recombinant xylose utilizing Saccharomyces cerevisiae strains were evaluated for their fermentation performance in hydrolysates of steam pretreated corncobs. Two strains, RHD-15 and KE6-12 with highest ethanol yield and lowest xylitol yield, respectively were further screened in SSCF using the whole slurry from pretreatment. Similar ethanol yields were reached with both strains, however, KE6-12 was chosen as the preferred strain since it produced 26% lower xylitol from consumed xylose compared to RHD-15. Model SSCF experiments with glucose or hydrolysate feed in combination with prefermentation resulted in 79% of xylose consumption and more than 75% of the theoretical ethanol yield on available glucose and xylose in lab and PDU scales. The results suggest that for an efficient xylose conversion to ethanol controlled release of glucose from enzymatic hydrolysis and low levels of glucose concentration must be maintained throughout the SSCF. Fed-batch SSCF in PDU with addition of enzymes at three different time points facilitated controlled release of glucose and hence co-consumption of glucose and xylose was observed yielding 76% of the theoretical ethanol yield on available glucose and xylose at 7.9% water insoluble solids (WIS). With a fed-batch SSCF in combination with prefermentation and a feed of substrate and enzymes 47 and 40 g l-1 of ethanol corresponding to 68% and 58% of the theoretical ethanol yield on available glucose and xylose were produced at 10.5% WIS in PDU and demo scale, respectively. The strain KE6-12 was able to

  9. CenC, a multidomain thermostable GH9 processive endoglucanase from Clostridium thermocellum: cloning, characterization and saccharification studies.

    PubMed

    Haq, Ikram ul; Akram, Fatima; Khan, Mahmood Ali; Hussain, Zahid; Nawaz, Ali; Iqbal, Kaleem; Shah, Ali Javed

    2015-11-01

    The growing demands of bioenergy has led to the emphasis on novel cellulases to improve efficiency of biodegradation process of plant biomass. Therefore, a thermostable cellulolytic gene (CenC) with 3675 bp was cloned from Clostridium thermocellum and over-expressed in Escherichia coli strain BL21 CodonPlus. It was attested that CenC belongs to glycoside hydrolase family 9 (GH9) with four binding domains, a processive endoglucanase. CenC was purified to homogeneity, producing a single band on SDS-PAGE corresponding to 137.11 kDa, by purification steps of heat treatment combined with ion-exchange chromatography. Purified enzyme displayed optimal activity at pH 6.0 and 70 °C. CenC had a half-life of 24 min at 74 °C, was stable up to 2 h at 60 °C and over a pH range of 5.5-7.5. Enzyme showed high affinity towards various substrates and processively released cellobiose from cellulosic substrates. It efficiently hydrolyzed carboxymethyl cellulose (30 U/mg), β-Glucan Barley (94 U/mg); also showed activity towards p-nitrophenyl-β-D-cellobioside (18 U/mg), birchwood xylan (19 U/mg), beechwood xylan (17.5 U/mg), avicel (9 U/mg), whatman filter paper (11 U/mg) and laminarin (3.3 U/mg). CenC exhibited Km, Vmax, Kcat, Vmax Km(-1) and Kcat Km(-1) of 7.14 mM, 52.4 µmol mg(-1) min(-1), 632.85 s(-1), 7.34 min(-1) and 88.63, respectively used CMC as substrate. Recombinant CenC saccharified pretreated wheat straw and bagasse to 5.12 and 7.31%, respectively at pH 7.0 and 45 °C after 2 h incubation. Its thermostability, high catalytic efficiency and independence of inhibitors make CenC enzyme an appropriate candidate for industrial applications and cost-effective saccharification process. PMID:26250549

  10. Ionic liquid/ultrasound pretreatment and in situ enzymatic saccharification of bagasse using biocompatible cholinium ionic liquid.

    PubMed

    Ninomiya, Kazuaki; Kohori, Asami; Tatsumi, Mai; Osawa, Koji; Endo, Takatsugu; Kakuchi, Ryohei; Ogino, Chiaki; Shimizu, Nobuaki; Takahashi, Kenji

    2015-01-01

    Choline acetate (ChOAc), a cholinium ionic liquid (IL), showed almost the same bagasse pretreatment capability as 1-ethyl-3-methylimidazolium acetate (EmimOAc), a conventional imidazolium IL used for biomass pretreatment. Moreover, ChOAc showed less of an inhibitory effect on cellulase than EmimOAc. Thus, ChOAc was used for IL/ultrasound-assisted pretreatment and in situ enzymatic saccharification, where IL was not washed out from the pretreated bagasse but diluted with the addition of a buffer solution. When in situ saccharification was performed for 48h in the presence of 10% ChOAc, the cellulose and hemicellulose saccharification percentages were 80% and 72%, respectively. When ChOAc was increased to 20%, the saccharification percentages were 72% and 53%, respectively. However, the values were just 28% and 2%, respectively, in case of 20% EmimOAc. A glucose/xylose solution free from IL and ChOAc aqueous solution without these sugars could be recovered separately by electrodialysis of the hydrolysate of in situ saccharification. PMID:25460999

  11. The deubiquitinating enzyme USP5 modulates neuropathic and inflammatory pain by enhancing Cav3.2 channel activity.

    PubMed

    García-Caballero, Agustin; Gadotti, Vinicius M; Stemkowski, Patrick; Weiss, Norbert; Souza, Ivana A; Hodgkinson, Victoria; Bladen, Chris; Chen, Lina; Hamid, Jawed; Pizzoccaro, Anne; Deage, Mickael; François, Amaury; Bourinet, Emmanuel; Zamponi, Gerald W

    2014-09-01

    T-type calcium channels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Cav3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Cav3.2 ubiquitination, decreases Cav3.2 protein levels, and reduces Cav3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T-type channel activity and their role in nociceptive signaling. PMID:25189210

  12. A new beta-glucosidase producing yeast for lower-cost cellulosic ethanol production from xylose-extracted corncob residues by simultaneous saccharification and fermentation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conventional cellulose-to-ethanol conversion by simultaneous saccharification and fermentation (SSF)requires enzymatic saccharification using both cellulase and ß-glucosidase allowing cellulose utilization by common ethanologenic yeast. Here we report a new yeast strain of Clavispora NRRL Y-50464 th...

  13. Enhanced production of polygalacturonase in solid-state fermentation: selection of the process conditions, isolation and partial characterization of the enzyme.

    PubMed

    Zaslona, Halina; Trusek-Holownia, Anna

    2015-01-01

    Polygalacturonase (PG) production by Penicillium chrysogenum during solid-state fermentation was accompanied by decomposition of orange peels. A leaching procedure was developed through the selection of solvent, time and intensity of stirring. A maximum PG activity was observed after 48 h peel inoculation. Further cultivation decreased the enzyme activity significantly, up to 60% of the maximum PG activity. During fermentation, a rapid acidification of the solid medium which inhibited the pectinolytic enzyme, was observed. Buffering agents with different pH values and different ionic strengths were examined to identify the most suitable medium to avoid this problem. Buffer addition counteracted acidification and enhanced active protein production, which was observed for all of the applied pH values (6.5-8.0) of the buffering agent. The most satisfactory results were obtained when using the highest pH at 8.0. The protein content and PG activity increased from 3.5 mg/g and 1.09 U/g to 7.7 mg/g and 7.11 U/g during cultivation, with uncontrolled and pH-controlled medium, respectively. Measurements at wide pH and temperature ranges indicated an optimum for PG activity at pH 5.0 and 43°C; however, high thermal stability corresponded to lower temperatures, and a temperature of 37°C is thus recommended. Under these conditions, the operational stability was determined to be t1/2=570 h. PMID:26505127

  14. Aggregated silver nanoparticles based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecules.

    PubMed

    Liang, Jiajie; Liu, Hongwu; Huang, Caihong; Yao, Cuize; Fu, Qiangqiang; Li, Xiuqing; Cao, Donglin; Luo, Zhi; Tang, Yong

    2015-06-01

    Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac. PMID:25928837

  15. Robust enzyme-silica composites made from enzyme nanocapsules.

    PubMed

    Li, Jie; Jin, Xin; Liu, Yang; Li, Fan; Zhang, Linlin; Zhu, Xianyuan; Lu, Yunfeng

    2015-06-14

    Novel enzyme composites are synthesized first by in situ polymerization around enzymes and a subsequent sol-gel process. Both the polymer shell and the silica shell with desired functional moieties provide not only great enzyme protection but also a favorable microenvironment, resulting in significantly enhanced activity and stability. PMID:25971337

  16. Isolation, screening, and identification of potential cellulolytic and xylanolytic producers for biodegradation of untreated oil palm trunk and its application in saccharification of lemongrass leaves.

    PubMed

    Ang, S K; Yahya, Adibah; Abd Aziz, Suraini; Md Salleh, Madihah

    2015-01-01

    This study presents the isolation and screening of fungi with excellent ability to degrade untreated oil palm trunk (OPT) in a solid-state fermentation system (SSF). Qualitative assay of cellulases and xylanase indicates notable secretion of both enzymes by 12 fungal strains from a laboratory collection and 5 strains isolated from a contaminated wooden board. High production of these enzymes was subsequently quantified in OPT in SSF. Aspergillus fumigates SK1 isolated from cow dung gives the highest xylanolytic activity (648.448 U g(-1)), generally high cellulolytic activities (CMCase: 48.006, FPase: 6.860, beta-glucosidase: 16.328 U g(-1)) and moderate lignin peroxidase activity (4.820 U/g), and highest xylanolytic activity. The xylanase encoding gene of Aspergillus fumigates SK1 was screened using polymerase chain reaction by a pair of degenerate primers. Through multiple alignment of the SK1 strain's xylanase nucleotide sequences with other published xylanases, it was confirmed that the gene belonged to the xylanase glycoside hydrolase family 11 (GH11) with a protein size of 24.49 kD. Saccharification of lemongrass leaves using crude cellulases and xylanase gives the maximum reducing sugars production of 6.84 g/L with glucose as the major end product and traces of phenylpropanic compounds (vanillic acid, p-coumaric acid, and ferulic acid). PMID:24960316

  17. Liver microsomal drug-metabolizing enzyme activity: enhancement by blockade of degradative processes in promethazine-treated rats.

    PubMed Central

    Fernández, G.; Villarruel, M. C.; Bernacchi, A.; de Castro, C. R.; Castro, J. A.

    1981-01-01

    Daily injection of promethazine over 4 days significantly increased the liver cytochrome P-450 content and ethyl morphine N-demethylase activity. These increases were evident after the first dose and were prevented by puromycin or actinomycin D administration. Repeated administration of promethazine does not increase the liver's ability to incorporate [14]C DL-leucine in microsomes but slows down the decay of radioactivity in microsomes previously labelled with ([14C]-guanidino) arginine. Repeated treatment with promethazine leads to a marked proliferation of the rough endoplasmic reticulum (RER) and a slight increase in the smooth endoplasmic reticulum (SER). Our findings suggest that the enhancement of P-450 and EM-ase activity result from the decelerating effect of promethazine on protein degradation. Images Fig. 1 Fig. 2 PMID:7295538

  18. Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor

    PubMed Central

    Núñez-López, Lizeth; Aguirre-Cruz, Andrés

    2015-01-01

    Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200–300% more glucose, adult vegetative plants yielded 40–90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production. PMID:25780769

  19. Distinct Geographical Distribution of the Miscanthus Accessions with Varied Biomass Enzymatic Saccharification

    PubMed Central

    Li, Xukai; Liao, Haofeng; Fan, Chunfen; Hu, Huizhen; Li, Ying; Li, Jing; Yi, Zili; Cai, Xiwen; Peng, Liangcai; Tu, Yuanyuan

    2016-01-01

    Miscanthus is a leading bioenergy candidate for biofuels, and it thus becomes essential to characterize the desire natural Miscanthus germplasm accessions with high biomass saccharification. In this study, total 171 natural Miscanthus accessions were geographically mapped using public database. According to the equation [P(H/L| East) = P(H/L∩East)/P(East)], the probability (P) parameters were calculated on relationships between geographical distributions of Miscanthus accessions in the East of China, and related factors with high(H) or low(L) values including biomass saccahrification under 1% NaOH and 1% H2SO4 pretreatments, lignocellulose features and climate conditions. Based on the maximum P value, a golden cutting line was generated from 42°25’ N, 108°22’ E to 22°58’ N, 116°28’ E on the original locations of Miscanthus accessions with high P(H|East) values (0.800–0.813), indicating that more than 90% Miscanthus accessions were originally located in the East with high biomass saccharification. Furthermore, the averaged insolation showed high P (H|East) and P(East|H) values at 0.782 and 0.754, whereas other climate factors had low P(East|H) values, suggesting that the averaged insolation is unique factor on Miscanthus distributions for biomass saccharification. In terms of cell wall compositions and wall polymer features, both hemicelluloses level and cellulose crystallinity (CrI) of Miscanthus accessions exhibited relative high P values, suggesting that they should be the major factors accounting for geographic distributions of Miscanthus accessions with high biomass digestibility. PMID:27532636

  20. Distinct Geographical Distribution of the Miscanthus Accessions with Varied Biomass Enzymatic Saccharification.

    PubMed

    Li, Xukai; Liao, Haofeng; Fan, Chunfen; Hu, Huizhen; Li, Ying; Li, Jing; Yi, Zili; Cai, Xiwen; Peng, Liangcai; Tu, Yuanyuan

    2016-01-01

    Miscanthus is a leading bioenergy candidate for biofuels, and it thus becomes essential to characterize the desire natural Miscanthus germplasm accessions with high biomass saccharification. In this study, total 171 natural Miscanthus accessions were geographically mapped using public database. According to the equation [P(H/L| East) = P(H/L∩East)/P(East)], the probability (P) parameters were calculated on relationships between geographical distributions of Miscanthus accessions in the East of China, and related factors with high(H) or low(L) values including biomass saccahrification under 1% NaOH and 1% H2SO4 pretreatments, lignocellulose features and climate conditions. Based on the maximum P value, a golden cutting line was generated from 42°25' N, 108°22' E to 22°58' N, 116°28' E on the original locations of Miscanthus accessions with high P(H|East) values (0.800-0.813), indicating that more than 90% Miscanthus accessions were originally located in the East with high biomass saccharification. Furthermore, the averaged insolation showed high P (H|East) and P(East|H) values at 0.782 and 0.754, whereas other climate factors had low P(East|H) values, suggesting that the averaged insolation is unique factor on Miscanthus distributions for biomass saccharification. In terms of cell wall compositions and wall polymer features, both hemicelluloses level and cellulose crystallinity (CrI) of Miscanthus accessions exhibited relative high P values, suggesting that they should be the major factors accounting for geographic distributions of Miscanthus accessions with high biomass digestibility. PMID:27532636

  1. Effect of ionic liquid weight ratio on pretreatment of bamboo powder prior to enzymatic saccharification.

    PubMed

    Ninomiya, Kazuaki; Soda, Hiroshi; Ogino, Chiaki; Takahashi, Kenji; Shimizu, Nobuaki

    2013-01-01

    The pretreatment efficiency of weight ratios ranging from 0 to 10 of the ionic liquid, cholinum IL, to bamboo powder was investigated. An IL/biomass ratio of 3g/g was critical to obtain a cellulose saccharification ratio of 80%. At this ratio, the treated bamboo powder remained as a solid. The solid-state pretreatment required a minimum amount of cholinium IL, which could reduce the cost of IL-assisted pretreatment and reduce the amount of wastewater generated in the process. PMID:23196237

  2. Optimization of the simultaneous saccharification and fermentation process using thermotolerant yeasts.

    PubMed

    Ballesteros, I; Oliva, J M; Ballesteros, M; Carrasco, J

    1993-01-01

    Different treatments to improve the thermotolerance of fermenting yeasts for simultaneous ethanol saccharification and fermentation process of cellulosic materials have been examined. Yeasts of the genera Saccharomyces and Kluyveromyces were tested for growth and fermentation at progressively higher temperatures in the range of 42-47 degrees C. The best results were obtained with K. marxianus LG, which was then submitted to different treatments in order to achieve thermotolerant clones. A total of 35 new clones were obtained that dramatically improved the SSF of 10% Solka-floc substrate at 45 degrees C when compared to the original strain, some with ethanol concentrations as high as 33 g/L. PMID:8323260

  3. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  4. Monitoring enzyme kinetic behavior of enzyme-quantum dot bioconjugates

    NASA Astrophysics Data System (ADS)

    Claussen, Jonathan C.; Walper, Scott A.; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

    2014-05-01

    Luminescent semiconductor nanocrystals or quantum dots (QDs) hold tremendous promise for in vivo biosensing, cellular imaging, theranostics, and smart molecular sensing probes due to their small size and favorable photonic properties such as resistance to photobleaching, size-tunable PL, and large effective Stokes shifts. Herein, we demonstrate how QD-based bioconjugates can be used to enhance enzyme kinetics. Enzyme-substrate kinetics are analyzed for solutions containing both alkaline phosphatase enzymes and QDs with enzyme-to- QD molar ratios of 2, 12, and 24 as well as for a solution containing the same concentration of enzymes but without QDs. The enzyme kinetic paramters Vmax, KM, and Kcat/KM are extracted from the enzyme progress curves via the Lineweaver-Burk plot. Results demonstrate an approximate increase in enzyme efficiency of 5 - 8% for enzymes immobilized on the QD versus free in solution without QD immobilization.

