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Sample records for reesei enzymatic hydrolysis

  1. Enzymatic hydrolysis of cocoa pod husks. [Trichoderma reesei

    SciTech Connect

    Simpson, B.K.; Oldham, J.H.; Martin, A.M

    1984-07-01

    Laboratory results are presented of the bioconversion of cellulose from cocoa pod husks, utilizing cellulase from three mutants of Trichoderma reesei. Total reducing sugars in filtered hydrolysates were estimated by the dinitrosalicylic acid method. The sugars present were identified by paper chromatography as glucose and xylose.

  2. Enzymatic hydrolysis of cellulose in aqueous two-phase systems. 1. Partition of cellulases from Trichoderma reesei

    SciTech Connect

    Tjerneld, F.; Persson, J.; Albertsson, P.A.; Hahn-Haegerdal, B.

    1985-07-01

    The partitioning of endo-..beta..-glucanase, exo-..beta..-glucananse, and ..beta..-glucosidase from Trichoderma reesei QM 9414 in aqueous two-phase systems has been studied with the object of designing a phase system for continuous bioconversion of cellulose. The partitioning of the enzymes in two-phase systems composed of various water soluble polymeric compounds were studied. Systems based on dextran and polyethylene glycol (PEG) were optimal for one sidedly partitioning of the enzymes to the bottom phase. The influence of polymer molecular weights, polymer concentration, ionic composition of the medium, pH, temperature, and adsorption of the enzymes to cellulose on the enyzme partition coefficients (K) were studied. By combining the effects of polymer molecular weight and adsorption to cellulose, K values could be reduced for endo-..beta..-glucanase to 0.02 and for ..beta..-glucosidase to 0.005 at 20 degrees C in a phase system of Dvalues could be reduced for endo-..beta..-glucanase to 0.02 and for ..beta..-glucosidase to 0.005 at 20 degrees C in a phase system of Dextran 40-PEG 40000 in the presence of excess cellulose. At 50 degrees C, K values were increased by a factor of two. In a phase system based on inexpensive crude dextran and PEG, the partition coefficient for endo-..beta..-glucanase was 0.16 and for beta-glucosidase was 0.14 at 20 degrees C with excess cellulose present.

  3. Optimization of enzymatic hydrolysis of steam-exploded corn stover by two approaches: response surface methodology or using cellulase from mixed cultures of Trichoderma reesei RUT-C30 and Aspergillus niger NL02.

    PubMed

    Fang, Hao; Zhao, Chen; Song, Xiang-Yang

    2010-06-01

    To optimize enzymatic hydrolysis of steam-exploded corn stover (SECS), two approaches, response surface methodology (RSM) and utilization of the cellulase from mixed cultures of Trichoderma reesei RUT-C30 and Aspergillus niger NL02, were introduced in this work. The RSM, the first approach, was consisted of Plackett-Burman Design (PBD) and Central Composite Design (CCD). After the optimization of RSM, a model was proposed to predict the optimum value 79.6% confirmed by the experimental result 80.1%. Mixed culture of T. reesei and A. niger was found to be an effective method to enhance cellulolytic enzymes production. Using the cellulase from mixed culture to optimize enzymatic hydrolysis was the second approach. The yield of 85.6% was obtained by the second approach using 25IU/g glucan cellulase. The two approaches were compared and it was found that the second approach was a better one with higher hydrolysis yield and less enzyme dosage. PMID:20149642

  4. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

    Activities in this project are aimed at overcoming barriers associated with high capital and operating costs and sub-optimal sugar yields resulting from pretreatment and subsequent enzymatic hydrolysis of biomass.

  5. Enzymatic hydrolysis and fermentation of agricultural residues to ethanol

    SciTech Connect

    Mes-Hartree, M.; Hogan, C.M.; Saddler, J.N.

    1984-01-01

    A combined enzymatic hydrolysis and fermentation process was used to convert steam-treated wheat and barley straw to ethanol. Maximum conversion efficiencies were obtained when the substrates were steamed for 90 s. These substrates could yield over 0.4 g ethanol/g cellulose following a combined enzymatic hydrolysis and fermentation process procedure using culture filtrates derived from Trichoderma harzianum E58. When culture filtrates from Trichoderma reesei C30 and T. reesei QM9414 were used, the ethanol yields obtained were 0.32 and 0.12 g ethanol/g cellulose utilized, respectively. The lower ethanol yields obtained with these strains were attributed to the lower amounts of ..beta..-glucosidase detected in the T. reesei culture filtrates.

  6. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-08-22

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  7. Enzymatic hydrolysis of organic phosphorus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Orthophosphate-releasing enzymatic hydrolysis is an alternative means for characterizing organic phosphorus (Po) in animal manure. The approach is not only simple and fast, but can also provide information difficult to obtain by other methods. Currently, commercially available phosphatases are mainl...

  8. Economics of enzymatic hydrolysis processes

    SciTech Connect

    Wright, J.D.

    1988-02-01

    Enzymatic hydrolysis processes have the ability to produce high yields of sugars for fermentation to fuel ethanol from lignocellulosic biomass. However, these systems have been plagued with yields, product concentrations, and reactions rates far below those that are theoretically possible. Engineering and economic analyses are presented on several fungal enzyme hydrolysis processes to illustrate the effects of the important process parameters, to quantify the progress that has been made to date, and to estimate the cost reductions that can be made through research improvements. All enzymatic hydrolysis processes require pretreatment, hydrolysis, fermentation, and enzyme production. The key effect of pretreatment is to allow access of the enzymes to the substrate. Pretreatments have been devised that make the biomass completely digestible that increase the xylose yield and concentration, and that integrate pretreatment with lignin utilization. Major improvements in enzyme activity and use of simultaneous saccharification and fermentation (SSF) have greatly reduced the inhibition of the enzymes. It now appears that ethanol inhibition of the yeast is the limiting factor. Enzyme production costs have been dramatically reduced because use of SSF has reduced enzyme loading. However, further improvements may be possible by using soluble carbon sources for production. Over the past decade, the predicted cost of ethanol from such processes has dropped from more than $4.00/gallon to approximately $1.60. Research is currently under way in the United States and has the potential to reduce the projected cost to less than $1.00/gallon. 65 refs., 16 figs., 1 tab.

  9. Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes for the hydrolysis of steam-exploded wheat straw

    PubMed Central

    2012-01-01

    Background An efficient hydrolysis of lignocellulosic substrates to soluble sugars for biofuel production necessitates the interplay and synergistic interaction of multiple enzymes. An optimized enzyme mixture is crucial for reduced cost of the enzymatic hydrolysis step in a bioethanol production process and its composition will depend on the substrate and type of pretreatment used. In the present study, an experimental design was used to determine the optimal composition of a Trichoderma reesei enzyme mixture, comprising the main cellulase and hemicellulase activities, for the hydrolysis of steam-exploded wheat straw. Methods Six enzymes, CBH1 (Cel7a), CBH2 (Cel6a), EG1 (Cel7b), EG2 (Cel5a), as well as the xyloglucanase Cel74a and the xylanase XYN1 (Xyl11a) were purified from a T. reesei culture under lactose/xylose-induced conditions. Sugar release was followed in milliliter-scale hydrolysis assays for 48 hours and the influence of the mixture on initial conversion rates and final yields is assessed. Results The developed model could show that both responses were strongly correlated. Model predictions suggest that optimal hydrolysis yields can be obtained over a wide range of CBH1 to CBH2 ratios, but necessitates a high proportion of EG1 (13% to 25%) which cannot be replaced by EG2. Whereas 5% to 10% of the latter enzyme and a xylanase content above 6% are required for highest yields, these enzymes are predicted to be less important in the initial stage of hydrolysis. Conclusions The developed model could reliably predict hydrolysis yields of enzyme mixtures in the studied domain and highlighted the importance of the respective enzyme components in both the initial and the final hydrolysis phase of steam-exploded wheat straw. PMID:22373423

  10. Review: Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-07-16

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  11. Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail.

    PubMed

    Bussamra, Bianca Consorti; Freitas, Sindelia; Costa, Aline Carvalho da

    2015-01-01

    The aim of this work was to study cocktail supplementation for sugar cane bagasse hydrolysis, where the enzymes were provided from both commercial source and microorganism cultivation (Trichoderma reesei and genetically modified Escherichia coli), followed by purification. Experimental simplex lattice mixture design was performed to optimize the enzymatic proportion. The response was evaluated through hydrolysis microassays validated here. The optimized enzyme mixture, comprised of T. reesei fraction (80%), endoglucanase (10%) and β-glucosidase (10%), converted, theoretically, 72% of cellulose present in hydrothermally pretreated bagasse, whereas commercial Celluclast 1.5L converts 49.11%±0.49. Thus, a rational enzyme mixture designed by using synergism concept and statistical analysis was capable of improving biomass saccharification. PMID:25846188

  12. Enzymatic hydrolysis of lignocellulosic biomass from Onopordum nervosum.

    PubMed

    Martín, C; Negro, M J; Alfonsel, M; Sáez, R

    1988-07-20

    Some properties of the cellulolytic complex obtained from Trichoderma reesei QM 9414 grown on Solka floc as carbon source and its ability to hydrolyze the lignocellulosic biomass of Onopordum nervosum Boiss were studied. The optimum enzyme activity was found at temperatures between 50 and 55 degrees C and pH ranging from 4.3 to 4.8. Hydrolysis of 4-nitropnenyl-beta-D-glucopyranoside (4-NPG) and cellobiose by the beta-glucosidase of the complex, showed competitive inhibition by glucose with a K(i) value of 0.8 mM for 4-NPG and 2. 56 mM for cellobiose. Enzymatic hydrolysis yield of Onopordum nervosum, evaluated as glucose production after 48 h, showed a threefold increase by pretreating the lignocellulosic substrate with alkali. When the loss of glucose incurred by de pretreatment was taken into account, a 160% increase in the final cellulose to glucose conversion was found to be due to the pretreatment. PMID:18584755

  13. Regenerating cellulose from ionic liquids for an accelerated enzymatic hydrolysis

    SciTech Connect

    Zhao, Hua; Jones, Cecil L; Baker, Gary A; Xia, Shuqian; Olubajo, Olarongbe; Person, Vernecia

    2009-01-01

    The efficient conversion of lignocellulosic materials into fuel ethanol has become a research priority in producing affordable and renewable energy. The pretreatment of lignocelluloses is known to be key to the fast enzymatic hydrolysis of cellulose. Recently, certain ionic liquids (ILs)were found capable of dissolving more than 10 wt% cellulose. Preliminary investigations [Dadi, A.P., Varanasi, S., Schall, C.A., 2006. Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnol. Bioeng. 95, 904 910; Liu, L., Chen, H., 2006. Enzymatic hydrolysis of cellulose materials treated with ionic liquid [BMIM]Cl. Chin. Sci. Bull. 51, 2432 2436; Dadi, A.P., Schall, C.A., Varanasi, S., 2007. Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment. Appl. Biochem. Biotechnol. 137 140, 407 421] suggest that celluloses regenerated from IL solutions are subject to faster saccharification than untreated substrates. These encouraging results offer the possibility of using ILs as alternative and nonvolatile solvents for cellulose pretreatment. However, these studies are limited to two chloride-based ILs: (a) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), which is a corrosive, toxic and extremely hygroscopic solid (m.p. 70 C), and (b) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), which is viscous and has a reactive side-chain. Therefore, more in-depth research involving other ILs is much needed to explore this promising pretreatment route. For this reason, we studied a number of chloride- and acetate-based ILs for cellulose regeneration, including several ILs newly developed in our laboratory. This will enable us to select inexpensive, efficient and environmentally benign solvents for processing cellulosic biomass. Our data confirm that all regenerated celluloses are less crystalline (58 75% lower) and more accessible to cellulase (>2 times) than untreated substrates. As a result, regenerated Avicel

  14. Palm Date Fibers: Analysis and Enzymatic Hydrolysis

    PubMed Central

    Shafiei, Marzieh; Karimi, Keikhosro; Taherzadeh, Mohammad J.

    2010-01-01

    Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes. PMID:21151438

  15. Pretreatment and enzymatic hydrolysis of lignocellulosic biomass

    NASA Astrophysics Data System (ADS)

    Corredor, Deisy Y.

    The performance of soybean hulls and forage sorghum as feedstocks for ethanol production was studied. The main goal of this research was to increase fermentable sugars' yield through high-efficiency pretreatment technology. Soybean hulls are a potential feedstock for production of bio-ethanol due to their high carbohydrate content (≈50%) of nearly 37% cellulose. Soybean hulls could be the ideal feedstock for fuel ethanol production, because they are abundant and require no special harvesting and additional transportation costs as they are already in the plant. Dilute acid and modified steam-explosion were used as pretreatment technologies to increase fermentable sugars yields. Effects of reaction time, temperature, acid concentration and type of acid on hydrolysis of hemicellulose in soybean hulls and total sugar yields were studied. Optimum pretreatment parameters and enzymatic hydrolysis conditions for converting soybean hulls into fermentable sugars were identified. The combination of acid (H2SO4, 2% w/v) and steam (140°C, 30 min) efficiently solubilized the hemicellulose, giving a pentose yield of 96%. Sorghum is a tropical grass grown primarily in semiarid and dry parts of the world, especially in areas too dry for corn. The production of sorghum results in about 30 million tons of byproducts mainly composed of cellulose, hemicellulose, and lignin. Forage sorghum such as brown midrib (BMR) sorghum for ethanol production has generated much interest since this trait is characterized genetically by lower lignin concentrations in the plant compared with conventional types. Three varieties of forage sorghum and one variety of regular sorghum were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and X-Ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and enzymatic hydrolysis

  16. Quantitative Secretomic Analysis of Trichoderma reesei Strains Reveals Enzymatic Composition for Lignocellulosic Biomass Degradation*

    PubMed Central

    Adav, Sunil S.; Chao, Lim Tze; Sze, Siu Kwan

    2012-01-01

    Trichoderma reesei is a mesophilic, filamentous fungus, and it is a major industrial source of cellulases, but its lignocellulolytic protein expressions on lignocellulosic biomass are poorly explored at present. The extracellular proteins secreted by T. reesei QM6a wild-type and hypercellulolytic mutant Rut C30 grown on natural lignocellulosic biomasses were explored using a quantitative proteomic approach with 8-plex high throughput isobaric tags for relative and absolute quantification (iTRAQ) and analyzed by liquid chromatography tandem mass spectrometry. We quantified 230 extracellular proteins, including cellulases, hemicellulases, lignin-degrading enzymes, proteases, protein-translocating transporter, and hypothetical proteins. Quantitative iTRAQ results suggested that the expressions and regulations of these lignocellulolytic proteins in the secretome of T. reesei wild-type and mutant Rut C30 were dependent on both nature and complexity of different lignocellulosic carbon sources. Therefore, we discuss here the essential lignocellulolytic proteins for designing an enzyme mixture for optimal lignocellulosic biomass hydrolysis. PMID:22355001

  17. Pretreatment and enzymatic hydrolysis of corn fiber

    SciTech Connect

    Grohmann, K.; Bothast, R.J.

    1996-10-01

    Corn fiber is a co-product of the corn wet milling industry which is usually marketed as a low value animal feed ingredient. Approximately 1.2 x 10{sup 6} dry tons of this material are produced annually in the United States. The fiber is composed of kernel cell wall fractions and a residual starch which can all be potentially hydrolyzed to a mixture of glucose, xylose, arabinose and galactose. We have investigated a sequential saccharification of polysaccharides in corn fiber by a treatment with dilute sulfuric acid at 100 to 160{degrees}C followed by partial neutralization and enzymatic hydrolysis with mixed cellulose and amyloglucosidase enzymes at 45{degrees}C. The sequential treatment achieved a high (approximately 85%) conversion of all polysaccharides in the corn fiber to monomeric sugars, which were in most cases fermentable to ethanol by the recombinant bacterium Escherichia coli KOll.

  18. Evaluation of Enzymatic Hydrolysis of CELSS Wheat Residue Cellulose at a Scale Environment to NASA's KSC Breadboard Project

    NASA Technical Reports Server (NTRS)

    Strayer, Richard F.

    1993-01-01

    Biomass processing at the Kennedy Space Center CELSS breadboard project has focused on the evaluation of breadboard-scale enzymatic hydrolysis of wheat residue cellulose (25%, w/w). Five replicate runs of cellulase production by Trichoderma reesei (QM9414) and enzymatic hydrolysis of residue cellulose were completed. Enzymes were produced in 1 0 days (5 L, 25 g (dry weight) residue). Cellulose hydrolysis (12 L, 50 g (dry weight) residue) using these enzymes produced 5.5 to 6.0 g glucose liter(exp -1) in 7 days. Cellulose conversion efficiency was 29%. These processes are feasible technically on a breadboard scale, but would only increase the edible wheat yield 10%.

  19. Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction.

    PubMed Central

    Nidetzky, B; Steiner, W; Hayn, M; Claeyssens, M

    1994-01-01

    The hydrolysis of Whatman no. 1 filter paper by purified cellulolytic components from Trichoderma reesei and the synergistic action of binary combinations of these enzymes on the same substrate were investigated. At 20 milligrams filter paper, enzyme concentrations needed to obtain half-maximal hydrolysis rates (KE values) were in the 3-4 microM range for the cellobiohydrolases (CBHs) and 0.05-0.10 microM for the endoglucanases (EGs). Catalytic-core proteins of CBH I and EG III, lacking the cellulose-binding domain, exhibit KE values 2.3 and 5.1 times higher than those of the intact enzymes. In synergistic combinations of two cellulases, the KE value of at least one enzyme was 3-10-fold reduced. CBH I/CBH II and CBH I/EG III combinations showed the most powerful synergism, and optimal ratios were a function of the total protein concentration. Results obtained in activity and adsorption assays using filter paper pretreated with one component, followed by inactivation and subsequent hydrolysis with the same or another cellulase component, point to a sequential enzymic attack of the cellulose and seems consistent with the mathematical model presented. PMID:8141786

  20. Expression of Trichoderma reesei β-Mannanase in Tobacco Chloroplasts and Its Utilization in Lignocellulosic Woody Biomass Hydrolysis

    PubMed Central

    Agrawal, Pankaj; Verma, Dheeraj; Daniell, Henry

    2011-01-01

    Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost-effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, the man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds in the selection medium showed inheritance of transgenes into the progeny without any Mendelian segregation. Expression of endo-β-mannanase for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-β-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-β-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40°C to 70°C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6–7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different plant-derived enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without

  1. Cellulose hydrolysis and binding with Trichoderma reesei Cel5A and Cel7A and their core domains in ionic liquid solutions.

