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Sample records for enhanced enzymic saccharification

  1. Enhanced saccharification of rice straw and hull by microwave-alkali pretreatment and lignocellulolytic enzyme production.

    PubMed

    Singh, Anita; Tuteja, Shuchi; Singh, Namita; Bishnoi, Narsi R

    2011-01-01

    In this study, statistical design of experiments was employed to plan experiments and optimize the microwave-alkali pretreatment of rice straw and hulls. Process parameters important in pretreatment of biomass were identified by a Plackett-Burman design and the parameters with significant effects were optimized using a box-behnken design (BBD). Experimental results show that alkali concentration (AC), irradiation time (IT) and substrate concentration (SC) were main factors governing the saccharification of rice straw and hulls. Optimum conditions of pretreatment were AC 2.75%, IT 22.50 min and SC 30 g/L, as optimized by BBD. The growth and production of lignocellulolytic enzymes from Aspergillus heteromorphus, solid state fermentation (SSF) was performed using rice straw and hulls pretreated under optimum conditions. Cellulases and xylanase reached the highest enzyme activity at 6th day of fermentation while maximum manganese peroxidase (MnP) and laccase activity occurred at 12th day. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  3. Enzymic saccharification of pretreated wheat straw.

    PubMed

    Vallander, L; Eriksson, K E

    1985-05-01

    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.

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

    PubMed

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

    2014-08-13

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

  5. Agricultural wastes as substrates for β-glucosidase production by Talaromyces thermophilus: Role of these enzymes in enhancing waste paper saccharification.

    PubMed

    Mallek-Fakhfakh, Hanen; Fakhfakh, Jawhar; Masmoudi, Najla; Rezgui, Fatma; Gargouri, Ali; Belghith, Hafedh

    2017-04-21

    In the present study, we investigated a potent extracellular β-glucosidases secreted by the thermophilic fungal strain AX4 of Talaromyces thermophilus, isolated from Tunisian soil samples. This strain was selected referring to the highest thermostability of its β-glucosidases compared to the other fungal isolates. The β-glucosidase production was investigated by submerged fermentation. The optimal temperature and initial pH for maximum β-glucosidase production were 50°C and 7.0, respectively. Several carbon sources were assayed for their effects on β-glucosidase production, significant yields were obtained in media containing lactose 1% (3.0 ± 0.36 U/ml) and wheat bran 2% (4.0 ± 0.4 U/ml). The combination of wheat bran at 2% and lactose at 0.8% as carbon source enhanced β-glucosidase production, which reached 8.5 ± 0.28 U/ml. Furthermore, the β-glucosidase-rich enzymatic juice of T. thermophilus exhibited significant synergism with Trichoderma reesei (Rut C30) cellulases for pretreated waste paper (PWP) hydrolysis. Interestingly, the use of this optimal enzymatic cocktail increased 4.23 fold the glucose yield after saccharification of waste paper. A maximum sugar yield (94%) was reached when using low substrate (2%) and enzyme loading (EC1).

  6. Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production.

    PubMed

    Trakarnpaiboon, Srisakul; Srisuk, Nantana; Piyachomkwan, Kuakoon; Sakai, Kenji; Kitpreechavanich, Vichien

    2017-09-14

    In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8 g:10 g:2 g yielded the highest enzyme production of 201.6 U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5 × 10(6) spores/mL inoculum, which gave the highest enzyme activity of 389.5 U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2 g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300 g raw cassava chips/L with cane molasses.

  7. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  9. 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.

  10. 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.

  11. Catalase improves saccharification of lignocellulose by reducing lytic polysaccharide monooxygenase-associated enzyme inactivation.

    PubMed

    Scott, Brian R; Huang, Hong Zhi; Frickman, Jesper; Halvorsen, Rune; Johansen, Katja S

    2016-03-01

    Efficient enzymatic saccharification of plant cell wall material is key to industrial processing of agricultural and forestry waste such as straw and wood chips into fuels and chemicals. Saccharification assays were performed on steam-pretreated wheat straw under ambient and O2-deprived environments and in the absence and presence of a lytic polysaccharide monooxygenase (LPMO) and catalase. A kinetic model was used to calculate catalytic rate and first-order inactivation rate constants of the cellulases from reaction progress curves. The addition of a LPMO significantly (P < 0.01, Student's T test) enhanced the rate of glucose release from 2.8 to 6.9 h(-1) under ambient O2 conditions. However, this also significantly (P < 0.01, Student's T test) increased the rate of inactivation of the enzyme mixture, thereby reducing the performance half-life from 65 to 35 h. Decreasing O2 levels or, strikingly, the addition of catalase significantly reduced (P < 0.01, Student's T test) enzyme inactivation and, as a consequence, higher efficiency of the cellulolytic enzyme cocktail was achieved. Oxidative inactivation of commercial cellulase mixtures is a significant factor influencing the overall saccharification efficiency and the addition of catalase can be used to protect these mixtures from inactivation.

  12. Saccharification and hydrolytic enzyme production of alkali pre-treated wheat bran by Trichoderma virens under solid state fermentation.

    PubMed

    El-Shishtawy, Reda M; Mohamed, Saleh A; Asiri, Abdullah M; Gomaa, Abu-Bakr M; Ibrahim, Ibrahim H; Al-Talhi, Hasan A

    2015-05-28

    In continuation of our previously interest in the saccharification of agriculture wastes by Bacillus megatherium in solid state fermentation (SSF), we wish to report an investigation and comparative evaluation among Trichoderma sp. for the saccharification of four alkali-pretreated agricultural residues and production of hydrolytic enzymes, carboxymethyl cellulase (CMCase), filter paperase (FPase), pectinase (PGase) and xylanase (Xylase) in SSF. The optimization of the physiological conditions of production of hydrolytic enzymes and saccharification content from Trichoderma virens using alkali-pretreated wheat bran was the last goal. The physico-chemical parameters of SSF include incubation time, incubation temperature, moisture content of the substrate, incubation pH, supplementation with carbon and nitrogen sources were optimized. Saccharification of different solid state fermentation sources wheat bran, date's seeds, grass and palm leaves, were tested for the production of fermentable sugar by Trichoderma sp. The maximum production of hydrolytic enzymes CMCase, FPase, PGase and Xylase and saccharification content were obtained on wheat bran. Time course, moisture content, optimum temperature, optimum pH, supplementation with carbon and nitrogen sources were optimized to achieve the maximum production of the hydrolytic enzymes, protein and total carbohydrate of T. virens using alkali pre-treated wheat bran. The maximum production of CMCase, FPase, PGase, Xylase, protein and carbohydrate content was recorded at 72 h of incubation, 50-70 % moisture, temperature 25-35 °C and pH 5. The influence of supplementary carbon and nitrogen sources was studied. While lactose and sucrose enhanced the activity of PGase from 79.2 to 582.9 and 632.6 U/g, starch inhibited all other enzymes. This was confirmed by maximum saccharification content. Among the nitrogen sources, yeast extract and urea enhanced the saccharification content and CMCase, PGase and Xylase. The results of

  13. 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.

  14. Exploring glycoside hydrolases and accessory proteins from wood decay fungi to enhance sugarcane bagasse saccharification.

    PubMed

    Valadares, Fernanda; Gonçalves, Thiago A; Gonçalves, Dayelle S P O; Segato, Fernando; Romanel, Elisson; Milagres, Adriane M F; Squina, Fabio M; Ferraz, André

    2016-01-01

    Glycoside hydrolases (GHs) and accessory proteins are key components for efficient and cost-effective enzymatic hydrolysis of polysaccharides in modern, biochemically based biorefineries. Currently, commercialized GHs and accessory proteins are produced by ascomycetes. However, the role of wood decay basidiomycetes proteins in biomass saccharification has not been extensively pursued. Wood decay fungi degrade polysaccharides in highly lignified tissues in natural environments, and are a promising enzyme source for improving enzymatic cocktails that are designed for in vitro lignocellulose conversion. GHs and accessory proteins were produced by representative brown- and white-rot fungi, Laetiporus sulphureus and Pleurotus ostreatus, respectively. Concentrated protein extracts were then used to amend commercial enzymatic cocktails for saccharification of alkaline-sulfite pretreated sugarcane bagasse. The main enzymatic activities found in the wood decay fungal protein extracts were attributed to endoglucanases, xylanases and β-glucosidases. Cellobiohydrolase (CBH) activities in the L. sulphureus and P. ostreatus extracts were low and nonexistent, respectively. The initial glucan conversion rates were boosted when the wood decay fungal proteins were used to replace half of the enzymes from the commercial cocktails. L. sulphureus proteins increased the glucan conversion levels, with values above those observed for the full load of commercial enzymes. Wood decay fungal proteins also enhanced the xylan conversion efficiency due to their high xylanase activities. Proteomic studies revealed 104 and 45 different proteins in the P. ostreatus and L. sulphureus extracts, respectively. The enhancement of the saccharification of alkaline-pretreated substrates by the modified enzymatic cocktails was attributed to the following protein families: GH5- and GH45-endoglucanases, GH3-β-glucosidases, and GH10-xylanases. The extracellular proteins produced by wood decay fungi provide

  15. 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

  16. 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.

  17. Xylanases of Bacillus spp. isolated from ruminant dung as potential accessory enzymes for agro-waste saccharification.

    PubMed

    Thite, V S; Nerurkar, A S

    2015-05-01

    Polysaccharide hydrolase producing bacteria were isolated for biomass saccharification step in two-step bioethanol production. Xylanolytic bacteria were found to be common in ruminant dung. Seven Bacillus dung isolates exhibited high xylanolytic activity; three of which were identified as Bacillus safensis and four as Bacillus altitudinis, based on 16S rDNA and gyrB gene sequencing. Interestingly, comparison of activity profiles for B. safensis M35 and B. altitudinis R31 and J208 crude xylanases showed activity in similar temperature and pH ranges of 40-60 °C and 6·0-9·0, respectively, even though they were isolated from three different dung sources. Furthermore, 22-28% viscosity reduction of beechwood xylan substrate by all the three xylanases points towards their endo-acting nature. Endo-acting xylanases are envisaged as accessory enzymes which help expose the cellulose fibres for the subsequent action of the core enzyme cellulases. In this study, when supplemented to the commercial cellulase as a cocktail, the accessory role of the crude xylanases from the selected Bacillus strains was established as 1·3, 2·33 and 1·9 fold increase in saccharification of barley husk, sugarcane bagasse and wheat husk was achieved, respectively. The uncontrolled use of fossil fuels and concerns about its future availability, have invoked interest over unconventional alternative energy sources like solar, hydropower, geothermal, nuclear and biomass. Plants, being largest renewable biomass on earth, have received consideration as a source of biofuels. Ruminant dung isolates M35, R31 and J208 belonging to Bacillus sp. produces majorly endo-xylanase when induced with wheat bran. Such plant cell wall degrading endo-xylanases with broad pH optima and mesophilic nature can act as accessory enzymes with cellulases to enhance the saccharification of plant biomass in biofuel industries. © 2015 The Society for Applied Microbiology.

  18. 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.

  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. Investigating the enzyme-lignin binding with surfactants for improved saccharification of pilot scale pretreated wheat straw.

    PubMed

    Agrawal, Ruchi; Satlewal, Alok; Kapoor, Manali; Mondal, Sujit; Basu, Biswajit

    2017-01-01

    In this study, commercial surfactants have been investigated at economically viable dosage to enhance the enzymatic saccharification of pretreated wheat straw at high solid loadings. Twenty one surfactants were evaluated with pilot scale pretreated wheat straw and mechanism of surfactant action has been elucidated. One surfactant has improved the saccharification of dilute acid wheat straw (DAWS) by 26.4% after 24h and 23.1% after 48h while, steam exploded wheat straw (SEWS) saccharification was increased by 51.2% after 24h and 36.4% after 48h at 10% solid loading. At 20% solid loading, about 31% increase in yield was obtained on DAWS and about 55% on SEWS after 48h. Further, lignin was isolated from pretreated wheat straws and characterized which revealed that SEWS derived lignin was more hydrophobic than DAWS lignin. This investigation suggests that surfactant supplementation during saccharification is an effective strategy to achieve higher saccharification yield.

  1. Empirical evaluation of inhibitory product, substrate, and enzyme effects during the enzymatic saccharification of lignocellulosic biomass.

    PubMed

    Smith, Benjamin T; Knutsen, Jeffrey S; Davis, Robert H

    2010-05-01

    The cellulose hydrolysis kinetics during batch enzymatic saccharification are typified by a rapid initial rate that subsequently decays, resulting in incomplete conversion. Previous studies suggest that changes associated with the solution, substrate, or enzymes may be responsible. In this work, kinetic experiments were conducted to determine the relative magnitude of these effects. Pretreated corn stover (PCS) was used as a lignocellulosic substrate likely to be found in a commercial saccharification process, while Avicel and Kraft lignin were used to create model substrates. Glucose inhibition was observed by spiking the reaction slurry with glucose during initial-rate experiments. Increasing the glucose concentration from 7 to 48 g/L reduced the cellulose conversion rate by 94%. When product sugars were removed using ultrafiltration with a 10 kDa membrane, the glucose-based conversion increased by 9.5%. Reductions in substrate reactivity with conversion were compared directly by saccharifying PCS and Avicel substrates that had been pre-reacted to different conversions. Reaction of substrate with a pre-conversion of 40% resulted in about 40% reduction in the initial rate of saccharification, relative to fresh substrate with identical cellulose concentration. Overall, glucose inhibition and reduced substrate reactivity appear to be dominant factors, whereas minimal reductions of enzyme activity were observed.

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

    PubMed

    El-Shishtawy, Reda M; Mohamed, Saleh A; Asiri, Abdullah M; Gomaa, Abu-bakr M; Ibrahim, Ibrahim H; Al-Talhi, Hasan A

    2014-04-24

    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. 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. 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 nitrogen sources.

  3. 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

  4. Construction of Aspergillus niger integrated with cellulase gene from Ampullaria gigas Spix for improved enzyme production and saccharification of alkaline-pretreated rice straw.

    PubMed

    Yang, Peizhou; Zhang, Haifeng; Cao, Lili; Zheng, Zhi; Jiang, Shaotong

    2016-12-01

    Aspergillus niger is an important microorganism that has been used for decades to produce extracellular enzymes. In this study, a novel Aspergillus niger strain integrated with a eukaryotic expression vector harboring the gpd-Shi promoter of shiitake mushrooms and cellulase gene of Ampullaria gigas Spix was engineered to improve cellulase production for the achievement of highly efficient saccharification of agricultural residues. In one strain, designated ACShi27, which exhibited the highest total cellulase expression, total cellulase, endoglucanase, exoglucanase, and xylanase expression levels were 1.73, 16.23, 17.73, and 150.83 U ml(-1), respectively; these values were 14.5, 22.3, 24.6, and 17.3% higher than those of the wild-type Aspergillus niger M85 using wheat bran as an induction substrate. Production of cellulases and xylanase by solid-state fermentation followed by in situ saccharification of ACShi27 was investigated with alkaline-pretreated rice straw as a substrate. After 2 days of enzyme induction at 30 °C, followed by 48 h of saccharification at 50 °C, the conversion rate of carbon polymers into reducing sugar reached 293.2 mg g(-1), which was 1.23-fold higher than that of the wild-type strain. The expression of sestc in Aspergillus niger can improve the total cellulase and xylanase activity and synergism, thereby enhancing the lignocellulose in situ saccharification.

  5. Simultaneous saccharification and fermentation of enzyme pretreated Lantana camara using S. cerevisiae.

    PubMed

    Kuila, Arindam; Banerjee, Rintu

    2014-10-01

    Lantana camara, an abundantly available non-edible lignocellulosic biomass has been found to be a potential feedstock for ethanol production. The substrate was first pretreated with laccase followed by simultaneous saccharification and fermentation using cellulase and Saccharomyces cerevisiae, respectively. Laccase was produced from Pleurotus sp. and carbohydratases (cellulase and xylanase) were produced from Trichoderma reesei Rut C30. Using pretreated substrate simultaneous saccharification and fermentation was optimized through central composite design-based response surface methodology. Maximum bioethanol concentration of 5.14 % (v/v) was obtained at optimum process conditions of substrate concentration 17 % (w/v), inoculum volume 9 % (v/v), inoculum age 60 and 144 h of incubation time. To enhance ethanol yield, S. cerevisiae was treated with ethyl methane sulfonate, a chemical mutagenic agent which induced mutagenesis. A maximum bioethanol concentration of 6.01 % (v/v) was obtained using the mutated strain of S. cerevisiae (CM5).

  6. 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

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

    SciTech Connect

    Kumar, Manoj

    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.

  8. 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.

  9. Enzyme production by wood-rot and soft-rot fungi cultivated on corn fiber followed by simultaneous saccharification and fermentation.

    PubMed

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

    2009-05-27

    This research aims at developing a biorefinery platform to convert lignocellulosic corn fiber into fermentable sugars at a moderate temperature (37 °C) with minimal use of chemicals. White-rot (Phanerochaete chrysosporium), brown-rot (Gloeophyllum trabeum), and soft-rot (Trichoderma reesei) fungi were used for in situ enzyme production to hydrolyze cellulosic and hemicellulosic components of corn fiber into fermentable sugars. Solid-substrate fermentation of corn fiber by either white- or brown-rot fungi followed by simultaneous saccharification and fermentation (SSF) with coculture of Saccharomyces cerevisiae has shown a possibility of enhancing wood rot saccharification of corn fiber for ethanol fermentation. The laboratory-scale fungal saccharification and fermentation process incorporated in situ cellulolytic enzyme induction, which enhanced overall enzymatic hydrolysis of hemi/cellulose components of corn fiber into simple sugars (mono-, di-, and trisaccharides). The yeast fermentation of the hydrolyzate yielded 7.8, 8.6, and 4.9 g ethanol per 100 g corn fiber when saccharified with the white-, brown-, and soft-rot fungi, respectively. The highest ethanol yield (8.6 g ethanol per 100 g initial corn fiber) is equivalent to 35% of the theoretical ethanol yield from starch and cellulose in corn fiber. This research has significant commercial potential to increase net ethanol production per bushel of corn through the utilization of corn fiber. There is also a great research opportunity to evaluate the remaining biomass residue (enriched with fungal protein) as animal feed.

  10. 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

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

    PubMed

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

    2014-10-07

    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.

  12. 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.

  13. Cationic polyacrylamides enhance rates of starch and cellulose saccharification.

    PubMed

    Reye, John T; Maxwell, Kendra; Rao, Swati; Lu, Jian; Banerjee, Sujit

    2009-10-01

    Adding a cationic polyacrylamide (c-PAM) to either the amylase mediated hydrolysis of corn starch or the hydrolysis of wood fiber by cellulase can enhance the initial hydrolysis rates, although a rate decrease can occur under some conditions. Several c-PAMs can serve as catalysts and the same c-PAM can improve the efficiency of both amylase and cellulase. The initial amylase rate approximately doubles; the analogous cellulase hydrolysis rate increases by about 40%. c-PAMs increase the binding of enzyme to substrate.

  14. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. AtCesA8-driven OsSUS3 expression leads to largely enhanced biomass saccharification and lodging resistance by distinctively altering lignocellulose features in rice.

    PubMed

    Fan, Chunfen; Feng, Shengqiu; Huang, Jiangfeng; Wang, Yanting; Wu, Leiming; Li, Xukai; Wang, Lingqiang; Tu, Yuanyuan; Xia, Tao; Li, Jingyang; Cai, Xiwen; Peng, Liangcai

    2017-01-01

    Biomass recalcitrance and plant lodging are two complex traits that tightly associate with plant cell wall structure and features. Although genetic modification of plant cell walls can potentially reduce recalcitrance for enhancing biomass saccharification, it remains a challenge to maintain a normal growth with enhanced biomass yield and lodging resistance in transgenic plants. Sucrose synthase (SUS) is a key enzyme to regulate carbon partitioning by providing UDP-glucose as substrate for cellulose and other polysaccharide biosynthesis. Although SUS transgenic plants have reportedly exhibited improvement on the cellulose and starch based traits, little is yet reported about SUS impacts on both biomass saccharification and lodging resistance. In this study, we selected the transgenic rice plants that expressed OsSUS3 genes when driven by the AtCesA8 promoter specific for promoting secondary cell wall cellulose synthesis in Arabidopsis. We examined biomass saccharification and lodging resistance in the transgenic plants and detected their cell wall structures and wall polymer features. During two-year field experiments, the selected AtCesA8::SUS3 transgenic plants maintained a normal growth with slightly increased biomass yields. The four independent transgenic lines exhibited much higher biomass enzymatic saccharification and bioethanol production under chemical pretreatments at P < 0.01 levels, compared with the controls of rice cultivar and empty vector transgenic line. Notably, all transgenic lines showed a consistently enhanced lodging resistance with the increasing extension and pushing forces. Correlation analysis suggested that the reduced cellulose crystallinity was a major factor for largely enhanced biomass saccharification and lodging resistance in transgenic rice plants. In addition, the cell wall thickenings with the increased cellulose and hemicelluloses levels should also contribute to plant lodging resistance. Hence, this study has proposed a

  16. Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse.

    PubMed

    van den Brink, Joost; Maitan-Alfenas, Gabriela Piccolo; Zou, Gen; Wang, Chengshu; Zhou, Zhihua; Guimarães, Valéria Monteze; de Vries, Ronald P

    2014-10-01

    Plant-degrading enzymes can be produced by fungi on abundantly available low-cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two "second generation" substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi-)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non-washed SCB. The sensitivity to non-washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification.

  17. Peracetic acid-ionic liquid pretreatment to enhance enzymatic saccharification of lignocellulosic biomass.

    PubMed

    Uju; Abe, Kojiro; Uemura, Nobuyuki; Oshima, Toyoji; Goto, Masahiro; Kamiya, Noriho

    2013-06-01

    To enhance enzymatic saccharification of pine biomass, the pretreatment reagents peracetic acid (PAA) and ionic liquid (IL) were validated in single reagent pretreatments or combination pretreatments with different sequences. In a 1h saccharification, 5-25% cellulose conversion was obtained from the single pretreatment of PAA or IL. In contrast, a marked enhancement in conversion rates was achieved by PAA-IL combination pretreatments (45-70%). The PAA followed by IL (PAA+IL) pretreatment sequence was the most effective for preparing an enzymatic digestible regenerated biomass with 250-fold higher glucose formation rates than untreated biomass and 2- to 12-fold higher than single pretreatments with PAA or IL alone. Structural analysis confirmed that this pretreatment resulted in biomass with highly porous structural fibers associated with the reduction of lignin content and acetyl groups. Using the PAA+IL sequence, biomass loading in the pretreatment step can be increased from 5% to 15% without significant decrease in cellulose conversion.

  18. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    PubMed

    Gong, Zhiwei; Shen, Hongwei; Wang, Qian; Yang, Xiaobing; Xie, Haibo; Zhao, Zongbao K

    2013-03-05

    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. 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. 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 improve the efficiency of microbial

  20. 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

  1. 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.

  2. Correlating physical changes and enhanced enzymatic saccharification of pine flour pretreated by Ν-Methylmorpholine-Ν-oxide

    Treesearch

    Ye Liu; Qixin Zhong; Siqun Wang; Zhiyong Cai

    2011-01-01

    Pretreatment of lignocellulosic biomass by Ν-methylmorpholine-Ν-oxide (NMMO), a solvent used in the textile industry to dissolve cellulose for production of regenerated cellulose fibers, was observed to enhance significantly enzymatic saccharification and fermentation. The enhancement was speculated to have been caused by reduced cellulose crystallinity...

  3. 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.

  4. 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.

  5. 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.

  6. pH-Induced Lignin Surface Modification to Reduce Nonspecific Cellulase Binding and Enhance Enzymatic Saccharification of Lignocelluloses

    Treesearch

    Hongming Lou; J.Y. Zhu; Tian Qing Lan; Huranran Lai; Xueqing Qiu

    2013-01-01

    We studied the mechanism of the significant enhancement in the enzymatic saccharification of lignocelluloses at an elevated pH of 5.5–6.0. Four lignin residues with different sulfonic acid contents were isolated from enzymatic hydrolysis of lodgepole pine pretreated by either dilute acid (DA) or sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL...

  7. Cobalt-60 gamma-ray irradiation pretreatment and sludge protein for enhancing enzymatic saccharification of hybrid poplar sawdust.

    PubMed

    Xiang, Yulin; Xiang, Yuxiu; Wang, Lipeng

    2016-12-01

    In order to improve the enzymatic saccharification of hybrid poplar sawdust, gamma irradiation pretreatment and enzymatic hydrolysis in the presence of sludge protein were investigated. The cellulose crystallinity index were significantly decreased after irradiation pretreatment, and adding sludge protein improved enzyme activity and increased the reducing sugar yield. The conditions of irradiation pretreatment and enzymatic hydrolysis in the presence of sludge protein were systematically examined. The maximum reducing sugar yield was 519mg/g under an irradiation dose of 300kGy, a sludge protein dosage of 2mg/mL, an enzymatic hydrolysis temperature of 45°C, an enzymatic hydrolysis time of 84h, and a 90FPU/g enzyme loading. This work indicated that the combined method of gamma irradiation pretreatment and enzymatic hydrolysis in the presence of sludge protein was a promising potential for the saccharification of hybrid poplar sawdust.

  8. 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

  9. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2017-02-01

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

  11. Lignosulfonate To Enhance Enzymatic Saccharification of Lignocelluloses: Role of Molecular Weight and Substrate Lignin

    Treesearch

    Haifeng Zhou; Hongming Lou; Dongjie Yang; J.Y. Zhu; Xueqing Qiu

    2013-01-01

    This study conducted an investigation of the effect of lignosulfonate (LS) on enzymatic saccharification of lignocelluloses. Two commercial LSs and one laboratory sulfonated kraft lignin were applied to Whatman paper, dilute acid and SPORL (sulfite pretreatment to overcome recalcitrance of lignocelluloses) pretreated aspen, and kraft alkaline and SPORL pretreated...

  12. 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).

  13. Gaseous ammonia pretreatment lowers the required energy input for fine milling-enhanced enzymatic saccharification of switchgrass.

    PubMed

    Diner, Bruce A; Lasio, Jelena; Camp, Carl E; David Rosenfeld, H; Fan, Janine; Fox, Bradley C

    2015-01-01

    as the ends of the cellulose fibrils are accessible to cellulolytic enzymes. The combination of knife milling, low moisture gaseous ammonia pretreatment followed by attritor milling that costs only ~5 % of the energy content of the biomass for a total energy input of ~11 % of the biomass energy content, is capable of delivering high sugar titers upon enzymatic saccharification. These results show, therefore, how to better integrate a mechanochemical step into the pretreatment of switchgrass in a commercial biomass to biofuels conversion process.

  14. 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.

  15. 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

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

    SciTech Connect

    Kumar, Manoj

    2011-05-09

    These are a set of slides from this conference. 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.

  17. Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings

    PubMed Central

    2014-01-01

    Background Economically feasible cellulosic ethanol production requires that the process can be operated at high solid loadings, which currently imparts technical challenges including inefficient mixing leading to heat and mass transfer limitations and high concentrations of inhibitory compounds hindering microbial activity during simultaneous saccharification and fermentation (SSF) process. Consequently, there is a need to develop cost effective processes overcoming the challenges when working at high solid loadings. Results In this study we have modified the yeast cultivation procedure and designed a SSF process to address some of the challenges at high water insoluble solids (WIS) content. The slurry of non-detoxified pretreated spruce when used in a batch SSF at 19% (w/w) WIS was found to be inhibitory to Saccharomyces cerevisiae Thermosacc that produced 2 g l-1 of ethanol. In order to reduce the inhibitory effect, the non-washed solid fraction containing reduced amount of inhibitors compared to the slurry was used in the SSF. Further, the cells were cultivated in the liquid fraction of pretreated spruce in a continuous culture wherein the outflow of cell suspension was used as cell feed to the SSF reactor in order to maintain the metabolic state of the cell. Enhanced cell viability was observed with cell, enzyme and substrate feed in a SSF producing 40 g l-1 ethanol after 96 h corresponding to 53% of theoretical yield based on available hexose sugars compared to 28 g l-1 ethanol in SSF with enzyme and substrate feed but no cell feed resulting in 37% of theoretical yield at a high solids loading of 20% (w/w) WIS content. The fed-batch SSF also significantly eased the mixing, which is usually challenging in batch SSF at high solids loading. Conclusions A simple modification of the cell cultivation procedure together with a combination of yeast, enzyme and substrate feed in a fed-batch SSF process, made it possible to operate at high solids loadings in a

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

    PubMed

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

    2016-02-01

    Simultaneous saccharification and fermentation (SSF) process was applied for biobutanol production by Clostridium saccharobutylicum DSM 13864 from corn stover (CS). The key influential factors in SSF process, including corn steep liquor concentration, dry biomass and enzyme loading, SSF temperature, inoculation size and pre-hydrolysis time were optimized. In 5-L bioreactor with SSF process, butanol titer and productivity of 12.3 g/L and 0.257 g/L/h were achieved at 48 h, which were 20.6% and 21.2% higher than those in separate hydrolysis and fermentation (SHF), respectively. The butanol yield reached 0.175 g/g pretreated CS in SSF, representing 50.9% increase than that in SHF (0.116 g/g pretreated CS). This study proves the feasibility of efficient and economic production of biobutanol from CS by SSF. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Steam-exploded biomass saccharification is predominately affected by lignocellulose porosity and largely enhanced by Tween-80 in Miscanthus.

    PubMed

    Sun, Dan; Alam, Aftab; Tu, Yuanyuan; Zhou, Shiguang; Wang, Yanting; Xia, Tao; Huang, Jiangfeng; Li, Ying; Zahoor; Wei, Xiaoyang; Hao, Bo; Peng, Liangcai

    2017-09-01

    In this study, total ten Miscanthus accessions exhibited diverse cell wall compositions, leading to largely varied hexoses yields at 17%-40% (% cellulose) released from direct enzymatic hydrolysis of steam-exploded (SE) residues. Further supplied with 2% Tween-80 into the enzymatic digestion, the Mis7 accession showed the higher hexose yield by 14.8-fold than that of raw material, whereas the Mis10 had the highest hexoses yield at 77% among ten Miscanthus accessions. Significantly, this study identified four wall polymer features that negatively affect biomass saccharification as p<0.05 or 0.01 in the SE residues, including cellulose DP, Xyl and Ara of hemicellulose, and S-monomer of lignin. Based on Simons' stain, the SE porosity (defined by DY/DB) was examined to be the unique positive factor on biomass enzymatic digestion. Hence, this study provides the potential strategy to enhance biomass saccharification using optimal biomass process technology and related genetic breeding in Miscanthus and beyond. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Liquid nitrogen pretreatment of eucalyptus sawdust and rice hull for enhanced enzymatic saccharification.