  5. Resveratrol protects the ovary against chromium-toxicity by enhancing endogenous antioxidant enzymes and inhibiting metabolic clearance of estradiol.

    PubMed

    Banu, Sakhila K; Stanley, Jone A; Sivakumar, Kirthiram K; Arosh, Joe A; Burghardt, Robert C

    2016-07-15

    Resveratrol (RVT), a polyphenolic component in grapes and red wine, has been known for its cytoprotective actions against several diseases. However, beneficial effects of RVT against early exposure to endocrine disrupting chemicals (EDCs) have not been understood. EDCs are linked to several ovarian diseases such as premature ovarian failure, polycystic ovary syndrome, early menopause and infertility in women. Hexavalent chromium (CrVI) is a heavy metal EDC, and widely used in >50 industries. Environmental contamination with CrVI in the US is rapidly increasing, predisposing the human to several illnesses including cancers and still birth. Our lab has been involved in determining the molecular mechanism of CrVI-induced female infertility and intervention strategies to mitigate CrVI effects. Lactating mother rats were exposed to CrVI (50ppm potassium dichromate) from postpartum days 1-21 through drinking water with or without RVT (10mg/kg body wt., through oral gavage daily). During this time, F1 females received respective treatments through mother's milk. On postnatal day (PND) 25, blood and the ovary, kidney and liver were collected from the F1 females for analyses. CrVI increased atresia of follicles by increasing cytochrome C and cleaved caspase-3; decreasing antiapoptotic proteins; decreasing estradiol (E2) biosynthesis and enhancing metabolic clearance of E2, increasing oxidative stress and decreasing endogenous antioxidants. RVT mitigated the effects of CrVI by upregulating cell survival proteins and AOXs; and restored E2 levels by inhibiting hydroxylation, glucuronidation and sulphation of E2. This is the first study to report the protective effects of RVT against any toxicant in the ovary. PMID:27129868

  6. Dietary antioxidant supplementation enhances lipid and protein oxidative stability of chicken broiler meat through promotion of antioxidant enzyme activity1

    PubMed Central

    Delles, Rebecca M.; Xiong, Youling L.; True, Alma D.; Ao, Touying; Dawson, Karl A.

    2014-01-01

    Recent nutrigenomic studies have shown that animal nutrition can have a major influence on tissue gene expression. Dietary antioxidant supplements can enhance the quality of meat through modification of tissue metabolic processes. This study investigated the influence of dietary antioxidants and quality of oil on the oxidative and enzymatic properties of chicken broiler breast meat stored in an oxygen-enriched package (HiOx: 80% O2/20% CO2) in comparison with air-permeable polyvinylchloride (PVC) or skin packaging systems during retail display at 2 to 4°C for up to 21 d. Broilers were fed either a diet with a low-oxidized (peroxide value 23 mEq of O2/kg) or high-oxidized (peroxide value 121 mEq of O2/kg) oil, supplemented with or without an algae-based Se yeast and organic mineral antioxidant pack for 42 d. Lipid and protein oxidation and tissue enzymatic activity were analyzed. In all packaging systems, lipid oxidation (TBA reactive substances) was inhibited by up to 32.5% (P < 0.05) with an antioxidant-supplemented diet when compared with diets without antioxidants, particularly in the HiOx and PVC systems. Protein sulfhydryls were significantly protected by antioxidant diets (e.g., by 14.6 and 17.8% for low-and high-oxidized dietary groups, respectively, in PVC d 7 samples). Glutathione peroxidase, catalase, and superoxide dismutase activities were significantly higher (P < 0.05) in antioxidant-supplemented diets compared with the basal diet, regardless of oil quality. Also, serum carbonyls were lower in broilers fed a low-oxidized antioxidant-supplemented treatment. The results demonstrate that dietary antioxidants can minimize the oxidative instability of proteins and lipids, and the protection may be linked to improved cellular antioxidant enzymatic activity. PMID:24879706

  7. Dietary antioxidant supplementation enhances lipid and protein oxidative stability of chicken broiler meat through promotion of antioxidant enzyme activity.

    PubMed

    Delles, Rebecca M; Xiong, Youling L; True, Alma D; Ao, Touying; Dawson, Karl A

    2014-06-01

    Recent nutrigenomic studies have shown that animal nutrition can have a major influence on tissue gene expression. Dietary antioxidant supplements can enhance the quality of meat through modification of tissue metabolic processes. This study investigated the influence of dietary antioxidants and quality of oil on the oxidative and enzymatic properties of chicken broiler breast meat stored in an oxygen-enriched package (HiOx: 80% O2/20% CO2) in comparison with air-permeable polyvinylchloride (PVC) or skin packaging systems during retail display at 2 to 4°C for up to 21 d. Broilers were fed either a diet with a low-oxidized (peroxide value 23 mEq of O2/kg) or high-oxidized (peroxide value 121 mEq of O2/kg) oil, supplemented with or without an algae-based Se yeast and organic mineral antioxidant pack for 42 d. Lipid and protein oxidation and tissue enzymatic activity were analyzed. In all packaging systems, lipid oxidation (TBA reactive substances) was inhibited by up to 32.5% (P < 0.05) with an antioxidant-supplemented diet when compared with diets without antioxidants, particularly in the HiOx and PVC systems. Protein sulfhydryls were significantly protected by antioxidant diets (e.g., by 14.6 and 17.8% for low-and high-oxidized dietary groups, respectively, in PVC d 7 samples). Glutathione peroxidase, catalase, and superoxide dismutase activities were significantly higher (P < 0.05) in antioxidant-supplemented diets compared with the basal diet, regardless of oil quality. Also, serum carbonyls were lower in broilers fed a low-oxidized antioxidant-supplemented treatment. The results demonstrate that dietary antioxidants can minimize the oxidative instability of proteins and lipids, and the protection may be linked to improved cellular antioxidant enzymatic activity. PMID:24879706

  8. Biocatalyst Enhancement

    EPA Science Inventory

    The increasing availability of enzyme collections has assisted attempts by pharmaceutical producers to adopt green chemistry approaches to manufacturing. A joint effort between an enzyme producer and a pharmaceutical manufacturer has been enhanced over the past three years by ena...

  9. Enzyme-Controlled Intracellular Self-Assembly of 18F Nanoparticles for Enhanced MicroPET Imaging of Tumor

    PubMed Central

    Liu, Yaling; Miao, Qingqing; Zou, Pei; Liu, Longfei; Wang, Xiaojing; An, Linna; Zhang, Xiaoliu; Qian, Xiangping; Luo, Shineng; Liang, Gaolin

    2015-01-01

    Herein, we report the development of a new “smart” radioactive probe (i.e., 1) which can undergo furin-controlled condensation and self-assembly of radioactive nanoparticles (i.e., 1-NPs) in tumor cells and its application for enhanced microPET imaging of tumors in nude mice co-injected with its cold analog (i.e., 1-Cold). Furin-controlled condensation of 1-Cold and self-assembly of its nanoparticles (i.e., 1-Cold-NPs) in vitro were validated and characterized with HPLC, mass spectra, SEM, and TEM analyses. Cell uptake studies showed that both 1 and 1-Cold have good cell permeability. TEM images of 1-Cold-treated MDA-MB-468 cells directly uncovered that the intracellular 1-Cold-NPs were at/near the location of furin (i.e., Golgi bodies). MTT results indicated that 50 µM 1-Cold did not impose cytotoxicity to MDA-MB-468 cells up to 12 hours. MicroPET imaging of MDA-MB-468 tumor-bearing mice indicated that mice co-injected with 1 and 1-Cold showed higher uptake and longer attenuation of the radioactivity in tumors than those mice only injected with same dosage of 1. Tumor uptake ratios of 1 between these two groups of mice reached the maximum of 8.2 folds at 240 min post injection. Biodistribution study indicated that the uptake ratios of 1 in kidneys between these two groups continuously increased and reached 81.9 folds at 240 min post injection, suggesting the formation of radioactive NPs (i.e., 1-NPs) in MDA-MB-468 tumors of mice co-injected with 1 and 1-Cold. And the nanoparticles were slowly digested and secreted from the tumors, accumulating in the kidneys. Our ''smart'' probe (i.e., 1), together with the strategy of co-injection, might help researchers trace the biomarkers of interest within a longer time window. PMID:26199645

  10. Enhanced Gastrointestinal Expression of Cytosolic Malic Enzyme (ME1) Induces Intestinal and Liver Lipogenic Gene Expression and Intestinal Cell Proliferation in Mice

    PubMed Central

    Al-Dwairi, Ahmed; Brown, Adam R.; Pabona, John Mark P.; Van, Trang H.; Hamdan, Hamdan; Mercado, Charles P.; Quick, Charles M.; Wight, Patricia A.; Simmen, Rosalia C. M.; Simmen, Frank A.

    2014-01-01

    The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue’s metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies. PMID:25402228

  11. Cholesterol enhances amyloid {beta} deposition in mouse retina by modulating the activities of A{beta}-regulating enzymes in retinal pigment epithelial cells

    SciTech Connect

    Wang, Jiying; Ohno-Matsui, Kyoko; Morita, Ikuo

    2012-08-10

    age-matched mice fed standard rodent chow diet did not. Activities and mRNA levels of NEP and {alpha}-secretase were significantly lower in native RPE cells freshly isolated from cholesterol-enriched chow fed mice compared to standard rodent chow fed mice. These findings suggest that cholesterol enhances subretinal A{beta} accumulation by modulating the activities of enzymes degrading and processing A{beta} in RPE cells in senescent subjects.

  12. Characterization of Cellulase Enzyme Inhibitors Formed During the Chemical Pretreatments of Rice Straw

    NASA Astrophysics Data System (ADS)

    Rajan, Kalavathy

    Production of fuels and chemicals from a renewable and inexpensive resource such as lignocellulosic biomass is a lucrative and sustainable option for the advanced biofuel and bio-based chemical platform. Agricultural residues constitute the bulk of potential feedstock available for cellulosic fuel production. On a global scale, rice straw is the largest source of agricultural residues and is therefore an ideal crop model for biomass deconstruction studies. Lignocellulosic biofuel production involves the processes of biomass conditioning, enzymatic saccharification, microbial fermentation and ethanol distillation, and one of the major factors affecting its techno-economic feasibility is the biomass recalcitrance to enzymatic saccharification. Preconditioning of lignocellulosic biomass, using chemical, physico-chemical, mechanical and biological pretreatments, is often practiced such that biomass becomes available to downstream processing. Pretreatments, such as dilute acid and hot water, are effective means of biomass conversion. However, despite their processing importance, preconditioning biomass also results in the production of carbohydrate and lignin degradation products that are inhibitory to downstream saccharification enzymes. The saccharification enzyme cocktail is made up of endo-cellulase, exo-cellulase and beta-glucosidase enzymes, whose role is to cleave cellulose polymers into glucose monomers. Specifically, endo-cellulase and exo-cellulase enzymes cleave cellulose chains in the middle and at the end, resulting in cellobiose molecules, which are hydrolyzed into glucose by beta-glucosidase. Unfortunately, degradation compounds generated during pretreatment inhibit the saccharification enzyme cocktail. Various research groups have identified specific classes of inhibitors formed during biomass pretreatment and have studied their inhibitory effect on the saccharification cocktail. These various research groups prepared surrogate solutions in an attempt to

  13. Conversion of steam-exploded cedar into ethanol using simultaneous saccharification, fermentation and detoxification process.

    PubMed

    Asada, Chikako; Sasaki, Chizuru; Takamatsu, Tomoki; Nakamura, Yoshitoshi

    2015-01-01

    In this study, we investigated the simultaneous saccharification, fermentation and detoxification SSDF process of steam-exploded cedar using a detoxification microorganism, Ureibacillus thermosphaericus A1, to facilitate efficient ethanol production. Steam explosion was applied as a pretreatment before enzymatic saccharification followed by alcohol fermentation. The highest glucose conversion rate was observed in the sample pretreated with a steam pressure of 45atm for 5min. Alcohol production by a heat-tolerant yeast, Saccharomyces cerevisiae BA11, was inhibited strongly by inhibitory materials present in the steam-exploded cedar, such as formic acid, furfural, and 5-hydroxymethylfurfural. The maximum amount of ethanol, i.e., 0.155g ethanol/g dry steam-exploded cedar, which corresponded to 74% of the theoretical ethanol yield, was obtained using the SSDF when U. thermosphaericus A1 degraded the inhibitory materials. A fed batch SSDF culture, in which U. thermosphaericus A1 was used to maintain low concentrations of inhibitory materials, was effective for increasing the ethanol concentration. PMID:25461004

  14. Study of chemical pretreatment and enzymatic saccharification for producing fermentable sugars from rice straw.

    PubMed

    Chen, Wen-Hsing; Chen, Yi-Chun; Lin, Jih-Gaw

    2014-07-01

    This study evaluated a cost-effective approach for the conversion of rice straw into fermentable sugars. The composition of rice straw pretreated with 1 % sulfuric acid or 1 % sodium hydroxide solution was compared to rice straw with no chemical pretreatment. Enzymatic saccharification experiments on non-pretreated rice straw (NPRS), pretreated rice straw (PRS), and pretreated rice straw with acid hydrolysate (PRSAH) were conducted in a series of batch reactors. The results indicated that pretreating the rice straw with dilute acid and base increased the cellulose content from 38 % to over 50 %. During enzymatic saccharification, straight aliphatic cellulose was hydrolyzed before branched hemicellulose, and glucose was the major hydrolysis product. The glucose yield was 0.52 g glucose/g for NPRS and was comparable to the yields of 0.50 g glucose/g for PRS and 0.58 g glucose/g for PRSAH. The hydrolysis of rice straw to produce glucose can be described by a first-order reaction with a rate constant of 0.0550 d(-1) for NPRS, 0.0653 d(-1) for PRSAH, and 0.0654 d(-1) for PRS. Overall, the production of fermentable sugars from ground rice straw will be more cost effective if the straw is not pretreated with chemicals. PMID:24346765

  15. Grape marc as a source of carbohydrates for bioethanol: Chemical composition, pre-treatment and saccharification.

    PubMed

    Corbin, Kendall R; Hsieh, Yves S Y; Betts, Natalie S; Byrt, Caitlin S; Henderson, Marilyn; Stork, Jozsef; DeBolt, Seth; Fincher, Geoffrey B; Burton, Rachel A

    2015-10-01

    Global grape production could generate up to 13 Mt/yr of wasted biomass. The compositions of Cabernet Sauvignon (red marc) and Sauvignon Blanc (white marc) were analyzed with a view to using marc as raw material for biofuel production. On a dry weight basis, 31-54% w/w of the grape marc consisted of carbohydrate, of which 47-80% was soluble in aqueous media. Ethanol insoluble residues consisted mainly of polyphenols, pectic polysaccharides, heteroxylans and cellulose. Acid and thermal pre-treatments were investigated for their effects on subsequent cellulose saccharification. A 0.5M sulfuric acid pre-treatment yielded a 10% increase in the amount of liberated glucose after enzymatic saccharification. The theoretical amount of bioethanol that could be produced by fermentation of grape marc was up to 400 L/t. However, bioethanol from only soluble carbohydrates could yield 270 L/t, leaving a polyphenol enriched fraction that may be used in animal feed or as fertilizer. PMID:26117238

  16. Epigallocatechin gallate incorporation into lignin enhances the alkaline delignification and enzymatic saccharification of cell walls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epigallocatechin gallate (EGCG) was evaluated as a potential lignin bioengineering target for rendering biomass more amenable to processing for biofuel production. In vitro peroxidase-catalyzed polymerization experiments revealed that both gallate and pyrogalloyl (B-ring) moieties in EGCG underwent ...