    PubMed

    Wahlström, Ronny; Rahikainen, Jenni; Kruus, Kristiina; Suurnäkki, Anna

    2014-04-01

    Ionic liquids (ILs) dissolve lignocellulosic biomass and have a high potential as pretreatment prior to total enzymatic hydrolysis. ILs are, however, known to inactivate cellulases. In this article, enzymatic hydrolysis of microcrystalline cellulose (MCC) and enzyme binding onto the cellulosic substrate were studied in the presence of cellulose-dissolving ILs. Two different ILs, 1,3-dimethylimidazolium dimethylphosphate ([DMIM]DMP) and 1-ethyl-3-methylimidazolium acetate ([EMIM]AcO), and two monocomponent cellulases, Trichoderma reesei cellobiohydrolase Cel7A and endoglucanase Cel5A, were used in the study. The role and IL sensitivity of the carbohydrate-binding module (CBM) were studied by performing hydrolysis and binding experiments with both the intact cellulases, and their respective core domains (CDs). Based on hydrolysis yields and substrate binding experiments for the intact enzymes and their CDs in the presence of ILs, the function of the CBM appeared to be very IL sensitive. Binding data suggested that the CBM was more important for the substrate binding of endoglucanase Cel5A than for the binding of cellobiohydrolase Cel7A. The CD of Cel7A was able to bind well to cellulose even without a CBM, whereas Cel5A CD had very low binding affinity. Hydrolysis also occurred with Cel5A CD even if this protein had very low binding affinity in all the studied matrices. Binding and hydrolysis were less affected by the studied ILs for Cel7A than for Cel5A. To our knowledge, this is the first systematic study of IL effects on cellulase substrate binding. PMID:24258388

  2. Enzymatic hydrolysis of biomass from wood.

    PubMed

    Álvarez, Consolación; Reyes-Sosa, Francisco Manuel; Díez, Bruno

    2016-03-01

    Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta-xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta-mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2-20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood. PMID:26833542

  3. Enzymatic hydrolysis and recrystallization behavior of initially amorphous cellulose.

    PubMed

    Bertran, M S; Dale, B E

    1985-02-01

    Cellulose samples from cotton and wood pulps with varying low degrees of crystallinity (mechanically decrystallized) were studied. The influence of initial cellulose crystallinity on sugar yield after enzymatic hydrolysis was determined by two different methods. As expected, samples with low crystallinity were much more accessible to enzymatic attack and glucose yields were higher than were samples of high initial crystallinity. Hydrolysis of cellulose seems more dependent on cellulose crystallinity than on the source of cellulose. It is known that decrystallized or amorphous cellulose can recrystallize under proper conditions, e.g., during acid hydrolysis. The data reported here also reveal some recrystallization during enzymatic hydrolysis which probably occurs simulataneously with a selective enzymatic attack on the amorphous regions of cellulose. In all cases, the amorphous celluloses recrystallized in the original lattice form, that of native cellulose. PMID:18553653

  4. A kinetic study of Trichoderma reesei Cel7B catalyzed cellulose hydrolysis.

    PubMed

    Song, Xiangfei; Zhang, Shujun; Wang, Yefei; Li, Jingwen; He, Chunyan; Yao, Lishan

    2016-06-01

    One prominent feature of Trichoderma reesei (Tr) endoglucanases catalyzed cellulose hydrolysis is that the reaction slows down quickly after it starts (within minutes). But the mechanism of the slowdown is not well understood. A structural model of Tr- Cel7B catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. The 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. This slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amount of reducing sugars released after 24h by Cel7B in phosphoric acid swollen cellulose, Avicel and filter paper cellulose hydrolysis is correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ∼15-35% more reducing sugars than the wild type (WT) Cel7B in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose. The activity of Tr- Cel7B against the insoluble substrate cellulose is determined by the enzyme's capability in hydrolyzing the soluble substrate. PMID:27178789

  5. Ultrasound Enhancement of Enzymatic Hydrolysis of Cellulose Plant Matter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The work reported here is based on acceleration of enzymatic hydrolysis of plant biomass substrate by introduction of low intensity, uniform ultrasound field into a reaction chamber (bio-reactor). This method may serve as improvement of rates in the hydrolysis of cellulosic materials to sugars, whi...

  6. Sugarcane bagasse enzymatic hydrolysis: rheological data as criteria for impeller selection.

    PubMed

    Pereira, Leonardo Tupi Caldas; Pereira, Lucas Tupi Caldas; Teixeira, Ricardo Sposina Sobral; Bon, Elba Pinto da Silva; Freitas, Suely Pereira

    2011-08-01

    The aim of this work was to select an efficient impeller to be used in a stirred reactor for the enzymatic hydrolysis of sugar cane bagasse. All experiments utilized 100 g (dry weight)/l of steam-pretreated bagasse, which is utilized in Brazil for cattle feed. The process was studied with respect to the rheological behavior of the biomass hydrolysate and the enzymatic conversion of the bagasse polysaccharides. These parameters were applied to model the power required for an impeller to operate at pilot scale (100 l) using empirical correlations according to Nagata [16]. Hydrolysis experiments were carried out using a blend of cellulases, β-glucosidase, and xylanases produced in our laboratory by Trichoderma reesei RUT C30 and Aspergillus awamori. Hydrolyses were performed with an enzyme load of 10 FPU/g (dry weight) of bagasse over 36 h with periodic sampling for the measurement of viscosity and the concentration of glucose and reducing sugars. The mixture presented pseudoplastic behavior. This rheological model allowed for a performance comparison to be made between flat-blade disk (Rushton turbine) and pitched-blade (45°) impellers. The simulation showed that the pitched blade consumed tenfold less energy than the flat-blade disk turbine. The resulting sugar syrups contained 22 g/l of glucose, which corresponded to 45% cellulose conversion. PMID:20844924

  7. Enzymatic Hydrolysis of Hydrotropic Pulps at Different Substrate Loadings.

    PubMed

    Denisova, Marina N; Makarova, Ekaterina I; Pavlov, Igor N; Budaeva, Vera V; Sakovich, Gennady V

    2016-03-01

    Enzymatic hydrolysis of cellulosic raw materials to produce nutrient broths for microbiological synthesis of ethanol and other valuable products is an important field of modern biotechnology. Biotechnological processing implies the selection of an effective pretreatment technique for raw materials. In this study, the hydrotropic treatment increased the reactivity of the obtained substrates toward enzymatic hydrolysis by 7.1 times for Miscanthus and by 7.3 times for oat hulls. The hydrotropic pulp from oat hulls was more reactive toward enzymatic hydrolysis compared to that from Miscanthus, despite that the substrates had similar compositions. As the initial substrate loadings were raised during enzymatic hydrolysis of the hydrotropic Miscanthus and oat hull pulps, the concentration of reducing sugars increased by 34 g/dm(3) and the yield of reducing sugars decreased by 31 %. The findings allow us to predict the efficiency of enzymatic hydrolysis of hydrotropic pulps from Miscanthus and oat hulls when scaling up the process by volume. PMID:26634840

  8. Enzymatic hydrolysis of steryl glycosides for their analysis in foods.

    PubMed

    Münger, Linda H; Nyström, Laura

    2014-11-15

    Steryl glycosides (SG) contribute significantly to the total intake of phytosterols. The standard analytical procedure involving acid hydrolysis fails to reflect the correct sterol profile of SG due to isomerization of some of the labile sterols. Therefore, various glycosylases were evaluated for their ability to hydrolyse SG under milder conditions. Using a pure SG mixture in aqueous solution, the highest glycolytic activity, as demonstrated by the decrease in SG and increase in free sterols was achieved using inulinase preparations (decrease of >95%). High glycolytic activity was also demonstrated using hemicellulase (63%). The applicability of enzymatic hydrolysis using inulinase preparations was further verified on SG extracted from foods. For example in potato peel Δ(5)-avenasteryl glucoside, a labile SG, was well preserved and contributed 26.9% of the total SG. Therefore, enzymatic hydrolysis is suitable for replacing acid hydrolysis of SG in food lipid extracts to accurately determine the sterol profile of SG. PMID:24912717

  9. [Enhanced enzymatic hydrolysis of excess sludge by surfactant].

    PubMed

    Yu, Jing; Luo, Kun; Yang, Qi; Li, Xiao-Ming; Xie, Bing-Xin; Yang, Guo-Jing; Mo, Chuang-Rong

    2011-08-01

    In order to enhance the efficiency of enzymatic hydrolysis of excess sludge, sodium dodecyl sulfate (SDS) was added to the system to explore the feasibility of promotion the enzyme hydrolysis. The results showed that the enzymatic hydrolysis of excess sludge could be greatly improved by SDS, and the mixed enzymes system was more effective than that by single enzyme system. SCOD releasing increased linearly with the increase of SDS dosage at the mixed enzymes concentration of 0.06 g/g. SCOD/TCOD increased from 1.3% to 54.3% and VSS reduction achieved to 43.2% at the SDS dosage of 0.20 g/g. Further studies indicated that SDS could improve the activity of external enzymes. At SDS dosage of 0.10 g/g, the protease activity of SDS + protease showed a 2. 3-time increase and the amylase activity of SDS + amylase showed a 1.2-time increase compared with enzymatic treatment. After 4 h hydrolysis, the concentration of protein, NH4+ -N and soluble sugar in SDS + mixed enzymes system were improved by 85.4%, 92.5% and 64.0%, respectively. Correspondingly, sludge hydrolysis within prior 4 h was consistent with first-order reaction dynamics. The reaction rate constant (K) of soluble sugar increased from 0.23 to 0.41, which indicated that the reaction rate of hydrolysis increased significantly. PMID:22619958

  10. Pretreatment of sallow prior to enzymatic hydrolysis

    SciTech Connect

    Galbe, M.; Zacchi, G.; Scott, C.D.

    1986-01-01

    Pretreatment of fast-growing sallow by steam explosion prior to enzymic hydrolysis was investigated to find optimum conditions regarding pretreatment temperature and time. Some preliminary experiments with impregnation of the material with H/sub 2/SO/sub 4/ or Na/sub 2/SO/sub 3/ were performed to reduce the byproduct formation and to increase the xylose yield. A temperature of 220 degrees for 15 minutes gave the highest yield, approximately 80% of the glucose available based on raw material. The xylose recovered was equal to or less than 20% when no chemicals were added. Impregnation with Na/sub 2/SO/sub 3/ gave an improvement compared with the unimpregnated material. About 30% of the xylose content could thus be recovered after the enzymic hydrolysis. The results are promising. (Refs. 5).

  11. The Preparation and Enzymatic Hydrolysis of a Library of Esters

    ERIC Educational Resources Information Center

    Sanford, Elizabeth M.; Smith, Traci L.

    2008-01-01

    An investigative case study involving the preparation of a library of esters using Fischer esterification and alcoholysis of acid chlorides and their subsequent enzymatic hydrolysis by pig liver esterase and orange peel esterase is described. Students work collaboratively to prepare and characterize the library of esters and complete and evaluate…

  12. Effect of particle size on enzymatic hydrolysis of pretreated Miscanthus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Particle size reduction is a crucial factor in transportation logistics as well as cellulosic conversion. The effect of particle size on enzymatic hydrolysis of pretreated Miscanthus x giganteus was determined. Miscanthus was ground using a hammer mill equipped with screens having 0.08, 2.0 or 6.0...

  13. Enzymatic hydrolysis of fractionated products from oil thermally oxidated

    SciTech Connect

    Yashida, H.; Alexander, J.C.

    1983-01-01

    Enzymatic hydrolysis of the acylglycerol products obtained from thermally oxidized vegetable oils was studied. Corn, sunflower and soybean oils were heated in the laboratory at 180/sup 0/C for 50, 70 and 100 hr with aeration and directly fractionated by silicic acid column chromatography. By successive elution with 20%, then 60% isopropyl ether in n-hexane, and diethyl ether, the thermally oxidized oils were separated into three fractions: the nonpolar fraction (monomeric compounds), slightly polar fraction (dimeric compounds), and polar fraction comprising oligomeric compounds. Enzymatic hydrolysis with pancreatic lipase showed that the monomers were hydrolyzed as rapidly as the corresponding unheated oils, the dimers much more slowly, and the oligomeric compounds barely at all. Overall, the hydrolysis of the dimers was less than 23% of that for the monomers, with small differences among the oils. Longer heating periods resulted in greater reductions in hydrolysis of the dimeric compounds. These results suggest that the degree of enzymatic hydrolysis of the fractionated acylglycerol compounds is related to differences in the thermal oxidative deterioration, and amounts of polar compounds in the products. (33 Refs.)

  14. Enzymatic hydrolysis of PTT polymers and oligomers.

    PubMed

    Eberl, A; Heumann, S; Kotek, R; Kaufmann, F; Mitsche, S; Cavaco-Paulo, A; Gübitz, G M

    2008-05-20

    Oligomers and polymers (film, fabrics) of the linear aromatic polyester poly(trimethylene terephthalate) (PTT) were treated with polyesterases from Thermomyces lanuginosus, Penicillium citrinum, Thermobifida fusca and Fusarium solani pisi. The cutinase from T. fusca was found to release the highest amounts of hydrolysis products from PTT materials and was able to open and hydrolyse a cyclic PTT dimer according to RP-HPLC-UV detection. In contrast, the lipase from T. lanuginosus also showed activity on the PTT fibres and on bis(3-hydroxypropyl) terephthalate (BHPT) but was not able to hydrolyse the polymer film, mono(3-hydroxypropyl) terephthalate (MHPT) nor the cyclic dimer of PTT. As control enzymes inhibited with mercury chloride were used. Surface hydrophilicity changes were investigated with contact angle measurements and the degree of crystallinity changes were determined with DSC. PMID:18405994

  15. Enzymatic hydrolysis of fructans in the tequila production process.

    PubMed

    Avila-Fernández, Angela; Rendón-Poujol, Xóchitl; Olvera, Clarita; González, Fernando; Capella, Santiago; Peña-Alvarez, Araceli; López-Munguía, Agustín

    2009-06-24

    In contrast to the hydrolysis of reserve carbohydrates in most plant-derived alcoholic beverage processes carried out with enzymes, agave fructans in tequila production have traditionally been transformed to fermentable sugars through acid thermal hydrolysis. Experiments at the bench scale demonstrated that the extraction and hydrolysis of agave fructans can be carried out continuously using commercial inulinases in a countercurrent extraction process with shredded agave fibers. Difficulties in the temperature control of large extraction diffusers did not allow the scaling up of this procedure. Nevertheless, batch enzymatic hydrolysis of agave extracts obtained in diffusers operating at 60 and 90 degrees C was studied at the laboratory and industrial levels. The effects of the enzymatic process on some tequila congeners were studied, demonstrating that although a short thermal treatment is essential for the development of tequila's organoleptic characteristics, the fructan hydrolysis can be performed with enzymes without major modifications in the flavor or aroma, as determined by a plant sensory panel and corroborated by the analysis of tequila congeners. PMID:19473003

  16. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    PubMed Central

    Thygesen, Lisbeth G.; Thybring, Emil E.; Johansen, Katja S.; Felby, Claus

    2014-01-01

    Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood. Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry, particularly when it comes to up-scaling of processes based on insoluble feed stocks. PMID:25232741

  17. Ultrasound-enhanced enzymatic hydrolysis of poly(ethylene terephthalate).

    PubMed

    Pellis, Alessandro; Gamerith, Caroline; Ghazaryan, Gagik; Ortner, Andreas; Herrero Acero, Enrique; Guebitz, Georg M

    2016-10-01

    The application of ultrasound was found to enhance enzymatic hydrolysis of poly(ethylene terephthalate) (PET). After a short activation phase up to 6.6times increase in the amount of released products was found. PET powder with lower crystallinity of 8% was hydrolyzed faster when compared to PET with 28% crystallinity. Ultrasound activation was found to be around three times more effective on powders vs. films most likely due to a larger surface area accessible to the enzyme. PMID:27481467

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

    PubMed

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

    2016-08-01

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

  19. Alkaline polyol pulping and enzymatic hydrolysis of hardwood: effect of pulping severity and pulp composition on cellulase activity and overall sugar yield.

    PubMed

    Hundt, Martin; Schnitzlein, Klaus; Schnitzlein, Michael G

    2013-05-01

    The saccharification of beech wood using alkaline polyol pulping (AlkaPolP) and enzymatic hydrolysis was investigated. It will be demonstrated that the AlkaPolP process yields high quality pulps which can easily be hydrolyzed by cellulases. In order to find optimum reaction conditions chips of Fagus sylvatica were pretreated by alkaline glycerol at temperatures between 190 and 230 °C for 15, 20, and 25 min. The impacts of temperature and time were expressed using a severity factor R0. The dependencies of the conversion during enzymatic hydrolysis on severity, pulp yield, delignification and pulp composition are shown. In further experiments it was investigated if the sugar yields can be increased by the application of ultrasound or surfactants before enzyme addition. Up to 95% of the initial cellulose in wood were converted into glucose using cellulases from Trichoderma reesei and β-glucosidase from Aspergillus niger. PMID:23570715

  20. Alcohol fermentation of sweet potato. Membrane reactor in enzymatic hydrolysis

    SciTech Connect

    Azhar, A.; Hamdy, M.K.

    1981-06-01

    Use of ultrafiltration membrane systems in stirred cell and in thin-channel systems for immobilizing enzyme (sweet potato intrinsic and crystalline /beta/-amylase) in hydrolysis of sweet potato through a continuous operation mode were studied. Both the filtration rate and reducing sugars, produced as the result of enzymatic hydrolysis, decreased with the filtration time. THe immobilized enzymes in the thin-channel system showed a much better performance compared to that in the stirred cell system. Addition of crystalline sweet potato /beta/-amylase to the sweet potato increased both the filtration rate and reducing-sugars content. Alcoholic fermentation of the filtrate resulted in an alcohol content of 4.2%. This represented fermentation of 95% of the sugars with an efficiency of 88%. 17 refs.

  1. Enzymatic hydrolysis of haloperidol decanoate and its inhibition by proteins.

    PubMed

    Nambu, K; Miyazaki, H; Nakanishi, Y; Oh-e, Y; Matsunaga, Y; Hashimoto, M

    1987-05-15

    When [14C]haloperidol decanoate, a long-acting neuroleptic and an ester of haloperidol and decanoic acid, was incubated in human whole blood and plasma and in rat plasma and homogenates of rat brain, lung, liver, kidney, pancreas and muscle, no hydrolysis of the ester was seen. Although the decanoate was hydrolyzed by partially purified carboxylesterase, addition of rat plasma or liver homogenate to the enzymic reaction mixture resulted in marked inhibition of hydrolysis, whereas addition of the defatted residues of plasma or liver produced only partial inhibition. The enzymic hydrolysis was inhibited also by beta-lipoprotein and albumin, depending on their concentrations. The assumption that interaction between haloperidol decanoate and protein resulted in inhibition of the hydrolytic reaction mediated by the enzyme was validated by kinetic models and experimental data. The kinetics were apparently competitive. Based on the kinetic analysis, the interaction between the decanoate and albumin or beta-lipoprotein was investigated by measuring their equilibrium constants and extent of protein binding. Haloperidol decanoate appeared to interact with several proteins; this was exemplified by other measures of protein binding, an increasing effect of proteins on the solubility, and the partition ratio of the ester. The interaction between haloperidol decanoate and proteins caused marked stabilization of this ester against enzymatic hydrolysis and, thereby, influenced its metabolism. PMID:3593395

  2. Structural modifications of lignocellulosics by pretreatments to enhance enzymatic hydrolysis

    SciTech Connect

    Gharpuray, M.M.; Lee, Y.F.; Fan, L.T.

