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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. 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.

  3. 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...

  4. Cooperation of Aspergillus nidulans enzymes increases plant polysaccharide saccharification.

    PubMed

    Tramontina, Robson; Robl, Diogo; Maitan-Alfenas, Gabriela Piccolo; de Vries, Ronald P

    2016-07-01

    Efficient polysaccharide degradation depends on interaction between enzymes acting on the main chain and the side chains. Previous studies demonstrated cooperation between several enzymes, but not all enzyme combinations have been explored. A better understanding of enzyme cooperation would enable the design of better enzyme mixtures, optimally profiting from synergistic effects. In this study, we analyzed the cooperation of several enzymes involved in the degradation of xylan, glucan, xyloglucan and crude plant biomass from Aspergillus nidulans by single and combined incubations with their polymeric substrate. Positive effects were observed between most enzymes, although not always to the same extent. Moreover, the tailor made cocktails formulated in this study resulted in efficient release of glucose from plant biomass. This study also serves as an example for the complex cooperation that occurs between enzymes in plant biomass saccharification and how expression in easily-accessible hosts, such as Pichia pastoris, can help in revealing these effects. PMID:26848939

  5. Enzymic saccharification of pretreated wheat straw. [Trichoderma reesei

    SciTech Connect

    Vallander, L.; Eriksson, K.E.

    1985-01-01

    Studies of pretreatment of wheat and its subsequent saccharification by Trichoderma reesei cellulases are reported. Steam explosion was found to be the most effective of the pretreatment methods tested. Data are presented describing the effect of enzyme and substrate concentration on the rate and degree of hydrolysis. Significant inhibition of the cellulases was observed when sugar concentrations were 6% or higher. This inhibition increased when glucose and ethanol were present simultaneously. Adsorption of enzymes to the substrate was followed during a 24-h hydrolysis period. An initial rapid and extensive adsorption occurred, followed by a short desorption period that was followed in turn by a further increased adsorption peaking after 3 h. Intermediate removal of hydrolysate, particularly in combination with a second addition of enzyme, clearly improved the yield of saccharification compared to an uninterrupted hydrolysis over a 24-h period. Thus, a 74% yield of reducing sugars was obtained. Furthermore, an increase in the amount of recoverable enzymes was observed under these conditions. Evidence is presented that suggests that a countercurrent technique, whereby free enzymes in recovered hydrolysate are adsorbed onto new substrate, may provide a means of recirculating dissolved enzymes.

  6. 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

  7. 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.

  8. 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...

  9. 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

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

    PubMed

    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

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

    PubMed

    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.

  12. 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

  13. 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

  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. Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures.

    PubMed

    Harrison, Mark D; Zhang, Zhanying; Shand, Kylie; O'Hara, Ian M; Doherty, William O S; Dale, James L

    2013-11-01

    Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5L (15-25FPU/g glucan) was in the following order: glycerol/HCl>HCl>H2SO4>NaOH, with the glycerol system achieving ≈ 100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6-6%) obtained with mixtures of endo-1,4-β-glucanase (EG) and β-glucosidase (βG) for the NaOH substrate were 2-6 times that of acid substrates. However, glucan conversions (15-60%) obtained with mixtures of cellobiohydrolase (CBH I) and βG on acidified glycerol substrate were 10-30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics. PMID:24045198

  16. Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures.

    PubMed

    Harrison, Mark D; Zhang, Zhanying; Shand, Kylie; O'Hara, Ian M; Doherty, William O S; Dale, James L

    2013-11-01

    Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5L (15-25FPU/g glucan) was in the following order: glycerol/HCl>HCl>H2SO4>NaOH, with the glycerol system achieving ≈ 100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6-6%) obtained with mixtures of endo-1,4-β-glucanase (EG) and β-glucosidase (βG) for the NaOH substrate were 2-6 times that of acid substrates. However, glucan conversions (15-60%) obtained with mixtures of cellobiohydrolase (CBH I) and βG on acidified glycerol substrate were 10-30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics.

  17. Mixtures of thermostable enzymes show high performance in biomass saccharification.

    PubMed

    Kallioinen, Anne; Puranen, Terhi; Siika-aho, Matti

    2014-07-01

    Optimal enzyme mixtures of six Trichoderma reesei enzymes and five thermostable enzyme components were developed for the hydrolysis of hydrothermally pretreated wheat straw, alkaline oxidised sugar cane bagasse and steam-exploded bagasse by statistically designed experiments. Preliminary studies to narrow down the optimization parameters showed that a cellobiohydrolase/endoglucanase (CBH/EG) ratio of 4:1 or higher of thermostable enzymes gave the maximal CBH-EG synergy in the hydrolysis of hydrothermally pretreated wheat straw. The composition of optimal enzyme mixtures depended clearly on the substrate and on the enzyme system studied. The optimal enzyme mixture of thermostable enzymes was dominated by Cel7A and required a relatively high amount of xylanase, whereas with T. reesei enzymes, the high proportion of Cel7B appeared to provide the required xylanase activity. The main effect of the pretreatment method was that the required proportion of xylanase was higher and the proportion of Cel7A lower in the optimized mixture for hydrolysis of alkaline oxidised bagasse than steam-exploded bagasse. In prolonged hydrolyses, less Cel7A was generally required in the optimal mixture. Five-component mixtures of thermostable enzymes showed comparable hydrolysis yields to those of commercial enzyme mixtures.

  18. 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).

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. Enhanced enzymatic hydrolysis of waste paper for ethanol production using separate saccharification and fermentation.

    PubMed

    Guerfali, Mohamed; Saidi, Adel; Gargouri, Ali; Belghith, Hafedh

    2015-01-01

    Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum-based liquid fuels. In this study, the feasibility of ethanol production from waste paper using the separate hydrolysis and fermentation (SHF) was investigated. Two types of waste paper materials, newspaper and office paper, were evaluated for their potential to be used as a renewable feedstock for the production of fermentable sugars via enzymatic hydrolysis of their cellulose fractions. Hydrolysis step was conducted with a mixture of cellulolytic enzymes produced locally by Trichoderma reesei Rut-C30 (cellulase-overproducing mutant) and Aspergillus niger F38 cultures. Surfactant pretreatment effect on waste paper enzymatic digestibility was studied and Triton X-100 at 0.5 % (w w(-1)) has improved the digestibility of newspaper about 45 %. The effects of three factors (dry matter quantity, phosphoric acid pretreatment and hydrolysis time) on the extent of saccharification were also assessed and quantified by using a methodical approach based on response surface methodology. Under optimal hydrolysis conditions, maximum degrees of saccharification of newspaper and office paper were 67 and 92 %, respectively. Sugars released from waste paper were subsequently converted into ethanol (0.38 g ethanol g(-1) sugar) with Saccharomyces cerevisiae CTM-30101.

  15. Bioconversion of hemicellulose: aspects of hemicellulose production by Trichod reesei qm 9414 and enzymic saccharification of hemicellulose

    SciTech Connect

    Dekker, R.F.H.

    1983-04-01

    The growth of Trichoderma reesei QM9414 in shake flasks at 28 degrees C on hemicellulose substrates and bagasse resulted in rather low yields of hemicellulolytic enzymes (1.0-1.5 units/mL xylanase and 0.05-0.08 units/mL beta-xylosidase). The influence of pH on the synthesis of beta-xylosidase was greater than on the synthesis of xylanase. Both xylanase and beta-xylosidase showed optimal activity at pH 4-5 and 55-60 degrees C. Xylanase was stable at pH 2-10 but was heat labile and totally inactivated after one hour at 65 degrees C. Enzyme stability towards heat could be increased in the presence of bovine serum albumin. The beta-xylosidase was more tolerant to heat, but stable over a pH range 2.5-6.0. The D-xylose inhibited both enzymes in a competitive manner. Hemicellulose (heteroxylan) was degraded to the extent of 30-40% within 24 hours. The degree of hydrolysis decreased as the substrate concentration increased and increased with increased amounts of enzyme. Multiple enzyme doses resulted in increased saccharification in reduced times. The degree of hydrolysis was influenced by the amount of beta-xylosidase present in the hemicellulolytic enzyme preparation. The beta-xylosidase was demonstrated to play an important role in the overall conversion of heteroxylan into xylose that is analogous to the role of beta- glucosidase in the saccharification of cellulose by cellulases. (Refs. 26).

  16. 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

  17. 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.

  18. 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.

  19. Corn fiber, cobs and stover: enzyme-aided saccharification and co-fermentation after dilute acid pretreatment.

    PubMed

    Van Eylen, David; van Dongen, Femke; Kabel, Mirjam; de Bont, Jan

    2011-05-01

    Three corn feedstocks (fibers, cobs and stover) available for sustainable second generation bioethanol production were subjected to pretreatments with the aim of preventing formation of yeast-inhibiting sugar-degradation products. After pretreatment, monosaccharides, soluble oligosaccharides and residual sugars were quantified. The size of the soluble xylans was estimated by size exclusion chromatography. The pretreatments resulted in relatively low monosaccharide release, but conditions were reached to obtain most of the xylan-structures in the soluble part. A state of the art commercial enzyme preparation, Cellic CTec2, was tested in hydrolyzing these dilute acid-pretreated feedstocks. The xylose and glucose liberated were fermented by a recombinant Saccharomyces cerevisiae strain. In the simultaneous enzymatic saccharification and fermentation system employed, a concentration of more than 5% (v/v) (0.2g per g of dry matter) of ethanol was reached. PMID:21392979

  20. 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

  1. Lignin extraction distinctively enhances biomass enzymatic saccharification in hemicelluloses-rich Miscanthus species under various alkali and acid pretreatments.

    PubMed

    Si, Shengli; Chen, Yan; Fan, Chunfen; Hu, Huizhen; Li, Ying; Huang, Jiangfeng; Liao, Haofeng; Hao, Bo; Li, Qing; Peng, Liangcai; Tu, Yuanyuan

    2015-05-01

    In this study, one- and two-step pretreatments with alkali and acid were performed in the three Miscanthus species that exhibit distinct hemicelluloses levels. As a result, one-step with 4% NaOH or two-step with 2% NaOH and 1% H2SO4 was examined to be optimal for high biomass saccharification, indicating that alkali was the main effecter of pretreatments. Notably, both one- and two-step pretreatments largely enhanced biomass digestibility distinctive in hemicelluloses-rich samples by effectively co-extracting hemicelluloses and lignin. However, correlation analysis further indicated that the effective lignin extraction, other than the hemicelluloses removals, predominately determined biomass saccharification under various alkali and acid pretreatments, leading to a significant alteration of cellulose crystallinity. Hence, this study has suggested the potential approaches in bioenergy crop breeding and biomass process technology.

  2. Evaluation of enzyme cost for simultaneous saccharification and fermentation of cellulose to ethanol

    SciTech Connect

    Becker, D.K.; Emert, G.H.

    1983-01-01

    The enzymes involved in the conversion of cellulose to glucose are US -1,4-endoglucanase, US -1,4-exo-cellobiohydrolase, and cellobiase. These enzymes can be obtained from a number of microbes including strains of Trichoderma reesei. Due to product inhibition of enzyme activities, it is advantageous to utilize them in a manner which maintains concentrations of low hydrolysis products but yields high fermentation products. A method of accomplishing this is saccharifying the cellulose to glucose and simultaneously allowing a second microbe to ferment the glucose to ethanol (SSF). Development of the SSF technology, from bench level to pilot-plant operation and commercial-plant design, has allowed identification of important process, engineering, and economic factors. The key step in the utilization of these feedstocks is the conversion of the cellulose to glucose. This paper will define the specific capital and operating cost inputs of enzyme production and utilization to the projected economics of the SSF process. 10 references, 1 figure, 2 tables.

  3. Saccharification of Kans grass using enzyme mixture from Trichoderma reesei for bioethanol production.

    PubMed

    Kataria, Rashmi; Ghosh, Sanjoy

    2011-11-01

    Bioethanol is one of the alternatives of the conventional fossil fuel. In present study, effect of different carbon sources on the production of cellulolytic enzyme (CMCase) from Trichoderma reesei at different temperatures, duration and pH were investigated and conditions were optimized. Acid treated Kans grass (Saccharum sponteneum) was subjected to enzymatic hydrolysis to produce fermentable sugars which was then fermented to bioethanol using Saccharomyces cerevisiae. The maximum CMCase production was found to be 1.46 U mL(-1) at optimum condition (28°C, pH 5 and cellulose as carbon source). The cellulases and xylanase activity were found to be 1.12 FPU g(-1) and 6.63 U mL(-1), respectively. Maximum total sugar was found to be 69.08 mg/g dry biomass with 20 FPU g(-1) dry biomass of enzyme dosage under optimum condition. Similar results were obtained when it was treated with pure enzyme. Upon fermentation of enzymatic hydrolysate, the yield of ethanol was calculated to be 0.46 g g(-1).

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. Simultaneous saccharification and fermentation of Kanlow switchgrass by thermotolerant Kluyveromyces marxianus IMB3: the effect of enzyme loading, temperature and higher solid loadings.

    PubMed

    Pessani, Naveen K; Atiyeh, Hasan K; Wilkins, Mark R; Bellmer, Danielle D; Banat, Ibrahim M

    2011-11-01

    Switchgrass (Panicum virgatum) was subjected to hydrothermolysis pretreatment and then used to study the effect of enzyme loading and temperature in a simultaneous saccharification and fermentation (SSF) with the thermotolerant yeast strain Kluyveromyces marxianus IMB3 at 8% solid loading. Various loadings of Accellerase 1500 between 0.1 and 1.1 mL g(-1) glucan were tested in SSF at 45 °C (activity of enzyme was 82.2 FPU mL(-1)). The optimum enzyme loading was 0.7 mL g(-1) glucan based on the six different enzyme loadings tested. SSFs were performed at 37, 41 and 45 °C with an enzyme loading of 0.7 mL g(-1) glucan. The highest ethanol concentration of 22.5 g L(-1) was obtained after 168 h with SSF at 45 °C, which was equivalent to 86% yield. Four different batch and fed-batch strategies were evaluated using a total solid loading of 12% (dry basis). About 32 g L(-1) ethanol was produced with the four strategies, which was equivalent to 82% yield.

  9. 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%.

  10. 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.

  11. Production of a lignocellulolytic enzyme system for simultaneous bio-delignification and saccharification of corn stover employing co-culture of fungi.

    PubMed

    Ma, Kedong; Ruan, Zhiyong

    2015-01-01

    Aiming at improving the efficiency of transferring corn stover into sugars, an efficient lignocellulolytic enzyme system was developed and investigated by co-cultivation of the Coprinus comatus with Trichoderma reesei in a single bioreactor. The results showed that the lignocellulolytic enzyme activities of the co-culture exceeded that of the monoculture, suggesting synergistic interaction between two fungi. The highest laccase activity from the co-culture was 2.6-fold increase over that of the C. comatus monoculture and reached a peak 3days earlier. The maximum delignification obtained was 66.5% and about 82% of the original polysaccharides were converted into fermentable sugars by simultaneous bio-delignification and saccharification process. Correlation analysis showed that sugar yields were directly proportional to the lignin degradation. Our results suggested that co-fungi cultivation was a valuable technique for corn stover bioconversion, which could produce high efficiency of lignocellulolytic enzyme system as a cheaper alternative to commercial enzymes for industrial utilization. PMID:25459871

  12. 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

  13. 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

  14. Steam explosion distinctively enhances biomass enzymatic saccharification of cotton stalks by largely reducing cellulose polymerization degree in G. barbadense and G. hirsutum.

    PubMed

    Huang, Yu; Wei, Xiaoyang; Zhou, Shiguang; Liu, Mingyong; Tu, Yuanyuan; Li, Ao; Chen, Peng; Wang, Yanting; Zhang, Xuewen; Tai, Hongzhong; Peng, Liangcai; Xia, Tao

    2015-04-01

    In this study, steam explosion pretreatment was performed in cotton stalks, leading to 5-6 folds enhancements on biomass enzymatic saccharification distinctive in Gossypium barbadense and Gossypium hirsutum species. Sequential 1% H2SO4 pretreatment could further increase biomass digestibility of the steam-exploded stalks, and also cause the highest sugar-ethanol conversion rates probably by releasing less inhibitor to yeast fermentation. By comparison, extremely high concentration alkali (16% NaOH) pretreatment with raw stalks resulted in the highest hexoses yields, but it had the lowest sugar-ethanol conversion rates. Characterization of wall polymer features indicated that biomass saccharification was enhanced with steam explosion by largely reducing cellulose DP and extracting hemicelluloses. It also showed that cellulose crystallinity and arabinose substitution degree of xylans were the major factors on biomass digestibility in cotton stalks. Hence, this study has provided the insights into cell wall modification and biomass process technology in cotton stalks and beyond.

  15. Cost-effective production of cellulose hydrolysing enzymes from Trichoderma sp. RCK65 under SSF and its evaluation in saccharification of cellulosic substrates.

    PubMed

    Chakraborty, Subhojit; Gupta, Rishi; Jain, Kavish Kumar; Kuhad, Ramesh Chander

    2016-11-01

    Trichoderma sp. is a potential cellulase producing mesophilic fungi which grow under mild acidic condition. In this study, growth and nutritional conditions were manipulated for the maximum and cost-effective production of cellulase using lab strain Trichoderma sp. RCK65 and checked for its efficiency in hydrolysis of Prosopis juliflora (a woody substrate). Preliminary studies suggested that when 48 h old secondary fungal culture (20 % v/w) was inoculated in wheat bran moistened with mineral salt solution (pH 4.5 and 1:3 solid to moisture ratio), incubated at 30 °C and after 72 h, it produced maximum cellulase (CMCase 145 U/gds, FPase 38 U/gds and β-glucosidase 105 U/gds). However, using statistical approach a S:L ratio (1:1) was surprisingly found to be optimum that improved cellulase that is CMCase activity by 6.21 %, FPase activity by 23.68 % and β-glucosidase activity by 37.28 %. The estimated cost of crude enzyme (Rs. 5.311/1000 FPase units) seems to be economically feasible which may be due to high enzyme titre, less cultivation time and low media cost. Moreover, when the crude enzyme was used to saccharify pretreated Prosopis juliflora (a woody substrate), it resulted up to 83 % (w/w) saccharification. PMID:27344316

  16. 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...

  17. 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.

  18. 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

  19. Modification of plant cell wall structure accompanied by enhancement of saccharification efficiency using a chemical, lasalocid sodium

    PubMed Central

    Okubo-Kurihara, Emiko; Ohtani, Misato; Kurihara, Yukio; Kakegawa, Koichi; Kobayashi, Megumi; Nagata, Noriko; Komatsu, Takanori; Kikuchi, Jun; Cutler, Sean; Demura, Taku; Matsui, Minami

    2016-01-01

    The cell wall is one major determinant of plant cell morphology, and is an attractive bioresource. Here, we report a novel strategy to modify plant cell wall property by small molecules. Lasalocid sodium (LS) was isolated by chemical screening to identify molecules that affect the cell morphology of tobacco BY-2 cells. LS treatment led to an increase in cell wall thickness, whilst the quantity and sugar composition of the cell wall remained unchanged in BY-2 cells. The chemical also disordered the cellular arrangement of hypocotyls of Arabidopsis plants, resulting in a decrease in hypocotyl length. LS treatment enhanced enzymatic saccharification efficiency in both BY-2 cells and Arabidopsis plants. Microarray analysis on Arabidopsis showed that exposure to LS upregulated type III peroxidase genes, of which some are involved in lignin biogenesis, and jasmonic acid response genes, and phloroglucinol staining supported the activation of lignification by the LS treatment. As jasmonic acid-mediated lignification is a typical reaction to cell wall damage, it is possible that LS induces cell wall loosening, which can trigger cell wall damage response. Thus, LS is a unique chemical for modification of cell wall and morphology through changes in cell wall architecture. PMID:27694977

  20. 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.

  1. Effect of reduction in yeast and enzyme concentrations in a simultaneous- saccharification-and-fermentation-based bioethanol process: technical and economic evaluation.

    PubMed

    Wingren, Anders; Galbe, Mats; Roslander, Christian; Rudolf, Andreas; Zacchi, Guido

    2005-01-01

    The ethanol production cost in a simultaneous saccharification and fermentation-based bioethanol process is influenced by the requirements for yeast production and for enzymes. The main objective of this study was to evaluate--technically and economically--the influence of these two factors on the production cost. A base case with 5 g/L of baker's yeast and an initial concentration of water-insoluble solids of 5% resulted in an experimental yield of 85%. When these data were implemented in Aspen Plus, yeast was assumed to be produced from sugars in the hydrolysate, reducing the overall ethanol yield to 69%. The ethanol production cost was 4.80 SEK/L (2.34 US$/gal). When adapted yeast was used at 2 g/L, an experimental yield of 74% was achieved and the estimated ethanol production cost was the same as in the base case. A 50% reduction in enzyme addition resulted in an increased production cost, to 5.06 SEK/L (2.47 US$/gal) owing to reduced ethanol yield.

  2. 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).

  3. 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

  4. Enhanced saccharification of sugarcane bagasse using soluble cellulase supplemented with immobilized β-glucosidase.

    PubMed

    Borges, Diogo Gontijo; Baraldo, Anderson; Farinas, Cristiane Sanchez; Giordano, Raquel de Lima Camargo; Tardioli, Paulo Waldir

    2014-09-01

    The β-glucosidase (BG) enzyme plays a vital role in the hydrolysis of lignocellulosic biomass. Supplementation of the hydrolysis reaction medium with BG can reduce inhibitory effects, leading to greater conversion. In addition, the inclusion of immobilized BG can be a useful way of increasing enzyme stability and recyclability. BG was adsorbed on polyacrylic resin activated by carboxyl groups (BG-PC) and covalently attached to glyoxyl-agarose (BG-GA). BG-PC exhibited similar behavior to soluble BG in the hydrolysis of cellobiose, while BG-GA hydrolyzed the same substrate at a lower rate. However, the thermal stability of BG-GA was higher than that of free BG. Hydrolysis of pretreated sugarcane bagasse catalyzed by soluble cellulase supplemented with immobilized BG improved the conversion by up to 40% after 96 h of reaction. Both derivatives remained stable up to the third cycle and losses of activity were less than 50% after five cycles.

  5. Enhancement of enzymatic saccharification of sugarcane bagasse by liquid hot water pretreatment.

    PubMed

    Hongdan, Zhang; Shaohua, Xu; Shubin, Wu

    2013-09-01

    Lignocellulosic biomass can be utilized to produce promising biofuels. In this study, liquid hot water pretreatments were performed to break the intricate structure of sugarcane bagasse, which resists the enzyme accessibility to cellulose. The effects of temperatures and times on the hemicellulose degradation (including the yields of pentoses and hexoses, the proportion of monomers and oligosaccharides, as well as limited inhibitors) and cellulose enzymatic digestibility were evaluated. The results indicated that the maximum xylose yields (combined 3.85 g xylose and 13.21 g xylo-oligosaccharides per 100 g raw material) in prehydrolyzate liquid were obtained at 180 °C and 30 min. Due to the effective removal of hemicellulose, the maximum glucose yield in enzyme hydrolyzate reached 37.27 g per 100 g raw material, representing 90.13% of glucose in the sugarcane bagasse. The maximal total sugars yield (combined prehydrolyzate and enzymatic hydrolyzate) were 53.65 g based on 100 g raw material.

  6. Comparison of organosolv and hydrotropic pretreatments of eucalyptus for enhancing enzymatic saccharification.

    PubMed

    Mou, Hongyan; Wu, Shubin

    2016-11-01

    The objective of this study was to investigate the effects of organosolv and hydrotropic pretreatments on improving enzymatic hydrolysis of eucalyptus. The chemical composition of the fiber surface was analyzed using X-ray photoelectron spectroscopy (XPS) to determine the surface characteristics of pretreated eucalyptus. Other than the significant decrease of surface coverage by lignin, hydrotropic pretreatment was more effective in removing the lignin and xylose from fiber cell walls than organosolv pretreatment. The restriction of acetyl and phenolic groups in pretreated substrates was typically eliminated by hydrotropic pretreatments. Moreover, fiber structure and morphology after pretreatments were more suitable for enzymatic hydrolysis. Cellulase adsorption capacity was notably improved by hydrotropic pretreatment, which indicating the better enzyme accessibility of cellulose in pretreated substrates. Eventually, higher glucose yield was obtained with hydrotropic pretreatment. In addition, the precipitated lignin as an important by-product of pretreatments was characterized by Fourier transforms infrared spectroscopy (FTIR) also. PMID:27590575

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. The effect of different ethoxylations for sorbitan monolaurate on enhancing simultaneous saccharification and fermentation (SSF) of wheat straw to ethanol.

    PubMed

    Badawi, A M; Fahmy, A A; Mohamed, Karima A; Noor El-Din, M R; Riad, M G

    2012-01-01

    In this paper, four nonionic surfactants with different hydrophilic-lipophilic balance (HLB) based on sorbitan monolaurate were synthesized by introducing ethylene oxide gas (n = 20, 40, 60, and 80 ethylene oxide units). The chemical structure of the prepared ethoxylated surfactants was confirmed using Fourier transform-infrared and (1)H NMR spectroscopes. The surface tension and thermodynamic properties of the prepared surfactants have been studied. The simultaneous saccharification and fermentation (SSF) process for ethanol production from microwave/alkali pretreated wheat straw has been assayed using nonionic surfactants have different ethylene oxide units. Ethanol yield was 82% and 61% for Kluyveromyces marxianus and Saccharomyces cerevisiae, respectively, with the addition of 2.5 g/l of the prepared nonionic surfactant (HLB = 18.2). Results show that the production of ethanol from microwave/alkali pretreated wheat straw increased with increasing the (HLB) value of the nonionic surfactant. PMID:21984384

  12. Alkaline peroxide pretreatment of rapeseed straw for enhancing bioethanol production by Same Vessel Saccharification and Co-Fermentation.

    PubMed

    Karagöz, Pinar; Rocha, Indre V; Özkan, Melek; Angelidaki, Irini

    2012-01-01

    Alkaline peroxide pretreatment of rapeseed straw was evaluated for conversion of cellulose and hemicellulose to fermentable sugars. After pretreatment, a liquid phase called pretreatment liquid and a solid phase were separated by filtration. The neutralized pretreatment liquids were used in a co-fermentation process, with Saccharomyces cerevisiae and Pichia stipitis. The solid fraction was used for simultaneous saccharification and co-fermentation process in the same vessel. The effects of various operating variables were investigated. Pretreatment with 5% (v/v) H(2)O(2) at 50 °C for 1h was found to be the optimal pretreatment combination with respect to overall ethanol production. At this condition, 5.73 g ethanol was obtained from pretreatment liquid and 14.07 g ethanol was produced by co-fermentation of solid fraction with P. stipitis. Optimum delignification was observed when 0.5 M MgSO(4) was included in the pretreatment mixture, and it resulted in 0.92% increase in ethanol production efficiency. PMID:22104093

  13. Alkaline peroxide pretreatment of rapeseed straw for enhancing bioethanol production by Same Vessel Saccharification and Co-Fermentation.