    PubMed

    Castoldi, Rafael; Correa, Vanesa G; de Morais, Gutierrez Rodrigues; de Souza, Cristina G M; Bracht, Adelar; Peralta, Rosely A; Peralta-Muniz Moreira, Regina F; Peralta, Rosane M

    2017-01-01

    In this work, liquid nitrogen was used for the first time in the pretreatment of plant biomasses for purposes of enzymatic saccharification. After treatment (cryocrushing), the initial rates of the enzymatic hydrolysis of eucalyptus sawdust and rice hull were increased more than ten-fold. Cryocrushing did not modify significantly the contents of cellulose, hemicellulose and lignin in both eucalyptus sawdust and rice hulls. However, substantial disorganization of the lignocellulosic materials in consequence of the pretreatment could be observed by electron microscopy. Cryocrushing was highly efficient in improving the saccharification of the holocellulose component of the plant biomasses (from 4.3% to 54.1% for eucalyptus sawdust and from 3.9% to 40.6% for rice hull). It is important to emphasize that it consists in a simple operation with low requirements of water and chemicals, no corrosion, no release of products such as soluble phenolics, furfural and hydroxymethylfurfural and no waste generation. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2016-04-01

    To improve the reducing sugar production from catalpa sawdust, thermo-chemical pretreatments were examined and the chemicals used including NaOH, Ca(OH)2, H2SO4, and HCl. The hemicellulose solubilization and cellulose crystallinity index (CrI) were significantly increased after thermo-alkaline pretreatments, and the thermo-Ca(OH)2 pretreatment showed the best improvement for reducing sugar production comparing to other three pretreatments. The conditions of thermo-Ca(OH)2 pretreatment and enzymatic hydrolysis were systematically optimized. Under the optimal conditions, the reducing sugar yield increased by 1185.7% comparing to the control. This study indicates that the thermo-Ca(OH)2 pretreatment is ideal for the saccharification of catalpa sawdust and that catalpa sawdust is a promising raw material for biofuel. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. 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.

  3. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Enzyme recycling in a simultaneous and separate saccharification and fermentation of corn stover: a comparison between the effect of polymeric micelles of surfactants and polypeptides.

    PubMed

    Eckard, Anahita Dehkhoda; Muthukumarappan, Kasiviswanathan; Gibbons, William

    2013-03-01

    The efficacy of enzyme recycling in simultaneous (SSF) and separate (SHF) saccharification and fermentation of corn stover was evaluated with the use of novel enzyme stabilizers of casein, Tween20 and polymeric micelles (PMs) of polyethylene glycol (PEG)-casein and PEG-Tween20. Amphiphiles were added to maximize the percentage of enzyme remaining in fermented liquor that could be recycled twice back into the process. With no additive, in SHF the ethanol yield was declined by 64.0% and 80.0% after the first and second recycling, respectively. Application of PMs of PEG-casein in one cycle of SHF significantly improved the theoretical ethanol yield from 0.49 ± 0.00 to 0.91 ± 0.00 g/g compared to when only casein (0.66 ± 0.00 g/g), Tween 20 (0.53 ± 0.00 g/g) and Tween 20-PEG (0.77 ± 0.08 g/g) were used. PMs of PEG-Tween and PEG-casein also improved enzyme recycling, such that the ethanol yield was improved by 50% and 108% beyond that obtained with Tween and casein, respectively.

  5. 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

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

    PubMed

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

    2015-01-01

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

  7. 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.

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

    PubMed Central

    2014-01-01

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

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

    PubMed

    Rico, Alejandro; Rencoret, Jorge; Del Río, José C; Martínez, Angel T; Gutiérrez, Ana

    2014-01-08

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

  10. 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%.

  11. Bacterial cellulose production from cotton-based waste textiles: enzymatic saccharification enhanced by ionic liquid pretreatment.

    PubMed

    Hong, Feng; Guo, Xiang; Zhang, Shuo; Han, Shi-fen; Yang, Guang; Jönsson, Leif J

    2012-01-01

    Cotton-based waste textiles were explored as alternative feedstock for production of bacterial cellulose (BC) by Gluconacetobacter xylinus. The cellulosic fabrics were treated with the ionic liquid (IL) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl). [AMIM]Cl caused 25% inactivation of cellulase activity at a concentration as low as of 0.02 g/mL and decreased BC production during fermentation when present in concentrations higher than 0.0005 g/mL. Therefore, removal of residual IL by washing with hot water was highly beneficial to enzymatic saccharification as well as BC production. IL-treated fabrics exhibited a 5-7-fold higher enzymatic hydrolysis rate and gave a seven times larger yield of fermentable sugars than untreated fabrics. BC from cotton cloth hydrolysate was obtained at an yield of 10.8 g/L which was 83% higher than that from the culture grown on glucose-based medium. The BC from G. xylinus grown on IL-treated fabric hydrolysate had a 79% higher tensile strength than BC from glucose-based culture medium which suggests that waste cotton pretreated with [AMIM]Cl has potential to serve as a high-quality carbon source for BC production.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. 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.

  14. 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.

  15. Microwave assisted alkaline pretreatment to enhance enzymatic saccharification of catalpa sawdust.

    PubMed

    Jin, Shuguang; Zhang, Guangming; Zhang, Panyue; Li, Fan; Wang, Siqi; Fan, Shiyang; Zhou, Shuqiong

    2016-12-01

    Catalpa sawdust, a promising biofuel production biomass, was pretreated by microwave-water, -NaOH, and -Ca(OH)2 to enhance enzymatic digestibility. After 48h enzymatic hydrolysis, microwave-Ca(OH)2 pretreated sample showed the highest reducing sugar yield. The content of hemicellulose and lignin in catalpa sawdust decreased after microwave-alkali pretreatment. SEM observation showed that the catalpa sawdust surface with microwave-Ca(OH)2 pretreatment suffered the most serious erosion. Crystallinity index of catalpa sawdust increased after all three kinds of pretreatment. The optimum conditions of microwave-Ca(OH)2 pretreatment were particle size of 40mesh, Ca(OH)2 dosage of 2.25% (w/v), microwave power of 400W, pretreatment time of 6min, enzyme loading of 175FPU/g, and hydrolysis time of 96h, and the reducing sugar yield of microwave-Ca(OH)2 pretreated catalpa sawdust reached 402.73mg/g, which increased by 682.15% compared with that of raw catalpa sawdust. The catalpa sawdust with microwave-Ca(OH)2 pretreatment is promising for biofuel production with great potential.

  16. 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

  17. 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

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

    PubMed

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

    2016-10-03

    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.

  19. 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.

  20. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Pervez, Sidra; Aman, Afsheen; Iqbal, Samina; Siddiqui, Nadir Naveed; Ul Qader, Shah Ali

    2014-05-29

    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. 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. 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.

  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. Optimization of saccharification potential of recombinant xylanase from Bacillus licheniformis.

    PubMed

    Aftab, Muhammad N; Zafar, Asma; Iqbal, Irfana; Kaleem, Afshan; Zia, Khalid M; Awan, Ali R

    2017-09-08

    Saccharification potential of xylanase enzyme cloned from Bacillus licheniformis into E. coli BL21 (DE3) was evaluated against plant biomass for the production of bioethanol. The expression of cloned gene was studied and conditions were optimized for its large scale production. The parameters effecting enzyme production were examined in a fermenter. Recombinant xylanase has the ability to breakdown birchwood xylan to release xylose as well as the potential to treat plant biomass, such as wheat straw, rice straw, and sugarcane bagass. The saccharification ability of this enzyme was optimized by studying various parameters. The maximum saccharification percentage (84%) was achieved when 20 units of recombinant xylanase were used with 8% sugarcane bagass at 50°C and 120 rpm after 6 hours of incubation. The results indicated that the bioconversion of natural biomass by recombinant xylanase into simple sugars can be used for biofuel (bioethanol) production. This process can replace the use of fossil fuels, and the use of bioethanol can significantly reduce the emission of toxic gases. Future directions regarding pre-treatment of cellulosic and hemicellulosic biomass and other processes that can reduce the cost and enhance the yield of biofuels are briefly discussed.

  5. Mechanism of rate enhancement of wood fiber saccharification by cationic polyelectrolytes.

    PubMed

    Mora, Sandeep; Lu, Jian; Banerjee, Sujit

    2011-09-01

    Cationic polyelectrolytes can increase the cellulase-induced hydrolysis rates of bleached wood fiber. We show that the polymer associates mainly with the amorphous region of fiber and acts principally on endoglucanase. Fiber/water partitioning of the enzyme follows a Langmuir isotherm for the untreated fiber but a Freundlich isotherm is obeyed for the polymer-treated fiber.

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

    Treesearch

    Zhaojiang Wang; JY Zhu; Yingjuan Fu; Menghua Qin; Zhiyong Shao; Jungang Jiang; Fang Yang

    2013-01-01

    Thermochemical pretreatment of lignocellulose is crucial to bioconversion in the fields of biorefinery and biofuels. However, the enzyme inhibitors in pretreatment hydrolysate make solid substrate washing and hydrolysate detoxification indispensable prior to enzymatic hydrolysis. Sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL) is a relatively...

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

    PubMed

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

    2012-06-01

    Lignocellulosic biomass is utilized as a renewable feedstock in various agro-industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these 'DP reducers' is achieved by expressing a bacterial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side-chain of hydroxycinnamoyl-CoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild-type plants. However, cell wall characterization of extract-free stems by thioacidolysis and by 2D-NMR revealed an increased amount of unusual C₆C₁ lignin monomers most likely linked with lignin as end-groups. Moreover the analysis of lignin isolated from these plants using size-exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C₆C₁ monomers ('DP reducers') into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis. © 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied

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

    USDA-ARS?s Scientific Manuscript database

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

  9. Enhanced biomass delignification and enzymatic saccharification of canola straw by steam-explosion pretreatment.

    PubMed

    Garmakhany, Amir Daraei; Kashaninejad, Mahdi; Aalami, Mehran; Maghsoudlou, Yahya; Khomieri, Mortza; Tabil, Lope G

    2014-06-01

    In recent decades, bioconversion of lignocellulosic biomass to biofuel (ethanol and biodiesel) has been extensively investigated. The three main chemical constituents of biomass are cellulose, hemicellulose and lignin. Cellulose and hemicellulose are polysaccharides of primarily fermentable sugars, glucose and xylose respectively. Hemicellulose also includes small fermentable fractions of arabinose, galactose and mannose. The main issue in converting lignocellulosic biomass to fuel ethanol is the accessibility of the polysaccharides for enzymatic breakdown into monosaccharides. This study focused on the use of steam explosion as the pretreatment method for canola straw as lignocellulosic biomass. Result showed that steam explosion treatment of biomass increased cellulose accessibility and it hydrolysis by enzyme hydrolysis. Following 72 h of enzyme hydrolysis, a maximum cellulose conversion to glucose yield of 29.40% was obtained for the steam-exploded sample while the control showed 11.60% glucose yields. Steam explosion pretreatment increased glucose production and glucose yield by 200% and 153.22%, respectively, compared to the control sample. The crystalline index increased from 57.48% in untreated canola straw to 64.72% in steam-exploded samples. Steam explosion pretreatment of biomass increased cellulose accessibility, and enzymatic hydrolysis increased glucose production and glucose yield of canola straw. © 2013 Society of Chemical Industry.

  10. 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

  11. Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification. [Trichoderma reesei

    SciTech Connect

    Gould, J.M.

    1984-01-01

    Approximately one-half of the lignin and most of the hemicellulose present in agricultural residues such as wheat straw and corn stover are solubilized when the residue is treated at 25/sup 0/C in an alkaline solution of hydrogen peroxide. The delignification reaction is most efficient when the ratio of hydrogen peroxide to substrate is at least 0.25 (w/w) and the pH is 11.5. The supernatant fraction from a given pretreatment, after addition of makeup peroxide and readjustment of the pH, can be recycled to treat at least six additional batches of substrate, resulting in a substantial concentration of hemicellulose and soluble lignin degradation products. Hydrolysis of the insoluble fraction with Trichoderma reesei cellulase after alkline peroxide treatment yields glucose with almost 100% efficiency, based upon the cellulose content of the residue before treatment. These data indicate that alkaline peroxide pretreatment is a simple and efficient method for enhancing the enzymatic digestibility of lignocellulosic crop residues to levels approaching the theoretical maximum.

  12. OsCESA9 conserved-site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice

    DOE PAGES

    Li, Fengcheng; Xie, Guosheng; Huang, Jiangfeng; ...

    2017-03-15

    Genetic modification of plant cell walls has been posed to reduce lignocellulose recalcitrance for enhancing biomass saccharification. Several dozen CESA mutants have been reported since cellulose synthase (CESA) gene was first identified, but almost all mutants exhibit the defective phenotypes in plant growth and development. Here, the rice (Oryza sativa) Osfc16 mutant with substitutions (W481C, P482S) at P-CR conserved site in CESA9 shows a slightly affected plant growth and higher biomass yield by 25%–41% compared with wild type (Nipponbare, a japonica variety). Chemical and ultrastructural analyses indicate that Osfc16 has a significantly reduced cellulose crystallinity (CrI) and thinner secondary cellmore » walls compared with wild type. CESA co-IP detection, together with implementations of a proteasome inhibitor (MG132) and two distinct cellulose inhibitors (Calcofluor, CGA), shows that CESA9 mutation could affect integrity of CESA4/7/9 complexes, which may lead to rapid CESA proteasome degradation for low-DP cellulose biosynthesis. These may reduce cellulose CrI, which improves plant lodging resistance, a major and integrated agronomic trait on plant growth and grain production, and enhances biomass enzymatic saccharification by up to 2.3-fold and ethanol productivity by 34%–42%. Our study has for the first time reported a direct modification for the low-DP cellulose production that has broad applications in biomass industries.« less

  13. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharina japonica.

    PubMed

    Jang, Ji-Suk; Cho, YuKyeong; Jeong, Gwi-Taek; Kim, Sung-Koo

    2012-01-01

    Ethanol was produced using the simultaneous saccharification and fermentation (SSF) method with macroalgae polysaccharide from the seaweed Saccharina japonica (Sea tangle, Dasima) as biomass. The seaweed was dried by hot air, ground with a hammer mill and filtered with a 200-mesh sieve prior to pretreatment. Saccharification was carried out by thermal acid hydrolysis with H(2)SO(4) and the industrial enzyme, Termamyl 120 L. To increase the yield of saccharification, isolated marine bacteria were used; the optimal saccharification conditions were 10% (w/v) seaweed slurry, 40 mM H(2)SO(4) and 1 g dcw/L isolated Bacillus sp. JS-1. Using this saccharification procedure, the reducing sugar concentration and viscosity were 45.6 ± 5.0 g/L and 24.9 cp, respectively, and the total yield of the saccharification with optimal conditions and S. japonica was 69.1%. Simultaneous saccharification and fermentation was carried out for ethanol production. The highest ethanol concentration, 7.7 g/L (9.8 ml/L) with a theoretical yield of 33.3%, was obtained by SSF with 0.39 g dcw/L Bacillus sp. JS-1 and 0.45 g dcw/L of the yeast, Pichia angophorae KCTC 17574.

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

    PubMed

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

    2013-10-10

    Malt hydrolyzing enzymes and yeast glycolytic and fermentation enzymes in the waste from beer fermentation broth (WBFB) were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A new 'one-pot consecutive batch strategy' was developed for efficient bio-ethanol production by simultaneous saccharification and fermentation (SSF) using WBFB without additional enzymes, microbial cells, or carbohydrates. Bio-ethanol production was conducted in batches using WBFB supernatant in the first phase at 25-67°C and 50rpm, followed by the addition of 3% WBFB solid residue to the existing culture broth in the second phase at 67°C. The ethanol production increased from 50 to 102.5g/L when bare supernatant was used in the first phase, and then to 219g ethanol/L in the second phase. The amount of ethanol obtained using this strategy was almost equal to that obtained using the original WBFB containing 25% solid residue at 33°C, and more than double that obtained when bare supernatant was used. Microscopic and gel electrophoresis studies revealed yeast cell wall degradation and secretion of cellular material into the surrounding medium. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) supported the existence of enzymes in WBFB involved in bioethanol production at elevated temperatures. The results of this study will provide insight for the development of new strategies for biofuel production. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. 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.

  17. Enhancement of In Situ Enzymatic Saccharification of Corn Stover by a Stepwise Sodium Hydroxide and Organic Acid Pretreatment.

    PubMed

    Qing, Qing; Guo, Qi; Zhou, Linlin; He, Yucai; Wang, Liqun; Zhang, Yue

    2017-01-01

    A stepwise pretreatment method that combines sodium hydroxide and organic acid pretreatments was proposed and investigated to maximize the recovery of main constituents of lignocellulose. The sodium hydroxide pretreatment was firstly optimized by a designed orthogonal experiment with the optimum pretreatment conditions determined as 1 wt% NaOH at 70 °C for 1 h, and 60.42 % of lignin was successfully removed during this stage. In the second stage, 0.5 % acetic acid was selected to pretreat the first-stage solid residue at 80 °C for 40 min in order to decompose hemicelluloses to soluble oligomers or monomers. Then, the whole slurry was subjected to in situ enzymatic saccharification by cellullase with a supplementation of xylanase to further degrade the xylooligosaccharides generated during the acetic acid pretreatment. The maximum reducing sugar and glucose yields achieved were 20.74 and 12.03 g/L, respectively. Furthermore, rapid ethanol fermentation and a yield of 80.3 % also testified this pretreatment method, and the in situ saccharification did not bring any negative impact on ethanol fermentation and has a broad application prospect.

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

    SciTech Connect

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

    2010-01-01

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

  19. Fast enzymatic saccharification of switchgrass after pretreatment with ionic liquids.

    PubMed

    Zhao, Hua; Baker, Gary A; Cowins, Janet V

    2010-01-01

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

  20. OsCESA9 conserved-site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice.

    PubMed

    Li, Fengcheng; Xie, Guosheng; Huang, Jiangfeng; Zhang, Ran; Li, Yu; Zhang, Miaomiao; Wang, Yanting; Li, Ao; Li, Xukai; Xia, Tao; Qu, Chengcheng; Hu, Fan; Ragauskas, Arthur J; Peng, Liangcai

    2017-09-01

    Genetic modification of plant cell walls has been posed to reduce lignocellulose recalcitrance for enhancing biomass saccharification. Since cellulose synthase (CESA) gene was first identified, several dozen CESA mutants have been reported, but almost all mutants exhibit the defective phenotypes in plant growth and development. In this study, the rice (Oryza sativa) Osfc16 mutant with substitutions (W481C, P482S) at P-CR conserved site in CESA9 shows a slightly affected plant growth and higher biomass yield by 25%-41% compared with wild type (Nipponbare, a japonica variety). Chemical and ultrastructural analyses indicate that Osfc16 has a significantly reduced cellulose crystallinity (CrI) and thinner secondary cell walls compared with wild type. CESA co-IP detection, together with implementations of a proteasome inhibitor (MG132) and two distinct cellulose inhibitors (Calcofluor, CGA), shows that CESA9 mutation could affect integrity of CESA4/7/9 complexes, which may lead to rapid CESA proteasome degradation for low-DP cellulose biosynthesis. These may reduce cellulose CrI, which improves plant lodging resistance, a major and integrated agronomic trait on plant growth and grain production, and enhances biomass enzymatic saccharification by up to 2.3-fold and ethanol productivity by 34%-42%. This study has for the first time reported a direct modification for the low-DP cellulose production that has broad applications in biomass industries. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  1. 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. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Saccharification of Okara fiber by plant dietary fiber hydrolases.

    PubMed

    Matsuo, Masako

    2004-08-01

    In this paper, the saccharification process of okara fiber with pectinase, xylanase and cellulase was investigated as a preliminary attempt to effectively utilize okara fiber. The solubilization of okara fiber was raised in proportion to the number of enzymes used; that is, by a single enzyme, two enzymes and three enzymes, in that order. The saccharification of okara fiber was much more completed by a combination of pectinase, xylanase and cellulase than by using individual preparations. This multi-enzyme system liberated sugars equivalent to 80% of the original okara fiber by weight. Moreover the structure of okara fiber gradually disintegrated by hydrolytic reactions with pectinase, xylanase and cellulase in turns. These results suggest that the saccharification of okara fiber progresses by the cooperative action of pectinase, xylanase and cellulase.

  3. Enhancement in ionic liquid tolerance of cellulase immobilized on PEGylated graphene oxide nanosheets: Application in saccharification of lignocellulose.

    PubMed

    Xu, Jiaxing; Sheng, Zhenhuan; Wang, Xinfeng; Liu, Xiaoyan; Xia, Jun; Xiong, Peng; He, Bingfang

    2016-01-01

    The objective of the present work was to improve ionic liquid (IL) tolerance of cellulase based on the exploration of functional nanoscale carriers for potential application in lignocellulosic biorefinery. PEGylated graphene oxide (GO) composite was successfully fabricated by chemical binding of 4-arm-PEG-NH2 and GO and applied to the immobilization of cellulase. The PEGylated GO-Cellulase retained 61% of the initial activity in 25% (w/v) 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) while free cellulase only retained 2%. The IL stability was enhanced more than 30 times. The relatively minor change in Km value (from 2.7 to 3.2mgmL(-1)) after the immobilization suggested that PEGylated GO-Cellulase was capable of closely mimicking the performance of free enzyme. After treating rice straw with [Bmim][Cl] and dilution to a final IL concentration of 15% (w/v), the slurry was directly hydrolyzed using PEGylated GO-Cellulase without IL removing and a high hydrolysis rate of 87% was achieved. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. 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.

  5. Expression of a mutated SPT15 gene in Saccharomyces cerevisiae enhances both cell growth and ethanol production in microaerobic batch, fed-batch, and simultaneous saccharification and fermentations.

    PubMed

    Seong, Yeong-Je; Park, Haeseong; Yang, Jungwoo; Kim, Soo-Jung; Choi, Wonja; Kim, Kyoung Heon; Park, Yong-Cheol

    2017-05-01

    The SPT15 gene encodes a Saccharomyces cerevisiae TATA-binding protein, which is able to globally control the transcription levels of various metabolic and regulatory genes. In this study, a SPT15 gene mutant (S42N, S78R, S163P, and I212N) was expressed in S. cerevisiae BY4741 (BSPT15-M3), of which effects on fermentative yeast properties were evaluated in a series of culture types. By applying different nitrogen sources and air supply conditions in batch culture, organic nitrogen sources and microaerobic condition were decided to be more favorable for both cell growth and ethanol production of the BSPT15-M3 strain than the control S. cerevisiae BY4741 strain expressing the SPT15 gene (BSPT15wt). Microaerobic fed-batch cultures of BSPT15-M3 with glucose shock in the presence of high ethanol content resulted in a 9.5-13.4% higher glucose consumption rate and ethanol productivity than those for the BSPT15wt strain. In addition, BSPT15-M3 showed 4.5 and 3.9% increases in ethanol productivity from cassava hydrolysates and corn starch in simultaneous saccharification and fermentation processes, respectively. It was concluded that overexpression of the mutated SPT15 gene would be a potent strategy to develop robust S. cerevisiae strains with enhanced cell growth and ethanol production abilities.

  6. 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.

  7. Enzymes Enhance Biofilm Removal Efficiency of Cleaners.

    PubMed

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

    2016-06-01

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

  8. A comparison of the production of ethanol between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using unpretreated cassava pulp and enzyme cocktail.

    PubMed

    Zhu, Mingjun; Li, Ping; Gong, Xinfang; Wang, Jufang

    2012-01-01

    The processes of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were employed using Saccharomyces cerevisiae for the production of ethanol from cassava pulp without any pretreatment. A combination of amylase, cellulase, cellobiase, and glucoamylase produced the highest levels of ethanol production in both the SHF and the SSF method. A temperature of 37 °C, a pH of 5.0, and an inoculum size of 6% were the optimum conditions for SSF. For the batch process at a pulp concentration of 20%, ethanol production levels from SHF and SSF were the highest, at 23.51 and 34.67 g L(-1) respectively, but in the fed-batch process, the levels of ethanol production from SHF and SSF rose to 29.39 and 43.25 g L(-1) respectively, which were 25% and 24.7% higher than those of the batch process. Thus SSF using the fed-batch provided a more efficient method for the utilization of cassava pulp.

  9. 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.

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

    PubMed

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

    2016-04-20

    Cotton stalk is a potential biomass for bioethanol production, while the conversion of direct saccharification or biotransformation of cotton stalk is extremely low due to the recalcitrant nature of lignocellulose. To enhance the enzymatic conversion of cotton stalks, the enzymatic saccharification parameters of high pressure assist-alkali pretreatment (HPAP) cotton stalk were optimized in the present study. Results indicated that a maximum reducing sugar yield of 54.7g/100g dry biomass cellulose was achieved at a substrate concentration of 2%, 100rpm agitation, 0.6g/g enzyme loading, 40°C hydrolysis temperature, 50h saccharification time, and pH 5.0. Scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy were used to identify structural changes in native, pretreated biomass and hydrolyzed residues. Structural analysis revealed large part of amorphous cellulose and partial crystalline cellulose in the HPAP cotton stalk were hydrolyzed during enzymatic treatment. HPAP cotton stalk can be used as a potential feed stock for bioethanol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. 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.

  12. Substrate catalysis enhances single-enzyme diffusion.

    PubMed

    Muddana, Hari S; Sengupta, Samudra; Mallouk, Thomas E; Sen, Ayusman; Butler, Peter J

    2010-02-24

    We show that diffusion of single urease enzyme molecules increases in the presence of urea in a concentration-dependent manner and calculate the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concentrations ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local molecular pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme molecule and lay the foundation for a further understanding of biological force generation and the development of enzyme-driven nanomotors.

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

    PubMed

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

    2015-11-01

    The hydrolysis/fermentation of wheat straw and the adsorption/desorption/deactivation of cellulases were studied using Cellic(®) CTec2 (Cellic) and Celluclast mixed with Novozyme 188. The distribution of enzymes - cellobiohydrolase I (Cel7A), endoglucanase I (Cel7B) and β-glucosidase - of the two formulations between the residual substrate and supernatant during the course of enzymatic hydrolysis and fermentation was investigated. The potential of recyclability using alkaline wash was also studied. The efficiency of hydrolysis with an enzyme load of 10 FPU/g cellulose reached >98% using Cellic(®) CTec2, while for Celluclast a conversion of 52% and 81%, was observed without and with β-glucosidase supplementation, respectively. The decrease of Cellic(®) CTec2 activity observed along the process was related to deactivation of Cel7A rather than of Cel7B and β-glucosidase. The adsorption/desorption profiles during hydrolysis/fermentation revealed that a large fraction of active enzymes remained adsorbed to the solid residue throughout the process. Surprisingly, this was the case of Cel7A and β-glucosidase from Cellic, which remained adsorbed to the solid fraction along the entire process. Alkaline washing was used to recover the enzymes from the solid residue. This method allowed efficient recovery of Celluclast enzymes; however, this may be achieved only when minor amounts of cellulose remain present. Regarding the Cellic formulation, neither the presence of cellulose nor lignin restricted an efficient desorption of the enzymes at alkaline pH. This work shows that the recycling strategy must be customized for each particular formulation, since the enzymes found e.g. in Cellic and Celluclast bear quite different behaviour regarding the solid-liquid distribution, stability and cellulose and lignin affinity. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. 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.

  15. A mechanistic model for enzymatic saccharification of cellulose using continuous distribution kinetics II: cooperative enzyme action, solution kinetics, and product inhibition.

    PubMed

    Griggs, Andrew J; Stickel, Jonathan J; Lischeske, James J

    2012-03-01

    The projected cost for the enzymatic hydrolysis of cellulosic biomass continues to be a barrier for the commercial production of liquid transportation fuels from renewable feedstocks. Predictive models for the kinetics of the enzymatic reactions will enable an improved understanding of current limitations, such as the slow-down of the overall conversion rate, and may point the way for more efficient utilization of the enzymes in order to achieve higher conversion yields. A mechanistically based kinetic model for the enzymatic hydrolysis of cellulose was recently reported in Griggs et al. (2011) (Part I). In this article (Part II), the enzyme system is expanded to include solution-phase kinetics, particularly cellobiose-to-glucose conversion by β-glucosidase (βG), and novel adsorption and product inhibition schemes have been incorporated, based on current structural knowledge of the component enzymes. Model results show cases of cooperative and non-cooperative hydrolysis for an enzyme system consisting of EG(I) and CBH(I). The model is used to explore various potential rate-limiting phenomena, such as substrate accessibility, product inhibition, sterically hindered enzyme adsorption, and the molecular weight of the cellulose substrate.

  16. 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.

  17. 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.

  18. Enhancing Enzyme Activity and Immobilization in Nanostructured Inorganic-Enzyme Complexes.

    PubMed

    Lang, Xuye; Zhu, Lingling; Gao, Yingning; Wheeldon, Ian

    2017-09-12

    Understanding the chemical and physical interactions at the interface of protein surfaces and inorganic crystals has important implications in the advancement of immobilized enzyme catalysis. Recently, enzyme-inorganic hybrid complexes have been demonstrated as effective materials for enzyme immobilization. The precipitation of phosphate nanocrystals in the presence of enzymes creates enzyme-Cu3(PO4)2·3H2O particles with high surface-to-volume ratios, enhanced activity, and increased stability. Here, we begin to develop a mechanistic understanding of enzyme loading in such complexes. Using a series of enzymes including horseradish peroxidase (HRP), a thermostable alcohol dehydrogenase (AdhD), diaphorase, catalase, glucose oxidase (GOx), and the protein bovine serum albumin (BSA), we identified a correlation between particle synthesis temperature, overall enzyme charge, and enzyme loading. The model enzyme HRP has a high predicted pI of ∼7.5 and maintains an overall positive charge under the synthesis conditions, phosphate buffer pH 7.4. HRP loading in HRP-Cu3(PO4)2 complexes was enhanced by 4.2-fold when synthesis was carried out at 37 °C in comparison with synthesis at 4 °C. HRP loading was further enhanced with synthesis at pH 8.0, correlating with a decrease in overall enzyme charge. Proteins with lower predicted pI values and negative overall charge (AdhD, pI of 5.6; diaphorase, pI of 6.8; GOx, pI of 5.2; catalase, pI of 6.9; and, BSA, pI of 5.7) exhibited higher enzyme loadings with 4 °C synthesis, 2.7-, 2.6-, 2.5-, 1.8-, and 1.7-fold protein loading enhancements, respectively. Using HRP as a model system, we also demonstrate that activity increased concomitantly with enzyme loading, and that particle nanostructure was minimally affected by synthesis temperature. Combined, the results presented here demonstrate the control of enzyme loading in enzyme-inorganic particles opening up new possibilities in enzyme and multienzyme catalysis.