  17. Enhanced enzymatic saccharification of sugarcane bagasse pretreated by combining O2 and NaOH.

    PubMed

    Bi, Shuaizhu; Peng, Lincai; Chen, Keli; Zhu, Zhengliang

    2016-08-01

    Sugarcane bagasse pretreated by combining O2 and NaOH with different variables was conducted to improve its enzymatic digestibility and sugar recovery, and the results were compared with sole NaOH pretreatment. Lignin removal for O2-NaOH pretreatment was around 10% higher than that for sole NaOH pretreatment under the same conditions, and O2-NaOH pretreatment resulted in higher glucan recovery in the solid remain. Subsequently, O2-NaOH pretreated sugarcane bagasse presented more efficient enzymatic digestibility than sole NaOH pretreatment. Under the moderate pretreatment conditions of combining 1% NaOH and 0.5MPa O2 at 80°C for 120min, a high glucan conversion of 95% was achieved after 48h enzymatic hydrolysis. Coupled with the operations of pretreatment and enzymatic hydrolysis, an admirable total sugar recovery of 89% (glucose recovery of 93% and xylose recovery of 84%) was obtained. The susceptibility of the substrates to enzymatic digestibility was explained by their physical and chemical characteristics. PMID:27208740

  18. 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

  19. Biomimetic cell wall model studies to identify new lignin bioengineering targets for improving biomass susceptibility to pretreatment and enzymatic saccharification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasingly, bioengineering of lignin to contain atypical building blocks from other metabolic pathways is being pursued to custom-design lignin that is easier to remove by chemical pretreatments and less inhibitory toward polysaccharide saccharification. Because plants produce such a diverse array...

  20. Simultaneous saccharification and fermentation of pretreated wheat straw to fuel ethanol using a recombinant bacterium for improved efficiency

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Typically four process steps are involved in the production of fuel ethanol from any lignocellulosic feedstock – pretreatment, enzymatic saccharification, fermentation, and ethanol recovery. Integration of two or more steps is important for lowering the ethanol production cost from lignocellulose. M...

  1. Effects of tea saponin on glucan conversion and bonding behaviour of cellulolytic enzymes during enzymatic hydrolysis of corncob residue with high lignin content

    PubMed Central

    2013-01-01

    Background Recently, interest in the utilization of corncob residue (CCR, with high lignin of 45.1%) as a feedstock for bioethanol has been growing. Surfactants have been one of the most popular additives intended to prevent the inhibitory effect of lignin on cellulolytic enzymes, thereby improving hydrolysis. In this study, the effects of biosurfactant tea saponin (TS) on the enzymatic hydrolysis of CCR and the bonding behavior of cellulolytic enzymes to the substrate were investigated. The surface tension in the supernatant was also detected to obtain information about the characteristics and stability of TS. Results The glucose concentration was 17.15 mg/mL at 120 hours of hydrolysis with the low loading of cellulolytic enzymes (7.0 FPU/g cellulose and 10.5 BGU/g cellulose) and 5% CCR. The optimal dosage of TS was its critical micelle concentration (cmc, 1.80 mg/mL). The glucose yield was enhanced from 34.29 to 46.28 g/100 g dry matter by TS. The results indicate that TS can promote the adsorption of cellulolytic enzymes on the substrate and mediate the release of adsorbed enzymes. Meanwhile, TS improves the recovery of the cellulolytic enzymes after a hydrolysis cycle and prevents deactivation of the enzymes during the intense shaking process. The surface tension in supernatants of digested CCR with TS remained at 50.00 mN/m during the course of hydrolysis. It is interesting to note that biosurfactant TS can maintain the surface tension in supernatants, despite its digestibility by cellulolytic enzymes. Conclusions Serving as an accelerant of lignocellulose hydrolysis, TS can also be degraded by the cellulolytic enzymes and release glucose while retaining stability, which reduces the cost of both the cellulolytic enzymes and the additive. As the glucose from the TS could be utilized by yeast, further efforts will investigate the mechanism of function and the application of TS in the production of ethanol by simultaneous saccharification and fermentation

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

    PubMed

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

    2014-10-01

    Understanding the process of biomass degradation by cellulolytic enzymes is of urgent importance for biofuel and chemical production. Optimizing pretreatment conditions and improving enzyme formulations both require assays to quantify saccharification products on solid substrates. Typically, such assays are performed using freely diffusing fluorophores or dyes that measure reducing polysaccharide chain ends. These methods have thus far not allowed spatial localization of hydrolysis activity to specific substrate locations with identifiable morphological features. Here we describe a hydrogel reagent signaling (HyReS) system that amplifies saccharification products and initiates crosslinking of a hydrogel that localizes to locations of cellulose hydrolysis, allowing for imaging of the degradation process in real time. Optical detection of the gel in a rapid parallel format on synthetic and natural pretreated solid substrates was used to quantify activity of T. emersonii and T. reesei enzyme cocktails. When combined with total internal reflection fluorescence microscopy and AFM imaging, the reagent system provided a means to visualize enzyme activity in real-time with high spatial resolution (<2 μm). These results demonstrate the versatility of the HyReS system in detecting cellulolytic enzyme activity and suggest new opportunities in real-time chemical imaging of biomass depolymerization. PMID:25116339

  3. High Fat Diet Enhances β-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting β-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation.

    PubMed

    Maesako, Masato; Uemura, Maiko; Tashiro, Yoshitaka; Sasaki, Kazuki; Watanabe, Kiwamu; Noda, Yasuha; Ueda, Karin; Asada-Utsugi, Megumi; Kubota, Masakazu; Okawa, Katsuya; Ihara, Masafumi; Shimohama, Shun; Uemura, Kengo; Kinoshita, Ayae

    2015-01-01

    Obesity and type 2 diabetes are risk factors of Alzheimer's disease (AD). We reported that a high fat diet (HFD) promotes amyloid precursor protein (APP) cleavage by β-site APP cleaving enzyme 1 (BACE1) without increasing BACE1 levels in APP transgenic mice. However, the detailed mechanism had remained unclear. Here we demonstrate that HFD promotes BACE1/Adaptor protein-2 (AP-2)/clathrin complex formation by increasing AP-2 levels in APP transgenic mice. In Swedish APP overexpressing Chinese hamster ovary (CHO) cells as well as in SH-SY5Y cells, overexpression of AP-2 promoted the formation of BACE1/AP-2/clathrin complex, increasing the level of the soluble form of APP β (sAPPβ). On the other hand, mutant D495R BACE1, which inhibits formation of this trimeric complex, was shown to decrease the level of sAPPβ. Overexpression of AP-2 promoted the internalization of BACE1 from the cell surface, thus reducing the cell surface BACE1 level. As such, we concluded that HFD may induce the formation of the BACE1/AP-2/clathrin complex, which is followed by its transport of BACE1 from the cell surface to the intracellular compartments. These events might be associated with the enhancement of β-site cleavage of APP in APP transgenic mice. Here we present evidence that HFD, by regulation of subcellular trafficking of BACE1, promotes APP cleavage. PMID:26414661

  4. Task-specific enhancement of hippocampus-dependent learning in mice deficient in monoacylglycerol lipase, the major hydrolyzing enzyme of the endocannabinoid 2-arachidonoylglycerol

    PubMed Central

    Kishimoto, Yasushi; Cagniard, Barbara; Yamazaki, Maya; Nakayama, Junko; Sakimura, Kenji; Kirino, Yutaka; Kano, Masanobu

    2015-01-01

    Growing evidence indicates that the endocannabinoid system is important for the acquisition and/or extinction of learning and memory. However, it is unclear which endocannabinoid(s) play(s) a crucial role in these cognitive functions, especially memory extinction. To elucidate the physiological role of 2-arachidonoylglycerol (2-AG), a major endocannabinoid, in behavioral and cognitive functions, we conducted a comprehensive behavioral test battery in knockout (KO) mice deficient in monoacylglycerol lipase (MGL), the major hydrolyzing enzyme of 2-AG. We found age-dependent increases in spontaneous physical activity (SPA) in MGL KO mice. Next, we tested the MGL KO mice using 5 hippocampus-dependent learning paradigms (i.e., Morris water maze (MWM), contextual fear conditioning, novel object recognition test, trace eyeblink conditioning, and water-finding test). In the MWM, MGL KO mice showed normal acquisition of reference memory, but exhibited significantly faster extinction of the learned behavior. Moreover, they showed faster memory acquisition on the reversal-learning task of the MWM. In contrast, in the contextual fear conditioning, MGL KO mice tended to show slower memory extinction. In the novel object recognition and water-finding tests, MGL KO mice exhibited enhanced memory acquisition. Trace eyeblink conditioning was not altered in MGL KO mice throughout the acquisition and extinction phases. These results indicate that 2-AG signaling is important for hippocampus-dependent learning and memory, but its contribution is highly task-dependent. PMID:26082696

  5. MLN4924, a Novel NEDD8-activating enzyme inhibitor, exhibits antitumor activity and enhances cisplatin-induced cytotoxicity in human cervical carcinoma: in vitro and in vivo study

    PubMed Central

    Lin, Wei-Chou; Kuo, Kuan-Lin; Shi, Chung-Sheng; Wu, June-Tai; Hsieh, Ju-Ton; Chang, Hong-Chiang; Liao, Shih-Ming; Chou, Chien-Tso; Chiang, Chih-Kang; Chiu, Wei-Shuo; Chiu, Tzu-Yuan; Pu, Yeong-Shiau; Ho, I-Lin; Wang, Zuo-He; Chang, Shih-Chen; Liu, Shing-Hwa; Jeng, Yung-Ming; Huang, Kuo-How

    2015-01-01

    MLN4924, an inhibitor of NEDD8 activating enzyme (NAE), has been reported to have activity against various malignancies. Here, we investigated the antitumor properties of MLN4924 and MLN4924 in combination with cisplatin on human cervical carcinoma (CC) in vitro and in vivo. Two human CC cell lines, ME-180 and HeLa, were used in this study. The cytotoxic effects of MLN4924 and/or cisplatin were measured by cell viability (MTT), proliferation (BrdU incorporation), apoptosis (flow cytometry with annexin V-FITC labeling), and the expression of cell apoptosis-related proteins (Western blotting). In vivo efficacy was determined in Nu/Nu nude mice with ME-180 and HeLa xenografts. The results showed that MLN4924 elicited viability inhibition, anti-proliferation and apoptosis in human CC cells, accompanied by activations of apoptosis-related molecules and Bid, Bcl-2 phosphorylation interruption, and interference with cell cycle regulators. Moreover, MLN4924 caused an endoplasmic reticulum stress response (caspase-4, ATF-4 and CHOP activations) and expression of other cellular stress molecules (JNK and c-Jun activations). Additionally, MLN4924 suppressed growth of CC xenografts in nude mice. Furthermore, we demonstrated that MLN4924 potentiated cisplatin-induced cytotoxicity in CC cells with activation of caspases. Consistently with this, MLN4924 significantly enhanced cisplatin-induced growth inhibition of CC xenografts. Together, these findings suggest that MLN4924 alone or in combination with cisplatin is of value in treating human CCs. PMID:26807316

  6. Purification and characterisation of processive-type endoglucanase and β-glucosidase from Aspergillus ochraceus MTCC 1810 through saccharification of delignified coir pith to glucose.

    PubMed

    Asha, P; Divya, Jose; Bright Singh, I S

    2016-08-01

    The study describes purification and characterisation of processive-type endoglucanase and β-glucosidase from Aspergillus ochraceus MTCC 1810 through bioconversion of delignified coir pith to fermentable glucose. The purified processive endoglucanase (AS-HT-Celuz A) and β-glucosidase (AS-HT-Celuz B) were found to have molecular mass of ≈78-kDa and 43-kDa respectively with optimum endoglucanase (35.63U/ml), total cellulase (28.15FPU/ml) and β-glucosidase (15.19U/ml) activities at 40°C/pH 6. The unique feature of AS-HT-Celuz A is the multiple substrate specificity and processivity towards both amorphous and crystalline cellulose. Zymogram indicated both endo and exoglucanase activities residing in different binding sites of a single protein exhibiting sequential synergy with its own β-glucosidase. Accordingly, the identified enzymes could be implemented as synergistic cellulases for complete cellulose saccharification which still considered an unresolved issue in bio-refineries. PMID:26976061

  7. Mutation in Brachypodium caffeic acid O-methyltransferase 6 alters stem and grain lignins and improves straw saccharification without deteriorating grain quality

    PubMed Central

    Ho-Yue-Kuang, Séverine; Alvarado, Camille; Antelme, Sébastien; Bouchet, Brigitte; Cézard, Laurent; Le Bris, Philippe; Legée, Frédéric; Maia-Grondard, Alessandra; Yoshinaga, Arata; Saulnier, Luc; Guillon, Fabienne; Sibout, Richard; Lapierre, Catherine; Chateigner-Boutin, Anne-Laure

    2016-01-01

    Cereal crop by-products are a promising source of renewable raw material for the production of biofuel from lignocellulose. However, their enzymatic conversion to fermentable sugars is detrimentally affected by lignins. Here the characterization of the Brachypodium Bd5139 mutant provided with a single nucleotide mutation in the caffeic acid O-methyltransferase BdCOMT6 gene is reported. This BdCOMT6-deficient mutant displayed a moderately altered lignification in mature stems. The lignin-related BdCOMT6 gene was also found to be expressed in grains, and the alterations of Bd5139 grain lignins were found to mirror nicely those evidenced in stem lignins. The Bd5139 grains displayed similar size and composition to the control. Complementation experiments carried out by introducing the mutated gene into the AtCOMT1-deficient Arabidopsis mutant demonstrated that the mutated BdCOMT6 protein was still functional. Such a moderate down-regulation of lignin-related COMT enzyme reduced the straw recalcitrance to saccharification, without compromising the vegetative or reproductive development of the plant. PMID:26433202

  8. Systematic optimization of fed-batch simultaneous saccharification and fermentation at high-solid loading based on enzymatic hydrolysis and dynamic metabolic modeling of Saccharomyces cerevisiae.

    PubMed

    Unrean, Pornkamol; Khajeeram, Sutamat; Laoteng, Kobkul

    2016-03-01

    An integrative simultaneous saccharification and fermentation (SSF) modeling is a useful guiding tool for rapid process optimization to meet the techno-economic requirement of industrial-scale lignocellulosic ethanol production. In this work, we have developed the SSF model composing of a metabolic network of a Saccharomyces cerevisiae cell associated with fermentation kinetics and enzyme hydrolysis model to quantitatively capture dynamic responses of yeast cell growth and fermentation during SSF. By using model-based design of feeding profiles for substrate and yeast cell in the fed-batch SSF process, an efficient ethanol production with high titer of up to 65 g/L and high yield of 85 % of theoretical yield was accomplished. The ethanol titer and productivity was increased by 47 and 41 %, correspondingly, in optimized fed-batch SSF as compared to batch process. The developed integrative SSF model is, therefore, considered as a promising approach for systematic design of economical and sustainable SSF bioprocessing of lignocellulose. PMID:26610806

  9. Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol▿ †

    PubMed Central

    Wen, Fei; Sun, Jie; Zhao, Huimin

    2010-01-01

    By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of Saccharomyces cerevisiae strains displaying a series of uni-, bi-, and trifunctional minicellulosomes. These minicellulosomes consist of (i) a miniscaffoldin containing a cellulose-binding domain and three cohesin modules, which was tethered to the cell surface through the yeast a-agglutinin adhesion receptor, and (ii) up to three types of cellulases, an endoglucanase, a cellobiohydrolase, and a β-glucosidase, each bearing a C-terminal dockerin. Cell surface assembly of the minicellulosomes was dependent on expression of the miniscaffoldin, indicating that formation of the complex was dictated by the high-affinity interactions between cohesins and dockerins. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme synergy and enzyme proximity synergy. More importantly, surface display of the trifunctional minicellulosomes gave yeast cells the ability to simultaneously break down and ferment phosphoric acid-swollen cellulose to ethanol with a titer of ∼1.8 g/liter. To our knowledge, this is the first report of a recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes. The strain reported here represents a useful engineering platform for developing CBP-enabling microorganisms and elucidating principles of cellulosome construction and mode of action. PMID:20023102

  10. Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth.