    PubMed

    Karagöz, Pinar; Rocha, Indre V; Özkan, Melek; Angelidaki, Irini

    2012-01-01

    Alkaline peroxide pretreatment of rapeseed straw was evaluated for conversion of cellulose and hemicellulose to fermentable sugars. After pretreatment, a liquid phase called pretreatment liquid and a solid phase were separated by filtration. The neutralized pretreatment liquids were used in a co-fermentation process, with Saccharomyces cerevisiae and Pichia stipitis. The solid fraction was used for simultaneous saccharification and co-fermentation process in the same vessel. The effects of various operating variables were investigated. Pretreatment with 5% (v/v) H(2)O(2) at 50 °C for 1h was found to be the optimal pretreatment combination with respect to overall ethanol production. At this condition, 5.73 g ethanol was obtained from pretreatment liquid and 14.07 g ethanol was produced by co-fermentation of solid fraction with P. stipitis. Optimum delignification was observed when 0.5 M MgSO(4) was included in the pretreatment mixture, and it resulted in 0.92% increase in ethanol production efficiency.

  14. Enhancement of ethanol production by simultaneous saccharification and fermentation (SSF) of rice straw using ethoxylated span 20.

    PubMed

    Badawi, A M; Fahmy, A A; Mohamed, Karima A; Noor El-Din, M R; Riad, M G

    2012-01-01

    In this work, four nonionic surfactants based on sorbitan monolaurate (Span 20) were synthesized by introducing ethylene oxide gas (n = 20, 40, 60, 80 ethylene oxide units) into Span 20 to give four new surfactants with different hydrophilic-lipophilic balance (HLB), namely, E(20), E(40), E(60), and E(80). The structures of the prepared nonionic surfactants were elucidated using Fourier-transform infrared (FT-IR) and (1)H-nuclear magnetic resonance (NMR) spectroscopy. The surface-tension measurements were recorded. The effects of the prepared nonionic surfactants on the simultaneous saccharification and fermentation (SSF) of microwave/alkali-pretreated rice straw to produce ethanol were investigated. From the obtained data, it was found that the addition of the nonionic surfactants at 2.5 g/L had a positive effect on SSF. The maximum ethanol yield (76 and 55%) was obtained after 72 hr for rice straw using Kluyveromyces marxianus and Saccharomyces cerevisiae, respectively. Also, it was found that the ethanol yield increases with increasing HLB of the prepared nonionic surfactants by increasing ethylene oxide units. The adsorption of nonionic surfactants on lignocelluloses is proposed to be due to hydrophobic and hydrogen bonding interactions between nonionic surfactants and the lignin part in the lignocelulose. It can be concluded that additions of surface-active compounds, such as nonionic surfactants, increase enzymatic conversion of rice straw for bioethanol purposes. PMID:22239707

  15. Enhanced ethanol production from Kinnow mandarin (Citrus reticulata) waste via a statistically optimized simultaneous saccharification and fermentation process.

    PubMed

    Oberoi, Harinder Singh; Vadlani, Praveen V; Nanjundaswamy, Ananda; Bansal, Sunil; Singh, Sandeep; Kaur, Simranjeet; Babbar, Neha

    2011-01-01

    Dried, ground, and hydrothermally pretreated Kinnow mandarin (Citrus reticulata) waste was used to produce ethanol via simultaneous saccharification and fermentation (SSF). Central composite design was used to optimize cellulase and pectinase concentrations, temperature, and time for SSF. The D-limonene concentration determined with high-performance liquid chromatography (HPLC) for fresh, dried, and pretreated biomass was 0.76%, 0.32%, and 0.09% (v/w), respectively. Design Expert software suggested that the first-order effect of all four factors and the second-order effect of cellulase and pectinase concentrations were significant for ethanol production. The validation experiment using 6 FPU gds(-1) cellulase and 60 IU gds(-1) pectinase at 37 °C for 12 h in a laboratory batch fermenter resulted in ethanol concentration and productivity of 42 g L(-1) and 3.50 g L(-1) h(-1), respectively. Experiments using optimized parameters resulted in an ethanol concentration similar to that predicted by the model equation and also helped reduce fermentation time. PMID:20863699

  16. Simultaneous saccharification and co-fermentation (SSCF) of AFEX(TM) pretreated corn stover for ethanol production using commercial enzymes and Saccharomyces cerevisiae 424A(LNH-ST).

    PubMed

    Jin, Mingjie; Gunawan, Christa; Balan, Venkatesh; Lau, Ming W; Dale, Bruce E

    2012-04-01

    Xylose consumption by Saccharomyces cerevisiae 424A(LNH-ST) during simultaneous saccharification and co-fermentation (SSCF) of AFEX(TM) pretreated switchgrass was inhibited by unhydrolyzed solids. Such inhibitory effects were not found in unhydrolyzed solids from AFEX(TM) pretreated corn stover (AFEX(TM)-CS). However, the xylose consumption was still unsatisfactory during 6h pre-hydrolysis SSCF. By extending the pre-hydrolysis time to 24h or longer, the xylose consumption was improved significantly. In order to better understand the reasons for such improvement, the hydrolysate slurries after 6h pre-hydrolysis and 24h pre-hydrolysis were studied and compared. We found that the glucose concentration after pre-hydrolysis was the critical factor that determined cell viability and hence xylose consumption during SSCF. Low temperature (30°C) and ethanol inhibition were shown to be the factors limiting hydrolysis rate and hence productivity during SSCF. PMID:22361075

  17. 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

  18. Saccharification and ethanol fermentation of apple pomace

    SciTech Connect

    Miller, J.E.; Weathers, P.J.; McConville, F.X.; Goldberg, M.

    1982-01-01

    Apple pomace (the pulp residue from pressing apple juice) is an abundant waste product and presents an expensive disposal problem. A typical (50,000 gal. juice/day) apple juice company in central Massachusetts produces 100 tons of pomace per day. Some of it is used as pig feed, but it is poor quality feed because of its low protein content. Most of the pomace is hauled away (at a cost of $4/ton) and landfilled (at a cost of $10/ton). If 5% (w/w) conversion of pomace to ethanol could be achieved, the need for this company to purchase No. 6 fuel oil (1000 gal/day) for cooking during processing would be eliminated. Our approach was to saccharify the pomace enzymatically, and then to carry out a yeast fermentation on the hydrolysate. We chose to use enzymatic hydrolysis instead of dilute acid hydrolysis in order to minimize pH control problems both in the fermentation phase and in the residue. The only chemical studies have concerned small subfractions of apple material: for example, cell walls have been analyzed but they constitute only 1 to 2% of the fresh weight of the apple (about 15 to 30% of the pomace fraction). Therefore, our major problems were: (1) to optimize hydrolysis by enzyme mixtures, using weight loss and ultimate ethanol production as optimization criteria; (2) to optimize ethanol production from the hydrolysate by judicious choice of yeast strains and fermentation conditions; and (3) achieve these optimizations consistent with minimum processing cost and energy input. We have obtained up to 5.1% (w/w) of ethanol without saccharification. We show here that hydrolysis with high levels of enzyme can enhance ethanol yield by up to 27%, to a maximum level of 6% (w/w); however, enzyme treament may be cost-effective only a low levels, for improvement of residue compaction. 3 figures, 4 tables.

  19. 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.

  20. Enhanced enzyme stability through site-directed covalent immobilization.

    PubMed

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

    2015-01-10

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

  1. 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.

  2. 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...

  3. Celluclast and Cellic® CTec2: Saccharification/fermentation of wheat straw, solid-liquid partition and potential of enzyme recycling by alkaline washing.

    PubMed

    Rodrigues, Ana Cristina; Haven, Mai Østergaard; Lindedam, Jane; Felby, Claus; Gama, Miguel

    2015-11-01

    The hydrolysis/fermentation of wheat straw and the adsorption/desorption/deactivation of cellulases were studied using Cellic(®) CTec2 (Cellic) and Celluclast mixed with Novozyme 188. The distribution of enzymes - cellobiohydrolase I (Cel7A), endoglucanase I (Cel7B) and β-glucosidase - of the two formulations between the residual substrate and supernatant during the course of enzymatic hydrolysis and fermentation was investigated. The potential of recyclability using alkaline wash was also studied. The efficiency of hydrolysis with an enzyme load of 10 FPU/g cellulose reached >98% using Cellic(®) CTec2, while for Celluclast a conversion of 52% and 81%, was observed without and with β-glucosidase supplementation, respectively. The decrease of Cellic(®) CTec2 activity observed along the process was related to deactivation of Cel7A rather than of Cel7B and β-glucosidase. The adsorption/desorption profiles during hydrolysis/fermentation revealed that a large fraction of active enzymes remained adsorbed to the solid residue throughout the process. Surprisingly, this was the case of Cel7A and β-glucosidase from Cellic, which remained adsorbed to the solid fraction along the entire process. Alkaline washing was used to recover the enzymes from the solid residue. This method allowed efficient recovery of Celluclast enzymes; however, this may be achieved only when minor amounts of cellulose remain present. Regarding the Cellic formulation, neither the presence of cellulose nor lignin restricted an efficient desorption of the enzymes at alkaline pH. This work shows that the recycling strategy must be customized for each particular formulation, since the enzymes found e.g. in Cellic and Celluclast bear quite different behaviour regarding the solid-liquid distribution, stability and cellulose and lignin affinity. PMID:26320717

  4. 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.

  5. 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

  6. Increased saccharification yields from aspen biomass upon treatment with enzymatically generated peracetic acid.

    PubMed

    Duncan, Shona; Jing, Qing; Katona, Adrian; Kazlauskas, Romas J; Schilling, Jonathan; Tschirner, Ulrike; Aldajani, Waleed Wafa

    2010-03-01

    The recalcitrance of lignocellulosic biomass to enzymatic release of sugars (saccharification) currently limits its use as feedstock for biofuels. Enzymatic hydrolysis of untreated aspen wood releases only 21.8% of the available sugars due primarily to the lignin barrier. Nature uses oxidative enzymes to selectively degrade lignin in lignocellulosic biomass, but thus far, natural enzymes have been too slow for industrial use. In this study, oxidative pretreatment with commercial peracetic acid (470 mM) removed 40% of the lignin (from 19.9 to 12.0 wt.% lignin) from aspen and enhanced the sugar yields in subsequent enzymatic hydrolysis to about 90%. Increasing the amount of lignin removed correlated with increasing yields of sugar release. Unfortunately, peracetic acid is expensive, and concentrated forms can be hazardous. To reduce costs and hazards associated with using commercial peracetic acid, we used a hydrolase to catalyze the perhydrolysis of ethyl acetate generating 60-70 mM peracetic acid in situ as a pretreatment to remove lignin from aspen wood. A single pretreatment was insufficient, but multiple cycles (up to eight) removed up to 61.7% of the lignin enabling release of >90% of the sugars during saccharification. This value corresponds to a predicted 581 g of fermentable sugars from 1 kg of aspen wood. Improvements in the enzyme stability are needed before the enzymatically generated peracetic acid is a commercially viable alternative.

  7. Chestnut shell as unexploited source of fermentable sugars: effect of different pretreatment methods on enzymatic saccharification.

    PubMed

    Maurelli, Luisa; Ionata, Elena; La Cara, Francesco; Morana, Alessandra

    2013-07-01

    Chestnut shell (CS) is an agronomic residue mainly used for extraction of antioxidants or as adsorbent of metal ions. It also contains some polysaccharide that has not been considered as potential source of fermentable sugars for biofuel production until now. In this study, the effect of different pretreatment methods on CS was evaluated in order to obtain the greatest conversion of cellulose and xylan into fermentable sugars. Hot acid impregnation, steam explosion (acid-catalysed or not), and aqueous ammonia soaking (AAS) were selected as pretreatments. The pretreated biomass was subjected to saccharification with two enzyme cocktails prepared from commercial preparations, and evaluation of the best pretreatment and enzyme cocktail was based on the yield of fermentable sugars produced. As AAS provided the best result after preliminary experiments, enhancement of sugar production was attempted by changing the concentrations of ammonium hydroxide, enzymes, and CS. The optimal pretreatment condition was 10 % ammonium hydroxide, 70 °C, 22 h with CS at 5 % solid loading. After saccharification of the pretreated CS for 72 h at 50 °C and pH 5.0 with a cocktail containing cellulase (Accellerase 1500), beta-glucosidase (Accellerase BG), and xylanase (Accellerase XY), glucose and xylose yields were 67.8 and 92.7 %, respectively.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. Scaffoldless engineered enzyme assembly for enhanced methanol utilization

    DOE PAGESBeta

    Price, J. Vincent; Chen, Long; Whitaker, W. Brian; Papoutsakis, Eleftherios; Chen, Wilfred

    2016-10-24

    Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

  18. Simultaneous saccharification and fermentation of cellulose to ethanol

    SciTech Connect

    Shea, P.T.

    1981-01-01

    Simultaneous saccharification and fermentation (SSF) of cullulose (untreated BW-200 Solka Floc) to ethanol utilizing the cellulase enzyme complex of Trichoderma reesei Rut C-30 and the yeast Saccharomyces cerevisiae QM 8226, has resulted in increased rates and longer times of hydrolysis when compared to simple saccharifications. Additionally, two schemes for ethanol removal during hydrolysis, nitrogen sparging and vacuum operation, have also shown increased rates and longer times of saccharification of cellulose when compared to the simple SSF. Both early and delayed yeast additions, different lengths of SSF operations, and different sparging techniques were investigated. The beta-glucosidase fraction of the T. ressei Rut C-30 cellulase enzyme system was able to convert cellobiose to glucose in the presence of ethyl alcohol eliminating the strong inhibition of celloboise on cellulase while the yeast converted glucose to ethanol by glucolysis eliminating the inhibition of glucose on beta-glucosidase. The hydrolysis curves did not fit either simple or competitive product inhibition Michaelis-Menten type kinetic analysis. An enzyme deactivation-inhibition model seems necessary to fit the data. The yield parameter for ethanol/substrate (Yp/s) varied from .42g/g to .47g/g (theoretical .51g/g) with the majority of glucose being converted to ethanol in less than 15 hours.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. Controlled expression of pectic enzymes in Arabidopsis thaliana enhances biomass conversion without adverse effects on growth.

    PubMed

    Tomassetti, Susanna; Pontiggia, Daniela; Verrascina, Ilaria; Reca, Ida Barbara; Francocci, Fedra; Salvi, Gianni; Cervone, Felice; Ferrari, Simone

    2015-04-01

    Lignocellulosic biomass from agriculture wastes is a potential source of biofuel, but its use is currently limited by the recalcitrance of the plant cell wall to enzymatic digestion. Modification of the wall structural components can be a viable strategy to overcome this bottleneck. We have previously shown that the expression of a fungal polygalacturonase (pga2 from Aspergillus niger) in Arabidopsis and tobacco plants reduces the levels of de-esterified homogalacturonan in the cell wall and significantly increases saccharification efficiency. However, plants expressing pga2 show stunted growth and reduced biomass production, likely as a consequence of an extensive loss of pectin integrity during the whole plant life cycle. We report here that the expression in Arabidopsis of another pectic enzyme, the pectate lyase 1 (PL1) of Pectobacterium carotovorum, under the control of a chemically inducible promoter, results, after induction of the transgene, in a saccharification efficiency similar to that of plants expressing pga2. However, lines with high levels of transgene induction show reduced growth even in the absence of the inducer. To overcome the problem of plant fitness, we have generated Arabidopsis plants that express pga2 under the control of the promoter of SAG12, a gene expressed only during senescence. These plants expressed pga2 only at late stages of development, and their growth was comparable to that of WT plants. Notably, leaves and stems of transgenic plants were more easily digested by cellulase, compared to WT plants, only during senescence. Expression of cell wall-degrading enzymes at the end of the plant life cycle may be therefore a useful strategy to engineer crops unimpaired in biomass yield but improved for bioconversion.

  5. The effect of nonenzymatic protein on lignocellulose enzymatic hydrolysis and simultaneous saccharification and fermentation.

    PubMed

    Wang, Hui; Kobayashi, Shinichi; Hiraide, Hatsue; Cui, Zongjun; Mochidzuki, Kazuhiro

    2015-01-01

    Nonenzymatic protein was added to cellulase hydrolysis and simultaneous saccharification and fermentation (SSF) of different biomass materials. Adding bovine serum albumin (BSA) and corn steep before cellulase enhanced enzyme activity in solution and increased cellulose and xylose conversion rates. The cellulose conversion rate of filter paper hydrolysis was increased by 32.5 % with BSA treatment. When BSA was added before cellulase, the remaining activity in the solution was higher than that in a control without BSA pretreatment. During SSF with pretreated rice straw as the substrate, adding 1.0 mg/mL BSA increased the ethanol yield by 13.6 % and final xylose yield by 42.6 %. The results indicated that lignin interaction is not the only mechanism responsible for the positive BSA effect. BSA had a stabilizing effect on cellulase and relieved cumulative sugar inhibition of enzymatic hydrolysis of biomass materials. Thus, nonenzymatic protein addition represents a promising strategy in the biorefining of lignocellulose materials.

  6. Pretreatment and saccharification of red macroalgae to produce fermentable sugars.

    PubMed

    Yun, Eun Ju; Kim, Hee Taek; Cho, Kyung Mun; Yu, Sora; Kim, Sooah; Choi, In-Geol; Kim, Kyoung Heon

    2016-01-01

    Red macroalgae are currently considered as renewable resources owing to their high carbohydrate and low lignin and hemicellulose contents. However, utilization of red macroalgae has been limited owing to the lack of established methods for pretreatment and an effective saccharification system. Furthermore, marine red macroalgae consist of the non-favorable mixed sugars for industrial microorganisms. In this review, we suggest strategies for converting red macroalgae to bio-based products, focusing on the pretreatment and saccharification of red macroalgae to produce fermentable sugars and the microbial fermentation of these sugars by industrial microorganisms. In particular, some recent breakthroughs for the efficient utilization of red macroalgae include the discovery of key enzymes for the complete monomerization of red macroalgal carbohydrate and the catabolic pathway of 3,6-anhydro-l-galactose, the most abundant sugar in red macroalgae. This review provides a comprehensive perspective for the efficient utilization of red macroalgae as sustainable resources to produce bio-based products.

  7. 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.

  8. 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.

  9. 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

  10. Effects of fungal pretreatment and steam explosion pretreatment on enzymatic saccharification of plant biomass.

    PubMed

    Sawada, T; Nakamura, Y; Kobayashi, F; Kuwahara, M; Watanabe, T

    1995-12-20

    The effects of consecutive treatments by a lignin-degrading fungus Phanerochaete chrysosporium and by steam explosion for the enzymatic saccharification of plant biomass were studied experimentally, and the optimal operational conditions for obtaining the maximum saccharification were evaluated. Beech wood-meal was treated by the fungus for 98 days and then by high steam temperatures of 170-230 degrees C with steaming times of 0-10 min. The treatment of the wood-meal by fungus prior to steam explosion enhanced the saccharification of wood-meal. The treated wood-meal was separated into holo-cellulose, water soluble material, methanol soluble lignin, and Klason lignin. The saccharification decreased linearly with the increase in the amount of Klason lignin. It was estimated by the equation for the saccharification of exploded wood-meal expressed as a function of steam temperature and steaming time that the maximum saccharification of wood-meal was obtained by consecutive treatments such as fungal treatment for 28 days and then steam explosion at a steam temperature of 215 degrees C and a steaming time of 6.5 min. (c) 1995 John Wiley & Sons, Inc.

  11. Cognitive enhancers (nootropics). Part 2: drugs interacting with enzymes.

    PubMed

    Froestl, Wolfgang; Muhs, Andreas; Pfeifer, Andrea

    2013-01-01

    Cognitive enhancers (nootropics) are drugs to treat cognition deficits in patients suffering from Alzheimer's disease, schizophrenia, stroke, attention deficit hyperactivity disorder, or aging. Cognition refers to a capacity for information processing, applying knowledge, and changing preferences. It involves memory, attention, executive functions, perception, language, and psychomotor functions. The term nootropics was coined in 1972 when memory enhancing properties of piracetam were observed in clinical trials. In the meantime, hundreds of drugs have been evaluated in clinical trials or in preclinical experiments. To classify the compounds, a concept is proposed assigning drugs to 19 categories according to their mechanism(s) of action, in particular drugs interacting with receptors, enzymes, ion channels, nerve growth factors, re-uptake transporters, antioxidants, metal chelators, and disease modifying drugs meaning small molecules, vaccines, and monoclonal antibodies interacting with amyloid-β and tau. For drugs whose mechanism of action is not known, they are either classified according to structure, e.g., peptides, or their origin, e.g., natural products. This review covers the evolution of research in this field over the last 25 years.

  12. 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...

  13. 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

  14. 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.

  15. 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.

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

    PubMed

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

    2016-02-10

    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.

  17. 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

  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. 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.

  20. A recombinant human enzyme for enhanced interstitial transport of therapeutics.

    PubMed

    Bookbinder, L H; Hofer, A; Haller, M F; Zepeda, M L; Keller, G-A; Lim, J E; Edgington, T S; Shepard, H M; Patton, J S; Frost, G I

    2006-08-28

    Subcutaneously injected therapeutics must pass through the interstitial matrix of the skin in order to reach their intended targets. This complex, three-dimensional structure limits the type and quantity of drugs that can be administered by local injection. Here we found that depolymerization of the viscoelastic component of the interstitial matrix in animal models with a highly purified recombinant human hyaluronidase enzyme (rHuPH20) increased the dispersion of locally injected drugs, across a broad range of molecular weights without tissue distortion. rHuPH20 increased infusion rates and the pattern and extent of appearance of locally injected drugs in systemic blood. In particular, rHuPH20 changed the pharmacokinetic profiles and significantly augmented the absolute bioavailability of locally injected large protein therapeutics. Importantly, within 24 h of injection, the interstitial viscoelastic barriers were restored without histologic alterations or signs of inflammation. rHuPH20 may function as an interstitial delivery enhancing agent capable of increasing the dispersion and bioavailability of coinjected drugs that may enable subcutaneous administration of therapeutics and replace intravenous delivery.

  1. 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.

  2. 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 ...

  3. Assessing Cellulase Performance on Pretreated Lignocellulosic Biomass Using Saccharification and Fermentation-Based Protocols

    NASA Astrophysics Data System (ADS)

    Dowe, Nancy

    Cellulase enzyme is a key cost component in the production of fuels and chemicals from lignocellulosic biomass. Cellulolytic ability of the enzyme preparation is often measured by activity assays using model substrates such as filter paper. Using lignocellulosic biomass as the substrate to assess enzyme performance has the potential of being more process relevant. We describe two procedures that use washed pretreated cellulosic material to measure the efficacy of cellulase enzymes. First, a saccharification assay that measures glucose yield as a function of the amount of cellulase used in the process. And second, the simultaneous saccharification and fermentation (SSF) assay measures cellulase performance by the amount of ethanol produced from enzymatic hydrolysis of the cellulosic material. You can use both assays to screen cellulases under a variety of substrate types, loadings, and process conditions.

  4. Lignin as a facilitator, not a barrier, during saccharification by brown rot fungi

    SciTech Connect

    Schilling, Jonathan S.; Tschirner, Ulrike; Blanchette, Robert A; Filley, Timothy

    2012-11-28

    This research focused on the biology of a group of wood-degrading fungi that cause brown rot in wood, with particular attention to the potential to mimic this biological approach ex situ for bioprocessing lignocellulosic biomass. Supported by the long-standing theory that these fungi use a two-step oxidative/enzymatic approach during brown rot, our team’s objectives were as follows: 1) to determine the discrete timing of lignin modifications, 2) to correlate these alterations with biocatalyst efficiency and ingress into plant cell walls, and 3) to reproduce modifications prior to saccharification for efficient bioprocessing. The core findings of our research were that 1) lignin modifications occur nearly coincident with enzyme secretion during brown rot and 2) there is no specificity to the benefit that a brown rot pretreatment has on the efficacy of cellulases – it is a general enhancement best predicted by chemical changes to lignin and side-chain hemicellulose sugars. In our work, this meant we could attain and predict broad improvements in saccharification using commercial cellulase cocktails, in some cases more than three-fold of that in untreated biomass. This project was completed with minimal variance from the original project management plan (PMP), resulting in fourteen presentations and posters, four peer-reviewed publications, and one additional publication now in review. The publications have been valuable to other scientists working toward similar goals and have been cited in thirteen peer-reviewed publications written by others since 2010. We are working with ADM to advance application options for industry, building on the lessons learned during this DOE award period.

  5. 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).

  6. Simultaneous saccharification and fermentation of cellulose: effect of ethanol on enzymatic saccharification of cellulose. [Trichoderma reesei

    SciTech Connect

    Ooshima, H.; Ishitani, Y.; Harano, Y.

    1985-01-01

    It was confirmed that simultaneous saccharification and fermentation are effective for accelerating enzymatic saccharification of cellulose. In this work, the effects of ethanol on the saccharification of tissue paper by Trichoderma cellulase (Meicelase CEPB) have been investigated. The following results were obtained. 1) Saccharification was inhibited by at least 0.2M ethanol. 2) Less than 4M ethanol did not affect the enzymatic activities of ..beta..-glucosidase and endoglucanase (C/sub x/) at all. The thermal stability of endoglucanase was not also varied by ethanol. 3) It is suggested that ethanol depresses the adsorption of exoglucanase on cellulose. 4) The rate expression of saccharification of cellulose in the presence of ethanol is proposed. 5) The inhibititory effect of ethanol was found to become more significant in the later stages of the reaction than just the initial stage.

  7. Engineered pentafunctional minicellulosome for simultaneous saccharification and ethanol fermentation in Saccharomyces cerevisiae.

    PubMed

    Liang, Youyun; Si, Tong; Ang, Ee Lui; Zhao, Huimin

    2014-11-01

    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.

  8. 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.

  9. 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.

  10. 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%.

  11. 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…

  12. Use of new membrane-reactor saccharification assay to evaluate the performance of cellulases under simulated SSF conditions

    SciTech Connect

    Baker, J.O.; Vinzant, T.B.; Ehrman, C.I.

    1997-12-31

    A new saccharification assay has been devised, in which a continuously buffer-swept membrane reactor is used to remove the solubilized saccharification products, thus allowing high extents of substrate conversion without significant inhibitory effects from the buildup of either cellobiose or glucose. This diafiltration saccharification assay (DSA) can, therefore, be used to obtain direct measurements of the performance of combinations of cellulose and substrate under simulated SSF conditions, without the saccharification results being complicated by factors that may influence the subsequent fermentation step. This assay has been used to compare the effectiveness of commercial and special in-house-produced Trichoderma reesei cellulose preparations in the saccharification of a standardized microcrystalline (Sigmacell) substrate and a dilute-acid pretreated lignocellulosic substrate. Initial results strongly suggest that enzyme preparations produced in the presence of the targeted lignocellulosic substrate will saccharify that substrate more effectively. These results call into question the widespread use of the {open_quotes}filter paper assay{close_quotes} as a reliable predictor of enzyme performance in the extensive hydrolysis of substrates that are quite different from filter paper in both physical properties and chemical composition. 14 refs., 6 figs.

  13. 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.

  14. Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes.

    PubMed

    Wu, Fei; Pelster, Lindsey N; Minteer, Shelley D

    2015-01-25

    Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

  15. 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.