  19. 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

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

    PubMed

    Bey, Mathieu; Berrin, Jean-Guy; Poidevin, Laetitia; Sigoillot, Jean-Claude

    2011-12-28

    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. 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. We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

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

    PubMed

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

    2016-08-01

    In this work, substrates prepared from thermo-mechanical treatment of Pinus radiata chips were vibratory ball milled for different times. In subsequent enzymatic hydrolysis, percent glucan conversion passed through a maximum value at a milling time of around 120min and then declined. Scanning electron microscopy revealed breakage of fibers to porous fragments in which lamellae and fibrils were exposed during ball milling. Over-milling caused compression of the porous fragments to compact globular particles with a granular texture, decreasing accessibility to enzymes. Carbon-13 NMR spectroscopy showed partial loss of interior cellulose in crystallites, leveling off once fiber breakage was complete. A mathematical model based on observed micromorphological changes supports ball milling mechanism. At a low enzyme loading of 2FPU/g of substrate and milling time of 120min gave a total monomeric sugar yield of 306g/kg of pulp which is higher than conventional pretreatment method such as steam exploded wood. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Enhanced bio-ethanol production from cellulosic materials by semi-simultaneous saccharification and fermentation using high temperature resistant Saccharomyces cerevisiae TJ14.

    PubMed

    Shahsavarani, Hosein; Hasegawa, Daisuke; Yokota, Daiki; Sugiyama, Minetaka; Kaneko, Yoshinobu; Boonchird, Chuenchit; Harashima, Satoshi

    2013-01-01

    The capability of multi-stress-tolerant Saccharomyces cerevisiae diploid strain TJ14 for the production of cellulosic bio-ethanol by semi-simultaneous saccharification and fermentation (SSSF) technology was evaluated under high-temperature conditions. At 39°C, the TJ14 produced 45 g/l ethanol by SSSF of 100 g (w/v)/l cellulose - a significantly higher concentration than reported in prevailing literature. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. 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.

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

    PubMed

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

    2007-03-06

    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.

  5. 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

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

    PubMed

    Alrumman, Sulaiman A

    2016-01-01

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

  7. Enhanced simultaneous saccharification and fermentation of pretreated beech wood by in situ treatment with the white rot fungus Irpex lacteus in a membrane aerated biofilm reactor.

    PubMed

    Brethauer, Simone; Robert Lawrence, Shahab; Michael Hans-Peter, Studer

    2017-03-18

    The aim of the present study was to investigate the combination of steam pretreatment and biological treatment with lignin degrading fungal strains in order to enable efficient bioprocessing of beech wood to ethanol. In a sequential process of steam and fungal pretreatment followed by enzymatic hydrolysis, Irpex lacteus almost doubled the glucose yield for mildly pretreated beech wood, but could not improve yields for more severely pretreated substrates. However, when simultaneous saccharification and fermentation is combined with in situ I. lacteus treatment, which is enabled by the application of a membrane aerated biofilm reactor, ethanol yields of optimally steam pretreated beech could be improved from 65 to 80%. Generally, in situ fungal treatment during bioprocessing of lignocellulose is an interesting method to harness the versatile abilities of white rot fungi.

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

    PubMed

    Sharma, Sandeep; Horn, Svein Jarle

    2016-08-01

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

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

    PubMed

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

    2014-01-01

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

  10. 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

  11. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Scaffoldless engineered enzyme assembly for enhanced methanol utilization

    DOE PAGES

    Price, J. Vincent; Chen, Long; Whitaker, W. Brian; ...

    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

  13. Scaffoldless engineered enzyme assembly for enhanced methanol utilization.

    PubMed

    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 channeling 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.

  14. Enzymatic membrane reactor for full saccharification of ionic liquid-pretreated microcrystalline cellulose.

    PubMed

    Lozano, Pedro; Bernal, Berenice; Jara, Antonio G; Belleville, Marie-Pierre

    2014-01-01

    Ultrafiltration reactors based on polymeric or ceramic membranes were shown to be suitable catalytic systems for fast enzymatic saccharification of cellulose, allowing the full recovery and reuse of enzymes. By pre-treating cellulose with the IL 1-butyl-3-methylimidazolium chloride, the suitability of this substrate for enzymatic saccharification in a reactor based on polymeric ultrafiltration membranes was demonstrated, leading to 95% cellulose hydrolysis in 4h at 50°C. The filtration process gave a clear glucose solution (up to 113 mM) at constant permeate flow (24.7 L h(-1) m(-2)), allowing the enzyme to be reused for 9 operation cycles under semi-continuous operation, without any loss of enzyme activity. Under continuous operation mode and using ceramic ultrafiltration membranes at different residence times, the enzymatic reactor showed constant profiles in both the permeate flow rate and the glucose concentration, demonstrating the excellent suitability of the proposed approach for the saccharification of cellulose.

  15. 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

  16. Fed-Batch Enzymatic Saccharification of High Solids Pretreated Lignocellulose for Obtaining High Titers and High Yields of Glucose.

    PubMed

    Jung, Young Hoon; Park, Hyun Min; Kim, Dong Hyun; Yang, Jungwoo; Kim, Kyoung Heon

    2017-01-11

    To reduce the distillation costs of cellulosic ethanol, it is necessary to produce high sugar titers in the enzymatic saccharification step. To obtain high sugar titers, high biomass loadings of lignocellulose are necessary. In this study, to overcome the low saccharification yields and the low operability of high biomass loadings, a fed-batch saccharification process was developed using an enzyme reactor that was designed and built in-house. After optimizing the cellulase and biomass feeding profiles and the agitation speed, 132.6 g/L glucose and 76.0% theoretical maximum glucose were obtained from the 60 h saccharification of maleic acid-pretreated rice straw at a 30% (w/v) solids loading with 15 filter paper units (FPU) of Cellic CTec2/g glucan. This study demonstrated that through the proper optimization of fed-batch saccharification, both high sugar titers and high saccharification yields are possible, even with using the high solids loading (i.e., ≥30%) with the moderate enzyme loading (i.e., <15 FPU/g glucan). These results could be contributed to improving economic feasibility of the high solids saccharification process in cellulosic fuel and chemical production.

  17. 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.

  18. 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.

  19. Enhanced Diffusion of Enzymes that Catalyze Exothermic Reactions.

    PubMed

    Golestanian, Ramin

    2015-09-04

    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.

  20. 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.

  1. 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.

  2. Enhancement of photoassimilate utilization by manipulation of starch regulatory enzymes

    SciTech Connect

    Okita, Thomas W.

    2016-05-11

    maturation to a starch granule. Although Pho1 catalyzes a reversible reaction, our DoE supported studies clearly demonstrated that the kinetic properties of this enzyme strongly favor synthesis of starch and that these catalytic properties are independent of the L80 peptide, a structural domain that is absent in phosphorylases from other organisms. Interesting expression of a Pho1 lacking the L80 peptide enhanced plant growth and seed yields, suggesting that Pho1 has a second function in controlling growth. Overall, results from these biochemical and physiological studies have increased our fundamental understanding on how these important starch regulatory enzymes operate at the molecular level and in planta, which will collectively aid in efforts to increase the utilization of higher plants as a renewable source of energy.

  3. Limiting factors in the simultaneous saccharification and fermentation process for conversion of cellulosic biomass to fuel ethanol

    SciTech Connect

    Philippidis, G.P.; Smith, T.K.

    1995-12-31

    The cellulosic fraction of biomass feedstocks can be converted to ethanol, a promising alternative fuel, using the simultaneous saccharification and fermentation (SSF) process. This process integrates the enzymatic hydrolysis of cellulose to glucose with the fermentation of glucose to ethanol. Its performance depends on the characteristics of the biomass, the quality of the cellulose enzyme complex, and the behavior of the fermentative organism. This study of cellulose conversion progress in batch mode indicates that during the early stage of SSF, cell growth is the rate-determining step in ethanol production. At later times, however, and for most of the duration of the SSF process, enzymatic hydrolysis becomes the limiting factor. Further experimental probing has shown that cellulose accessibility to the enzyme is the key cause of the decreased rate of cellulose hydrolysis. It is therefore concluded that accessibility needs to be enhanced to improve the productivity of the SSF process.

  4. 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.

  5. Modelling of pretreatment and saccharification with different feedstocks and kinetic modeling of sorghum saccharification.

    PubMed

    Prathyusha, N; Kamesh, Reddi; Rani, K Yamuna; Sumana, C; Sridhar, S; Prakasham, R S; Yashwanth, V V N; Sheelu, G; Kumar, M Pradeep

    2016-12-01

    Experiments have been performed for pretreatment of sorghum, wheat straw and bamboo through high temperature alkali pretreatment with different alkaline loading and temperatures, and the data on extent of delignification in terms of the final compositions of cellulose, hemicellulose and lignin have been generated. Further, enzymatic saccharification has been carried out in all the cases to find the extent of conversion possible after 72h. The effect of different operating parameters on the extent of delignification and cellulose conversion are evaluated. This data is employed to develop a generalized multi-feedstock and individual feedstock based models which can be used to determine the extent of delignification and cellulose conversion for any and specific biomass respectively with alkaline pretreatment and similar enzyme conditions as considered in the present study. Also, a kinetic model is developed and validated for sorghum for cellulosic conversion.

  6. Multiple effects of swelling by sodium bicarbonate after delignification on enzymatic saccharification of rice straw.

    PubMed

    Kahar, Prihardi; Taku, Kazuo; Tanaka, Shuzo

    2013-12-01

    The multiple effects of pretreatments by chemical delignification using acidified sodium chlorite (ASC) and swelling using sodium bicarbonate (SB) for enzymatic saccharification of rice straw in bioethanol production have been investigated in this study. The treatment with the combination of ASC three times (3× ASC) first and SB later resulted in the significant reduction in Klason lignin content up to 90% (wt./wt.). By the saccharification of the pretreated rice straw with cellulase enzymes, it was confirmed that SB treatment was an important step in the pretreatment process not only to disintegrate the cellulose structure but also to facilitate the amorphization of the crystalline cellulose as well as the extended removal of integrated lignin. Furthermore, FTIR analyses revealed that the crystal type of cellulose appeared to be changed from type I to type II by SB treatment, thereby increasing the cellulose surface area and making it more accessible to the cellulase enzyme. Conversion rate to sugar was remarkably increased when 3× ASC + SB treatments were applied to untreated rice straw, even though the saccharification of the treated rice straw was performed at a low enzyme loading (1/100, wt.-enzymes/wt.-substrate). Conclusively, rice straw could be saccharified at high yield in short time at low cellulase loading, enables the enzymatic saccharification to be more feasible for practical bioethanol production using rice straw as a substrate. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

    DOEpatents

    Lu, Jue [Okemos, MI; Okeke, Benedict [Montgomery, AL

    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.

  8. 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.

  9. 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.

  10. Modeling and experimental studies on intermittent starch feeding and citrate addition in simultaneous saccharification and fermentation of starch to flavor compounds.

    PubMed

    Chavan, Abhijit R; Raghunathan, Anuradha; Venkatesh, K V

    2009-04-01

    Simultaneous saccharification and fermentation (SSF) is a combined process of saccharification of a renewable bioresource and fermentation process to produce products, such as lactic acid and ethanol. Recently, SSF has been extensively used to convert various sources of cellulose and starch into fermentative products. Here, we present a study on production of buttery flavors, namely diacetyl and acetoin, by growing Lactobacillus rhamnosus on a starch medium containing the enzyme glucoamylase. We further develop a structured kinetics for the SSF process, which includes enzyme and growth kinetics. The model was used to simulate the effect of pH and temperature on the SSF process so as to obtain optimum operating conditions. The model was experimentally verified by conducting SSF using an initial starch concentration of 100 g/L. The study demonstrated that the developed kinetic was able to suggest strategies for improved productivities. The developed model was able to accurately predict the enhanced productivity of flavors in a three stage process with intermittent addition of starch. Experimental and simulations demonstrated that citrate addition can also lead to enhanced productivity of flavors. The developed optimal model for SSF was able to capture the dynamics of SSF in batch mode as well as in a three stage process. The structured kinetics was also able to quantify the effect of multiple substrates present in the medium. The study demonstrated that structured kinetic models can be used in the future for design and optimization of SSF as a batch or a fed-batch process.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag

    PubMed Central

    Shen, Aimee; Lupardus, Patrick J.; Morell, Montse; Ponder, Elizabeth L.; Sadaghiani, A. Masoud; Garcia, K. Christopher; Bogyo, Matthew

    2009-01-01

    We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP6), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms. PMID:19956581

  16. 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.

  17. Enzyme

    MedlinePlus

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

  18. 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.

  19. Engineering the cell wall by reducing de-methyl-esterified homogalacturonan improves saccharification of plant tissues for bioconversion

    PubMed Central

    Lionetti, Vincenzo; Francocci, Fedra; Ferrari, Simone; Volpi, Chiara; Bellincampi, Daniela; Galletti, Roberta; D’Ovidio, Renato; De Lorenzo, Giulia; Cervone, Felice

    2010-01-01

    Plant cell walls represent an abundant, renewable source of biofuel and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification), to be subsequently converted by microorganisms into ethanol or other products, is their recalcitrance to enzymatic saccharification. We investigated whether the structure of pectin that embeds the cellulose-hemicellulose network affects the exposure of cellulose to enzymes and consequently the process of saccharification. Reduction of de-methyl-esterified homogalacturonan (HGA) in Arabidopsis plants through the expression of a fungal polygalacturonase (PG) or an inhibitor of pectin methylesterase (PMEI) increased the efficiency of enzymatic saccharification. The improved enzymatic saccharification efficiency observed in transformed plants could also reduce the need for acid pretreatment. Similar results were obtained in PG-expressing tobacco plants and in PMEI-expressing wheat plants, indicating that reduction of de-methyl-esterified HGA may be used in crop species to facilitate the process of biomass saccharification. PMID:20080727

  20. 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).

  1. The effect of mixing on the liquefaction and saccharification of cellulosic fibers.

    PubMed

    Lavenson, David M; Tozzi, Emilio J; Karuna, Nardrapee; Jeoh, Tina; Powell, Robert L; McCarthy, Michael J

    2012-05-01

    The enzymatic hydrolysis of cellulosic material is a key step in the biochemical routes for production of renewable fuels and chemicals. This must be performed at high solids to be economically viable. High solids operations creates numerous processing challenges, most importantly the limitations due to mass transfer and poor mixing of enzymes in the cellulose suspensions. We use magnetic resonance imaging (MRI), a cylindrical penetrometer, and HPLC to demonstrate the importance of spatial homogeneity in the distribution of enzyme on the rates of liquefaction of the substrate and in the suspension mechanical strength. Our results show that the largest mechanical strength changes occur in a narrow interval of time during the initial stages of conversion. Differences in enzyme concentration distribution occurring at the centimeter-scale produced order of magnitude differences in liquefaction and saccharification rates, supporting the hypothesis that mixing quality has a major influence in both liquefaction and saccharification rates.

  2. 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%.

  3. Understanding the effects of lignosulfonate on enzymatic saccharification of pure cellulose

    Treesearch

    Hongming Lou; Haifeng Zhou; Xiuli Li; Mengxia Wang; J.Y. Zhu; Xueqing Qiu

    2014-01-01

    The effects of lignosulfonate (LS) on enzymatic saccharification of pure cellulose were studied. Four fractions of LS with different molecular weight (MW) prepared by ultrafiltration of a commercial LS were applied at different loadings to enzymatic hydrolysis of Whatman paper under different pH. Using LS fractions with low MW and high degree of sulfonation can enhance...

  4. Ethanosolv Pretreatment of Bamboo with Dilute Acid for Efficient Enzymatic Saccharification

    Treesearch

    Zhiqiang Li; Zehui Jiang; Benhua Fei; Zhiyong Cai; Xuejun Pan

    2012-01-01

    Bamboo is a potential lignocellulosic biomass for the production of bioethanol because of its high cellulose and hemicelluloses content. In this research, ethanosolv pretreatment catalyzed by sulfuric acid was studied in order to enhance enzymatic saccharification of moso bamboo. The addition of 2% (w/w on bamboo) sulfuric acid in water or 75% (v/v) ethanol was...

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

    PubMed Central

    Liang, Youyun; Si, Tong

    2014-01-01

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

  6. 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.

  7. 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.

  8. 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…

  9. 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…

  10. The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production.

    PubMed

    Kim, Hee Taek; Lee, Saeyoung; Kim, Kyoung Heon; Choi, In-Geol

    2012-03-01

    A sugar platform equipped with acetic acid, multiple agarases and neoagarobiose hydrolase (NABH) converted recalcitrant agar polysaccharide into monosugars, which was evaluated by simultaneous saccharification and fermentation (SSF). The sugar platform was divided into chemical liquefaction and enzymatic saccharification. The chemical liquefaction was carried out in mild conditions (using a dilute acetic acid at 80°C for 1-6h) to avoid the production of fermentation inhibitors and hence the highest degree of liquefaction of 95.6% (w/w) was obtained. We mimicked the natural agarolytic pathway using three microbial agarases (Aga16B, Aga50D and DagA) and NABH, and the enzyme system converted 79.1% of agarose to monosugars. The chemical liquefaction and SSF of 30 g/l agarose resulted in 4.4 g/l ethanol concentration and 49.3% of the theoretical ethanol yield to d-galactose. This is the first report on the complete enzymatic conversion of agarose into its monosugars and the SSF of agarose into ethanol.

  11. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. 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.

  13. Temperature-responsive enzyme-polymer nanoconjugates with enhanced catalytic activities in organic media.

    PubMed

    Zhu, Jingying; Zhang, Yifei; Lu, Diannan; Zare, Richard N; Ge, Jun; Liu, Zheng

    2013-07-11

    A general approach for preparing enzyme-polymer nanoconjugates that respond to temperature in organic media is presented. These nanoconjugates readily dissolve in organic solvents for homogenous catalysis at 40 °C and showed greatly enhanced apparent catalytic activities. The recovery of the soluble enzyme-polymer nanoconjugates is accomplished by temperature-induced precipitation.

  14. 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.

  15. Automated saccharification assay for determination of digestibility in plant materials

    PubMed Central

    2010-01-01

    Background Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. Results We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared. Conclusions The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered

  16. Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers.

    PubMed

    Kricka, L J; Schmerfeld-Pruss, D; Edwards, B

    1991-01-01

    Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proenhancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para-iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picomole, respectively. The proenhancer strategy was effective in assays for beta-D-galactosidase, beta-D-glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement.

  17. 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.

  18. Enhancement of enzyme activity in supercritical carbon dioxide via changes in acid-base conditions.

    PubMed

    Harper, Neil; Barreiros, Susana

    2002-01-01

    Enzyme performance is often impaired in supercritical carbon dioxide. We were able to enhance enzyme activity in this medium via changes in acid-base conditions by using ion-exchange materials (solid H(+)/Na(+) buffer pairs and a zeolite), which were selected on the basis of the response of an organosoluble acid-base indicator. The concentration of ion-exchange materials had an important effect on the catalytic activity of subtilisin Carlsberg cross-linked enzyme crystals (CLECs), and this was related to the protonation and hydration states of the enzyme. The buffer Na(2)CO(3)/NaHCO(3) gave the highest enhancement in enzyme activity (by a factor of 54), probably as a result of its high basicity and capacity to counteract the deleterious effect of carbonic acid to a greater extent than the other materials tested.

  19. Exothermicity Is Not a Necessary Condition for Enhanced Diffusion of Enzymes

    NASA Astrophysics Data System (ADS)

    Illien, Pierre; Zhao, Xi; Dey, Krishna K.; Butler, Peter J.; Sen, Ayusman; Golestanian, Ramin

    2017-07-01

    Recent experiments have revealed that the diffusivity of exothermic and fast enzymes is enhanced when they are catalytically active, and different physical mechanisms have been explored and quantified to account for this observation. We perform measurements on the endothermic and relatively slow enzyme aldolase, which also shows substrate-induced enhanced diffusion. We propose a new physical paradigm, which reveals that the diffusion coefficient of a model enzyme hydrodynamically coupled to its environment increases significantly when undergoing changes in conformational fluctuations in a substrate-dependent manner, and is independent of the overall turnover rate of the underlying enzymatic reaction. Our results show that substrate-induced enhanced diffusion of enzyme molecules can be explained within an equilibrium picture, and that the exothermicity of the catalyzed reaction is not a necessary condition for the observation of this phenomenon.

  20. 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.

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

    PubMed

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

    2016-01-01

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

  2. The relation between xyr1 overexpression in Trichoderma harzianum and sugarcane bagasse saccharification performance.

    PubMed

    da Silva Delabona, Priscila; Rodrigues, Gisele Nunes; Zubieta, Mariane Paludetti; Ramoni, Jonas; Codima, Carla Aloia; Lima, Deise Juliana; Farinas, Cristiane Sanchez; da Cruz Pradella, José Geraldo; Seiboth, Bernhard

    2017-03-20

    This work investigates the influence of the positive regulator XYR1 of Trichoderma harzianum on the production of cellulolytic enzymes, using sugarcane bagasse as carbon source. Constitutive expression of xyr1 was achieved under the control of the strong Trichoderma reesei pki1 promoter. Five clones with xyr1 overexpression achieved higher xyr1 expression and greater enzymatic productivity when cultivated under submerged fermentation, hence validating the genetic construction for T. harzianum. Clone 5 presented a relative expression of xyr1 26-fold higher than the parent strain and exhibited 66, 37, and 36% higher values for filter paper activity, xylanase activity, and β-glucosidase activity, respectively, during cultivation in a stirred-tank bioreactor. The overexpression of xyr1 in T. harzianum resulted in an enzymatic complex with significantly improved performance in sugarcane bagasse saccharification, with an enhancement of 25% in the first 24h. Our results also show that constitutive overexpression of xyr1 leads to the induction of several important players in biomass degradation at early (24h) and also late (48h) timepoints of inoculation. However, we also observed that the carbon catabolite repressor CRE1 was upregulated in xyr1 overexpression mutants. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression and suggest an attractive approach for increasing total cellulase productivity in T. harzianum.

  3. Thermophilic fungi as new sources for production of cellulases and xylanases with potential use in sugarcane bagasse saccharification.

    PubMed

    de Cassia Pereira, J; Paganini Marques, N; Rodrigues, A; Brito de Oliveira, T; Boscolo, M; da Silva, R; Gomes, E; Bocchini Martins, D A

    2015-04-01

    To obtain new cellulases and xylanases from thermophilic fungi; evaluate their potential for sugarcane bagasse saccharification. Thirty-two heat-tolerant fungi were isolated from the environment, identified (morphological/molecular tools) and the production of the enzymes was evaluated by solid state fermentation using lignocellulosic materials as substrates. Myceliophthora thermophila JCP 1-4 was the best producer of endoglucanase (357·51 U g(-1) ), β-glucosidase (45·42 U g(-1) ), xylanase (931·11 U g(-1) ) and avicelase (3·58 U g(-1) ). These enzymes were most active at 55-70°C and stable at 30-60°C. Using crude enzymatic extract from M. thermophila JCP 1-4 to saccharify sugarcane bagasse pretreated with microwaves and glycerol, glucose and xylose yields obtained were 15·6 and 35·13% (2·2 and 1·95 g l(-1) ), respectively. All isolated fungi have potential to produce the enzymes; M. thermophila JCP 1-4 enzymatic extract have potential to be better explored in saccharification experiments. Pretreatment improved enzymatic saccharification, as sugar yields were much higher than those obtained from in natura bagasse. Myceliophthora thermophila JCP 1-4 produces avicelase (not commonly found among fungi; important to hydrolyse crystalline cellulose) and a β-glucosidase resistant to glucose inhibition, interesting characteristics for saccharification experiments. © 2015 The Society for Applied Microbiology.

  4. Carboxymethyl cellulase production optimization from newly isolated thermophilic Bacillus subtilis K-18 for saccharification using response surface methodology.

    PubMed

    Irfan, Muhammad; Mushtaq, Qudsia; Tabssum, Fouzia; Shakir, Hafiz Abdullah; Qazi, Javed Iqbal

    2017-12-01

    In this study, a novel thermophilic strain was isolated from soil and used for cellulase production in submerged fermentation using potato peel as sole carbon source. The bacterium was identified by 16S rRNA gene sequencing technology. Central composite design was applied for enhanced production using substrate concentration, inoculum size, yeast extract and pH as dependent variables. Highest enzyme titer of 3.50 ± 0.11 IU/ml was obtained at 2% substrate concentration, 2% inoculum size, 1% yeast extract, pH 5.0, incubation temperature of 50 °C for 24 h of fermentation period. The crude enzyme was characterized having optimum pH and temperature of 7.0 and 50 °C, respectively. The efficiency of enzyme was checked by enzymatic hydrolysis of acid/alkali treated pine needles which revealed that 54.389% saccharification was observed in acid treated pine needles. These results indicated that the cellulase produced by the Bacillus subtilis K-18 (KX881940) could be effectively used for industrial processes particularly for bioethanol production.

  5. 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.

  6. 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.

  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. Enhanced intracellular stability of dextran-horse radish peroxidase conjugate: an approach to enzyme replacement therapy.

    PubMed

    Mumtaz, S; Bachhawat, B K

    1992-09-15

    Horse radish peroxidase (HRP), a mannose-containing glycoprotein was covalently modified by conjugation with dextran. The rapid uptake of HRP by the liver is markedly inhibited by mannan. The uptake of dextran-HRP conjugate by the liver, though lower compared to that of the free enzyme, is also partially inhibited by mannan. Liposomes were therefore used as carriers for delivering the free and the modified HRP to the liver. The dextran-HRP conjugate showed greater stability intracellularly as compared to the free enzyme. The enhanced stability of enzymes upon their extensive glycosylation with nondegradable sugar polymers would be of importance in extending the catalytic life of therapeutically active enzymes and thereby improve their therapeutic potential for the treatment of certain enzyme deficiency disorders.

  9. Enzyme-Enhanced Extraction of Antioxidant Ingredients from Algae.

    PubMed

    Adalbjörnsson, Björn V; Jónsdóttir, Rósa

    2015-01-01

    Marine algae are not only a rich source of dietary fibre, proteins, vitamins, and minerals, but also contain a great variety of secondary metabolites with diverse biological activities. Marine macroalgae are a rich source of various natural antioxidants such as polyphenols, especially phlorotannins (made of polyphloroglucinol units) derived from brown algae, which play an important role in preventing lipid peroxidation. In recent years, a number of potent antioxidant compounds have been isolated and identified from different types of edible seaweeds. Extraction methods commonly used for the isolation of antioxidants are based on conventional water or organic solvent extractions. However, recent advances have shown that enzymatic hydrolysis can achieve higher yield of bioactive compounds from algae. Here we describe a method based on enzymatic hydrolysis which both increases yield and decreases cost associated with organic solvents. This method achieves cell wall disruption and breakdown of internal storage components for more effective release of intracellular bioactive compounds. In addition, hydrolysis of proteins produces peptides which may have antioxidant properties, thus enhancing the bioactivity of the algal extract. The method described can be used for production of extracts from red and brown macroalgal species.

  10. 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.

  11. Pipeline transport and simultaneous saccharification of corn stover.

    PubMed

    Kumar, Amit; Cameron, Jay B; Flynn, Peter C

    2005-05-01

    Pipeline transport of corn stover delivered by truck from the field is evaluated against a range of truck transport costs. Corn stover transported by pipeline at 20% solids concentration (wet basis) or higher could directly enter an ethanol fermentation plant, and hence the investment in the pipeline inlet end processing facilities displaces comparable investment in the plant. At 20% solids, pipeline transport of corn stover costs less than trucking at capacities in excess of 1.4 M drytonnes/yr when compared to a mid range of truck transport cost (excluding any credit for economies of scale achieved in the ethanol fermentation plant from larger scale due to multiple pipelines). Pipelining of corn stover gives the opportunity to conduct simultaneous transport and saccharification (STS). If current enzymes are used, this would require elevated temperature. Heating of the slurry for STS, which in a fermentation plant is achieved from waste heat, is a significant cost element (more than 5 cents/l of ethanol) if done at the pipeline inlet unless waste heat is available, for example from an electric power plant located adjacent to the pipeline inlet. Heat loss in a 1.26 m pipeline carrying 2 M drytonnes/yr is about 5 degrees C at a distance of 400 km in typical prairie clay soils, and would not likely require insulation; smaller pipelines or different soil conditions might require insulation for STS. Saccharification in the pipeline would reduce the need for investment in the fermentation plant, saving about 0.2 cents/l of ethanol. Transport of corn stover in multiple pipelines offers the opportunity to develop a large ethanol fermentation plant, avoiding some of the diseconomies of scale that arise from smaller plants whose capacities are limited by issues of truck congestion.

  12. 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.

  13. 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.

  14. 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

    2016-08-03

    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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. 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.