    PubMed

    Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon

    2013-06-01

    A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system. PMID:23377262

  11. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  12. Understanding Enzymes.

    ERIC Educational Resources Information Center

    Sinnott, M. L.

    1979-01-01

    Describes the way enzymes operate through reaction energetics, and explains that most of the catalytic power of enzymes lies in the strong noncovalent forces responsible for initial binding of substrate, which are only manifested at the transition state of the reaction. (Author/GA)

  13. Soil Enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The functionality and resilience of natural and managed ecosystems mainly rely on the metabolic abilities of microbial communities, the main source of enzymes in soils. Enzyme mediated reactions are critical in the decomposition of organic matter, cycling of nutrients, and in the breakdown of herbic...

  14. Simultaneous saccharification and fermentation of cassava to succinic acid by Escherichia coli NZN111.

    PubMed

    Chen, Cuixia; Ding, Shaopeng; Wang, Dezheng; Li, Zhimin; Ye, Qin

    2014-07-01

    In this study, the production of succinic acid from cassava starch and raw cassava instead of glucose by Escherichia coli NZN111 was investigated. During the two-stage fermentation, simultaneous saccharification and fermentation (SSF) was applied in the anaerobic stage. The results showed that both the productivity and specific productivity in the process conducted at 40°C were higher than those in the cultivation conducted at 37°C. The yield of succinic acid based on the amount of added starch reached the highest level 0.86 g/g and cassava starch was almost totally hydrolyzed in the SSF process. With the improved cell density, 127.13 g/L of succinic acid was obtained. When the liquefied crude cassava powder was used directly in SSF, 106.17 g/L of succinic acid was formed. The result showed that crude cassava powder could be another cheap raw material for succinic acid formation. PMID:24787322

  15. Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol.

    PubMed

    Ballesteros, I; Ballesteros, M; Cabañas, A; Carrasco, J; Martín, C; Negro, M J; Saez, F; Saez, R

    1991-01-01

    A total of 27 yeast strains belonging to the groups Candida, Saccharomyces, and Kluyveromyces were screened for their ability to grow and ferment glucose at temperatures ranging 32-45 degrees C. K. marxianus and K. fragilis were found to be the best ethanol producing organisms at the higher temperature tested and, so, were selected for subsequent simultaneous saccharification and fermentation (SSF) studies. SSF experiments were performed at 42 and 45 degrees C, utilizing Solkafloc (10%) as cellulose substrate and a cellulase loading of 15 FPU/g substrate. Best results were achieved at 42 degrees C with K. marxianus L. G. and K. fragilis L. G., both of which produced close to 38 g/L ethanol and 0.5 ethanol yield, in 78 h. PMID:1929369

  16. Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol

    SciTech Connect

    Ballesteros, I.; Ballesteros, M.; Cabanas, A.

    1991-12-31

    A total of 27 yeast strains belonging to the groups Candida, Saccharomyces, and Kluyveromyces were screened for their ability to grow and ferment glucose at temperatures ranging 32-45{degrees}C. K. marxianus and K. fragilis were found to be the best ethanol producing organisms at the higher temperature tested and, so, were selected for subsequent simultaneous saccharification and fermentation (SSF) studies. SSF experiments were performed at 42 and 45{degrees}C, utilizing Solkafloc (10%) as cellulose substrate and a cellulose loading of 15 FPU/g substrate. Best results were achieved at 42{degrees}C with K. marxianus L. G. and K. fragilis L. G., both of which produced close to 38 g/L ethanol and 0.5 ethanol yield, in 78 h.

  17. Saccharification of natural lignocellulose biomass and polysaccharides by highly negatively charged heteropolyacids in concentrated aqueous solution.

    PubMed

    Ogasawara, Yoshiyuki; Itagaki, Shintaro; Yamaguchi, Kazuya; Mizuno, Noritaka

    2011-04-18

    Highly negatively charged heteropolyacids (HPAs), in particular H(5) BW(12) O(40) , efficiently promoted saccharification of crystalline cellulose into water-soluble saccharides in concentrated aqueous solutions (e.g., 82 % total yield and 77 % glucose yield, based on cellulose with a 0.7 M H(5) BW(12) O(40) solution); the performance was much better than those of previously reported systems with commonly utilized mineral acids (e.g., H(2) SO(4) and HCl) and HPAs (e.g., H(3) PW(12) O(40) and H(4) SiW(12) O(40)). Besides crystalline cellulose, the present system was applicable to the selective transformation of cellobiose, starch, and xylan to the corresponding monosaccharides such as glucose and xylose. In addition, one-pot synthesis of levulinic acid and sorbitol directly from cellulose was realized by using concentrated HPA solutions. The present system, concentrated aqueous solutions of highly negatively charged HPAs, was further applicable to saccharification of natural (non-purified) lignocellulose biomass, such as "rice plant straw", "oil palm empty fruit bunch (palm EFB) fiber", and "Japanese cedar sawdust", giving a mixture of the corresponding water-soluble saccharides, such as glucose (main product), galactose, mannose, xylose, arabinose, and cellobiose, in high yields (≥77 % total yields of saccharides based on holocellulose). Separation of the saccharides and H(5) BW(12) O(40) was easy, and the retrieved H(5) BW(12) O(40) could repeatedly be used without appreciable loss of the high performance. PMID:21404445

  18. Aqueous Ionic Liquids and Deep Eutectic Solvents for Cellulosic Biomass Pretreatment and Saccharification

    PubMed Central

    Xia, Shuqian; Baker, Gary A.; Li, Hao; Ravula, Sudhir; Zhao, Hua

    2014-01-01

    Ionic liquids (ILs) have proven effective solvents for pretreating lignocellulose, leading to the fast saccharification of cellulose and hemicellulose. However, the high current cost of most ILs remains a major barrier to commercializing this recent approach at a practical scale. As a strategic detour, aqueous solutions of ILs are also being explored as less costly alternatives to neat ILs for cellulose pretreatment. However, limited studies on a few select IL systems are known and there remains no systematic survey of various ILs, eluding an in-depth understanding of pretreatment mechanisms afforded by aqueous IL systems. As a step toward filling this gap, this study presents results for Avicel cellulose pretreatment by neat and aqueous solutions (1.0 and 2.0 M) of 20 different ILs and three deep eutectic solvents, correlating enzymatic hydrolysis rates of pretreated cellulose with various IL properties such as hydrogen-bond basicity, polarity, Hofmeister ranking, and hydrophobicity. The pretreatment efficiencies of neat ILs may be loosely correlated to the hydrogen-bond basicity of the constituent anion and IL polarity; however, the pretreatment efficacies for aqueous ILs are more complicated and cannot be simply related to any single IL property. Several aqueous IL systems have been identified as effective alternatives to neat ILs in lignocellulose pretreatment. In particular, this study reveals that aqueous solutions of 1-butyl-3-methylimidazolium methanesulfonate ([BMIM][MeSO3]) are effective for pretreating switchgrass (Panicum virgatum), resulting in fast saccharification of both cellulose and hemicellulose. An integrated analysis afforded by X-ray diffraction, scanning electron microscopy, thermogravimetric analysis and cellulase adsorption isotherm of lignocellulose samples is further used to deliver a more complete view of the structural changes attending aqueous IL pretreatment. PMID:24729865

  19. Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose.

    PubMed

    Pawar, Prashant Mohan-Anupama; Derba-Maceluch, Marta; Chong, Sun-Li; Gómez, Leonardo D; Miedes, Eva; Banasiak, Alicja; Ratke, Christine; Gaertner, Cyril; Mouille, Grégory; McQueen-Mason, Simon J; Molina, Antonio; Sellstedt, Anita; Tenkanen, Maija; Mellerowicz, Ewa J

    2016-01-01

    Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1-expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production. PMID:25960248

  20. Engineering Cellulase Enzymes for Bioenergy

    NASA Astrophysics Data System (ADS)

    Atreya, Meera Elizabeth

    methods. Protein engineering targets to improve cellulases include reducing enzyme inhibition, improving inter-enzyme synergy, and increasing enzyme thermotolerance. Ameliorating enzyme inhibition could improve catalytic activity and thus the speed of conversion from biomass to fermentable sugars. Improved enzyme synergy could reduce the enzyme loading required to achieve equivalent biomass conversion. Finally, thermostable enzymes could enable more biomass to be processed at a time, due to high temperatures decreasing the viscosity of biomass slurries. A high-temperature enzyme saccharification reaction could also decrease the risk of contamination in the resulting concentrated sugar solution. Throughout my PhD, I have explored research projects broadly across all of these topics, with the most success in addressing the issue of enzyme inhibition. Cellulase enzyme Cel7A is the most abundant cellulase employed by natural systems for cellulose hydrolysis. Cellobiohydrolase enzymes like Cel7A break down cellulose into cellobiose (two glucose molecules). Unfortunately, upon cleavage, this product molecule interferes with continued hydrolysis activity of Cel7A; the strong binding of cellobiose in the active site can obstruct the enzyme from processing down the cellulase chain. This phenomenon, known as product inhibition, is a bottleneck to efficient biomass breakdown. Using insights from computational protein modeling studies, I experimentally generated and tested mutant Cel7A enzymes for improved tolerance to cellobiose. Indeed, this strategy yielded Cel7A enzymes exhibiting reduced product inhibition, including some mutants completely impervious to cellobiose. The improvements in tolerance to cellobiose, however, resulted in an overall reduction of enzyme activity for the mutants tested. Nevertheless, my findings substantiated computational reports with experimental evidence and pinpointed an amino acid residue in the Cel7A product binding site that is of interest for

  1. Impact of grey zone sample testing by enzyme-linked immunosorbent assay in enhancing blood safety: Experience at a tertiary care hospital in North India

    PubMed Central

    Solanki, Archana; Singh, Abhay; Chaudhary, Rajendra

    2016-01-01

    Background: Enzyme-linked immunosorbent assay (ELISA) used for screening blood donors for transfusion transmitted infections (TTIs) can sometimes fail to detect blood donors who are recently infected or possessing the low strength of pathogen. Estimation of a grey zone in ELISA testing and repeat testing of grey zone samples can further help in reducing the risks of TTI in countries where nucleic acid amplification testing for TTIs is not feasible. Materials and Methods: Grey zone samples with optical density (OD) lying between cut-off OD and 10% below the cut-off OD (cut-off OD × 0.9) were identified during routine ELISA testing. On performing repeat ELISA testing on grey zone samples in duplicate, the samples showing both OD value below grey zone were marked nonreactive, and samples showing one or both OD value in the grey zone were marked indeterminate. The samples on repeat testing showing one or both OD above cut-off value were marked positive. Results: About 119 samples (77 for hepatitis B virus [HBV], 23 for human immunodeficiency virus [HIV], and 19 for hepatitis C virus [HCV]) were found to be in grey zone. On repeat testing of these samples in duplicate, 70 (58.8%) samples (45 for HBV, 12 for HIV, and 13 for HCV) were found to be reactive. Six (5%) samples (four for HBV, one for HIV, and one for HCV) were found to be indeterminate. Conclusion: Seventy donors initially screened negative, were found out to be potentially infectious on repeat grey zone testing. Thus, estimation of grey zone samples with repeat testing can further enhance the safety of blood transfusion. PMID:27011675

  2. Celastrol enhances Nrf2 mediated antioxidant enzymes and exhibits anti-fibrotic effect through regulation of collagen production against bleomycin-induced pulmonary fibrosis.

    PubMed

    Divya, Thomas; Dineshbabu, Vadivel; Soumyakrishnan, Syamala; Sureshkumar, Anandasadagopan; Sudhandiran, Ganapasam

    2016-02-25

    Pulmonary fibrosis (PF) is characterized by excessive accumulation of extracellular matrix components in the alveolar region which distorts the normal lung architecture and impairs the respiratory function. The aim of this study is to evaluate the anti-fibrotic effect of celastrol, a quinine-methide tri-terpenoid mainly found in Thunder God Vine root extracts against bleomycin (BLM)-induced PF through the enhancement of antioxidant defense system. A single intratracheal instillation of BLM (3 U/kg.bw) was administered in rats to induce PF. Celastrol (5 mg/kg) was given intraperitoneally, twice a week for a period of 28 days. BLM-induced rats exhibits declined activities of enzymatic and non-enzymatic antioxidants which were restored upon treatment with celastrol. BLM-induced rats show increased total and differential cell counts as compared to control and celastrol treated rats. Histopathological analysis shows increased inflammation and alveolar damage; while assay of hydroxyproline and Masson's trichrome staining shows an increased collagen deposition in BLM-challenged rats that were decreased upon celastrol treatment. Celastrol also reduces inflammation in BLM-induced rats as evidenced by decrease in the expressions of mast cells, Tumor necrosis factor-alpha (TNF- α) and matrix metalloproteinases (MMPs) 2 and 9. Further, Western blot analysis shows that celastrol is a potent inducer of NF-E2-related factor 2 (Nrf2) and it restores the activities of Phase II enzymes such as hemoxygenase-1 (HO-1), glutathione-S-transferase (GSTs) and NADP(H): quinine oxidoreductase (NQO1) which were declined upon BLM administration. The results of this study show evidence on the protective effect of celastrol against BLM-induced PF through its antioxidant and anti-fibrotic effects. PMID:26768587

  3. Kinetic studies on novel plasminogen activators. Demonstration of fibrin enhancement for hybrid enzymes comprising the A-chain of plasmin (Lys-78) and B-chain of tissue-type plasminogen activator (Ile-276) or urokinase (Ile-159).

    PubMed Central

    Fears, R; Dodd, I; Ferres, H; Robinson, J H

    1990-01-01

    The activation of plasminogen by two novel hybrid enzymes, constructed from the A-chain of plasmin and the B-chains of tissue-type plasminogen activator (t-PA) or urokinase, was compared with the activation by the parent enzymes. Basal kinetic constants for 'Lys-plasminogen' (human plasminogen with N-terminal lysine) and 'Glu-plasminogen' (human plasminogen with N-terminal glutamic acid) activation were similar to those of the parent activators. The Km for plasminogen turnover for both hybrid enzymes was considerably decreased in the presence of both soluble fibrin and a mimic, a CNBr digest of fibrinogen. These enhancements and the related apparent negative co-operativity are similar to the behaviour of t-PA itself. The results are discussed with regard to the molecular features involved in the mechanism of fibrin stimulation. PMID:2139324

  4. High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

    SciTech Connect

    Chou, Hong L.; Dai, Ziyu; Hsieh, Chia W.; Ku, Maurice S.

    2011-12-10

    Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. In this study, the cellulose hydrolytic enzyme {beta}-1, 4-endoglucanase (E1) from the thermophilic bacterium Acidothermus cellulolyticus was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer with the signal peptide of tobacco pathogenesis-related protein for targeting the protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial enzyme were obtained, which expressed the gene at high levels with a normal phenotype. The specific activities of E1 in the leaves of the highest expressing transgenic rice lines were about 20 fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. Zymogram and temperature-dependent activity analyses demonstrated the thermostability of the enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid yielded almost twice more reducing sugars than wild type straw. Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in

  5. Coniferyl ferulate incorporation into lignin dramatically enhances the delignification and enzymatic hydrolysis of maize cell walls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Incorporating ester interunit linkages into plant lignins could enhance the delignification and saccharification of fiber for fermentation into ethanol or other industrial products. In this study, we examined how substitution of coniferyl alcohol (a normal monolignol) with 0 to 60% coniferyl ferulat...

  6. Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation

    PubMed Central

    Elliston, Adam; Wilson, David R.; Wellner, Nikolaus; Collins, Samuel R.A.; Roberts, Ian N.; Waldron, Keith W.