  16. Enhanced diffusion, chemotaxis, and pumping by active enzymes: progress toward an organizing principle of molecular machines.

    PubMed

    Astumian, R Dean

    2014-12-23

    Active enzymes diffuse more rapidly than inactive enzymes. This phenomenon may be due to catalysis-driven conformational changes that result in "swimming" through the aqueous solution. Recent additional work has demonstrated that active enzymes can undergo chemotaxis toward regions of high substrate concentration, whereas inactive enzymes do not, and, further, that active enzymes immobilized at surfaces can directionally pump liquids. In this Perspective, I will discuss these phenomena in light of Purcell's work on directed motion at low Reynold's number and in the context of microscopic reversibility. The conclusions suggest that a deep understanding of catalytically driven enhanced diffusion of enzymes and related phenomena can lead toward a general organizing principle for the design, characterization, and operation of molecular machines.

  17. Protoplast fusion enhances lignocellulolytic enzyme activities in Trichoderma reesei.

    PubMed

    Cui, Yu-xiao; Liu, Jia-jing; Liu, Yan; Cheng, Qi-yue; Yu, Qun; Chen, Xin; Ren, Xiao-dong

    2014-12-01

    Protoplast fusion was used to obtain a higher production of lignocellulolytic enzymes with protoplast fusion in Trichoderma reesei. The fusant strain T. reesei JL6 was obtained from protoplast fusion from T. reesei strains QM9414, MCG77, and Rut C-30. Filter paper activity of T. reesei JL6 increased by 18% compared with that of Rut C-30. β-Glucosidase, hemicellulase and pectinase activities of T. reesei JL6 were also higher. The former activity was 0.39 Uml(-1), while those of QM9414, MCG77, and Rut C-30 were 0.13, 0.11, and 0.16 Uml(-1), respectively. Pectinase and hemicellulase activities of JL6 were 5.4 and 15.6 Uml(-1), respectively, which were slightly higher than those of the parents. The effects of corn stover and wheat bran carbon sources on the cellulase production and growth curve of T. reesei JL6 were also investigated.

  18. Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes.

    PubMed

    Parolo, Claudio; de la Escosura-Muñiz, Alfredo; Merkoçi, Arben

    2013-02-15

    The use of gold nanoparticles (AuNPs) as labeling carriers in combination with the enzymatic activity of the horseradish peroxidase (HRP) in order to achieve an improved optical lateral flow immunoassay (LFIA) performance is presented here. Briefly in a LFIA with an immune-sandwich format AuNPs are functionalized with a detection antibody already modified with HRP, obtaining an 'enhanced' label. Two different detection strategies have been tested: the first one following just the red color of the AuNPs and the second one using a substrate for the HRP (3 different substrates are evaluated), which produces a darker color that enhances the intensity of the previous red color of the unmodified AuNPs. In such very simple way it is gaining sensitivity (up to 1 order of magnitude) without losing the simplicity of the LFIA format, opening the way to other LFIA applications including their on-demand performance tuning according to the analytical scenario.

  19. Ethanol production via simultaneous saccharification and fermentation of sodium hydroxide treated corn stover using Phanerochaete chrysosporium and Gloeophyllum trabeum.

    PubMed

    Vincent, Micky; Pometto, Anthony L; van Leeuwen, J Hans

    2014-04-01

    Ethanol was produced via the simultaneous saccharification and fermentation (SSF) of dilute sodium hydroxide treated corn stover. Saccharification was achieved by cultivating either Phanerochaete chrysosporium or Gloeophyllum trabeum on the treated stover, and fermentation was then performed by using either Saccharomyces cerevisiae or Escherichia coli K011. Ethanol production was highest on day 3 for the combination of G. trabeum and E. coli K011 at 6.68 g/100g stover, followed by the combination of P. chrysosporium and E. coli K011 at 5.00 g/100g stover. SSF with S. cerevisiae had lower ethanol yields, ranging between 2.88 g/100g stover at day 3 (P. chrysosporium treated stover) and 3.09 g/100g stover at day 4 (G. trabeum treated stover). The results indicated that mild alkaline pretreatment coupled with fungal saccharification offers a promising bioprocess for ethanol production from corn stover without the addition of commercial enzymes.

  20. 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.

  1. Cognitive enhancers (nootropics). Part 2: drugs interacting with enzymes. Update 2014.

    PubMed

    Froestl, Wolfgang; Muhs, Andreas; Pfeifer, Andrea

    2014-01-01

    Scientists working in the field of Alzheimer's disease and, in particular, cognitive enhancers are very productive. The review on Drugs interacting with Enzymes was accepted in August 2012. However, this field is very dynamic. New potential targets for the treatment of Alzheimer's disease were identified. This update describes drugs interacting with 60 enzymes versus 43 enzymes in the first paper. Some compounds progressed in their development, while many others were discontinued. The present review covers the evolution of research in this field through April 2014.

  2. Rapid and enhanced proteolytic digestion using electric-field-oriented enzyme reactor.

    PubMed

    Zhou, Yu; Yi, Tie; Park, Sung-Soo; Chadwick, Wayne; Shen, Rong-Fong; Wu, Wells W; Martin, Bronwen; Maudsley, Stuart

    2011-06-10

    We have created a novel enzyme reactor using electric field-mediated orientation and immobilization of proteolytic enzymes (trypsin/chymotrypsin) on biocompatible PVDF membranes in a continuous flow-through chamber. Using less than 5min, this reactor in various enzyme combinations can produce enhanced rapid digestion for standardized prototypic proteins, hydrophilic proteins and hydrophobic transmembrane proteins when compared to in-solution techniques. With improved digestive efficiency, our reactor improved the overall functional analysis of lipid raft proteomes by identifying more closely functionally linked proteins and elucidated a richer set of biological processes and pathways linked to the proteins than traditional in-solution methods. PMID:21338726

  3. Cognitive enhancers (nootropics). Part 2: drugs interacting with enzymes. Update 2014.

    PubMed

    Froestl, Wolfgang; Muhs, Andreas; Pfeifer, Andrea

    2014-01-01

    Scientists working in the field of Alzheimer's disease and, in particular, cognitive enhancers are very productive. The review on Drugs interacting with Enzymes was accepted in August 2012. However, this field is very dynamic. New potential targets for the treatment of Alzheimer's disease were identified. This update describes drugs interacting with 60 enzymes versus 43 enzymes in the first paper. Some compounds progressed in their development, while many others were discontinued. The present review covers the evolution of research in this field through April 2014. PMID:24903780

  4. 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

  5. [Responses of enzymes in terrestrial plants to enhanced UV-B radiation].

    PubMed

    Yao, Xiaoqin; Liu, Qing

    2006-05-01

    With the destruction of ozone layer, ultraviolet-B (UV-B, 280 to approximately 320nm) radiation has being enhanced at the earth's surface. The energy of UV-B irradiation is far higher than that of visible light, which could be absorbed by biomacromolecules such as protein and nuclei acid. Enzyme is a sort of protein catalyzing the biochemical processes, and its content and activity in plant have strong responses to enhanced UV-B radiation. This paper summarized the research advances in the effects of enhanced UV-B radiation on the key enzymes, mainly including antioxidant enzymes, ribulose-1, 5-diphosphoscarboxylase, nitrate reductase and glutamine synthetase in terrestrial plants. Some suggestions for future research in this field were put forward.

  6. Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

    PubMed

    Gao, Yingning; Roberts, Christopher C; Toop, Aaron; Chang, Chia-En A; Wheeldon, Ian

    2016-08-01

    Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems.

  7. 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

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

    PubMed

    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 2(2) 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

  9. High-throughput Saccharification assay for lignocellulosic materials.

    PubMed

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

    2011-07-03

    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

  10. 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.

  11. Improving ethanol production by membrane technology: The continuous saccharification reactor

    SciTech Connect

    Cheryan, M.; Escobar, J.

    1993-12-31

    The saccharification of liquefied starch is typically done in a batch mode, taking 30--72 hours and requiring large quantities of enzyme (since each dose of enzyme is used only once). The process can be improved considerably by using a membrane reactor in which the reaction vessel is connected in a semi-closed loop configuration to a membrane module of the appropriate chemical nature and physical configuration. The continuous membrane reactor (CMR) concept was first evaluated with a dead-end cell, and later scaled-up to a cross-flow recycle configuration using hollow fibers or spiral wound modules. The CMR results in a dramatic reduction in reaction time to 5--10 hours, and reduces overall enzyme usage by 50--70%. In addition, the dextrose stream is crystal clear with little or no suspended particles, protein or fat, thus potentially reducing downstream costs. The CMR has been scaled up to a pilot-scale system of 1,500 liters with a membrane capacity of 30--65 cm{sup 2}, which is presently undergoing on-site trials at a large ethanol plant. The economics of this operation appear to be quite attractive.

  12. Ethanol production via in situ fungal saccharification and fermentation of mild alkali and steam pretreated corn fiber.

    PubMed

    Shrestha, Prachand; Khanal, Samir Kumar; Pometto, Anthony L; Hans van Leeuwen, J

    2010-11-01

    The effect of mild alkali and steam pretreatments on fungal saccharification and sequential simultaneous-saccharification and fermentation (SSF) of corn fiber to ethanol was studied. The corn fiber was pretreated with: (i) 2% NaOH (w/w) at 30 degrees C for 2h and (ii) steaming at 100 degrees C for 2h. Ethanol yields were 2.6g, 2.9g and 5.5g ethanol/100g of corn fiber, respectively, for Phanerochaete chrysosporium, Gloeophyllum trabeum and Trichoderma reesei saccharification and sequential SSFs. SSF with commercial cellulase enzyme - Spezyme-CP had 7.7g ethanol/100g corn fiber. Mild alkali pretreatment resulted in higher glucose yields following fungal saccharification of corn fiber. However, the ethanol yields were comparatively similar for untreated and mild alkali pretreated corn fiber. Solid-substrate fermentation of corn fiber with fungi can be improved to either eliminate or reduce the dosage of commercial cellulase enzymes during SSF.

  13. 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

  14. Fundamental challenges in mechanistic enzymology: progress toward understanding the rate enhancements of enzymes.

    PubMed

    Herschlag, Daniel; Natarajan, Aditya

    2013-03-26

    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 multifaceted 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.

  15. 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

  16. Simultaneous saccharification: fermentation with Zymomonas mobilis

    SciTech Connect

    Spangler, D.J.; Emert, G.H.

    1986-01-01

    In recent years, an ethanol production process has been developed which utilizes Trichoderma reesei cellulase and Candida brassicae IFO 1664 in the simultaneous saccharification/fermentation (SSF) of cellulose to ethanol. The direct production of ethanol from cellulose in an SSF process alleviates the problem of end production inhibition. Glucose does not accumulate in this system, but rather is fermented to ethanol immediately following saccharification. The result is an increase in yield of 25% or greater as compared with separate processes of saccharification and fermentation. An alternative organisms which might be used in place of yeasts in ethanol production processes is Zymomonas mobilis. The optimum temperature for hydrolysis of cellulose by Trichoderma reesei cellulases is 50/sup 0/C. Since this hydrolysis is the rate limiting step in the SSF process, it is advantageous to utilize the most temperature tolerant ethanol producer available. Candida brassicae is currently the organism of choice due to its ability to produce ethanol efficiently at 40/sup 0/C. This investigation reports on the screening of Zymomonas strains and evaluating the feasibility of utilizing the most temperature tolerant strain in place of C. brassicae in SSF.

  17. 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...

  18. Associating cooking additives with sodium hydroxide to pretreat bamboo residues for improving the enzymatic saccharification and monosaccharides production.

    PubMed

    Huang, Caoxing; He, Juan; Wang, Yan; Min, Douyong; Yong, Qiang

    2015-10-01

    Cooking additive pulping technique is used in kraft mill to increase delignification degree and pulp yield. In this work, cooking additives were firstly applied in the sodium hydroxide pretreatment for improving the bioconversion of bamboo residues to monosaccharides. Meanwhile, steam explosion and sulfuric acid pretreatments were also carried out on the sample to compare their impacts on monosaccharides production. Results indicated that associating anthraquinone with sodium hydroxide pretreatment showed the best performance in improving the original carbohydrates recovery, delignification, enzymatic saccharification, and monosaccharides production. After consecutive pretreatment and enzymatic saccharification process, 347.49 g, 307.48 g, 142.93 g, and 87.15 g of monosaccharides were released from 1000 g dry bamboo residues pretreated by sodium hydroxide associating with anthraquinone, sodium hydroxide, steam explosion and sulfuric acid, respectively. The results suggested that associating cooking additive with sodium hydroxide is an effective pretreatment for bamboo residues to enhance enzymatic saccharification for monosaccharides production.

  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. Comparison and evaluation of concurrent saccharification and anaerobic digestion of Napier grass after pretreatment by three microbial consortia.

    PubMed

    Wen, Boting; Yuan, Xufeng; Li, Qing X; Liu, Jingjing; Ren, Jiwei; Wang, Xiaofen; Cui, Zongjun

    2015-01-01

    Napier grass is potentially a viable feedstock for biofuel production. The present study investigated biological pretreatment of Napier grass by three microbial consortia followed by saccharification and anaerobic digestion. The pretreatment efficiencies of three microbial consortia were compared in terms of degradation ability, saccharide and biogas yield. The lignocellulose loss rates of Napier grass varied largely. The biomass pretreated by the consortium WSD-5 gave 43.4% and 66.2% total sugar yield under low and moderate loadings of commercial enzyme mixtures, while the highest yield was 83.2% pretreated by the consortium MC1 under a high enzyme loading. The maximum methane yield of pretreated samples by the consortia MC1, WSD-5 and XDC-2 were 259, 279, 247ml/g VS, respectively, which were 1.39, 1.49 and 1.32times greater than the values of the untreated controls. This study showed that pretreatments by MC1, WSD-5 and XDC-2 were capable of significantly enhancing both the saccharide and methane yields from Napier grass.

  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. 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.

  3. 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

  4. Enhancing enzyme stability and metabolic functional ability of β-galactosidase through functionalized polymer nanofiber immobilization.

    PubMed

    Misson, Mailin; Jin, Bo; Chen, Binghui; Zhang, Hu

    2015-10-01

    A functionalized polystyrene nanofiber (PSNF) immobilized β-galactosidase assembly (PSNF-Gal) was synthesized as a nanobiocatalyst aiming to enhance the biocatalyst stability and functional ability. The PSNF fabricated by electrospinning was functionalized through a chemical oxidation method for enzyme binding. The bioengineering performance of the enzyme carriers was further evaluated for bioconversion of lactose to galacto-oligosaccharides (GOS). The modified PSNF-Gal demonstrated distinguished performances to preserve the same activity as the free β-galactosidase at the optimum pH of 7.0, and to enhance the enzyme stability of PSNF-Gal in an alkaline condition up to pH 10. The PSNF assembly demonstrated improved thermal stability from 37 to 60 °C. The nanobiocatalyst was able to retain 30 % of its initial activity after ninth operation cycles comparing to four cycles with the unmodified counterpart. In contrast with free β-galactosidase, the modified PSNF-Gal enhanced the GOS yield from 14 to 28 %. These findings show the chemically modified PSNF-based nanobiocatalyst may be pertinent for various enzyme-catalysed bioprocessing applications.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. Parameter determination and validation for a mechanistic model of the enzymatic saccharification of cellulose-Iβ.

    PubMed

    Nag, Ambarish; Sprague, Michael A; Griggs, Andrew J; Lischeske, James J; Stickel, Jonathan J; Mittal, Ashutosh; Wang, Wei; Johnson, David K

    2015-01-01

    Cost-effective production of fuels and chemicals from lignocellulosic biomass often involves enzymatic saccharification, which has been the subject of intense research and development. Recently, a mechanistic model for the enzymatic saccharification of cellulose has been developed that accounts for distribution of cellulose chain lengths, the accessibility of insoluble cellulose to enzymes, and the distinct modes of action of the component cellulases [Griggs et al. (2012) Biotechnol. Bioeng., 109(3):665-675; Griggs et al. (2012) Biotechnol. Bioeng., 109(3):676-685]. However, determining appropriate values for the adsorption, inhibition, and rate parameters required further experimental investigation. In this work, we performed several sets of experiments to aid in parameter estimation and to quantitatively validate the model. Cellulosic materials differing in degrees of polymerization and crystallinity (α-cellulose-Iβ and highly crystalline cellulose-Iβ ) were digested by component enzymes (EGI/CBHI/βG) and by mixtures of these enzymes. Based on information from the literature and the results from these experiments, a single set of model parameters was determined, and the model simulation results using this set of parameters were compared with the experimental data of total glucan conversion, chain-length distribution, and crystallinity. Model simulations show significant agreement with the experimentally derived glucan conversion and chain-length distribution curves and provide interesting insights into multiple complex and interacting physico-chemical phenomena involved in enzymatic hydrolysis, including enzyme synergism, substrate accessibility, cellulose chain length distribution and crystallinity, and inhibition of cellulases by soluble sugars.

  12. 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.

  13. 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

  14. Wood saccharification by enzyme systems without prior delignification

    SciTech Connect

    Koshijima, T.; Yaku, F.; Muraki, E.; Tanaka, T.; Azuma, J.

    1983-01-01

    Around 80% of the polysaccharides contained in Akamatsu (Pinus densiflora) wood was hydrolyzed by using Cellulosin AP originated from Aspergillus niger without prior delignification when wood meal had been previously finely divided for 24 h by a vibration-type ball mill. The substrate concentration used in the enzymatic hydrolysis was 1 g/100 ml. It has been found that the hydrolysis rate increases to 86% by using a 1:1 mixture of Cellulosin AP and Onozuka R-10 cellulases and wood milled for 2 h. When the hydrolysis is performed at the substrate concentration 4 g/100 ml, however, 2 h of milling allows only a 42.5% hydrolysis rate, and it is necessary to take more than 24 h to degrade 80% of the polysaccharides contained. The reaction rate of enzymatic hydrolysis increased with increasing number of roll millings, thus 22 times of roll milling resulted in a twofold increase in reaction rate of the enzymatic hydrolysis compared with that of no milling. Plotting of the reaction rate V of the enzymatic hydrolysis against substrate concentration (S) showed that V leveled off at 10 g/100 ml of (S) in the 22 times roll-milled wood, and the value was only 4 g/100 ml in case of the untreated wood. The enzymatic hydrolysis rate has been compared for cellulose and wood, both of which were previously milled to different extents. In the case of cellulose, the hydrolysis rate showed a maximum at 24 h of milling, then decreased thereafter. This is not the case with woodmeal, where the existence of lignin as a radical scavenger may prevent the radicals formed in cellulose molecules from acting as retardant for enzymatic hydrolysis of cellulose. 9 references, 9 figures, 7 tables.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. Enzymatic saccharification of solid residue of olive mill in a batch reactor.

    PubMed

    Abdi; Hamdache; Belhocine; Grib; Lounici; Piron; Mameri

    2000-12-01

    This paper describes the enzymatic hydrolysis of solid residue of olive mill (OMRS) in a batch reactor with the Trichoderma reesei enzyme. Before enzymatic saccharification, crude lignocellulosic material is submitted to alkaline pre-treatment with NaOH. Optimum conditions of the pre-treatment (temperature of T=100 degrees C and OMRS-NaOH concentration ratio of about R=20) were determined. The optimum enzymatic conditions determined were as follows: pH of about 5, temperature of T=50 degrees C and enzyme to mass substrate mass ratio E/S=0.1g enzyme (g OMRS)(-1). The maximum saccharification yield obtained at optimum experimental conditions was about 50%. The experimental results agree with Lineweaver Burk's formula for low substrate concentrations. At substrate concentrations greater than 40gdm(-3), inhibitory effects were encountered. The kinetic constants obtained for the batch reactor were K(m)=0.1gdm(-3)min(-1) and V(m)=800gdm(-3).

  20. 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.

  1. Simultaneous Saccharification and Fermentation of Sugar Beet Pulp for Efficient Bioethanol Production

    PubMed Central

    Berłowska, Joanna; Balcerek, Maria; Dziekońska-Kubczak, Urszula; Patelski, Piotr; Dziugan, Piotr

    2016-01-01

    Sugar beet pulp, a byproduct of sugar beet processing, can be used as a feedstock in second-generation ethanol production. The objective of this study was to investigate the effects of pretreatment, of the dosage of cellulase and hemicellulase enzyme preparations used, and of aeration on the release of fermentable sugars and ethanol yield during simultaneous saccharification and fermentation (SSF) of sugar beet pulp-based worts. Pressure-thermal pretreatment was applied to sugar beet pulp suspended in 2% w/w sulphuric acid solution at a ratio providing 12% dry matter. Enzymatic hydrolysis was conducted using Viscozyme and Ultraflo Max (Novozymes) enzyme preparations (0.015–0.02 mL/g dry matter). Two yeast strains were used for fermentation: Ethanol Red (S. cerevisiae) (1 g/L) and Pichia stipitis (0.5 g/L), applied sequentially. The results show that efficient simultaneous saccharification and fermentation of sugar beet pulp was achieved. A 6 h interval for enzymatic activation between the application of enzyme preparations and inoculation with Ethanol Red further improved the fermentation performance, with the highest ethanol concentration reaching 26.9 ± 1.2 g/L and 86.5 ± 2.1% fermentation efficiency relative to the theoretical yield. PMID:27722169

  2. 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.

  3. 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

  4. 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.

  5. [Electricity generation of surplus sludge microbial fuel cells enhanced by additional enzyme].

    PubMed

    Yang, Hui; Liu, Zhi-Hu; Li, Xiao-Ming; Yang, Qi; Fang, Li; Huang, Hua-Jun; Zeng, Guang-Ming; Li, Shuo

    2012-01-01

    In this paper the feasibility of enhanced electricity generation of microbial fuel cell fed surplus sludge by additional enzymes (neutral protease and alpha-amylase) was discussed. The effect of dosage of additional enzyme on characteristics of electricity generation of the surplus sludge microbial fuel cell (SSMFC) and the reduction of surplus sludge were investigated. The results indicated that the maximum output power destiny of the group of experiment was higher than that of control under the same condition. Moreover, the maximum output power density, coulomb efficiency, efficiency of reducing TCOD, efficiency of reducing TSS and efficiency of reducing VSS reached up to 507 W x m(-2) (700 mW x m(-2)), 3.98% (5.11%), 88.31% (94.09%), 83.18% (98.02%) and 89.03% (98.80%) respectively for protease (alpha-amylase) at the dosage of 10 mg x g(-1). This study demonstrated that additional enzyme greatly enhanced the electricity generation of MFC with simultaneous accomplishments of sludge treatment, providing a novel approach for the practical application of microbial fuel cell.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. Histone deacetylase enzyme silencing using shRNAs enhances radiosensitivity of SW579 thyroid cancer cells

    PubMed Central

    Wang, Ye; Jin, Tao; Dai, Xueming; Yan, Dongwang; Peng, Zhihai

    2016-01-01

    The aim of the present study was to screen the enzymes that are associated with the radiosensitivity of SW579 thyroid cancer cells, and investigate whether radiation, combined with specific RNA interference on the screened enzymes, enhances radiosensitivity of SW579 thyroid cancer cells. Quantitative polymerase chain reaction (qPCR) was used to analyze epigenetic enzyme expression changes before and after radiotherapy, and four enzymes, histone deacetylase 1 (HDAC1), HDAC2, HDAC4 and HDAC6 were screened. Western blot analysis was performed to analyze the change in HDAC1, HDAC2, HDAC4 and HDAC6 protein expression following radiotherapy. Short hairpin RNA (ShRNA)-HDAC1, shRNA-HDAC2, shRNA-HDAC4 and shRNA-HDAC6 plasmids were constructed and SW579 cells were transfected with corresponding shRNA-HDACs. Reverse transcription-qPCR was used to detect whether downregulation of HDAC mRNAs had been effective. In addition, shRNA and shRNA negative control (NC) pools were established and transfected into the SW579 cells. The samples were divided into four groups; control, trichostatin A, shRNA pool and shRNA NC pool, to analyze the effective enhancement of specific shRNA on radiosensitivity in thyroid cancer cells. The morphological changes were observed in the SW579 cells, and the number of tumor cells decreased markedly in the shRNA pool group compared with that of the other three groups. Therefore, it was concluded that HDACs present a potential target for increasing the sensitivity of thyroid cancer cells to radiotherapy, and shRNA-HDAC interference combined with radiotherapy promotes the radiosensitivity of tumors. PMID:27600599

  11. 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

  12. Enhancing biodistribution of therapeutic enzymes in vivo by modulating surface coating and concentration of ICAM-1-targeted nanocarriers.

    PubMed

    Hsu, Janet; Bhowmick, Tridib; Burks, Scott R; Kao, Joseph P Y; Muro, Silvia

    2014-02-01

    Coupling therapeutic proteins to targeted nanocarriers can enhance their biodistribution. This is the case for enzyme replacement therapies where intravenously injected enzymes must avoid prolonged blood exposure while reaching body organs. We have shown enhanced tissue targeting of various lysosomal enzymes by coupling to nanocarriers targeted to intercellular adhesion molecule-1 (ICAM-1). Here, we varied design parameters to modify tissue enzyme levels without affecting specific targeting and relative biodistribution. We coupled a-galactosidase (aGal; affected in Fabry disease) to model polymer nanocarriers and varied enzyme load (50 vs. 500 molecules/particle), anti-ICAM surface density (80 vs. 180 molecules/particle), and nanocarrier concentration (1.6 x 1013 vs. 2.4 x 1013 carriers/kg) to render three formulations (45, 449, 555 microg alphaGal/kg). Naked alpha Gal preferentially distributed in blood vs. organs, while nanocarriers shifted biodistribution from blood to tissues. Accumulation in brain, kidneys, heart, liver, lungs, and spleen did not vary among nanocarrier formulations, with enhanced specific tissue accumulation compared to naked aGal. The highest specificity was associated with lowest antibody density and nanocarrier concentration, but highest enzyme load; possibly because of synergistic enzyme affinity toward cell-surface markers. Variation of these parameters significantly increased absolute enzyme accumulation. This strategy may help optimize delivery of lysosomal enzyme replacement and, likely, other protein delivery approaches.

  13. Considering water availability and the effect of solute concentration on high solids saccharification of lignocellulosic biomass.

    PubMed

    Selig, Michael J; Hsieh, Chia-Wen Carmen; Thygesen, Lisbeth G; Himmel, Michael E; Felby, Claus; Decker, Stephen R

    2012-01-01

    Milliliter scale (ligno)cellulose saccharifications suggest general solute concentration and its impact on water availability plays a significant role in detrimental effects associated with high solids lignocellulose conversions. A microtumbler developed to enable free-fall mixing at dry solids loadings up to 35% (w/w) repeatedly produced known detrimental conversion trends on cellulose, xylan and pretreated lignocellulose with commercial enzymes. Despite this, high concentrations of insoluble nonhydrolysable dextrans did not depress saccharification extents in 5% (w/w) cellulose slurries suggesting mass transfer limitations may not significantly limit hydrolysis extents at high solids loadings. Interestingly, cellulose saccharification by purified cellulases showed increased conversions with increasing dry solids loadings. This prompted investigations into impacts the concentration of soluble species, such as sugar alcohols, low molecular weight enzyme preparation components, and monomer hydrolysis products, have on the hydrolysis environment. Such substances significantly depress conversion rates and were shown to correlatively lower water activity (A(w) ) in the hydrolysis environment while high insoluble solids concentrations did not. Furthermore, low-field NMR on concentrated slurries of insoluble complex carbohydrates, including the nonhydrolysable dextrans, showed all solids constrained water significantly more than high concentrations of soluble species (inhibitory) suggesting water constraint may not be as problematic an issue at high solids loadings compared to the availability of water in the system. Additionally, the introduction of soluble species lessened overall water constraint in high solids systems and appears to shift the distribution of water away from insoluble surfaces. This is potentially a critical issue for industrial processes operating at high dry solids levels.