  16. Enhancement of Drug Delivery: Enzyme-replacement Therapy for Murine Morquio A Syndrome

    PubMed Central

    Tomatsu, Shunji; Montaño, Adriana M; Dung, Vu Chi; Ohashi, Amiko; Oikawa, Hirotaka; Oguma, Toshihiro; Orii, Tadao; Barrera, Luis; Sly, William S

    2010-01-01

    Mucopolysaccharidosis IVA (MPS IVA, Morquio A disease) is an inherited lysosomal storage disorder that features skeletal chondrodysplasia caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Human GALNS was bioengineered with the N-terminus extended by the hexaglutamate sequence (E6) to improve targeting to bone (E6-GALNS). We initially assessed blood clearance and tissue distribution. Next, to assess the effectiveness of storage clearance and reversal of pathological phenotype, a dose of 250 U/g of enzyme was given weekly to Morquio A mice (adults: 12 or 24 weeks, newborn: 8 weeks). Sulfatase modifier factor 1 (SUMF1) was co-transfected to activate the enzyme fully. The E6-GALNS tagged enzyme had markedly prolonged clearance from circulation, giving over 20 times exposure time in blood, compared to untagged enzyme. The tagged enzyme was retained longer in bone, with residual enzyme activity demonstrable at 48 hours after infusion. The pathological findings in adult mice treated with tagged enzyme showed substantial clearance of the storage materials in bone, bone marrow, and heart valves, especially after 24 weekly infusions. Mice treated from the newborn period showed marked reduction of storage materials in tissues investigated. These findings indicate the feasibility of using tagged enzyme to enhance delivery and pathological effectiveness in Morquio A mice. PMID:20332769

  17. Enhancement of drug delivery: enzyme-replacement therapy for murine Morquio A syndrome.

    PubMed

    Tomatsu, Shunji; Montaño, Adriana M; Dung, Vu Chi; Ohashi, Amiko; Oikawa, Hirotaka; Oguma, Toshihiro; Orii, Tadao; Barrera, Luis; Sly, William S

    2010-06-01

    Mucopolysaccharidosis IVA (MPS IVA, Morquio A disease) is an inherited lysosomal storage disorder that features skeletal chondrodysplasia caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Human GALNS was bioengineered with the N-terminus extended by the hexaglutamate sequence (E6) to improve targeting to bone (E6-GALNS). We initially assessed blood clearance and tissue distribution. Next, to assess the effectiveness of storage clearance and reversal of pathological phenotype, a dose of 250 U/g of enzyme was given weekly to Morquio A mice (adults: 12 or 24 weeks, newborn: 8 weeks). Sulfatase modifier factor 1 (SUMF1) was co-transfected to activate the enzyme fully. The E6-GALNS tagged enzyme had markedly prolonged clearance from circulation, giving over 20 times exposure time in blood, compared to untagged enzyme. The tagged enzyme was retained longer in bone, with residual enzyme activity demonstrable at 48 hours after infusion. The pathological findings in adult mice treated with tagged enzyme showed substantial clearance of the storage materials in bone, bone marrow, and heart valves, especially after 24 weekly infusions. Mice treated from the newborn period showed marked reduction of storage materials in tissues investigated. These findings indicate the feasibility of using tagged enzyme to enhance delivery and pathological effectiveness in Morquio A mice.

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

    PubMed

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

    1996-10-01

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

  19. Potential of Mangrove-Associated Endophytic Fungi for Production of Carbohydrolases with High Saccharification Efficiency.

    PubMed

    Maroldi, M M C; Vasconcellos, V M; Lacava, P T; Farinas, C S

    2017-09-02

    The endophytic fungi represent a potential source of microorganisms for enzyme production. However, there have been only few studies exploiting their potential for the production of enzymes of industrial interest, such as the (hemi)cellulolytic enzymatic cocktail required in the hydrolysis of lignocellulosic biomass. Here, a collection of endophytic fungi isolated from mangrove tropical forests was evaluated for the production of carbohydrolases and performance on the hydrolysis of cellulose. For that, 41 endophytic strains were initially screened using a plate assay containing crystalline cellulose as the sole carbon source and the selected strains were cultivated under solid-state fermentation for endoglucanase, β-glucosidase, and xylanase enzyme quantification. The hydrolysis of a cellulosic material with the enzymes from endophytic strains of the Aspergillus genus resulted in glucose and conversion values more than twofold higher than the reference strains (Aspergillus niger F12 and Trichoderma reesei Rut-C30). Particularly, the enzymes from strains A. niger 56 (3) and A. awamori 82 (4) showed a distinguished saccharification performance, reaching cellulose conversion values of about 35% after 24 h. Linking hydrolysis performance to the screening steps played an important role towards finding potential fungal strains for producing enzymatic cocktails with high saccharification efficiency. These results indicate the potential of mangrove-associated endophytic fungi for production of carbohydrolases with efficient performance in the hydrolysis of biomass, thus contributing to the implementation of future biorefineries.

  20. 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.

  1. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

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

    PubMed

    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

    2015-05-14

    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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. 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

  6. Effect of thermostable α-amylase injection on mechanical and physiochemical properties for saccharification of extruded corn starch.

    PubMed

    Myat, Lin; Ryu, Gi-Hyung

    2014-01-30

    In industry, a jet cooker is used to gelatinize starch by mixing the starch slurry with steam under pressure at 100-175 °C. A higher degree of starch hydrolysis in an extruder is possible with glucoamylase. Unfortunately, it is difficult to carry out liquefaction and saccharification in parallel, because the temperature of gelatinization will be too high and will inactivate glucoamylase. Since the temperature for liquefaction and saccharification is different, it is hard to change the temperature from high (required for liquefaction) to low (required for saccharification). The industrial gelatinization process is usually carried out with 30-35% (w/w) dry solids starch slurry. Conventional jet cookers cannot be used any more at high substrate concentrations owing to higher viscosity. In this study, therefore, corn starch was extruded at different melt temperatures to overcome these limitations and to produce the highest enzyme-accessible starch extrudates. Significant effects on physical properties (water solubility index, water absorption index and color) and chemical properties (reducing sugar and % increase in reducing sugar after saccharification) were achieved by addition of thermostable α-amylase at melt temperatures of 115 and 135 °C. However, there was no significant effect on % increase in reducing sugar of extruded corn starch at 95 °C. The results show the great potential of extrusion with thermostable α-amylase injection at 115 and 135 °C as an effective pretreatment for breaking down starch granules, because of the significant increase (P < 0.05) in % reducing sugar and enzyme-accessible extrudates for saccharification yield. © 2013 Society of Chemical Industry.

  7. In muro deacetylation of xylan affects lignin properties and improves saccharification of aspen wood.

    PubMed

    Pawar, Prashant Mohan-Anupama; Derba-Maceluch, Marta; Chong, Sun-Li; Gandla, Madhavi Latha; Bashar, Shamrat Shafiul; Sparrman, Tobias; Ahvenainen, Patrik; Hedenström, Mattias; Özparpucu, Merve; Rüggeberg, Markus; Serimaa, Ritva; Lawoko, Martin; Tenkanen, Maija; Jönsson, Leif J; Mellerowicz, Ewa J

    2017-01-01

    Lignocellulose from fast growing hardwood species is a preferred source of polysaccharides for advanced biofuels and "green" chemicals. However, the extensive acetylation of hardwood xylan hinders lignocellulose saccharification by obstructing enzymatic xylan hydrolysis and causing inhibitory acetic acid concentrations during microbial sugar fermentation. To optimize lignocellulose for cost-effective saccharification and biofuel production, an acetyl xylan esterase AnAXE1 from Aspergillus niger was introduced into aspen and targeted to cell walls. AnAXE1-expressing plants exhibited reduced xylan acetylation and grew normally. Without pretreatment, their lignocellulose yielded over 25% more glucose per unit mass of wood (dry weight) than wild-type plants. Glucose yields were less improved (+7%) after acid pretreatment, which hydrolyses xylan. The results indicate that AnAXE1 expression also reduced the molecular weight of xylan, and xylan-lignin complexes and/or lignin co-extracted with xylan, increased cellulose crystallinity, altered the lignin composition, reducing its syringyl to guaiacyl ratio, and increased lignin solubility in dioxane and hot water. Lignin-associated carbohydrates became enriched in xylose residues, indicating a higher content of xylo-oligosaccharides. This work revealed several changes in plant cell walls caused by deacetylation of xylan. We propose that deacetylated xylan is partially hydrolyzed in the cell walls, liberating xylo-oligosaccharides and their associated lignin oligomers from the cell wall network. Deacetylating xylan thus not only increases its susceptibility to hydrolytic enzymes during saccharification but also changes the cell wall architecture, increasing the extractability of lignin and xylan and facilitating saccharification.

  8. Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability.

    PubMed

    Moraïs, Sarah; Stern, Johanna; Kahn, Amaranta; Galanopoulou, Anastasia P; Yoav, Shahar; Shamshoum, Melina; Smith, Matthew A; Hatzinikolaou, Dimitris G; Arnold, Frances H; Bayer, Edward A

    2016-01-01

    The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components

  9. 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.

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

    PubMed

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

    2012-10-01

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

  11. 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.

  12. 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.

  13. 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

    2015-01-08

    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.

  14. 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

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

    DOE PAGES

    Riedel, Clement; Gabizon, Ronen; Wilson, Christian A. M.; ...

    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

  16. High titer ethanol production from simultaneous enzymatic saccharification and fermentation of aspen at high solids : a comparison between SPORL and dilute acid pretreatments

    Treesearch

    J.Y. Zhu; R. Gleisner; C.T. Scott; X.L. Luo; S. Tian

    2011-01-01

    Native aspen (Populus tremuloides) was pretreated using sulfuric acid and sodium bisulfite (SPORL) and dilute sulfuric acid alone (DA). Simultaneous enzymatic saccharification and fermentation (SSF) was conducted at 18% solids using commercial enzymes with cellulase loadings ranging from 6 to 15 FPU/g glucan and Saccharomyces cerevisiae Y5. Compared with DA...

  17. An evaluation of chemical pretreatment methods for improving enzymatic saccharification of chili postharvest residue.

    PubMed

    Preeti, Varghese Elizabeth; Sandhya, Soolamkandath Variem; Kuttiraja, Mathiyazhakan; Sindhu, Raveendran; Vani, Sankar; Kumar, Sukumaran Rajeev; Pandey, Ashok; Binod, Parameswaran

    2012-07-01

    Residue of chili plants left in the field after harvesting is a major lignocellulosic resource that is underexploited. India has over 0.6 million tons of this residue available as surplus annually which projects it as a potent feedstock for conversion to bioethanol. The cellulose, hemicellulose and lignin content of the chili residues are subject to variations with type of cultivar, geographical region and the season of cultivation, and the composition is critical in developing strategies for its conversion to bioalcohol(s). As with any lignocellulosic biomass, this feedstock needs pretreatment to make it more susceptible to hydrolysis by enzymes which is the most efficient method for generating sugars which can, then, be fermented to alcohol. Pretreatment of chili postharvest residue (CPHR) is, therefore, important though very little study has addressed this challenge. Similarly, enzymatic saccharification of pretreated chili biomass is another area which needs dedicated R&D because the combination of enzyme preparations and the conditions for saccharification are different in different biomass types. The present study was undertaken to develop an optimal process for pretreatment and enzymatic saccharification of CPHR that will yield high amount of free sugars. Dilute acid and alkali pretreatment of the biomass was studied at high temperatures (120-180 °C), with mixing (50-200 rpm) in a high pressure reactor. The holding time was adjusted between 15 and 60 min, and the resultant biomass was evaluated for its susceptibility to enzymatic hydrolysis. Similarly, the conditions for hydrolysis including biomass and enzyme loadings, mixing and incubation time were studied using a Taguchi method of experimentation and were optimized to obtain maximal yield of sugars. Efficiency of pretreatment was gauged by observing the changes in composition and the physicochemical properties of native and pretreated biomass which were analyzed by SEM and XRD analyses. The studies

  18. 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.

  19. 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.

  20. Grapefruit and oroblanco enhance hepatic detoxification enzymes in rats: possible role in protection against chemical carcinogenesis.

    PubMed

    Hahn-Obercyger, Michal; Stark, Aliza H; Madar, Zecharia

    2005-03-09

    Citrus fruits are considered to be functional foods that promote good health. This study was carried out to assess the effect of oroblanco and grapefruit consumption on hepatic detoxification enzymes. Male Sprague-Dawley rats were provided with either regular drinking water (control) or experimental treatments of oroblanco juice, grapefruit juice, or a sugar mix for 6 weeks. After 1 week of treatment, half the animals in each group were injected with the procarcinogen 1,2-dimethylhydrazine. Grapefruit juice significantly increased activity and expression of the hepatic phase I enzyme, cytochrome P450 CYP1A1, with a marked trend toward enhanced NAD(P)H:quinone reductase (QR) activity. Oroblanco juice significantly increased glutathione S-transferase phase II enzyme activity along with CYP1A1 expression and a notable trend toward increased activity of both CYP1A1 and QR. These results suggest that these citrus fruits are bifunctional inducers, modulating both phase I and phase II drug-metabolizing enzymes to enhance hepatic detoxification.

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

    PubMed

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

    2015-06-01

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

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

    PubMed

    Fan, C Y; Krishnamurthy, S

    1995-06-01

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

  3. Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce

    PubMed Central

    Bertilsson, Magnus; Olofsson, Kim; Lidén, Gunnar

    2009-01-01

    Background Simultaneous saccharification and fermentation (SSF) is a promising process option for ethanol production from lignocellulosic materials. However, both the overall ethanol yield and the final ethanol concentration in the fermentation broth must be high. Hence, almost complete conversion of both hexoses and pentoses must be achieved in SSF at a high solid content. A principal difficulty is to obtain an efficient pentose uptake in the presence of high glucose and inhibitor concentrations. Initial glucose present in pretreated spruce decreases the xylose utilization by yeast, due to competitive inhibition of sugar transport. In the current work, prefermentation was studied as a possible means to overcome the problem of competitive inhibition. The free hexoses, initially present in the slurry, were in these experiments fermented before adding the enzymes, thereby lowering the glucose concentration. Results This work shows that a high degree of xylose conversion and high ethanol yields can be achieved in SSF of pretreated spruce with a xylose fermenting strain of Saccharomyces cerevisiae (TMB3400) at 7% and 10% water insoluble solids (WIS). Prefermentation and fed-batch operation, both separately and in combination, improved xylose utilization. Up to 77% xylose utilization and 85% of theoretical ethanol yield (based on total sugars), giving a final ethanol concentration of 45 g L-1, were obtained in fed-batch SSF at 10% WIS when prefermentation was applied. Conclusion Clearly, the mode of fermentation has a high impact on the xylose conversion by yeast in SSF. Prefermentation enhances xylose uptake most likely because of the reduced transport inhibition, in both batch and fed-batch operation. The process significance of this will be even greater for xylose-rich feedstocks. PMID:19356227

  4. Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate.

    PubMed

    Tao, Xiaoqi; Wang, Wenjun; Wang, Zhanhui; Cao, Xingyuan; Zhu, Jinghui; Niu, Lanlan; Wu, Xiaoping; Jiang, Haiyang; Shen, Jianzhong

    2014-06-01

    In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N-azoles as secondary enhancer was also presented.

  5. 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

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

    SciTech Connect

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

    1991-12-31

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

  7. Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor.

    PubMed

    Zhang, Jian; Chu, Deqiang; Huang, Juan; Yu, Zhanchun; Dai, Gance; Bao, Jie

    2010-03-01

    The higher ethanol titer inevitably requires higher solids loading during the simultaneous enzymatic saccharification and fermentation (SSF) using lignocellulose as the feedstock. The mixing between the solid lignocellulose and the liquid enzyme is crucially important. In this study, a bioreactor with a novel helical impeller was designed and applied to the SSF operation of the steam explosion pretreated corn stover under different solids loadings and different enzyme dosages. The performances using the helical impeller and the common Rushton impeller were compared and analyzed by measuring rheological properties and the mixing energy consumption. The results showed that the new designed stirring system had better performances in the saccharification yield, ethanol titer, and energy cost than those of the Rushton impeller stirring. The mixing energy consumption under different solids loadings and enzyme dosages during SSF operation were analyzed and compared to the thermal energy in the ethanol produced. A balance for achieving the optimal energy cost between the increased mixing energy cost and the reduced distillation energy cost at the high solids loading should be made. The potentials of the new bioreactor were tested under various SSF conditions for obtaining optimal ethanol yield and titer.

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

    PubMed

    Berłowska, Joanna; Pielech-Przybylska, Katarzyna; Balcerek, Maria; Dziekońska-Kubczak, Urszula; Patelski, Piotr; Dziugan, Piotr; Kręgiel, Dorota

    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.

  9. 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

  10. Filamentous fungi in good shape: microparticles for tailor-made fungal morphology and enhanced enzyme production.

    PubMed

    Driouch, Habib; Roth, Andreas; Dersch, Petra; Wittmann, Christoph

    2011-01-01

    Filamentous fungi such as Aspergillus niger are important biocatalysts for industrial production of various enzymes as well as organic acids or antibiotics. In suspended culture these microorganisms exhibit a complex morphology which typically has a strong influence on their production properties. In this regard, we have recently shown that the addition of inorganic micro particles to the culture medium is a straightforward and elegant approach to precisely tame fungal morphology. For A. niger a full range of morphological forms from pellets with different diameters to free mycelium could be adjusted by supplementation with talc powder. Aluminium oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition. Exemplified for different recombinant A. niger strains enzyme production could be strongly enhanced by the addition of microparticles. This was demonstrated for the production of fructofuranosidase, an important high-value biocatalyst for pre-biotic fructo-oligosaccharides, by recombinant A. niger. In a microparticle enhanced fed-batch process, a highly productive mycelium could be achieved. The enzyme titre of 2800 U/mL finally reached was more then tenfold higher then that of any other process reported so far. Here we provide additional insights into the novel production process. This includes the confirmation of the highly selective production of the target enzyme fructofuranosidase using MALDI-TOF MS analysis. Moreover, we show that the obtained enzyme suspension can be efficiently used with minimal pre-treatment for the biosynthesis of short chain fructooligosaccharides of the inulin type, such as 1-kestose and 1-nystose, prebiotics with substantial commercial interest. In particular, these compounds are highly attractive for human consumption, since they have been shown to reduce the risk of colon cancer. In summary, the use of microparticles opens a new avenue of engineering

  11. 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.

  12. 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

  13. 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.

  14. Enhancing polyphenol extraction from unripe apples by carbohydrate-hydrolyzing enzymes*

    PubMed Central

    Zheng, Hu-zhe; Hwang, In-Wook; Chung, Shin-Kyo

    2009-01-01

    The effects of process variables such as enzyme types, enzyme ratio, reaction temperature, pH, time, and ethanol concentration on the extraction of unripe apple polyphenol were investigated. The results indicated that Viscozyme L had the strongest effect on polyphenols extraction and was selected to study the polyphenol composition. The ratio of enzyme (Viscozyme L) to substrate (2 fungal beta-glucanase units (FBG)) at 0.02, reaction at pH 3.7, 50 °C for 12 h, and ethanol concentration of 70% were chosen as the most favorable extraction condition. Total phenolic content (TPC), reducing sugar content (RSC), and extraction yield increased by about 3, 1.5, and 2 times, respectively, compared with control. The contents of p-coumaric acid, ferulic acid, and caffeic acid increased to 8, 4, and 32 times, respectively. The enzyme-aided polyphenol extraction process from unripe apples might be applied to food industry for enhancing bioactive compound production. PMID:19946955

  15. [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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. Enhanced immunogenicity of a functional enzyme by T cell epitope modification

    PubMed Central

    Mucha, Jeanette M; Stickler, Marcia M; Poulose, AJ; Ganshaw, Grant; Saldajeno, Mae; Collier, Kathy; Huang, Manley T; Harding, Fiona A

    2002-01-01

    Background T helper epitopes are necessary for the induction of high titers of antigen-specific IgG antibodies. We are interested in the epitope modification of intact proteins as a method to enhance their immunogenicity for the generation of recombinant protein-based vaccines. Results Hartley strain guinea pig T cell epitopes were mapped for two related bacterial proteases. Two T cell epitopes were found in one of the proteases, while a comparatively reduced immunogenicity protease had no detectable T cell epitopes. A T cell epitope sequence homologous to the immunogenic protease was created in the less immunogenic protease by changing a single amino acid. Proliferative responses to the whole protein parent enzyme were two-fold higher in splenocyte cultures from variant-immunized animals. We found that the single amino acid change in the variant resulted in a protein immunogen that induced higher titers of antigen-specific IgG antibody at low doses and at early time points during the immunization protocol. The serum from parent- and variant-immunized guinea pigs cross-reacted at both the protein and the peptide level. Finally, animals primed to the variant but boosted with the parent enzyme had higher levels of antigen-specific IgG than animals immunized with the parent enzyme alone. Conclusions With a single amino acid change we have introduced a T cell epitope into a comparatively low-immunogenic enzyme and have increased its immunogenicity while retaining the enzyme's original proteolytic function. The ability to immunomodulate proteins while leaving their function intact has important implication for the development of recombinant vaccines and protein-based therapeutics. PMID:11869454

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2012-02-01

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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. Angiotensin-converting enzyme overexpression in myelocytes enhances the immune response.

    PubMed

    Bernstein, Kenneth E; Gonzalez-Villalobos, Romer A; Giani, Jorge F; Shah, Kandarp; Bernstein, Ellen; Janjulia, Tea; Koronyo, Yosef; Shi, Peng D; Koronyo-Hamaoui, Maya; Fuchs, Sebastien; Shen, Xiao Z

    2014-10-01

    Angiotensin-converting enzyme (ACE) plays an important role in blood pressure control. ACE also has effects on renal function, reproduction, hematopoiesis, and several aspects of the immune response. ACE 10/10 mice overexpress ACE in monocytic cells; macrophages from ACE 10/10 mice demonstrate increased polarization toward a proinflammatory phenotype. As a result, ACE 10/10 mice have a highly effective immune response following challenge with melanoma, bacterial infection, or Alzheimer disease. As shown in ACE 10/10 mice, enhanced monocytic function greatly contributes to the ability of the immune response to defend against a wide variety of antigenic and non-antigenic challenges.

  10. Enhancement of Rooibos (Aspalathus linearis) aqueous extract and antioxidant yield with fungal enzymes.

    PubMed

    Pengilly, Mia; Joubert, Elizabeth; van Zyl, Willem H; Botha, Alfred; Bloom, Marinda

    2008-06-11

    The leaves and stems of the Rooibos plant ( Aspalathus linearis) are used for the production of an herbal tea known for its health promoting properties, which have been linked to its flavonoid content but which is substantially reduced by the traditional processing method employed. Selected food-grade fungi were screened for their potential to improve the yield of soluble matter extracted from rooibos plant material. Fungal cocktails of hydrolyzing enzymes enhanced either the yield of soluble solids ( Lentinula edodes and Rhizopus oryzae cultured in yeast peptone-wheat straw medium) or the yield in antioxidants from fermented rooibos ( R. oryzae cultured in potato dextrose or yeast peptone-wheat straw medium). When applied to green rooibos, L. edodes (cultured in yeast peptone-wheat straw medium) enhanced the release of soluble solids as well as color formation, leading to semifermented rooibos with a relatively high aspalathin content, compared to fermented rooibos.

  11. Gd(III)-induced Supramolecular Hydrogelation with Enhanced Magnetic Resonance Performance for Enzyme Detection

    PubMed Central

    Hua, Yongquan; Pu, Guojuan; Ou, Caiwen; Zhang, Xiaoli; Wang, Ling; Sun, Jiangtao; Yang, Zhimou; Chen, Minsheng

    2017-01-01

    Here we report a supramolecular hydrogel based on Gd(III)-peptide complexes with dramatically enhanced magnetic resonance (MR) performance. The hydrogelations were formed by adding Gd(III) ion to the nanofiber dispersion of self-assembling peptides naphthalene-Gly-Phe-Phe-Tyr-Gly-Arg-Gly-Asp (Nap-GFFYGRGD) or naphthalene-Gly-Phe-Phe-Tyr-Gly-Arg-Gly-Glu (Nap-GFFYGRGE). We further showed that, by adjusting the molar ratio between Gd(III) and the corresponding peptide, the mechanical property of resulting gels could be fine-tuned. The longitudinal relaxivity (r1) of the Nap-GFFYGRGE-Gd(III) was 58.9 mM−1 S−1, which to our knowledge is the highest value for such peptide-Gd(III) complexes so far. Such an enhancement of r1 value could be applied for enzyme detection in aqueous solutions and cell lysates. PMID:28074904

  12. Gd(III)-induced Supramolecular Hydrogelation with Enhanced Magnetic Resonance Performance for Enzyme Detection

    NASA Astrophysics Data System (ADS)

    Hua, Yongquan; Pu, Guojuan; Ou, Caiwen; Zhang, Xiaoli; Wang, Ling; Sun, Jiangtao; Yang, Zhimou; Chen, Minsheng

    2017-01-01

    Here we report a supramolecular hydrogel based on Gd(III)-peptide complexes with dramatically enhanced magnetic resonance (MR) performance. The hydrogelations were formed by adding Gd(III) ion to the nanofiber dispersion of self-assembling peptides naphthalene-Gly-Phe-Phe-Tyr-Gly-Arg-Gly-Asp (Nap-GFFYGRGD) or naphthalene-Gly-Phe-Phe-Tyr-Gly-Arg-Gly-Glu (Nap-GFFYGRGE). We further showed that, by adjusting the molar ratio between Gd(III) and the corresponding peptide, the mechanical property of resulting gels could be fine-tuned. The longitudinal relaxivity (r1) of the Nap-GFFYGRGE-Gd(III) was 58.9 mM‑1 S‑1, which to our knowledge is the highest value for such peptide-Gd(III) complexes so far. Such an enhancement of r1 value could be applied for enzyme detection in aqueous solutions and cell lysates.

  13. 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.

  14. Signal enhancement strategy for a micro-arrayed polydiacetylene (PDA) immunosensor using enzyme-catalyzed precipitation.

    PubMed

    Lee, Jong Uk; Jeong, Ji Hoon; Lee, Doo Sung; Sim, Sang Jun

    2014-11-15

    This paper describes a signal enhancement strategy to improve the sensitivity of an antibody-based immunosensor that uses polydiacetylene (PDA) liposomes to detect a target protein (human immunoglobulin E [hIgE]). To achieve ultrasensitive detection, multiple stimuli applied to PDA immunosensor chips offer a signal enhancement method that combines the primary immune reaction between antigen and antibody with the sandwich method of polyclonal antibody (pAb)-conjugated horseradish peroxidase (HRP). In the second step, fluorescence is enhanced by the mechanical pressure from the precipitate formed by enzyme catalysis. In order to detect hIgE, the surface of immobilized PDA liposomes was conjugated with monoclonal antibodies against hIgE, and fluorescence signals were detected after the antigen-antibody reaction. In this step, hIgE concentrations as low as 10 ng/mL were detected. Fluorescence signals slightly increased when anti-hIgE pAb-HRP was used as an amplifying agent after primary immunoresponse. After secondary immunoresponse, HRP-catalyzed oxidation of 3,3'-diaminobenzidine produced an insoluble precipitate that strongly stimulated PDA liposomes by their weight and pressure, thereby dramatically increasing the fluorescence signal. Thus, PDA liposome immunosensor could detect hIgE concentrations as low as 0.01 ng/mL, representing a 1000-fold increase in sensitivity over the signal generated by the primary immunoresponse. This study indicates that increasing the external mechanical force applied to PDA liposomes by enzyme-catalyzed precipitate formation enhanced the sensitivity of the PDA liposome immunosensor chip. This strategy can be applied to the detection of other biomolecules in experimental or clinical settings where ultrasensitive and highly specific biosensing is required. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Experiment of Enzyme Kinetics Using Guided Inquiry Model for Enhancing Generic Science Skills

    NASA Astrophysics Data System (ADS)

    Amida, N.; Supriyanti, F. M. T.; Liliasari

    2017-02-01

    This study aims to enhance generic science skills of students using guided inquiry model through experiments of enzyme kinetics. This study used quasi-experimental methods, with pretest-posttestnonequivalent control group design. Subjects of this study were chemistry students enrolled in biochemistry lab course, consisted of 18 students in experimental class and 19 students in control class. Instrument in this study were essay test that involves 5 indicators of generic science skills (i.e. direct observation, causality, symbolic language, mathematical modeling, and concepts formation) and also student worksheets. The results showed that the experiments of kinetics enzyme using guided inquiry model have been enhance generic science skills in high category with a value of average of 0.77. Four indicators classified in the high category are direct observation, causality, symbolic language, and mathematical modeling with the value of 0,73 0,70; 0,96; dan 0,85. Meanwhile, indicator of concepts formation in the medium category with a value of 0.62

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

    PubMed

    Liu, Gang; Zhang, Yan; Xing, Miao

    2006-03-01

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

  17. Enhanced ethanol fermentation of brewery wastewater using the genetically modified strain E. coli KO11.

    PubMed

    Rao, Kripa; Chaudhari, Vaibhav; Varanasi, Sasidhar; Kim, Dong-Shik

    2007-02-01

    We have used liquid waste obtained from a beer brewery process to produce ethanol. To increase the productivity, genetically modified organism, Escherichia coli KO11, was used for ethanol fermentation. Yeast was also used to produce ethanol from the same feed stock, and the ethanol production rates and resulting concentrations of sugars and ethanol were compared with those of KO11. In the experiments, first the raw wastewater was directly fermented using two strains with no saccharification enzymes added. Then, commercial enzymes, alpha-amylase, pectinase, or a combination of both, were used for simultaneous saccharification and fermentation, and the results were compared with those of the no-enzyme experiments for KO11 and yeast. Under the given conditions with or without the enzymes, yeast produced ethanol more rapidly than E. coli KO11, but the final ethanol concentrations were almost the same. For both yeast and KO11, the enzymes were observed to enhance the ethanol yields by 61-84% as compared to the fermentation without enzymes. The combination of the two enzymes increased ethanol production the most for the both strains. The advantages of using KO11 were not demonstrated clearly as compared to the yeast fermentation results.

  18. Simultaneous saccharification and fermentation of fungal pretreated cornstalk for hydrogen production using Thermoanaerobacterium thermosaccharolyticum W16.

    PubMed

    Zhao, Lei; Cao, Guang-Li; Wang, Ai-Jie; Guo, Wan-Qian; Ren, Hong-Yu; Ren, Nan-Qi

    2013-10-01

    In this research, environmentally friendly fungal pretreatment was first adopted for deconstruction of cornstalk. Then the fungal-pretreated cornstalk was employed to produce hydrogen in simultaneous saccharification and fermentation (SSF) using crude enzyme from Trichoderma viride and Thermoanaerobacterium thermosaccharolyticum W16. The influence of various factors including substrate concentration, initial pH, and enzyme loading on hydrogen production were evaluated. The highest hydrogen yield of 89.3 ml/g-cornstalk was obtained with an initial pH 6.5, 0.75% substrate concentration, and 34 FPU/g cellulose. Compared the result with SSF of physical or chemical pretreated lignocellulosic materials, this research suggested an economic and efficient way for hydrogen production from lignocellulosic biomass. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. 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.