    1983-01-01

    In this work an evaluation was made of a wide variety of single and multiple pretreatment methods for enhancing the rate of enzymatic hydrolysis of wheat straw. A multiple pretreatment consisted of a physical pretreatment followed by a chemical pretreatment. The structural features of wheat straw, including the specific surface area, crystallinity index, and lignin content, were measured to understand the mechanism of the enhancement in the hyrolysis rate upon pretreatment. It has been found that, in general, multiple pretreatments were not promising, since the hydrolysis rates rarely exceeded those achieved by single pretreatments. Ball-milling pretreatment was found to be effective in increasing the specific surface area and decreasing the crystallinity index. Treatment with ethylene glycol was highly effective in increasing the specific surface area, in addition to a high degree of delignification. Peracetic acid pretreatment was highly effective in delignifying substrate. Among multiple pretreatments, those involving peracetic acid treatment generally had lower crystallinity indices and lignin content values. The relationship between the hydrolysis rate and the set of structural features indicated that an increase in surface area and a decrease in the crystallinity and lignin content enhance the hydrolysis; the specific surface area is the most influential of the structural features, followed by the lignin content. (Refs. 23).

  3. β-cyclodextrin assistant flavonoid glycosides enzymatic hydrolysis

    PubMed Central

    Jin, Xin; Zhang, Zhen-hai; Sun, E.; Jia, Xiao-Bin

    2013-01-01

    Background: The content of icaritin and genistein in herba is very low, preparation with relatively large quantities is an important issue for extensive pharmacological studies. Objective: This study focuses on preparing and enzymic hydrolysis of flavonoid glycosides /β-cyclodextrin inclusion complex to increase the hydrolysis rate. Materials and Methods: The physical property of newly prepared inclusion complex was tested by differential scanning calorimetry (DSC). The conditions of enzymatic hydrolysis were optimized for the bioconversion of flavonoid glycosides /β-cyclodextrin inclusion complex by mono-factor experimental design. The experiments are using the icariin and genistein as the model drugs. Results: The solubility of icariin and genistein were increased almost 17 times from 29.2 μg/ml to 513.5 μg/ml at 60°C and 28 times from 7.78 μg/ml to 221.46 μg/ml at 50°C, respectively, demonstrating that the inclusion complex could significantly increase the solubility of flavonoid glycosides. Under the optimal conditions, the reaction time of icariin and genistin decreased by 68% and 145%, when compared with that without β-CD inclusion. By using this enzymatic condition, 473 mg icaritin (with the purity of 99.34%) and 567 mg genistein(with the purity of 99.46%), which was finally determined by melt point, ESI-MS, UV, IR, 1H NMR and 13C NMR, was obtained eventually by transforming the inclusion complex(contains 1.0 g substrates). Conclusion: This study can clearly indicate a new attempt to improve the speed of enzyme-hydrolysis of poorly water-soluble flavonoid glycosides and find a more superior condition which is used to prepare icaritin and genistein. PMID:24143039

  4. Effect of melanin on enzymatic hydrolysis of cellulosic waste.

    PubMed

    Ray, R M; Desai, J D

    1984-07-01

    Wood waste powder from Tectona grandis containing melanin was less susceptible to enzymatic hydrolysis than powder without melanin. About a 53% increase in saccharification was noted when melanin was removed. Melanin caused inhibition to all cellulolytic enzymes, but in different degrees. Endo-beta-1,4-glucanase and beta-glucosidase were markedly inhibited when melanin was preincubated with enzyme, while exo-beta-1,4-glucanase was severely inhibited when melanin was preincubated with substrate. The latter was found to be dependent on the contact time. The activities of endo-beta-1,4-glucanase and beta-glucosidase were noncompetitively inhibited by melanin. PMID:18553434

  5. Optimization of reaction conditions for enzymatic viscosity reduction and hydrolysis of wheat arabinoxylan in an industrial ethanol fermentation residue.

    PubMed

    Sørensen, Hanne R; Pedersen, Sven; Meyer, Anne S

    2006-01-01

    This study examined enzyme-catalyzed viscosity reduction and evaluated the effects of substrate dry matter concentration on enzymatic degradation of arabinoxylan in a fermentation residue, "vinasse", resulting from industrial ethanol manufacture on wheat. Enzymatic catalysis was accomplished with a 50:50 mixture of an enzyme preparation from Humicola insolens, Ultraflo L, and a cellulolytic enzyme preparation from Trichoderma reesei, Celluclast 1.5 L. This enzyme mixture was previously shown to exhibit a synergistic action on arabinoxylan degradation. The viscosity of vinasse decreased with increased enzyme dosage and treatment time at pH 5, 50 degrees C, 5 wt % vinasse dry matter. After 24 h of enzymatic treatment, 76-84%, 75-80%, and 43-47%, respectively, of the theoretically maximal arabinose, xylose, and glucose releases were achieved, indicating that the viscosity decrease was a result of enzyme-catalyzed hydrolysis of arabinoxylan, beta-glucan, and cellulose. In designed response surface experiments, the optimal enzyme reaction conditions with respect to pH and temperature of the vinasse, the vinasse supernatant (mainly soluble material), and the vinasse sediment (mainly insoluble substances) varied from pH 5.2-6.4 and 41-49 degrees C for arabinose release and from pH 4.9-5.3 and 42-46 degrees C for xylose release. Even though only limited hydrolysis of the arabinoxylan in the vinasse sediment fraction was obtained, the results indicated that the same enzyme activities acted on the arabinoxylan in the different vinasse fractions irrespective of the state of solubility of the substrate material. The levels of liberated arabinose and xylose increased with increased dry matter concentration during enzymatic hydrolysis in the vinasse and the vinasse supernatant, but at the same time, increased substrate dry matter concentrations gave corresponding linear decreases in the hydrolytic efficiency as evaluated from levels of monosaccharide release per weight unit dry

  6. Enzymatic hydrolysis of defatted mackerel protein with low bitter taste

    NASA Astrophysics Data System (ADS)

    Hou, Hu; Li, Bafang; Zhao, Xue

    2011-03-01

    Ultrasound-assisted solvent extraction was confirmed as a novel, effective method for separating lipid from mackerel protein, resulting in a degreasing rate (DR) of 95% and a nitrogen recovery (NR) of 88.6%. To obtain protein hydrolysates with high nitrogen recovery and low bitter taste, enzymatic hydrolysis was performed using eight commercially available proteases. It turned out that the optimum enzyme was the `Mixed enzymes for animal proteolysis'. An enzyme dosage of 4%, a temperature of 50°, and a hydrolysis time of 300 min were found to be the optimum conditions to obtain high NR (84.28%) and degree of hydrolysis (DH, 16.18%) by orthogonal experiments. Glutamic acid was the most abundant amino acid of MDP (defatted mackerel protein) and MDPH (defatted mackerel protein hydrolysates). Compared with the FAO/WHO reference protein, the essential amino acid chemical scores (CS) were greater than 1.0 (1.0-1.7) in MDPH, which is reflective of high nutritional value. This, coupled with the light color and slight fishy odor, indicates that MDPH would potentially have a wide range of applications such as nutritional additives, functional ingredients, and so on.

  7. Mechanistic investigation in ultrasound induced enhancement of enzymatic hydrolysis of invasive biomass species.

    PubMed

    Borah, Arup Jyoti; Agarwal, Mayank; Poudyal, Manisha; Goyal, Arun; Moholkar, Vijayanand S

    2016-08-01

    This study has assessed four invasive weeds, viz. Saccharum spontaneum (SS), Mikania micrantha (MM), Lantana camara (LC) and Eichhornia crassipes (EC) for enzymatic hydrolysis prior to bioalcohol fermentation. Enzymatic hydrolysis of pretreated biomasses of weeds has been conducted with mechanical agitation and sonication under constant (non-optimum) conditions. Profiles of total reducible sugar release have been fitted to HCH-1 model of enzymatic hydrolysis using Genetic Algorithm. Trends in parameters of this model reveal physical mechanism of ultrasound-induced enhancement of enzymatic hydrolysis. Sonication accelerates hydrolysis kinetics by ∼10-fold. This effect is contributed by several causes, attributed to intense micro-convection generated during sonication: (1) increase in reaction velocity, (2) increase in enzyme-substrate affinity, (3) reduction in product inhibition, and (4) enhancement of enzyme activity due to conformational changes in its secondary structure. Enhancement effect of sonication is revealed to be independent of conditions of enzymatic hydrolysis - whether optimum or non-optimum. PMID:26898160

  8. Periodic peristalsis releasing constrained water in high solids enzymatic hydrolysis of steam exploded corn stover.

    PubMed

    Liu, Zhi-Hua; Chen, Hong-Zhang

    2016-04-01

    Periodic peristalsis was used to release water constraint and increase high solids enzymatic hydrolysis efficiency. Glucan and xylan conversion in periodic peristalsis enzymatic hydrolysis (PPEH) at 21% solid loading increased by 5.2-6.4% and 6.8-8.8% compared with that in incubator shaker enzymatic hydrolysis (ISEH), respectively. Hydrolysis kinetics suggested that sugars conversion significantly increased within 24h in PPEH compared with ISEH. The peak height of main water pool increased by 7.7-43.1% within 24h in PPEH compared with ISEH. The increases in peak height of main water pool were consistent with the increases in glucan conversion. Submicroscopic particulates and macro granule residues contributed greatly to water constraint compared with glucose, xylose, ethanol, and Tween 80. Smaller particle size and longer residence time resulted in lower water constraint and facilitated the enzymatic hydrolysis performance. Periodic peristalsis was an effective method to reduce water constraint and increase high solids enzymatic hydrolysis efficiency. PMID:26826953

  9. 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.

  10. Chemical structures of corn stover and its residue after dilute acid prehydrolysis and enzymatic hydrolysis: Insight into factors limiting enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Advanced solid-state NMR techniques and wet chemical analyses were applied to investigate untreated corn stover (UCS) and its residues after dilute acid prehydrolysis (DAP) and enzymatic hydrolysis (RES) to provide evidence for the limitations to the effectiveness of enzyme hydrolysis. Advanced soli...

  11. Free Energy Diagram for the Heterogeneous Enzymatic Hydrolysis of Glycosidic Bonds in Cellulose*

    PubMed Central

    Sørensen, Trine Holst; Cruys-Bagger, Nicolaj; Borch, Kim; Westh, Peter

    2015-01-01

    Kinetic and thermodynamic data have been analyzed according to transition state theory and a simplified reaction scheme for the enzymatic hydrolysis of insoluble cellulose. For the cellobiohydrolase Cel7A from Hypocrea jecorina (Trichoderma reesei), we were able to measure or collect relevant values for all stable and activated complexes defined by the reaction scheme and hence propose a free energy diagram for the full heterogeneous process. For other Cel7A enzymes, including variants with and without carbohydrate binding module (CBM), we obtained activation parameters for the association and dissociation of the enzyme-substrate complex. The results showed that the kinetics of enzyme-substrate association (i.e. formation of the Michaelis complex) was almost entirely entropy-controlled and that the activation entropy corresponded approximately to the loss of translational and rotational degrees of freedom of the dissolved enzyme. This implied that the transition state occurred early in the path where the enzyme has lost these degrees of freedom but not yet established extensive contact interactions in the binding tunnel. For dissociation, a similar analysis suggested that the transition state was late in the path where most enzyme-substrate contacts were broken. Activation enthalpies revealed that the rate of dissociation was far more temperature-sensitive than the rates of both association and the inner catalytic cycle. Comparisons of one- and two-domain variants showed that the CBM had no influence on the transition state for association but increased the free energy barrier for dissociation. Hence, the CBM appeared to promote the stability of the complex by delaying dissociation rather than accelerating association. PMID:26183776

  12. Effects of fibrillation on the wood fibers' enzymatic hydrolysis enhanced by mechanical refining.

    PubMed

    Liu, Wei; Wang, Bing; Hou, Qingxi; Chen, Wei; Wu, Ming

    2016-04-01

    The hardwood bleached kraft pulp (HBKP) fibers were pretreated by PFI mill to obtain the substrates, the effects of fibrillation on HBKP fibers' enzymatic hydrolysis was studied. The results showed that the enzymatic hydrolysis efficiency was enhanced obviously by mechanical refining. The mechanical refining alterated the fibers' characteristics such as fibrillation degree, specific surface area, swelling ability, crystallinity, fiber length and fines content. All these factors correlating to the enzymatic hydrolysis were evaluated through mathematical analysis. Among these factors, the fibrillation degree has the profoundest impact on the enzymatic hydrolysis of wood fibers. Consequently, the mechanical refining aiming for a high fibrillation degree was feasible to enhance the enzymatic hydrolysis of lignocellulosic biomass. PMID:26851576

  13. Factors limiting the enzymatic hydrolysis of wheat gluten.

    PubMed

    Giesler, L; Linke, D; Berger, R G

    2014-05-21

    The enzymatic hydrolysis of wheat gluten for the production of seasonings using mixtures of endo- and exopeptidases results in yields typically below 40%. Possible limiting parameters, such as an increasing product inhibition, autopeptidolysis of the enzymes, and lack of cleavage sites, were studied using novel peptidases from Flammulina velutipes or the commercial Flavourzyme preparation. Seven intermittent electrodialysis steps (10 g/L gluten and 10 kaU/mL) for the in situ removal of amino acids minimized the product inhibition. During 16 h, hydrolysis progressed nearly linearly. Compared to the batch control, a 3-fold yield of amino acids released was obtained indicating that an integrated product removal alleviates the problem of product inhibition. Autopeptidolysis, as shown using sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme activity assays, was suppressed with increasing concentrations of competing gluten substrate. Peptidases of F. velutipes showed product inhibition only, whereas a combined effect of product inhibition and lack of cleavage sites was observed for Flavourzyme. PMID:24787755

  14. MATHEMATICAL MODELING OF ENZYMATIC HYDROLYSIS OF STARCH: APPLICATION TO FUEL ETHANOL PRODUCTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymatic hydrolysis of starch in corn is an important step that determines fermentation efficiency. Corn genetics, post harvest handling and process conditions are factors that affect starch hydrolysis. There is a lack of mathematical models for starch hydrolysis in the dry grind corn process tha...

  15. A cellulose-binding module of the Trichoderma reesei beta-mannanase Man5A increases the mannan-hydrolysis of complex substrates.

    PubMed

    Hägglund, Per; Eriksson, Torny; Collén, Anna; Nerinckx, Wim; Claeyssens, Marc; Stålbrand, Henrik

    2003-02-27

    Endo-beta-1,4-D-mannanases (beta-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The beta-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5ADeltaCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5ADeltaCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei. PMID:12523968

  16. Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hydrolysis of cellulose in presence of xylans.

    PubMed

    Dondelinger, Eve; Aubry, Nathalie; Ben Chaabane, Fadhel; Cohen, Céline; Tayeb, Jean; Rémond, Caroline

    2016-03-01

    Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemicellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes developed in the context of lignocellulosic biorefinery. PMID:27001439

  17. Effect of non-enzymatic proteins on enzymatic hydrolysis and simultaneous saccharification and fermentation of different lignocellulosic materials.

    PubMed

    Wang, Hui; Kobayashi, Shinichi; Mochidzuki, Kazuhiro

    2015-08-01

    Non-enzymatic proteins were added during hydrolysis of cellulose and simultaneous saccharification and fermentation (SSF) of different biomass materials. Bovine serum albumin (BSA), a model non-enzymatic protein, increased cellulose and xylose conversion efficiency and also enhanced the ethanol yield during SSF of rice straw subjected to varied pretreatments. Corn steep liquor, yeast extract, and peptone also exerted a similar effect as BSA and enhanced the enzymatic hydrolysis of rice straw. Compared to the glucose yields obtained after enzymatic hydrolysis of rice straw in the absence of additives, the glucose yields after 72h of hydrolysis increased by 12.7%, 13.5%, and 13.7% after addition of the corn steep liquor, yeast extract, and peptone, respectively. This study indicated the use of BSA as an alternative to intensive pretreatment of lignocellulosic materials for enhancing enzymatic digestibility. The utilization of non-enzymatic protein additives is promising for application in glucose and ethanol production from lignocellulosic materials. PMID:25974351

  18. Impacts of microalgae pre-treatments for improved anaerobic digestion: thermal treatment, thermal hydrolysis, ultrasound and enzymatic hydrolysis.

    PubMed

    Ometto, Francesco; Quiroga, Gerardo; Pšenička, Pavel; Whitton, Rachel; Jefferson, Bruce; Villa, Raffaella

    2014-11-15

    Anaerobic digestion (AD) of microalgae is primarily inhibited by the chemical composition of their cell walls containing biopolymers able to resist bacterial degradation. Adoption of pre-treatments such as thermal, thermal hydrolysis, ultrasound and enzymatic hydrolysis have the potential to remove these inhibitory compounds and enhance biogas yields by degrading the cell wall, and releasing the intracellular algogenic organic matter (AOM). This work investigated the effect of four pre-treatments on three microalgae species, and their impact on the quantity of soluble biomass released in the media and thus on the digestion process yields. The analysis of the composition of the soluble COD released and of the TEM images of the cells showed two main degradation actions associated with the processes: (1) cell wall damage with the release of intracellular AOM (thermal, thermal hydrolysis and ultrasound) and (2) degradation of the cell wall constituents with the release of intracellular AOM and the solubilisation of the cell wall biopolymers (enzymatic hydrolysis). As a result of this, enzymatic hydrolysis showed the greatest biogas yield increments (>270%) followed by thermal hydrolysis (60-100%) and ultrasounds (30-60%). PMID:25150520

  19. Numerical prediction of kinetic model for enzymatic hydrolysis of cellulose using DAE-QMOM approach

    NASA Astrophysics Data System (ADS)

    Jamil, N. M.; Wang, Q.

    2016-06-01

    Bioethanol production from lignocellulosic biomass consists of three fundamental processes; pre-treatment, enzymatic hydrolysis, and fermentation. In enzymatic hydrolysis phase, the enzymes break the cellulose chains into sugar in the form of cellobiose or glucose. A currently proposed kinetic model for enzymatic hydrolysis of cellulose that uses population balance equation (PBE) mechanism was studied. The complexity of the model due to integrodifferential equations makes it difficult to find the analytical solution. Therefore, we solved the full model of PBE numerically by using DAE-QMOM approach. The computation was carried out using MATLAB software. The numerical results were compared to the asymptotic solution developed in the author's previous paper and the results of Griggs et al. Besides confirming the findings were consistent with those references, some significant characteristics were also captured. The PBE model for enzymatic hydrolysis process can be solved using DAE-QMOM method. Also, an improved understanding of the physical insights of the model was achieved.

  20. Enzymatic hydrolysis and characterization of waste lignocellulosic biomass produced after dye bioremediation under solid state fermentation.