  14. An enzyme-assisted nanoparticle crosslinking approach to enhance the mechanical strength of peptide-based supramolecular hydrogels.

    PubMed

    Li, Ying; Ding, Yin; Qin, Meng; Cao, Yi; Wang, Wei

    2013-10-01

    In this work we reported an enzyme-assisted nanoparticle crosslinking (EANC) strategy to enhance the mechanical stability of peptide-based supramolecular hydrogels by more than 3000 times. PMID:23948779

  15. Impact of bleaching on subcritical water- and Formosolv-pretreated tulip tree to enhance enzyme accessibility.

    PubMed

    Myint, Aye Aye; Kim, Dae Sung; Lee, Hun Wook; Yoon, Junho; Choi, In-Gyu; Choi, Joon Weon; Lee, Youn-Woo

    2013-10-01

    A novel method was developed for fractionating cellulose microfibrils from forest residue (tulip tree sawdust) to enhance cellulose digestibility, particularly at minimum enzyme loadings. This method involved three main stages: selective hemicellulose solubilization by subcritical water (SCW) pretreatment, delignification of the SCW-pretreated solids using the Formosolv process, and deformylation/bleaching of the cellulose pulp with alkaline hydrogen peroxide solution. This process produced nearly 98% white cellulose microfibrils with 23-fold higher conversion to glucose as compared to the raw substrate after 72 h of enzymatic hydrolysis. This study showed that cellulose swelling had the greatest effect on the enzymatic hydrolysis efficiency of delignified pulp obtained by the Formosolv process.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Bench-scale bioethanol production from eucalyptus by high solid saccharification and glucose/xylose fermentation method.

    PubMed

    Fujii, Tatsuya; Murakami, Katsuji; Endo, Takashi; Fujimoto, Shinji; Minowa, Tomoaki; Matsushika, Akinori; Yano, Shinichi; Sawayama, Shigeki

    2014-04-01

    In the bioethanol production process, high solid saccharification and glucose/xylose co-fermentation are important technologies for obtaining increased ethanol concentrations; however, bench-scale studies using combinations of these methods are limited. In this study, we hydrolyzed high solid concentration of milled eucalyptus using commercial enzymes and obtained 138.4 g/L total monomeric sugar concentration. These sugars were fermented to 53.5 g/L of ethanol by a xylose-utilizing recombinant Saccharomyces cerevisiae strain, MA-R4. These experiments were performed in bench scale (using 50 L scale solid mixer and 70 L scale fermenter). The results obtained in this study were comparable to our previous results in laboratory scale, indicating that we successfully achieved an efficient high solid saccharification and glucose/xylose co-fermentation system in bench scale. PMID:23917411

  2. Kinetic modeling of multi-feed simultaneous saccharification and co-fermentation of pretreated birch to ethanol.

    PubMed

    Wang, Ruifei; Koppram, Rakesh; Olsson, Lisbeth; Franzén, Carl Johan

    2014-11-01

    Fed-batch simultaneous saccharification and fermentation (SSF) is a feasible option for bioethanol production from lignocellulosic raw materials at high substrate concentrations. In this work, a segregated kinetic model was developed for simulation of fed-batch simultaneous saccharification and co-fermentation (SSCF) of steam-pretreated birch, using substrate, enzymes and cell feeds. The model takes into account the dynamics of the cellulase-cellulose system and the cell population during SSCF, and the effects of pre-cultivation of yeast cells on fermentation performance. The model was cross-validated against experiments using different feed schemes. It could predict fermentation performance and explain observed differences between measured total yeast cells and dividing cells very well. The reproducibility of the experiments and the cell viability were significantly better in fed-batch than in batch SSCF at 15% and 20% total WIS contents. The model can be used for simulation of fed-batch SSCF and optimization of feed profiles.

  3. 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

  4. 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

  5. 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.

  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. Brassinosteroid alleviates polychlorinated biphenyls-induced oxidative stress by enhancing antioxidant enzymes activity in tomato.

    PubMed

    Ahammed, Golam Jalal; Ruan, Yi-Ping; Zhou, Jie; Xia, Xiao-Jian; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan

    2013-03-01

    Polychlorinated biphenyls (PCBs) are persistent organic pollutants often found in the atmosphere. Phytoremediation of airborne PCBs is an emerging new concept to minimize potential human exposure. However, effects of atmospheric PCBs on plant growth, photosynthesis and antioxidant defence system are poorly understood area. Brassinosteroids have been reported to alleviate different abiotic stresses including organic pollutants-induced stress. Hence, we studied the effects of PCBs and 24-epibrassinolide (EBR) on biomass accumulation, photosynthetic machinery and antioxidant system in tomato plants. PCBs (0.4, 2.0 and 10 μg/l) mist spray significantly decreased dry weight, photosynthesis, chlorophyll contents in a dose dependent manner. Both stomatal and non-stomatal factors were involved in PCBs-induced photosynthetic inhibition. Likewise, the maximal photochemical efficiency of PSII (Fv/Fm), the quantum efficiency of PSII photochemistry (Φ(PSII)) and photochemical quenching coefficient were increasingly decreased by various levels of PCBs, suggesting an induction of photoinhibition. Increased accumulation of H(2)O(2) and O(2)(-) accompanied with high lipid peroxidation confirmed occurrence of oxidative stress upon PCBs exposure. Meanwhile, antioxidant enzymes activity was decreased following exposure to PCBs. Foliar application of EBR (100 nM) increased biomass, photosynthetic capacity, chlorophyll contents and alleviated photoinhibition by enhancing Fv/Fm, Φ(PSII) and qP. EBR significantly decreased harmful ROS accumulation and lipid peroxidation through the induction of antioxidant enzymes activity. Our results suggest a protective role of EBR against PCBs stress which may strengthen phytoremediation approaches by enhancing plant tolerance.

  8. Simultaneous saccharification and fermentation of delignified lignocellulosic biomass at high solid loadings by a newly isolated thermotolerant Kluyveromyces sp. for ethanol production.

    PubMed

    Narra, Madhuri; James, Jisha P; Balasubramanian, Velmurugan

    2015-03-01

    Simultaneous saccharification and fermentation studies were carried out using thermotolerant newly isolated Kluyveromyces sp. with three different delignified lignocellulosic biomass viz. rice straw, wheat straw and sugarcane bagasse at 5-15% solid loading and 6-12 FPU g(-1) substrate enzyme loading for different time intervals 0-72 h at 42°C. Maximum ethanol achieved from rice straw, wheat straw and sugarcane bagasse with in-house crude cellulases from Aspergillus terreus was 23.23, 18.29 and 17.91 mg mL(-1) at 60 h with 10% solid load and 9 FPU g(-1) substrate enzyme loading. Tween 80 1% (v/v) enhanced the ethanol yield by 8.39%, 9.26% and 8.14% in rice straw, wheat straw and sugarcane bagasse, respectively. External supplementation of β-glucosidase to the crude as well commercial cellulases produced maximum theoretical ethanol yield of 71.76%, 63.77%, 57.15% and 84.56%, 72.47%, 70.55% from rice straw, wheat straw and sugarcane bagasse, respectively.

  9. Simultaneous saccharification and microbial lipid fermentation of corn stover by oleaginous yeast Trichosporon cutaneum.

    PubMed

    Liu, Wei; Wang, Yumei; Yu, Zhanchun; Bao, Jie

    2012-08-01

    Simultaneous saccharification and fermentation (SSF) is the most commonly practiced operation in lignocellulose bioconversion to avoid the sugar product inhibition to cellulase enzymes. In this study, for the first time SSF was tested on microbial lipid fermentation using the diluted acid pretreated and biodetoxified corn stover. The results show that SSF was effective than the separate hydrolysis and fermentation (SHF) on lipid accumulation of Trichosporon cutaneum CX1 cells in both the small scale (5L) and the enlarged scale (50 L) bioreactors. The solutions for the oxygen transfer and the lipid extraction in SSF practically worked well. The process parameters were optimized and the lipid yield obtained were 3.03 g/L in the 5L, and 3.23 g/L in the 50 L, respectively. The result also shows that the cellulase enzyme could be partially recycled in the SSF. The study provided a practical and efficient way for microbial lipid production from lignocellulose material. PMID:22695140

  10. Succinic acid production from corn stover by simultaneous saccharification and fermentation using Actinobacillus succinogenes.

    PubMed

    Zheng, Pu; Fang, Lin; Xu, Yan; Dong, Jin-Jun; Ni, Ye; Sun, Zhi-Hao

    2010-10-01

    Simultaneous saccharification and fermentation (SSF) technique was applied for succinic acid production by Actinobacillus succinogenes in a 5-l stirred bioreactor with corn stover as the raw material. The process parameters of SSF, including corn stover pretreatment condition, substrate concentration, enzyme loading and fermentation temperature were investigated. Results indicated that pretreating corn stover with diluted alkaline was beneficial for the succinic acid production, and succinic acid yield could be significantly increased when adding the cellulase supplemented with cellobiase. The maximal succinic acid concentration and yield could reach 47.4 g/l and 0.72 g/g-substrate, respectively. The corresponding operation conditions were summarized as follows: SSF operation at 38 °C for 48 h, diluted alkaline pretreated corn stover as substrate with concentration of 70 g/l, enzyme loading of 20FPU cellulase and 10 U cellobiase per gram substrate. This result suggested an industrial potential of succinic acid production by using SSF and corn stover.

  11. DNA Enzyme-Decorated DNA Nanoladders as Enhancer for Peptide Cleavage-Based Electrochemical Biosensor.

    PubMed

    Kou, Bei-Bei; Zhang, Li; Xie, Hua; Wang, Ding; Yuan, Ya-Li; Chai, Ya-Qin; Yuan, Ruo

    2016-09-01

    Herein, we developed a label-free electrochemical biosensor for sensitive detection of matrix metalloproteinase-7 (MMP-7) based on DNA enzyme-decorated DNA nanoladders as enhancer. A peptide and single-stranded DNA S1-modified platinum nanoparticles (P1-PtNPs-S1), which served as recognition nanoprobes, were first immobilized on electrode. When target MMP-7 specifically recognized and cleaved the peptide, the PtNPs-S1 bioconjugates were successfully released from electrode. The remaining S1 on electrode then hybridized with ssDNA1 (I1) and ssDNA2 (I2), which could synchronously trigger two hybridization chain reactions (HCRs), resulting in the in situ formation of DNA nanoladders. The desired DNA nanoladders not only were employed as ideal nanocarriers for enzyme loading, but also maintained its catalytic activity. With the help of hydrogen peroxide (H2O2), manganese porphyrin (MnPP) with peroxidase-like activity accelerated the 4-chloro-1-naphthol (4-CN) oxidation with generation of insoluble precipitation on electrode, causing a very low differential pulse voltammetry (DPV) signal for quantitative determination of MMP-7. Under optimal conditions, the developed biosensor exhibited a wide linear ranging from 0.2 pg/mL to 20 ng/mL, and the detection limit was 0.05 pg/mL. This work successfully realized the combination of DNA signal amplification technique with artificial mimetic enzyme-catalyzed precipitation reaction in peptide cleavage-based protein detection, offering a promising avenue for the detection of other proteases. PMID:27532492

  12. 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.

  13. Enzyme-Enhanced Extraction of Phenolic Compounds and Proteins from Flaxseed Meal

    PubMed Central

    Ribeiro, Bernardo Dias; Barreto, Daniel Weingart; Coelho, Maria Alice Zarur

    2013-01-01

    Flaxseed (Linum usitatissimum) meal, the main byproduct of the flaxseed oil extraction process, is composed mainly of proteins, mucilage, and phenolic compounds. The extraction methods of phenolics either commonly employed the use of mixed solvents (dioxane/ethanol, water/acetone, water/methanol, and water/ethanol) or are done with the aid of alkaline, acid, or enzymatic hydrolysis. This work aimed at the study of optimal conditions for a clean process, using renewable solvents and enzymes, for the extraction of phenolics and proteins from flaxseed meal. After a screening of the most promising commercial preparations based on different carbohydrases and proteases, a central composite rotatable design and a mixture design were applied, achieving as optimal results a solution containing 6.6 and 152 g kg−1 meal of phenolics and proteins, respectively. The statistical approach used in the present study for the enzyme-enhanced extraction of phenolics and proteins from the major flaxseed byproduct was effective. By means of the sequential experimental design methodology, the extraction of such compounds was increased 10-fold and 14-fold, when compared to a conventional nonenzymatic extraction. PMID:25969774

  14. 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.

  15. Employing bifunctional enzymes for enhanced extraction of bioactives from plants: flavonoids as an example.

    PubMed

    Xu, Ming-Shu; Chen, Shuo; Wang, Wen-Quan; Liu, Si-Qin

    2013-08-21

    A cost-effective and environmentally friendly approach was developed to improve the extraction of active ingredients from plants, in which a bifunctional enzyme was employed for not only facilitating cell wall degradation but also increasing the bioactivity of target compounds in the extract. In the aqueous extraction of flavonoids from Glycyrrhizae radix, Trichoderma viride cellulase, a commercial cell-wall-degrading enzyme, was found to efficiently deglycosylate liquiritin and isoliquiritin, which are of high content but low bioactivity, into their aglycones that have much higher physiological activities for dietary and medicinal uses. Under optimized conditions, the extraction yield of liquiritigenin and isoliquiritigenin aglycones reached 4.23 and 0.39 mg/g of dry weight (dw) with 6.51- and 3.55-fold increases, respectively. The same approach was expanded to the extraction of flavonoids from Scutellariae radix using Penicillium decumbens naringinase, where enhanced production of more bioactive bacalein and wogonin was achieved via enzymatic deglycosylation of bacalin and wogonoside. PMID:23869387

  16. Employing bifunctional enzymes for enhanced extraction of bioactives from plants: flavonoids as an example.

    PubMed

    Xu, Ming-Shu; Chen, Shuo; Wang, Wen-Quan; Liu, Si-Qin

    2013-08-21

    A cost-effective and environmentally friendly approach was developed to improve the extraction of active ingredients from plants, in which a bifunctional enzyme was employed for not only facilitating cell wall degradation but also increasing the bioactivity of target compounds in the extract. In the aqueous extraction of flavonoids from Glycyrrhizae radix, Trichoderma viride cellulase, a commercial cell-wall-degrading enzyme, was found to efficiently deglycosylate liquiritin and isoliquiritin, which are of high content but low bioactivity, into their aglycones that have much higher physiological activities for dietary and medicinal uses. Under optimized conditions, the extraction yield of liquiritigenin and isoliquiritigenin aglycones reached 4.23 and 0.39 mg/g of dry weight (dw) with 6.51- and 3.55-fold increases, respectively. The same approach was expanded to the extraction of flavonoids from Scutellariae radix using Penicillium decumbens naringinase, where enhanced production of more bioactive bacalein and wogonin was achieved via enzymatic deglycosylation of bacalin and wogonoside.

  17. 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

  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

    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. 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.

  1. 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

  2. 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.

  3. Enhancing bioplastic-substrate interaction via pore induction and directed migration of enzyme location.

    PubMed

    Lele, Bhalchandra S; Papworth, Glenn; Katsemi, Vicky; Rüterjans, Heinz; Martyano, Igor; Klabunde, Kenneth J; Russell, Alan J

    2004-06-20

    We demonstrate two novel approaches to enhance interactions of polymer-immobilized biomolecules with their substrates. In the first approach, diisopropylfluorophosphatase (DFPase) containing poly(urethane) (PU) coatings were made microporous by incorporating, then extracting, poly(ethylene glycol)-based diesters as porogens. Incorporation of 2% w/w porogen increased the effective diffusion coefficient of diisopropylfluorophosphate (DFP) through the coatings by 30% and increased the apparent turnover number of immobilized DFPase 3-fold. In the second approach, prior to immobilization, hydrophobic modification of DFPase was achieved through its conjugation with a dimer/trimer mixture of a uretdione based on 1,6-diisocyanatohexane. When the hydrophobically modified DFPase was immobilized in coatings, catalytic activity was 4-fold higher than that of the equivalent, immobilized, native DFPase. This activity enhancement was independent of the presence or absence of pores. Confocal microscopy images of coatings containing fluorescently labeled lysozyme show that the native enzyme is distributed uniformly over the entire thickness of the coatings. Hydrophobically modified and fluorescently labeled lysozyme is accumulated only in the upper 10 microm cross-sectional layer of a 100 microm-thick coating. Interactions of bioplastics with their substrates are tunable either by pore induction in a polymer or by directed migration of the hydrophobically modified biomolecule to the desired location. The latter approach has broad implications, including overcoming mass transfer limitations experienced by immobilized biocatalysts.

  4. 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.

  5. 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.

  6. Phytoestrogens enhance antioxidant enzymes after swimming exercise and modulate sex hormone plasma levels in female swimmers.

    PubMed

    Mestre-Alfaro, Antonia; Ferrer, Miguel D; Sureda, Antoni; Tauler, Pedro; Martínez, Elisa; Bibiloni, Maria M; Micol, Vicente; Tur, Josep A; Pons, Antoni

    2011-09-01

    Our aim was to investigate the effects of diet supplementation with phytoestrogens on sex hormone levels, antioxidant adaptive responses and oxidative damage induced by exercise. Ten female swimmers participated for 26 days in a diet intervention with either a functional beverage rich in vitamins C and E or the same beverage but also supplemented with Lippia citriodora extract (PLX) containing 20 mg/100 ml verbascoside. After the intervention all subjects participated in a swimming session for 30 min maintaining the intensity at about 75-80% of their individual best performance time for a 50-m swim. In lymphocytes, the superoxide dismutase activity increased after exercise, with a higher increase in the PLX group. Swimming increased the erythrocyte activity of glutathione peroxidase and glutathione reductase in the PLX group. Purified glutathione reductase activity increased after an in vitro incubation with PLX. No effects were observed on the lymphocyte levels of malondialdehyde and carbonyls, but exercise increased the percentage of high-damaged lymphocytes 2.8 times in the placebo group and 1.5 times in the PLX group. PLX decreased the levels of 17-β-estradiol and testosterone and increased the levels of the sex hormone binding globulin. In conclusion, supplementation with phytoestrogens enhances the glutathione-dependent enzyme activities in erythrocytes and the superoxide dismutase activity in lymphocytes in response to exercise. PLX also shows direct antioxidant properties, by increasing glutathione reductase enzyme activity in vitro. Supplementation with phytoestrogens also decreases the plasma steroid hormone levels, pointing towards a possible agonistic effect of verbascoside in the hypothalamic regulation of estradiol synthesis.

  7. 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

  8. 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

  9. 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).

  10. Effect of enzyme secreting bacterial pretreatment on enhancement of aerobic digestion potential of waste activated sludge interceded through EDTA.

    PubMed

    Kavitha, S; Adish Kumar, S; Yogalakshmi, K N; Kaliappan, S; Rajesh Banu, J

    2013-12-01

    In this study, the effect of Ethylene diamine tetra acetic acid (EDTA) on Extracellular polymeric substance (EPS) removal tailed with bacterial enzymatic pretreatment on aerobic digestion of activated sludge was studied. In order to enhance the accessibility of sludge to the enzyme secreting bacteria; the extracellular polymeric substances were removed using EDTA. EDTA efficiently removed the EPS with limited cell lysis and enhanced the sludge enzyme activity at its lower concentration of 0.2 g/g SS. The sludge was then subjected to bacterial pretreatment to enhance the aerobic digestion. In aerobic digestion the best results in terms of Suspended solids (SS) reduction (48.5%) and COD (Chemical oxygen demand) solubilization (47.3%) was obtained in experimental reactor than in control. These results imply that aerobic digestion can be enhanced efficiently through bacterial pretreatment of EPS removed sludge.

  11. Nitric oxide induces specific isoforms of antioxidant enzymes in soybean leaves subjected to enhanced ultraviolet-B radiation.

    PubMed

    Santa-Cruz, Diego M; Pacienza, Natalia A; Zilli, Carla G; Tomaro, Maria L; Balestrasse, Karina B; Yannarelli, Gustavo G

    2014-12-01

    Antioxidant enzymes play a key role in plant tolerance to different types of stress, including ultraviolet-B (UV-B) radiation. Here we report that nitric oxide (NO) enhances antioxidant enzymes gene expression and increases the activity of specific isoforms protecting against UV-B radiation. Pre-treatments with sodium nitroprussiate (SNP), a NO-donor, prevented lipid peroxidation, ion leakage and H2O2 and superoxide anion accumulation in leaves of UV-B-treated soybean plants. Transcripts levels of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were significantly induced by SNP. These data correlated with the enhancement of particular antioxidant enzyme isoforms, such as one CAT isoform and two APX isoforms. Moreover, SNP induced the expression of three new isoforms of SOD, identified as Mn-SOD subclass. Further results showed that total activities of SOD, CAT and APX significantly increased by 2.2-, 1.8- and 2.1-fold in SNP-treated plants compared to controls, respectively. The protective effect of SNP against UV-B radiation was negated by addition of the specific NO scavenger cPTIO, indicating that NO released by SNP mediates the enhancement of antioxidant enzymes activities. In conclusion, NO is involved in the signaling pathway that up-regulates specific isoforms of antioxidant enzymes protecting against UV-B-induced oxidative stress.

  12. Regulating yeast flavor metabolism by controlling saccharification reaction rate in simultaneous saccharification and fermentation of Chinese Maotai-flavor liquor.

    PubMed

    Wu, Qun; Chen, Bi; Xu, Yan

    2015-05-01

    Maotai-flavor liquor is produced by simultaneous saccharification and fermentation (SSF), in which filamentous fungi produce hydrolases to degrade the starch into fermentable sugar. Saccharomyces cerevisiae simultaneously transforms the sugars to ethanol and flavor compounds. The saccharification rate plays an important role in regulating the liquor yield and flavor profile. This work investigated the effect of saccharification rate on fermentation by regulating the inoculation ratio (1:0.1, 1:0.5, 1:1, 1:5, 1:10) of S. cerevisiae and Aspergillus oryzae, the main saccharification agent. We found no significant difference in reducing sugar content among the mixed cultures with different ratios. This indicated a balance of the saccharification rate and the sugar consumption rate, in which the former was controlled by the interaction between A. oryzae and S. cerevisiae, and the latter controlled the metabolism of the two species. The ethanol yield was the highest in ratios of 1:0.5, 1:1, and 1:5, while the total production of flavor compounds was the highest for the ratio of 1:0.5, which was mainly attributed to the vigorous metabolism of S. cerevisiae. The inoculum ratio of 1:10 produced the second highest content of flavor compounds in which a large number of alcohols and esters were derived from the vigorous metabolism of A. oryzae. This indicated that the saccharification rate significantly influenced the flavor metabolism. This study improves understanding of the interaction and cooperation between A. oryzae and S. cerevisiae in co-culture fermentation for Chinese liquor making.

  13. Cognitive enhancers (Nootropics). Part 3: drugs interacting with targets other than receptors or enzymes. Disease-modifying drugs. Update 2014.

    PubMed

    Froestl, Wolfgang; Pfeifer, Andrea; Muhs, Andreas

    2014-01-01

    Scientists working in the field of Alzheimer's disease and, in particular, cognitive enhancers, are very productive. The review "Drugs interacting with Targets other than Receptors or Enzymes. Disease-modifying Drugs" was accepted in October 2012. In the last 20 months, new targets for the potential treatment of Alzheimer's disease were identified. Enormous progress was realized in the pharmacological characterization of natural products with cognitive enhancing properties. This review covers the evolution of research in this field through May 2014.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

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

    PubMed Central

    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

    2016-01-01

    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

  3. Hydrolytic potential of Trichoderma sp. strains evaluated by microplate-based screening followed by switchgrass saccharification.

    PubMed

    Cianchetta, Stefano; Galletti, Stefania; Burzi, Pier Luigi; Cerato, Claudio

    2012-05-10

    Bioconversion of lignocellulosic biomass to fuel requires a hydrolysis step to obtain fermentable sugars, generally accomplished by fungal enzymes. Large-scale screening of different microbial strains would provide optimal enzyme cocktails for any target feedstock. The aim of this study was to screen a large collection of Trichoderma sp. strains for the hydrolytic potential towards switchgrass (Panicum virgatum L.). Strains were cultivated in a small-scale system and assayed in micro-plates for xylanase and cellulase activities. The population distributions of these traits are reported after growth on switchgrass in comparison with cellulose. The distribution profiles suggest that the growth on switchgrass strongly promotes xylanase production. The IK4 strain displayed the highest xylanase activity after growth on switchgrass (133U/mL). Enzymes (10FPU/g substrate) from IK4 were compared with those from 2 cellulolytic Trichoderma strains and a commercial enzyme in saccharification time-course experiments on untreated and pretreated switchgrass and on an artificial substrate. Samples were analysed by DNS assay and by an oxygraphic method for sugar equivalent or glucose concentration. On the untreated substrate, IK4 enzymes even outperformed a 5-fold load of commercial enzyme, suggesting that xylanase or accessory enzymes are a limiting factor on this type of recalcitrant substrate. On the other substrates, IK4 preparations showed intermediate behaviour if compared with the commercial enzyme at 10FPU/g substrate and at 5-fold load. IK4 also nearly halved the time to release 50% of the hydrolysable sugar equivalents (T(50%)), with respect to the other preparations at the same enzymatic load. DNS assay and oxygraphic method gave highly correlated results for the 3 saccharified substrates. The study suggests that accessory enzymes like xylanase play a key role in improving the performance of cellulase preparations on herbaceous lignocellulosic feedstocks like switchgrass.

  4. 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.

  5. 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

  6. 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

  7. Starch saccharification and fermentation of uncooked sweet potato roots for fuel ethanol production.

    PubMed

    Zhang, Peng; Chen, Caifa; Shen, Yanhu; Ding, Tielin; Ma, Daifu; Hua, Zichun; Sun, Dongxu

    2013-01-01

    An energy-saving ethanol fermentation technology was developed using uncooked fresh sweet potato as raw material. A mutant strain of Aspergillus niger isolated from mildewed sweet potato was used to produce abundant raw starch saccharification enzymes for treating uncooked sweet potato storage roots. The viscosity of the fermentation paste of uncooked sweet potato roots was lower than that of the cooked roots. The ethanol fermentation was carried out by Zymomonas mobilis, and 14.4 g of ethanol (87.2% of the theoretical yield) was produced from 100g of fresh sweet potato storage roots. Based on this method, an energy-saving, high efficient and environment-friendly technology can be developed for large-scale production of fuel ethanol from sweet potato roots.