  20. Substrate-Related Factors Affecting Enzymatic Saccharification of Lignocelluloses: Our Recent Understanding

    Treesearch

    Shao-Yuan Leu; J.Y. Zhu

    2013-01-01

    Enzymatic saccharification of cellulose is a key step in conversion of plant biomass to advanced biofuel and chemicals. Many substrate-related factors affect saccharification. Rather than examining the role of each individual factor on overall saccharification efficiency, this study examined how each factor affects the three basic processes of a heterogeneous...

  1. 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.

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

    PubMed

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

    2007-11-01

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

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

    PubMed Central

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

    2007-01-01

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

  4. 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.

  5. Iron deficiency enhances the levels of ascorbate, glutathione, and related enzymes in sugar beet roots.

    PubMed

    Zaharieva, Tatiana B; Abadía, Javier

    2003-06-01

    The effects of Fe deficiency stress on the levels of ascorbate and glutathione, and on the activities of the enzymes ferric chelate reductase, glutathione reductase (EC 1.6.4.2), ascorbate free-radical reductase (EC 1.6.5.4) and ascorbate peroxidase (EC 1.11.1.11), have been investigated in sugar beet ( Beta vulgaris L.) roots. Plasma membrane vesicles and cytosolic fractions were isolated from the roots of the plants grown in nutrient solutions in the absence or presence of Fe for two weeks. Plants responded to Fe deficiency not only with a 20-fold increase in root ferric chelate reductase activity, but also with moderately increased levels of the general reductants ascorbate (2-fold) and glutathione (1.6-fold). The enzymes of the ascorbate-glutathione cycle in roots were also affected by Fe deficiency. Glutathione reductase activity was enhanced 1.4-fold with Fe deficiency, associated to an increased ratio of reduced to oxidized glutathione, from 3.1 to 5.2. The plasma membrane fraction from iron-deficient roots showed 1.7-fold higher ascorbate free-radical reductase activity, whereas in the cytosolic fraction the enzyme activity was not affected by Fe deficiency. The activity of the cytosolic hemoprotein ascorbate peroxidase decreased approximately by 50% with Fe deprivation. These results show that sugar beet responds to Fe deficiency with metabolic changes affecting components of the ascorbate-glutathione cycle in root cells. This suggests that the ascorbate-glutathione cycle would play certain roles in the general Fe deficiency stress responses in strategy I plants.

  6. 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

    2016-09-07

    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.

  7. Graphene immobilized enzyme/polyethersulfone mixed matrix membrane: Enhanced antibacterial, permeable and mechanical properties

    NASA Astrophysics Data System (ADS)

    Duan, Linlin; Wang, Yuanming; Zhang, Yatao; Liu, Jindun

    2015-11-01

    Enzyme immobilization has been developed to address lots of issues of free enzyme, such as instability, low activity and difficult to retain. In this study, graphene was used as an ideal carrier for lysozyme immobilization, including graphene oxide (GO) immobilized lysozyme (GO-Ly) and chemically reduced graphene oxide (CRGO) immobilized lysozyme (CRGO-Ly). Herein, lysozyme as a bio-antibacterial agent has excellent antibacterial performance and the products of its catalysis are safety and nontoxic. Then the immobilized lysozyme materials were blended into polyethersulfone (PES) casting solution to prepare PES ultrafiltration membrane via phase inversion method. GO and CRGO were characterized by Fourier transform infrared spectroscopy (FTIR), Ultraviolet-visible spectrum (UV), X-ray diffraction (XRD), and transmission electron microscopy (TEM) and the immobilized lysozyme composites were observed by fluorescent microscopy. The results revealed that GO and CRGO were successfully synthesized and lysozyme was immobilized on their surfaces. The morphology, hydrophilicity, mechanical properties, separation properties and antibacterial activity of the hybrid membranes were characterized in detail. The hydrophilicity, water flux and mechanical strength of the hybrid membranes were significantly enhanced after adding the immobilized lysozyme. In the antibacterial experiment, the hybrid membranes exhibited an effective antibacterial performance against Escherichia coli (E. coli).

  8. 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.

  9. 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

  10. 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

  11. Angiotensin converting enzyme over expression in myelocytes enhances the immune response

    PubMed Central

    Bernstein, Kenneth E.; Gonzalez-Villalobos, Romer A.; Giani, Jorge F.; Shah, Kandarp; Bernstein, Ellen; Janjulia, Tea; Koronyo, Yosef; Shi, Peng D.; Koronyo-Hamaoui, Maya; Fuchs, Sebastien; Shen, Xiao Z.

    2015-01-01

    Angiotensin converting enzyme (ACE) plays an important role in blood pressure control. ACE also has effects on renal function, reproduction, hematopoiesis and several aspects of the immune response. ACE 10/10 mice over express ACE in monocytic cells; macrophages from ACE 10/10 mice demonstrate increased polarization towards a proinflammatory phenotype. As a result, ACE 10/10 mice have a highly effective immune response following challenge with either melanoma, bacterial infection or Alzheimer’s disease. The ACE 10/10 mice suggest that enhanced monocytic function greatly contributes to the ability of the immune response to defend against a wide variety of antigenic and non-antigenic challenges. PMID:24633750

  12. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. 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.

  14. 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.

  15. Improving enzymatic saccharification of bamboo shoot shell by alkalic salt pretreatment with H2O2.

    PubMed

    Qing, Qing; Zhou, Linlin; Huang, Meizi; Guo, Qi; He, Yucai; Wang, Liqun; Zhang, Yue

    2016-02-01

    Pretreatment of bamboo shoot shell (BSS) by a combination of alkalic salts with hydrogen peroxide (H2O2) was evaluated for its delignification effect and for its ability to enhance enzymatic saccharification of pretreated solids. By comparing different alkalic salts, the combination of 9% Na3PO4·12H2O and 0.3g/g H2O2 (ASHP) was identified as an effective system that showed the highest delignification of 87.7% and the total reducing sugar yield of 97.1% when pretreated BSS at a solid to liquid ratio of 1/20 (w/w) at 80°C for 2h. The delignification effect and the disruption of the lignocelluloses structure by this novel pretreatment method were deduced to be the main reasons that led to enhanced enzymatic saccharification as supported by the chemical composition analysis and the results of SEM, FTIR and XRD analyses of the untreated and alkalic salt pretreated BSS.

  16. 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).

  17. Continuous enzymatic liquefaction of starch for saccharification.

    PubMed

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

    1982-11-01

    A process was explored for continuous enzymatic liquefaction of corn starch at high concentration and subsequently 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 variable such as starch concentration (40-70 degrees ), 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 min). 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 process conditions were achieved by using 50-60% starch concentration, at 95 degrees C, with 0.4% alpha-amylase, and a 6-min residence period in the mixture. Under these conditions, rate and extents of glucose obtained after glucoamylase treatment approached those obtained in longer laboratory batch liquefactions. The amount of glucose formed in 24h 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).

  18. 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

    2015-05-04

    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. Copyright © 2015. Published by Elsevier B.V.

  19. 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.

  20. Defying the activity-stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability.

    PubMed

    Siddiqui, Khawar Sohail

    2017-05-01

    The biotechnological applications of enzymes are limited due to the activity-stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity-stability trade-off can be overcome. In this review, the origin of the activity-stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity-stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel-Danson equilibrium model, catalytic antibodies, and life on cold planets.

  1. In-vitro evaluation of enteric coated insulin tablets containing absorption enhancer and enzyme inhibitor.

    PubMed

    Wong, Chun Y; Martinez, Jorge; Carnagarin, Revathy; Dass, Crispin R

    2017-03-01

    The aim of this study was to develop an enteric coated insulin tablet formulation using polymers, absorption enhancer and enzyme inhibitor, which protect the tablets in acidic pH and enhance systemic bioavailability. In this study, the influence of coating by cellulose acetate hydrogen phthalate solution and chosen excipients on Glut-4 transporter translocation in C2C12 skeletal muscle cells was examined. Following the determination of optimum number of coating layers, two dissolution buffers such as 0.01 m hydrochloric acid, pH 2, and 50 mm phosphate, pH 7.4, were employed to determine the in-vitro release of insulin. Insulin was protected by the coating during the dissolution process. Five (5-CL) coating layers and eight (8-CL) coating layers had minimal insulin release in hydrochloric acid, but not three (3-CL) coating layers. Glut-4 translocation in C2C12 cells was promoted by the chosen excipients. No detrimental metabolic effects were observed in these cells. To date, limited studies combine the overall effectiveness of multiple excipients. Our study showed that the coated tablets have an immediate release effect in phosphate buffer. In Glut-4 translocation assay, insulin was still functional after releasing from the tablet. Such tablet formulation can be potentially beneficial to type 1 diabetes patients. © 2017 Royal Pharmaceutical Society.

  2. 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.

  3. 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.

  4. Enhanced levels of mitochondrial enzyme 17beta-hydroxysteroid dehydrogenase type 10 in patients with Alzheimer disease and multiple sclerosis.

    PubMed

    Kristofiková, Zdena; Bocková, Markéta; Hegnerová, Katerina; Bartos, Ales; Klaschka, Jan; Rícný, Jan; Rípová, Daniela; Homola, Jirí

    2009-10-01

    The multifunctional mitochondrial enzyme 17beta-hydroxysteroid dehydrogenase type 10 might play a role in the development of Alzheimer disease via its high-affinity binding to amyloid beta peptides and its neuronal over-expression. It is suggested that the cerebrospinal fluid levels of the enzyme, free or bound to amyloid beta peptides, are a potential specific biomarker of Alzheimer disease. However, mitochondrial dysfunction seems to play a role in many neurological diseases including multiple sclerosis. In this study, the specificity of changes in relation to the enzyme over-expression was evaluated using enzyme-linked immunosorbent and surface plasmon resonance sensors. The data indicated pronounced increases in the enzyme levels, specifically to 179% in multiple sclerosis and to 573% in Alzheimer disease when compared to the age-matched controls. Although the differences between both diseases were statistically significant, enzyme levels do not appear to be a highly specific biomarker of Alzheimer disease. On the other hand, enhancement in levels of the enzyme bound to amyloid beta peptides was only observed in people with Alzheimer disease, which suggests that the complex should be further considered as a possible biomarker. In patients with multiple sclerosis, our results are the first to demonstrate significant changes in enzyme expression and to suggest possible alterations in amyloid beta peptides.

  5. Engineering of the yeast antioxidant enzyme Mpr1 for enhanced activity and stability.

    PubMed

    Iinoya, Kaoru; Kotani, Tetsuya; Sasano, Yu; Takagi, Hiroshi

    2009-06-01

    The budding yeast Saccharomyces cerevisiae Sigma1278b has the MPR1 gene, which confers resistance to the proline analogue azetidine-2-carboxylate (AZC). This gene encodes an N-acetyltransferase Mpr1 that detoxifies AZC, and the homologous genes have been found in many yeasts. Recently, we found that Mpr1 protects yeast cells by reducing the intracellular reactive oxygen species (ROS) levels under oxidative stresses, such as heat-shock, freezing, or ethanol treatment. Unlike the known antioxidant enzymes, Mpr1 is thought to acetylate toxic metabolite(s) involved in ROS generation via oxidative events. To improve the enzymatic functions of Mpr1, we applied PCR random mutagenesis to MPR1. The mutagenized plasmid library was introduced into the S. cerevisiae S288C strain lacking MPR1, and we successfully isolated two Mpr1 variants with higher AZC resistance (K63R and F65L/L117V). Interestingly, overexpression of the K63R variant was found to increase cell viability or decrease intracellular ROS levels after exposure to H(2)O(2) or ethanol compared with the wild-type Mpr1. In vitro studies with the recombinant enzymes showed that the catalytic efficiency of the K63R variant for AZC and acetyl-CoA was higher than that of the wild-type Mpr1 and that the F65L mutation greatly enhanced the thermal stability. The mutational analysis and molecular modeling suggest that an alpha-helix containing Lys63 and Phe65 has important roles in the function of Mpr1. In addition, the wild-type and K63R variant Mpr1 reduced intracellular ROS levels under ethanol stress conditions on haploid sake yeast cells. These results suggest that engineering Mpr1 might be useful in breeding oxidative stress-tolerant yeast strains.

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

    PubMed

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

    2014-02-01

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

  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. How cell wall complexity influences saccharification efficiency in Miscanthus sinensis

    DOE PAGES

    De Souza, Amanda P.; Kamei, Claire L. Alvim; Torres, Andres F.; ...

    2015-04-23

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

  10. 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

  11. 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.

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

    PubMed Central

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

    2015-01-01

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

  13. 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.

  14. Aptamer capturing of enzymes on magnetic beads to enhance assay specificity and sensitivity.

    PubMed

    Zhao, Qiang; Li, Xing-Fang; Le, X Chris

    2011-12-15

    Activity and specificity of enzyme molecules are important to enzymatic reactions and enzyme assays. We describe an aptamer capturing approach that improves the specificity and the sensitivity of enzyme detection. An aptamer recognizing the target enzyme molecule is conjugated on a magnetic bead, increasing the local concentration, and serves as an affinity probe to capture and separate minute amounts of the enzyme. The captured enzymes catalyze the subsequent conversion of fluorogenic substrate to fluorescent products, enabling a sensitive measure of the active enzyme. The feasibility of this technique is demonstrated through assays for human alpha thrombin and human neutrophil elastase (HNE), two important enzymes. Thrombin (2 fM) and 100 fM HNE can be detected. The incorporation of two binding events, substrate recognition and aptamer binding, greatly improves assay specificity. With its simplicity, this approach is applicable to biosensing and detection of disease biomarkers.

  15. 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

    2011-04-07

    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.

  16. Cellulosic ethanol production on temperature-shift simultaneous saccharification and fermentation using the thermostable yeast Kluyveromyces marxianus CHY1612.

    PubMed

    Kang, Hyun-Woo; Kim, Yule; Kim, Seung-Wook; Choi, Gi-Wook

    2012-01-01

    In cellulosic ethanol production, use of simultaneous saccharification and fermentation (SSF) has been suggested as the favorable strategy to reduce process costs. Although SSF has many advantages, a significant discrepancy still exists between the appropriate temperature for saccharification (45-50 °C) and fermentation (30-35 °C). In the present study, the potential of temperature-shift as a tool for SSF optimization for bioethanol production from cellulosic biomass was examined. Cellulosic ethanol production of the temperature-shift SSF (TS-SSF) from 16 w/v% biomass increased from 22.2 g/L to 34.3 g/L following a temperature shift from 45 to 35 °C compared with the constant temperature of 45 °C. The glucose conversion yield and ethanol production yield in the TS-SSF were 89.3% and 90.6%, respectively. At higher biomass loading (18 w/v%), ethanol production increased to 40.2 g/L with temperature-shift time within 24 h. These results demonstrated that the temperature-shift process enhances the saccharification ratio and the ethanol production yield in SSF, and the temperature-shift time for TS-SSF process can be changed according to the fermentation condition within 24 h.

  17. 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.

  18. 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.

  19. Expression of thermostable β-xylosidase in Escherichia coli for use in saccharification of plant biomass.

    PubMed

    Aftab, Muhammad N; Zafar, Asma; Awan, Ali R

    2017-01-31

    The present work is aimed to evaluate the saccharification potential of a thermostable β-xylosidase cloned from Bacillus licheniformis into Escherichia coli for production of bioethanol from plant biomass. Recombinant β-xylosidase enzyme possesses the ability of bioconversion of plant biomass like wheat straw, rice straw and sugarcane bagass. By using this approach, plant biomass that mainly constitute cellulose can be converted to reducing sugars that could then be easily converted to bioethanol by simple fermentation process. The production of bioethanol will help to overcome energy requirements due to depleting fossil fuels and will also help to protect environment by reducing greenhouse gas emission. In the end, future directions are briefly mentioned that can be utilized to reduce the cost and increase the yield of biofuels.

  20. Sequential enzymatic saccharification and fermentation of ionic liquid and organosolv pretreated agave bagasse for ethanol production

    SciTech Connect

    Pérez-Pimienta, Jose A.; Vargas-Tah, Alejandra; López-Ortega, Karla M.; Medina-López, Yessenia N.; Mendoza-Pérez, Jorge A.; Avila, Sayeny; Singh, Seema; Simmons, Blake A.; Loaces, Inés; Martinez, Alfredo

    2016-11-16

    Agave bagasse (AGB) has gained recognition as a drought-tolerant biofuel feedstock with high productivity in semiarid regions. A comparative analysis of ionic liquid (IL) and organosolv (OV) pretreatment technologies in AGB was performed using a sequential enzymatic saccharification and fermentation (SESF) strategy with cellulolytic enzymes and the ethanologenic Escherichia coli strain MS04. After pretreatment, 86% of xylan and 45% of lignin were removed from OV-AGB, whereas IL-AGB reduced lignin content by 28% and xylan by 50% when compared to the untreated biomass. High glucan ( > 90%) and xylan ( > 83%) conversion was obtained with both pretreated samples. During the fermentation stage (48 h), 12.1 and 12.7 kg of ethanol were produced per 100 kg of untreated AGB for IL and OV, respectively. These comparative analyses showed the advantages of SESF using IL and OV in a biorefinery configuration where a better understanding of AGB recalcitrance is key for future applications.

  1. Sequential enzymatic saccharification and fermentation of ionic liquid and organosolv pretreated agave bagasse for ethanol production.

    PubMed

    Pérez-Pimienta, Jose A; Vargas-Tah, Alejandra; López-Ortega, Karla M; Medina-López, Yessenia N; Mendoza-Pérez, Jorge A; Avila, Sayeny; Singh, Seema; Simmons, Blake A; Loaces, Inés; Martinez, Alfredo

    2017-02-01

    Agave bagasse (AGB) has gained recognition as a drought-tolerant biofuel feedstock with high productivity in semiarid regions. A comparative analysis of ionic liquid (IL) and organosolv (OV) pretreatment technologies in AGB was performed using a sequential enzymatic saccharification and fermentation (SESF) strategy with cellulolytic enzymes and the ethanologenic Escherichia coli strain MS04. After pretreatment, 86% of xylan and 45% of lignin were removed from OV-AGB, whereas IL-AGB reduced lignin content by 28% and xylan by 50% when compared to the untreated biomass. High glucan (>90%) and xylan (>83%) conversion was obtained with both pretreated samples. During the fermentation stage (48h), 12.1 and 12.7kg of ethanol were produced per 100kg of untreated AGB for IL and OV, respectively. These comparative analyses showed the advantages of SESF using IL and OV in a biorefinery configuration where a better understanding of AGB recalcitrance is key for future applications.

  2. 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.

  3. 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.

  4. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. 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

  6. Chromium functionalized diglyme plasma polymer coating enhances enzyme-linked immunosorbent assay performance.

    PubMed

    Welch, Nicholas G; Madiona, Robert M T; Easton, Christopher D; Scoble, Judith A; Jones, Robert T; Muir, Benjamin W; Pigram, Paul J

    2016-11-10

    Ensuring the optimum orientation, conformation, and density of substrate-bound antibodies is critical for the success of sandwich enzyme-linked immunosorbent assays (ELISAs). In this work, the authors utilize a diethylene glycol dimethyl ether plasma polymer (DGpp) coating, functionalized with chromium within a 96 well plate for the enhanced immobilization of a capture antibody. For an equivalent amount of bound antibody, a tenfold improvement in the ELISA signal intensity is obtained on the DGpp after incubation with chromium, indicative of improved orientation on this surface. Time-of-flight secondary-ion-mass-spectrometry (ToF-SIMS) and principal component analysis were used to probe the molecular species at the surface and showed ion fragments related to lysine, methionine, histidine, and arginine coupled to chromium indicating candidate antibody binding sites. A combined x-ray photoelectron spectroscopy and ToF-SIMS analysis provided a surface molecular characterization that demonstrates antibody binding via the chromium complex. The DGpp+Cr surface treatment holds great promise for improving the efficacy of ELISAs.

  7. Pomegranate peel extract decreases small intestine lipid peroxidation by enhancing activities of major antioxidant enzymes.

    PubMed

    Al-Gubory, Kaïs H; Blachier, François; Faure, Patrice; Garrel, Catherine

    2016-08-01

    Pomegranate peel extract (PPE) contains several compounds with antioxidative properties. PPE added to foods may interact with endogenous antioxidants and promote health. However, little is known about the biochemical mechanisms by which PPE exerts their actions on tissues of biological systems in vivo. The purpose of this study was to determine the effects of PPE on activities of antioxidant enzymes. Mice were used to investigate the effects of PPE on plasma levels of malondialdehyde (MDA), tissue MDA content and activities of superoxide dismutase 1 (SOD1), SOD2 and glutathione peroxidase (GPX) in the small intestine, liver and skeletal muscle - different tissues involved in the digestion, absorption and metabolism of dietary nutrients. Control mice were fed a standard diet, whereas treated mice were fed for 40 days with the standard diet containing 5% or 10% PPE. Mice fed the 10% PPE diet exhibited lower plasma MDA concentrations, reduced content of MDA in the small intestine and liver and higher levels of SOD1 and GPX activities in the small intestine compared to mice fed the control diet. These findings demonstrate that intake of PPE in diet attenuates small intestine lipid peroxidation and strengthens the first line of small intestine antioxidant defense by enhancing enzymatic antioxidative pathways. PPE is worthy of further study as a therapeutic approach to prevent peroxidative stress-induced gut pathogenesis. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  8. 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.

  9. Evaluation of Ethanol Production Activity by Engineered Saccharomyces cerevisiae Fermenting Cellobiose through the Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation of Cellulose.

    PubMed

    Lee, Won-Heong; Jin, Yong-Su

    2017-09-28

    In simultaneous saccharification and fermentation (SSF) for production of cellulosic biofuels, engineered Saccharomyces cerevisiae capable of fermenting cellobiose has provided several benefits, such as lower enzyme costs and faster fermentation rate compared with wild-type S. cerevisiae fermenting glucose. In this study, the effects of an alternative intracellular cellobiose utilization pathway-a phosphorolytic pathway based on a mutant cellodextrin transporter (CDT-1 (F213L)) and cellobiose phosphorylase (SdCBP)-was investigated by comparing with a hydrolytic pathway based on the same transporter and an intracellular β-glucosidase (GH1-1) for their SSF performances under various conditions. Whereas the phosphorolytic and hydrolytic cellobiose-fermenting S. cerevisiae strains performed similarly under the anoxic SSF conditions, the hydrolytic S. cerevisiae performed slightly better than the phosphorolytic S. cerevisiae under the microaerobic SSF conditions. Nonetheless, the phosphorolytic S. cerevisiae expressing the mutant CDT-1 showed better ethanol production than the glucose-fermenting S. cerevisiae with an extracellular β-glucosidase, regardless of SSF conditions. These results clearly prove that introduction of the intracellular cellobiose metabolic pathway into yeast can be effective on cellulosic ethanol production in SSF. They also demonstrate that enhancement of cellobiose transport activity in engineered yeast is the most important factor affecting the efficiency of SSF of cellulose.

  10. 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.

  11. 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.

  12. Anti-BlyS antibody reduces the immune reaction against enzyme and enhances the efficacy of enzyme replacement therapy in Fabry disease model mice.

    PubMed

    Sato, Yohei; Ida, Hiroyuki; Ohashi, Toya

    2017-02-02

    Formation of antibodies against a therapeutic enzyme is an important complication during enzyme replacement therapy (ERT) for lysosomal storage diseases. Fabry disease (FD) is caused by a deficiency of alpha-galactosidase (GLA), which results in the accumulation of globotriaosylceramide (GL-3). We have shown immune tolerance induction (ITI) during ERT in FD model mice by using an anti-B lymphocyte stimulator (anti-BlyS) antibody (belimumab). A single dose of the anti-BlyS antibody temporarily lowered the percentage of B cells and IgG antibody titer against recombinant human GLA. Administration of a low maintenance dose of the anti-BlyS antibody suppressed the B cell population and immunotolerance was induced in 20% of mice, but antibody formation could not be prevented. We then increased the maintenance dose of the anti-BlyS antibody and immunotolerance was induced in 50% of mice. Therapeutic enzyme distribution and clearance of GL-3 were also enhanced by a high maintenance dose of the anti-BlyS antibody.

  13. 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.

  14. 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.

  15. 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.

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

    PubMed Central

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

    2013-01-01

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

  17. Cinnamaldehyde enhances Nrf2 nuclear translocation to upregulate phase II detoxifying enzyme expression in HepG2 cells.

    PubMed

    Huang, Tzou-Chi; Chung, Yu-Ling; Wu, Mei-Li; Chuang, Show-Mei

    2011-05-11

    Cinnamaldehyde has been demonstrated to stimulate glutathione production and the expression of phase II detoxifying enzymes in HepG2 cells. The mechanism underlying this cinnamaldehyde-mediated gene expression relies on Nrf2 transcriptional activity. Therefore, the molecular signaling events in cinnamaldehyde-mediated detoxifying enzyme expression were further investigated in this study. Cinnamaldehyde activated ERK1/2, Akt, and JNK signaling pathways, but not the p38 MAP kinase pathway, subsequently leading to Nrf2 nuclear translocation and eventually increasing phase II enzyme expression. In contrast, inhibition of ERK1/2, Akt, or JNK pathways attenuated Nrf2 nuclear translocation and phase II enzyme expression. Depletion of Nrf2 by small RNA interference (si-RNA) showed that the protein levels of phase II enzymes were no longer induced by cinnamaldehyde. A luciferase reporter assay and an electrophoretic mobility shift assay (EMSA) also demonstrated that cinnamaldehyde-activated signaling resulted in the increased transcriptional activity of Nrf2 through binding to the ARE4 enhancer sequence. Altogether, these data suggest that ERK1/2, Akt, and JNK pathways activated by cinnamaldehyde collectively control Nrf2 nuclear translocation and transcriptional activity, leading to the increase of phase II enzyme expression. Application of an appropriate chemopreventive agent such as cinnamaldehyde could potentially be an alternative strategy for cancer chemoprevention.

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

    USDA-ARS?s Scientific Manuscript database

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

  19. 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.

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

    PubMed Central

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

    2017-01-01

    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 hyper-stable 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. PMID:27071357

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

    USDA-ARS?s Scientific Manuscript database

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

  2. 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

  3. 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.

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

    PubMed

    Bu, Lingxi; Xing, Yang; Yu, Hailong; Gao, Yuxia; Jiang, Jianxin

    2012-12-04

    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. 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. 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.

  5. 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.

  6. Ethanol production from sunflower meal biomass by simultaneous saccharification and fermentation (SSF) with Kluyveromyces marxianus ATCC 36907.

    PubMed

    Camargo, Danielle; Gomes, Simone D; Sene, Luciane

    2014-11-01

    The lignocellulosic materials are considered promising renewable resources for ethanol production, but improvements in the processes should be studied to reduce operating costs. Thus, the appropriate enzyme loading for cellulose saccharification is critical for process economics. This study aimed at evaluating the concentration of cellulase and β-glucosidase in the production of bioethanol by simultaneous saccharification and fermentation (SSF) of sunflower meal biomass. The sunflower biomass was pretreated with 6% H2SO4 (w/v), at 121 °C, for 20 min, for hemicellulose removal and delignificated with 1% NaOH. SSF was performed with Kluyveromyces marxianus ATCC 36907, at 38 °C, 150 rpm, for 72 h, with different enzyme concentrations (Cellulase Complex NS22086-10, 15 and 20 FPU/gsubstrate and β-Glucosidase NS22118, with a cellulase to β-glucosidase ratio of 1.5:1; 2:1 and 3:1). The best condition for ethanol production was cellulase 20 FPU/gsubstrate and β-glucosidase 13.3 CBU/gsubstrate, resulting in 27.88 g/L ethanol, yield of 0.47 g/g and productivity of 0.38 g/L h. Under this condition the highest enzymatic conversion of cellulose to glucose was attained (87.06%).

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

    PubMed

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

    2011-06-01

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

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

    PubMed Central

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

    2011-01-01

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

  9. 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.

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

    PubMed Central

    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

  11. Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media.

    PubMed

    Akbar, Umar; Aschenbrenner, Carl D; Harper, Michael R; Johnson, Harvey R; Dordick, Jonathan S; Clark, Douglas S

    2007-04-15

    A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed "direct solubilization," this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation.

  12. 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

  13. Metal accumulation by sunflower (Helianthus annuus L.) and the efficacy of its biomass in enzymatic saccharification.

    PubMed

    Dhiman, Saurabh Sudha; Zhao, Xin; Li, Jinglin; Kim, Dongwook; Kalia, Vipin C; Kim, In-Won; Kim, Jae Young; Lee, Jung-Kul

    2017-01-01

    Accumulation of metal contaminants in soil as a result of various industrial and anthropogenic activities has reduced soil fertility significantly. Phytoextraction of metal contaminants can improve soil fertility and provide inexpensive feedstock for biorefineries. We investigated the hyperaccumulation capacity of sunflower (Helianthus annuus) biomass by cultivating these plants in various concentrations of metal contaminants. Sunflowers were grown in soils contaminated with various levels of heavy metals (10-2,000 mg/kg dry soil). The degree of metal uptake by different parts of the biomass and the residual concentration in the soil were estimated through inductively coupled plasma mass spectrometry. An almost 2.5-fold hyperaccumulation of Zn2+ was observed in the leaf and flower biomass compared with the concentration in the soil. For the subsequent saccharification of biomass with hyperaccumulated contaminants, a fungal lignocellulosic consortium was used. The fungal consortium cocktail retained more than 95% filter paper activity with 100 mM Ni2+ ions even after 36 h. The highest saccharification yield (SY, 87.4%) was observed with Ni2+ as the contaminant (10 mg/kg dry wt), whereas Pb2+ (251.9 mg/kg dry wt) was the strongest inhibitor of biomass hydrolysis, resulting in only a 30% SY. Importantly, the enzyme cocktail produced by the fungal consortium resulted in almost the same SY (%) as that obtained from a combination of commercial cellulase and β-glucosidase. Significant sugar conversion (61.7%) from H. annuus biomass hydrolysate occurred, resulting in the production of 11.4 g/L of bioethanol. This is the first study to assess the suitability of phytoremediated sunflower biomass for bioethanol production.