    2015-01-01

    This study evaluated steam (SE) explosion on the saccharification and simultaneous saccharification and fermentation (SSF) of waste copier paper. SE resulted in a colouration, a reduction in fibre thickness and increased water absorption. Changes in chemical composition were evident at severities greater than 4.24 resulting in a loss of xylose and the production of breakdown products known to inhibit fermentation (particularly formic acid and acetic acid). SE did not improve final yields of glucose or ethanol, and at severities 4.53 and 4.83 reduced yields probably due to the effect of breakdown products and fermentation inhibitors. However, at moderate severities of 3.6 and 3.9 there was an increase in initial rates of hydrolysis which may provide a basis for reducing processing times. Co-steam explosion of waste copier paper and wheat straw attenuated the production of breakdown products, and may also provide a basis for improving SSF of lignocellulose. PMID:25846183

  7. Ag43-mediated display of a thermostable β-glucosidase in Escherichia coli and its use for simultaneous saccharification and fermentation at high temperatures

    PubMed Central

    2014-01-01

    Background The autotransporter (AT) system can potentially be used in the secretion of saccharolytic enzymes for the production of lignocellulosic biofuels and chemicals using Escherichia coli. Although ATs share similar structural characteristics, their capacity for secreting heterologous proteins widely varies. Additionally, the saccharolytic enzyme selected to be secreted should match the cell growth or cell fermentation conditions of E. coli. Results In the search for an AT that suits the physiological performance of the homo-ethanologenic E. coli strain MS04, an expression plasmid based on the AT antigen 43 (Ag43) from E. coli was developed. The β-glucosidase BglC from the thermophile bacterium Thermobifida fusca was displayed on the outer membrane of the E. coli strain MS04 using the Ag43 system (MS04/pAg43BglC). This strain was used to hydrolyze and ferment 40 g/L of cellobiose in mineral media to produce 16.65 g/L of ethanol in 48 h at a yield of 81% of the theoretical maximum. Knowing that BglC shows its highest activity at 50°C and retains more than 70% of its activity at pH 6, therefore E. coli MS04/pAg43BglC was used to ferment crystalline cellulose (Avicel) in a simultaneous saccharification and fermentation (SSF) process using a commercial cocktail of cellulases (endo and exo) at pH 6 and at a relatively high temperature for E. coli (45°C). As much as 22 g/L of ethanol was produced in 48 h. Conclusions The Ag43-BglC system can be used in E. coli strains without commercial β-glucosidases, reducing the quantities of commercial enzymes needed for the SSF process. Furthermore, the present work shows that E. coli cells are able to ferment sugars at 45°C during the SSF process using 40 g/L of Avicel, reducing the gap between the working conditions of the commercial saccharolytic enzymes and ethanologenic E. coli. PMID:25078445

  8. In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

    PubMed Central

    Hattori, Ai; Unno, Hideaki; Goda, Shuichiro; Motoyama, Kento; Yoshimura, Tohru

    2015-01-01

    ABSTRACT In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of β-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus. IMPORTANCE This study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance. PMID:26303832

  9. Saccharification and ethanol fermentation from cholinium ionic liquid-pretreated bagasse with a different number of post-pretreatment washings.

    PubMed

    Ninomiya, Kazuaki; Omote, Sayuri; Ogino, Chiaki; Kuroda, Kosuke; Noguchi, Mana; Endo, Takatsugu; Kakuchi, Ryohei; Shimizu, Nobuaki; Takahashi, Kenji

    2015-01-01

    Choline acetate (ChOAc), a cholinium ionic liquid (IL), was compared with 1-ethyl-3-methylimidazolium acetate (EmimOAc) with regard to biomass pretreatment, inhibition on cellulase and yeast, residuals in pretreated biomass, and saccharification and fermentation of pretreated biomass. Irrespective of ChOAc and EmimOAc, cellulose and hemicellulose saccharification of the IL-pretreated bagasse were over 90% and 60%, respectively. Median effective concentrations (EC50) based on cellulase activity were 32 wt% and 16 wt% for ChOAc and EmimOAc, respectively. The EC50 based on yeast growth were 3.1 wt% and 0.3 wt% for ChOAc and EmimOAc respectively. The residuals in IL-pretreated bagasse were 10% and 23% for ChOAc and EmimOAc, respectively, when washed 2 times after pretreatment. Ethanol yield on a bagasse basis were 60% and 24% for ChOAc and EmimOAc, respectively, in the saccharification and fermentation of IL-pretreated bagasse when washed 2 times. ChOAc-pretreated bagasse could be saccharified and fermented with fewer wash times than EmimOAc-pretreated bagasse. PMID:25898080

  10. Comparison of alkali treatments for efficient release of p-coumaric acid and enzymatic saccharification of sorghum pith.

    PubMed

    Jiang, Kankan; Li, Lulu; Long, Liangkun; Ding, Shaojun

    2016-05-01

    Two separate temperature and time ranges were respectively conducted for optimizing release of p-coumaric acid and enzymatic saccharification of sorghum pith by NaOH pretreatment using response surface methodology. Two desirable pretreatment conditions were selected as follows: 37°C, 2% NaOH and 12h, and 100°C, 1.75% NaOH and 37min in the low and high temperature ranges, respectively. Under these conditions, the enzymatic glucose yields were 85.6% and 90.4% respectively, whereas p-coumaric acid yields were 95.1% and 98.1% respectively. The final recovery of esterified p-coumaric acid reached 82.8% and 87.4% respectively after further separation with HP-20 resin. Interestingly, strong linear correlations exist between p-coumaric acid release with glucan saccharification yield and lignin dissolution. These results indicate that sorghum pith could be an attractive source for natural p-coumaric acid and efficient release of p-coumaric acid and enzymatic saccharification of sorghum pith can be achieved by mild NaOH pretreatment. PMID:26868149

  11. Characteristics of Corn Stover Pretreated with Liquid Hot Water and Fed-Batch Semi-Simultaneous Saccharification and Fermentation for Bioethanol Production

    PubMed Central

    Li, Xuezhi; Lu, Jie; Zhao, Jian; Qu, Yinbo

    2014-01-01

    Corn stover is a promising feedstock for bioethanol production because of its abundant availability in China. To obtain higher ethanol concentration and higher ethanol yield, liquid hot water (LHW) pretreatment and fed-batch semi-simultaneous saccharification and fermentation (S-SSF) were used to enhance the enzymatic digestibility of corn stover and improve bioconversion of cellulose to ethanol. The results show that solid residues from LHW pretreatment of corn stover can be effectively converted into ethanol at severity factors ranging from 3.95 to 4.54, and the highest amount of xylan removed was approximately 89%. The ethanol concentrations of 38.4 g/L and 39.4 g/L as well as ethanol yields of 78.6% and 79.7% at severity factors of 3.95 and 4.54, respectively, were obtained by fed-batch S-SSF in an optimum conditions (initial substrate consistency of 10%, and 6.1% solid residues added into system at the prehydrolysis time of 6 h). The changes in surface morphological structure, specific surface area, pore volume and diameter of corn stover subjected to LHW process were also analyzed for interpreting the possible improvement mechanism. PMID:24763192

  12. High β-glucosidase secretion in Saccharomyces cerevisiae improves the efficiency of cellulase hydrolysis and ethanol production in simultaneous saccharification and fermentation.

    PubMed

    Tang, Hongting; Hou, Jin; Shen, Yu; Xu, Lili; Yang, Hui; Fang, Xu; Bao, Xiaoming

    2013-11-28

    Bioethanol production from lignocellulose is considered as a sustainable biofuel supply. However, the low cellulose hydrolysis efficiency limits the cellulosic ethanol production. The cellulase is strongly inhibited by the major end product cellobiose, which can be relieved by the addition of β-glucosidase. In this study, three β-glucosidases from different organisms were respectively expressed in Saccharomyces cerevisiae and the β-glucosidase from Saccharomycopsis fibuligera showed the best activity (5.2 U/ml). The recombinant strain with S. fibuligera β-glucosidase could metabolize cellobiose with a specific growth rate similar to the control strain in glucose. This recombinant strain showed higher hydrolysis efficiency in the cellulose simultaneous saccharification and fermentation, when using the Trichoderma reesei cellulase, which is short of the β-glucosidase activity. The final ethanol concentration was 110% (using Avicel) and 89% (using acid-pretreated corncob) higher than the control strain. These results demonstrated the effect of β-glucosidase secretion in the recombinant S. cerevisiae for enhancing cellulosic ethanol conversion. PMID:23928840

  13. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  14. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  15. Production of cellulase enzyme by Trichoderma reesei Cefl9 and its application in the production of bio-ethanol.

    PubMed

    Kumar, M Ravi; Kumaran, M D Bala; Balashanmugam, P

    2014-05-01

    The present study aimed at the production of cellulase enzyme from the cellulolytic fungi Trichoderma reesei CEF19 and subsequent application of the cellulase for the fermentation of ethanol. For the same, the cellulolytic fungi, Trichoderma reesei CEF19 was isolated and was allowed to produce cellulase enzyme using optimized conditions. The cellulase enzyme was extracted and purified with the help of ammonium sulphate precipitation, dialysis followed by ion exchange chromatography with DEAE-Sephadex column. The purified cellulase enzyme was characterized using SDS-PAGE analysis. The saccharification of the cellulosic substrates was done using the cellulase enzyme. The fermentation of saccharified cellulosic substrates into ethanol was carried out using Saccharomyces cerevisiae. From the results obtained, rice straw was found to be the better source for the ethanol production when compared to the other substrates. PMID:26031010

  16. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    SciTech Connect

    Resch, M.

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution imaging techniques. Also, translating findings between model substrates to intact biomass is critical for evaluating enzyme performance. Here we employ a fungal free enzyme cocktail, a complexed cellulosomal system, and a combination of the two to investigate saccharification mechanisms on cellulose I, II and III along with corn stover from Clean Fractionation (CF), which is an Organosolv pretreatment. The insoluble Cellulose Enriched Fraction (CEF) from CF contains mainly cellulose with minor amounts of residual hemicellulose and lignin, the amount of which depends on the CF pretreatment severity. Enzymatic digestions at both low and high-solids loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated, dilute acid pretreated corn stover. Transmission and scanning electron microscopy of the biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and complexed enzyme systems, and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate for the first time. These results also demonstrate that the presence of lignin, rather than cellulose morphology, is more detrimental to cellulosome action than to free cellulases. As enzyme costs are a major economic driver for biorefineries, this study provides key inputs for the evaluation of CF as a pretreatment method for biomass conversion.

  17. Production of lactic acid from the mixture of softwood pre-hydrolysate and paper mill sludge by simultaneous saccharification and fermentation.

    PubMed

    Shi, Suan; Kang, Li; Lee, Y Y

    2015-03-01

    Paper mill sludge is a solid waste material composed of pulp residues and ash generated from pulping and paper making process. The carbohydrate portion of the sludges from Kraft/Recycle paper mill has chemical and physical characteristics similar to those of commercial wood pulp. Because of its high carbohydrate content and well-dispersed structure, the sludge can be biologically converted to value-added products without pretreatment. In bioconversion of solid feedstock such as paper mill sludge, a certain amount of water must be present to attain fluidity. In this study, hemicellulose pre-hydrolysate, in place of water, was added to the sludge to increase the concentration of the final product. Pre-hydrolysate was obtained by hot-water treatment of pine wood in which the total sugar concentration reached 4 wt.%. The mixture was processed by simultaneous saccharification and fermentation (SSF) using enzymes (cellulase and pectinase) and Lactobacillus rhamnosus (ATCC-10863). Pectinase was added to hydrolyze mannose oligomers in the pre-hydrolysate to monomers. During the SSF of the mixture, calcium carbonate in the paper sludge acted as a buffer, yielding calcium lactate as the final product. External pH control was unnecessary due to the buffer action of calcium carbonate that maintained the pH near optimum for the SSF. The lactic acid yield in the range of 80-90 % of the theoretical maximum was obtained. Use of the mixed feed of pre-hydrolysate and pulp mill sludges in the SSF raised the product concentration to 60 g of lactate/L. PMID:25561054

  18. Structural evaluation and bioethanol production by simultaneous saccharification and fermentation with biodegraded triploid poplar

    PubMed Central

    2013-01-01

    Background Pretreatment is a key step to decrease the recalcitrance of lignocelluloses and then increase the digestibility of cellulose in second-generation bioethanol production. In this study, wood chips from triploid poplar were biopretreated with white rot fungus Trametes velutina D10149. The effects of incubation duration on delignification efficiency and structural modification of cellulose were comparably studied, as well as the digestibility of cellulose by simultaneous saccharification and fermentation (SSF). Results Although microbial pretreatments did not significantly introduce lignin degradation, the data from SSF exhibited higher cellulose conversion (21-75% for biopretreated samples for 4–16 weeks) as compared to the untreated poplar (18%). In spite of the essential maintain of crystallinity, the modification of lignin structure during fungal treatment undoubtedly played a key role in improving cellulose bioconversion rates. Finally, the ethanol concentration of 5.16 g/L was detected in the fermentation broth from the cellulosic sample biodegraded for 16 weeks after 24 h SSF, achieving 34.8% cellulose utilization in poplar. Conclusion The potential fungal pretreatment with Trametes velutina D10149 was firstly explored in this study. It is found that the biopretreatment process had a significant effect on the digestibility of substrate probably due to the removal and unit variation of lignin, since the crystallinities of substrates were rarely changed. Additional investigation is still required especially to improve the selectivity for lignin degradation and optimize the digestibility of cellulose. PMID:23517540

  19. Biobutanediol-mediated liquefaction of empty fruit bunch saccharification residues to prepare lignin biopolyols.

    PubMed

    Lee, Jae Hoon; Lee, Eun Yeol

    2016-05-01

    Saccharification residue from empty fruit bunch (EFB) was liquefied with bio-butanediol to produce lignin biopolyols for the preparation of biopolyurethane. To substitute petroleum-derived polyhydric alcohols, butanediol isomers (1,4-butanediol, levo-2,3-bio-butanediol, and meso-2,3-bio-butanediol) or PEG#400-blended butanediol isomers were used as liquefaction solvents in the presence of sulfuric acid catalyst. Lignin biopolyols with a conversion of 63.3%, a hydroxyl number of 582.7 mg KOH/g and an acid number of 21.7 mg KOH/g were obtained under the optimal condition consisting of 25% biomass loading, 3% acid loading, and a temperature of 150°C for 120 min when liquefied with 1,4-butanediol/PEG#400 blended solvent (9/1, w/w). When the levo-2,3-bio-butanediol solvent was used in the absence of PEG#400, the highest conversion, 68.9%, was obtained. Lignin biopolyol-based biopolyurethanes were synthesized with toluene diisocyanate. FT-IR analysis revealed that EFB lignin biopolyols liquefied with bio-butanediols were suitable monomers for the preparation of biopolyurethane. PMID:26918835

  20. Ethanol production from banana peels using statistically optimized simultaneous saccharification and fermentation process.