  8. 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.

  9. Fractal kinetic analysis of the enzymatic saccharification of CO2 laser pretreated corn stover.

    PubMed

    Tian, Shuang-Qi; Ma, Sen; Wang, Xin-Wei; Zhang, Zheng-Nan

    2013-10-15

    The enzymatic hydrolyses of laser pretreated corn stover as a novel pretreatment method were examined to establish a simplified kinetic model for the complicated hydrolysis process. The time dependence of the total reducing sugars amount was closely related to the amounts of cellulosic materials and amounts of cellulase. The evaluated model fitted very well with the experimental data of enzymatic hydrolysis of laser pretreated corn stover under different conditions, including cellulase loading, nature of substrate, substrate loading in the reaction medium. The results indicated that the complex kinetics of cellulase enzymatic saccharification could be assessed with the fractal kinetic model. The cellulase enzymatic reaction process was effectively predicted and controlled with the kinetic model. The result showed that the model could effectively reflect dynamic process of enzyme hydrolysis.

  10. Simultaneous saccharification and fermentation of acid-pretreated rapeseed meal for succinic acid production using Actinobacillus succinogenes.

    PubMed

    Chen, Kequan; Zhang, Han; Miao, Yelian; Wei, Ping; Chen, Jieyu

    2011-04-01

    Rapeseed meal was evaluated for succinic acid production by simultaneous saccharification and fermentation using Actinobacillus succinogenes ATCC 55618. Diluted sulfuric acid pretreatment and subsequent hydrolysis with pectinase was used to release sugars from rapeseed meal. The effects of culture pH, pectinase loading and yeast extract concentration on succinic acid production were investigated. When simultaneous saccharification and fermentation of diluted acid pretreated rapeseed meal with a dry matter content of 12.5% (w/v) was performed at pH 6.4 and a pectinase loading of 2% (w/w, on dry matter) without supplementation of yeast extract, a succinic acid concentration of 15.5 g/L was obtained at a yield of 12.4 g/100g dry matter. Fed-batch simultaneous saccharification and fermentation was carried out with supplementation of concentrated pretreated rapeseed meal and pectinase at 18 and 28 h to yield a final dry matter content of 20.5% and pectinase loading of 2%, with the succinic acid concentration enhanced to 23.4 g/L at a yield of 11.5 g/100g dry matter and a productivity of 0.33 g/(Lh). This study suggests that rapeseed meal may be an alternative substrate for the efficient production of succinic acid by A. succinogenes without requiring nitrogen source supplementation.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. Kinetic modeling of simultaneous saccharification and fermentation of corn starch for ethanol production.

    PubMed

    Białas, Wojciech; Czerniak, Adrian; Szymanowska-Powałowska, Daria

    2014-01-01

    Fuel ethanol production, using a simultaneous saccharification and fermentation process (SSF) of native starch from corn flour, has been performed using Saccharomyces cerevisiae and a granular starch hydrolyzing enzyme. The quantitative effects of mash concentration, enzyme dose and pH were investigated with the use of a Box-Wilson central composite design protocol. Proceeding from results obtained in optimal fermentation conditions, a kinetics model relating the utilization rates of starch and glucose as well as the production rates of ethanol and biomass was tested. Moreover, scanning electron microscopy (SEM) was applied to investigate corn starch granule surface after the SFF process. A maximum ethanol concentration of 110.36 g/l was obtained for native corn starch using a mash concentration of 25%, which resulted in ethanol yield of 85.71%. The optimal conditions for the above yield were found with an enzyme dose of 2.05 ml/kg and pH of 5.0. These results indicate that by using a central composite design, it is possible to determine optimal values of the fermentation parameters for maximum ethanol production. The investigated kinetics model can be used to describe SSF process conducted with granular starch hydrolyzing enzymes. The SEM micrographs reveal randomly distributed holes on the surface of granules.

  16. 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.

  17. Simultaneous saccharification of inulin and starch using commercial glucoamylase and the subsequent bioconversion to high titer sorbitol and gluconic acid.

    PubMed

    An, Kehong; Hu, Fengxian; Bao, Jie

    2013-12-01

    A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.

  18. Enhanced Heat Stability of α-Chymotrypsin through Single-Enzyme Confinement in Attoliter Liposomes.

    PubMed

    Yoshimoto, Makoto; Yamada, Jun; Baba, Misaki; Walde, Peter

    2016-07-01

    The entrapment of α-chymotrypsin (α-CT) within 70-140 nm liposomes formed from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) leads to an unexpected and remarkable increase in the thermal stability of the enzyme. This finding is based on the observation that heating aqueous suspensions of α-CT-containing POPC liposomes to 80 °C for 30 minutes resulted in partial enzyme inactivation, whereas the same treatment of aqueous solutions of free α-CT inactivated the enzyme completely. The stabilizing effect of enzyme confinement in the attoliter volumes of the liposomes was found to increase with decreasing numbers of α-CT molecules per liposome. Single-enzyme confinement was particularly effective, as intermolecular interactions between heat-denatured α-CT molecules (causing irreversible inactivation) are not possible. PMID:27124158

  19. Enhanced enzymatic hydrolysis of langostino shell chitin with mixtures of enzymes from bacterial and fungal sources.

    PubMed

    Donzelli, Bruno G G; Ostroff, Gary; Harman, Gary E

    2003-09-01

    A combination of enzyme preparations from Trichoderma atroviride and Serratia marcescens was able to completely degrade high concentrations (100 g/L) of chitin from langostino crab shells to N-acetylglucosamine (78%), glucosamine (2%), and chitobiose (10%). The result was achieved at 32 degrees C in 12 days with no pre-treatment (size reduction or swelling) of the substrate and without removal of the inhibitory end-products from the mixture. Enzymatic degradation of three forms of chitin by Serratia/Trichoderma and Streptomyces/Trichoderma blends was carried out according to a simplex-lattice mixture design. Fitted polynomial models indicated that there was synergy between prokaryotic and fungal enzymes for both hydrolysis of crab chitin and reduction of turbidity of colloidal chitin (primarily endo-type activity). Prokaryotic/fungal enzymes were not synergistic in degrading chitosan. Enzymes from prokaryotic sources had much lower activity against chitosan than enzymes from T. atroviride.

  20. Optimization of parameters for enhanced oil recovery from enzyme treated wild apricot kernels.

    PubMed

    Rajaram, Mahatre R; Kumbhar, Baburao K; Singh, Anupama; Lohani, Umesh Chandra; Shahi, Navin C

    2012-08-01

    Present investigation was undertaken with the overall objective of optimizing the enzymatic parameters i.e. moisture content during hydrolysis, enzyme concentration, enzyme ratio and incubation period on wild apricot kernel processing for better oil extractability and increased oil recovery. Response surface methodology was adopted in the experimental design. A central composite rotatable design of four variables at five levels was chosen. The parameters and their range for the experiments were moisture content during hydrolysis (20-32%, w.b.), enzyme concentration (12-16% v/w of sample), combination of pectolytic and cellulolytic enzyme i.e. enzyme ratio (30:70-70:30) and incubation period (12-16 h). Aspergillus foetidus and Trichoderma viride was used for production of crude enzyme i.e. pectolytic and cellulolytic enzyme respectively. A complete second order model for increased oil recovery as the function of enzymatic parameters fitted the data well. The best fit model for oil recovery was also developed. The effect of various parameters on increased oil recovery was determined at linear, quadric and interaction level. The increased oil recovery ranged from 0.14 to 2.53%. The corresponding conditions for maximum oil recovery were 23% (w.b.), 15 v/w of the sample, 60:40 (pectolytic:cellulolytic), 13 h. Results of the study indicated that incubation period during enzymatic hydrolysis is the most important factor affecting oil yield followed by enzyme ratio, moisture content and enzyme concentration in the decreasing order. Enzyme ratio, incubation period and moisture content had insignificant effect on oil recovery. Second order model for increased oil recovery as a function of enzymatic hydrolysis parameters predicted the data adequately. PMID:23904657

  1. 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

  2. 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.

  3. 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

  4. Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

    PubMed Central

    2012-01-01

    Background Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites. PMID:23148661

  5. Sodium chloride enhances cadmium tolerance through reducing cadmium accumulation and increasing anti-oxidative enzyme activity in tobacco.

    PubMed

    Zhang, Bing-Lin; Shang, Sheng-Hua; Zhang, Hai-Tao; Jabeen, Zahra; Zhang, Guo-Ping

    2013-06-01

    The effect of sodium chloride (NaCl) on cadmium (Cd) uptake, translocation, and oxidative stress was investigated using 2 tobacco cultivars differing in Cd tolerance. The growth inhibition of the tobacco plants exposed to Cd toxicity was in part alleviated by moderate addition of NaCl in the culture solution. Cadmium concentration of shoots and roots in the 2 cultivars increased with increasing Cd levels in the solution and decreased with the addition of NaCl. The addition of NaCl could alleviate the oxidative stress caused by Cd toxicity, as reflected by reduced production of malondialdehyde and recovered or enhanced activities of antioxidative enzymes catalase and glutathione peroxidase. The results also showed that the enhancement of antioxidative enzyme activity by NaCl for the tobacco plants exposed to Cd stress is related to induced Ca signaling.

  6. 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

  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. 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

  9. Expression of a fungal glucuronoyl esterase in Populus: effects on wood properties and saccharification efficiency.

    PubMed

    Latha Gandla, Madhavi; Derba-Maceluch, Marta; Liu, Xiaokun; Gerber, Lorenz; Master, Emma R; Mellerowicz, Ewa J; Jönsson, Leif J

    2015-04-01

    The secondary walls of angiosperms contain large amounts of glucuronoxylan that is thought to be covalently linked to lignin via ester bonds between 4-O-methyl-α-D-glucuronic acid (4-O-Me-GlcA) moieties in glucuronoxylan and alcohol groups in lignin. This linkage is proposed to be hydrolysed by glucuronoyl esterases (GCEs) secreted by wood-degrading fungi. We report effects of overexpression of a GCE from the white-rot basidiomycete Phanerochaete carnosa, PcGCE, in hybrid aspen (Populus tremula L. x tremuloides Michx.) on the wood composition and the saccharification efficiency. The recombinant enzyme, which was targeted to the plant cell wall using the signal peptide from hybrid aspen cellulase PttCel9B3, was constitutively expressed resulting in the appearance of GCE activity in protein extracts from developing wood. Diffuse reflectance FT-IR spectroscopy and pyrolysis-GC/MS analyses showed significant alternation in wood chemistry of transgenic plants including an increase in lignin content and S/G ratio, and a decrease in carbohydrate content. Sequential wood extractions confirmed a massive (+43%) increase of Klason lignin, which was accompanied by a ca. 5% decrease in cellulose, and ca. 20% decrease in wood extractives. Analysis of the monosaccharide composition using methanolysis showed a reduction of 4-O-Me-GlcA content without a change in Xyl contents in transgenic lines, suggesting that the covalent links between 4-O-Me-GlcA moieties and lignin protect these moieties from degradation. Enzymatic saccharification without pretreatment resulted in significant decreases of the yields of Gal, Glc, Xyl and Man in transgenic lines, consistent with their increased recalcitrance caused by the increased lignin content. In contrast, the enzymatic saccharification after acid pretreatment resulted in Glc yields similar to wild-type despite of their lower cellulose content. These data indicate that whereas PcGCE expression in hybrid aspen increases lignin deposition

  10. An ionically tagged water-soluble artificial enzyme promotes the dephosphorylation reaction with nitroimidazole: enhanced ionic liquid effect and mechanism.

    PubMed

    Ferreira, José G L; Ramos, Luciana M; de Oliveira, Aline L; Orth, Elisa S; Neto, Brenno A D

    2015-06-01

    In this paper, we describe a novel synthesized ionically tagged water-soluble artificial enzyme (PI) that can efficiently cleave phosphate esters, with enhanced an ionic liquid effect through cooperative effects for the substrate activation and further nucleophilic reaction. The dephosphorylation reaction with PI was evaluated in the presence and absence of 2-methyl-4(5)-nitroimidazole, showing impressive rate enhancements of up to 2 × 10(6)-fold, ascribed to the imidazolide species known as excellent nucleophiles, and formed favorably at lower pH values in the presence of PI.

  11. 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

  12. 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

  13. 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.

  14. Enhanced production of polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins

    PubMed Central

    Hayashi, Shohei; Satoh, Yasuharu; Ujihara, Tetsuro; Takata, Yusuke; Dairi, Tohru

    2016-01-01

    In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. These ACPs were previously shown to be important for PUFA productivity. In this study, we examined their function in more detail. PUFA productivities increased depending on the number of ACPs without profile changes in each subunit A of eukaryotic and prokaryotic PUFA synthases. We also constructed derivative enzymes from subunit A with 5 × ACPs. Enzymes possessing one inactive ACP at any position produced ~30% PUFAs compared with the parental enzyme but unexpectedly had ~250% productivity compared with subunit A with 4 × ACPs. Enzymes constructed by replacing the 3rd ACP with an inactive ACP from another subunit A or ACP-unrelated sequences produced ~100% and ~3% PUFAs compared with the parental 3rd ACP-inactive enzyme, respectively. These results suggest that both the structure and number of ACP domains are important for PUFA productivity. PMID:27752094

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. Enhancement of tofu isoflavone recovery by pretreatment of soy milk with koji enzyme extract.

    PubMed

    Wu, Meng-Lung; Chang, Ju-Chun; Lai, Yu-Hsuan; Cheng, Sung-Lang; Chiou, Robin Y-Y

    2004-07-28

    Isoflavones are novel nutraceutical constituents of soybeans, but considerable amounts are lost in the whey during conventional tofu manufacturing. In this study, in a small-scale process, 2 mL of koji enzyme extract (soybean koji/deionized water, 1/3, w/v) was combined with 600 mL of soy milk, and 30 mL aliquots were incubated at 35 degrees C for 0, 30, 60, 120, and 300 min, for enzyme pretreatment. After each treatment time, soy milk was heated to 85 degrees C, CaSO4 was added to aggregate protein, and the mixture was centrifuged to separate the solids (tofu) from the whey. The tofu yield and moisture contents from soy milk treated for 30 or 60 min were higher than those from soy milk treated for 0 (control), 120, or 300 min. The protein content of freeze-dried tofu varied in a limited range, and native PAGE and SDS-PAGE patterns revealed slight quantitative and qualitative variations among products. Soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased as the time of enzyme pretreatment of the soy milk increased. After 30 min of pretreatment, daidzin, genistin, daidzein, and genistein contents recovered in tofu products were higher than those of the control. In a pilot-scale process, aliquots (3 L) of soy milk were enzyme-treated for 30 min, aggregated with CaSO4, and hydraulically pressed to remove the whey. As in pretreatments, soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased. In a comparison of the control and enzyme-treated tofu products, the total recoveries of daidzin, genistin, daidzein, and genistein in the tofu products increased from 54.9% to 64.2%. When the tofu products were subjected to a sensory panel test, both products were judged acceptable.

  3. Continuous SSCF of AFEX™ pretreated corn stover for enhanced ethanol productivity using commercial enzymes and Saccharomyces cerevisiae 424A (LNH-ST).

    PubMed

    Jin, Mingjie; Gunawan, Christa; Balan, Venkatesh; Yu, Xiurong; Dale, Bruce E

    2013-05-01

    High productivity processes are critical for commercial production of cellulosic ethanol. One high productivity process-continuous hydrolysis and fermentation-has been applied in corn ethanol industry. However, little research related to this process has been conducted on cellulosic ethanol production. Here, we report and compare the kinetics of both batch SHF (separate hydrolysis and co-fermentation) and SSCF (simultaneous saccharification and co-fermentation) of AFEX™ (Ammonia Fiber Expansion) pretreated corn stover (AFEX™-CS). Subsequently, we designed a SSCF process to evaluate continuous hydrolysis and fermentation performance on AFEX™-CS in a series of continuous stirred tank reactors (CSTRs). Based on similar sugar to ethanol conversions (around 80% glucose-to-ethanol conversion and 47% xylose-to-ethanol conversion), the overall process ethanol productivity for continuous SSCF was 2.3- and 1.8-fold higher than batch SHF and SSCF, respectively. Slow xylose fermentation and high concentrations of xylose oligomers were the major factors limiting further enhancement of productivity. PMID:23192401

  4. Continuous SSCF of AFEX™ pretreated corn stover for enhanced ethanol productivity using commercial enzymes and Saccharomyces cerevisiae 424A (LNH-ST).

    PubMed

    Jin, Mingjie; Gunawan, Christa; Balan, Venkatesh; Yu, Xiurong; Dale, Bruce E

    2013-05-01

    High productivity processes are critical for commercial production of cellulosic ethanol. One high productivity process-continuous hydrolysis and fermentation-has been applied in corn ethanol industry. However, little research related to this process has been conducted on cellulosic ethanol production. Here, we report and compare the kinetics of both batch SHF (separate hydrolysis and co-fermentation) and SSCF (simultaneous saccharification and co-fermentation) of AFEX™ (Ammonia Fiber Expansion) pretreated corn stover (AFEX™-CS). Subsequently, we designed a SSCF process to evaluate continuous hydrolysis and fermentation performance on AFEX™-CS in a series of continuous stirred tank reactors (CSTRs). Based on similar sugar to ethanol conversions (around 80% glucose-to-ethanol conversion and 47% xylose-to-ethanol conversion), the overall process ethanol productivity for continuous SSCF was 2.3- and 1.8-fold higher than batch SHF and SSCF, respectively. Slow xylose fermentation and high concentrations of xylose oligomers were the major factors limiting further enhancement of productivity.

  5. Effective saccharification of kraft pulp by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae.

    PubMed

    Yamada, Ryosuke; Yoshie, Toshihide; Sakai, Shoji; Wakai, Satoshi; Asai-Nakashima, Nanami; Okazaki, Fumiyoshi; Ogino, Chiaki; Hisada, Hiromoto; Tsutsumi, Hiroko; Hata, Yoji; Kondo, Akihiko

    2015-01-01

    Kraft pulp is a promising feedstock for bioproduction. The efficiency of kraft pulp saccharification was improved by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae. Application of the cellulase cocktail was demonstrated by simultaneous saccharification and fermentation, using kraft pulp and non-cellulolytic yeast. Such application would make possible to do an efficient production of other chemicals from kraft pulp.

  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. 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.

  8. Plasmon-enhanced enzyme-linked immunosorbent assay on large arrays of individual particles made by electron beam lithography.

    PubMed

    Chen, Si; Svedendahl, Mikael; Antosiewicz, Tomasz J; Käll, Mikael

    2013-10-22

    Ultrasensitive biosensing is one of the main driving forces behind the dynamic research field of plasmonics. We have previously demonstrated that the sensitivity of single nanoparticle plasmon spectroscopy can be greatly enhanced by enzymatic amplification of the refractive index footprint of individual protein molecules, so-called plasmon-enhanced enzyme-linked immunosorbent assay (ELISA). The technique, which is based on generation of an optically dense precipitate catalyzed by horseradish peroxidase at the metal surface, allowed for colorimetric analysis of ultralow molecular surface coverages with a limit of detection approaching the single molecule limit. However, the plasmonic response induced by a single enzyme can be expected to vary for a number of reasons, including inhomogeneous broadening of the sensing properties of individual particles, variation in electric field enhancement over the surface of a single particle and variation in size and morphology of the enzymatic precipitate. In this report, we discuss how such inhomogeneities affect the possibility to quantify the number of molecules bound to a single nanoparticle. The discussion is based on simulations and measurements of large arrays of well-separated gold nanoparticles fabricated by electron beam lithography (EBL). The new data confirms the intrinsic single-molecule sensitivity of the technique but we were not able to clearly resolve the exact number of adsorbed molecules per single particle. The results indicate that the main sources of uncertainty come from variations in sensitivity across the surface of individual particles and between different particles. There is also a considerable uncertainty in the actual precipitate morphology produced by individual enzyme molecules. Possible routes toward further improvements of the methodology are discussed.

  9. Parameter estimation for simultaneous saccharification and fermentation of food waste into ethanol using Matlab Simulink.

    PubMed

    Davis, Rebecca Anne

    2008-03-01

    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 degrees 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.

  10. 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.

  11. Production of bio-ethanol from pretreated agricultural byproduct using enzymatic hydrolysis and simultaneous saccharification.

    PubMed

    Gomathi, D; Muthulakshmi, C; Kumar, D Guru; Ravikumar, G; Kalaiselvi, M; Uma, C

    2012-01-01

    Global warming alerts and threats are on the rise due to the utilization of fossil fuels. Alternative fuel sources like bio-ethanol and biodiesel are being produced to combat against these threats. Bio-ethanol can be produced from a range of substrate. The present study is aimed at the Production of bioethanol from pretreated agricultural substrate using enzymatic hydrolysis and simultaneous saccharification with the addition of purified fungal enzyme. Most cellulosic biomass is not fermentable without appropriate pretreatment methods and so dilute sulfuric acid pretreatment was applied to make the cellulose contained in the waste susceptible to endoglucanase enzyme. A range of acid pretreatment of wheat bran was made in which the sample that was pretreated with 1% dilute sulfuric acid gave maximum yield of ethanol in both methods such as 5.83 g L(-1) and 5.27 g L(-1), respectively. Ethanol produced from renewable and cheap agricultural products (wheat bran) provides reduction in green house gas emission, carbon monoxide, sulfur, and helps to eliminate smog from the environment. PMID:22693831

  12. Production of bio-ethanol from pretreated agricultural byproduct using enzymatic hydrolysis and simultaneous saccharification.

    PubMed

    Gomathi, D; Muthulakshmi, C; Kumar, D Guru; Ravikumar, G; Kalaiselvi, M; Uma, C

    2012-01-01

    Global warming alerts and threats are on the rise due to the utilization of fossil fuels. Alternative fuel sources like bio-ethanol and biodiesel are being produced to combat against these threats. Bio-ethanol can be produced from a range of substrate. The present study is aimed at the Production of bioethanol from pretreated agricultural substrate using enzymatic hydrolysis and simultaneous saccharification with the addition of purified fungal enzyme. Most cellulosic biomass is not fermentable without appropriate pretreatment methods and so dilute sulfuric acid pretreatment was applied to make the cellulose contained in the waste susceptible to endoglucanase enzyme. A range of acid pretreatment of wheat bran was made in which the sample that was pretreated with 1% dilute sulfuric acid gave maximum yield of ethanol in both methods such as 5.83 g L(-1) and 5.27 g L(-1), respectively. Ethanol produced from renewable and cheap agricultural products (wheat bran) provides reduction in green house gas emission, carbon monoxide, sulfur, and helps to eliminate smog from the environment.

  13. Efficient pretreatment of Vietnamese rice straw by soda and sulfate cooking methods for enzymatic saccharification.

    PubMed

    Dien, Le Quang; Phuong, Nguyen Thi Minh; Hoa, Doan Thai; Hoang, Phan Huy

    2015-02-01

    This manuscript presents a study on alkaline pretreatment of Vietnamese rice (Oryza sativa L.) straw that grows in Northern Vietnam for enzymatic saccharification. The NaOH pretreatment (soda cooking) and NaOH/Na2S pretreatment (sulfate cooking) were applied for rice straw pretreatment, which have relatively similar condition with industrial pulping processes but at lower temperature. Pretreated biomass solid was then enzymatic hydrolyzed by commercial enzyme Cellic®CTec2 (Novozymes) with enzyme dosage of 35 FPU/g to achieve reducing sugars. The suitable condition for pretreatment was found at temperature of about 100 °C, pretreatment time of 2 h, and solid/liquid ratio of 1:10 with active alkali dosage of 20 % of dry rice straw. Under this pretreatment condition, sugar yield in enzymatic hydrolysis up to 45.33 and 48.92 % over dry rice straw could be obtained after soda cooking and sulfate cooking pretreatment, respectively. Moreover, the changes of components of rice straw after pretreatment were also studied. The crystallinity of cellulose in pretreated biomass solid was calculated from XRD pattern. And the fibril morphology after treatment was revealed by the microscopic observations performed by scanning electron microscope (SEM).

  14. Kinetic modeling of multi-feed simultaneous saccharification and co-fermentation of pretreated birch to ethanol.

    PubMed

    Wang, Ruifei; Koppram, Rakesh; Olsson, Lisbeth; Franzén, Carl Johan

    2014-11-01

    Fed-batch simultaneous saccharification and fermentation (SSF) is a feasible option for bioethanol production from lignocellulosic raw materials at high substrate concentrations. In this work, a segregated kinetic model was developed for simulation of fed-batch simultaneous saccharification and co-fermentation (SSCF) of steam-pretreated birch, using substrate, enzymes and cell feeds. The model takes into account the dynamics of the cellulase-cellulose system and the cell population during SSCF, and the effects of pre-cultivation of yeast cells on fermentation performance. The model was cross-validated against experiments using different feed schemes. It could predict fermentation performance and explain observed differences between measured total yeast cells and dividing cells very well. The reproducibility of the experiments and the cell viability were significantly better in fed-batch than in batch SSCF at 15% and 20% total WIS contents. The model can be used for simulation of fed-batch SSCF and optimization of feed profiles. PMID:25270046

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

    PubMed

    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

    2014-01-14

    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.

  16. 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

  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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Determination of rotavirus serotype-specific antibodies in sera by competitive enhanced enzyme immunoassay.

    PubMed

    Beards, G M; Desselberger, U

    1989-01-01

    A method is described for the specific detection of antibody to individual rotavirus serotypes in sera. A competitive enzyme immunoassay (EIA) was developed in which rotavirus serotype-specific monoclonal antibodies against VP7 compete with antibodies in test sera for rotavirus serotype-specific antigen bound to a solid phase. There was an excellent correlation between serotype-specific EIA results and serotype-specific neutralization titres (r = 0.915, P = less than 0.001). The value of this method for rotavirus epidemiology and vaccine trials is discussed.

  3. 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

  4. Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide.

    PubMed

    Inokuma, Kentaro; Bamba, Takahiro; Ishii, Jun; Ito, Yoichiro; Hasunuma, Tomohisa; Kondo, Akihiko

    2016-11-01

    Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc. PMID:27183011

  5. 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

  6. 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.

  7. Enhancement of PVA-degrading enzyme production by the application of pH control strategy.

    PubMed

    Li, Min; Zhang, Dongxu; Du, Guocheng; Chen, Jian

    2012-02-01

    In batch culture for Poly(vinyl alcohol) (PVA)-degrading enzyme (PVAase) production by a mixed culture, higher pH (pH 7.5) was favorable for PVAase production at the prophase of cultivation, but lower pH (pH 7.0) was favorable at the anaphase. This situation was caused by the fact that the optimum pH for different key enzymes [PVA dehydrogenase (PVADH) and oxidized PVA hydrolase (OPH)] production is various. The activity and average specific production rate of PVADH reached the highest values at constant pH 7.5, whereas those of OPH appeared at pH 7.0. A two-stage pH control strategy was therefore developed and compared for its potential in improving PVAase production. By using this strategy, the maximal PVAase activity reached 2.05 U/ml, which increased by 15.2% and 24.2% over the fermentation at constant pH 7.5 and 7.0.