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

    PubMed

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

    2011-02-01

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

  15. Metal accumulation by sunflower (Helianthus annuus L.) and the efficacy of its biomass in enzymatic saccharification

    PubMed Central

    Dhiman, Saurabh Sudha; Zhao, Xin; Li, Jinglin; Kim, Dongwook; Kalia, Vipin C.; Kim, In-Won; Kim, Jae Young; Lee, Jung-Kul

    2017-01-01

    Accumulation of metal contaminants in soil as a result of various industrial and anthropogenic activities has reduced soil fertility significantly. Phytoextraction of metal contaminants can improve soil fertility and provide inexpensive feedstock for biorefineries. We investigated the hyperaccumulation capacity of sunflower (Helianthus annuus) biomass by cultivating these plants in various concentrations of metal contaminants. Sunflowers were grown in soils contaminated with various levels of heavy metals (10–2,000 mg/kg dry soil). The degree of metal uptake by different parts of the biomass and the residual concentration in the soil were estimated through inductively coupled plasma mass spectrometry. An almost 2.5-fold hyperaccumulation of Zn2+ was observed in the leaf and flower biomass compared with the concentration in the soil. For the subsequent saccharification of biomass with hyperaccumulated contaminants, a fungal lignocellulosic consortium was used. The fungal consortium cocktail retained more than 95% filter paper activity with 100 mM Ni2+ ions even after 36 h. The highest saccharification yield (SY, 87.4%) was observed with Ni2+ as the contaminant (10 mg/kg dry wt), whereas Pb2+ (251.9 mg/kg dry wt) was the strongest inhibitor of biomass hydrolysis, resulting in only a 30% SY. Importantly, the enzyme cocktail produced by the fungal consortium resulted in almost the same SY (%) as that obtained from a combination of commercial cellulase and β-glucosidase. Significant sugar conversion (61.7%) from H. annuus biomass hydrolysate occurred, resulting in the production of 11.4 g/L of bioethanol. This is the first study to assess the suitability of phytoremediated sunflower biomass for bioethanol production. PMID:28437478

  16. 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.

  17. Elucidation of Factors Effecting Enzymatic Saccharification using Transgenic Hardwoods

    NASA Astrophysics Data System (ADS)

    Min, Douyong

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

  18. 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. Copyright © 2013 SETAC.

  19. Saccharification of Douglas-fir wood by a combination of prehydrolysis and pyrolysis

    SciTech Connect

    Shafizadeh, F.; Stevenson, T.T.

    1982-12-01

    A process has been investigated for the saccharification of wood, involving prehydrolysis, lignocellulose pyrolysis, and tar hydrolysis. In this process, ground wood was first prehydrolyzed to remove the more readily hydrolyzable hemicelluloses. The residual lignocellulose was then pyrolyzed rapidly to provide a tar containing levoglucosan and its condensation products. The tar was hydrolyzed to convert these products to glucose. Laboratory experiments have shown that this process can convert a common softwood such as Douglas-fir to 14% char and 42% hexoses. This amounts to a 59% recovery of the hexoses: 32% from prehydrolysis and 27% from pyrolysis. The prehydrolysis served not only to remove hemicelluloses, but also to increase the yield of glucose from pyrolysis and subsequent tar hydrolysis. It has been shown that this enhancement is due to the removal of inorganic ash and the catalytic effect of trace amounts of acid remaining in the lignocellulose. 3 figures, 3 tables.

  20. Sorghum husk biomass as a potential substrate for production of cellulolytic and xylanolytic enzymes by Nocardiopsis sp. KNU.

    PubMed

    Kshirsagar, Siddheshwar D; Bhalkar, Bhumika N; Waghmare, Pankajkumar R; Saratale, Ganesh D; Saratale, Rijuta G; Govindwar, Sanjay P

    2017-07-01

    Nocardiopsis sp. KNU was found to degrade various lignocellulosic waste materials, namely, sorghum husk, sugarcane tops and leaves, wheat straw, and rice husk very efficiently. The strain was found to produce high amounts of cellulase and hemicellulase. Augmentation of cotton seed cake as an organic nitrogen source revealed inductions in activities of endoglucanase, glucoamylase, and xylanase up to 70.03, 447.89, and 275.10 U/ml, respectively. Nonionic surfactant Tween-80 addition was found to enhance the activity of endoglucanase enzyme. Cellulase produced by Nocardiopsis sp. KNU utilizing sorghum husk as a substrate was found to retain its stability in various surfactants up to 90%. The produced enzyme was further tested for saccharification of mild alkali pretreated rice husk. The changes in morphology and functional group were analyzed using scanning electron microscopy and Fourier transform infrared spectroscopy. Enzymatic saccharification confirmed the hydrolytic potential of crude cellulase. The hydrolysate products were analyzed by high-performance thin layer chromatography.

  1. 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.

  2. Rapid and Complete Enzyme Hydrolysis of Lignocellulosic Nanofibrils

    Treesearch

    Raquel Martin-Sampedro; Ilari Filpponen; Ingrid C. Hoeger; J.Y. Zhu; Janne Laine; Orlando J. Rojas

    2012-01-01

    Rapid enzymatic saccharification of lignocellulosic nanofibrils (LCNF) was investigated by monitoring nanoscale changes in mass via quartz crystal microgravimetry and also by measuring reducing sugar yields. In only a few minutes LCNF thin films were completely hydrolyzed upon incubation in multicomponent enzyme systems. Conversion to sugars and oligosaccharides of...

  3. Continuous ethanol production from cassava through simultaneous saccharification and fermentation by self-flocculating yeast Saccharomyces cerevisiae CHFY0321.

    PubMed

    Choi, Gi-Wook; Kang, Hyun-Woo; Moon, Se-Kwon; Chung, Bong-Woo

    2010-03-01

    In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h(-1). Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h(-1). At dilution rates in the range of 0.014 h(-1) to 0.042 h(-1), high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h(-1), the ethanol volumetric productivity and ethanol yield were 3.58 g/L x h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.

  4. 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

  5. Production of lignocellulolytic enzymes and enhancement of in vitro digestibility during solid state fermentation of wheat straw by Phlebia floridensis.

    PubMed

    Sharma, Rakesh Kumar; Arora, Daljit Singh

    2010-12-01

    Degradation by white rot fungi has the potential to increase digestibility of wheat straw and thus improve its value as animal feed. To optimize conditions for production of lignocellulolytic enzymes by Phlebia floridensis during solid state fermentation of wheat straw along with enhancement of in vitro digestibility, a response surface methodology (RSM) based experiment was designed. Effect of moisture content, inorganic nitrogen source (NH(4)Cl) and malt extract on lignocellulolytic enzymes, changes in chemical constituents and digestibility of wheat straw was evaluated. With increase in moisture content, laccase production increased up to 34-fold, while Manganese peroxidase was optimally produced in the presence of almost equal amount (50-55 mg/g of WS) of NH(4)Cl and malt extract. These supplements also significantly (p<0.05) enhanced the production of CMCase and xylanase. In vitro digestibility was increased by almost 50% with a loss of 27.6% and 14.6% in lignin and total organic matter, respectively. The present findings revealed P. floridensis to be an efficient organism for lignocellulolytic enzymes production and simultaneous enhancement in in vitro digestibility of wheat straw. 2010 Elsevier Ltd. All rights reserved.

  6. Corn stover saccharification with concentrated sulfuric acid: effects of saccharification conditions on sugar recovery and by-product generation.

    PubMed

    Liu, Ze-Shen; Wu, Xiao-Lei; Kida, Kenji; Tang, Yue-Qin

    2012-09-01

    Although concentrated sulfuric acid saccharification is not a novel method for breaking down lignocellulosic biomass, the process by which saccharification affects biomass decomposition, sugar recovery, and by-product generation is not well studied. The present study employed Taguchi experimental design to study the effects of seven parameters on corn stover concentrated sulfuric acid saccharification. The concentration of sulfuric acid and the temperature of solubilization significantly affect corn stover decomposition. They also have significant effects on glucose and xylose recoveries. Low generation of furfural and 5-hydroxymethyl-2-furfural (5HMF) was noted and organic acids were the main by-products detected in the hydrolysate. Temperature also significantly affected the generation of levulinic acid and formic acid; however, acetic acid generation was not significantly influenced by all seven parameters. The ratio of acid to feedstock significantly affected glucose recovery, but not total sugar recovery. The corn stover hydrolysate was well fermented by both glucose- and xylose-fermenting yeast strains. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. 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.

  8. 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

  9. 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.

  10. Study on optimization of process parameters for enhancing the multi-hydrolytic enzyme activity in garbage enzyme produced from preconsumer organic waste.

    PubMed

    Arun, C; Sivashanmugam, P

    2017-02-01

    The garbage enzymes produced from preconsumer organic waste containing multi hydrolytic enzyme activity which helps to solubilize the waste activated sludge. The continuous production of garbage enzyme and its scaling up process need a globe optimized condition. In present study the effect of fruit peel composition and sonication time on enzyme activity were investigated. Garbage enzyme produced from 6g pineapple peels: 4g citrus peels pre-treated with ultrasound for 20min shows higher hydrolytic enzymes activity. Simultaneously statistical optimization tools were used to model garbage enzyme production with higher activity of amylase, lipase and protease. The maximum activity of amylase, lipase and protease were predicted to be 56.409, 44.039, 74.990U/ml respectively at optimal conditions (pH (6), temperature (37°C), agitation (218 RPM) and fermentation duration (3days)). These optimized conditions can be successfully used for large scale production of garbage enzyme with higher hydrolytic enzyme activity.

  11. 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.

  12. Production of l(+)-lactic acid from acid pretreated sugarcane bagasse using Bacillus coagulans DSM2314 in a simultaneous saccharification and fermentation strategy.

    PubMed

    van der Pol, Edwin C; Eggink, Gerrit; Weusthuis, Ruud A

    2016-01-01

    Sugars derived from lignocellulose-rich sugarcane bagasse can be used as feedstock for production of l(+)-lactic acid, a precursor for renewable bioplastics. In our research, acid-pretreated bagasse was hydrolysed with the enzyme cocktail GC220 and fermented by the moderate thermophilic bacterium Bacillus coagulans DSM2314. Saccharification and fermentation were performed simultaneously (SSF), adding acid-pretreated bagasse either in one batch or in two stages. SSF was performed at low enzyme dosages of 10.5-15.8 FPU/g DW bagasse. The first batch SSF resulted in an average productivity of 0.78 g/l/h, which is not sufficient to compete with lactic acid production processes using high-grade sugars. Addition of 1 g/l furfural to precultures can increase B. coagulans resistance towards by-products present in pretreated lignocellulose. Using furfural-containing precultures, productivity increased to 0.92 g/l/h, with a total lactic acid production of 91.7 g in a 1-l reactor containing 20% W/W DW bagasse. To increase sugar concentrations, bagasse was solubilized with a liquid fraction, obtained directly after acid pretreatment. Solubilizing the bagasse fibres with water increased the average productivity to 1.14 g/l/h, with a total lactic acid production of 84.2 g in a 1-l reactor. Addition of bagasse in two stages reduced viscosity during SSF, resulting in an average productivity in the first 23 h of 2.54 g/l/h, similar to productivities obtained in fermentations using high-grade sugars. Due to fast accumulation of lactic acid, enzyme activity was repressed during two-stage SSF, resulting in a decrease in productivity and a slightly lower total lactic acid production of 75.6 g. In this study, it is shown that an adequate production of lactic acid from lignocellulose was successfully accomplished by a two-stage SSF process, which combines acid-pretreated bagasse, B. coagulans precultivated in the presence of furfural as microorganism, and GC220 as enzyme

  13. 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.

  14. Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse.

    PubMed

    Mishra, Vartika; Jana, Asim K; Jana, Mithu Maiti; Gupta, Antriksh

    2017-07-01

    The objective of this work was to study the increase in multiple lignolytic enzyme productions through the use of supplements in combination in pretreatment of sweet sorghum bagasse (SSB) by Coriolus versicolor such that enzymes act synergistically to maximize the lignin degradation and selectivity. Enzyme activities were enhanced by metallic salts and phenolic compound supplements in SSF. Supplement of syringic acid increased the activities of LiP, AAO and laccase; gallic acid increased MnP; CuSO4 increased laccase and PPO to improve the lignin degradations and selectivity individually, higher than control. Combination of supplements optimized by RSM increased the production of laccase, LiP, MnP, PPO and AAO by 17.2, 45.5, 3.5, 2.4 and 3.6 folds respectively for synergistic action leading to highest lignin degradation (2.3 folds) and selectivity (7.1 folds). Enzymatic hydrolysis of pretreated SSB yielded ∼2.43 times fermentable sugar. This technique could be widely applied for pretreatment and enzyme productions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. 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.

  16. Metagenomics for the development of new biocatalysts to advance lignocellulose saccharification for bioeconomic development.

    PubMed

    Montella, Salvatore; Amore, Antonella; Faraco, Vincenza

    2016-12-01

    The world economy is moving toward the use of renewable and nonedible lignocellulosic biomasses as substitutes for fossil sources in order to decrease the environmental impact of manufacturing processes and overcome the conflict with food production. Enzymatic hydrolysis of the feedstock is a key technology for bio-based chemical production, and the identification of novel, less expensive and more efficient biocatalysts is one of the main challenges. As the genomic era has shown that only a few microorganisms can be cultured under standard laboratory conditions, the extraction and analysis of genetic material directly from environmental samples, termed metagenomics, is a promising way to overcome this bottleneck. Two screening methodologies can be used on metagenomic material: the function-driven approach of expression libraries and sequence-driven analysis based on gene homology. Both techniques have been shown to be useful for the discovery of novel biocatalysts for lignocellulose conversion, and they enabled identification of several (hemi)cellulases and accessory enzymes involved in (hemi)cellulose hydrolysis. This review summarizes the latest progress in metagenomics aimed at discovering new enzymes for lignocellulose saccharification.

  17. 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.

  18. 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.

  19. 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).

  20. 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.

  1. Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance

    PubMed Central

    Gusakov, Alexander V.; Nemashkalov, Vitaly A.; Satrutdinov, Aidar D.; Sinitsyn, Arkady P.

    2017-01-01

    Background Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases. Results Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL), and eglIV, encoding LPMO (formerly endoglucanase IV) from Trichoderma reesei (TrLPMO), were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3) varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples). The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10–43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1. Conclusions The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a

  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. Copyright 2004 American Chemical Society

  3. 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.

  4. 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.

  5. 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

  6. Comparison of two-stage acid-alkali and alkali-acid pretreatments on enzymatic saccharification ability of the sweet sorghum fiber and their physicochemical characterizations.

    PubMed

    Li, Ping; Cai, Di; Zhang, Changwei; Li, Shufeng; Qin, Peiyong; Chen, Changjing; Wang, Yong; Wang, Zheng

    2016-12-01

    Two-stage acid/alkali pretreatment was used to enhance the saccharification efficiency of sweet sorghum fiber. The physicochemical characterizations of the pretreated fibers were evaluated by SEM, FTIR and XRD. The acid and alkali sequence in the two-stage pretreatment process was compared, and their dosage was optimized. The results indicated that the two-stage pretreatment showed better saccharification performance when compared with conventional single stage pretreatment. And compared with the acid-alkali sequence, the alkali-acid sequence achieved higher glucose yield (0.23g·g(-1)) under the optimized conditions, which was 1.64 and 1.21 times higher than that of the single stage and the acid-alkali pretreatments, respectively. Overall, the two-stage pretreatment process is a promising approach to achieve high fermentable glucose conversion rate of cellulosic material. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Immobilization of indigenous holocellulase on iron oxide (Fe2O3) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw.

    PubMed

    Kumar, Ajay; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

    2017-03-01

    The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher Vmax and lower Km than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw.

  8. EFICAz2: enzyme function inference by a combined approach enhanced by machine learning.

    PubMed

    Arakaki, Adrian K; Huang, Ying; Skolnick, Jeffrey

    2009-04-13

    We previously developed EFICAz, an enzyme function inference approach that combines predictions from non-completely overlapping component methods. Two of the four components in the original EFICAz are based on the detection of functionally discriminating residues (FDRs). FDRs distinguish between member of an enzyme family that are homofunctional (classified under the EC number of interest) or heterofunctional (annotated with another EC number or lacking enzymatic activity). Each of the two FDR-based components is associated to one of two specific kinds of enzyme families. EFICAz exhibits high precision performance, except when the maximal test to training sequence identity (MTTSI) is lower than 30%. To improve EFICAz's performance in this regime, we: i) increased the number of predictive components and ii) took advantage of consensual information from the different components to make the final EC number assignment. We have developed two new EFICAz components, analogs to the two FDR-based components, where the discrimination between homo and heterofunctional members is based on the evaluation, via Support Vector Machine models, of all the aligned positions between the query sequence and the multiple sequence alignments associated to the enzyme families. Benchmark results indicate that: i) the new SVM-based components outperform their FDR-based counterparts, and ii) both SVM-based and FDR-based components generate unique predictions. We developed classification tree models to optimally combine the results from the six EFICAz components into a final EC number prediction. The new implementation of our approach, EFICAz2, exhibits a highly improved prediction precision at MTTSI < 30% compared to the original EFICAz, with only a slight decrease in prediction recall. A comparative analysis of enzyme function annotation of the human proteome by EFICAz2 and KEGG shows that: i) when both sources make EC number assignments for the same protein sequence, the assignments tend to

  9. EFICAz2: enzyme function inference by a combined approach enhanced by machine learning

    PubMed Central

    Arakaki, Adrian K; Huang, Ying; Skolnick, Jeffrey

    2009-01-01

    Background We previously developed EFICAz, an enzyme function inference approach that combines predictions from non-completely overlapping component methods. Two of the four components in the original EFICAz are based on the detection of functionally discriminating residues (FDRs). FDRs distinguish between member of an enzyme family that are homofunctional (classified under the EC number of interest) or heterofunctional (annotated with another EC number or lacking enzymatic activity). Each of the two FDR-based components is associated to one of two specific kinds of enzyme families. EFICAz exhibits high precision performance, except when the maximal test to training sequence identity (MTTSI) is lower than 30%. To improve EFICAz's performance in this regime, we: i) increased the number of predictive components and ii) took advantage of consensual information from the different components to make the final EC number assignment. Results We have developed two new EFICAz components, analogs to the two FDR-based components, where the discrimination between homo and heterofunctional members is based on the evaluation, via Support Vector Machine models, of all the aligned positions between the query sequence and the multiple sequence alignments associated to the enzyme families. Benchmark results indicate that: i) the new SVM-based components outperform their FDR-based counterparts, and ii) both SVM-based and FDR-based components generate unique predictions. We developed classification tree models to optimally combine the results from the six EFICAz components into a final EC number prediction. The new implementation of our approach, EFICAz2, exhibits a highly improved prediction precision at MTTSI < 30% compared to the original EFICAz, with only a slight decrease in prediction recall. A comparative analysis of enzyme function annotation of the human proteome by EFICAz2 and KEGG shows that: i) when both sources make EC number assignments for the same protein sequence, the

  10. Plasmon-Enhanced Enzymatic Reactions: A Study of Nanoparticle-Enzyme Distance- and Nanoparticle Loading-Dependent Enzymatic Activity

    PubMed Central

    Abel, Biebele; Akinsule, Alice; Andrews, Canisha; Aslan, Kadir

    2011-01-01

    A detailed investigation of the dependence of the efficiency of plasmon-enhanced enzymatic reactions on the distance between silver island films (SIFs) and horse radish peroxidase (HRP) enzyme and on the loading of SIFs on glass surfaces is presented. Three different extent of loading of SIFs on glass slides were used: 1) low, 2) medium and 3) high, which was characterized by using optical absorption spectroscopy and scanning electron microscopy. Streptavidin-linked HRP enzyme was deposited onto SIFs and glass slides by using three different strategies: strategy 1: biotin-avidin protein assay (distance between SIFs and HRP = 4–8 nm), strategy 2: self assembled monolayers (SAMs) (1–5 nm), strategy 3: polymer layer (1–5 nm). The efficiency of enzymatic conversion of O-phenylenediamine dihydrochloride (OPD) to a colored product by HRP on SIFs and glass surfaces was assessed by optical absorption spectroscopy. The distance between SIFs and HRP and the extent of loading of SIFs on the glass surfaces were shown to have significant effect on the efficiency of plasmon-enhanced enzymatic reactions. In this regard, up to an %250 increase in enzymatic conversion of OPD was observed from SIFs with high loading using strategy 1. In addition, we have studied the potential of repeated use of SIFs in plasmon-enhanced enzymatic reactions. PMID:21949594

  11. Plasmon-Enhanced Enzymatic Reactions: A Study of Nanoparticle-Enzyme Distance- and Nanoparticle Loading-Dependent Enzymatic Activity.

    PubMed

    Abel, Biebele; Akinsule, Alice; Andrews, Canisha; Aslan, Kadir

    2011-01-01

    A detailed investigation of the dependence of the efficiency of plasmon-enhanced enzymatic reactions on the distance between silver island films (SIFs) and horse radish peroxidase (HRP) enzyme and on the loading of SIFs on glass surfaces is presented. Three different extent of loading of SIFs on glass slides were used: 1) low, 2) medium and 3) high, which was characterized by using optical absorption spectroscopy and scanning electron microscopy. Streptavidin-linked HRP enzyme was deposited onto SIFs and glass slides by using three different strategies: strategy 1: biotin-avidin protein assay (distance between SIFs and HRP = 4-8 nm), strategy 2: self assembled monolayers (SAMs) (1-5 nm), strategy 3: polymer layer (1-5 nm). The efficiency of enzymatic conversion of O-phenylenediamine dihydrochloride (OPD) to a colored product by HRP on SIFs and glass surfaces was assessed by optical absorption spectroscopy. The distance between SIFs and HRP and the extent of loading of SIFs on the glass surfaces were shown to have significant effect on the efficiency of plasmon-enhanced enzymatic reactions. In this regard, up to an %250 increase in enzymatic conversion of OPD was observed from SIFs with high loading using strategy 1. In addition, we have studied the potential of repeated use of SIFs in plasmon-enhanced enzymatic reactions.

  12. 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.

  13. Enhanced antioxidant enzymes are associated with reduced hydrogen peroxide in barley roots under saline stress.

    PubMed

    Kim, Sang Yong; Lim, Jung-Hyun; Park, Myoung Ryoul; Kim, Young Jin; Park, Tae Il; Seo, Yong Won; Choi, Kyeong Gu; Yun, Song Joong

    2005-03-31

    Antioxidant enzymes are related to the resistance to various abiotic stresses including salinity. Barley is relatively tolerant to saline stress among crop plants, but little information is available on barley antioxidant enzymes under salinity stress. We investigated temporal and spatial responses of activities and isoform profiles of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), non-specific peroxidase (POX), and glutathione reductase (GR) to saline stress in barley seedlings treated with 200 mM NaCl for 0, 1, 2, 5 days, respectively. In the control plant, hydrogen peroxide content was about 2-fold higher in the root than in the shoot. Under saline stress, hydrogen peroxide content was decreased drastically by 70% at 2 d after NaCl treatment (DAT) in the root. In the leaf, however, the content was remained unchanged by 2 DAT and increased about 14 % at 5 DAT. In general, the activities of antioxidant enzymes were increased in the root and shoot under saline stress. But the increase was more significant and consistent in the root. The activities of SOD, CAT, APX, POX, and GR were increased significantly in the root within 1 DAT, and various elevated levels were maintained by 5 DAT. Among the antioxidant enzymes, CAT activity was increased the most drastically. The significant increase in the activities of SOD, CAT, APX, POX, and GR in the NaCl-stressed barley root was highly correlated with the increased expression of the constitutive isoforms as well as the induced ones. The hydrogen peroxide content in the root.

  14. 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.

  15. Phenolic acids in some cereal grains and their inhibitory effect on starch liquefaction and saccharification.

    PubMed

    Kandil, Amin; Li, Jihong; Vasanthan, Thava; Bressler, David C

    2012-08-29

    The presence of phenolic acids in cereal grain is thought to influence starch hydrolysis during liquefaction and saccharification of grain flours in the bioethanol industry. As a basis for remodeling starch hydrolysis systems and understanding inhibition mechanisms, the composition and concentration of phenolic acids in whole grain flours of triticale, wheat, barley, and corn were analyzed by high-performance liquid chromatography. The total phenolic acid contents (sum of nine phenolic acids) in the four grains were 1.14, 1.70, 0.90, and 1.25 mg/g, respectively, with more than 90% found in the bound form. Ferulic, coumaric, and protocatechuic acids were the major phenolic acids in triticale and wheat. Gallic acid was also rich in triticale. Ferulic, coumaric, hydroxybenzoic, and gallic acids were predominant in barley. In corn, ferulic, coumaric, gallic, and syringic acids were abundant. On the basis of these profiles, pure phenolic acids were added individually and collectively to isolated starches at amounts either equivalent to or 3 times those in the whole grains for hydrolysis. The degree of starch hydrolysis with α-amylase and amyloglucosidase decreased up to 8% when individual phenolic acids were present in cooked starch slurry. The decreases were more pronounced when phenolic acids were added collectively (4-5% with α-amylase and 9-13% with sequential α-amylase and amyloglucosidase). The study of a phenolic acid-starch-enzyme model system indicated that the interactions of phenolic acid-enzyme and phenolic acid-starch significantly contributed to the inhibitory effect of starch hydrolysis. Heating facilitated the interactions. Phenolic acids thus play a significant role in the resistance of starch to enzyme and/or the loss of enzyme activity during starch hydrolysis.

  16. Creation of a putative third metal binding site in type II dihydroorotases significantly enhances enzyme activity.

    PubMed

    Huang, Yen-Hua; Huang, Cheng-Yang

    2015-01-01

    Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. DHOase is divided into two types (I and II). Type II DHOase generally contains a binuclear metal center in its active site. Recently, the crystal structure of DHOase domain in human CAD protein (huDHOase) has revealed three metal ions in the protein's active site. However, whether type II DHOase can have the critical third metal ion, as observed in huDHOase, remains unknown. In the present study, the putative third metal binding site in type II enzymes, such as the prokaryotic Salmonella enterica serovar Typhimurium LT2 DHOase (StDHOase) and the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase), was created and identified. StDHOase T198E and ScDHOase T208E mutants had higher activities compared with their wild-type enzymes. The need for a higher DHOase stability and activity may drive creation of the third metal ion binding site in huDHOase, which can be achieved by mutating a highly conserved position T in type II dihydroorotases to E, similar to that in huDHOase.

  17. Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403.

    PubMed

    Abicht, Helge K; Gonskikh, Yulia; Gerber, Simon D; Solioz, Marc

    2013-06-01

    Lactococcus lactis possesses a pronounced extracellular Cu(2+)-reduction activity which leads to the accumulation of Cu(+) in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductase-deficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The ΔmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the ΔmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the ΔmenE mutant, due to the production of Cu(+) ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu(+) was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu(+) toxicity in L. lactis.

  18. 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.

  19. Microwave-assisted inorganic salt pretreatment of sugarcane leaf waste: Effect on physiochemical structure and enzymatic saccharification.

    PubMed

    Moodley, Preshanthan; Kana, E B Gueguim

    2017-07-01

    This paper presents a method to pretreat sugarcane leaf waste using microwave-assisted (MA) inorganic salt to enhance enzymatic saccharification. The effects of process parameters of salt concentration, microwave power intensity and pretreatment time on reducing sugar yield from sugarcane leaf waste were investigated. Pretreatment models based on MA-NaCl, MA-ZnCl2 and MA-FeCl3 were developed with high coefficients of determination (R(2) >0.8) and optimized. Maximum reducing sugar yield of 0.406g/g was obtained with 2M FeCl3 at 700W for 3.5min. Scanning electron microscopy (SEM), Fourier Transform Infrared analysis (FTIR) and X-ray diffraction (XRD) showed major changes in lignocellulosic structure after MA-FeCl3 pretreatment with 71.5% hemicellulose solubilization. This regime was further assessed on sorghum leaves and Napier grass under optimal MA-FeCl3 conditions. A 2-fold and 3.1-fold increase in sugar yield respectively were observed compared to previous reports. This pretreatment was highly effective for enhancing enzymatic saccharification of lignocellulosic biomass. Copyright © 2017. Published by Elsevier Ltd.

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

    PubMed Central

    Marx, Ailie; Galilee, Meytal; Alian, Akram

    2015-01-01

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

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

    PubMed

    Marx, Ailie; Galilee, Meytal; Alian, Akram

    2015-12-18

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

  2. Enhanced ELISA using a handheld pH meter and enzyme-coated microparticles for the portable, sensitive detection of proteins.

    PubMed

    Zhang, Yun; Yang, Jiani; Nie, Jinfang; Yang, Juanhua; Gao, Dong; Zhang, Lang; Li, Jianping

    2016-02-28

    This work describes a general methodology for enhanced enzyme-linked immunosorbent assay (ELISA) that integrates enzyme-coated microparticle probes for robust yet highly efficient signal amplification and a handheld pH meter for a simple, portable, quantitative readout. Its utility is well demonstrated with the detection of the target protein with a 14-fold enhancement of sensitivity in comparison with the conventional optical ELISA.

  3. Enhancement of the activity of enzyme immobilized on polydopamine-coated iron oxide nanoparticles by rational orientation of formate dehydrogenase.

    PubMed

    Gao, Xin; Ni, Kefeng; Zhao, Chengcheng; Ren, Yuhong; Wei, Dongzhi

    2014-10-20

    Immobilization of enzymes onto nanoparticles and retention of their structure and activity, which may be related to the orientation of enzymes on nanoparticles, remain a challenge. Here, we developed a novel enzyme-orientation strategy to enhance the activity of formate dehydrogenase immobilized on polydopamine-coated iron oxide nanoparticles via site-directed mutation. Seven mutants were constructed based on homology modeling of formate dehydrogenase and immobilized on polydopamine-coated iron oxide nanoparticles to investigate the influence of these mutations on immobilization. The immobilized mutant C242A/C275V/C363V/K389C demonstrated the highest immobilization yield and retained 90% of its initial activity, which was about 3-fold higher than that of wild-type formate dehydrogenase. Moreover, co-immobilization of formate dehydrogenase and leucine dehydrogenase was performed for the synthesis of l-tert-leucine. The catalytic efficiency of the co-immobilized mutant C242A/C275V/C363V/K389C and leucine dehydrogenase increased by more than 4-fold compared to that of co-immobilized wild-type formate dehydrogenase and leucine dehydrogenase. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. 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.