    PubMed

    Waghmare, Pankajkumar R; Kadam, Avinash A; Saratale, Ganesh D; Govindwar, Sanjay P

    2014-09-01

    Sugarcane bagasse (SCB) adsorbes 60% Reactive Blue172 (RB172). Providensia staurti EbtSPG able to decolorize SCB adsorbed RB172 up to 99% under solid state fermentation (SSF). The enzymatic saccharification efficiency of waste biomass after bioremediation of RB172 process (ddSCB) has been evaluated. The cellulolyitc crude enzyme produced by Phanerochaete chrysosporium used for enzymatic hydrolysis of native SCB and ddSCB which produces 0.08 and 0.3 g/L of reducing sugars respectively after 48 h of incubation. The production of hexose and pentose sugars during hydrolysis was confirmed by HPTLC. The effect of enzymatic hydrolysis on SCB and ddSCB has been evaluated by FTIR, XRD and SEM analysis. Thus, during dye biodegradation under SSF causes biological pretreatment of SCB which significantly enhanced its enzymatic saccharification. Adsorption of dye on SCB, its bioremediation under SSF produces wastes biomass and which further utilized for enzymatic saccharification for biofuel production. PMID:24656486

  1. Fuzzy logic feedback control for fed-batch enzymatic hydrolysis of lignocellulosic biomass.

    PubMed

    Tai, Chao; Voltan, Diego S; Keshwani, Deepak R; Meyer, George E; Kuhar, Pankaj S

    2016-06-01

    A fuzzy logic feedback control system was developed for process monitoring and feeding control in fed-batch enzymatic hydrolysis of a lignocellulosic biomass, dilute acid-pretreated corn stover. Digested glucose from hydrolysis reaction was assigned as input while doser feeding time and speed of pretreated biomass were responses from fuzzy logic control system. Membership functions for these three variables and rule-base were created based on batch hydrolysis data. The system response was first tested in LabVIEW environment then the performance was evaluated through real-time hydrolysis reaction. The feeding operations were determined timely by fuzzy logic control system and efficient responses were shown to plateau phases during hydrolysis. Feeding of proper amount of cellulose and maintaining solids content was well balanced. Fuzzy logic proved to be a robust and effective online feeding control tool for fed-batch enzymatic hydrolysis. PMID:26915095

  2. Three-stage enzymatic hydrolysis of steam-exploded corn stover at high substrate concentration.

    PubMed

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

    2011-04-01

    The feasibility of three-stage hydrolysis of steam-exploded corn stover at high-substrate concentration was investigated. When substrate concentration was 30% and enzyme loading was 15-30 FPU/g cellulose, three-stage (9+9+12 h) hydrolysis could reach a hydrolysis yield of 59.9-81.4% in 30 h. Compared with one-stage hydrolysis for 72 h, an increase of 34-37% in hydrolysis yield could be achieved. When steam-exploded corn stover was used as the substrate for enzyme synthesis and hydrolysis was conducted at a substrate concentration of 25% with an enzyme loading of 20 FPU/g cellulose, a hydrolysis yield of 85.1% was obtained, 19% higher than that the commercial cellulase could reach under the same conditions. The removal of end products was suggested to improve the adsorption of cellulase on the substrate and enhance the productivity of enzymatic hydrolysis. PMID:21300538

  3. Low Intensity Uniform Ultrasound Accelerates Enzymatic Hydrolysis of Cellulose Plant Matter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The work reported here is based on acceleration of enzymatic hydrolysis of plant biomass substrate by introduction of low intensity, uniform ultrasound field into a reaction chamber (bio-reactor). This method may serve as an improvement of rates in the hydrolysis of cellulosic materials to sugars, ...

  4. Enzymatic hydrolysis of rice protein with papain and antioxidation activity of hydrolysate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzymatic hydrolysis technology of rice protein and the antioxidant activity of the hydrolysate were studied. Substrate concentration,enzyme dose,pH value and temperature were selected as factors to optimize the hydrolysis parameters with single—factor and orthogonal tests. Results show the opti...

  5. Use of an algal hydrolysate to improve enzymatic hydrolysis of anaerobically digested fiber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study investigated the use of acid hydrolyzed algae to enhance the enzymatic hydrolysis of cellulosic biomass. We first characterized wastewater-grown algal samples and determined the optimal conditions (acid concentration, reaction temperature, and reaction time) for algal hydrolysis using di...

  6. Improving enzymatic hydrolysis of industrial hemp ( Cannabis sativa L.) by electron beam irradiation

    NASA Astrophysics Data System (ADS)

    Shin, Soo-Jeong; Sung, Yong Joo

    2008-09-01

    The electron beam irradiation was applied as a pretreatment of the enzymatic hydrolysis of hemp biomass with doses of 150, 300 and 450 kGy. The higher irradiation dose resulted in the more extraction with hot-water extraction or 1% sodium hydroxide solution extraction. The higher solubility of the treated sample was originated from the chains scission during irradiation, which was indirectly demonstrated by the increase of carbonyl groups as shown in diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) spectra. The changes in the micro-structure of hemp resulted in the better response to enzymatic hydrolysis with commercial cellulases (Celluclast 1.5L and Novozym 342). The improvement in enzymatic hydrolysis by the irradiation was more evident in the hydrolysis of the xylan than in that of the cellulose.

  7. Enzyme feeding strategies for better fed-batch enzymatic hydrolysis of empty fruit bunch.

    PubMed

    Sugiharto, Yohanes Eko Chandra; Harimawan, Ardiyan; Kresnowati, Made Tri Ari Penia; Purwadi, Ronny; Mariyana, Rina; Andry; Fitriana, Hana Nur; Hosen, Hauna Fathmadinda

    2016-05-01

    Lignin inhibitory becomes a major obstacle for enzymatic hydrolysis of empty fruit bunch conducted in high solid loading. Since current technology required high enzyme loading, surfactant application could not effectively used since it is only efficient in low enzyme loading. In addition, it will increase final operation cost. Hence, another method namely "proportional enzyme feeding" was investigated in this paper. In this method, enzyme was added to reactor proportionally to substrate addition, different from conventional method ("whole enzyme feeding") where whole enzyme was added prior to hydrolysis process started. Proportional enzyme feeding could increase enzymatic digestibility and glucose concentration up to 26% and 12% respectively, compared to whole enzyme feeding for hydrolysis duration more than 40h. If enzymatic hydrolysis was run less than 40h (25% solid loading), whole enzyme feeding is preferable. PMID:26881335

  8. Intensification of enzymatic hydrolysis of waste newspaper using ultrasound for fermentable sugar production.

    PubMed

    Subhedar, Preeti B; Babu, Narmadha R; Gogate, Parag R

    2015-01-01

    An effective conversion of lignocellulose into fermentable sugars is a key step in producing bioethanol in an eco-friendly and cost effective manner. In this study, the effect of ultrasound on enzymatic hydrolysis of newspaper, a potential feedstock for bioethanol production due to its high cellulosic content, was investigated. The effect of substrate loading, enzyme loading, temperature, ultrasonic power and duty cycle on the hydrolysis has been studied. Optimum conditions for conventional enzymatic hydrolysis were substrate loading of 5% (w/v), enzyme loading of 0.14% (w/v), temperature of 323K, and under these conditions and 72h of hydrolysis, reducing sugar yield of 11.569g/L was obtained. In case of ultrasound-assisted enzymatic hydrolysis approach, optimum conditions obtained were substrate loading of 3% (w/v), enzyme loading of 0.8% (w/v), sonication power of 60W, duty cycle of 70%, hydrolysis time of 6.5h and the reducing sugar yield obtained under these conditions was 27.6g/L. Approximately 2.4 times increase in the release of reducing sugar concentration was obtained by the ultrasound-assisted enzymatic hydrolysis approach. Results indicate that there is a synergistic effect obtained from the combination of ultrasound and enzymes which lowers the diffusion-limiting barrier to enzyme/substrate binding and results in an increase in reaction rate. The experimental data were also fitted in a simple three parameter kinetic model. PMID:25060116

  9. Hydrogel coated monoliths for enzymatic hydrolysis of penicillin G

    PubMed Central

    Smeltink, M. W.; Straathof, A. J. J.; Paasman, M. A.; van de Sandt, E. J. A. X.; Kapteijn, F.; Moulijn, J. A.

    2008-01-01

    The objective of this work was to develop a hydrogel-coated monolith for the entrapment of penicillin G acylase (E. coli, PGA). After screening of different hydrogels, chitosan was chosen as the carrier material for the preparation of monolithic biocatalysts. This protocol leads to active immobilized biocatalysts for the enzymatic hydrolysis of penicillin G (PenG). The monolithic biocatalyst was tested in a monolith loop reactor (MLR) and compared with conventional reactor systems using free PGA, and a commercially available immobilized PGA. The optimal immobilization protocol was found to be 5 g l−1 PGA, 1% chitosan, 1.1% glutaraldehyde and pH 7. Final PGA loading on glass plates was 29 mg ml−1 gel. For 400 cpsi monoliths, the final PGA loading on functionalized monoliths was 36 mg ml−1 gel. The observed volumetric reaction rate in the MLR was 0.79 mol s−1 m−3monolith. Apart from an initial drop in activity due to wash out of PGA at higher ionic strength, no decrease in activity was observed after five subsequent activity test runs. The storage stability of the biocatalysts is at least a month without loss of activity. Although the monolithic biocatalyst as used in the MLR is still outperformed by the current industrial catalyst (immobilized preparation of PGA, 4.5 mol s−1 m−3catalyst), the rate per gel volume is slightly higher for monolithic catalysts. Good activity and improved mechanical strength make the monolithic bioreactor an interesting alternative that deserves further investigation for this application. Although moderate internal diffusion limitations have been observed inside the gel beads and in the gel layer on the monolith channel, this is not the main reason for the large differences in reactor performance that were observed. The pH drop over the reactor as a result of the chosen method for pH control results in a decreased performance of both the MLR and the packed bed reactor compared to the batch system. A different

  10. Bioabatement with hemicellulase supplementation to reduce enzymatic hydrolysis inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Removal of inhibitory compounds by bioabatement, combined with xylan hydrolysis, enables effective cellulose hydrolysis of pretreated corn stover, for fermentation of the sugars to fuel ethanol or other products. The fungus Coniochaeta ligniaria NRRL30616 eliminates most enzyme and fermentation inhi...

  11. Comparison of enzymatic and acid hydrolysis of bound flavor compounds in model system and grapes.

    PubMed

    Dziadas, Mariusz; Jeleń, Henryk H

    2016-01-01

    Four synthesized terpenyl-ß-D-glycopyranosides (geranyl, neryl, citronellyl, myrtenyl) were subjected to enzymatic (AR 2000, pH 5.5) and acid (citric buffer, pH 2.5) hydrolysis. Decrease of glycosides was measured by HPLC and the volatiles released--by comprehensive gas chromatography-mass spectrometry (GC × GC-ToF-MS). Enzymatic hydrolysis performed for 21 h yielded 100% degree of hydrolysis for all glycosides but citronellyl (97%). Degree of acid hydrolysis was highly dependent on type of aglycone and the conditions. The highest degree was achieved for geraniol, followed by citronellol and nerol. Myrtenylo-ß-D-glycopyranoside was the most resistant glycoside to hydrolysis. Acid hydrolysis degree was also related to temperature/time combination, the highest being for 100 °C and 2 h. In a result of enzymatic hydrolysis 85-91% of total peak areas was terpene aglycone, whereas for acid hydrolysis the area of released terpene aglycone did not exceed 1.3% of total peak area indicating almost complete decomposition/transformation of terpenyl aglycone. PMID:26212990

  12. On-site enzymes produced from Trichoderma reesei RUT-C30 and Aspergillus saccharolyticus for hydrolysis of wet exploded corn stover and loblolly pine.

    PubMed

    Rana, Vandana; Eckard, Anahita D; Teller, Philip; Ahring, Birgitte K

    2014-02-01

    Cellulase production by two filamentous fungi Trichoderma reesei RUT-C30 and novel fungal strain, Aspergillus saccharolyticus on pretreated corn stover was investigated. Cellulase production was followed by the hydrolysis of two feedstocks, wet-exploded corn stover (WECS) and wet-exploded loblolly pine (WELP) by on-site produced enzyme cocktails containing cellulase from T. reesei RUT-C30 and β-glucosidase from A. saccharolyticus. The sugar yields using the on-site enzyme cocktails were compared with commercial enzymes preparations, Celluclast 1.5L and Novozym 188 at two substrate concentrations, 5% and 10% (w/w) and enzyme loading at 5 and 15 FPU/g glucan for WECS and WELP. The highest sugar yields were obtained at 5% (w/w) substrate concentration and 15 FPU/g glucan for both feedstocks. Glucose yields of 81% and 88% were obtained from on-site and commercial enzymes, respectively using WECS as feed stock. The sugar yields were 55% and 58% for WELP samples hydrolyzed with on-site and commercial enzymes, respectively. PMID:24412480

  13. Effects of chemical and enzymatic treatments on the hydrolysis of swine wastewater.

    PubMed

    Lee, Y H; Chung, Y-C; Jung, J-Y

    2008-01-01

    Slow degradation of swine wastewater mainly due to the particulate and recalcitrant organic matters is a main disadvantage of anaerobic digestion leading to very long solids retention time. Therefore, to accelerate hydrolysis known as the rate-limiting step of the overall digestion process, chemical treatment processes under various acidic and alkaline conditions as well as enzymatic treatment processes using cellulase and protease enzymes were tested for the hydrolysis of the swine wastewater. The effectiveness of various treatment processes was compared mainly by means of an increment of soluble organics in the treated swine wastewater. Among various treatment processes tested in this study, cellulase enzymatic treatment resulted in the most efficient hydrolysis of the swine wastewater. For the cellulase enzymatic hydrolysis, the observed hydrolytic constant value was 0.42 d(-1) and 26.6% of soluble organics in the swine wastewater increased within 12 hr. Compared to untreated swine wastewater, pre-treated swine wastewater by cellulase enzymatic process showed 10.7% higher anaerobic digestibility at the end of 20 d incubation and 29% higher initial methane production rate. These results further confirmed the transformation of particulate and recalcitrant organic compounds in the swine wastewater into soluble and relatively easily biodegradable organic products by the cellulase enzymatic hydrolysis. PMID:18957769

  14. Continuous enzymatic hydrolysis of lignocellulosic biomass with simultaneous detoxification and enzyme recovery.

    PubMed

    Gurram, Raghu N; Menkhaus, Todd J

    2014-07-01

    Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical conversion of lignocellulosic materials into renewable biofuels and biochemicals. Here, we show that enzymatic hydrolysis of acid-pretreated pine wood with continuous and/or fed-batch enzyme addition improved sugar conversion efficiencies by over sixfold. In addition, specific activity of the hydrolysis enzymes (cellulases, hemicellulases, etc.) increased as a result of continuously washing the residual solids with removal of glucose (avoiding the end product inhibition) and other enzymatic inhibitory compounds (e.g., furfural, hydroxymethyl furfural, organic acids, and phenolics). As part of the continuous hydrolysis, anion exchange resin was tested for its dual application of simultaneous enzyme recovery and removal of potential enzymatic and fermentation inhibitors. Amberlite IRA-96 showed favorable adsorption profiles of inhibitors, especially furfural, hydroxymethyl furfural, and acetic acid with low affinity toward sugars. Affinity of hydrolysis enzymes to adsorb onto the resin allowed for up to 92 % of the enzymatic activity to be recovered using a relatively low-molar NaCl wash solution. Integration of an ion exchange column with enzyme recovery into the proposed fed-batch hydrolysis process can improve the overall biorefinery efficiency and can greatly reduce the production costs of lignocellulosic biorenewable products. PMID:24793195

  15. Optimization of dilute acid pretreatment of water hyacinth biomass for enzymatic hydrolysis and ethanol production

    PubMed Central

    Idrees, Muhammad; Adnan, Ahmad; Sheikh, Shahzad; Qureshic, Fahim Ashraf

    2013-01-01

    The present study was conducted for the optimization of pretreatment process that was used for enzymatic hydrolysis of lignocellulosic biomass (Water Hyacinth, WH), which is a renewable resource for the production of bioethanol with decentralized availability. Response surface methodology has been employed for the optimization of temperature (oC), time (hr) and different concentrations of maleic acid (MA), sulfuric acid (SA) and phosphoric acid (PA) that seemed to be significant variables with P < 0.05. High F and R2 values and low P-value for hydrolysis yield indicated the model predictability. The pretreated biomass producing 39.96 g/l, 39.86 g/l and 37.9 g/l of reducing sugars during enzymatic hydrolysis with yield 79.93, 78.71 and 75.9 % from PA, MA and SA treated respectively. The order of catalytic effectiveness for hydrolysis yield was found to be phosphoric acid > maleic acid > sulfuric acid. Mixture of sugars was obtained during dilute acid pretreatment with glucose being the most prominent sugar while pure glucose was obtained during enzymatic hydrolysis. The resulting sugars, obtained during enzymatic hydrolysis were finally fermented to ethanol, with yield 0.484 g/g of reducing sugars which is 95 % of theoretical yield (0.51 g/g glucose) by using commercial baker's yeast (Sacchromyces cerveasiae). PMID:26417215

  16. Enhancing fermentable sugar yield from cassava pulp for bioethanol production: microwave-coupled enzymatic hydrolysis approach.