  8. 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

  9. 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

  10. Probing fundamental film parameters of immobilized enzymes--towards enhanced biosensor performance. Part II-Electroanalytical estimation of immobilized enzyme performance.

    PubMed

    Fogel, R; Limson, J L

    2011-07-10

    The method of immobilization of a protein has a great influence on the overall conformation, and hence, functioning of the protein. Thus, a greater understanding of the events undergone by the protein during immobilization is key to manipulating the immobilization method to produce a strategy that influences the advantages of immobilization while minimizing their disadvantages in biosensor design. In this, the second paper of a two-part series, we have assessed the kinetic parameters of thin-film laccase monolayers, covalently attached to SAMs differing in spacer-arm length and lateral density of spacer arms. This was achieved using chronoamperometry and an electroactive product (p-benzoquinone), which was modeled in a non-linear regressional fashion to extract the relevant parameters. Finally, comparisons between the kinetic parameters presented in this paper and the rheological parameters of laccase monolayers immobilized in the same manner (Part I of this two paper series) were performed. Improvements in the maximal enzyme-catalysed current, i(max), the apparent Michaelis-Menten constant, K(m) and the apparent biosensor sensitivity were noted for most of the surfaces with increasing linker length. Decreasing the lateral density of the spacer-arms brought about a general improvement in these parameters, which is attributed to the decrease in multiple points of immobilization undergone by functional proteins. Finally, comparisons between rheological data and kinetics data showed that the degree of viscosity exhibited by protein films has a negative influence on attached protein layers, while enhanced protein hydration levels (assessed piezoelectrically from data obtained in Paper 1) has a positive effect on those surfaces comprising rigidly bound protein layers.

  11. 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.

  12. 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.

  13. 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

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

    PubMed

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

    2016-02-04

    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.

  15. 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

  16. 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

  17. Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana

    PubMed Central

    2013-01-01

    Background Second-generation biofuels are generally produced from the polysaccharides in the lignocellulosic plant biomass, mainly cellulose. However, because cellulose is embedded in a matrix of other polysaccharides and lignin, its hydrolysis into the fermentable glucose is hampered. The senesced inflorescence stems of a set of 20 Arabidopsis thaliana mutants in 10 different genes of the lignin biosynthetic pathway were analyzed for cell wall composition and saccharification yield. Saccharification models were built to elucidate which cell wall parameters played a role in cell wall recalcitrance. Results Although lignin is a key polymer providing the strength necessary for the plant’s ability to grow upward, a reduction in lignin content down to 64% of the wild-type level in Arabidopsis was tolerated without any obvious growth penalty. In contrast to common perception, we found that a reduction in lignin was not compensated for by an increase in cellulose, but rather by an increase in matrix polysaccharides. In most lignin mutants, the saccharification yield was improved by up to 88% cellulose conversion for the cinnamoyl-coenzyme A reductase1 mutants under pretreatment conditions, whereas the wild-type cellulose conversion only reached 18%. The saccharification models and Pearson correlation matrix revealed that the lignin content was the main factor determining the saccharification yield. However, also lignin composition, matrix polysaccharide content and composition, and, especially, the xylose, galactose, and arabinose contents influenced the saccharification yield. Strikingly, cellulose content did not significantly affect saccharification yield. Conclusions Although the lignin content had the main effect on saccharification, also other cell wall factors could be engineered to potentially increase the cell wall processability, such as the galactose content. Our results contribute to a better understanding of the effect of lignin perturbations on plant cell

  18. Optimization of enzymatic hydrolysis for ethanol production by simultaneous saccharification and fermentation of wastepaper.

    PubMed

    Sangkharak, Kanokphorn

    2011-11-01

    The present study investigated the development of high sugar production by optimization of an enzymatic hydrolysis process using both conventional and statistical methods, as well as the production of ethanol by the selected wastepaper source. Among four sources of pretreated wastepaper including office paper, newspaper, handbills and cardboard, office paper gave the highest values of cellulose (87.12%) and holocelluloses (89.07%). The effects of the amount of wastepaper, the pretreatment method and the type of enzyme on reducing sugar production from office paper were studied using conventional methods. The highest reducing sugar production (1851.28 µg L(-1); 37.03% conversion of glucose) was obtained from the optimal condition containing 40 mg of office paper, pretreated with stream explosion and hydrolysed with the combination of cellulase from Aspergillus niger and Trichoderma viride at the fixed loading rate of 20 FPU g(-1) sample. The effects of interaction of wastepaper amount and enzyme concentration as well as incubation time were studied by a statistical method using central composite design. The optimal medium composition consisted of 43.97 µg L(-1), 28.14 FPU g(-1) sample and 53.73 h of wastepaper, enzyme concentration and incubation time, respectively, and gave the highest amount of sugar production (2184.22 µg L(-1)) and percentage conversion of glucose (43.68%). The ethanol production from pretreated office paper using Saccharomyces cerevisiae in a simultaneous saccharification and fermentation process was 21.02 g L(-1) after 36 h of cultivation, corresponding to an ethanol volumetric production rate of 0.58 g ethanol L(-1) h(-1). PMID:21242181

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

    PubMed

    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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. Uncouplers stimulate photosynthesis in intact chloroplasts by enhancing light-activation of enzymes regulated by the ferredoxin-thioredoxin system.

    PubMed

    Rosa, L; Whatley, F R

    1981-08-01

    Some uncouplers stimulate CO(2)-dependent O(2) 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 H(2)O(2), 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 H(2)O(2). 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications.

    PubMed

    Branchini, Bruce R; Southworth, Tara L; Fontaine, Danielle M; Kohrt, Dawn; Talukder, Munya; Michelini, Elisa; Cevenini, Luca; Roda, Aldo; Grossel, Martha J

    2015-09-01

    Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega's luc2 for reporter and imaging applications.

  16. Cognitive enhancers (nootropics). Part 3: drugs interacting with targets other than receptors or enzymes. disease-modifying drugs.

    PubMed

    Froestl, Wolfgang; Pfeifer, Andrea; Muhs, Andreas

    2013-01-01

    Cognitive enhancers (nootropics) are drugs to treat cognition deficits in patients suffering from Alzheimer's disease, schizophrenia, stroke, attention deficit hyperactivity disorder, or aging. Cognition refers to a capacity for information processing, applying knowledge, and changing preferences. It involves memory, attention, executive functions, perception, language, and psychomotor functions. The term nootropics was coined in 1972 when memory enhancing properties of piracetam were observed in clinical trials. In the meantime, hundreds of drugs have been evaluated in clinical trials or in preclinical experiments. To classify the compounds, a concept is proposed assigning drugs to 19 categories according to their mechanism(s) of action, in particular drugs interacting with receptors, enzymes, ion channels, nerve growth factors, re-uptake transporters, antioxidants, metal chelators, and disease modifying drugs, meaning small molecules, vaccines, and monoclonal antibodies interacting with amyloid-β and tau. For drugs, whose mechanism of action is not known, they are either classified according to structure, e.g., peptides, or their origin, e.g., natural products. The review covers the evolution of research in this field over the last 25 years.

  17. Rice Endosperm Starch Phosphorylase (Pho1) Assembles with Disproportionating Enzyme (Dpe1) to Form a Protein Complex That Enhances Synthesis of Malto-oligosaccharides.

    PubMed

    Hwang, Seon-Kap; Koper, Kaan; Satoh, Hikaru; Okita, Thomas W

    2016-09-16

    Starch synthesis in cereal grain endosperm is dependent on the concerted actions of many enzymes. The starch plastidial phosphorylase (Pho1) plays an important role in the initiation of starch synthesis and in the maturation of starch granule in developing rice seeds. Prior evidence has suggested that the rice enzyme, OsPho1, may have a physical/functional interaction with other starch biosynthetic enzymes. Pulldown experiments showed that OsPho1 as well as OsPho1 devoid of its L80 region, a peptide unique to higher plant phosphorylases, captures disproportionating enzyme (OsDpe1). Interaction of the latter enzyme form with OsDpe1 indicates that the putative regulatory L80 is not responsible for multienzyme assembly. This heterotypic enzyme complex, determined at a molar ratio of 1:1, was validated by reciprocal co-immunoprecipitation studies of native seed proteins and by co-elution chromatographic and co-migration electrophoretic patterns of these enzymes in rice seed extracts. The OsPho1-OsDpe1 complex utilized a broader range of substrates for enhanced synthesis of larger maltooligosaccharides than each individual enzyme and significantly elevated the substrate affinities of OsPho1 at 30 °C. Moreover, the assembly with OsDpe1 enables OsPho1 to utilize products of transglycosylation reactions involving G1 and G3, sugars that it cannot catalyze directly. PMID:27502283

  18. 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

  19. 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

  20. 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

  1. 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

  2. Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

    PubMed

    Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2012-06-01

    We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

  3. 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

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

    2012-09-01

    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.

  4. 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.

  5. Optimization of Xylanase Production through Response Surface Methodology by Fusarium sp. BVKT R2 Isolated from Forest Soil and Its Application in Saccharification

    PubMed Central

    Ramanjaneyulu, Golla; Rajasekhar Reddy, Bontha

    2016-01-01

    Xylanses are hydrolytic enzymes with wide applications in several industries like biofuels, paper and pulp, deinking, food, and feed. The present study was aimed at hitting at high yield xylanase producing fungi from natural resources. Two highest xylanase producing fungal isolates—Q12 and L1 were picked from collection of 450 fungal cultures for the utilization of xylan. These fungal isolates—Q12 and L1 were identified basing on ITS gene sequencing analysis as Fusarium sp. BVKT R2 (KT119615) and Fusarium strain BRR R6 (KT119619), respectively with construction of phylogenetic trees. Fusarium sp. BVKT R2 was further optimized for maximum xylanase production and the interaction effects between variables on production of xylanase were studied through response surface methodology. The optimal conditions for maximal production of xylanase were sorbitol 1.5%, yeast extract 1.5%, pH of 5.0, Temperature of 32.5°C, and agitation of 175 rpm. Under optimal conditions, the yields of xylanase production by Fusarium sp. BVKT R2 was as high as 4560 U/ml in SmF. Incubation of different lignocellulosic biomasses with crude enzyme of Fusarium sp. BVKT R2 at 37°C for 72 h could achieve about 45% saccharification. The results suggest that Fusarium sp. BVKT R2 has potential applications in saccharification process of biomass. PMID:27713726

  6. Hydrothermal pretreatment conditions to enhance ethanol production from poplar biomass.

    PubMed

    Negro, Maria José; Manzanares, Paloma; Ballesteros, Ignacio; Oliva, Jose Miguel; Cabañas, Araceli; Ballesteros, Mercedes

    2003-01-01

    Pretreatment has been recognized as a key step in enzyme-based conversion processes of lignocellulose biomass to ethanol. The aim of this study is to evaluate two hydrothermal pretreatments (steam explosion and liquid hot water) to enhance ethanol production from poplar (Populus nigra) biomass by a simultaneous saccharification and fermentation (SSF) process. The composition of liquid and solid fractions obtained after pretreatment, enzymatic digestibility, and ethanol production of poplar biomass pretreated at different experimental conditions was analyzed. The best results were obtained in steam explosion pretreatment at 210 C and 4 min, taking into account cellulose recovery above 95%, enzymatic hydrolysis yield of about 60%, SSF yield of 60% of theoretical, and 41% xylose recovery in the liquid fraction. Large particles can be used for poplar biomass in both pretreatments, since no significant effect of particle size on enzymatic hydrolysis and SSF was obtained.

  7. Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose

    PubMed Central

    2013-01-01

    Background Plant expansins and fungal swollenin that can disrupt crystalline cellulose have great potential for applications in conversion of biomass. Recent studies have been mainly focused on Trichoderma reesei swollenin that show relatively low activity in the promotion of cellulosic hydrolysis. Our aim was to isolate a novel swollenin with greater disruptive activity, to establish an efficient way of producing recombinant swollenin, and to optimize the procedure using swollenin in facilitation of cellulosic hydrolysis. Results A novel gene encoding a swollenin-like protein, POSWOI, was isolated from the filamentous fungus Penicillium oxalicum by Thermal Asymmetric Interlaced PCR (TAIL-PCR). It consisted of a family 1 carbohydrate-binding module (CBM1) followed by a linker connected to a family 45 endoglucanase-like domain. Using the cellobiohydrolase I promoter, recombinant POSWOI was efficiently produced in T. reesei with a yield of 105 mg/L, and showed significant disruptive activity on crystalline cellulose. Simultaneous reaction with both POSWOI and cellulases enhanced the hydrolysis of crystalline cellulose Avicel by approximately 50%. Using a POSWOI-pretreatment procedure, cellulases can produce nearly twice as many reducing sugars as without pretreatment. The mechanism by which POSWOI facilitates the saccharification of cellulose was also studied using a cellulase binding assay. Conclusion We present a novel fungal swollenin with considerable disruptive activity on crystalline cellulose, and develop a better procedure for using swollenin in facilitating cellulosic hydrolysis. We thus provide a new approach for the effective bioconversion of cellulosic biomass. PMID:23688024

  8. Phenyllactic acid production by simultaneous saccharification and fermentation of pretreated sorghum bagasse.

    PubMed

    Kawaguchi, Hideo; Teramura, Hiroshi; Uematsu, Kouji; Hara, Kiyotaka Y; Hasunuma, Tomohisa; Hirano, Ko; Sazuka, Takashi; Kitano, Hidemi; Tsuge, Yota; Kahar, Prihardi; Niimi-Nakamura, Satoko; Oinuma, Ken-ichi; Takaya, Naoki; Kasuga, Shigemitsu; Ogino, Chiaki; Kondo, Akihiko

    2015-04-01

    Dilute acid-pretreated sorghum bagasse, which was predominantly composed of glucan (59%) and xylose (7.2%), was used as a lignocellulosic feedstock for d-phenyllactic acid (PhLA) production by a recombinant Escherichia coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens. During fermentation with enzymatic hydrolysate of sorghum bagasse as a carbon source, the PhLA yield was reduced by 35% compared to filter paper hydrolysate, and metabolomics analysis revealed that NAD(P)H regeneration and intracellular levels of erythrose-4-phosphate and phosphoenolpyruvate for PhLA biosynthesis markedly reduced. Compared to separate hydrolysis and fermentation (SHF) with sorghum bagasse hydrolysate, simultaneous saccharification and fermentation (SSF) of sorghum bagasse under glucose limitation conditions yielded 4.8-fold more PhLA with less accumulation of eluted components, including p-coumaric acid and aldehydes, which inhibited PhLA fermentation. These results suggest that gradual enzymatic hydrolysis during SSF enhances PhLA production under glucose limitation and reduces the accumulation of fermentation inhibitors, collectively leading to increased PhLA yield.

  9. Enhanced Gene Detection Assays for Fumarate-Adding Enzymes Allow Uncovering of Anaerobic Hydrocarbon Degraders in Terrestrial and Marine Systems

    PubMed Central

    von Netzer, Frederick; Pilloni, Giovanni; Kleindienst, Sara; Krüger, Martin; Knittel, Katrin; Gründger, Friederike

    2013-01-01

    The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. Fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) have already been established as specific functional marker genes for anaerobic hydrocarbon degraders. Several recent studies based on pure cultures and laboratory enrichments have shown the existence of new and deeply branching FAE gene lineages, such as clostridial benzylsuccinate synthases and homologues, as well as naphthylmethylsuccinate synthases. However, established FAE gene detection assays were not designed to target these novel lineages, and consequently, their detectability in different environments remains obscure. Here, we present a new suite of parallel primer sets for detecting the comprehensive range of FAE markers known to date, including clostridial benzylsuccinate, naphthylmethylsuccinate, and alkylsuccinate synthases. It was not possible to develop one single assay spanning the complete diversity of FAE genes alone. The enhanced assays were tested with a range of hydrocarbon-degrading pure cultures, enrichments, and environmental samples of marine and terrestrial origin. They revealed the presence of several, partially unexpected FAE gene lineages not detected in these environments before: distinct deltaproteobacterial and also clostridial bssA homologues as well as environmental nmsA homologues. These findings were backed up by dual-digest terminal restriction fragment length polymorphism diagnostics to identify FAE gene populations independently of sequencing. This allows rapid insights into intrinsic degrader populations and degradation potentials established in aromatic and aliphatic hydrocarbon-impacted environmental systems. PMID:23124238

  10. Enhanced production of thrombinase by Streptomyces venezuelae: kinetic studies on growth and enzyme production of mutant strain.

    PubMed

    Naveena, Balakrishnan; Gopinath, Kannapan Panchamoorthy; Sakthiselvan, Punniavan; Partha, Nagarajan

    2012-05-01

    This investigation provides the enhanced production of thrombinase, a fibrinolytic enzyme using mutant Streptomyces venezuelae. Initially the mutagenesis of the marine isolate was done by UV and Ethyl methane sulfonate (EMS) and their mutational efficiencies were compared. The mutants were selected based on their high thrombinase activity and used for further studies. The mutant was found to be more halo and thermo tolerant comparing to wild. The effect of Dissolved oxygen level was also determined and the mutant offered the maximum specific growth rate as 0.2404 (h(-1)). The mutant showed high resistance to higher initial lactose concentration and the inhibition concentration was found to be 155.1mg/mL. The effect of S(0)/X(0) ratio on specific substrate consumption and production rate were also investigated. Both mutant and wild showed increase in specific substrate consumption and production rate at higher S(0)/X(0) ratio but the mutant showed better values than the wild strain. PMID:22401714

  11. 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

  12. Quantitative characterization of the impact of pulp refining on enzymatic saccharification of the alkaline pretreated corn stover.

    PubMed

    Xu, Huanfei; Li, Bin; Mu, Xindong; Yu, Guang; Liu, Chao; Zhang, Yuedong; Wang, Haisong

    2014-10-01

    In this work, corn stover was refined by a pulp refining instrument (PFI refiner) after NaOH pretreatment under varied conditions. The quantitative characterization of the influence of PFI refining on enzymatic hydrolysis was studied, and it was proved that the enhancement of enzymatic saccharification by PFI refining of the pretreated corn stover was largely due to the significant increment of porosity of substrates and the reduction of cellulose crystallinity. Furthermore, a linear relationship between beating degree and final total sugar yields was found, and a simple way to predict the final total sugar yields by easily testing the beating degree of PFI refined corn stover was established. Therefore, this paper provided the possibility and feasibility for easily monitoring the fermentable sugar production by the simple test of beating degree, and this will be of significant importance for the monitoring and controlling of industrial production in the future.

  13. Simultaneous saccharification and co-fermentation of aqueous ammonia pretreated corn stover with an engineered Saccharomyces cerevisiae SyBE005.

    PubMed

    Zhu, Jia-Qing; Qin, Lei; Li, Bing-Zhi; Yuan, Ying-Jin

    2014-10-01

    Co-fermentation of glucose and xylose from lignocelluloses is an efficient approach to increasing ethanol production. Simultaneous saccharification and co-fermentation (SSCF) of corn stover pretreated with aqueous ammonia was performed using engineered yeast with xylose utilization pathway. Thus far, the effect of the several key factors on SSCF was investigated, including temperature, inoculation size, pre-hydrolysis and pH. Ethanol concentration was achieved to 36.5 g/L during SSCF process with 6% glucan loading. The addition of Tween 20 reduced enzyme loading, i.e., from 15 to 7.5 FPU/gglucan with the same final ethanol concentration. The ethanol concentration was achieved to 70.1g/L at 12% glucan loading. Yeast feeding, combined with substrate and enzyme feeding, was proved to be an efficient approach for SSCF with high solid loading. PMID:25016219

  14. 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

  15. 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

  16. The Histone Methyltransferase Enzyme Enhancer of Zeste Homolog 2 Protects against Podocyte Oxidative Stress and Renal Injury in Diabetes.

    PubMed

    Siddiqi, Ferhan S; Majumder, Syamantak; Thai, Kerri; Abdalla, Moustafa; Hu, Pingzhao; Advani, Suzanne L; White, Kathryn E; Bowskill, Bridgit B; Guarna, Giuliana; Dos Santos, Claudia C; Connelly, Kim A; Advani, Andrew

    2016-07-01

    Epigenetic regulation of oxidative stress is emerging as a critical mediator of diabetic nephropathy. In diabetes, oxidative damage occurs when there is an imbalance between reactive oxygen species generation and enzymatic antioxidant repair. Here, we investigated the function of the histone methyltransferase enzyme enhancer of zeste homolog 2 (EZH2) in attenuating oxidative injury in podocytes, focusing on its regulation of the endogenous antioxidant inhibitor thioredoxin interacting protein (TxnIP). Pharmacologic or genetic depletion of EZH2 augmented TxnIP expression and oxidative stress in podocytes cultured under high-glucose conditions. Conversely, EZH2 upregulation through inhibition of its regulatory microRNA, microRNA-101, downregulated TxnIP and attenuated oxidative stress. In diabetic rats, depletion of EZH2 decreased histone 3 lysine 27 trimethylation (H3K27me3), increased glomerular TxnIP expression, induced podocyte injury, and augmented oxidative stress and proteinuria. Chromatin immunoprecipitation sequencing revealed H3K27me3 enrichment at the promoter of the transcription factor Pax6, which was upregulated on EZH2 depletion and bound to the TxnIP promoter, controlling expression of its gene product. In high glucose-exposed podocytes and the kidneys of diabetic rats, the lower EZH2 expression detected coincided with upregulation of Pax6 and TxnIP. Finally, in a gene expression array, TxnIP was among seven of 30,854 genes upregulated by high glucose, EZH2 depletion, and the combination thereof. Thus, EZH2 represses the transcription factor Pax6, which controls expression of the antioxidant inhibitor TxnIP, and in diabetes, downregulation of EZH2 promotes oxidative stress. These findings expand the extent to which epigenetic processes affect the diabetic kidney to include antioxidant repair.

  17. Simultaneous saccharification and cofermentation of peracetic acid-pretreated biomass.

    PubMed

    Teixeira, L C; Linden, J C; Schroeder, H A

    2000-01-01

    Previous work in our laboratories has demonstrated the effectiveness of peracetic acid for improving enzymatic digestibility of lignocellulosic materials. The use of dilute alkali solutions as a pre-pretreatment prior to peracetic acid lignin oxidation increased carbohydrate hydrolysis yields in a synergistic as opposed to additive manner. Deacetylation of xylan is easily achieved using dilute alkali solutions under mild conditions. In this article, we evaluate the effectiveness of peracetic acid combined with an alkaline pre-pretreatment through simultaneous saccharification and cofermentation (SSCF) of pretreated hybrid poplar wood and sugar cane bagasse. Respective ethanol yields of 92.8 and 91.9% of theoretical are achieved using 6% NaOH/15% peracetic acid-pretreated substrates and recombinant Zymomonas mobilis CP4/pZB5. Reduction of acetyl groups of the lignocellulosic materials is demonstrated following alkaline pre-pretreatments. Such processing may be helpful in reducing peracetic acid requirements. The influence of deacetylation is more significant in combined pretreatments using lower peracetic acid loadings.

  18. 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...

  19. 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

  20. Α-L-arabinofuranosidase 3 from Penicillium purpurogenum (ABF3): potential application in the enhancement of wine flavour and heterologous expression of the enzyme.

    PubMed

    Ravanal, Maria Cristina; Rosa, Lorena; Eyzaguirre, Jaime

    2012-09-15

    An α-l-arabinofuranosidase (ABF3) from Penicillium purpurogenum was purified and its possible biotechnological application in the enhancement of wine flavour combined with P. purpurogenum β-glucosidase was studied. A must from Muscat of Alexandria was used to isolate the glycosides. The total monosaccharide (glucose, arabinose and xylose) levels in the glycosides were determined after acid hydrolysis, and were compared with the result of enzymatic hydrolysis. These results were analogous to those obtained in similar experiments using a commercial preparation, thus suggesting that the enzyme from P. purpurogenum may prove useful in this particular application. This prompted us to express the enzyme heterologously. The abf3 gene was thus expressed in Pichia pastoris. The recombinant enzyme was purified and it shows the same properties of the native ABF3 (substrate specificity, kinetic constants, pH and temperature optima and antibody cross-reactivity).

  1. A Chaperone Enhances Blood α-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy

    PubMed Central

    Parenti, Giancarlo; Fecarotta, Simona; la Marca, Giancarlo; Rossi, Barbara; Ascione, Serena; Donati, Maria Alice; Morandi, Lucia Ovidia; Ravaglia, Sabrina; Pichiecchio, Anna; Ombrone, Daniela; Sacchini, Michele; Pasanisi, Maria Barbara; De Filippi, Paola; Danesino, Cesare; Della Casa, Roberto; Romano, Alfonso; Mollica, Carmine; Rosa, Margherita; Agovino, Teresa; Nusco, Edoardo; Porto, Caterina; Andria, Generoso

    2014-01-01

    Enzyme replacement therapy is currently the only approved treatment for Pompe disease, due to acid α-glucosidase deficiency. Clinical efficacy of this approach is variable, and more effective therapies are needed. We showed in preclinical studies that chaperones stabilize the recombinant enzyme used for enzyme replacement therapy. Here, we evaluated the effects of a combination of enzyme therapy and a chaperone on α-glucosidase activity in Pompe disease patients. α-Glucosidase activity was analyzed by tandem-mass spectrometry in dried blood spots from patients treated with enzyme replacement therapy, either alone or in combination with the chaperone N-butyldeoxynojirimycin given at the time of the enzyme infusion. Thirteen patients with different presentations (3 infantile-onset, 10 late-onset) were enrolled. In 11 patients, the combination treatment resulted in α-glucosidase activities greater than 1.85-fold the activities with enzyme replacement therapy alone. In the whole patient population, α-glucosidase activity was significantly increased at 12 hours (2.19-fold, P = 0.002), 24 hours (6.07-fold, P = 0.001), and 36 hours (3.95-fold, P = 0.003). The areas under the curve were also significantly increased (6.78-fold, P = 0.002). These results suggest improved stability of recombinant α-glucosidase in blood in the presence of the chaperone. PMID:25052852

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Flavonoid rich fraction of Dioscorea bulbifera Linn. (Yam) enhances mitochondrial enzymes and antioxidant status and thereby protects heart from isoproterenol induced myocardial infarction.

    PubMed

    Jayachandran, K S; Vasanthi, Hannah R; Rajamanickama, G V

    2010-12-01

    With recent advances in nutrition sciences, natural products and health-promoting foods have received extensive attention from both health professionals and the common population. The flavonoid rich fraction (FRF) of Dioscorea bulbifera Linn. has a strong free radical scavenging activity. FRF (150 mg/kg) when intervened for a period of 35 days prior to isoproterenol (ISO) challenge to rats maintained the creatine kinase - MB (CK-MB) activity in serum without elevation. Alterations in the antioxidant status in the mitochondria were recognized in the heart tissue of ISO induced rats. ISO induced rats pretreated with FRF (150 mg/kg) ameliorated the lipid peroxidation and thereby enhanced the antioxidant status as evidenced by the increase in the reduced glutathione (GSH) content and the activity of antioxidant enzymes. Moreover, the tricarboxylic acid cycle enzymes such isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and α-ketoglutarate dehydrogenase (α-KGDH), which were found decreased in the ISO induced rats showed an enhanced activity in FRF (150 mg/kg) pretreated rats. The activity of NADH dehydrogenase and cytochrome-C-oxidase the enzymes, which transfer the electron in the electron transport chain (ETC) was also increased significantly (p<0.05) in FRF (150 mg/kg) pretreated rats, when compared with ISO induced rats. These results suggest the cardioprotective effect of FRF of Dioscorea bulbifera Linn. in ISO induced MI by attenuating the lipid peroxidation by scavenging free radicals and modulating the energy producing mitochondrial enzymes. PMID:20874686

  8. The enhancement of phase 2 enzyme activities by sodium butyrate in normal intestinal epithelial cells is associated with Nrf2 and p53.