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

    PubMed Central

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

    2013-01-01

    Simultaneous saccharification and fermentation (SSF) studies of steam exploded and alkali pretreated different leafy biomass were accomplished by recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for bioethanol production. The recombinant C. thermocellum GH5 cellulase and GH43 hemicellulase genes expressed in Escherichia coli cells were grown in repetitive batch mode, with the aim of enhancing the cell biomass production and enzyme activity. In batch mode, the cell biomass (A 600 nm) of E. coli cells and enzyme activities of GH5 cellulase and GH43 hemicellulase were 1.4 and 1.6 with 2.8 and 2.2 U·mg−1, which were augmented to 2.8 and 2.9 with 5.6 and 3.8 U·mg−1 in repetitive batch mode, respectively. Steam exploded wild grass (Achnatherum hymenoides) provided the best ethanol titres as compared to other biomasses. Mixed enzyme (GH5 cellulase, GH43 hemicellulase) mixed culture (Saccharomyces cerevisiae, Candida shehatae) system gave 2-fold higher ethanol titre than single enzyme (GH5 cellulase) single culture (Saccharomyces cerevisiae) system employing 1% (w/v) pretreated substrate. 5% (w/v) substrate gave 11.2 g·L−1 of ethanol at shake flask level which on scaling up to 2 L bioreactor resulted in 23 g·L−1 ethanol. 91.6% (v/v) ethanol was recovered by rotary evaporator with 21.2% purification efficiency. PMID:24089676

  6. 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

  7. 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.

  8. Enhanced biomolecular detection based on localized surface plasmon resonance (LSPR) using enzyme-precipitation reaction.

    PubMed

    Lee, Seung-Woo; Ahn, Junhyoung; Kim, Min-Gon; Shin, Yong-Beom; Lee, Jae Jong; Lim, Ki-Pil; Kim, Ki-Bum

    2010-05-01

    An enzyme-catalyzed precipitation reaction was employed as a means to increase the change in the LSPR signal after intermolecular bindings between antigens and antibodies occurred on gold nanodot surfaces. The gold nanodot array with an diameter of 175 nm and a thickness of 20 nm was fabricated on a glass wafer using thermal nanoimprint lithography. The human interleukin (hIL) 5 antibody was immobilized on the gold nanodot, followed by binding of hIL 5 to the anti-hIL 5. Subsequently, a biotinylated anti-hIL 5 and a alkaline phosphatase conjugated with streptavidin were simultaneously introduced. A mixture of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP) and nitro blue tetrazolium (NBT) was then used for precipitation, which resulted from the biocatalytic reaction of the alkaline phosphatase on gold nanodot. The LSPR spectra were obtained after each binding process. Using this analysis, the enzyme-catalyzed precipitation reaction on gold nanodots was found to be effective in amplifying the change in the peak wavelength of LSPR after molecular bindings.

  9. 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.

  10. Enzyme augmentation therapy enhances the therapeutic efficacy of bone marrow transplantation in mucopolysaccharidosis type II mice.

    PubMed

    Akiyama, Kazumasa; Shimada, Yohta; Higuchi, Takashi; Ohtsu, Makoto; Nakauchi, Hiromitsu; Kobayashi, Hiroshi; Fukuda, Takahiro; Ida, Hiroyuki; Eto, Yoshikatsu; Crawford, Brett E; Brown, Jillian R; Ohashi, Toya

    2014-02-01

    Before the availability of an enzyme replacement therapy (ERT) for mucopolysaccharidosis type II (MPS II), patients were treated by bone marrow transplantation (BMT). However, the effectiveness of BMT for MPS II was equivocal, particularly at addressing the CNS manifestations. To study this further, we subjected a murine model of MPS II to BMT and evaluated the effect at correcting the biochemical and pathological aberrations in the viscera and CNS. Our results indicated that BMT reduced the accumulation of glycosaminoglycans (GAGs) in a variety of visceral organs, but not in the CNS. With the availability of an approved ERT for MPS II, we investigated and compared the relative merits of the two strategies either as a mono or combination therapy. We showed that the combination of BMT and ERT was additive at reducing tissue levels of GAGs in the heart, kidney and lung. Moreover, ERT conferred greater efficacy if the immunological response against the infused recombinant enzyme was low. Finally, we showed that pathologic GAGs might potentially represent a sensitive biomarker to monitor the therapeutic efficacy of therapies for MPS II. © 2013.

  11. 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

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

    PubMed

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

    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.

  13. 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

  14. 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.

  15. Antitumor Synergism and Enhanced Survival with a Tumor Vasculature-Targeted Enzyme Prodrug System, Rapamycin, and Cyclophosphamide.

    PubMed

    Krais, John J; Virani, Needa; McKernan, Patrick H; Nguyen, Quang; Fung, Kar-Ming; Sikavitsas, Vassilios I; Kurkjian, Carla; Harrison, Roger G

    2017-09-01

    Mutant cystathionine gamma-lyase was targeted to phosphatidylserine exposed on tumor vasculature through fusion with Annexin A1 or Annexin A5. Cystathionine gamma-lyase E58N, R118L, and E338N mutations impart nonnative methionine gamma-lyase activity, resulting in tumor-localized generation of highly toxic methylselenol upon systemic administration of nontoxic selenomethionine. The described therapeutic system circumvents systemic toxicity issues using a novel drug delivery/generation approach and avoids the administration of nonnative proteins and/or DNA required with other enzyme prodrug systems. The enzyme fusion exhibits strong and stable in vitro binding with dissociation constants in the nanomolar range for both human and mouse breast cancer cells and in a cell model of tumor vascular endothelium. Daily administration of the therapy suppressed growth of highly aggressive triple-negative murine 4T1 mammary tumors in immunocompetent BALB/cJ mice and MDA-MB-231 tumors in SCID mice. Treatment did not result in the occurrence of negative side effects or the elicitation of neutralizing antibodies. On the basis of the vasculature-targeted nature of the therapy, combinations with rapamycin and cyclophosphamide were evaluated. Rapamycin, an mTOR inhibitor, reduces the prosurvival signaling of cells in a hypoxic environment potentially exacerbated by a vasculature-targeted therapy. IHC revealed, unsurprisingly, a significant hypoxic response (increase in hypoxia-inducible factor 1 α subunit, HIF1A) in the enzyme prodrug-treated tumors and a dramatic reduction of HIF1A upon rapamycin treatment. Cyclophosphamide, an immunomodulator at low doses, was combined with the enzyme prodrug therapy and rapamycin; this combination synergistically reduced tumor volumes, inhibited metastatic progression, and enhanced survival. Mol Cancer Ther; 16(9); 1855-65. ©2017 AACR. ©2017 American Association for Cancer Research.

  16. Targeting Heparan Sulfate Proteoglycans and their Modifying Enzymes to Enhance Anticancer Chemotherapy Efficacy and Overcome Drug Resistance.

    PubMed

    Lanzi, Cinzia; Zaffaroni, Nadia; Cassinelli, Giuliana

    2017-01-01

    Targeting heparan sulfate proteoglycans (HSPGs) and enzymes involved in heparan sulfate (HS) chain editing is emerging as a new anticancer strategy. The involvement of HSPGs in tumor cell signaling, inflammation, angiogenesis and metastasis indicates that agents able to inhibit aberrant HSPG functions can potentially act as multitarget drugs affecting both tumor cell growth and the supportive boost provided by the microenvironment. Moreover, accumulating evidence supports that an altered expression or function of HSPGs, or of the complex enzyme system regulating their activities, can also depress the tumor response to anticancer treatments in several tumor types. Thereby, targeting HSPGs or HSPG modifying enzymes appears an appealing approach to enhance chemotherapy efficacy. A great deal of effort from academia and industry has led to the development of agents mimicking HS, and/or inhibiting HSPG modifying enzymes. Inhibitors of Sulf-2, an endosulfatase that edits the HS sulfation pattern, and inhibitors of heparanase, the endoglycosidase that produces functional HS fragments, appear particularly promising. In fact, a Sulf-2 inhibitor (OKN-007), and two heparanase inhibitors/HS mimics (roneparstat, PG545) are currently under early clinical investigation. In this review, we summarized preclinical studies in experimental tumor models of the main chemical classes of Sulf-2 and heparanase inhibitors. We described examples of different mechanisms through which heparanase and HSPGs, often in cooperation, may impact tumor sensitivity to various antitumor agents. Finally, we reported a few preclinical studies showing increased antitumor efficacy obtained with the use of candidate clinical HS mimics in combination regimens. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. 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. © 2016 Wiley Periodicals, Inc.

  18. Effects of quinolone derivatives on eukaryotic topoisomerase II. A novel mechanism for enhancement of enzyme-mediated DNA cleavage.

    PubMed

    Robinson, M J; Martin, B A; Gootz, T D; McGuirk, P R; Moynihan, M; Sutcliffe, J A; Osheroff, N

    1991-08-05

    The effects of two novel quinolone derivatives, CP-67,804 and CP-115,953 (the 1-ethyl and 1-cyclopropyl derivatives of 6,8-difluoro-7-(4-hydroxyphenyl)-4-quinolone-3-carboxylic acid, respectively), on the enzymatic activities of Drosophila melanogaster topoisomerase II were examined. Both drugs enhanced the enzyme's pre- and post-strand passage DNA cleavage activities. CP-67,804 was nearly as potent an enhancer as etoposide, while CP-115,953 was approximately 2 times more potent than this topoisomerase II-targeted antineoplastic drug. In contrast to etoposide, which stabilizes enzyme-DNA cleavage complexes primarily by inhibiting topoisomerase II-mediated DNA religation, neither quinolone impaired the enzyme's ability to religate cleaved DNA. To further assess the characteristics of these unusual quinolone derivatives, the cytotoxic effects of CP-67,804 and CP-115,953 toward wild-type Chinese hamster ovary cells and VpmR-5 cells (an epipodophyllotoxin-resistant Chinese hamster ovary line) were examined. Both quinolones were cytotoxic to the wild-type cells. CP-115,953 was the more potent agent and displayed a level of cytotoxicity similar to that of etoposide. Finally, the VpmR-5 line showed cross-resistance to CP-67,804 (approximately 3.7-fold) and CP-115,953 (approximately 1.3-fold). Although quinolone cross-resistance was less pronounced than observed for etoposide (approximately 12-fold), it indicates that topoisomerase II is a physiological target for CP-67,804 and CP-115,953 in mammalian cells. These findings strongly suggest that these quinolone derivatives represent a novel class of topoisomerase II-targeted drugs which have potential as antineoplastic agents.

  19. Antioxidant enzymes in Spodoptera littoralis (Boisduval): are they enhanced to protect gut tissues during oxidative stress?

    PubMed

    Krishnan, Natraj; Kodrík, Dalibor

    2006-01-01

    The Egyptian armyworm Spodoptera littoralis is a polyphagous insect attacking a number of plant species including those belonging to the Solanaceae and Cruciferaceae families. Its digestive physiology must therefore adapt to the food plant to ensure maximum extraction of nutrients with minimum trade-off in terms of growth retardation by pro-oxidant allelochemicals. To investigate this, the caterpillars of S. littoralis were fed on a semi-artificial diet (Manduca Premix-Heliothis Premix) and for 24 h on potato plants (Solanum tuberosum), respectively, at the mature 6th instar, and the levels of oxidative radicals and antioxidant enzymes in their guts were compared. The gut pH, standard redox potential (Eh) and electron availability (pe) revealed that oxidizing conditions prevail which promote oxidation of pro-oxidant allelochemicals in foliage. Oxidative stress in the foregut and midgut tissue and the gut contents was assessed from the generation of superoxide radical, total peroxide content and protein carbonyl content. Antioxidant defense was measured by the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX) and glutathione S-transferase peroxidase (GSTpx). A significant (p < 0.001) increase in the superoxide radical production (in foregut tissue, foregut and midgut contents), concomitant with an increase in total peroxide (in foregut contents) and protein carbonyl levels (in foregut and midgut tissue) were noted in larvae fed on the plants in contrast to those fed the semi-artificial diet. Similarly, a significant up-regulation of antioxidant enzymes SOD (in midgut tissues), CAT (in foregut, midgut tissue and contents), APOX (in foregut contents, midgut tissue and contents) and GSTpx (in foregut tissues) was recorded on the plant diet in comparison to the semi-artificial diet. The pro-oxidant allelochemicals in the plant diet are thus eliminated by the insect at the expense of up-regulation of antioxidative enzymes in response

  20. High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol.

    PubMed

    Varga, Enikõ; Klinke, Helene B; Réczey, Kati; Thomsen, Anne Belinda

    2004-12-05

    In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.

  1. Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes.

    PubMed

    Ko, Ja Kyong; Bak, Jin Seop; Jung, Min Woo; Lee, Hee Jin; Choi, In-Geol; Kim, Tae Hyun; Kim, Kyoung Heon

    2009-10-01

    Rice straw was pretreated using aqueous-ammonia solution at moderate temperatures to enable production of the maximum amount of fermentable sugars from enzymatic hydrolysis. The effects of various operating variables including pretreatment temperature, pretreatment time, the concentration of ammonia and the solid-to-liquid ratio on the degree of lignin removal and the enzymatic digestibility were optimized using response surface methodology. The optimal reaction conditions, which resulted in an enzymatic digestibility of 71.1%, were found to be 69 degrees C, 10h and an ammonia concentration of 21% (w/w). The effects of different commercial cellulases and the additional effect of a non-cellulolytic enzyme, xylanase, were also evaluated. Additionally, simultaneous saccharification and fermentation was conducted with rice straw to assess the ethanol production yield and productivity.

  2. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

    PubMed

    Salvat, Regina S; Verma, Deeptak; Parker, Andrew S; Kirsch, Jack R; Brooks, Seth A; Bailey-Kellogg, Chris; Griswold, Karl E

    2017-06-27

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 10(9) variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

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

    PubMed

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

    2015-04-15

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

  4. Biological pretreatment of rice straw with Streptomyces griseorubens JSD-1 and its optimized production of cellulase and xylanase for improved enzymatic saccharification efficiency.

    PubMed

    Zhang, Dan; Luo, Yanqing; Chu, Shaohua; Zhi, Yuee; Wang, Bin; Zhou, Pei

    2016-08-17

    Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L(-1) for rice straw, wheat bran, peptone, and CaCO3, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL(-1) were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.

  5. Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification.

    PubMed

    Cara, Cristóbal; Ruiz, Encarnación; Oliva, José Miguel; Sáez, Felicia; Castro, Eulogio

    2008-04-01

    The production of fermentable sugars from olive tree biomass was studied by dilute acid pretreatment and further saccharification of the pretreated solid residues. Pretreatment was performed at 0.2%, 0.6%, 1.0% and 1.4% (w/w) sulphuric acid concentrations while temperature was in the range 170-210 degrees C. Attention is paid to sugar recovery both in the liquid fraction issued from pretreatment (prehydrolysate) and that in the water-insoluble solid (WIS). As a maximum, 83% of hemicellulosic sugars in the raw material were recovered in the prehydrolysate obtained at 170 degrees C, 1% sulphuric acid concentration, but the enzyme accessibility of the corresponding pretreated solid was not very high. In turn, the maximum enzymatic hydrolysis yield (76.5%) was attained from a pretreated solid (at 210 degrees C, 1.4% acid concentration) in which cellulose solubilization was detected; moreover, sugar recovery in the prehydrolysate was the poorest one among all the experiments performed. To take account of fermentable sugars generated by pretreatment and the glucose released by enzymatic hydrolysis, an overall sugar yield was calculated. The maximum value (36.3 g sugar/100 g raw material) was obtained when pretreating olive tree biomass at 180 degrees C and 1% sulphuric acid concentration, representing 75% of all sugars in the raw material. Dilute acid pretreatment improves results compared to water pretreatment.

  6. 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.

  7. 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.

  8. Optimization of simultaneous saccharification and fermentation conditions with amphipathic lignin derivatives for concentrated bioethanol production.

    PubMed

    Cheng, Ningning; Koda, Keiichi; Tamai, Yutaka; Yamamoto, Yoko; Takasuka, Taichi E; Uraki, Yasumitsu

    2017-05-01

    Amphipathic lignin derivatives (A-LDs) prepared from the black liquor of soda pulping of Japanese cedar are strong accelerators for bioethanol production under a fed-batch simultaneous enzymatic saccharification and fermentation (SSF) process. To improve the bioethanol production concentration, conditions such as reaction temperature, stirring program, and A-LDs loadings were optimized in both small scale and large scale fed-batch SSF. The fed-batch SSF in the presence of 3.0g/L A-LDs at 38°C gave the maximum ethanol production and a high enzyme recovery rate. Furthermore, a jar-fermenter equipped with a powerful mechanical stirrer was designed for 1.5L-scale fed-batch SSF to achieve rigorous mixing during high substrate loading. Finally, the 1.5L fed-batch SSF with a substrate loading of 30% (w/v) produced a high ethanol concentration of 87.9g/L in the presence of A-LDs under optimized conditions. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Conversion of aqueous ammonia-treated corn stover to lactic acid by simultaneous saccharification and cofermentation.

    PubMed

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

    2007-04-01

    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.

  10. Enhancement of toxicity and enzyme-repressing activity of p-dioxane by chlorination: stereoselective effects.

    PubMed

    Woo, Y T; Neuburger, B J; Arcos, J C; Argus, M F; Nishiyama, K; Griffin, G W

    1980-01-01

    The acute toxicity of p-dioxane may be enhanced up to 1000-fold by chlorination of the compound. The effect was stereoselective. Of the stereoisomers tested, tetrachloro-p-dioxane, isomer I (2r, 3t, 5t, 6c) was over 80 times more toxic than isomer II (2r, 3c, 5t, 6t). The latter compound was also a potent repressor of hepatic dimethylnitrosamine-demethylase I (DMN-d) and aryl hydrocarbon hydroxylase (AHH).

  11. 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.

  12. 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

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

    PubMed Central

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

    2015-01-01

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

  14. Enhanced Sensitivity for Detection of Plasmodium falciparum gametocytes by magnetic nanoparticles combined with enzyme substrate system.

    PubMed

    Tangchaikeeree, Tienrat; Sawaisorn, Piamsiri; Somsri, Sangdao; Polpanich, Duangporn; Putaporntip, Chaturong; Tangboriboonrat, Pramuan; Udomsangpetch, Rachanee; Jangpatarapongsa, Kulachart

    2017-03-01

    The highly sensitive and specific detection of Pfg377 gene of Plasmodium falciparum gametocyte using Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay (MELGA) was successfully developed. The MELGA included amplification of the Pfg377 gene by polymerase chain reaction (PCR) using magnetic nanoparticles (MNPs)-conjugated forward primer and biotinylated reverse primer, followed by post-analytical process using horseradish peroxidase (HRP)-conjugated streptavidin (SA). The complexes of MELGA product were incubated with the peroxidase substrate and hydrogen peroxide to produce the signal for colorimetric measurement. Altogether, the MELGA technique provided a highly sensitive and specific detection at 1 P. falciparum gametocyte/µL, which was more efficient than that of microscopic examination and rapid diagnostic tests (RDTs). Additionally, the MELGA could detect target gene at femtogram level, which was greater sensitive than the conventional PCR, nested PCR and loop-mediated isothermal amplification (LAMP). The MELGA technique could become a novel and practical method that overcome limitation of traditional gametocyte detection.

  15. Enhanced production of n-alkanes in Escherichia coli by spatial organization of biosynthetic pathway enzymes.

    PubMed

    Rahmana, Ziaur; Sung, Bong Hyun; Yi, Ji-Yeun; Bui, Le Minh; Lee, Jun Hyoung; Kim, Sun Chang

    2014-12-20

    Alkanes chemically mimic hydrocarbons found in petroleum, and their demand as biofuels is steadily increasing. Biologically, n-alkanes are produced from fatty acyl-ACPs by acyl-ACP reductases (AARs) and aldehyde deformylating oxygenases (ADOs). One of the major impediments in n-alkane biosynthesis is the low catalytic turnover rates of ADOs. Here, we studied n-alkane biosynthesis in Escherichia coli using a chimeric ADO-AAR fusion protein or zinc finger protein-guided ADO/AAR assembly on DNA scaffolds to control their stoichiometric ratios and spatial arrangements. Bacterial production of n-alkanes with the ADO-AAR fusion protein was increased 4.8-fold (24 mg/L) over a control strain expressing ADO and AAR separately. Optimal n-alkane biosynthesis was achieved when the ADO:AAR binding site ratio on a DNA scaffold was 3:1, yielding an 8.8-fold increase (44 mg/L) over the control strain. Our findings indicate that the spatial organization of alkane-producing enzymes is critical for efficient n-alkane biosynthesis in E. coli.

  16. Overexpression of bacterial ethylene-forming enzyme gene in Trichoderma reesei enhanced the production of ethylene

    PubMed Central

    Chen, Xi; Liang, Yong; Hua, Jing; Tao, Li; Qin, Wensheng; Chen, Sanfeng

    2010-01-01

    In order to efficiently utilize natural cellulose materials to produce ethylene, three expression vectors containing the ethylene-forming enzyme (efe) gene from Pseudomonas syringae pv. glycinea were constructed. The target gene was respectively controlled by different promoters: cbh I promoter from Trichoderma reesei cellobiohydrolases I gene, gpd promoter from Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene and pgk I promoter from T. reesei 3-phosphoglycerate kinase I gene. After transforming into T. reesei QM9414, 43 stable transformants were obtained by PCR amplification and ethylene determination. Southern blot analysis of 14 transformants demonstrated that the efe gene was integrated into chromosomal DNA with copy numbers from 1 to 4. Reverse transcription polymerase chain reaction (RT-PCR) analysis of 6 transformants showed that the heterologous gene was transcribed. By using wheat straw as a carbon source, the ethylene production rates of aforementioned 14 transformants were measured. Transformant C30-3 with pgk I promoter had the highest ethylene production (4,012 nl h-1 l-1). This indicates that agricultural wastes could be used to produce ethylene in recombinant filamentous fungus T. reesei. PMID:20150979

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

    PubMed

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

    2000-08-01

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

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

    USDA-ARS?s Scientific Manuscript database

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

  19. Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA)

    PubMed Central

    Ciaurriz, Paula; Fernández, Fátima; Tellechea, Edurne; Moran, Jose F

    2017-01-01

    The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased. PMID:28243563

  20. 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.

  1. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2014-05-01

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

  3. Enzyme-Sensitive and Amphiphilic PEGylated Dendrimer-Paclitaxel Prodrug-Based Nanoparticles for Enhanced Stability and Anticancer Efficacy.

    PubMed

    Li, Ning; Cai, Hao; Jiang, Lei; Hu, Jiani; Bains, Ashika; Hu, Jesse; Gong, Qiyong; Luo, Kui; Gu, Zhongwei

    2017-03-01

    In this study, we prepared a smart polymeric vehicle for the hydrophobic drug paclitaxel (PTX) that allowed a maximum steady-state circulation and a fast intracellular release in tumors. PTX was linked to the Janus PEGylated (PEG = poly(ethylene glycol)) peptide dendrimer via an enzyme-sensitive linker glycylphenylalanylleucylglycine tetrapeptide by efficient click reaction, resulting in Janus dendritic prodrug with 20.9% PTX content. The prodrug self-assembled into nanoscale particles with appropriate nanosizes, compact morphology, and negative surface charge. In addition to high stability during circulation, as demonstrated by protein adsorption assays and drug release studies in the cancer's intracellular environment, the nanoparticles were able to quickly release the drug intact in its original molecular structure, as verified via high-performance liquid chromatography and mass spectrometry analyses. Compared to free PTX, the enzyme-responsive feature of nanoparticles promoted higher cytotoxicity against 4T1 cancer cells and much lower cytotoxicity against normal cells. The nanoparticles accumulated in the tumor and were retained for an extended period of time, as confirmed by fluorescence imaging. Therefore, these nanoparticles exhibited significantly enhanced antitumor efficiency in the 4T1 breast cancer model as indicated by the observed inhibition of angiogenesis and proliferation as well as induction of apoptosis. Moreover, the nanoparticles reduced the occurrence of side effects, particularly dose-limited toxicities, as monitored by body weight and hematological features. Hence, our Janus PEGylated dendrimer-PTX prodrug-based nanoparticles may potentially serve as nanoscale vehicles for breast cancer therapy.

  4. 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

  5. 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.

  6. Leucine accelerates blood ethanol oxidation by enhancing the activity of ethanol metabolic enzymes in the livers of SHRSP rats.

    PubMed

    Murakami, Hitoshi; Ito, Michiko; Furukawa, Yuji; Komai, Michio

    2012-12-01

    Chronic ethanol consumption induces liver diseases, such as alcoholic hepatitis and cirrhosis. The enhancement of alcohol oxidation is important in the prevention of these liver diseases. Chronic supplementation with branched chain amino acids (BCAAs) prevents liver cirrhosis. Therefore, BCAAs may be associated with enhanced ethanol oxidation. To evaluate this hypothesis, we investigated the effect of the administration of individual BCAAs on ethanol oxidation and changes in alcohol-metabolizing enzyme activities following acute alcohol intake in rats. Blood ethanol concentrations and the activities of alcohol-metabolizing enzymes, such as alcohol dehydrogenase (ADH) and low and high Km aldehyde dehydrogenase (ALDH), were measured in the liver following acute ethanol administration in rats; the ethanol was administered 30 min after the treatment with amino acids [such as leucine (Leu), isoleucine (Ile), valine (Val) or alanine (Ala)]. Leu significantly decreased the blood ethanol concentration 1 h after ethanol administration compared to the water-treated control (C) [C 0.46 ± 0.09, Leu 0.18 ± 0.04, Ile 0.27 ± 0.09, Val 0.46 ± 0.1, Ala 0.43 ± 0.06, mean ± SEM (g/l), P < 0.05]. In addition, leucine significantly stimulated ADH activity 30 min after ethanol intake [C 0.042 ± 0.014, Leu 0.090 ± 0.016, Ile 0.042 ± 0.008, Val 0.022 ± 0.010, Ala 0.070 ± 0.016, mean ± SEM (unit/mg protein), P < 0.05] and low Km ALDH activity 15 min after ethanol intake [C 0.51 ± 0.63, Leu 3.72 ± 0.66, Ile 1.26 ± 0.89, Val: ND, Ala 1.86 ± 1.57, mean ± SEM (unit/mg protein), P < 0.05]. However, leucine and its metabolite α-keto-isocaproic acid did not enhance ethanol clearance in isolated rat hepatocytes. These results indicate that leucine accelerates ethanol oxidation by indirectly enhancing ADH and low Km ALDH activities in the liver.

  7. 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

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

    SciTech Connect

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

    2009-01-01

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

  9. Enzyme-responsive peptide dendrimer-gemcitabine conjugate as a controlled-release drug delivery vehicle with enhanced antitumor efficacy.

    PubMed

    Zhang, Chengyuan; Pan, Dayi; Li, Jin; Hu, Jiani; Bains, Ashika; Guys, Nicholas; Zhu, Hongyan; Li, Xiaohui; Luo, Kui; Gong, Qiyong; Guc, Zhongwei

    2017-03-01

    Stimuli-responsive peptide dendrimer-drug conjugates have presented significant potential for cancer therapy. To develop an effective nanoscale chemotherapeutic prodrug, we developed a novel enzyme-responsive PEGylated lysine peptide dendrimer-gemcitabine conjugate (Dendrimer-GEM) based nanoparticle via the highly efficient click reaction. Owing to the glycylphenylalanylleucylglycine tetra-peptide (GFLG) as an enzyme-cleavable linker to conjugate gemcitabine (GEM), the prepared nanoparticles were able to release drug significantly faster in the tumor cellular environments, which specifically contains secreted Cathepsin B, quantifiably more than 80% GEM was released with Cathepsin B compared to the condition without Cathepsin B at 24h. This nanoparticle demonstrated enhanced antitumor efficacy in a 4T1 murine breast cancer model without obvious systemic toxicity, resulting in significantly suppressed relative tumor volumes (86.17 ± 38.27%) and a 2-fold higher value of tumor growth inhibition (~90%) than GEM∙HCl treatment. These results suggest that the PEGylated peptide dendrimer-gemcitabine conjugate can be an effective antitumor agent for breast cancer therapy. Statement of significance We found that the functionalized dendrimer based nanoscale drug delivery vehicles exhibited enhanced therapeutic indexes and reduced toxicity as compared to the free drug gemcitabine. Compared with current nanoparticles, such as dendritic anticancer drug delivery systems, the new design was capable of self-assembling into nanoscale particles with sizes of about 80-110 nm, which is suitable as antitumor drug delivery vehicle due to the potential longer intravascular half-life and higher accumulation in tumor tissue via EPR effect. Owing to the optimized architecture, the system was given the enzyme-responsive drug release feature, and showed excellent antitumor activity on the 4T1 breast tumor model due to the evidences from tumor growth curves, immunohistochemical analysis and

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

    SciTech Connect

    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 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

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

    DOE PAGES

    Montella, Salvatore; Balan, Venkatesh; da Costa Sousa, Leonardo; ...

    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

  12. 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

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

    PubMed

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

    2016-01-01

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

  14. 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.

  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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2015-01-01

    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 have previously reported the ~2-fold enhanced activity and 1.4-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 femtomol levels of ATP. Additionally, 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 in living cells offering an improved alternative to Promega’s luc2 for reporter and imaging applications. PMID:26049097

  17. Enhanced expression of retinoic acid-metabolizing enzyme CYP26A1 in sunlight-damaged human skin.

    PubMed

    Osanai, Makoto; Lee, Gang-Hong

    2011-12-01

    Vitamin A deficiency (VAD) is associated with increased susceptibility to carcinogenesis. CYP26A1, the gene encoding a cytochrome P450 enzyme specifically involved in metabolic inactivation of retinoic acid (RA), the most active vitamin A derivative, has been shown to result in a state of functional VAD of the cell. Recently, we demonstrated that CYP26A1 efficiently promotes cell survival properties and eventually contributes to the carcinogenic process, implying roles as an oncogene. To clarify the possible association between VAD caused by CYP26A1 expression and the development of human epithelial neoplasia, we examined whether enhanced expression of CYP26A1 might be observed in various lesions of human skin. We report here that basal keratinocytes showed only weak positivity of CYP26A1 in sunlight-nonexposed areas, whereas strong positive staining was observed in skin from chronically sunexposed body areas and in epidermis that had the dysplastic changes known as actinic keratosis. However, we found no expression of constitutive CYP26A1 in skin malignancies such as squamous cell carcinomas. Our observation suggests an involvement of enhanced CYP26A1 expression causing a functional VAD state in skin that can potentially lead to neoplastic transformation of keratinocytes in an early phase during skin carcinogenesis.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. Optimization of bioethanol production during simultaneous saccharification and fermentation in very high-gravity cassava mash.