    PubMed

    Sudha, A; Sivakumar, V; Sangeetha, V; Devi, K S Priyenka

    2015-08-01

    Cassava pulp, a potential biological feedstock for ethanol production has been subjected to microwave-assisted alkali pretreatment and microwave-coupled enzymatic hydrolysis. Microwave pretreatment may be a good alternative as it can reduce the pretreatment time and improve the enzymatic activity during hydrolysis. Liquid to solid ratio for the pretreatment of cassava pulp was found to be 20:1. Cassava pulp was pretreated at various NaOH concentration, microwave temperature and gave maximum yield of reducing sugar with 1.5% NaOH at 90 °C in 30 min than conventional alkali pretreatment after enzymatic hydrolysis. The subsequent enzymatic saccharification of pretreated cassava pulp using α amylase dosage of 400 IU at microwave temperature of 90 °C resulted in highest reducing sugar yield of 723 mg/g pulp. Microwave-assisted alkali pretreatment improved the enzymatic saccharification of cassava pulp by increasing its accessibility to hydrolytic enzymes. Microwave-assisted alkali pretreatment and microwave-coupled enzymatic hydrolysis are found to be efficient for improving the yield of reducing sugar. PMID:25832789

  17. Study of enzymatic hydrolysis of fructans from Agave salmiana characterization and kinetic assessment.

    PubMed

    Michel-Cuello, Christian; Ortiz-Cerda, Imelda; Moreno-Vilet, Lorena; Grajales-Lagunes, Alicia; Moscosa-Santillán, Mario; Bonnin, Johanne; González-Chávez, Marco Martín; Ruiz-Cabrera, Miguel

    2012-01-01

    Fructans were extracted from Agave salmiana juice, characterized and subjected to hydrolysis process using a commercial inulinase preparation acting freely. To compare the performance of the enzymatic preparation, a batch of experiments were also conducted with chicory inulin (reference). Hydrolysis was performed for 6 h at two temperatures (50, 60 °C) and two substrate concentrations (40, 60 mg/ml). Hydrolysis process was monitored by measuring the sugars released and residual substrate by HPLC. A mathematical model which describes the kinetics of substrate degradation as well as fructose production was proposed to analyze the hydrolysis assessment. It was found that kinetics were significantly influenced by temperature, substrate concentration, and type of substrate (P < 0.01). The extent of substrate hydrolysis varied from 82 to 99%. Hydrolysis product was mainly constituted of fructose, obtaining from 77 to 96.4% of total reducing sugars. PMID:22629216

  18. Study of Enzymatic Hydrolysis of Fructans from Agave salmiana Characterization and Kinetic Assessment

    PubMed Central

    Michel-Cuello, Christian; Ortiz-Cerda, Imelda; Moreno-Vilet, Lorena; Grajales-Lagunes, Alicia; Moscosa-Santillán, Mario; Bonnin, Johanne; González-Chávez, Marco Martín; Ruiz-Cabrera, Miguel

    2012-01-01

    Fructans were extracted from Agave salmiana juice, characterized and subjected to hydrolysis process using a commercial inulinase preparation acting freely. To compare the performance of the enzymatic preparation, a batch of experiments were also conducted with chicory inulin (reference). Hydrolysis was performed for 6 h at two temperatures (50, 60°C) and two substrate concentrations (40, 60 mg/ml). Hydrolysis process was monitored by measuring the sugars released and residual substrate by HPLC. A mathematical model which describes the kinetics of substrate degradation as well as fructose production was proposed to analyze the hydrolysis assessment. It was found that kinetics were significantly influenced by temperature, substrate concentration, and type of substrate (P < 0.01). The extent of substrate hydrolysis varied from 82 to 99%. Hydrolysis product was mainly constituted of fructose, obtaining from 77 to 96.4% of total reducing sugars. PMID:22629216

  19. Sodium sulfite-formaldehyde pretreatment of mixed hardwoods and its effect on enzymatic hydrolysis.

    PubMed

    Jin, Yongcan; Yang, Linfeng; Jameel, Hasan; Chang, Hou-min; Phillips, Richard

    2013-05-01

    In this work, mixed hardwoods were pretreated by sodium sulfite-formaldehyde (SF). The effects of SF pretreatment on the chemical compositions and enzymatic hydrolysis of mixed hardwoods were investigated. SF pretreatment temperature had a significant effect on pulp yield and delignification, resulting in an increased efficiency of enzymatic hydrolysis. After 96 h of enzymatic hydrolysis at the cellulase loading of 40 FPU/g substrate, the yields of glucan and xylan on the basis of original wood were 37% and 11% for the pulp produced with 12% sulfite charge at 170 °C for 2 h. The total sugar recovery based on the sugar in original wood was 74%. These results indicate that sulfite-formaldehyde cooking is of great potential to be a pretreatment method for a greenfield mill to produce fuel ethanol from hardwood. PMID:23127844

  20. Comparison of Enzymatic Hydrolysis and Acid Hydrolysis of Sterol Glycosides from Foods Rich in Δ(7)-Sterols.

    PubMed

    Münger, Linda H; Jutzi, Sabrina; Lampi, Anna-Maija; Nyström, Laura

    2015-08-01

    In this study, we present the difference in sterol composition of extracted steryl glycosides (SG) hydrolyzed by either enzymatic or acid hydrolysis. SG were analyzed from foods belonging to the plant families Cucurbitaceae (melon and pumpkin seeds) and Amaranthaceae (amaranth and beetroot), both of which are dominated by Δ(7)-sterols. Released sterols were quantified by gas chromatography with a flame ionization detector (GC-FID) and identified using gas chromatography/mass spectrometry (GC-MS). All Δ(7)-sterols identified (Δ(7)-stigmastenyl, spinasteryl, Δ(7)-campesteryl, Δ(7)-avenasteryl, poriferasta-7,25-dienyl and poriferasta-7,22,25-trienyl glucoside) underwent isomerization under acidic conditions and high temperature. Sterols with an ethylidene or methylidene side chain were found to form multiple artifacts. The artifact sterols coeluted with residues of incompletely isomerized Δ(7)-sterols, or Δ(5)-sterols if present, and could be identified as Δ(8(14))-sterols on the basis of relative retention time, and their MS spectra as trimethylsilyl (TMS) and acetate derivatives. For instance, SG from melon were composed of 66% Δ(7)-stigmastenol when enzymatic hydrolysis was performed, whereas with acid hydrolysis only 8% of Δ(7)-stigmastenol was determined. The artifact of Δ(7)-stigmastenol coeluted with residual non-isomerized spinasterol, demonstrating the high risk of misinterpretation of compositional data obtained after acid hydrolysis. Therefore, the accurate composition of SG from foods containing sterols with a double bond at C-7 can only be obtained by enzymatic hydrolysis or by direct analysis of the intact SG. PMID:25757602

  1. Dynamic modeling and validation of a lignocellulosic enzymatic hydrolysis process--a demonstration scale study.

    PubMed

    Prunescu, Remus Mihail; Sin, Gürkan

    2013-12-01

    The enzymatic hydrolysis process is one of the key steps in second generation biofuel production. After being thermally pretreated, the lignocellulosic material is liquefied by enzymes prior to fermentation. The scope of this paper is to evaluate a dynamic model of the hydrolysis process on a demonstration scale reactor. The following novel features are included: the application of the Convection-Diffusion-Reaction equation to a hydrolysis reactor to assess transport and mixing effects; the extension of a competitive kinetic model with enzymatic pH dependency and hemicellulose hydrolysis; a comprehensive pH model; and viscosity estimations during the course of reaction. The model is evaluated against real data extracted from a demonstration scale biorefinery throughout several days of operation. All measurements are within predictions uncertainty and, therefore, the model constitutes a valuable tool to support process optimization, performance monitoring, diagnosis and process control at full-scale studies. PMID:24212094

  2. Enzymatic hydrolysis and fermentation of dilute acid pretreated cornstalk to biohydrogen

    NASA Astrophysics Data System (ADS)

    Pan, C. M.; Fan, Y. T.; Hou, H. W.

    2010-03-01

    The coupling method of acid pretreatment and enzymatic hydrolysis of cornstalk for hydrogen production was investigated in this study. Experimental results showed that temperature, pH and enzyme loading all had an individual significant influence on soluble sugar yield and Ps. The optimum condition for soluble sugar was close to that for Ps. The maximum hydrogen yield from cornstalk by anaerobic mixed microflora was 209.8 ml/g-TVS on the optimum enzymatic hydrolysis condition which was 52 °C of temperature, pH4.8 and 9.4 IU/g of enzyme loading.

  3. Contrasting effects of hardwood and softwood organosolv lignins on enzymatic hydrolysis of lignocellulose.

    PubMed

    Lai, Chenhuan; Tu, Maobing; Shi, Zhiqiang; Zheng, Ke; Olmos, Luis G; Yu, Shiyuan

    2014-07-01

    Identifying an appropriate parameter to elucidate effects of lignin on enzymatic hydrolysis is essential to understand the interactions between enzymes and lignin. Contrasting effects of hardwood organosolv lignin (EOL-SG) and softwood organosolv lignin (EOL-LP) on enzymatic hydrolysis were observed. The addition of EOL-SG (8 g/L) significantly improved the 72 h hydrolysis yields of organosolv pretreated sweetgum (OPSG) and loblolly pine (OPLP) from 49.3% to 68.6% and from 41.2% to 60.8%, respectively. In contrast, the addition of EOL-LP decreased the 72 h hydrolysis yields of OPSG and OPLP to 42.0% and 38.1%, respectively. A strong correlation between the distribution coefficients of cellulase enzymes on lignins and the changes of hydrolysis yields indicated that the inhibitory or stimulatory effects of organosolv lignins on enzymatic hydrolysis were governed by the distribution coefficients (R). The different R values probably were related to the electrostatic interactions, hydrophobic interactions and hydrogen bondings between enzymes and lignin. PMID:24835744

  4. Bioabatement with xylanase supplementation to reduce enzymatic hydrolysis inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bioabatement, using the fungus Coniochaeta ligniaria NRRL30616 can effectively eliminate enzyme inhibitors from pretreated biomass hydrolysis. However, our recent research suggested that bioabatement had no beneficial effect on removing xylo-oligomers which were identified as strong inhibitors to ce...

  5. Simultaneous pretreatment and enzymatic hydrolysis of forage biomass

    SciTech Connect

    Henk, L.; Linden, J.C.

    1993-12-31

    Sweet sorghum is an attractive fermentation feedstock because as much as 40% of the dry weight consists of readily femented sugars such as sucrose, glucose and frutose. Cellulose and hemicellulose comprise another 50%. However, if this material is to be used a year-round feedstock for ethanol production, a stable method of storage must be developed to maintain the sugar content. A modified version of the traditional ensiling process is made effective by the addition of cellulolytic/hemicellulolytic enzymes and lactic acid bacteria to freshly chopped sweet sorghum prior to the production of silage. In situ hydrolysis of cellulose and hemicellulose occurs concurrently with the acidic ensiling fementation. By hydolyzing the acetyl groups using acetyl xylan esterase and 3-0-methyl glucuronyl side chains using pectinase from hemicellulose, cellulose becomes accessible to hydrolysis by cellulase, both during in situ ensiling with enzymes and in the simultaneous saccharification and fermentation (SSF) to ethanol.

  6. Novel agents for enzymatic and fungal hydrolysis of stevioside

    PubMed Central

    Milagre, H.M.S.; Martins, L.R.; Takahashi, J.A.

    2009-01-01

    A comparative study on the potential of some biological agents to perform the hydrolysis of stevioside was carried out, aiming at establishing an alternative methodology to achieve the aglycon steviol or its rearranged derivative isosteviol, in high yields to be used in the preparation of novel bioactive compounds. Hydrolysis reactions were performed by using filamentous fungi (Aspergillus niger, Rhizopus stolonifer and Rhizopus arrhizus), a yeast (Saccharomyces cerevisiae) and enzymes (pancreatin and lipases PL250 and VFL 8000). Pancreatin showed the best hydrolytic activity, furnishing isosteviol at 93.9% of yield, at pH 4.0, using toluene as a co-solvent. Steviol was produced using both pancreatin at pH 7.0 (20.2% yield) and A. niger at pH 7 (20.8% yield). PMID:24031374

  7. Allergenicity of Peanut Proteins is Retained Following Enzymatic Hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rationale: Hydrolysis of peanut proteins by food-grade enzymes may reduce allergenicity and could lead to safer forms of immunotherapy. Methods: Light roasted peanut flour extracts were digested with pepsin (37°C, pH 2), Alcalase (60°C pH 8), or Flavourzyme (50°C, pH 7) up to 1 hr, or sequentially w...

  8. The enzymatic hydrolysis of leather waste with chromium recycling

    SciTech Connect

    Kim, M.S.; Clesceri, L.S.

    1996-11-01

    The work of Taylor et al. (1990) has shown the potential for alkaline hydrolase enzymes for the solubilization of waste from the tanning industry. The authors have carried this work further to examine the mechanism whereby enzymes release chromium from leather waste. An alkaline digest of waste leather was used in this work. Treatment with strong alkali produced a thick slurry that contained 7,000 ppm chromium. The objective of this work is to optimize a closed cycle system for the recycling of chromium salts for tanning as well as a chrome-free product for use as a fertilizer. The authors are able to track the progress of the leather protein hydrolysis with polyacrylamide gel electrophoresis (PAGE). By means of PAGE, it is possible to determine the relationship between chromium release and the extent of protein hydrolysis. Rate constant for hydrolysis and chromium release have been developed for various hydrolysis conditions. Chemical precipitation of chromium from the hydrolysate results in a purified product for reuse in tanning. The chrome-free hydrolysate can be applied as a fertilizer either directly or as a dried product. There are more than 56,000 metric tons of tannery waste produced annually in the US. The majority of the organic solids can be converted into high quality fertilizers. Since the nitrogen is organic rather than inorganic, release is at a controlled rate since the microbody in the soil must make the nitrogen available for plant growth. Leather manufacturing is a world-wide industry. Conversion of leather waste to fertilizers can improve global productivity as well as solve a waste problem.

  9. Effects of enzymatic hydrolysis of protein on the pasting properties of different types of wheat flour.

    PubMed

    Chen, J S; Wang, S Y; Deng, Z Y; Zhang, X Y; Feng, S L; Yuan, H Q; Tian, J C

    2012-05-01

    As one of the most effective methods to modify proteins, enzymatic hydrolysis is used widely in the preparation of wheat products in the food industry. During the same process, starch pasting occurs frequently. The effects of wheat protein hydrolysis with papain, pepsin, and trypsin on the pasting properties of 3 different kinds of flour were investigated in 5 concentrations. Results showed that the peak viscosity, trough, final, and integral area of pasting curve of these flours decreased with increasing enzymatic hydrolysis of protein, and decreased significantly with the increasing enzyme concentrations. Medium-gluten flour was the least sensitive to enzymatic activity and weak-gluten the most sensitive. Downtrends appeared with increasing papain and trypsin concentrations in the form of breakdown. Enzymes had no significant different effect on the peak times of strong- and medium-gluten flour, but prolonged peak time slightly in weak-gluten flour. The pasting time and temperature of strong- and medium-gluten flour were significantly increased in a concentration-dependent manner. However, there were no significant effects on the pasting times of weak-gluten flour. These results could supply a basis for utilization of enzymatic hydrolysis of wheat protein in food industry and for further studies into the interactions between hydrolyzed protein and starch in food or processing industries. PMID:22510095

  10. Enzymatic Hydrolysis of Peanut Flour Produces Bioactive Peptides with Reduced Allergenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peanut allergy is one of the most severe food allergies due to its life-threatening nature and persistency. Current immunotherapy methods, though effective, are often accompanied by allergic side-effects. Enzymatic hydrolysis of peanut flour has the potential to produce bioactive peptides with impro...

  11. Biological pretreatment of corn stover with white-rot fungus for improved enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biological pretreatment of lignocellulosic biomass by white-rot fungus can represent a low-cost and eco-friendly alternative to harsh physical, chemical or physico-chemical pretreatment methods to facilitate enzymatic hydrolysis. However, fungal pretreatment can cause carbohydrate loss and it is, th...

  12. Acceleration of the Enzymatic Hydrolysis of Cotton Waste Celluloses by Low Intensity Uniform Ultrasound Field

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cost-competitive production of bio-ethanol and other biofuels is currently impeded, mostly by high cost and low efficiency of enzymatic hydrolysis of feedstock biomass and especially plant celluloses. Despite substantial reduction in the cost of production of cellulolytic enzymes in recent times...

  13. Enhancement of enzymatic hydrolysis of sugar cane bagasse by steam explosion pretreatment

    SciTech Connect

    Kling, S.H.; Neto, C.C.; Ferrara, M.A.; Torres, J.C.R.; Magalhaes, D.B.; Ryu, D.D.Y.

    1987-01-01

    In this study, the possibility of applying a steam explosion pretreatment process to sugarcane bagasse was investigated, and the effectiveness of the pretreatment in terms of hemicellulose solubilization and enhancement of enzymatic hydrolysis was determined. The steam requirement for the pretreatment was also investigated at the pilot-plant scale, and these results are presented.

  14. Sugar loss and enzyme inhibition due to oligosaccharides accumulation during high solids-loading enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, ammonia fiber expansion: AFEX and extractive ammonia: EA). The methodology for large-scale separation of ...

  15. Analysis of Phosphorus in Soil Humic Acid Fractions by Enzymatic Hydrolysis and Ultraviolet Irradiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Humic acid is an important soil component which influences chemical, biological, and physical soil properties. In this study, we investigated lability of phosphorus (P) in the mobile humic acid (MHA) and calcium humate (CaHA) fractions of four soils by orthophosphate-releasing enzymatic hydrolysis a...

  16. Understanding the fundamental mechanism behind accumulation of oligosaccharides during high solids loading enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During enzymatic hydrolysis of biomass, polysaccharides are cleaved by glycosyl hydrolases to soluble oligosaccharides and further hydrolyzed by ß-glucosidase, ß-xylosidase and other enzymes to monomeric sugars. However, commercial enzyme mixtures do not hydrolyze all of these oligosaccharides and v...

  17. Inhibition of cellulose enzymatic hydrolysis by laccase-derived compounds from phenols.

    PubMed

    Oliva-Taravilla, Alfredo; Tomás-Pejó, Elia; Demuez, Marie; González-Fernández, Cristina; Ballesteros, Mercedes

    2015-01-01

    The presence of inhibitors compounds after pretreatment of lignocellulosic materials affects the saccharification and fermentation steps in bioethanol production processes. Even though, external addition of laccases selectively removes the phenolic compounds from lignocellulosic prehydrolysates, when it is coupled to saccharification step, lower hydrolysis yields are attained. Vanillin, syringaldehyde and ferulic acid are phenolic compounds commonly found in wheat-straw prehydrolysate after steam-explosion pretreatment. These three phenolic compounds were used in this study to elucidate the inhibitory mechanisms of laccase-derived compounds after laccase treatment. Reaction products derived from laccase oxidation of vanillin and syringaldehyde showed to be the strongest inhibitors. The presence of these products causes a decrement on enzymatic hydrolysis yield of a model cellulosic substrate (Sigmacell) of 46.6 and 32.6%, respectively at 24 h. Moreover, a decrease in more than 50% of cellulase and β-glucosidase activities was observed in presence of laccase and vanillin. This effect was attributed to coupling reactions between phenoxyl radicals and enzymes. On the other hand, when the hydrolysis of Sigmacell was performed in presence of prehydrolysate from steam-exploded wheat straw a significant inhibition on enzymatic hydrolysis was observed independently of laccase treatment. This result pointed out that the other components of wheat-straw prehydrolysate are affecting the enzymatic hydrolysis to a higher extent than the possible laccase-derived products. PMID:25740593

  18. Modeling enzymatic hydrolysis of lignocellulosic substrates using confocal fluorescence microscopy I: filter paper cellulose.