    PubMed

    Yaku, Keisuke; Enami, Yuka; Kurajyo, Chika; Matsui-Yuasa, Isao; Konishi, Yotaro; Kojima-Yuasa, Akiko

    2012-11-01

    Dietary fiber fermentation by the colonic bacterial flora produces short-chain fatty acids, acetate, propionate and butyrate. Among them, butyrate is considered to be the major energy substrate for colonocytes and, at least in rats, seems to protect against colonic carcinogenesis. In this study, we examined the effect and the mechanisms of short-chain fatty acids on the activity of phase 2 enzymes. Sodium butyrate increased phase 2 enzyme activities in normal rat small intestine epithelial cells, Glutathione S-transferase and NAD(P)H:quinone oxidoreductase (NQO) in a dose-dependent manner(;) however, other short-chain fatty acids did not increase them. The mechanism of the induction of phase 2 enzymes with sodium butyrate sodium butyrate, but not other short-chain fatty acids was related to the increase of NF-E2-related factor 2 (Nrf2) nuclear translocation and the decrease in the levels of nuclear fraction p53. Sodium butyrate also caused enhancement of Nrf2 mRNA levels and suppression of p53 mRNA levels. Sodium butyrate enhances the activities of phase 2 enzymes via an increase in the Nrf2 protein levels in the nucleus and a decrease in the mRNA and protein levels of p53.

  9. 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

  10. 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

  11. 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

  12. 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

  13. Effective approach to greatly enhancing selective secretion and expression of three cytoplasmic enzymes in Escherichia coli through synergistic effect of EDTA and lysozyme.

    PubMed

    Liu, Sen-Lin; Du, Kun; Chen, Wei-Zhao; Liu, Gang; Xing, Miao

    2012-09-01

    An effective approach to greatly enhancing the selective secretion and expression of recombinant cytoplasmic enzymes in Escherichia coli was successfully developed through the synergistic effect of ethylenediaminetetraacetate (EDTA) and lysozyme. The method was applied to two endoglucanases (EGs) and an amylase. The optimal culture conditions of temperature and concentration of isopropyl-β-D: -1-thiogalactopyranoside (IPTG) were 23-30 °C and 0.2 mM, respectively, under which the three enzymes could be expressed in active form. Among all the chemicals tested, EDTA was found to be most suitable for enhancing the secretion of EG-I-1A into the medium. Addition of lysozyme alone had little influence on the secretion and expression. In contrast, on the basis of the addition of 5 g EDTA/L at the induction time of 12 h, the simultaneous addition of 0.15 g lysozyme/L further significantly increased the secretion and expression of the three enzymes, demonstrating the synergistic effect of EDTA and lysozyme. The production of EG-I-1A in the culture medium by adding 5 g EDTA/L and 0.15 g lysozyme/L under the optimal culture conditions of 23 °C and 0.2 mM IPTG was over 260-fold higher than that without EDTA and lysozyme under the standard conditions of 37 °C and 1 mM IPTG. In summary, the advantage of this novel cultivation approach for secretion was that not only did it selectively enhance the secretion of the proteins of interest, but also greatly increased the expression of the three enzymes by over 80 %.

  14. Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method.

    PubMed

    Lin, C T; Lin, M T; Chen, Y T; Shaw, J F

    1995-05-01

    The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 degrees C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant kd calculated for the monomer at 85 degrees C was 0.029 min-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 degrees C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 degrees C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.

  15. 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

  16. 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.

  17. 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

  18. 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...

  19. 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...

  20. 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.

  1. 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...

  2. Hydrothermal pretreatment and enzymatic saccharification of corn stover for efficient ethanol production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corn stover used in this study contained 37.0±0.4% cellulose, 31.3±0.6% hemicellulose and 17.8±0.2% lignin on dry basis. Hydrothermal pretreatment and enzymatic saccharification were evaluated for conversion of corn stover cellulose and hemicellulose to fermentable sugars. Under the optimum condit...

  3. Enzyme-assisted extraction enhancing the phenolic release from cauliflower (Brassica oleracea L. var. botrytis) outer leaves.

    PubMed

    Huynh, Nguyen Thai; Smagghe, Guy; Gonzales, Gerard Bryan; Van Camp, John; Raes, Katleen

    2014-07-30

    Phenolic compounds are highly present in byproducts from the cauliflower (Brassica oleracea L. var. botrytis) harvest and are thus a valuable source for valorization toward phenolic-rich extracts. In this study, we aimed to optimize and characterize the release of individual phenolic compounds from outer leaves of cauliflower, using two commercially available polysaccharide-degrading enzymes, Viscozyme L and Rapidase. As major results, the optimal conditions for the enzyme treatment were: enzyme/substrate ratio of 0.2% for Viscozyme L and 0.5% for Rapidase, temperature 35 °C, and pH 4.0. Using a UPLC-HD-TOF-MS setup, the main phenolic compounds in the extracts were identified as kaempferol glycosides and their combinations with different hydroxycinnamic acids. The most abundant components were kaempferol-3-feruloyldiglucoside and kaempferol-3-glucoside (respectively, 37.8 and 58.4 mg rutin equiv/100 g dry weight). Incubation of the cauliflower outer leaves with the enzyme mixtures resulted in a significantly higher extraction yield of kaempferol-glucosides as compared to the control treatment.

  4. 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.

  5. Enhanced architecture of lipid-carbon nanotubes as langmuir-blodgett films to investigate the enzyme activity of phospholipases from snake venom.

    PubMed

    Caseli, Luciano; Tiburcio, Vera L B; Vargas, Frey F R; Marangoni, Sérgio; Siqueira, José R

    2012-11-15

    The immobilization of biomolecules in films with a controlled architecture permits the access of information on the molecular interactions, not only between film components, but also between the film and substances in the external environment. In this study, we investigated the immobilization of the phospholipase A(2) from snake venoms (4-nitro-3-(octanoyloxy)benzoic acid, OBZ) in solid supports as a Langmuir-Blodgett (LB) film, followed by incorporation of carbon nanotubes (CNTs). The hybrid film was characterized by infrared spectroscopy and the interactions with its catalytic substrate were investigated. The presence of CNTs leads to a structure with an adequate environment to preserve the enzyme properties, leading to an optimum catalytic activity. This enhanced architecture was exploited in terms of vibrational spectroscopy, which indicated changes in the secondary structure of the enzyme upon contact with the catalytic substrate.

  6. Optimized simultaneous saccharification and co-fermentation of rice straw for ethanol production by Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture using design of experiments.

    PubMed

    Suriyachai, Nopparat; Weerasaia, Khatiya; Laosiripojana, Navadol; Champreda, Verawat; Unrean, Pornkamol

    2013-08-01

    Herein an ethanol production process from rice straw was optimized. Simultaneous saccharification and co-fermentation (SSCF) using Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture was carried out to enhance ethanol production. The optimal saccharification solid loading was 5%. Key fermentation parameters for co-culture including cell ratio, agitation rate and temperature was rationally optimized using design of experiment (DoE). Optimized co-culture conditions for maximum ethanol production efficiency were at S. cerevisiae:S. stipitis cell ratio of 0.31, agitation rate of 116 rpm and temperature of 33.1°C. The optimized SSCF process reached ethanol titer of 15.2g/L and ethanol yield of 99% of theoretical yield, consistent with the DoE model prediction. Moreover, SSCF process under high biomass concentration resulted in high ethanol concentration of 28.6g/L. This work suggests the efficiency and scalability of the developed SSCF process which could provide an important basis for the economic feasibility of ethanol production from lignocelluloses. PMID:23735799

  7. Optimized simultaneous saccharification and co-fermentation of rice straw for ethanol production by Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture using design of experiments.

    PubMed

    Suriyachai, Nopparat; Weerasaia, Khatiya; Laosiripojana, Navadol; Champreda, Verawat; Unrean, Pornkamol

    2013-08-01

    Herein an ethanol production process from rice straw was optimized. Simultaneous saccharification and co-fermentation (SSCF) using Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture was carried out to enhance ethanol production. The optimal saccharification solid loading was 5%. Key fermentation parameters for co-culture including cell ratio, agitation rate and temperature was rationally optimized using design of experiment (DoE). Optimized co-culture conditions for maximum ethanol production efficiency were at S. cerevisiae:S. stipitis cell ratio of 0.31, agitation rate of 116 rpm and temperature of 33.1°C. The optimized SSCF process reached ethanol titer of 15.2g/L and ethanol yield of 99% of theoretical yield, consistent with the DoE model prediction. Moreover, SSCF process under high biomass concentration resulted in high ethanol concentration of 28.6g/L. This work suggests the efficiency and scalability of the developed SSCF process which could provide an important basis for the economic feasibility of ethanol production from lignocelluloses.

  8. 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.

  9. Production of L- and D-lactic acid from waste Curcuma longa biomass through simultaneous saccharification and cofermentation.

    PubMed

    Nguyen, Cuong Mai; Kim, Jin-Seog; Nguyen, Thanh Ngoc; Kim, Seul Ki; Choi, Gyung Ja; Choi, Yong Ho; Jang, Kyoung Soo; Kim, Jin-Cheol

    2013-10-01

    Simultaneous saccharification and cofermentation (SSCF) of Curcuma longa waste biomass obtained after turmeric extraction to L- and D-lactic acid by Lactobacillus coryniformis and Lactobacillus paracasei, respectively, was investigated. This is a rich, starchy, agro-industrial waste with potential for use in industrial applications. After optimizing the fermentation of the biomass by adjusting nitrogen sources, enzyme compositions, nitrogen concentrations, and raw material concentrations, the SSCF process was conducted in a 7-l jar fermentor at 140 g dried material/L. The maximum lactic acid concentration, average productivity, reducing sugar conversion and lactic acid yield were 97.13 g/L, 2.7 g/L/h, 95.99% and 69.38 g/100 g dried material for L-lactic acid production, respectively and 91.61 g/L, 2.08 g/L/h, 90.53% and 65.43 g/100 g dried material for D-lactic acid production, respectively. The simple and efficient process described in this study could be utilized by C. longa residue-based lactic acid industries without requiring the alteration of plant equipment.

  10. 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

  11. Post-anthesis alternate wetting and moderate soil drying enhances activities of key enzymes in sucrose-to-starch conversion in inferior spikelets of rice.

    PubMed

    Zhang, Hao; Li, Hongwei; Yuan, Liming; Wang, Zhiqin; Yang, Jianchang; Zhang, Jianhua

    2012-01-01

    This study tested the hypothesis that a post-anthesis moderate soil drying can improve grain filling through regulating the key enzymes in the sucrose-to-starch pathway in the grains of rice (Oryza sativa L.). Two rice cultivars were field grown and two irrigation regimes, alternate wetting and moderate soil drying (WMD) and conventional irrigation (CI, continuously flooded), were imposed during the grain-filling period. The grain-filling rate and activities of four key enzymes in sucrose-to-starch conversion, sucrose synthase (SuSase), adenosine diphosphate-glucose pyrophosphorylase (AGPase), starch synthase (StSase), and starch branching enzyme (SBE), showed no significant difference between WMD and CI regimes for the earlier flowering superior spikelets. However, they were significantly enhanced by the WMD for the later flowering inferior spikelets. The activities of both soluble and insoluble acid invertase in the grains were little affected by the WMD. The two cultivars showed the same tendencies. The activities of SuSase, AGPase, StSase, and SBE in grains were very significantly correlated with the grain-filling rate. The abscisic acid (ABA) concentration in inferior spikelets was remarkably increased in the WMD and very significantly correlated with activities of SuSase, AGPase, StSase, and SBE. Application of ABA on plants under CI produced similar results to those seen in plants receiving WMD. Applying fluridone, an indirect inhibitor of ABA synthesis, produced the opposite effect. The results suggest that post-anthesis WMD could enhance sink strength by regulating the key enzymes involved, and consequently, increase the grain-filling rate and grain weight of inferior spikelets. ABA plays an important role in this process.

  12. A joint analysis of transcriptomic and metabolomic data uncovers enhanced enzyme-metabolite coupling in breast cancer.

    PubMed

    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

  13. 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

  14. 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.

  15. 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 ...

  16. 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

  17. 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.

  18. 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

  19. 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

    2016-01-01

    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

  20. 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

  1. 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.

  2. 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

  3. Enhanced expression of an endoglucanase in Bacillus subtilis by using the sucrose-inducible sacB promoter and improved properties of the recombinant enzyme.

    PubMed

    Liu, Sen-Lin; Du, Kun

    2012-06-01

    An endoglucanase from Bacillus akibai I-1 was successfully overexpressed in Bacillus subtilis 168 and the expression level of the recombinant enzyme was greatly enhanced by using the sucrose-inducible sacB promoter. The endoglucanase activity in the culture supernatant of recombinant B. subtilis by using itself promoter (HpaII) in plasmid pMA5 was 3U/ml. Interestingly, with the addition of sacB promoter at downstream from the HpaII promoter or the replacement of HpaII promoter by the sacB promoter, the endoglucanase activities reached 62 and 60U/ml, respectively, under the optimal culture conditions. These results demonstrated that the sacB promoter might be more efficient for the expression of the endoglucanase than the HpaII promoter. More interestingly, the purified native enzyme had broad pH stability, good thermostability and resistibility to various metal ions and chelating agents examined, while the recombinant enzyme had improved resistibility to SDS, which was stable in 0.2% (w/v) laundry detergent and thus showed great potential in detergents industry.

  4. Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome.

    PubMed

    Davidi, Lital; Moraïs, Sarah; Artzi, Lior; Knop, Doriv; Hadar, Yitzhak; Arfi, Yonathan; Bayer, Edward A

    2016-09-27

    Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymatic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addition of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amount of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degradation of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into soluble sugars en route to alternative fuels production. PMID:27621442

  5. Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome.

    PubMed

    Davidi, Lital; Moraïs, Sarah; Artzi, Lior; Knop, Doriv; Hadar, Yitzhak; Arfi, Yonathan; Bayer, Edward A

    2016-09-27

    Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymatic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addition of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amount of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degradation of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into soluble sugars en route to alternative fuels production.

  6. Changes in plant cell-wall structure of corn stover due to hot compressed water pretreatment and enhanced enzymatic hydrolysis.

    PubMed

    Zhou, Wei; Yang, Maohua; Wang, Caixia; Liu, Jianfei; Xing, Jianmin

    2014-08-01

    Corn stover is a potential feedstock for biofuel production. This work investigated physical and chemical changes in plant cell-wall structure of corn stover due to hot compressed water (HCW) pretreatment at 170-190 °C in a tube reactor. Chemical composition analysis showed the soluble hemicellulose content increased with pretreatment temperature, whereas the hemicellulose content decreased from 29 to 7 % in pretreated solids. Scanning electron microscopy revealed the parenchyma-type second cell-wall structure of the plant was almost completely removed at 185 °C, and the sclerenchyma-type second cell wall was greatly damaged upon addition of 5 mmol/L ammonium sulfate during HCW pretreatment. These changes favored accessibility for enzymatic action. Enzyme saccharification of solids by optimized pretreatment with HCW at 185 °C resulted in an enzymatic hydrolysis yield of 87 %, an enhancement of 77 % compared to the yield from untreated corn stover.

  7. An enhanced in vivo stable isotope labeling by amino acids in cell culture (SILAC) model for quantification of drug metabolism enzymes.

    PubMed

    MacLeod, A Kenneth; Fallon, Padraic G; Sharp, Sheila; Henderson, Colin J; Wolf, C Roland; Huang, Jeffrey T-J

    2015-03-01

    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. Poxvirus decapping enzymes enhance virulence by preventing the accumulation of dsRNA and the induction of innate antiviral responses.

    PubMed

    Liu, Shin-Wu; Katsafanas, George C; Liu, Ruikang; Wyatt, Linda S; Moss, Bernard

    2015-03-11

    Poxvirus replication involves synthesis of double-stranded RNA (dsRNA), which can trigger antiviral responses by inducing phosphorylation-mediated activation of protein kinase R (PKR) and stimulating 2'5'-oligoadenylate synthetase (OAS). PKR inactivates the translation initiation factor eIF2α via phosphorylation, while OAS induces the endonuclease RNase L to degrade RNA. We show that poxvirus decapping enzymes D9 and D10, which remove caps from mRNAs, inhibit these antiviral responses by preventing dsRNA accumulation. Catalytic site mutations of D9 and D10, but not of either enzyme alone, halt vaccinia virus late protein synthesis and inhibit virus replication. Infection with the D9-D10 mutant was accompanied by massive mRNA reduction, cleavage of ribosomal RNA, and phosphorylation of PKR and eIF2α that correlated with a ∼ 15-fold increase in dsRNA compared to wild-type virus. Additionally, mouse studies show extreme attenuation of the mutant virus. Thus, vaccinia virus decapping, in addition to targeting mRNAs for degradation, prevents dsRNA accumulation and anti-viral responses.

  9. 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.

  10. The enhancement of antioxidant compounds extracted from Thymus vulgaris using enzymes and the effect of extracting solvent.

    PubMed

    Cerda, Alejandra; Martínez, María Eugenia; Soto, Carmen; Poirrier, Paola; Perez-Correa, Jose R; Vergara-Salinas, Jose R; Zúñiga, María Elvira

    2013-08-15

    We evaluate the total phenolic compounds (TPC) content and the antioxidant activity (AA) of extracts obtained from ground fresh thyme (FT) and depleted thyme (DT), a by-product of the process of essential oil extraction. In addition, enzymatic treatments were evaluated to improve the extraction yields of polyphenolic compounds from thyme. Extractions were performed using several solvents as methanol, ethanol, and water. Enzymes were applied prior to extraction or during the extraction process. The best results were obtained using a mixture of methanol and water, resulting in 2790 and 220 mg Gallic acid equivalent (GAE)/L of TPC for FT and DT, respectively. A similar result was observed for AA. With regard to enzymatic treatment, application of Grindamyl CA 150 enzyme as a pre-treatment resulted in the production of an extract from DT with 614 mg TE (trolox equivalent)/L of AA, 70% more than the control, and an AA of 621 mg TE/L (74% more than the control sample) was obtained using Grindamyl CA 150 during the extraction process. These results suggest that enzymatic treatment is an interesting alternative for producing antioxidant extracts from DT.

  11. Engineering of a high-throughput screening system to identify cellulosic biomass, pretreatments, and enzyme formulations that enhance sugar release.

    PubMed

    Studer, Michael H; DeMartini, Jaclyn D; Brethauer, Simone; McKenzie, Heather L; Wyman, Charles E

    2010-02-01

    The recalcitrance of cellulosic biomass, the only abundant, sustainable feedstock for making liquid fuels, is a primary obstacle to low cost biological processing, and development of more easily converted plants and more effective enzymes would be of great benefit. Because no single parameter describes recalcitrance, superior variants can only be identified by measuring sugar release from plants subjected to pretreatment and enzymatic hydrolysis. However, genetic modifications of plants coupled with molecular engineering of deconstruction proteins and definition of pretreatment conditions create a very large sample set, and previous methods for biomass pretreatment at elevated temperatures and pressures prevented use of a fully integrated high-throughput (HTP) screening pipeline. Herein, we report on the engineering of a novel HTP pretreatment system employing a 96 well-plate format that withstands extreme pretreatment conditions for rapid screening of biomass-enzyme-pretreatment combinations. This includes the development of new approaches to steam heating and water quenching the system that result in much faster heat up and cool down than previously possible and show consistent temperature histories across the multiwell plate. Coupled pretreatment and enzymatic hydrolysis performance of the well plate pretreatment system is shown to be consistent among the many wells in the device and also with performance of conventional tubular reactors.

  12. 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

  13. 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

  14. 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.

  15. 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.

  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. 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. 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%.

  19. Direct saccharification and ethanol fermentation of cello-oligosaccharides with recombinant yeast.

    PubMed

    Liang, Xianxiang; Yoshida, Takashi; Uryu, Toshiyuki

    2013-01-01

    Ethanol was produced at good rates by direct saccharification and fermentation of cello-oligosaccharides with pYBGA1 yeast, a recombinant laboratory yeast expressing β-glucosidase. Cellobiose in the concentration of 50 g/L was directly fermented for 60 h with 1×10(8) cells/mL of pYBGA1 yeast at 30 °C to give ethanol at an 80% theoretical conversion rate and a concentration of more than 20 g/L of concentration. Conversion to ethanol increased with increasing cellobiose concentration in the feed. When cellobiose was used at the concentration of 100g/L, ethanol conversion and concentration increased to 85% and 45 g/L, respectively, in 96 h incubation. Other cello-oligosaccharides, cellotriose, cellotetraose, and cellopentaose at the concentration of 50 g/L, respectively, were also fermented directly for 72 h with 1×10(8) cells/mL of pYBGA1 yeast to produce ethanol in the conversion rates and concentrations of 71-73% and 18.0-18.5 g/L, respectively. The direct saccharification and fermentation mechanism of cello-oligosaccharides with pYBGA1 yeast, as revealed by HPLC measurements, suggesting that cellotetraose, for example, was saccharificated to cellotriose, cellobiose, and glucose and then fermented to give ethanol. These results suggest that the direct saccharification and fermentation of cello-oligosaccharides with pYBGA1 has several advantages as a simple procedure and for time, cost, and energy consumptions.

  20. On-site cellulase production by Trichoderma reesei 3EMS35 mutant and same vessel saccharification and fermentation of acid treated wheat straw for ethanol production.

    PubMed

    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

  1. On-site cellulase production by Trichoderma reesei 3EMS35 mutant and same vessel saccharification and fermentation of acid treated wheat straw for ethanol production.

    PubMed

    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

  2. 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

  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 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

  5. 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.

  6. Saccharification behavior of cellulose acetate during enzymatic processing for microbial ethanol production.

    PubMed

    Hama, Shinji; Nakano, Kohsuke; Onodera, Kaoru; Nakamura, Masashi; Noda, Hideo; Kondo, Akihiko

    2014-04-01

    This study was conducted to realize the potential application of cellulose acetate to enzymatic processing, followed by microbial ethanol fermentation. To eliminate the effect of steric hindrance of acetyl groups on the action of cellulase, cellulose acetate was subjected to deacetylation in the presence of 1N sodium hydroxide and a mixture of methanol/acetone, yielding 88.8-98.6% at 5-20% substrate loadings during a 48h saccharification at 50°C. Ethanol fermentation using Saccharomyces cerevisiae attained a high yield of 92.3% from the initial glucose concentration of 44.2g/L; however, a low saccharification yield was obtained at 35°C, decreasing efficiency during simultaneous saccharification and fermentation (SSF). Presaccharification at 50°C prior to SSF without increasing the total process time attained the ethanol titers of 19.8g/L (5% substrate), 38.0g/L (10% substrate), 55.9g/L (15% substrate), and 70.9g/L (20% substrate), which show a 12.0-16.2% improvement in ethanol yield.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. Pevonedistat, a NEDD8-activating enzyme inhibitor, is active in mantle cell lymphoma and enhances rituximab activity in vivo.

    PubMed

    Czuczman, Natalie M; Barth, Matthew J; Gu, Juan; Neppalli, Vishala; Mavis, Cory; Frys, Sarah E; Hu, Qiang; Liu, Song; Klener, Pavel; Vockova, Petra; Czuczman, Myron S; Hernandez-Ilizaliturri, Francisco J

    2016-03-01

    Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 μM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-κB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-κB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL. PMID:26675347

  13. Verification of enzymes deterioration due to Cu(II) presence in an enhanced biological phosphorus removal system.

    PubMed

    Tsai, Yung-Pin; Tzeng, Huey-Fen; Lin, Jan-Wei; Lu, Meng-Shan; Lin, Jyun-Yen

    2013-04-01

    This study experimentally demonstrated that polyphosphate accumulating organisms (PAOs) losing the abilities of anaerobically synthesizing polyhydroxyalkanoates and aerobically taking up phosphate under Cu(II) presence was due to the inhibition of enzyme activities of acetyl-CoA synthases (ACS) and polyphosphate kinase (PPK), respectively. ACS activity tests showed the apparent maximum specific activity (Vmax) of ACS decreased with increasing Cu(II) concentration, revealing Cu(II) is a mixed inhibitor for ACS. Inhibition coefficients showed Cu(II) has a higher affinity for free ACS than for ACS-coenzyme A complex. PPK activity tests showed the Vmax substantially decreased with increasing Cu(II) concentration, revealing Cu(II) is also a mixed inhibitor for PPK. Inhibition coefficients showed Cu(II) more easily bound to free PPK than to PPK-Adenosine triphosphate complex. Experimental data also showed the aerobic mechanism of PAOs taking up phosphate was completely interrupted when 3mgL(-1) of Cu(II) was added.

  14. New ammunition for the proteomic reactor: strong anion exchange beads and multiple enzymes enhance protein identification and sequence coverage.

    PubMed

    Zhou, Hu; Hou, Weimin; Lambert, Jean-Philippe; Figeys, Daniel

    2010-08-01

    The enrichment and processing of proteomic samples prior to multi-dimensional chromatography remain a challenge in 'gel-free' proteomics. We previously reported the development of a microfluidic device called the "proteomic reactor" that relied on enriching proteins by using strong cation exchange (SCX) followed by trypsin digestion in an interstitial volume as little as 50 nL. Here, we report a novel proteomic reactor that is based on polymeric strong anion exchange (SAX) material to analyse proteomic samples. We also compare the performance of the SAX proteomic reactor to our previously reported SCX proteomic reactor for analysing complex yeast proteomes. Our results indicate that the SAX protein reactor preferentially identifies more acidic peptides and proteins compared to the SCX reactor. We show that the SAX and SCX reactors are complementary and that their combination increases the number of unique peptides and proteins identified by 50%. Furthermore, we show that the number of protein identified can be increased further by up to 40% using different proteolytic enzymes on the proteomic reactor.

  15. Selectively enhanced expression of prophenoloxidase activating enzyme 1 (PPAE1) at a bacteria clearance site in the white shrimp, Litopenaeus vannamei

    PubMed Central

    2011-01-01

    Background The prophenoloxidase-activating (PO activating) system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating enzyme (PPAE), which is the direct activator of prophenoloxidase (proPO). Despite their importance in crustacean PO activating system, the studies on them remain limited. Results Here we report on a PPAE of white shrimp, Litopenaeus vannamei (lvPPAE1), which showed 94% similarity to PPAE1 of Penaeus monodon. We found that lvPPAE1 in fluid hemocytes was down regulated after challenge by Vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. In vivo gene silence of lvPPAE1 by RNAi can significantly reduce the phenoloxidase activity (PO) and increase the susceptibility of shrimps to V. harveyi. Although lvPPAE1 was down-regulated in fluid hemocytes by Vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue. Conclusion Suppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. This is a novel feature for PPAE expression. The results will contribute to our understanding of the PO activating system in crustaceans. PMID:22208405

  16. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin-cadmium induced diabetic nephrotoxic rats.