    PubMed

    Yingling, Bao; Zongcheng, Yan; Honglin, Wang; Li, Chen

    2011-02-01

    Hydrolysis and fermentation conditions for production of ethanol from very high-gravity cassava mash by Saccharomyces cerevisiae during simultaneous saccharification and fermentation (SSF) processing were optimized using a statistical methodology. During the first part of the study, Placket-Burman design (PBD) was used to study 19 factors that could potentially influence ethanol production. Gravity, particle size, initial pH, and fermentation temperature were identified as key factors that significantly increased final ethanol concentration. The main and interaction effects of these factors were subsequently evaluated based on a quadratic equation generated by central composite design (CCD) using response-surface methodology (RSM). Under the optimized very high-gravity conditions, the final ethanol concentration obtained from experiment increased from 8.21% (wt.%) to 15.03% (wt.%) and was in good agreement with model prediction. By employing two other commercial Saccharomyces strains, similar results were obtained under the same optimized condition. Therefore, we conclude that final ethanol concentration, ethanol productivity (V (P/max)), glucose utilization (Y (G/s), Y (P/s)), and fermentation efficiency (η (f)) were enhanced or maintained under the optimized condition of 40% gravity, 390 μm particle size, initial pH 5.5, and 27°C fermentation temperature.

  3. Alkaline pretreatment improves saccharification and ethanol yield from waste money bills.

    PubMed

    Sheikh, Md Mominul Islam; Kim, Chul-Hwan; Park, Hyeon-Jin; Kim, Sung-Ho; Kim, Gyeong-Chul; Lee, Ji-Young; Sim, Sung-Woong; Kim, Jae Won

    2013-01-01

    Waste money bills (WMB) is a by-product of the money making process that consists of rich-cellulosic material for many biotechnological applications. This waste money bills is unusable and usually exhausted. Saccharification was improved using various concentrations of sodium hydroxide, NaOH (0.0, 0.5, 1.0, 2.0, 2.5, and 3.0% v/v) and various reaction times (20, 30, and 40 min) during pretreatment at 121 °C. Prior to ethanol fermentation, the highest glucose yield (62.2 mg/mL) was found by pretreatment consisting of 30 min at 2.0% NaOH, and it increased 33.8% as compared to an untreated sample. The highest amount of ethanol was obtained (26.1 mg/mL) during fermentation, and this was increased 95.3 and 22.5% as compared to aerobic and anaerobic conditions respectively during pretreatment with 2.0% NaOH for 30 min. Under anaerobic conditions, ethanol fermentation was enhanced by adding 0.4 mmol benzoic acid. Production of ethanol from waste money bills would cut waste management costs and make profitable.

  4. Bioconversion of corn stover derived pentose and hexose to ethanol using cascade simultaneous saccharification and fermentation (CSSF).

    PubMed

    Li, Xuan; Kim, Tae Hyun

    2012-01-01

    A cascade type of fermentation, designated the cascade simultaneous saccharification and fermentation (CSSF), was studied to convert corn stover derived pentose and hexose to ethanol with reduced enzyme input. In detail, each step of CSSF utilizes two sequential SSF phases operating on pentose and hexose, i.e., pentose conversion using xylanase, endo-glucanase, and recombinant Escherichia coli (KO11) with minimal glucose conversion in the first phase SSF, and hexose conversion in the second phase SSF using cellulase, β-glucosidase, and Saccharomyces cerevisiae (D(5)A). In this cascade scheme, multiple stages of 1st and 2nd phase SSF were performed in series; enzymes are recycled from the fermentation broth of the last stage for the use of the next stage. This bioconversion process yielded up to 60% of the theoretical maximum ethanol yield based on the total sugars in untreated corn stover, while enzyme loadings were reduced by 50% (v/v) and the final ethanol concentration reached 27 g/l.

  5. Statistical optimization of recycled-paper enzymatic hydrolysis for simultaneous saccharification and fermentation via central composite design.

    PubMed

    Liu, Qing; Cheng, Ke-ke; Zhang, Jian-an; Li, Jin-ping; Wang, Ge-hua

    2010-01-01

    A central composite design of the response surface methodology (RSM) was employed to study the effects of temperature, enzyme concentration, and stirring rate on recycled-paper enzymatic hydrolysis. Among the three variables, temperature and enzyme concentration significantly affected the conversion efficiency of substrate, whereas stirring rate was not effective. A quadratic polynomial equation was obtained for enzymatic hydrolysis by multiple regression analysis using RSM. The results of validation experiments were coincident with the predicted model. The optimum conditions for enzymatic hydrolysis were temperature, enzyme concentration, and stirring rate of 43.1 degrees C, 20 FPU g(-1) substrate, and 145 rpm, respectively. In the subsequent simultaneous saccharification and fermentation (SSF) experiment under the optimum conditions, the highest 28.7 g ethanol l(-1) was reached in the fed-batch SSF when 5% (w/v) substrate concentration was used initially, and another 5% added after 12 h fermentation. This ethanol output corresponded to 77.7% of the theoretical yield based on the glucose content in the raw material.

  6. Enhanced enzymatic hydrolysis of eucalyptus by synergy of zinc chloride hydrate pretreatment and bovine serum albumin.

    PubMed

    Wei, Weiqi; Wu, Shubin

    2017-08-24

    Enhancement of eucalyptus enzymatic saccharification by synergy of ZnCl2 hydrate pretreatment and bovine serum albumin (BSA) was investigated in this study. The result showed that the ZnCl2 hydrate pretreatment could not only selectively extract up to ∼100% of the hemicellulose from eucalyptus, but also convert portion of high crystalline cellulose I into low crystalline cellulose II, which both beneficial for enhancing subsequent pretreated solids enzymatic saccharification. The addition of BSA into enzymatic hydrolysis step could significantly promote the glucose release from pretreated solids, especially, under the low enzyme loading. Furthermore, the material balance indicated that the highest glucose yield of this study was 35.5g/100g raw material, which representing 90.3% of glucose in raw eucalyptus, combined with the xylose yield, 13.9g/100g eucalyptus, it can be concluded that ZnCl2 hydrate pretreatment offered the potential to co-produce xylose and glucose from eucalyptus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Epinephrine enhances lysosomal enzyme delivery across the blood brain barrier by up-regulation of the mannose 6-phosphate receptor.

    PubMed

    Urayama, Akihiko; Grubb, Jeffrey H; Banks, William A; Sly, William S

    2007-07-31

    Delivering therapeutic levels of lysosomal enzymes across the blood-brain barrier (BBB) has been a pivotal issue in treating CNS storage diseases, including the mucopolysaccharidoses. An inherited deficiency of beta-glucuronidase (GUS) causes mucopolysaccharidosis type VII that is characterized by increased systemic and CNS storage of glycosaminoglycans. We previously showed that the neonate uses the mannose 6-phosphate (M6P) receptor to transport phosphorylated GUS (P-GUS) across the BBB and that this transporter is lost with maturation. Induction of expression of this BBB transporter would make enzyme replacement therapy in the adult possible. Here, we tested pharmacological manipulation with epinephrine to restore functional transport of P-GUS across the adult BBB. Epinephrine (40 nmol) coinjected i.v. with (131)I-P-GUS induced the transport across the BBB in 8-week-old mice. The brain influx rate of (131)I-P-GUS (0.29 mul/g per min) returned to the level seen in neonates. Capillary depletion showed that 49% of the (131)I-P-GUS in brain was in brain parenchyma. No increases of influx rate or the vascular space for (125)I-albumin, a vascular marker, was observed with epinephrine (40 nmol), showing that enhanced passage was not caused by disruption of the BBB. Brain uptake of (131)I-P-GUS was significantly inhibited by M6P in a dose-dependent manner, whereas epinephrine failed to increase brain uptake of nonphosphorylated GUS. Thus, the effect of epinephrine on the transport of (131)I-P-GUS was ligand specific. These results indicate that epinephrine restores the M6P receptor-mediated functional transport of (131)I-P-GUS across the BBB in adults to levels seen in the neonate.

  8. 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.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2015-12-01

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

  11. Combined effect of improved cell yield and increased specific productivity enhances recombinant enzyme production in genome-reduced Bacillus subtilis strain MGB874.

    PubMed

    Manabe, Kenji; Kageyama, Yasushi; Morimoto, Takuya; Ozawa, Tadahiro; Sawada, Kazuhisa; Endo, Keiji; Tohata, Masatoshi; Ara, Katsutoshi; Ozaki, Katsuya; Ogasawara, Naotake

    2011-12-01

    Genome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novel Bacillus subtilis strain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of the rocDEF-rocR region, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of the rocDEF-rocR region caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without the rocDEF-rocR region, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of the rocDEF-rocR region and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement of B. subtilis reduced-genome strains as cell factories for the production of heterologous enzymes.

  12. Overexpression of a bifunctional enzyme, CrtS, enhances astaxanthin synthesis through two pathways in Phaffia rhodozyma.

    PubMed

    Chi, Shuang; He, Yanfeng; Ren, Jie; Su, Qian; Liu, Xingchao; Chen, Zhi; Wang, Mingan; Li, Ying; Li, Jilun

    2015-06-18

    A moderate-temperature, astaxanthin-overproducing mutant strain (termed MK19) of Phaffia rhodozyma was generated in our laboratory. The intracellular astaxanthin content of MK19 was 17-fold higher than that of wild-type. The TLC profile of MK19 showed a band for an unknown carotenoid pigment between those of β-carotene and astaxanthin. In the present study, we attempted to identify the unknown pigment and to enhance astaxanthin synthesis in MK19 by overexpression of the crtS gene that encodes astaxanthin synthase (CrtS). A crtS-overexpressing strain was constructed without antibiotic marker. A recombinant plasmid with lower copy numbers was shown to be stable in MK19. In the positive recombinant strain (termed CSR19), maximal astaxanthin yield was 33.5% higher than MK19, and the proportion of astaxanthin as a percentage of total carotenoids was 84%. The unknown carotenoid was identified as 3-hydroxy-3',4'-didehydro-β,Ψ-carotene-4-one (HDCO) by HPLC, mass spectrometry, and NMR spectroscopy. CrtS was found to be a bifunctional enzyme that helped convert HDCO to astaxanthin. Enhancement of crtS transcriptional level increased transcription levels of related genes (crtE, crtYB, crtI) in the astaxanthin synthesis pathway. A scheme of carotenoid biosynthesis in P. rhodozyma involving alternative bicyclic and monocyclic pathways is proposed. CrtS overexpression leads to up-regulation of synthesis-related genes and increased astaxanthin production. The transformant CSR19 is a stable, secure strain suitable for feed additive production. The present findings help clarify the regulatory mechanisms that underlie metabolic fluxes in P. rhodozyma carotenoid biosynthesis pathways.

  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.

  14. 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

  15. Insulin enhances metabolic capacities of cancer cells by dual regulation of glycolytic enzyme pyruvate kinase M2

    PubMed Central

    2013-01-01

    Background Insulin is tightly associated with cancer progression; however, mechanistic insights into such observations are poorly understood. Recent studies show that metabolic transformation is critical to cancer cell proliferation. Here, we attempt to understand the role of insulin in promotion of cancer metabolism. To this end, the role of insulin in regulating glycolytic enzyme pyruvate kinase M2 (PKM2) was examined. Results We observed that insulin up-regulated PKM2 expression, through PI3K/mTOR mediated HIF1α induction, but significantly reduced PKM2 activity independent of this pathway. Drop in PKM2 activity was attributed to subunit dissociation leading to formation of low activity PKM2 oligomers, as assessed by density gradient centrifugation. However, tyrosine 105 phosphorylation of PKM2, known for inhibiting PKM2 activity, remained unaffected on insulin treatment. Interestingly, insulin-induced ROS was found responsible for PKM2 activity reduction. The observed changes in PKM2 status led to augmented cancer metabolism. Insulin-induced PKM2 up-regulation resulted in enhanced aerobic glycolysis as confirmed by PKM2 knockdown studies. Further, PKM2 activity reduction led to characteristic pooling of glycolytic intermediates and increased accumulation of NADPH; suggesting diversion of glucose flux towards macromolecular synthesis, necessary for cancer cell growth. Conclusion The study identifies new PKM2-mediated effects of insulin on cancer metabolism, thus, advancing the understanding of insulin’s role in cancer. PMID:23837608

  16. Evaluation of the antioxidant properties of the angiotensin-converting enzyme inhibitor, captopril and the nucleotide enhancing agent, acadesine.

    PubMed

    Wasil, M; Kelly, F J

    1995-11-01

    The angiotensin-converting enzyme inhibitor, captopril and the nucleotide enhancing agent, acadesine, protect myocardial tissue from ischaemia/reperfusion-induced injury. Although both drugs have well established, independent mechanisms of cardiac protection, they may also have antioxidant activity which could contribute to their beneficial action. In this study we have examined the antioxidant activity of captopril and acadesine by examining their ability to scavenge ABTS radicals, formed from the interaction of ferryl metmyoglobin with phenothiazine in the presence of hydrogen peroxide. For comparison, we compared these results to those obtained for a range of other drugs commonly used for the treatment of cardiovascular disorders. These included verapamil (arrhythmia), isosorbide dinitrate (angina), atenolol (hypertension) and enalapril (congestive heart failure). The antioxidant properties of these drugs were then compared to the well characterised antioxidants, Trolox (a water soluble vitamin E analogue), ascorbate and glutathione. Captopril and acadesine were both shown to be efficient scavengers of ABTS radicals, importantly at drug concentrations expected to be found in vivo. These data confirm that the antioxidant potential of captopril and acadesine may be an important component of their mechanism of action, with both drugs probably protecting the myocardium against oxygen derived free radicals during ischaemia/reperfusion.

  17. Surface modification for enhancing antibody binding on polymer-based microfluidic device for enzyme-linked immunosorbent assay.

    PubMed

    Bai, Yunling; Koh, Chee Guan; Boreman, Megan; Juang, Yi-Je; Tang, I-Ching; Lee, L James; Yang, Shang-Tian

    2006-10-24

    A novel surface treatment method using poly(ethyleneimine) (PEI), an amine-bearing polymer, was developed to enhance antibody binding on the poly(methyl methacrylate) (PMMA) microfluidic immunoassay device. By treating the PMMA surface of the microchannel on the microfluidic device with PEI, 10 times more active antibodies can be bound to the microchannel surface as compared to those without treatment or treated with the small amine-bearing molecule, hexamethylenediamine (HMD). Consequently, PEI surface modification greatly improved the immunoassay performance of the microfluidic device, making it more sensitive and reliable in the detection of IgG. The improvement can be attributed to the spacer effect as well as the functional amine groups provided by the polymeric PEI molecules. Due to the smaller dimensions (140x125 microm) of the microchannel, the time required for antibody diffusion and adsorption onto the microchannel surface was reduced to only several minutes, which was 10 times faster than the similar process carried out in 96-well plates. The microchip also had a wider detection dynamic range, from 5 to 1000 ng/mL, as compared to that of the microtiter plate (from 2 to 100 ng/mL). With the PEI surface modification, PMMA-based microchips can be effectively used for enzyme linked immunosorbent assays (ELISA) with a similar detection limit, but much less reagent consumption and shorter assay time as compared to the conventional 96-well plate.

  18. Overexpression of a brassinosteroid biosynthetic gene Dwarf enhances photosynthetic capacity through activation of Calvin cycle enzymes in tomato.

    PubMed

    Li, Xiao-Jing; Guo, Xie; Zhou, Yan-Hong; Shi, Kai; Zhou, Jie; Yu, Jing-Quan; Xia, Xiao-Jian

    2016-01-28

    Genetic manipulation of brassinosteroid (BR) biosynthesis or signaling is a promising strategy to improve crop yield and quality. However, the relationships between the BR-promoted growth and photosynthesis and the exact mechanism of BR-regulated photosynthetic capacity are not clear. Here, we generated transgenic tomato plants by overexpressing Dwarf, a BR biosynthetic gene that encodes the CYP85A1, and compared the photosynthetic capacity with the BR biosynthetic mutant d (im) and wild type. Overexpression of Dwarf promoted net photosynthetic rate (P N), whereas BR deficiency in d (im) led to a significant inhibition in P N as compared with WT. The activation status of RuBisCO, and the protein content and activity of RuBisCO activase, but not the total content and transcripts of RuBisCO were closely related to the endogenous BR levels in different genotypes. However, endogenous BR positively regulated the expression and activity of fructose-1,6-bisphosphatase. Dwarf overexpression enhanced the activity of dehydroascorbate reductase and glutathione reductase, leading to a reduced redox status, whereas BR deficiency had the contrasting effects. In addition, BR induced a reduction of 2-cystein peroxiredoxin without altering the protein content. BR plays a role in the regulation of photosynthesis. BR can increase the photosynthetic capacity by inducing a reduced redox status that maintains the activation states of Calvin cycle enzymes.

  19. Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology.

    PubMed

    Lee, Jae-Won; Rodrigues, Rita C L B; Jeffries, Thomas W

    2009-12-01

    Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 2(3) full factorial design with six axial points. Temperatures ranged from 132 to 180 degrees C, time from 10 to 90 min and oxalic acid loadings from 0.01 to 0.038 g/g solids. Separate maxima were found for enzymatic saccharification and hemicellulose fermentation, respectively, with the condition for maximum saccharification being significantly more severe. Ethanol production was affected by reaction temperature more than by oxalic acid and reaction time over the ranges examined. The effect of reaction temperature was significant at a 95% confidence level in its effect on ethanol production. Oxalic acid and reaction time were statistically significant at the 90% level. The highest ethanol concentration (20 g/l) was obtained after 48 h with an ethanol volumetric production rate of 0.42 g ethanol l(-1) h(-1). The ethanol yield after SSF with P. stipitis was significantly higher than predicted by sequential saccharification and fermentation of substrate pretreated under the same condition. This was attributed to the secretion of beta-glucosidase by P. stipitis. During SSF, free extracellular beta-glucosidase activity was 1.30 pNPG U/g with P. stipitis, while saccharification without the yeast was 0.66 pNPG U/g.

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

    DOE PAGES

    Eudes, Aymerick; Sathitsuksanoh, Noppadon; Baidoo, Edward E. K.; ...

    2015-01-13

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

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

    PubMed

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

    2015-12-01

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

  2. 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.

  3. Liquefaction, saccharification, and fermentation of ammoniated corn to ethanol.

    PubMed

    Taylor, Frank; Kim, Tae Hyun; Abbas, Charles A; Hicks, Kevin B

    2008-01-01

    Treatment of whole corn kernels with anhydrous ammonia gas has been proposed as a way to facilitate the separation of nonfermentable coproducts before fermentation of the starch to ethanol, but the fermentability of ammoniated corn has not been thoroughly investigated. Also, it is intended that the added ammonia nitrogen in ammonia treated corn (approximately 1 g per kg corn) may satisfy the yeast nutritional requirement for free amino nitrogen (FAN). In this study, procedures for ammoniation, liquefaction, saccharification, and fermentation at two scales (12-L and 50-mL) were used to determine the fermentation rate, final ethanol concentration, and ethanol yield from starch in ammoniated or nonammoniated corn. The maximum achievable ethanol concentration at 50 h fermentation time was lower with ammoniated corn than with nonammoniated corn. The extra nitrogen in ammoniated corn satisfied some of the yeast requirements for FAN, thereby reducing the requirement for corn steep liquor. Based upon these results, ammoniation of corn does not appear to have a positive impact on the fermentability of corn to ethanol. Ammoniation may still be cost effective, if the advantages in terms of improved separations outweigh the disadvantages in terms of decreased fermentability.

  4. Enzymatic Saccharification of Lignocelluloses Should be Conducted at Elevated pH 5.2-6.2

    Treesearch

    T.Q. Lan; Hongming Lou; J.Y. Zhu

    2013-01-01

    This study revealed that cellulose enzymatic saccharification response curves of lignocellulosic substrates were very different from those of pure cellulosic substrates in terms of optimal pH and pH operating window. The maximal enzymatic cellulose saccharification of lignocellulosic substrates occurs at substrate suspension

  5. Comparative analysis of the secretomes of Schizophyllum commune and other wood-decay basidiomycetes during solid-state fermentation reveals its unique lignocellulose-degrading enzyme system.

    PubMed

    Zhu, Ning; Liu, Jiawen; Yang, Jinshui; Lin, Yujian; Yang, Yi; Ji, Lei; Li, Meng; Yuan, Hongli

    2016-01-01

    The genome of Schizophyllum commune encodes a diverse repertoire of degradative enzymes for plant cell wall breakdown. Recent comparative genomics study suggests that this wood decayer likely has a mode of biodegradation distinct from the well-established white-rot/brown-rot models. However, much about the extracellular enzyme system secreted by S. commune during lignocellulose deconstruction remains unknown and the underlying mechanism is poorly understood. In this study, extracellular proteins of S. commune colonizing Jerusalem artichoke stalk were analyzed and compared with those of two white-rot fungi Phanerochaete chrysosporium and Ceriporiopsis subvermispora and a brown-rot fungus Gloeophyllum trabeum. Under solid-state fermentation (SSF) conditions, S. commune displayed considerably higher levels of hydrolytic enzyme activities in comparison with those of P. chrysosporium, C. subvermispora and G. trabeum. During biodegradation process, this fungus modified the lignin polymer in a way which was consistent with a hydroxyl radical attack, similar to that of G. trabeum. The crude enzyme cocktail derived from S. commune demonstrated superior performance over a commercial enzyme preparation from Trichoderma longibrachiatum in the hydrolysis of pretreated lignocellulosic biomass at low enzyme loadings. Secretomic analysis revealed that compared with three other fungi, this species produced a higher diversity of carbohydrate-degrading enzymes, especially hemicellulases and pectinases acting on polysaccharide backbones and side chains, and a larger set of enzymes potentially supporting the generation of hydroxyl radicals. In addition, multiple non-hydrolytic proteins implicated in enhancing polysaccharide accessibility were identified in the S. commune secretome, including lytic polysaccharide monooxygenases (LPMOs) and expansin-like proteins. Plant lignocellulose degradation by S. commune involves a hydroxyl radical-mediated mechanism for lignocellulose modification

  6. 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

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

    PubMed Central

    2014-01-01

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

  8. Thermophilic Bacillus coagulans requires less cellulases for simultaneous saccharification and fermentation of cellulose to products than mesophilic microbial biocatalysts.

    PubMed

    Ou, Mark S; Mohammed, Nazimuddin; Ingram, L O; Shanmugam, K T

    2009-05-01

    Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g(-1) cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g(-1) cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.

  9. Microwave-Assisted Alkali Pre-Treatment, Densification and Enzymatic Saccharification of Canola Straw and Oat Hull

    PubMed Central

    Agu, Obiora S.; Tabil, Lope G.; Dumonceaux, Tim

    2017-01-01

    The effects of microwave-assisted alkali pre-treatment on pellets’ characteristics and enzymatic saccharification for bioethanol production using lignocellulosic biomass of canola straw and oat hull were investigated. The ground canola straw and oat hull were immersed in distilled water, sodium hydroxide and potassium hydroxide solutions at two concentrations (0.75% and 1.5% w/v) and exposed to microwave radiation at power level 713 W and three residence times (6, 12 and 18 min). Bulk and particle densities of ground biomass samples were determined. Alkaline-microwave pre-treated and untreated samples were subjected to single pelleting test in an Instron universal machine, pre-set to a load of 4000 N. The measured parameters, pellet density, tensile strength and dimensional stability were evaluated and the results showed that the microwave-assisted alkali pre-treated pellets had a significantly higher density and tensile strength compared to samples that were untreated or pre-treated by microwave alone. The chemical composition analysis showed that microwave-assisted alkali pre-treatment was able to disrupt and break down the lignocellulosic structure of the samples, creating an area of cellulose accessible to cellulase reactivity. The best enzymatic saccharification results gave a high glucose yield of 110.05 mg/g dry sample for canola straw ground in a 1.6 mm screen hammer mill and pre-treated with 1.5% NaOH for 18 min, and a 99.10 mg/g dry sample for oat hull ground in a 1.6 mm screen hammer mill and pre-treated with 0.75% NaOH for 18 min microwave-assisted alkali pre-treatments. The effects of pre-treatment results were supported by SEM analysis. Overall, it was found that microwave-assisted alkali pre-treatment of canola straw and oat hull at a short residence time enhanced glucose yield. PMID:28952504

  10. Microwave-Assisted Alkali Pre-Treatment, Densification and Enzymatic Saccharification of Canola Straw and Oat Hull.

    PubMed

    Agu, Obiora S; Tabil, Lope G; Dumonceaux, Tim

    2017-03-26

    The effects of microwave-assisted alkali pre-treatment on pellets' characteristics and enzymatic saccharification for bioethanol production using lignocellulosic biomass of canola straw and oat hull were investigated. The ground canola straw and oat hull were immersed in distilled water, sodium hydroxide and potassium hydroxide solutions at two concentrations (0.75% and 1.5% w/v) and exposed to microwave radiation at power level 713 W and three residence times (6, 12 and 18 min). Bulk and particle densities of ground biomass samples were determined. Alkaline-microwave pre-treated and untreated samples were subjected to single pelleting test in an Instron universal machine, pre-set to a load of 4000 N. The measured parameters, pellet density, tensile strength and dimensional stability were evaluated and the results showed that the microwave-assisted alkali pre-treated pellets had a significantly higher density and tensile strength compared to samples that were untreated or pre-treated by microwave alone. The chemical composition analysis showed that microwave-assisted alkali pre-treatment was able to disrupt and break down the lignocellulosic structure of the samples, creating an area of cellulose accessible to cellulase reactivity. The best enzymatic saccharification results gave a high glucose yield of 110.05 mg/g dry sample for canola straw ground in a 1.6 mm screen hammer mill and pre-treated with 1.5% NaOH for 18 min, and a 99.10 mg/g dry sample for oat hull ground in a 1.6 mm screen hammer mill and pre-treated with 0.75% NaOH for 18 min microwave-assisted alkali pre-treatments. The effects of pre-treatment results were supported by SEM analysis. Overall, it was found that microwave-assisted alkali pre-treatment of canola straw and oat hull at a short residence time enhanced glucose yield.

  11. 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.

  12. Synthetic CO2-fixation enzyme cascades immobilized on self-assembled nanostructures that enhance CO2/O2 selectivity of RubisCO.

    PubMed

    Satagopan, Sriram; Sun, Yuan; Parquette, Jon R; Tabita, F Robert

    2017-01-01

    With increasing concerns over global warming and depletion of fossil-fuel reserves, it is attractive to develop innovative strategies to assimilate CO2, a greenhouse gas, into usable organic carbon. Cell-free systems can be designed to operate as catalytic platforms with enzymes that offer exceptional selectivity and efficiency, without the need to support ancillary reactions of metabolic pathways operating in intact cells. Such systems are yet to be exploited for applications involving CO2 utilization and subsequent conversion to valuable products, including biofuels. The Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) play a pivotal role in global CO2 fixation. We hereby demonstrate the co-assembly of two RubisCO-associated multienzyme cascades with self-assembled synthetic amphiphilic peptide nanostructures. The immobilized enzyme cascades sequentially convert either ribose-5-phosphate (R-5-P) or glucose, a simpler substrate, to ribulose 1,5-bisphosphate (RuBP), the acceptor for incoming CO2 in the carboxylation reaction catalyzed by RubisCO. Protection from proteolytic degradation was observed in nanostructures associated with the small dimeric form of RubisCO and ancillary enzymes. Furthermore, nanostructures associated with a larger variant of RubisCO resulted in a significant enhancement of the enzyme's selectivity towards CO2, without adversely affecting the catalytic activity. The ability to assemble a cascade of enzymes for CO2 capture using self-assembling nanostructure scaffolds with functional enhancements show promise for potentially engineering entire pathways (with RubisCO or other CO2-fixing enzymes) to redirect carbon from industrial effluents into useful bioproducts.

  13. A novel film-pore-surface diffusion model to explain the enhanced enzyme adsorption of corn stover pretreated by ultrafine grinding.

    PubMed

    Zhang, Haiyan; Chen, Longjian; Lu, Minsheng; Li, Junbao; Han, Lujia

    2016-01-01

    Ultrafine grinding is an environmentally friendly pretreatment that can alter the degree of polymerization, the porosity and the specific surface area of lignocellulosic biomass and can, thus, enhance cellulose hydrolysis. Enzyme adsorption onto the substrate is a prerequisite for the enzymatic hydrolysis process. Therefore, it is necessary to investigate the enzyme adsorption properties of corn stover pretreated by ultrafine grinding. The ultrafine grinding pretreatment was executed on corn stover. The results showed that ultrafine grinding pretreatment can significantly decrease particle size [from 218.50 μm of sieve-based grinding corn stover (SGCS) to 17.45 μm of ultrafine grinding corn stover (UGCS)] and increase the specific surface area (SSA), pore volume (PV) and surface composition (SSA: from 1.71 m(2)/g of SGCS to 2.63 m(2)/g of UGCS, PV: from 0.009 cm(3)/g of SGCS to 0.024 m(3)/g of UGCS, cellulose surface area: from 168.69 m(2)/g of SGCS to 290.76 m(2)/g of UGCS, lignin surface area: from 91.46 m(2)/g of SGCS to 106.70 m(2)/g of UGCS). The structure and surface composition changes induced by ultrafine grinding increase the enzyme adsorption capacity from 2.83 mg/g substrate of SGCS to 5.61 mg/g substrate of UGCS. A film-pore-surface diffusion model was developed to simultaneously predict the enzyme adsorption kinetics of both the SGCS and UGCS. Satisfactory predictions could be made with the model based on high R (2) and low RMSE values (R (2) = 0.95 and RMSE = 0.16 mg/g for the UGCS, R (2) = 0.93 and RMSE = 0.09 mg/g for the SGCS). The model was further employed to analyze the rate-limiting steps in the enzy