    PubMed

    Oberoi, Harinder Singh; Vadlani, Praveen V; Saida, Lavudi; Bansal, Sunil; Hughes, Joshua D

    2011-07-01

    Dried and ground banana peel biomass (BP) after hydrothermal sterilization pretreatment was used for ethanol production using simultaneous saccharification and fermentation (SSF). Central composite design (CCD) was used to optimize concentrations of cellulase and pectinase, temperature and time for ethanol production from BP using SSF. Analysis of variance showed a high coefficient of determination (R(2)) value of 0.92 for ethanol production. On the basis of model graphs and numerical optimization, the validation was done in a laboratory batch fermenter with cellulase, pectinase, temperature and time of nine cellulase filter paper unit/gram cellulose (FPU/g-cellulose), 72 international units/gram pectin (IU/g-pectin), 37 °C and 15 h, respectively. The experiment using optimized parameters in batch fermenter not only resulted in higher ethanol concentration than the one predicted by the model equation, but also saved fermentation time. This study demonstrated that both hydrothermal pretreatment and SSF could be successfully carried out in a single vessel, and use of optimized process parameters helped achieve significant ethanol productivity, indicating commercial potential for the process. To the best of our knowledge, ethanol concentration and ethanol productivity of 28.2 g/l and 2.3 g/l/h, respectively from banana peels have not been reported to date. PMID:21376555

  1. The operable modeling of simultaneous saccharification and fermentation of ethanol production from cellulose.

    PubMed

    Shen, Jiacheng; Agblevor, Foster A

    2010-03-01

    An operable batch model of simultaneous saccharification and fermentation (SSF) for ethanol production from cellulose has been developed. The model includes four ordinary differential equations that describe the changes of cellobiose, glucose, yeast, and ethanol concentrations with respect to time. These equations were used to simulate the experimental data of the four main components in the SSF process of ethanol production from microcrystalline cellulose (Avicel PH101). The model parameters at 95% confidence intervals were determined by a MATLAB program based on the batch experimental data of the SSF. Both experimental data and model simulations showed that the cell growth was the rate-controlling step at the initial period in a series of reactions of cellulose to ethanol, and later, the conversion of cellulose to cellobiose controlled the process. The batch model was extended to the continuous and fed-batch operating models. For the continuous operation in the SSF, the ethanol productivities increased with increasing dilution rate, until a maximum value was attained, and rapidly decreased as the dilution rate approached the washout point. The model also predicted a relatively high ethanol mass for the fed-batch operation than the batch operation. PMID:19412687

  2. Comparison of different ionic liquids pretreatment for corn stover enzymatic saccharification.

    PubMed

    Mood, Sohrab Haghighi; Golfeshan, Amir Hossein; Tabatabaei, Meisam; Abbasalizadeh, Saeed; Ardjmand, Mehdi; Jouzani, Gholamreza Salehi

    2014-01-01

    Recently, application of ionic liquids (ILs) has received much attention due to their special solvency properties as a promising method of pretreatment for lignocellulosic biomass. Easy recovery of ionic liquids, chemical stability, temperature stability, nonflammability, low vapor pressure, and wide liquidus range are among those unique properties. These solvents are also known as green solvents due to their low vapor pressure. The present study was set to compare the effect of five different ILs, namely, 1-ethyl-3-methylimidazolium acetate ([EMIM][Ac]), 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]), 1-ethyl-3-methyl imidazolium diethyl phosphate ([EMIM][DEP]), 1-allyl-3-methylimidazolium chloride ([AMIM][Cl]), and 1-ethyl-3-methylimidazolium-hydrogen sulfate ([EMIM][HSO₄]), on corn stover in a bioethanol production process. The performance of ILs was evaluated based on the change observed in chemical structure, crystallinity index, cellulose digestibility, and glucose release. Overall, [EMIM][Ac]-pretreated corn stover led to significantly higher saccharification, with cellulose digestibility reaching 69% after 72 hr, whereas digestibility of untreated barley straw was measured at only 21%. PMID:24397717

  3. Butanol production from lignocellulose by simultaneous fermentation, saccharification, and pervaporation or vacuum evaporation.

    PubMed

    Díaz, Víctor Hugo Grisales; Tost, Gerard Olivar

    2016-10-01

    Techno-economic study of acetone, butanol and ethanol (ABE) fermentation from lignocellulose was performed. Simultaneous saccharification, fermentation and vacuum evaporation (SFS-V) or pervaporation (SFS-P) were proposed. A kinetic model of metabolic pathways for ABE fermentation with the effect of phenolics and furans in the growth was proposed based on published laboratory results. The processes were optimized in Matlab®. The end ABE purification was carried out by heat-integrated distillation. The objective function of the minimization was the total annualized cost (TAC). Fuel consumption of SFS-P using poly[1-(trimethylsilyl)-1-propyne] membrane was between 13.8 and 19.6% lower than SFS-V. Recovery of furans and phenolics for the hybrid reactors was difficult for its high boiling point. TAC of SFS-P was increased 1.9 times with supplementation of phenolics and furans to 3g/l each one for its high toxicity. Therefore, an additional detoxification method or an efficient pretreatment process will be necessary. PMID:27367813

  4. Mild pretreatment and enzymatic saccharification of cellulose with recycled ionic liquids towards one-batch process.

    PubMed

    Auxenfans, Thomas; Buchoux, Sébastien; Djellab, Karim; Avondo, Carine; Husson, Eric; Sarazin, Catherine

    2012-10-01

    The development of second-generation bioethanol involves minimizing the energy input throughout the processing steps. We report here that efficient ionic liquid pretreatments of cellulose can be achieved with short duration times (20 min) at mild temperature (45°C) with [Emim](+)[MeO(H)PO(2)](-) and at room temperature (25 °C) with [Emim](+)[CH(3)COO](-). In these conditions, yields of glucose were increased by a factor of 3. In addition, the recycling of these two imidazolium-based ILs can be performed in maintaining their efficiency to pretreat cellulose. The short time and mild temperature of cellulose solubilization allowed a one-batch processing of [Emim](+)[MeO(H)PO(2)](-) IL-pretreatment and saccharification. In the range from 0 to 100% IL in an aqueous enzymatic medium, the glucose yields were improved at IL proportions between 10 and 40%. The maximum yield at 10% IL is very promising to consider one batch process as efficient as two-step process. PMID:22840005

  5. Lignocellulose Recalcitrance Screening by Integrated High Throughput Hydrothermal Pretreatment and Enzymatic Saccharification

    SciTech Connect

    Selig, M. J.; Tucker, M. P.; Sykes, R. W.; Reichel, K. L.; Brunecky, R.; Himmel, M. E.; Davis, M. F.; Decker, S. R.

    2010-04-01

    We report a novel 96-well multiplate reactor system for comparative analysis of lignocellulose recalcitrance via integrated hydrothermal pretreatment and enzymatic saccharification. The system utilizes stackable nickel/gold-plated 96-well aluminum reactor plates, a clamping device fit to a standard Parr reactor, and robotics for efficient liquids and solids handling. A capacity of 20 plates allows up to 1,920 separate hydrothermal reactions per run. Direct and rapid analysis of key end-products, glucose and xylose, is facilitated by the use of glucose oxidase/peroxidase and xylose dehydrogenase-linked assays. To demonstrate efficacy, a set of 755 poplar core samples from the US Department of Energy's BioEnergy Science Center was tested. Total sugar release ranged from 0.17 to 0.64 g/g of biomass and correlated strongly with the ratio of syringyl to guaiacyl lignins in the samples. Variance among sample replicates was sufficiently minimal to permit clear assignment of differences in recalcitrance throughout this large sample set.

  6. Study on the bio-methane yield and microbial community structure in enzyme enhanced anaerobic co-digestion of cow manure and corn straw.

    PubMed

    Wang, Xuemei; Li, Zifu; Zhou, Xiaoqin; Wang, Qiqi; Wu, Yanga; Saino, Mayiani; Bai, Xue

    2016-11-01

    The use of enzymes to improve anaerobic co-digestion (AcoD) of cow manure and corn straw was explored in this study, including cellulase pretreatment and direct additions of amylase and protease. The effects of enzymes on microbial community structure were investigated though PCR-DGGE method. Results showed that AcoD with amylase achieved the highest methane yield of 377.63ml·CH4/g·VS, which was an increase of 110.79%. The methane increment consumed the amylase of 4.18×10(-5)g/ml·CH4. Enzymes mainly affected the bacteria in the hydrolysis stage rather than the bacteria in the hydrogenesis and acetogenesis stage and the archaea in the methanogenesis stage. However, the experimental results demonstrated that enzymes had no negative influence on microbial communities; the predominant microbial communities were similar. Therefore, AcoD with amylase was an effective way to improve the bio-methane yield of cow manure and corn straw. PMID:27484671

  7. Biomonitoring of Organophosphorus Agent Exposure by Reactivation of Cholinesterase Enzyme Based on Carbon Nanotube-Enhanced Flow-Injection Amperometric Detection

    SciTech Connect

    Du, Dan; Wang, Jun; Smith, Jordan N.; Timchalk, Charles; Lin, Yuehe

    2009-11-15

    A portable, rapid, and sensitive assessment of sub-clinical organophosphorus (OPs) agent exposure based on reactivation of cholinesterase (ChE) from OP-inhibited ChE using rat saliva (in vitro) was developed using an electrochemical sensor coupled with a microflow-injection system. The sensor was based on a carbon nanotube (CNT)-modified screen printed carbon electrode (SPE), which was integrated into a flow cell. Due to the extent of inter-individual ChE activity variability, ChE biomonitoring often requires an initial base-line determination (non-inhibited) of enzyme activity which is then directly compared with activity after OP exposure. This manuscript described an alternative strategy where reactivation of the phosphorylated enzyme was exploited to enable measurement of both inhibited and baseline ChE activity (i.e. after reactivation) in the same sample. The use of CNT makes the electrochemical detection of the products from enzymatic reactions more feasible with extremely high sensitivity and at low potentials. Paraoxon was selected as a model OP compound for in vitro inhibition studies. Some experiment parameters, (e.g. inhibition and reactivation times), have been optimized such that, 92 - 95% ChE reactivation can be achieved over a broad range of ChE inhibition (5 - 94 %) with paraoxon. The extent of enzyme inhibition using this electrochemical sensor correlates well with conventional enzyme activity measurements.

  8. Enzyme oscillation can enhance the thermodynamic efficiency of cellular metabolism: consequence of anti-phase coupling between reaction flux and affinity

    NASA Astrophysics Data System (ADS)

    Himeoka, Yusuke; Kaneko, Kunihiko

    2016-04-01

    Cells generally convert nutrient resources to products via energy transduction. Accordingly, the thermodynamic efficiency of this conversion process is one of the most essential characteristics of living organisms. However, although these processes occur under conditions of dynamic metabolism, most studies of cellular thermodynamic efficiency have been restricted to examining steady states; thus, the relevance of dynamics to this efficiency has not yet been elucidated. Here, we develop a simple model of metabolic reactions with anabolism–catabolism coupling catalyzed by enzymes. Through application of external oscillation in the enzyme abundances, the thermodynamic efficiency of metabolism was found to be improved. This result is in strong contrast with that observed in the oscillatory input, in which the efficiency always decreased with oscillation. This improvement was effectively achieved by separating the anabolic and catabolic reactions, which tend to disequilibrate each other, and taking advantage of the temporal oscillations so that each of the antagonistic reactions could progress near equilibrium. In this case, anti-phase oscillation between the reaction flux and chemical affinity through oscillation of enzyme abundances is essential. This improvement was also confirmed in a model capable of generating autonomous oscillations in enzyme abundances. Finally, the possible relevance of the improvement in thermodynamic efficiency is discussed with respect to the potential for manipulation of metabolic oscillations in microorganisms.

  9. Enhanced enzymatic hydrolysis of mild alkali pre-treated rice straw at high-solid loadings using in-house cellulases in a bench scale system.

    PubMed

    Narra, Madhuri; Balasubramanian, Velmurugan; James, Jisha P

    2016-06-01

    In the present study, scale-up systems for cellulase production and enzymatic hydrolysis of pre-treated rice straw at high-solid loadings were designed, fabricated and tested in the laboratory. Cellulase production was carried out using tray fermentation at 45 °C by Aspergillus terreus in a temperature-controlled humidity chamber. Enzymatic hydrolysis studies were performed in a horizontal rotary drum reactor at 50 °C with 25 % (w/v) solid loading and 9 FPU g(-1) substrate enzyme load using in-house as well commercial cellulases. Highly concentrated fermentable sugars up to 20 % were obtained at 40 h with an increased saccharification efficiency of 76 % compared to laboratory findings (69.2 %). These findings demonstrate that we developed a simple and less energy intensive bench scale system for efficient high-solid saccharification. External supplementation of commercial β-glucosidase and hemicellulase ensured better hydrolysis and further increased the saccharification efficiency by 14.5 and 20 %, respectively. An attempt was also made to recover cellulolytic enzymes using ultrafiltration module and nearly 79-84 % of the cellulases and more than 90 % of the sugars were recovered from the saccharification mixture. PMID:26941245

  10. Enzymes toughen up for chemical processing

    SciTech Connect

    Hairston, D.

    1995-05-01

    While enzymes have been making tremendous inroads into detergent formulation and food processing, the penetration of these protein-based catalysts into other chemical-process manufacture and hazardous waste treatment--where they are slated to replace heavy metal catalysts and other processing aids--has been relatively slow. Recently, however, enhancements in the enzyme`s properties are opening the door wider for such broadened usage. Some of these non-traditional uses of enzymes are described.

  11. Microbial cell-free extracts as sources of enzyme activities to be used for enhancement flavor development of ewe milk cheese.

    PubMed

    Calasso, Maria; Mancini, Leonardo; Di Cagno, Raffaella; Cardinali, Gianluigi; Gobbetti, Marco

    2015-09-01

    Freeze-dried cell-free extracts (CFE) from Lactobacillus casei LC01, Weissella cibaria 1XF5, Hafnia alvei Moller ATCC 51815, and Debaryomyces hansenii LCF-558 were used as sources of enzyme activities for conditioning the ripening of ewe milk cheese. Compared with control cheese (CC), CFE did not affect the gross composition and the growth of the main microbial groups of the cheeses. As shown through urea-PAGE electrophoresis of the pH 4.6-soluble nitrogen fraction and the analysis of free AA, the secondary proteolysis of the cheeses with CFE added was markedly differed from that of the CC. Compared with CC, several enzyme activities were higher in the water-soluble extracts from cheeses made with CFE. In agreement, the levels of 49 volatile compounds significantly differentiated CC from the cheeses made with CFE. The level of some alcohols, ketones, sulfur compounds, and furans were the lowest in the CC, whereas most aldehydes were the highest. Each CFE seemed to affect a specific class of chemical compounds (e.g., the CFE from H. alvei ATCC 51815 mainly influenced the synthesis of sulfur compounds). Apart from the microbial source used, the cheeses with the addition of CFE showed higher score for acceptability than the control cheese. Cheese ripening was accelerated or conditioned using CFE as sources of tailored enzyme activities. PMID:26142846

  12. 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.

  13. Simultaneous saccharification and fermentation (SSF) of jackfruit seed powder (JFSP) to l-lactic acid and to polylactide polymer.