    PubMed

    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.

  17. Hinokitiol Exerts Anticancer Activity through Downregulation of MMPs 9/2 and Enhancement of Catalase and SOD Enzymes: In Vivo Augmentation of Lung Histoarchitecture.

    PubMed

    Huang, Chien-Hsun; Jayakumar, Thanasekaran; Chang, Chao-Chien; Fong, Tsorng-Harn; Lu, Shing-Hwa; Thomas, Philip Aloysius; Choy, Cheuk-Sing; Sheu, Joen-Rong

    2015-09-25

    Melanoma is extremely resistant to chemotherapy and the death rate is increasing hastily worldwide. Extracellular matrix promotes the migration and invasion of tumor cells through the production of matrix metalloproteinase (MMP)-2 and -9. Evidence has shown that natural dietary antioxidants are capable of inhibiting cancer cell growth. Our recent studies showed that hinokitiol, a natural bioactive compound, inhibited vascular smooth muscle cell proliferation and platelets aggregation. The present study is to investigate the anticancer efficacy of hinokitiol against B16-F10 melanoma cells via modulating tumor invasion factors MMPs, antioxidant enzymes in vitro. An in vivo mice model of histological investigation was performed to study the patterns of elastic and collagen fibers. Hinokitiol inhibited the expression and activity of MMPs-2 and -9 in B16-F10 melanoma cells, as measured by western blotting and gelatin zymography, respectively. An observed increase in protein expression of MMPs 2/9 in melanoma cells was significantly inhibited by hinokitiol. Notably, hinokitiol (1-5 μM) increased the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) from the reduction in melanoma cells. Also, hinokitiol (2-10 µM) concentration dependently reduced in vitro Fenton reaction induced hydroxyl radical (OH·) formation. An in vivo study showed that hinokitiol treatment increased elastic fibers (EF), collagens dispersion, and improved alveolar alterations in the lungs of B16/F10 injected mice. Overall, our findings propose that hinokitiol may be a potent anticancer candidate through down regulation of MMPs 9/2, reduction of OH· production and enhancement of antioxidant enzymes SOD and CAT.

  18. Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates.

    PubMed

    Várnai, Anikó; Viikari, Liisa; Marjamaa, Kaisa; Siika-aho, Matti

    2011-01-01

    The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance.

  19. 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.

  20. Stress enhances the gene expression and enzyme activity of phenylalanine ammonia-lyase and the endogenous content of salicylic acid to induce flowering in pharbitis.

    PubMed

    Wada, Kaede C; Mizuuchi, Kaori; Koshio, Aya; Kaneko, Kentaro; Mitsui, Toshiaki; Takeno, Kiyotoshi

    2014-07-01

    The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Violet. The exogenously applied SA did not induce flowering under non-stress conditions but did promote flowering under weak stress conditions in both cultivars. These results suggest that stress-induced flowering in pharbitis is induced, at least partly, by SA, and the synthesis of SA is promoted by PAL.

  1. Chemical modification of turnip peroxidase with methoxypolyethylene glycol enhances activity and stability for phenol removal using the immobilized enzyme.

    PubMed

    Quintanilla-Guerrero, F; Duarte-Vázquez, M A; Tinoco, R; Gómez-Suárez, M; García-Almendárez, B E; Vazquez-Duhalt, R; Regalado, C

    2008-09-10

    Peroxidase from turnip roots (TP) was isolated followed by modification with methoxypolyethylene glycol (MPEG). The catalytic activity of the modified TP (MTP) on ABTS increased 2.5 times after 80 min of reaction. MTP showed a KM similar value to that of TP, but a significantly greater kcat for ABTS oxidation, in aqueous buffer. Chemical modification produced an enhanced stability in organic solvents and increased thermal stability of about 4 times that of TP, in aqueous buffer at 70 degrees C. Circular dichroism showed that MPEG modification decreased TP alpha-helical structure from 26 to 16% and increased beta-turns from 26 to 34%, resulting in an enhanced conformational stability. The temperature at the midpoint of thermal denaturation (melting temperature) increased from 57 to 63 degrees C after modification. MTP was immobilized in alginate beads (IMTP) and tested for oxidative polymerization of concentrated phenolic synthetic solutions, achieving 17 effective contact cycles removing >65% phenols. IMTP may be useful for the development of an enzymatic process for wastewater effluent treatment. PMID:18698787

  2. Chromium-Insulin Reduces Insulin Clearance and Enhances Insulin Signaling by Suppressing Hepatic Insulin-Degrading Enzyme and Proteasome Protein Expression in KKAy Mice.

    PubMed

    Wang, Zhong Q; Yu, Yongmei; Zhang, Xian H; Komorowski, James

    2014-01-01

    JDS-chromium-insulin (CRI)-003 is a novel form of insulin that has been directly conjugated with chromium (Cr) instead of zinc. Our hypothesis was that CRI enhances insulin's effects by altering insulin-degrading enzyme (IDE) and proteasome enzymes. To test this hypothesis, we measured hepatic IDE content and proteasome parameters in a diabetic animal model. Male KKAy mice were randomly divided into three groups (n = 8/group); Sham (saline), human regular insulin (Reg-In), and chromium conjugated human insulin (CRI), respectively. Interventions were initiated at doses of 2 U insulin/kg body weight daily for 8-weeks. Plasma glucose and insulin were measured. Hepatic IDE, proteasome, and insulin signaling proteins were determined by western blotting. Insulin tolerance tests at week 7 showed that both insulin treatments significantly reduced glucose concentrations and increased insulin levels compared with the Sham group, CRI significantly reduced glucose at 4 and 6 h relative to Reg-In (P < 0.05), suggesting the effects of CRI on reducing glucose last longer than Reg-In. CRI treatment significantly increased hepatic IRS-1 and Akt1 and reduced IDE, 20S as well as 19S protein abundance (P < 0.01, P < 0.05, and P < 0.001, respectively), but Reg-In only significantly increased Akt1 (P < 0.05). Similar results were also observed in Reg-In- and CRI-treated HepG2 cells. This study, for the first time, demonstrates that CRI reduces plasma insulin clearance by inhibition of hepatic IDE protein expression and enhances insulin signaling as well as prevents degradation of IRS-1 and IRS-2 by suppressing ubiquitin-proteasome pathway in diabetic mice.

  3. 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

  4. 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

  5. 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

  6. Proximity effect among cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface.

    PubMed

    Bae, Jungu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.

  7. 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...

  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. 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).

  10. 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

  11. 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

  12. 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.

  13. Enzyme-Controlled Intracellular Self-Assembly of (18)F Nanoparticles for Enhanced MicroPET Imaging of Tumor.

    PubMed

    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

  14. Hydrogen sulphide enhances photosynthesis through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings.

    PubMed

    Chen, Juan; Wu, Fei-Hua; Wang, Wen-Hua; Zheng, Chen-Juan; Lin, Guang-Hui; Dong, Xue-Jun; He, Jun-Xian; Pei, Zhen-Ming; Zheng, Hai-Lei

    2011-08-01

    Hydrogen sulphide (H(2)S) is emerging as a potential messenger molecule involved in modulation of physiological processes in animals and plants. In this report, the role of H(2)S in modulating photosynthesis of Spinacia oleracea seedlings was investigated. The main results are as follows. (i) NaHS, a donor of H(2)S, was found to increase the chlorophyll content in leaves. (ii) Seedlings treated with different concentrations of NaHS for 30 d exhibited a significant increase in seedling growth, soluble protein content, and photosynthesis in a dose-dependent manner, with 100 μM NaHS being the optimal concentration. (iii) The number of grana lamellae stacking into the functional chloroplasts was also markedly increased by treatment with the optimal NaHS concentration. (iv) The light saturation point (Lsp), maximum net photosynthetic rate (Pmax), carboxylation efficiency (CE), and maximal photochemical efficiency of photosystem II (F(v)/F(m)) reached their maximal values, whereas the light compensation point (Lcp) and dark respiration (Rd) decreased significantly under the optimal NaHS concentration. (v) The activity of ribulose-1,5-bisphosphate carboxylase (RuBISCO) and the protein expression of the RuBISCO large subunit (RuBISCO LSU) were also significantly enhanced by NaHS. (vi) The total thiol content, glutathione and cysteine levels, internal concentration of H(2)S, and O-acetylserine(thiol)lyase and L-cysteine desulphydrase activities were increased to some extent, suggesting that NaHS also induced the activity of thiol redox modification. (vii) Further studies using quantitative real-time PCR showed that the gene encoding the RuBISCO large subunit (RBCL), small subunit (RBCS), ferredoxin thioredoxin reductase (FTR), ferredoxin (FRX), thioredoxin m (TRX-m), thioredoxin f (TRX-f), NADP-malate dehydrogenase (NADP-MDH), and O-acetylserine(thiol)lyase (OAS) were up-regulated, but genes encoding serine acetyltransferase (SERAT), glycolate oxidase (GYX), and cytochrome

  15. 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

  16. Overexpression of VrUBC1, a Mung Bean E2 Ubiquitin-Conjugating Enzyme, Enhances Osmotic Stress Tolerance in Arabidopsis.

    PubMed

    Chung, Eunsook; Cho, Chang-Woo; So, Hyun-Ah; Kang, Jee-Sook; Chung, Young Soo; Lee, Jai-Heon

    2013-01-01

    The ubiquitin conjugating enzyme E2 (UBC E2) mediates selective ubiquitination, acting with E1 and E3 enzymes to designate specific proteins for subsequent degradation. In the present study, we characterized the function of the mung bean VrUBC1 gene (Vigna radiata UBC 1). RNA gel-blot analysis showed that VrUBC1 mRNA expression was induced by either dehydration, high salinity or by the exogenous abscisic acid (ABA), but not by low temperature or wounding. Biochemical studies of VrUBC1 recombinant protein and complementation of yeast ubc4/5 by VrUBC1 revealed that VrUBC1 encodes a functional UBC E2. To understand the function of this gene in development and plant responses to osmotic stresses, we overexpressed VrUBC1 in Arabidopsis (Arabidopsis thaliana). The VrUBC1-overexpressing plants displayed highly sensitive responses to ABA and osmotic stress during germination, enhanced ABA- or salt-induced stomatal closing, and increased drought stress tolerance. The expression levels of a number of key ABA signaling genes were increased in VrUBC1-overexpressing plants compared to the wild-type plants. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that VrUBC1 interacts with AtVBP1 (A. thalianaVrUBC1 Binding Partner 1), a C3HC4-type RING E3 ligase. Overall, these results demonstrate that VrUBC1 plays a positive role in osmotic stress tolerance through transcriptional regulation of ABA-related genes and possibly through interaction with a novel RING E3 ligase.

  17. Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor.

    PubMed

    Núñez-López, Lizeth; Aguirre-Cruz, Andrés; Barrera-Figueroa, Blanca Estela; Peña-Castro, Julián Mario

    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.

  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. 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.

  20. 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.

  1. 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)

  2. 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

  3. 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

  4. Saccharification of wheat-straw cellulose by enzymatic hydrolysis following fermentative and chemical pretreatment

    SciTech Connect

    Detroy, R.W.; Lindenfelser, L.A.; St. Julian, G. Jr.; Orton, W.L.

    1980-01-01

    In our investigations, wheat straw fermentations were conducted using the edible, white-rot fungus commonly known as the oyster mushroom, Pleurotus ostreatus (Jacq. ex Fr.) Kummer, as fermentation organism. Fermented substrates were evaluated for degree of lignin and cellulose degradation and saccharification. In addition, since our primary objective in the P. ostreatus fermentation was to increase the amount of availabile cellulose in straw for further fermentation, cellulose hydrolysis rates were determined. Cellulose conversion to fermentable sugar was also determined on chemically modified straws by subjecting them to enzymatic hydrolysis. Progress and extent of delignification was follwed also by scanning electron microscopy (SEM), and structural changes were determined in treated-straw substrates.

  5. 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.

  6. Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

    PubMed Central

    Hetzler, Stephan; Bröker, Daniel

    2013-01-01

    The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml−1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step. PMID:23793636

  7. 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

  8. 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

  9. 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

  10. 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 ...

  11. 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.

  12. 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

  13. Production of diosgenin from Dioscorea zingiberensis tubers through enzymatic saccharification and microbial transformation.

    PubMed

    Zhu, Yu-Ling; Huang, Wen; Ni, Jin-Ren; Liu, Wei; Li, Hui

    2010-02-01

    In order to develop a clean and effective approach for producing the valuable drug diosgenin from Dioscorea zingiberensis tubers, two successive processes, enzymatic saccharification and microbial transformation, were used. With enzymatic saccharification, 98.0% of starch was excluded from the raw herb, releasing saponins from the network structure of starch. Subsequently, the treated tubers were fermented with Trichoderma reesei under optimal conditions for 156 h. During microbial transformation, glycosidic bonds, which link beta-D-glucose or alpha-L-rhamnose with aglycone at the C-3 position in saponins, were broken down effectively to give a diosgenin yield of 90.6+/-2.45%, 42.4% higher than that obtained from bioconversion of raw tubers directly. Scaled up fermentation was conducted in a 5.0-l bioreactor and gave a diosgenin yield of 91.2+/-3.21%. This is the first report on the preparation of diosgenin from herbs through microbial transformation as well as utilizing other available components in the raw material, providing an environmentally friendly alternative to diosgenin production.

  14. Process integration for simultaneous saccharification, fermentation, and recovery (SSFR): Production of butanol from corn stover using Clostridium beijerinckii P260

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A simultaneous saccharification, fermentation, and recovery (SSFR) process was developed for production of acetone butanol ethanol (AB or ABE), of which butanol is the main product, from corn stover employing Clostridium beijerinckii P260. Of the 86 gL^-1^ corn stover, over 97% of the sugars were r...

  15. 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...

  16. Simultaneous saccharification and continuous fermentation of sludge-containing mash for bioethanol production by Saccharomyces cerevisiae CHFY0321.

    PubMed

    Moon, Se-Kwon; Kim, Seung Wook; Choi, Gi-Wook

    2012-02-20

    A continuous process was employed to improve the volumetric productivity of bioethanol production from cassava mash containing sludge and to simplify the process of ethanol production from cassava. After raw cassava powder was liquefied, it was used directly in a continuous process without sludge filtration or saccharification. A fermentor consisting of four linked stirrer tanks was used for simultaneous saccharification and continuous fermentation (SSCF). Although the mash contained sludge, continuous fermentation was successfully achieved. We chose the dilution rate on the basis of the maximum saccharification time; the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.028 h⁻¹. The volumetric productivity, final ethanol concentration, and % of theoretical ethanol yield were 2.41 g/Lh, 86.1g/L, and 91%, respectively. This SSCF process using the self-flocculating yeast Saccharomyces cerevisiae CHFY0321 illustrates the possibility of realizing cost-effective bioethanol production by eliminating additional saccharification and filtration processes. In addition, flocculent CHFY0321, which our group developed, showed excellent fermentation results under continuous ethanol production.

  17. 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

  18. The challenge of enzyme cost in the production of lignocellulosic biofuels.

    PubMed

    Klein-Marcuschamer, Daniel; Oleskowicz-Popiel, Piotr; Simmons, Blake A; Blanch, Harvey W

    2012-04-01

    With the aim of understanding the contribution of enzymes to the cost of lignocellulosic biofuels, we constructed a techno-economic model for the production of fungal cellulases. We found that the cost of producing enzymes was much higher than that commonly assumed in the literature. For example, the cost contribution of enzymes to ethanol produced by the conversion of corn stover was found to be $0.68/gal if the sugars in the biomass could be converted at maximum theoretical yields, and $1.47/gal if the yields were based on saccharification and fermentation yields that have been previously reported in the scientific literature. We performed a sensitivity analysis to study the effect of feedstock prices and fermentation times on the cost contribution of enzymes to ethanol price. We conclude that a significant effort is still required to lower the contribution of enzymes to biofuel production costs.

  19. Simultaneous saccharification and co-fermentation of paper sludge to ethanol by Saccharomyces cerevisiae RWB222--Part I: kinetic modeling and parameters.

    PubMed

    Zhang, Jiayi; Shao, Xiongjun; Townsend, Oliver V; Lynd, Lee R

    2009-12-01

    A kinetic model was developed to predict batch simultaneous saccharification and co-fermentation (SSCF) of paper sludge by the xylose-utilizing yeast Saccharomyces cerevisiae RWB222 and the commercial cellulase preparation Spezyme CP. The model accounts for cellulose and xylan enzymatic hydrolysis and competitive uptake of glucose and xylose. Experimental results show that glucan and xylan enzymatic hydrolysis are highly correlated, and that the low concentrations of xylose encountered during SSCF do not have a significant inhibitory effect on enzymatic hydrolysis. Ethanol is found to not only inhibit the specific growth rate, but also to accelerate cell death. Glucose and xylose uptake rates were found to be competitively inhibitory, but this did not have a large impact during SSCF because the sugar concentrations are low. The model was used to evaluate which constants had the greatest impact on ethanol titer for a fixed substrate loading, enzyme loading, and fermentation time. The cellulose adsorption capacity and cellulose hydrolysis rate constants were found to have the greatest impact among enzymatic hydrolysis related constants, and ethanol yield and maximum ethanol tolerance had the greatest impact among fermentation related constants.

  20. 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.

  1. Mutation in Brachypodium caffeic acid O-methyltransferase 6 alters stem and grain lignins and improves straw saccharification without deteriorating grain quality.

    PubMed

    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.

  2. 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

  3. 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

  4. Simultaneous saccharification and co-fermentation of crystalline cellulose and sugar cane bagasse hemicellulose hydrolysate to lactate by a thermotolerant acidophilic Bacillus sp.

    PubMed

    Patel, Milind A; Ou, Mark S; Ingram, Lonnie O; Shanmugam, K T

    2005-01-01

    Polylactides produced from renewable feedstocks, such as corn starch, are being developed as alternatives to plastics derived from petroleum. In addition to corn, other less expensive biomass resources can be readily converted to component sugars (glucose, xylose, etc.) by enzyme and/or chemical treatment for fermentation to optically pure lactic acid to reduce the cost of lactic acid. Lactic acid bacteria used by the industry lack the ability to ferment pentoses (hemicellulose-derived xylose and arabinose), and their growth and fermentation optima also differ from the optimal conditions for the activity of fungal cellulases required for depolymerization of cellulose. To reduce the overall cost of simultaneous saccharification and fermentation (SSF) of cellulose, we have isolated bacterial biocatalysts that can grow and ferment all sugars in the biomass at conditions that are also optimal for fungal cellulases. SSF of Solka Floc cellulose by one such isolate, Bacillus sp. strain 36D1, yielded l(+)-lactic acid at an optical purity higher than 95% with cellulase (Spezyme CE; Genencor International) added at about 10 FPU/g cellulose, with a product yield of about 90% of the expected maximum. Volumetric productivity of SSF to lactic acid was optimal between culture pH values of 4.5 and 5.5 at 50 degrees C. At a constant pH of 5.0, volumetric productivity of lactic acid was maximal at 55 degrees C. Strain 36D1 also co-fermented cellulose-derived glucose and sugar cane bagasse hemicellulose-derived xylose simultaneously (SSCF). In a batch SSCF of 40% acid-treated hemicellulose hydrolysate (over-limed) and 20 g/L Solka Floc cellulose, strain 36D1 produced about 35 g/L lactic acid in about 144 h with 15 FPU of Spezyme CE/g cellulose. The maximum volumetric productivity of lactic acid in this SSCF was 6.7 mmol/L (h). Cellulose-derived lactic acid contributed to about 30% of this total lactic acid. These results show that Bacillus sp. strain 36D1 is well-suited for

  5. Enzyme-linked immunosorbent assay by enhanced chemiluminescence detection for the standardization of estrogenic miroestrol in Pueraria candollei Graham ex Benth.

    PubMed

    Yusakul, Gorawit; Udomsin, Orapin; Tanaka, Hiroyuki; Morimoto, Satoshi; Juengwatanatrakul, Thaweesak; Putalun, Waraporn

    2015-08-01

    Miroestrol (ME) is a potent phytoestrogen from the P. candollei tuberous root. It has been approved for use in clinical trials due to its beneficial effect on disorders associated with estrogen deficiency. To ensure medical efficacy and safety, high performance analytical methods for ME analysis are required to standardize products from the P. candollei root. An enhanced chemiluminescence enzyme-linked immunosorbent assay (ECL-ELISA) was developed and validated using a polyclonal antibody against ME and a chemiluminescent system of luminol-H2 O2 -horseradish peroxidase-4-(1-imidazolyl) phenol. The ECL-ELISA system exhibited linearity over a concentration range of 0.31-10.00 ng mL(-1) , for which the relative standard variation (%RSD) was less than 10% for both intra- and interplate determinations. The ECL-ELISA is reliable for the determination of ME as reflected by the high recovery percentage (101.22-103.06%). As a comparative analysis, the ME content in each sample determined by ECL-ELISA was correlated with high coefficients of determination with colorimetric ELISA (R(2)  = 0.998) and high performance liquid chromatography (HPLC) (R(2)  = 0.998) methods. The ECL-ELISA method could be applied to all of the commercial products containing P. candollei root, when the products contain between 0.706 ± 0.046 and 13.123 ± 0.794 µg g(-1) dry wt. of ME. This method is useful as a high performance analytical method for the quantity control of ME in raw materials and end products at both the research and industrial levels.

  6. Cloning and characterization of two β-glucosidase/xylosidase enzymes from yak rumen metagenome.

    PubMed

    Bao, Lei; Huang, Qiang; Chang, Lei; Sun, Qingwen; Zhou, Jungang; Lu, Hong

    2012-01-01

    Two β-glucosidase/xylosidase genes, Rubg3A and Rubg3B, were cloned from yak rumen uncultured microorganisms by metagenome method and function-based screening. Recombinant RuBG3A and RuBG3B purified from Escherichia coli were characterized for enzymatic properties, and they exhibited activity against 4-nitrophenyl-β-D: -glucopyranoside and 4-nitrophenyl-β-D: -xylopyranoside, suggesting bifunctional β-glucosidase/xylosidase activity. Chromatography analysis showed that they could effectively hydrolyze cellooligosaccharide substrates, indicating the facilitation in saccharification of cellulose. RuBG3A and RuBG3B can also increase the reducing sugar released in xylan hydrolysis to 218% and 169%, respectively, through synergism with xylanase, suggesting their application in hemicellulose saccharification. Molecular modeling and substrate docking showed that there should be one active center responsible for the bifunctional activity in each enzyme, since the active site pocket is substantially wide to allow the entry of both β-glucosidic or β-xylosidic substrates, which elucidated the structure-function relationship in substrate specificities. Therefore, the enzymatic properties, the participation in hydrolysis of cellooligosaccharides, and the synergism with xylanase make RuBG3A and RuBG3B very interesting candidates for saccharification of both cellulose and hemicellulose.

  7. 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

  8. The effect of metal ions as co-catalysts on acidic ionic liquid catalyzed single-step saccharification of corn stover in water.

    PubMed

    Wiredu, Bernard; Amarasekara, Ananda S

    2015-01-01

    The effects of adding Cr(3+), Mn(2+), Fe(3+), Co(2+) Ni(2+), Cu(2+), Zn(2+) and La(3+) chlorides as co-catalysts to 1-(1-propylsulfonic)-3-methylimidazolium chloride acidic ionic liquid catalyzed saccharification of corn stover in aqueous medium was studied at 140-170 °C, by measuring the total reducing sugar (TRS) and glucose yields. The samples with Mn(2+), Fe(3+), Co(2+) as co-catalysts produced higher TRS yields compared to the sample without the metal ions. The Mn(2+) produced the highest catalytic effect enhancements and produced TRS yields of 68.0%, 72.9%, 90.2% and 87.9% at 140, 150, 160 and 170 °C respectively; whereas the corn stover samples without the Mn(2+) produced TRS yields of 42.9%, 52.3%, 54.4% and 53.5% at the same four temperatures. At higher temperatures of 160 and 170 °C, all metal ions studied produced significant enhancements in glucose yields, except Cr(3+). The addition of La(3+) as a co-catalyst produced the highest glucose yield improvement. PMID:25911191

  9. The effect of metal ions as co-catalysts on acidic ionic liquid catalyzed single-step saccharification of corn stover in water.

    PubMed

    Wiredu, Bernard; Amarasekara, Ananda S

    2015-01-01

    The effects of adding Cr(3+), Mn(2+), Fe(3+), Co(2+) Ni(2+), Cu(2+), Zn(2+) and La(3+) chlorides as co-catalysts to 1-(1-propylsulfonic)-3-methylimidazolium chloride acidic ionic liquid catalyzed saccharification of corn stover in aqueous medium was studied at 140-170 °C, by measuring the total reducing sugar (TRS) and glucose yields. The samples with Mn(2+), Fe(3+), Co(2+) as co-catalysts produced higher TRS yields compared to the sample without the metal ions. The Mn(2+) produced the highest catalytic effect enhancements and produced TRS yields of 68.0%, 72.9%, 90.2% and 87.9% at 140, 150, 160 and 170 °C respectively; whereas the corn stover samples without the Mn(2+) produced TRS yields of 42.9%, 52.3%, 54.4% and 53.5% at the same four temperatures. At higher temperatures of 160 and 170 °C, all metal ions studied produced significant enhancements in glucose yields, except Cr(3+). The addition of La(3+) as a co-catalyst produced the highest glucose yield improvement.

  10. 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

  11. 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

  12. Influence of the alkaline delignification on the simultaneous saccharification and fermentation (SSF) of sugar cane bagasse.

    PubMed

    Soares, Mariana Lucena; Gouveia, Ester Ribeiro

    2013-11-01

    Ethanol production from steam explosion alkaline delignified bagasse was investigated by saccharification and simultaneous fermentation. Non delignified bagasse (ND) contained 25% lignin, and after alkaline delignification, materials with 6% (D1 - NaOH 1% w/v) and 12% (D05 - NaOH 0.5% w/v) lignin, respectively, were obtained. Ethanol production increased 450% and 733% in relation to ND, when D05 and D1 material, respectively, were used. Higher productivity and EtOH/bagasse were observed for D1. However, higher enzymatic convertibility of cellulose was obtained with 0.5% w/v NaOH. Alkaline delignification increased the ethanol production despite decreased cellulose.

  13. 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.

  14. Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast.

    PubMed

    Hari Krishna, S; Janardhan Reddy, T; Chowdary, G V

    2001-04-01

    Simultaneous saccharification and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5-3.5% w/v) compared to S. cerevisiae (2.0-2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with beta-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka floc > A. leptopus > sugar cane.

  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.

  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. Steam explosion of oilseed rape straw: establishing key determinants of saccharification efficiency.

    PubMed

    Wood, Ian P; Elliston, Adam; Collins, Sam R A; Wilson, David; Bancroft, Ian; Waldron, Keith W

    2014-06-01<