    PubMed

    Luterbacher, Jeremy S; Moran-Mirabal, Jose M; Burkholder, Eric W; Walker, Larry P

    2015-01-01

    Enzymatic hydrolysis is one of the critical steps in depolymerizing lignocellulosic biomass into fermentable sugars for further upgrading into fuels and/or chemicals. However, many studies still rely on empirical trends to optimize enzymatic reactions. An improved understanding of enzymatic hydrolysis could allow research efforts to follow a rational design guided by an appropriate theoretical framework. In this study, we present a method to image cellulosic substrates with complex three-dimensional structure, such as filter paper, undergoing hydrolysis under conditions relevant to industrial saccharification processes (i.e., temperature of 50°C, using commercial cellulolytic cocktails). Fluorescence intensities resulting from confocal images were used to estimate parameters for a diffusion and reaction model. Furthermore, the observation of a relatively constant bound enzyme fluorescence signal throughout hydrolysis supported our modeling assumption regarding the structure of biomass during hydrolysis. The observed behavior suggests that pore evolution can be modeled as widening of infinitely long slits. The resulting model accurately predicts the concentrations of soluble carbohydrates obtained from independent saccharification experiments conducted in bulk, demonstrating its relevance to biomass conversion work. PMID:25042118

  19. Enzymatic hydrolysis of fractionated products from oils thermally oxidized in the laboratory.

    PubMed

    Yoshida, H; Alexander, J C

    1983-06-01

    Enzymatic hydrolysis of the acylglycerol products obtained from thermally oxidized vegetable oils was studied. Corn, sunflower and soybean oils were heated in the laboratory at 180 C for 50, 70 and 100 hr with aeration and directly fractionated by silicic acid column chromatography. By successive elution with 20%, then 60% isopropyl ether in n-hexane, and diethyl ether, the thermally oxidized oils were separated into three fractions: the nonpolar fraction (monomeric compounds), slightly polar fraction (dimeric compounds), and polar fraction comprising oligomeric compounds. Enzymatic hydrolysis with pancreatic lipase showed that the monomers were hydrolyzed as rapidly as the corresponding unheated oils, the dimers much more slowly, and the oligomeric compounds barely at all. Overall, the hydrolysis of the dimers was less than 23% of that for the monomers, with small differences among the oils. Longer heating periods resulted in greater reductions in hydrolysis of the dimeric compounds. These results suggest that the degree of enzymatic hydrolysis of the fractionated acylglycerol compounds is related to differences in the thermal oxidative deterioration, and amounts of polar compounds in the products. PMID:6877045

  20. Production of reducing sugar from Enteromorpha intestinalis by hydrothermal and enzymatic hydrolysis.

    PubMed

    Kim, Dong-Hyun; Lee, Sang-Bum; Jeong, Gwi-Taek

    2014-06-01

    In this work, to evaluate the efficacy of marine macro-algae Enteromorpha intestinalis as a potential bioenergy resource, the effects of reaction conditions (solid-to-liquid ratio, reaction temperature, and reaction time) on sugars produced by a combined process of hydrothermal and enzymatic hydrolysis were investigated. As a result of the hydrothermal hydrolysis, a 7.3g/L (8% yield) total reducing sugar was obtained under conditions including solid-to-liquid ratio of 1:10, reaction temperature of 170°C, and reaction time of 60min. By subsequent (post-hydrothermal) enzymatic hydrolysis of samples treated at 170°C for 30min, a 20.1g/L (22% yield) was achieved. PMID:24727694

  1. High-yield production of biosugars from Gracilaria verrucosa by acid and enzymatic hydrolysis processes.

    PubMed

    Kim, Se Won; Hong, Chae-Hwan; Jeon, Sung-Wan; Shin, Hyun-Jae

    2015-11-01

    Gracilaria verrucosa, the red alga, is a suitable feedstock for biosugar production. This study analyzes biosugar production by the hydrolysis of G. verrucosa conducted under various conditions (i.e., various acid concentrations, substrate concentrations, reaction times, and enzyme dosages). The acid hydrolysates of G. verrucosa yielded a total of 7.47g/L (37.4%) and 10.63g/L (21.26%) of reducing sugars under optimal small (30mL) and large laboratory-scale (1L) hydrolysis processes, respectively. Reducing sugar obtained from acid and enzymatic hydrolysates were 10% higher, with minimum by-products, than those reported in other studies. The mass balance for the small laboratory-scale process showed that the acid and enzymatic hydrolysates had a carbohydrate conversion of 57.2%. The mass balance approach to the entire hydrolysis process of red seaweed for biosugar production can be applied to other saccharification processes. PMID:26299978

  2. Enzymatic hydrolysis of poultry meal with endo- and exopeptidases.

    PubMed

    Nchienzia, H A; Morawicki, R O; Gadang, V P

    2010-10-01

    The objective of this research was to determine whether commercial proteases are capable of hydrolyzing denatured poultry by-product proteins that have gone through a rendering process. The material used for this research was low-ash poultry meal obtained from a local poultry processor. Samples of poultry meal were treated with the commercial proteases Alcalase, Flavourzyme, Protamex, and Liquipanol that were added individually or as a combination of 2 proteases, which were incorporated either simultaneously or sequentially. Temperature was controlled during the reaction to the optimal level for each enzyme, and pH was initially adjusted to the most favorable level for each enzyme and was maintained during the course of the reaction by addition of NaOH at established intervals. Consumption of NaOH was used to calculate the degree of hydrolysis. At the end of the hydrolysis, the molecular weight of selected hydrolysates was determined by size exclusion chromatography and by gel electrophoresis. In addition, amino acid analysis was performed on selected hydrolysates. Results show that the sequential treatment with Alcalase and Flavourzyme served best for the preparation of poultry meal hydrolysates with a maximum degree of hydrolysis of 11.1% and the highest hydrolyzable material recovered at 58%, which is attributed to the combined nature of the endo- and exocatalytic action of Alcalase and Flavourzyme, respectively. Hydrolysate with Flavourzyme or the combination of Flavourzyme and Alcalase were both good methods to produce significant amounts of free amino acids. This research shows the feasibility of hydrolyzing poultry by-products that went through a rendering process using different brands of commercial proteases. Findings from this research are important in the preparation of palatants, in which relatively inexpensive hydrolyzed poultry meal could be used to improve the flavor of companion animal food products. PMID:20852119

  3. Alkal treatment of corn stover to improve sugar production by enzymatic hydrolysis

    SciTech Connect

    MacDonald, D.G.; Bakhshi, N.N.; Mathews, J.F.; Roychowdhury, A.; Bajpai, P.; Moo-Young, M.

    1983-08-01

    Alkali treatment of corn stover improves the availability of cellulose and hemicellulose for enzymatic attack. Treatments were carried out for 1 to 60 min at temperatures and NaOH concentrations ranging from 100 to 150 degrees C and 0 to 2%, respectively. Solubilization of the stover and sugar production by enzymatic hydrolysis (Trichoderma viride cellulase) of the solid residue and the dissolved solids were used to measure the effect of caustic treatment. At 150 degrees C and 2% NaOH concentration, 65% of the original stover was dissolved after 5 min and 52% saccharification (g sugar/g stover) of the residue and dissolved solids by enzymatic hydrolysis was achieved compared to 20% for untreated corn stover. (6 Refs.)

  4. Effects of cutting orientation in poplar wood biomass size reduction on enzymatic hydrolysis sugar yield.

    PubMed

    Zhang, Meng; Ju, Xiaohui; Song, Xiaoxu; Zhang, Xiao; Pei, Z J; Wang, Donghai

    2015-10-01

    The aim of this study was to understand how cutting orientation in poplar wood biomass size reduction affects enzymatic hydrolysis sugar yield of wood particles. A metal cutting (milling) machine was used to produce poplar wood particles from three cutting orientations. Results showed that cutting orientation significantly affected enzymatic hydrolysis sugar yield of wood particles. In this study, size reduction from the optimum cutting orientation produced 50% more sugars than the other two cutting orientations. Particles from the cutting orientation with the highest sugar yield had a large enzyme accessible area (125 mg orange dye/g biomass, as evaluated by Simons' stain procedure) and low crystallinity (50% crystallinity index, as calculated by the Segal method). Furthermore, small particle size did not necessarily lead to improvement in enzymatic digestibility. PMID:26220047

  5. Enzymatic hydrolysis of cellulose pretreated with ionic liquids and N-methyl Morpholine N-Oxide

    NASA Astrophysics Data System (ADS)

    Yau Li, Elizabeth

    The effect of N-methyl Morpholine N-Oxide (NMMO), 1-ethyl-3-methyl-imidazolium acetate ([Emim]Ac) and 1-ethyl-3-methyl-imidazolium diethyl phosphate ([Emim]DEP) on pretreatment and enzymatic hydrolysis of dissolving pulp was studied. X-ray diffraction measurements of regenerated cellulose from these solvents showed that solvent pretreatment reduces the crystallinity of cellulose. However, crystallinity might not be a major factor affecting the in-situ enzymatic hydrolysis of cellulose in these solvents. Although regenerated cellulose from [Emim]DEP showed the lowest crystallinity index (˜15%), in-situ enzymatic hydrolysis of cellulose dissolved in NMMO showed the highest cellulose conversion (68% compared to 65% for [Emim]Ac and 37% for [Emim]DEP at enzyme loading of 122 FPU/g). Moreover, results showed that enzymes could tolerate up to NMMO concentration of 100 g/L and still yield full conversion of cellulose. Since it is not necessary to remove all the NMMO, less amount of water will be required for the washing step and thus the process will be more economical. The HCH-1 model was used in an attempt to model the enzymatic hydrolysis of cellulose in NMMO. With the incorporation of NMMO inhibition and a factor to account for unreacted cellulose, the model was able to correlate the experimental data of the enzymatic hydrolysis of cellulose (6.68 g/L) at various NMMO concentrations (0, 50, 100, 150 and 250 g/L). However, the experimental results also suggest that NMMO might be deactivating the enzymes rather than inhibiting them. More studies need to be done at varying cellulose, NMMO and enzyme concentrations to find the exact nature of this deactivation of NMMO.

  6. Physicochemical structural changes of poplar and switchgrass during biomass pretreatment and enzymatic hydrolysis

    DOE PAGESBeta

    Meng, Xianzhi; Sun, Qining; Kosa, Matyas; Huang, Fang; Pu, Yunqiao; Ragauskas, Arthur J.

    2016-07-27

    Converting lignocellulosics to simple sugars for second generation bioethanol is complicated due to biomass recalcitrance, and it requires a pretreatment stage prior to enzymatic hydrolysis. In this study, native, pretreated (acid and alkaline) and partially hydrolyzed poplar and switchgrass were characterized by using Simons’ staining for cellulose accessibility, GPC for degree of polymerization (DP), and FTIR for chemical structure of plant cell wall. The susceptibility of the pretreated biomass to enzymatic hydrolysis could not be easily predicted from differences in cellulose DP and accessibility. During hydrolysis, the most significant DP reduction occurred at the very beginning of hydrolysis, and themore » DP began to decrease at a significantly slower rate after this initial period, suggesting an existence of a synergistic action of endo- and exoglucanases that contribute to the occurrence of a “peeling off” mechanism. Cellulose accessibility was found to be increased at the beginning of hydrolysis, after reaching a maximum value then started to decrease. In conclusion, the fresh enzyme restart hydrolysis experiment along with the accessibility data indicated that the factors associated with the nature of enzyme such as irreversible nonspecific binding of cellulases by lignin and steric hindrance of enzymes should be responsible for the gradual slowing down of the reaction rate.« less

  7. Mechanistic modeling of enzymatic hydrolysis of cellulose integrating substrate morphology and cocktail composition.

    PubMed

    Huron, Maïté; Hudebine, Damien; Lopes Ferreira, Nicolas; Lachenal, Dominique

    2016-05-01

    A mechanistic model of enzymatic hydrolysis taking into account the morphology of the cellulosic particles and its evolution with time was developed. The individual behavior of the main enzymes involved in the reaction (cellobiohydrolases, endoglucanases, and β-glucosidases), as well as synergy effects, were also included. A large panel of experimental tests was done to fit and validate the model. This database included different enzymes mixtures and operating conditions and allowed to determine and compare with accuracy the adsorption and kinetic parameters of the different enzymes. Model predictions on short hydrolysis times were very satisfactory. On longer times, a deactivation constant was added to represent the hydrolysis slowdown. The model also allowed to predict the impact of enzymes ratios and initial substrate parameters (chain length distribution, polymerization degree) on hydrolysis, and to follow the evolution of these parameters with time. This model revealed general trends on the impact of cellulose morphology on hydrolysis. It is a useful tool to better understand the mechanisms involved in enzymatic hydrolysis of cellulose and to determine optimal cellulolytic cocktails for process design. Biotechnol. Bioeng. 2016;113: 1011-1023. © 2015 Wiley Periodicals, Inc. PMID:26524470

  8. Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities.

    PubMed

    Yuan, Jiao-Jiao; Wang, Cheng-Zhang; Ye, Jian-Zhong; Tao, Ran; Zhang, Yu-Si

    2015-01-01

    Oleuropein (OE), the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT) and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity) optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE) were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL) was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods. PMID:25679050

  9. Acid and enzymatic hydrolysis of pretreated cellulosic materials as an analytical tool

    SciTech Connect

    Ladisch, C.M.; Chiasson, C.M.; Tsao, G.T.

    1982-07-01

    A rapid and accurate procedure for the quantitative analysis of cellulose in textiles based on acid and enzymatic hydrolysis was investigated. Total hydrolysis was achieved by a two-step procedure: the cellulose in the sample was first dissolved in cadoxen and then reprecipitated. The material, thus pretreated, was then hydrolyzed with acid or enzyme catalytic agents. Hydrolysis products were detected and quantified by colorimetric, enzymic, and liquid chromatographic methods of analysis. Samples examined included cotton, rayon, Avicel, CF-11, and cotton/polyester blends. The specificity of the enzyme hydrolysis method allowed analysis of raw cotton without prior purification. Results of the analyses were compared to those obtained by existing methods of analysis.

  10. Enzymatic hydrolysis of microcrystalline cellulose in concentrated seawater.

    PubMed

    Grande, Philipp M; de María, Pablo Domínguez

    2012-01-01

    This communication explores the use of seawater (1X) and concentrated seawater (2X and 4X) as reaction media for the enzyme-catalyzed depolymerization of cellulose. The commercially available Accellerase-1500® - a "cocktail" of different glycosidases - is able to depolymerize several amorphous celluloses and microcrystalline cellulose Avicel® in these reaction media, at slightly lower rates (ca. 90%) than those observed when reactions are performed in pure citrate buffer (control reactions). Remarkably, at concentrated seawater effluents enzymes also display significant rates of cellulose hydrolysis. Considering the expected increasing shortages in accessibility to fresh drinkable water, the herein-reported concept may provide novel inspiring leads for a smart use of resources in an environmentally-friendly and efficient manner, and for the genetic development of cellulases highly active and stable in concentrated seawater solutions. PMID:22101072

  11. Controlled Enzymatic Hydrolysis: A New Strategy for the Discovery of Antimicrobial Peptides.

    PubMed

    Adje, Estelle Yaba; Balti, Rafik; Lecouturier, Didier; Kouach, Mostafa; Dhulster, Pascal; Guillochon, Didier; Nedjar-Arroume, Naïma

    2013-09-01

    The use of antimicrobial peptides (AMPs) is an alternative to traditional antibiotics. AMPs are obtained using different methods such as bacterial synthesis, chemical synthesis and controlled enzymatic hydrolysis. The later is an interesting approach that deserves our attention because of the yields gathered and peptides engineered. Usually, activities of AMPs obtained in such a way are tightly dependent on the hydrolysis mechanism used. This paper deals with the hydrolysis of hemoglobin mechanism as a potential source of AMPs. Production of AMPs from hemoglobin using enzymatic controlled system is linked to hemoglobin structure. Further, we show that bovine hemoglobin, which is sensitive to peptic hydrolysis, results upon enzymatic digestion as a great source of AMPs. The hemoglobin in native and denatured states was hydrolyzed by "one-by-one" and "zipper" mechanisms, respectively. Nevertheless, a new mechanism named "semi-zipper" mechanism is obtained when protein is in molten globule structural state, constituting an original strategy for AMPs production. Seventy seven percentage of the peptides obtained by this new strategy showed antibacterial activity against nine strains. PMID:26782986

  12. Liquid hot water pretreatment of multi feedstocks and enzymatic hydrolysis of solids obtained thereof.

    PubMed

    Michelin, Michele; Teixeira, José António

    2016-09-01

    Agricultural feedstocks (brewers' spent grain - BSG, corncob - CC, corn husk - CH, wheat straw - WS and Luffa sponge - LS) were pretreated by liquid hot water (LHW) in order to increase cellulose recovery and enzymatic saccharification. LHW-pretreatment resulted in hemicellulose solubilization, and solids enriched in cellulose. Chemical analysis showed different susceptibilities of the feedstocks to LHW-pretreatment and enzymatic hydrolysis. Pretreated feedstocks presented higher crystallinity (determined through X-ray diffraction) and thermal stability (determined through thermogravimetric analysis) than untreated feedstocks. SEM images confirmed the effect of LHW-pretreatment on structural changes. Moreover, enzymatic hydrolysis and cellulose conversion to glucose (CCG) were improved for pretreated feedstocks, with exception of LS. CCG (in relation to glucose potential on solids) followed the order: BSG>CH>WS>CC>LS. LHW-pretreatment showed to be a good technology to pretreat multi feedstocks and for improving the enzymatic hydrolysis of recalcitrant agricultural feedstocks to sugars, which can be further converted to ethanol-fuel and other value-added chemicals. PMID:27318165

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

    PubMed

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

    2010-07-01

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

  14. Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

    SciTech Connect

    Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.; Park, Sunkyu; Kim, Seong H.

    2015-10-14

    A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlate with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. Moreover, it was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component.

  15. Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

    DOE PAGESBeta

    Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.; Park, Sunkyu; Kim, Seong H.

    2015-10-14

    A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlatemore » with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. Moreover, it was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component.« less

  16. Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

    NASA Astrophysics Data System (ADS)

    Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.; Park, Sunkyu; Kim, Seong H.

    2015-10-01

    A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlate with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. It was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component.

  17. Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

    PubMed Central

    Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.; Park, Sunkyu; Kim, Seong H.

    2015-01-01

    A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlate with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. It was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component. PMID:26463274

  18. Improved enzymatic hydrolysis of wheat straw by combined use of gamma ray and dilute acid for bioethanol production

    NASA Astrophysics Data System (ADS)

    Hyun Hong, Sung; Taek Lee, Jae; Lee, Sungbeom; Gon Wi, Seung; Ju Cho, Eun; Singh, Sudhir; Sik Lee, Seung; Yeoup Chung, Byung

    2014-01-01

    Pretreating wheat straw with a combination of dilute acid and gamma irradiation was performed in an attempt to enhance the enzymatic hydrolysis for bioethanol production. The glucose yield was significantly affected by combined pretreatment (3% sulfuric acid-gamma irradiation), compared with untreated wheat straw and individual pretreatment. The increasing enzymatic hydrolysis after combined pretreatment is resulting from decrease in crystallinity of cellulose, loss of hemicelluloses, and removal or modification of lignin. Therefore, combined pretreatment is one of the most effective methods for enhancing the enzymatic hydrolysis of wheat straw biomass.

  19. Phosphoric acid pretreatment of Achyranthes aspera and Sida acuta weed biomass to improve enzymatic hydrolysis.

    PubMed

    Siripong, Premjet; Duangporn, Premjet; Takata, Eri; Tsutsumi, Yuji

    2016-03-01

    Achyranthes aspera and Sida acuta, two types of weed biomass are abundant and waste in Thailand. We focus on them as novel feedstock for bio-ethanol production because they contain high-cellulose content (45.9% and 46.9%, respectively) and unutilized material. Phosphoric acid (70%, 75%, and 80%) was employed for the pretreatment to improve by enzymatic hydrolysis. The pretreatment process removed most of the xylan and a part of the lignin from the weeds, while most of the glucan remained. The cellulose conversion to glucose was greater for pretreated A. aspera (86.2 ± 0.3%) than that of the pretreated S. acuta (82.2 ± 1.1%). Thus, the removal of hemicellulose significantly affected the efficiency of the enzymatic hydrolysis. The scanning electron microscopy images showed the exposed fibrous cellulose on the cell wall surface, and this substantial change of the surface structure contributed to improving the enzyme accessibility. PMID:26744804

  20. Total fractionation of green tea residue by microwave-assisted alkaline pretreatment and enzymatic hydrolysis.

    PubMed

    Tsubaki, Shuntaro; Azuma, Jun-ichi

    2013-03-01

    Total refinery of constituents of green tea residue was achieved by combination of microwave-assisted alkaline pretreatment and enzymatic hydrolysis. Alkaline pretreatment was effective at separating pectic polysaccharides, protein, phenolic compounds and aliphatic compounds (probably originating from cuticular components), and the solubilization rate was attained 64–74% by heating at 120–200 °C. The higher heating value (HHV) of alkali-soluble fraction attained 20.1 MJ/kg, indicating its usability as black-liquor-like biofuel. Successive cellulolytic enzymatic hydrolysis mainly converted cellulose into glucose and attained the maximum solubilization rate of 89%. Final residue was predominantly composed of aliphatic cuticular components with high proportion in 9,10,18-trihydroxyoctadecanoic acid (30.1–48.6%). These cuticular components are potential alternative feedstock for aliphatic compounds commonly found in oil plants. PMID:23384782

  1. Pretreatment of garden biomass by alkali-assisted ultrasonication: effects on enzymatic hydrolysis and ultrastructural changes

    PubMed Central

    2014-01-01

    The present investigation aims at studying the effectiveness of alkali-assisted ultrasonication on pretreatment of garden biomass (GB). Dry and powdered GB suspended in 1% NaOH was ultrasonicated for 15, 30 and 60 minutes at a frequency of 25 KHZ. The mode of action and effectiveness of alkali-assisted ultrasonication on GB was established through microscopic, scanning electron microscopic and X-ray diffraction studies. A perusal of results showed that alkali-assisted ultrasonication led to fibrillation of GB which ultimately facilitated enzymatic hydrolysis. The results also indicated that alkali-assisted ultrasonication is an efficient means of pretreatment of GB at moderate (45-50°C) working temperature and low (1%) concentration of alkali. The yield of reducing sugar after enzymatic hydrolysis increased almost six times as compared to control due to alkali-assisted ultrasonication. PMID:24843790

  2. Distinct roles of residual xylan and lignin in limiting enzymatic hydrolysis of organosolv pretreated loblolly pine and sweetgum.