    PubMed

    Nair, Nimisha Rajendran; Nampoothiri, K Madhavan; Banarjee, Rintu; Reddy, Gopal

    2016-08-01

    A newly isolated amylolytic lactic acid bacterium, Streptococcus equinus, was used for the production of l-lactic acid from jackfruit seed powder (JFSP) by simultaneous saccharification and fermentation (SSF). After optimization of shake flask fermentation by a response surface box-behnken design, the maximum lactate titer was 109g/L from 200g/L jackfruit seed powder. Amberlite IRA67, a weak base resin, was used to recover pure lactic acid from fermented broth and subsequently used for the synthesis of polylactic acid by direct condensation polymerization method with a yield of 62%. PMID:27005791

  14. Nutrient and sensory qualities of kunun zaki from different saccharification agents.

    PubMed

    Terna, Gaffa; Jideani, I A; Nkama, I

    2002-03-01

    Kunun zaki--a cereal-based non-alcoholic, non-carbonated beverage--was studied. The ratio of blends of major ingredients, nutrient, amino acid content and sensory qualities of kunun zaki generated with different saccharifying agents were investigated. The main ingredients of the formulations were malted rice, sweet potato, soybeans and Cadaba farinosa (Dangarafa or Legel in Hausa), each used separately with sorghum to produce a kunun zaki type. The weight ratios of the major ingredients were 8:91 for malted rice-sorghum, 7:92 for sweet potato-sorghum, 9:90 for soybean-sorghum and 4:95 for Cadaba farinosa-sorghum blends with ginger contributing 1% in each case as a spice. The nutrient composition of kunun zaki samples from different saccharifying agents ranged from 87 to 91% for moisture, 3.19 to 7.86% for crude protein, 0.37 to 0.75% for fat, 0.93 to 1.20% for ash and 2.69 to 5.84% for carbohydrate. Glutamic acid (4.49-11.66 g) was the most abundant amino acid in the samples while cysteine was the least abundant (0.34-1.45 g) in all the samples. The lowest concentration of all the essential amino acids except for tryptophan occurred when malted rice was used (0.44-1.40 g). Among the essential amino acids, cysteine, valine, isoleucine and methionine occurred in extremely low quantities compared with FAO/WHO reference protein values. The dual role (saccharification and enrichment) of soybean in kunun zaki processing is a desirable attribute and offers an advantage over the other agents. The different saccharifying agents had no significant effect (P > 0.05) on colour and flavour of kunun zaki but did influence sweetness, mouthfeel and overall acceptability. The beverage made with malted rice was most liked overall. PMID:11939105

  15. Overexpression of GA20-OXIDASE1 impacts plant height, biomass allocation and saccharification efficiency in maize.

    PubMed

    Voorend, Wannes; Nelissen, Hilde; Vanholme, Ruben; De Vliegher, Alex; Van Breusegem, Frank; Boerjan, Wout; Roldán-Ruiz, Isabel; Muylle, Hilde; Inzé, Dirk

    2016-03-01

    Increased biomass yield and quality are of great importance for the improvement of feedstock for the biorefinery. For the production of bioethanol, both stem biomass yield and the conversion efficiency of the polysaccharides in the cell wall to fermentable sugars are of relevance. Increasing the endogenous levels of gibberellic acid (GA) by ectopic expression of GA20-OXIDASE1 (GA20-OX1), the rate-limiting step in GA biosynthesis, is known to affect cell division and cell expansion, resulting in larger plants and organs in several plant species. In this study, we examined biomass yield and quality traits of maize plants overexpressing GA20-OX1 (GA20-OX1). GA20-OX1 plants accumulated more vegetative biomass than control plants in greenhouse experiments, but not consistently over two years of field trials. The stems of these plants were longer but also more slender. Investigation of GA20-OX1 biomass quality using biochemical analyses showed the presence of more cellulose, lignin and cell wall residue. Cell wall analysis as well as expression analysis of lignin biosynthetic genes in developing stems revealed that cellulose and lignin were deposited earlier in development. Pretreatment of GA20-OX1 biomass with NaOH resulted in a higher saccharification efficiency per unit of dry weight, in agreement with the higher cellulose content. On the other hand, the cellulose-to-glucose conversion was slower upon HCl or hot-water pretreatment, presumably due to the higher lignin content. This study showed that biomass yield and quality traits can be interconnected, which is important for the development of future breeding strategies to improve lignocellulosic feedstock for bioethanol production. PMID:26903034

  16. Enhancement of Cellulose Degradation by Cattle Saliva

    PubMed Central

    Seki, Yasutaka; Kikuchi, Yukiko; Kimura, Yoshihiro; Yoshimoto, Ryo; Takahashi, Masatoshi; Aburai, Kenichi; Kanai, Yoshihiro; Ruike, Tatsushi; Iwabata, Kazuki; Sugawara, Fumio; Sakai, Hideki; Abe, Masahiko; Sakaguchi, Kengo

    2015-01-01

    Saccharification of cellulose is a promising technique for producing alternative source of energy. However, the efficiency of conversion of cellulose into soluble sugar using any currently available methodology is too low for industrial application. Many additives, such as surfactants, have been shown to enhance the efficiency of cellulose-to-sugar conversion. In this study, we have examined first whether cattle saliva, as an additive, would enhance the cellulase-catalyzed hydrolysis of cellulose, and subsequently elucidated the mechanism by which cattle saliva enhanced this conversion. Although cattle saliva, by itself, did not degrade cellulose, it enhanced the cellulase-catalyzed degradation of cellulose. Thus, the amount of reducing sugar produced increased approximately 2.9-fold by the addition of cattle saliva. We also found that non-enzymatic proteins, which were present in cattle saliva, were responsible for causing the enhancement effect. Third, the mechanism of cattle saliva mediated enhancement of cellulase activity was probably similar to that of the canonical surfactants. Cattle saliva is available in large amounts easily and cheaply, and it can be used without further purification. Thus, cattle saliva could be a promising additive for efficient saccharification of cellulose on an industrial scale. PMID:26402242

  17. Enhancing the secretion efficiency and thermostability of a Bacillus deramificans pullulanase mutant (D437H/D503Y) by N-terminal domain truncation.

    PubMed

    Duan, Xuguo; Wu, Jing

    2015-03-01

    Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072-4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8- and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 × 10(8) and 449.3 × 10(8) U · mol(-1), respectively, which are 0.94- and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7- and 1.3-fold that of the DM enzyme. Kinetic studies showed that the Km values of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and the Kcat/Km values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced d-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes. PMID:25556190

  18. Enhancing the Secretion Efficiency and Thermostability of a Bacillus deramificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

    PubMed Central

    Duan, Xuguo

    2015-01-01

    Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072–4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8- and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 × 108 and 449.3 × 108 U · mol−1, respectively, which are 0.94- and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7- and 1.3-fold that of the DM enzyme. Kinetic studies showed that the Km values of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and the Kcat/Km values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced d-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes. PMID:25556190

  19. Mutual enhancement of the current density and the coulombic efficiency for a bioanode by entrapping bi-enzymes with Os-complex modified electrodeposition paints.

    PubMed

    Shao, Minling; Zafar, Muhammad Nadeem; Sygmund, Christoph; Guschin, Dmitrii A; Ludwig, Roland; Peterbauer, Clemens K; Schuhmann, Wolfgang; Gorton, Lo

    2013-02-15

    A bioanode with high current density and coulombic efficiency was developed by co-immobilization of pyranose dehydrogenase from Agaricus meleagris (AmPDH) with the dehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophiles (recDHCtCDH) expressed recombinantly in Escherichia coli. The two enzymes were entrapped in Os-complex modified electrodeposition polymers (Os-EDPs) with specifically adapted redox potential by means of chemical co-deposition. AmPDH oxidizes glucose at both the C2 and C3 positions whereas recDHCtCDH oxidizes glucose only at the C1 position. Electrochemical measurements reveal that maximally 6 electrons can be harvested from one glucose molecule at the two-enzyme anode via a cascade reaction, as AmPDH oxidizes the products formed from of the recDHCtCDH catalyzed substrate oxidation and vice versa. Furthermore, a significant increase in current density can be obtained by combining AmPDH and recDHCtCDH in a single modified electrode. We propose the use of this bioanode in biofuel cells with increased current density and coulombic efficiency. PMID:22959203

  20. Recyclable enzyme mimic of cubic Fe3O4 nanoparticles loaded on graphene oxide-dispersed carbon nanotubes with enhanced peroxidase-like catalysis and electrocatalysis

    SciTech Connect

    Wang, Hua; Li, Shuai; Si, Yanmei; Sun, Zhongzhao; Li, Shuying; Lin, Yuehe

    2014-01-01

    Fe3O4 nanoparticles as nanocatalysts may present peroxidase-like catalysis activities and high electrocatalysis if loaded on conductive carbon nanotube (CNT) supports; however, their catalysis performances in an aqueous system might still be challenged by the poor aqueous dispersion of hydrophobic carbon supports and/or low stability of loaded iron catalysts. In this work, amphiphilic graphene oxide nanosheets were employed as “surfactant” to disperse CNTs to create stable graphene oxide-dispersed CNT (GCNT) supports in water for covalently loading cubic Fe3O4 nanoparticles with improved distribution and binding efficiency. Compared with original Fe3O4 nanos and CNT-loaded Fe3O4 nanocomplex, the prepared GCNT–Fe3O4 nanocomposite could achieve higher aqueous stability and, especially, much stronger peroxidase-like catalysis and electrocatalysis to H2O2, presumably resulting from the synergetic effects of two conductive carbon supports and cubic Fe3O4 nanocatalysts effectively loaded. Colorimetric and direct electrochemical detections of H2O2 and glucose using the GCNT–Fe3O4 nanocomposite were conducted with high detection sensitivities, demonstrating the feasibility of practical sensing applications. Such a magnetically recyclable “enzyme mimic” may circumvent some disadvantages of natural protein enzymes and common inorganic catalysts, featuring the multi-functions of high peroxidase-like catalysis, strong electrocatalysis, magnetic separation/recyclability, environmental stability, and direct H2O2 electrochemistry.

  1. A Graphene-enhanced imaging of microRNA with enzyme-free signal amplification of catalyzed hairpin assembly in living cells.

    PubMed

    Liu, Haiyun; Tian, Tian; Ji, Dandan; Ren, Na; Ge, Shenguang; Yan, Mei; Yu, Jinghua

    2016-11-15

    In situ imaging of miRNA in living cells could help us to monitor the miRNA expression in real time and obtain accurate information for studying miRNA related bioprocesses and disease. Given the low-level expression of miRNA, amplification strategies for intracellular miRNA are imperative. Here, we propose an amplification strategy with a non-destructive enzyme-free manner in living cells using catalyzed hairpin assembly (CHA) based on graphene oxide (GO) for cellular miRNA imaging. The enzyme-free CHA exhibits stringent recognition and excellent signal amplification of miRNA in the living cells. GO is a good candidate as a fluorescence quencher and cellular carrier. Taking the advantages of the CHA and GO, we can monitor the miRNA at low level in living cells with a simple, sensitive and real-time manner. Finally, imaging of miRNAs in the different expression cells is realized. The novel method could supply an effective tool to visualize intracellular low-level miRNAs and help us to further understand the role of miRNAs in cellular processes. PMID:27315515

  2. Computational enzyme design

    NASA Astrophysics Data System (ADS)

    Bolon, Daniel N.

    2002-08-01

    The long-term objective of computational enzyme design is the ability to generate efficient protein catalysts for any chemical reaction. This thesis develops and experimentally validates a general computational approach for the design of enzymes with novel function. In order to include catalytic mechanism in protein design, a high-energy state (HES) rotamer (side chain representation) was constructed. In this rotamer, substrate atoms are in a HES. In addition, at least one amino acid side chain is positioned to interact favorably with substrate atoms in their HES and facilitate the reaction. Including an amino acid side chain in the HES rotamer automatically positions substrate relative to a protein scaffold and allows protein design algorithms to search for sequences capable of interacting favorably with the substrate. Because chemical similarity exists between the transition state and the high-energy state, optimizing the protein sequence to interact favorably with the HES rotamer should lead to transition state stabilization. In addition, the HES rotamer model focuses the subsequent computational active site design on a relevant phase space where an amino acid is capable of interacting in a catalytically active geometry with substrate. Using a HES rotamer model of the histidine mediated nucleophilic hydrolysis of p-nitrophenyl acetate, the catalytically inert 108 residue E. coli thioredoxin as a scaffold, and the ORBIT protein design software to compute sequences, an active site scan identified two promising active site designs. Experimentally, both candidate ?protozymes? demonstrated catalytic activity significantly above background. In addition, the rate enhancement of one of these ?protozymes? was the same order of magnitude as the first catalytic antibodies. Because polar groups are frequently buried at enzyme-substrate interfaces, improved modeling of buried polar interactions may benefit enzyme design. By studying native protein structures, rules have been

  3. Comparative evaluation of chemical and enzymatic saccharification of mixotrophically grown de-oiled microalgal biomass for reducing sugar production.

    PubMed

    Pancha, Imran; Chokshi, Kaumeel; Maurya, Rahulkumar; Bhattacharya, Sourish; Bachani, Pooja; Mishra, Sandhya

    2016-03-01

    For the commercialization of microalgal based biofuels, utilization of de-oiled carbohydrate rich biomass is important. In the present study, chemo-enzymatic hydrolysis of mixotrophically grown Scenedesmus sp. CCNM 1077 de-oiled biomass is evaluated. Among the chemical hydrolysis, use of 0.5M HCl for 45 min at 121°C resulted in highest saccharification yield of 37.87% w/w of de-oiled biomass. However, enzymatic hydrolysis using Viscozyme L at loading rate of 20 FBGU/g of de-oiled biomass, pH 5.5 and temperature 45°C for 72 h resulted in saccharification yield of 43.44% w/w of de-oiled biomass. Further, 78% ethanol production efficiency was achieved with enzymatically hydrolyzed de-oiled biomass using yeast Saccharomyces cerevisiae ATCC 6793. These findings of the present study show application of mixotrophically grown de-oiled biomass of Scenedesmus sp. CCNM 1077 as promising feedstock for bioethanol production. PMID:26771924

  4. Ethanol production of semi-simultaneous saccharification and fermentation from mixture of cotton gin waste and recycled paper sludge

    PubMed Central

    Agblevor, Foster A.

    2010-01-01

    Ethanol production from the steam-exploded mixture of 75% cotton gin waste and 25% recycled paper sludge in various conditions was investigated by semi-simultaneous saccharification and fermentation (SSSF) consisting of a pre-hydrolysis and a simultaneous saccharification and fermentation (SSF). Four cases were studied: 24-h pre-hydrolysis + 48-h SSF (SSSF 24), 12-h pre-hydrolysis + 60-h SSF (SSSF 12), 72-h SSF, and 48-h hydrolysis + 24-h fermentation (SHF). The ethanol concentration, yield, and productivity of SSSF 24 were higher than those of the other operations. A model of SSF was used to simulate the data for four components in SSF. The analysis of the reaction rates of cellobiose, glucose, cell, and ethanol using the model and the parameters from the experiments showed that there was a transition point of the rate-controlling step at which the cell growth control in the initial 2 h was changed to the cellobiose reaction control in later period during ethanol production of SSF from the mixture. PMID:20559849

  5. Improved enzymatic saccharification of steam exploded cotton stalk using alkaline extraction and fermentation of cellulosic sugars into ethanol.

    PubMed

    Keshav, Praveen K; Naseeruddin, Shaik; Rao, L Venkateswar

    2016-08-01

    Cotton stalk, a widely available and cheap agricultural residue lacking economic alternatives, was subjected to steam explosion in the range 170-200°C for 5min. Steam explosion at 200°C and 5min led to significant hemicellulose solubilization (71.90±0.10%). Alkaline extraction of steam exploded cotton stalk (SECOH) using 3% NaOH at room temperature for 6h led to 85.07±1.43% lignin removal with complete hemicellulose solubilization. Besides, this combined pretreatment allowed a high recovery of the cellulosic fraction from the biomass. Enzymatic saccharification was studied between steam exploded cotton stalk (SECS) and SECOH using different cellulase loadings. SECOH gave a maximum of 785.30±8.28mg/g reducing sugars with saccharification efficiency of 82.13±0.72%. Subsequently, fermentation of SECOH hydrolysate containing sugars (68.20±1.16g/L) with Saccharomyces cerevisiae produced 23.17±0.84g/L ethanol with 0.44g/g yield. PMID:27155264

  6. Disruption of thyroid hormone homeostasis in Ugt1a-deficient Gunn rats by microsomal enzyme inducers is not due to enhanced thyroxine glucuronidation

    SciTech Connect

    Richardson, Terrilyn A.; Klaassen, Curtis D.

    2010-10-01

    Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) are thought to increase glucuronidation of thyroxine (T{sub 4}), thus reducing serum T{sub 4}, and subsequently increasing thyroid stimulating hormone (TSH). Ugt1a1 and Ugt1a6 mediate T{sub 4} glucuronidation. Therefore, this experiment determined the involvement of Ugt1a enzymes in increased T{sub 4} glucuronidation, decreased serum T{sub 4}, and increased TSH after MEI treatment. Male Wistar and Ugt1a-deficient Wistar (Gunn) rats were fed a control diet or diet containing pregnenolone-16{alpha}-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum T{sub 4}, triiodothyronine (T{sub 3}), and TSH concentrations, hepatic T{sub 4}/T{sub 3} glucuronidation, and thyroid histology and follicular cell proliferation were investigated. PCN, 3-MC, and PCB treatments decreased serum T{sub 4}, whereas serum T{sub 3} was maintained in both Gunn and Wistar rats (except for PCB treatment). TSH was increased in Wistar and Gunn rats after PCN (130 and 277%) or PCB treatment (72 and 60%). T{sub 4} glucuronidation in Wistar rats was increased after PCN (298%), 3-MC (85%), and PCB (450%), but was extremely low in Gunn rats, and unchanged after MEI. T{sub 3} glucuronidation was increased after PCN (121%) or PCB (58%) in Wistar rats, but only PCN increased T{sub 3} glucuronidation in Gunn rats (43%). PCN treatment induced thyroid morphological changes and increased follicular cell proliferation in both strains. These data demonstrate that T{sub 4} glucuronidation cannot be increased in Ugt1a-deficient Gunn rats. Thus, the decrease in serum T{sub 4}, increase in TSH, and increase in thyroid cell proliferation after MEI are not dependent on increased T{sub 4} glucuronidation, and cannot be attributed to Ugt1a enzymes.