    PubMed

    Li, Mi; Tu, Maobing; Cao, Dongxu; Bass, Patrick; Adhikari, Sushil

    2013-01-23

    The interactions between xylan/lignin and cellulase enzymes play a key role in the effective hydrolysis of lignocellulosic biomass. Organosolv pretreated loblolly pine (OPLP) and sweetgum (OPSG) were used to quantitatively elucidate the distinct roles of residual xylan and lignin on enzymatic hydrolysis, based on the initial hydrolysis rates and the final hydrolysis yields. The initial hydrolysis rates of OPLP and OPSG were 1.45 (glucose) and 1.19 g/L/h (glucose), respectively, under the enzyme loading of 20 FPU/g glucan. The final glucan hydrolysis yields of OPLP and OPSG at 72 h were 76.4 and 98.9%, respectively. By correlating the amount of residual lignin and xylan to the initial hydrolysis rate and the final hydrolysis yield in OPLP and OPSG, a more accurate fundamental understanding of the roles of xylan and lignin in limiting the enzymatic hydrolysis has been developed. The higher amount of residual xylan (9.7%) in OPSG resulted in lower initial hydrolysis rate (1.19 g/L/h). The higher amount of residual lignin in OPLP (18.6%) resulted in lower final hydrolysis yield of glucan (76.4%). In addition, we observed in the simultaneous saccharification and fermentation (SSF) that ethyl xyloside was produced by the enzymatic catalysis of xylose/xylan and ethanol. PMID:23270516

  3. Effect of pH on cellulase production and morphology of Trichoderma reesei and the application in cellulosic material hydrolysis.

    PubMed

    Li, Chen; Yang, Zhenhua; Zhang, Ronglin He Can; Zhang, Dongyuan; Chen, Shulin; Ma, Lijuan

    2013-12-01

    A low-cost of cellulase achieved through improving fermentation technology remains a key requirement for commercialization of cellulosic biofuels and biochemicals. pH plays a very important role in the process of cellulase synthesis by Trichoderma reesei. In this work, effects of pH on the production and production rates of three cellulase components (endoglucanase, exoglucanase, β-glucosidase) and mycelial morphology were studied. Production rates of the cellulase components were kept highest and the mycelial morphology was maintained at the optimal status by developing a phased pH control strategy in order to improve cellulase production. Cellulase production in terms of filter paper activity and β-glucosidase production in batch fermentation increased 17.6% and 22%. Saccharification efficiency of the enzyme obtained by pH control was evaluated by hydrolyzing pretreated corn cob. Saccharification yield increased significantly (up to 26.2%) compared with that without pH control. These results add new knowledge on approach for improving cellulase production. PMID:24129314

  4. Kinetic study of enzymatic hydrolysis of acid-pretreated coconut coir

    NASA Astrophysics Data System (ADS)

    Fatmawati, Akbarningrum; Agustriyanto, Rudy

    2015-12-01

    Biomass waste utilization for biofuel production such as bioethanol, has become more prominent currently. Coconut coir is one of lignocellulosic food wastes, which is abundant in Indonesia. Bioethanol production from such materials consists of more than one step. Pretreatment and enzymatic hydrolysis is crucial steps to produce sugar which can then be fermented into bioethanol. In this research, ground coconut coir was pretreated using dilute sulfuric acid at 121°C. This pretreatment had increased the cellulose content and decreased the lignin content of coconut coir. The pretreated coconut coir was hydrolyzed using a mix of two commercial cellulase enzymes at pH of 4.8 and temperature of 50°C. The enzymatic hydrolysis was conducted at several initial coconut coir slurry concentrations (0.1-2 g/100 mL) and reaction times (2-72 hours). The reducing sugar concentration profiles had been produced and can be used to obtain reaction rates. The highest reducing sugar concentration obtained was 1,152.567 mg/L, which was produced at initial slurry concentration of 2 g/100 mL and 72 hours reaction time. In this paper, the reducing sugar concentrations were empirically modeled as a function of reaction time using power equations. Michaelis-Menten kinetic model for enzymatic hydrolysis reaction is adopted. The kinetic parameters of that model for sulfuric acid-pretreated coconut coir enzymatic hydrolysis had been obtained which are Vm of 3.587×104 mg/L.h, and KM of 130.6 mg/L.

  5. Structural Changes of Lignin after Liquid Hot Water Pretreatment and Its Effect on the Enzymatic Hydrolysis.

    PubMed

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

    2016-01-01

    During liquid hot water (LHW) pretreatment, lignin is mostly retained in the pretreated biomass, and the changes in the chemical and structural characteristics of lignin should probably refer to re-/depolymerization, solubilization, or glass transition. The residual lignin could influence the effective enzymatic hydrolysis of cellulose. The pure lignin was used to evaluate the effect of LHW process on its structural and chemical features. The surface morphology of LHW-treated lignin observed with the scanning electron microscopy (SEM) was more porous and irregular than that of untreated lignin. Compared to the untreated lignin, the surface area, total pore volume, and average pore size of LHW-treated lignin tested with the Brunner-Emmet-Teller (BET) measurement were increased. FTIR analysis showed that the chemical structure of lignin was broken down in the LHW process. Additionally, the impact of untreated and treated lignin on the enzymatic hydrolysis of cellulose was also explored. The LHW-treated lignin had little impact on the cellulase adsorption and enzyme activities and somehow could improve the enzymatic hydrolysis of cellulose. PMID:27563678

  6. Structural Changes of Lignin after Liquid Hot Water Pretreatment and Its Effect on the Enzymatic Hydrolysis

    PubMed Central

    Zhuang, Xinshu; Qi, Wei; Yu, Qiang; Wang, Qiong

    2016-01-01

    During liquid hot water (LHW) pretreatment, lignin is mostly retained in the pretreated biomass, and the changes in the chemical and structural characteristics of lignin should probably refer to re-/depolymerization, solubilization, or glass transition. The residual lignin could influence the effective enzymatic hydrolysis of cellulose. The pure lignin was used to evaluate the effect of LHW process on its structural and chemical features. The surface morphology of LHW-treated lignin observed with the scanning electron microscopy (SEM) was more porous and irregular than that of untreated lignin. Compared to the untreated lignin, the surface area, total pore volume, and average pore size of LHW-treated lignin tested with the Brunner-Emmet-Teller (BET) measurement were increased. FTIR analysis showed that the chemical structure of lignin was broken down in the LHW process. Additionally, the impact of untreated and treated lignin on the enzymatic hydrolysis of cellulose was also explored. The LHW-treated lignin had little impact on the cellulase adsorption and enzyme activities and somehow could improve the enzymatic hydrolysis of cellulose. PMID:27563678

  7. Enhancing enzymatic hydrolysis of xylan by adding sodium lignosulfonate and long-chain fatty alcohols.

    PubMed

    Lou, Hongming; Yuan, Long; Qiu, Xueqing; Qiu, Kexian; Fu, Jinguo; Pang, Yuxia; Huang, Jinhao

    2016-01-01

    Sodium lignosulfonate (SXSL) and long-chain fatty alcohols (LFAs) could enhance the enzymatic hydrolysis of xylan, and the compound of SXSL and LFAs have synergies on the enzymatic hydrolysis. SXSL shows a strong enhancement in buffer pH range from 4.0 to 6.0. The enhancement increased with the SXSL dosage and the xylanase loading. The cellulose and lignin in corncob substrate could not only adsorb xylanase nonproductively, but also seriously reduce the accessibility of xylanase on xylan to impede the enzymatic hydrolysis of xylan. Cellulase could break the plant cell wall structure of corncob and make additives work better. The xylose yield of corncob at 72h increased from 59.4% to 73.7% by adding the compound of 5g/L SXSL and 0.01% (v/v) n-decanol, which was higher than that without cellulase and additives by 30.7%. Meanwhile, the glucose yield at 72h of corncob increased from 45.8% to 62.3%. PMID:26476164

  8. Biological Pretreatment of Rubberwood with Ceriporiopsis subvermispora for Enzymatic Hydrolysis and Bioethanol Production

    PubMed Central

    Nazarpour, Forough; Abdullah, Dzulkefly Kuang; Abdullah, Norhafizah; Motedayen, Nazila; Zamiri, Reza

    2013-01-01

    Rubberwood (Hevea brasiliensis), a potential raw material for bioethanol production due to its high cellulose content, was used as a novel feedstock for enzymatic hydrolysis and bioethanol production using biological pretreatment. To improve ethanol production, rubberwood was pretreated with white rot fungus Ceriporiopsis subvermispora to increase fermentation efficiency. The effects of particle size of rubberwood (1 mm, 0.5 mm, and 0.25 mm) and pretreatment time on the biological pretreatment were first determined by chemical analysis and X-ray diffraction and their best condition obtained with 1 mm particle size and 90 days pretreatment. Further morphological study on rubberwood with 1 mm particle size pretreated by fungus was performed by FT-IR spectra analysis and SEM observation and the result indicated the ability of this fungus for pretreatment. A study on enzymatic hydrolysis resulted in an increased sugar yield of 27.67% as compared with untreated rubberwood (2.88%). The maximum ethanol concentration and yield were 17.9 g/L and 53% yield, respectively, after 120 hours. The results obtained demonstrate that rubberwood pretreated by C. subvermispora can be used as an alternative material for the enzymatic hydrolysis and bioethanol production. PMID:24167813

  9. Ethanol production by enzymatic hydrolysis: parametric analysis of a base-case process

    SciTech Connect

    Isaacs, S.H.

    1984-05-01

    A base-case flowsheet for an enzymatic hydrolysis process is presented. Included is a parametric sensitivity analysis to identify key research issues and an assessment of this technology. The plant discussed is a large-scale facility, producing 50 million gallons of ethanol per year. The plant design is based on the process originally conceived by the US National Army Command and consists of these process steps: pretreatment; enzyme production; enzyme hydrolysis; fermentation; and distillation. The base-case design parameters are based on recent laboratory data from Lawrence Berkeley Laboratories and the University of California at Berkeley. The selling price of ethanol is used to compare variations in the base-case operating parameters, which include hydrolysis efficiencies, capital costs, enzyme production efficiencies, and enzyme recycle. 28 references, 38 figures, 8 tables.

  10. Influence of fluid dynamic conditions on enzymatic hydrolysis of lignocellulosic biomass: Effect of mass transfer rate.

    PubMed

    Wojtusik, Mateusz; Zurita, Mauricio; Villar, Juan C; Ladero, Miguel; Garcia-Ochoa, Felix

    2016-09-01

    The effect of fluid dynamic conditions on enzymatic hydrolysis of acid pretreated corn stover (PCS) has been assessed. Runs were performed in stirred tanks at several stirrer speed values, under typical conditions of temperature (50°C), pH (4.8) and solid charge (20% w/w). A complex mixture of cellulases, xylanases and mannanases was employed for PCS saccharification. At low stirring speeds (<150rpm), estimated mass transfer coefficients and rates, when compared to chemical hydrolysis rates, lead to results that clearly show low mass transfer rates, being this phenomenon the controlling step of the overall process rate. However, for stirrer speed from 300rpm upwards, the overall process rate is controlled by hydrolysis reactions. The ratio between mass transfer and overall chemical reaction rates changes with time depending on the conditions of each run. PMID:27233094

  11. Oxidative processes during enzymatic hydrolysis of cod protein and their influence on antioxidant and immunomodulating ability.

    PubMed

    Halldorsdottir, Sigrun M; Sveinsdottir, Holmfridur; Freysdottir, Jona; Kristinsson, Hordur G

    2014-01-01

    Fish protein hydrolysates (FPH) have many desirable properties, however heating and shifts in pH can cause oxidation during enzymatic hydrolysis. The objective was to investigate oxidative processes during enzymatic hydrolysis of fish protein and the impact of oxidation on the antioxidant and immunomodulating ability of FPH. Protease P "Amano" 6 was used to hydrolyze cod protein in the presence and absence of pro-oxidants at pH 8 and 36°C to achieve 20% degree of hydrolysis. Results from thiobarbituric acid reactive substances (TBARS) and sensory analysis indicate that oxidation can develop rapidly during hydrolysis. A cellular antioxidant assay using a HepG2 cell model indicated a negative impact of oxidation products on antioxidant properties of the FPH while results obtained in chemical assays showed a negligible impact. Results from a dendritic cell model indicating that oxidation products may affect anti-inflammatory activity in the body. This study provides important information regarding bioactive FPH. PMID:24001832

  12. Enzymatic Hydrolysis Does Not Reduce the Biological Reactivity of Soybean Proteins for All Allergic Subjects.

    PubMed

    Panda, Rakhi; Tetteh, Afua O; Pramod, Siddanakoppalu N; Goodman, Richard E

    2015-11-01

    Many soybean protein products are processed by enzymatic hydrolysis to attain desirable functional food properties or in some cases to reduce allergenicity. However, few studies have investigated the effects of enzymatic hydrolysis on the allergenicity of soybean products. In this study the allergenicity of soybean protein isolates (SPI) hydrolyzed by Alcalase, trypsin, chymotrypsin, bromelain, or papain was evaluated by IgE immunoblots using eight soybean-allergic patient sera. The biological relevance of IgE binding was evaluated by a functional assay using a humanized rat basophilic leukemia (hRBL) cell line and serum from one subject. Results indicated that hydrolysis of SPI by the enzymes did not reduce the allergenicity, and hydrolysis by chymotrypsin or bromelain has the potential to increase the allergenicity of SPI. Two-dimensional (2D) immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the chymotrypsin-hydrolyzed samples indicated fragments of β-conglycinin protein are responsible for the apparent higher allergenic potential of digested SPI. PMID:26447491

  13. Effect of alkali lignins with different molecular weights from alkali pretreated rice straw hydrolyzate on enzymatic hydrolysis.

    PubMed

    Li, Yun; Qi, Benkun; Luo, Jianquan; Wan, Yinhua

    2016-01-01

    This study investigated the effect of alkali lignins with different molecular weights on enzymatic hydrolysis of lignocellulose. Different alkali lignins fractions, which were obtained from cascade ultrafiltration, were added into the dilute acid pretreated (DAP) and alkali pretreated (AP) rice straws respectively during enzymatic hydrolysis. The results showed that the addition of alkali lignins enhanced the hydrolysis and the enhancement for hydrolysis increased with increasing molecular weights of alkali lignins, with maximum enhancement being 28.69% for DAP and 20.05% for AP, respectively. The enhancement was partly attributed to the improved cellulase activity, and filter paper activity increased by 18.03% when adding lignin with highest molecular weight. It was found that the enhancement of enzymatic hydrolysis was correlated with the adsorption affinity of cellulase on alkali lignins, and the difference in surface charge and hydrophobicity of alkali lignins were responsible for the difference in affinity between cellulase and lignins. PMID:26496216

  14. Facile, room-temperature pre-treatment of rice husks with tetrabutylphosphonium hydroxide: Enhanced enzymatic and acid hydrolysis yields.

    PubMed

    Lau, B B Y; Luis, E T; Hossain, M M; Hart, W E S; Cencia-Lay, B; Black, J J; To, T Q; Aldous, L

    2015-12-01

    Aqueous solutions of tetrabutylphosphonium hydroxide have been evaluated as pretreatment media for rice husks, prior to sulphuric acid hydrolysis or cellulase enzymatic hydrolysis. Varying the water:tetrabutylphosphonium hydroxide ratio varied the rate of delignification, as well as silica, lignin and cellulose solubility. Pre-treatment with 60wt% hydroxide dissolved the rice husk and the regenerated material was thus heavily disrupted. Sulphuric acid hydrolysis of 60wt%-treated samples yielded the highest amount of glucose per gram of rice husk. Solutions with good lignin and silica solubility but only moderate to negligible cellulose solubility (10-40wt% hydroxide) were equally effective as pre-treatment media for both acid and enzymatic hydrolysis. However, pre-treatment with 60wt% hydroxide solutions was incompatible with downstream enzymatic hydrolysis. This was due to significant incorporation of phosphonium species in the regenerated biomass, which significantly inhibited the activity of the cellulase enzymes. PMID:26342336

  15. [Optimization of liquid ammonia treatment for enzymatic hydrolysis of Saccharum arundinaceum to fermentable sugars].

    PubMed

    Liu, Jianjun; Peng, Hehuan; Zhao, Xiangjun; Cheng, Cheng; Chen, Feng; Shao, Qianjun

    2013-03-01

    China has abundant available marginal land that can be used for cultivation of lignocellulosic energy plants. Saccharum arundinaceum Retz. is a potential energy crop with both high biomass yield and low soil fertility requirements. It can be planted widely as cellulosic ethanol feedstock in southern China. In the present work Saccharum arundinaceum was pretreated by liquid ammonia treatment (LAT) to overcome biomass recalcitrance, followed by enzymatic hydrolysis. The monosaccharide contents (glucose, xylose, and arabinose) of the enzymatic hydrolysate were determined by high performance liquid chromatography. Experimental results show that the optimal LAT pretreatment conditions were 130 0C, 2:1 (W/W) ammonia to biomass ratio, 80% moisture content (dry weight basis) and 5 min residence time. Approximately 69.34% glucan and 82.60% xylan were converted after 72 h enzymatic hydrolysis at 1% glucan loading using 15 FPU/(g of glucan) of cellulase. The yields of glucose and xylose were 573% and 1 056% higher than those of the untreated biomass, and the LAT-pretreated substrates obtained an 8-fold higher of total monosaccharide yield than untreated substrates. LAT pretreatment was an effective to increase the enzymatic digestibility of Saccharum arundinaceum compared to acid impregnated steam explosion and similar to that of acid treatment and ammonia fiber expansion treatment. PMID:23789274

  16. A Factorial Analysis Study on Enzymatic Hydrolysis of Fiber Pressed Oil Palm Frond for Bioethanol Production

    NASA Astrophysics Data System (ADS)

    Hashim, F. S.; Yussof, H. W.; Zahari, M. A. K. M.; Illias, R. M.; Rahman, R. A.