  7. Effects of selenium dietary enhancement on hatchery-reared coho salmon, Oncorhynchus kisutch (Walbaum), when compared with wild coho: hepatic enzymes and seawater adaptation evaluated.

    USGS Publications Warehouse

    Felton, S.P.; Landolt, M.L.; Grace, R.; Palmisano, A.N.

    1996-01-01

    Hatchery-reared coho salmon, Oncorhynchus kisutch (Walbaum), were fed elevated levels of selenium (as Na2SeO3) to raise eviscerated body burdens to the level measured in wild counterparts. The goal was to find a dietary concentration that would achieve the desired effect without causing damage to growth and normal development. To measure some indices of health, the detoxifying enzymes chosen were hepatic glutathione peroxidase (GSH-Px) and hepatic superoxide dismutase (SOD). Eviscerated body selenium (Se) concentration, GSH-Px and SOD levels were measured during and at the end of the 9 month freshwater feeding trial. Selenium retention and enzyme activity were also measured during 6 months’residence in sea water (SW). Selenium supplements were added to a commercial ration to give final concentrations of 1.1, 8.6, 11.1, 13.6 μg g-1 Se in the four respective diets. The results indicated that a dietary concentration of 8.6 μg g-1selenium was capable of inducing eviscerated body burdens similar to those found in wild fish. The elevated selenium levels persisted throughout the freshwater (FW) rearing phase, but declined when the fish were fed an unsupplemented ration upon SW entry. Superoxide dismutase levels did not increase above control levels. Glutathione peroxidase levels increased in fish fed the supplemented diets. GSH-Px activity declined in the higher supplemented dietary groups when all groups were reduced to the control group level of 1.1 μg g-1. Cumulative mortality in SW was 20% in fish fed either the 1.1 or the 8.6 μg g-1 Se diets. The 8.6 μg g-1 Se supplemented diets did produce healthy coho, comparable to their wild counterparts.

  8. [Effect of N-terminal truncation of Bacillus acidopullulyticus pullulanase on enzyme properties and functions].

    PubMed

    Chen, A'na; Liu, Xiuxia; Dai, Xiaofeng; Zhan, Jinling; Peng, Feng; Li, Lu; Wang, Fen; Li, Song; Yang, Yankun; Bai, Zhonghu

    2016-03-01

    We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 degrees C) with the wild-type pullulanase (WT). The K(m) of M1 and M5 were 1.42 mg/mL and 1.85 mg/mL, respectively, 2.4- and 3.1-fold higher than that of the WT. k(cat)/K(m) value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT. PMID:27349118

  9. Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant Escherichia coli strain FBR5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The WS used in this study contained 32.0±0% cellulose, 32.1±1.3% hemicellulo...

  10. Double clicking for site-specific coupling of multiple enzymes.

    PubMed

    Lim, Sung In; Cho, Jinhwan; Kwon, Inchan

    2015-09-14

    A method to site-specifically couple multiple enzymes is reported. The approach is based on the site-specific incorporation of a clickable non-natural amino acid into enzymes and two compatible click reactions. The multi-enzyme reaction system exhibited enhanced catalytic efficiency over the respective free enzymes. PMID:26191550

  11. Application of thermophilic enzymes and water jet system to cassava pulp.

    PubMed

    Chaikaew, Siriporn; Maeno, Yuka; Visessanguan, Wonnop; Ogura, Kota; Sugino, Gaku; Lee, Seung-Hwan; Ishikawa, Kazuhiko

    2012-12-01

    Co-production of fermentable sugars and nanofibrillated cellulose from cassava pulp was achieved by the combination of thermophilic enzymes (endoglucanase, β-glucosidase, and α-amylase) and a new atomization system (Star Burst System; SBS), which employs opposing water jets. The SBS represents a key technology for providing cellulose nanofibers and improving the enzymatic saccharification of cassava pulp. Depending on the enzymes used, the production of glucose from cassava pulp treated with the SBS was 1.2- to 2.5-fold higher than that from pulp not treated with the SBS. Nanofibrillated cellulose with the gel-like property in suspension was produced (yield was over 90%) by α-amylase treatment, which completely released trapped starch granules from the fibrous cell wall structure of cassava pulp pretreated with the SBS. The SBS provides an environmentally low-impact pretreatment system for processing biomass material into value-added products. PMID:23073093

  12. Improved Sugar Production by Optimizing Planetary Mill Pretreatment and Enzyme Hydrolysis Process

    PubMed Central

    Kwon, Jeong Heo; Lee, Siseon; Lee, Jae-Won; Hong, Youn-Woo; Chang, Jeong Ho; Sung, Daekyung; Kim, Sung Hyun; Sang, Byoung-In; Mitchell, Robert J.; Lee, Jin Hyung

    2015-01-01

    This paper describes an optimization of planetary mill pretreatment and saccharification processes for improving biosugar production. Pitch pine (Pinus rigida) wood sawdust waste was used as biomass feedstock and the process parameters optimized in this study were the buffering media, the milling time, the enzyme quantity, and the incubation time. Glucose yields were improved when acetate buffer was used rather than citrate buffer. Initially, with each process variable tests, the optimal values were 100 minutes of milling, an enzyme concentration of 16 FPU/g-biomass, and a 12-hour enzymatic hydrolysis. Typically, interactions between these experimental conditions and their effects on glucose production were next investigated using RSM. Glucose yields from the Pinus rigida waste exceeded 80% with several of the conditions tested, demonstrating that milling can be used to obtain high levels of glucose bioconversion from woody biomass for biorefinery purposes. PMID:26539475

  13. Designing Improved Enzymes of industrial application from marine microorganisms using Protein Engineering

    NASA Astrophysics Data System (ADS)

    Prajapati, A. S.; Panchal, K.; Subramanian, R. B.; Patel, D. H.; Sudhir, A. P.; Dave, B. R.

    2015-12-01

    Global demand for energy has grown with the development of new industries, requiring constant improvement and search for new sources of energy. One of the challenges today is releasing the energy of glucose that nature has cleverly locked into lignocellulosic biomass. Potent and efficient enzyme preparations need to be developed for the enzymatic saccharification process to be more economical. Approaches like enzyme engineering, reconstitution of enzyme mixtures and bioprospecting for superior enzymes are gaining importance. The ocean is considered to be a great reservoir of biodiversity. Because enzymes have unequalled advantages, many industries are keenly interested in adapting enzymatic methods for their processes. Microbial communities in marine environments are ecologically relevant as intermediaries of energy and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. The exploitation of marine bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. Several industrial enzymes are derived from terrestrial sources, whereas, marine environment which encompasses about 71 percent of the earth's surface and a vast resources for useful enzymes, remain unexplored. Marine microorganisms take active part in the mineralization of complex organic matter through degradative pathways of their metabolism. Bacteria from marine environments secrete different enzymes based on their habitat and their ecological functions. Therefore marine microbial enzymes have become the focal point of interest. Even though many of these enzymes are being isolated, the efficiency of hydrolysis is very poor. This could be overcome by altering the substrate specificity of lignocellulases. Protein engineering could prove to be useful to improve the catalytic function these enzymes.

  14. Cadmium telluride quantum dots (CdTe-QDs) and enhanced ultraviolet-B (UV-B) radiation trigger antioxidant enzyme metabolism and programmed cell death in wheat seedlings.

    PubMed

    Chen, Huize; Gong, Yan; Han, Rong

    2014-01-01

    Nanoparticles (NPs) are becoming increasingly widespread in the environment. Free cadmium ions released from commonly used NPs under ultraviolet-B (UV-B) radiation are potentially toxic to living organisms. With increasing levels of UV-B radiation at the Earth's surface due to the depletion of the ozone layer, the potential additive effect of NPs and UV-B radiation on plants is of concern. In this study, we investigated the synergistic effect of CdTe quantum dots (CdTe-QDs), a common form of NP, and UV-B radiation on wheat seedlings. Graded doses of CdTe-QDs and UV-B radiation were tested, either alone or in combination, based on physical characteristics of 5-day-old seedlings. Treatments of wheat seedlings with either CdTe-QDs (200 mg/L) or UV-B radiation (10 KJ/m(2)/d) induced the activation of wheat antioxidant enzymes. CdTe-QDs accumulation in plant root cells resulted in programmed cell death as detected by DNA laddering. CdTe-QDs and UV-B radiation inhibited root and shoot growth, respectively. Additive inhibitory effects were observed in the combined treatment group. This research described the effects of UV-B and CdTe-QDs on plant growth. Furthermore, the finding that CdTe-QDs accumulate during the life cycle of plants highlights the need for sustained assessments of these interactions. PMID:25329900

  15. SIRT5 Deficiency Enhances Susceptibility to Kainate-Induced Seizures and Exacerbates Hippocampal Neurodegeneration not through Mitochondrial Antioxidant Enzyme SOD2

    PubMed Central

    Li, Fengling; Liu, Lei

    2016-01-01

    Epilepsy is a common and serious neurological disorder characterized by occurrence of recurrent spontaneous seizures, and emerging evidences support the association of mitochondrial dysfunction with epilepsy. Sirtuin 5 (SIRT5), localized in mitochondrial matrix, has been considered as an important functional modulator of mitochondria that contributes to ageing and neurological diseases. Our data shows that SIRT5 deficiency strikingly increased mortality rate and severity of response to epileptic seizures, dramatically exacerbated hippocampal neuronal loss and degeneration in mice exposed to Kainate (KA), and triggered more severe reactive astrogliosis. We found that the expression of mitochondrial SIRT5 of injured hippocampus was relatively up-regulated, indicating its potential contribution to the comparably increased survival of these cells and its possible neuroprotective role. Unexpectedly, SIRT5 seems not to apparently alter the decline of antioxidant enzymes superoxide dismutase 2 (SOD2) and glutathione peroxidase (GPx) in hippocampus caused by KA exposure in our paradigm, which indicates the protective role of SIRT5 on seizures and cellular degeneration might through different regulatory mechanism that would be explored in the future. In the present study, we provided strong evidences for the first time to demonstrate the association between SIRT5 and epilepsy, which offers a new understanding of the roles of SIRT5 in mitochondrial functional regulation. The neuroprotection of SIRT5 in KA-induced epileptic seizure and neurodegeneration will improve our current knowledge of the nature of SIRT5 in central nervous system (CNS) and neurological diseases. PMID:27445698

  16. The Arachidonate 15-Lipoxygenase Enzyme Product 15-HETE Is Present in Heart Tissue from Patients with Ischemic Heart Disease and Enhances Clot Formation

    PubMed Central

    Lundqvist, Annika; Sandstedt, Mikael; Sandstedt, Joakim; Wickelgren, Ruth; Hansson, Göran I.; Jeppsson, Anders; Hultén, Lillemor Mattsson

    2016-01-01

    Ischemic heart disease is a major cause of death and morbidity and the search for novel therapeutic targets is still required. We have previously shown that the enzyme arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), is highly expressed in ischemic heart tissue, but its role in the pathogenesis of ischemic heart disease is unclear. Here we showed that expression of ALOX15, but not ALOX12 or ALOX15B, was increased in ischemic versus non-ischemic human heart biopsy samples. A similar ALOX expression pattern was found in hypoxic human cardiomyocytes and cardiac endothelial cells. We also showed that levels of 15-HETE were significantly higher in ischemic versus non-ischemic human heart biopsy samples and showed a tendency to increase in serum from the patients with ischemic heart disease. Moreover, hypoxia increased the production of 15-HETE levels from human cardiomyocytes and cardiac endothelial cells. The hypoxia-induced increase in 15-HETE levels from human cardiomyocytes was inhibited by the ALOX15 inhibitor baicalein. Finally, by using intrinsic rotational thromboelastometry, we showed that human whole blood clotted faster in the presence of 15-HETE. In summary, we propose that increased ALOX15 expression in heart tissue under ischemic conditions may lead to increased production of 15-HETE, potentially contributing to thrombosis. PMID:27552229

  17. Enhancing the Bioconversion of Winery and Olive Mill Waste Mixtures into Lignocellulolytic Enzymes and Animal Feed by Aspergillus uvarum Using a Packed-Bed Bioreactor.

    PubMed

    Salgado, José Manuel; Abrunhosa, Luís; Venâncio, Armando; Domínguez, José Manuel; Belo, Isabel

    2015-10-28

    Wineries and olive oil industries are dominant agro-industrial activities in southern European regions. Olive pomace, exhausted grape marc, and vine shoot trimmings are lignocellulosic residues generated by these industries, which could be valued biotechnologically. In the present work these residues were used as substrate to produce cellulases and xylanases through solid-state fermentation using Aspergillus uvarum MUM 08.01. For that, two factorial designs (3(2)) were first planned to optimize substrate composition, temperature, and initial moisture level. Subsequently, the kinectics of cellulolytic enzyme production, fungal growth, and fermented solid were characterized. Finally, the process was performed in a packed-bed bioreactor. The results showed that cellulase activity improved with the optimization processes, reaching 33.56 U/g, and with the packed-bed bioreactor aeration of 0.2 L/min, reaching 38.51 U/g. The composition of fermented solids indicated their potential use for animal feed because cellulose, hemicellulose, lignin, and phenolic compounds were partially degraded 28.08, 10.78, 13.3, and 28.32%, respectively, crude protein was increased from 8.47 to 17.08%, and the mineral contents meet the requirements of main livestock. PMID:26165254

  18. A multi-walled carbon nanotubes-poly(L-lysine) modified enantioselective immunosensor for ofloxacin by using multi-enzyme-labeled gold nanoflower as signal enhancer.

    PubMed

    He, Zuyu; Zang, Shuai; Liu, Yingju; He, Yuan; Lei, Hongtao

    2015-11-15

    The enantioselective detection of trace amounts of ofloxacin is very important in many fields. In this work, an enantioselective and sensitive electrochemical immunosensor was constructed for the detection of chiral antibiotic ofloxacin based on a dual amplification strategy using multiwall carbon nanotubes-poly(L-lysine) as a matrix to immobilize the antigen and multi-enzyme-antibody functionalized gold nanoflowers as an electrochemical detection label. The fabrication process of the dual-amplified immunosensor was characterized by scanning electron microscopy, cyclic voltammogram and electrochemical impedance spectroscopy, respectively. After the optimization of the experimental conditions, a competitive immunoassay, i.e., the association ability with the corresponding antibody between the captured antigen and free S-OFL or R-OFL in the solution, showed that the immunosensor exhibited a sensitive response to S-OFL in the range from 0.26 to 25.6 ng/mL with a detection limit of 0.15 ng/mL as well as a sensitive response to R-OFL in the range from 0.37 to 12.8 ng/mL with a detection limit of 0.30 ng/mL. Along with the acceptable sensitivity and stability, the S-OFL or R-OFL immunosensor showed selective ability to its corresponding enantiomer, suggesting this amplification strategy may hold a potential application in the detection of OFL in food or environment. PMID:26047998

  19. ORGANOPHOSPHATE DEGRADING ENZYMES - PHASE I

    EPA Science Inventory

    Agave BioSystems in collaboration with Carl A. Batt proposes to develop decon-nanoparticles, which will leverage ongoing opportunities in enzyme engineering and the fabrication of functionalized magnetic nanoparticles. Enhanced performance will be engineered into the system t...

  20. Enhancing the thermal robustness of an enzyme by directed evolution: least favorable starting points and inferior mutants can map superior evolutionary pathways.

    PubMed

    Gumulya, Yosephine; Reetz, Manfred T

    2011-11-01

    In a previous directed evolution study, the B-FIT approach to increasing the thermal robustness of proteins was introduced and applied to the lipase from Bacillus subtilis. It is based on the general concept of iterative saturation mutagenesis (ISM), according to which sites in an enzyme are subjected to saturation mutagenesis, the best hit of a given library is then used as a template for randomization at other sites, and the process is continued until the desired catalyst improvement has been achieved. The appropriate choice of the ISM sites is crucial; in the B-FIT method the criterion is residues characterized by highest B factors available from X-ray crystallography data. In the present study, B-FIT was employed in order to increase the thermal robustness of the epoxide hydrolase from Aspergillus niger. Several rounds of ISM resulted in the best variant showing a 21 °C increase in the T(60)(50) value, an 80-fold improvement in half-life at 60 °C, and a 44 kcal mol(-1) improvement in inactivation energy. Seven other variants were also evolved with moderate yet significant improvements; these were ch