    2016-03-01

    Different technologies have been developed to for the conversion of lignocellulosic biomass to suitable fermentation substrates for bioethanol production. The enzymatic conversion of cellulose seems to be the most promising technology as it is highly specific and does not produce substantial amounts of unwanted byproducts. The effects of agitation speed, enzyme loading, temperature, pH and reaction time on the conversion of glucose from fiber pressed oil palm frond (FPOPF) for bioethanol production were screened by statistical analysis using response surface methodology (RSM). A half fraction two-level factorial analysis with five factors was selected for the experimental design to determine the best enzymatic conditions that produce maximum amount of glucose. FPOPF was pre-treated with alkaline prior to enzymatic hydrolysis. The enzymatic hydrolysis was performed using a commercial enzyme Cellic CTec2. From this study, the highest yield of glucose concentration was 9.736 g/L at 72 hours reaction time at 35 °C, pH 5.6, and 1.5% (w/v) of enzyme loading. The model obtained was significant with p-value <0.0001. It is suggested that this model had a maximum point which is likely to be the optimum point and possible for the optimization process.

  17. The influence of solid/liquid separation techniques on the sugar yield in two-step dilute acid hydrolysis of softwood followed by enzymatic hydrolysis

    PubMed Central

    Monavari, Sanam; Galbe, Mats; Zacchi, Guido

    2009-01-01

    Background Two-step dilute acid hydrolysis of softwood, either as a stand-alone process or as pretreatment before enzymatic hydrolysis, is considered to result in higher sugar yields than one-step acid hydrolysis. However, this requires removal of the liquid between the two steps. In an industrial process, filtration and washing of the material between the two steps is difficult, as it should be performed at high pressure to reduce energy demand. Moreover, the application of pressure leads to more compact solids, which may affect subsequent processing steps. This study was carried out to investigate the influence of pressing the biomass, in combination with the effects of not washing the material, on the sugar yield obtained from two-step dilute acid hydrolysis, with and without subsequent enzymatic digestion of the solids. Results Washing the material between the two acid hydrolysis steps, followed by enzymatic digestion, resulted in recovery of 96% of the mannose and 81% of the glucose (% of the theoretical) in the liquid fraction, regardless of the choice of dewatering method (pressing or vacuum filtration). Not washing the solids between the two acid hydrolysis steps led to elevated acidity of the remaining solids during the second hydrolysis step, which resulted in lower yields of mannose, 85% and 74% of the theoretical, for the pressed and vacuum-filtered slurry, respectively, due to sugar degradation. However, this increase in acidity resulted in a higher glucose yield (94.2%) from pressed slurry than from filtered slurry (77.6%). Conclusion Pressing the washed material between the two acid hydrolysis steps had no significant negative effect on the sugar yields of the second acid hydrolysis step or on enzymatic hydrolysis. Not washing the material resulted in a harsher second acid hydrolysis step, which caused greater degradation of the sugars during subsequent acid hydrolysis of the solids, particularly in case of the vacuum-filtered solids. However

  18. Antioxidative activities of hydrolysates from edible birds nest using enzymatic hydrolysis

    NASA Astrophysics Data System (ADS)

    Muhammad, Nurul Nadia; Babji, Abdul Salam; Ayub, Mohd Khan

    2015-09-01

    Edible bird's nest protein hydrolysates (EBN) were prepared via enzymatic hydrolysis to investigate its antioxidant activity. Two types of enzyme (alcalase and papain) were used in this study and EBN had been hydrolysed with different hydrolysis time (30, 60, 90 and 120 min). Antioxidant activities in EBN protein hydrolysate were measured using DPPH, ABTS+ and Reducing Power Assay. From this study, increased hydrolysis time from 30 min to 120 min contributed to higher DH, as shown by alcalase (40.59%) and papain (24.94%). For antioxidant assay, EBN hydrolysed with papain showed higher scavenging activity and reducing power ability compared to alcalase. The highest antioxidant activity for papain was at 120 min hydrolysis time with ABTS (54.245%), DPPH (49.78%) and Reducing Power (0.0680). Meanwhile for alcalase, the highest antioxidant activity was at 30 min hydrolysis time. Even though scavenging activity for EBN protein hydrolysates were high, the reducing power ability was quite low as compared to BHT and ascorbic Acid. This study showed that EBN protein hydrolysate with alcalase and papain treatments potentially exhibit high antioxidant activity which have not been reported before.

  19. Process evaluation of enzymatic hydrolysis with filtrate recycle for the production of high concentration sugars.

    PubMed

    Xue, Ying; Rusli, Jannov; Chang, Hou-Min; Phillips, Richard; Jameel, Hasan

    2012-02-01

    Process simulation and lab trials were carried out to demonstrate and confirm the efficiency of the concept that recycling hydrolysate at low total solid enzymatic hydrolysis is one of the options to increase the sugar concentration without mixing problems. Higher sugar concentration can reduce the capital cost for fermentation and distillation because of smaller retention volume. Meanwhile, operation cost will also decrease for less operating volume and less energy required for distillation. With the computer simulation, time and efforts can be saved to achieve the steady state of recycling process, which is the scenario for industrial production. This paper, to the best of our knowledge, is the first paper discussing steady-state saccharification with recycling of the filtrate form enzymatic hydrolysis to increase sugar concentration. Recycled enzymes in the filtrate (15-30% of the original enzyme loading) resulted in 5-10% higher carbohydrate conversion compared to the case in which recycled enzymes were denatured. The recycled hydrolysate yielded 10% higher carbohydrate conversion compared to pure sugar simulated hydrolysate at the same enzyme loading, which indicated hydrolysis by-products could boost enzymatic hydrolysis. The high sugar concentration (pure sugar simulated) showed inhibition effect, since about 15% decrease in carbohydrate conversion was observed compared with the case with no sugar added. The overall effect of hydrolysate recycling at WinGEMS simulated steady-state conditions with 5% total solids was increasing the sugar concentration from 35 to 141 g/l, while the carbohydrate conversion was 2% higher for recycling at steady state (87%) compared with no recycling strategy (85%). Ten percent and 15% total solid processes were also evaluated in this study. PMID:22167689

  20. The slowdown of the endoglucanase Trichoderma reesei Cel5A-catalyzed cellulose hydrolysis is related to its initial activity.

    PubMed

    Shu, Zhiyu; Wang, Yefei; An, Liaoyuan; Yao, Lishan

    2014-12-01

    One important feature of hydrolysis of cellulose by cellulases is that the reaction slows down quickly after it starts. In this work, we investigate the slowdown mechanism at the early stage of the reaction using endoglucanase Tr. Cel5A-catalyzed phosphate acid-swollen cellulose (PASC) hydrolysis as a model system. Specifically, we focus on the effect of enzyme adsorption on the reaction slowdown. Nineteen single mutations are introduced (with the assistance of molecular dynamics simulations) to perturb the enzyme PASC interaction, yielding the adsorption partitioning coefficient Kr that ranged from 0.12 to 0.39 L/g, compared to that of the wild type (0.26 L/g). Several residues, including T18, K26, Y26, H229, and T300, are demonstrated to be important for adsorption of the enzyme to PASC. The kinetic measurements show that the slowdown of the hydrolysis is not correlated with the adsorption quantified by the partitioning coefficient Kr but is anticorrelated with the initial activity. This result suggests that the mutants with higher activity are more prone to being trapped or deplete the most reactive substrate faster and the adsorption plays no apparent role in the reaction slowdown. The initial activity of Cel5A against PASC is correlated with the enzyme specific activity against a soluble substrate p-nitrophenyl cellobioside. PMID:25423499

  1. Effects of agitation on particle-size distribution and enzymatic hydrolysis of pretreated spruce and giant reed

    PubMed Central

    2014-01-01

    Background Mixing is an energy demanding process which has been previously shown to affect enzymatic hydrolysis. Concentrated biomass slurries are associated with high and non-Newtonian viscosities and mixing in these systems is a complex task. Poor mixing can lead to mass and/or heat transfer problems as well as inhomogeneous enzyme distribution, both of which can cause possible yield reduction. Furthermore the stirring energy dissipation may impact the particle size which in turn may affect the enzymatic hydrolysis. The objective of the current work was to specifically quantify the effects of mixing on particle-size distribution (PSD) and relate this to changes in the enzymatic hydrolysis. Two rather different materials were investigated, namely pretreated Norway spruce and giant reed. Results Changes in glucan hydrolysis and PSD were measured as a function of agitation during enzymatic hydrolysis at fiber loadings of 7 or 13% water-insoluble solids (WIS). Enzymatic conversion of pretreated spruce was strongly affected by agitation rates at the higher WIS content. However, at low WIS content the agitation had almost no effect on hydrolysis. There was some effect of agitation on the hydrolysis of giant reed at high WIS loading, but it was smaller than that for spruce, and there was no measurable effect at low WIS loading. In the case of spruce, intense agitation clearly affected the PSD and resulted in a reduced mean particle size, whereas for giant reed the decrease in particle size was mainly driven by enzymatic action. However, the rate of enzymatic hydrolysis was not increased after size reduction by agitation. Conclusions The impact of agitation on the enzymatic hydrolysis clearly depends not only on feedstock but also on the solids loading. Agitation was found to affect the PSD differently for the examined pretreated materials spruce and giant reed. The fact that the reduced mean particle diameter could not explain the enhanced hydrolysis rates found for

  2. Effects of enzymatic hydrolysis on conformational and functional properties of chickpea protein isolate.

    PubMed

    Mokni Ghribi, Abir; Maklouf Gafsi, Ines; Sila, Assaâd; Blecker, Christophe; Danthine, Sabine; Attia, Hamadi; Bougatef, Ali; Besbes, Souhail

    2015-11-15

    The impact of enzymatic hydrolysis by Alcalase on the conformational and functional properties of chickpea protein isolate (CPI) was investigated. The physicochemical, interfacial tension and surface characteristics of CPI and their hydrolysates (CPH) according to the degree of hydrolysis (DH) were also determined. These parameters were then related to the changes in the emulsification activity (EAI) and stability (ESI). The enzymatic hydrolysis was found to improve protein recovery and solubility, leading to a reduction in the molecular weight bands with a concomitant increase in the intensity and appearance of protein bands having apparent molecular mass below 20 kDa. The interfacial tension decreased from ∼ 66.5 mN m(-1) for CPI to ∼ 59.1 m Nm(-1) for CPH. A similar trend was observed for the surface charge which declined from -27.55 mV to -16.4 mV for the CPI and CPH, respectively. These changes were found to have a detrimental effect on the EAI and ESI values. PMID:25977033

  3. Coupling of ultrafiltration and enzymatic hydrolysis aiming at valorizing shrimp wastewater.

    PubMed

    Tonon, Renata V; dos Santos, Bianca A; Couto, Cinthia C; Mellinger-Silva, Caroline; Brígida, Ana Iraidy S; Cabral, Lourdes M C

    2016-05-01

    The objective of this work was to obtain a protein hydrolysate from the wastewater generated during shrimp cooking, by coupling ultrafiltration and enzymatic hydrolysis processes. Initially, the effluent was concentrated by ultrafiltration, reaching a protein concentration factor of 3.2. The concentrated effluent was then enzymatically hydrolyzed, aiming at obtaining peptides with antioxidant capacity. The effects of some process variables--temperature (55-75 °C), pH (7-9) and enzyme/substrate (E/S) ratio (0.1-2.5%)--on the degree of hydrolysis and the antioxidant capacity were evaluated. The increase in temperature and pH resulted in lower degree of hydrolysis and higher antioxidant capacity. The conditions selected as the most suitable were: temperature of 75 °C, pH of 9.0 and E/S ratio of 0.1%. The hydrolysates produced at these conditions were also evaluated for total amino acid content and electrophoretic profile, showing a suitable amount of essential amino acids that covers the recommended daily needs. PMID:26769500

  4. An investigation on keratin extraction from wool and feather waste by enzymatic hydrolysis.

    PubMed

    Eslahi, Niloofar; Dadashian, Fatemeh; Nejad, Nahid Hemmati

    2013-01-01

    In this study, the possibility of keratin extraction from wool and feather by an enzymatic treatment along with a reducing agent has been investigated. The effects of different parameters, that is, enzyme loading, type of substrate and surfactant, hydrolysis time, and reducing agent concentration, have been examined in order to optimize the enzymatic hydrolysis. The optimal condition for maximum keratin extraction was attained by making use of 1 g/L sodium dodecyl sulfate (an anionic surfactant) and 2.6% (v/v) protease (Savinase), along with 8.6 and 6.4 g/L sodium hydrogen sulfite (a reducing agent) for wool and feathers, respectively, at liquor to fiber ratio of 25 mL/g for 4 hr. The obtained results indicated higher degradation of wool fiber in comparison with feathers, which might be due to the higher hydrophilic nature of the former. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) patterns revealed that the molecular weights of the extracted proteins from wool and feather were lower than those for the untreated fibers. Scanning electron micrographs showed fibers fibrillation and degradation upon enzymatic treatment. Besides, Fourier-transform infrared (FTIR) spectra indicated no evident changes in the chemical structure of the hydrolyzed fibers. However, wool and feather remainders were mostly composed of α-helix and β-sheets conformations, respectively. PMID:23768110

  5. Particle Concentration and Yield Stress of Biomass Slurries During Enzymatic Hydrolysis at High-Solids Loadings

    SciTech Connect

    Roche, C. M.; Dibble, C. J.; Knutsen, J. S.; Stickel, J. J.; Liberatore, M. W.

    2009-01-01

    Effective and efficient breakdown of lignocellulosic biomass remains a primary barrier for its use as a feedstock for renewable transportation fuels. A more detailed understanding of the material properties of biomass slurries during conversion is needed to design cost-effective conversion processes. A series of enzymatic saccharification experiments were performed with dilute acid pretreated corn stover at initial insoluble solids loadings of 20% by mass, during which the concentration of particulate solids and the rheological property yield stress ({tau}{sub y}) of the slurries were measured. The saccharified stover liquefies to the point of being pourable ({tau}{sub y} {le} 10 Pa) at a total biomass conversion of about 40%, after roughly 2 days of saccharification for a moderate loading of enzyme. Mass balance and semi-empirical relationships are developed to connect the progress of enzymatic hydrolysis with particle concentration and yield stress. The experimental data show good agreement with the proposed relationships. The predictive models developed here are based on established physical principles and should be applicable to the saccharification of other biomass systems. The concepts presented, especially the ability to predict yield stress from extent of conversion, will be helpful in the design and optimization of enzymatic hydrolysis processes that operate at high-solids loadings.

  6. Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis

    PubMed Central

    2010-01-01

    The efficient enzymatic saccharification of cellulose at low cellulase (protein) loadings continues to be a challenge for commercialization of a process for bioconversion of lignocellulose to ethanol. Currently, effective pretreatment followed by high enzyme loading is needed to overcome several substrate and enzyme factors that limit rapid and complete hydrolysis of the cellulosic fraction of biomass substrates. One of the major barriers faced by cellulase enzymes is their limited access to much of the cellulose that is buried within the highly ordered and tightly packed fibrillar architecture of the cellulose microfibrils. Rather than a sequential 'shaving' or 'planing' of the cellulose fibrils from the outside, it has been suggested that these inaccessible regions are disrupted or loosened by non-hydrolytic proteins, thereby increasing the cellulose surface area and making it more accessible to the cellulase enzyme complex. This initial stage in enzymatic saccharification of cellulose has been termed amorphogenesis. In this review, we describe the various amorphogenesis-inducing agents that have been suggested, and their possible role in enhancing the enzymatic hydrolysis of cellulose. PMID:20178562

  7. Rapid Online Non-Enzymatic Protein Digestion Combining Microwave Heating Acid Hydrolysis and Electrochemical Oxidation

    PubMed Central

    Basile, Franco; Hauser, Nicolas

    2010-01-01

    We report an online non-enzymatic method for site-specific digestion of proteins to yield peptides that are well suited for collision induced dissociation (CID) tandem mass spectrometry (MS/MS). The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochemical oxidation at tryptophan and tyrosine. The combined microwave/electrochemical (microwave/echem) digestion is reproducible and produces peptides with an average sequence length of 10 amino acids. This peptide length is similar to the average peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel non-enzymatic digestion method, when analyzed by ESI-MS, produce protonated molecules with mostly +1 and +2 charge states. The combination of these two non-enzymatic methods overcomes shortcomings with each individual method in that: i) peptides generated by the microwave-hydrolysis method have an average amino acid length of 16 amino acids, and ii) the inability of the electrochemical-cleavage method to reproducibly digest proteins with molecular masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min digestion time) on a series of standard peptides and proteins as well as an E. coli protein extract. PMID:21138252

  8. Comparative study on chemical pretreatments to accelerate enzymatic hydrolysis of aquatic macrophyte biomass used in water purification processes.

    PubMed

    Mishima, D; Tateda, M; Ike, M; Fujita, M

    2006-11-01

    In this study, enzymatic hydrolysis of two floating aquatic plants which are suitable for water purification, water hyacinth (Eichhornia crassipes) and water lettuce (Pistia stratiotes L.), was performed to produce sugars. Twenty chemical pretreatments were comparatively examined in order to improve the efficiency of enzymatic hydrolysis. As a result, the alkaline/oxidative (A/O) pretreatment, in which sodium hydroxide and hydrogen peroxide were used, was the most effective pretreatment in terms of improving enzymatic hydrolysis of the leaves of water hyacinth and water lettuce. The amount of reducing sugars in enzymatic hydrolysate of water lettuce leaves was 1.8 times higher than that of water hyacinth leaves, therefore water lettuce seems to be more attractive as a biomass resource than water hyacinth. Although roots of these plants contained large amounts of polysaccharides such as cellulose and hemicellulose, they generated less monosaccharides than from leaves, no matter which chemical pretreatment was tested. PMID:16309902

  9. Comparison of sodium carbonate pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce fermentable sugars.

    PubMed

    Jin, Yongcan; Huang, Ting; Geng, Wenhui; Yang, Linfeng

    2013-06-01

    The specific characteristics of biomass structure and chemical composition of straw stem and leaf may result in different behavior of pretreatment and enzymatic hydrolysis. In this work, sodium carbonate (SC) was employed as a pretreatment to improve the enzymatic digestibility of wheat straw. The chemical composition and enzymatic hydrolysis of wheat straw stem and leaf (sheath included) were investigated comparatively. Most of the polysaccharides are kept in the solid fractions after SC pretreatment, while the stem has better delignification selectivity than leaf at high temperature. The enzymatic hydrolysis efficiency of wheat straw leaf is significantly higher than that of stem. The maximum total sugar yield from SC pretreated leaf was about 16% higher than stem. The results show that sodium carbonate is of great potential to be used as a pretreatment for the production of bioethanol from straw handling waste in a straw pulp mill with a low feedstock cost. PMID:23587832

  10. Membrane-based recovery of glucose from enzymatic hydrolysis of ionic liquid pretreated cellulose.

    PubMed

    Abels, Christian; Thimm, Kristof; Wulfhorst, Helene; Spiess, Antje Christine; Wessling, Matthias

    2013-12-01

    In this work, a membrane-based downstream process for the recovery of glucose from cellulose hydrolysis is described and evaluated. The cellulose is pretreated with the ionic liquid 1,3-dimethyl-imidazolium dimethylphosphate to reduce its crystallinity. After enzymatic conversion of cellulose to glucose the hydrolysate is filtered with an ultrafiltration membrane to remove residual particulates and enzymes. Nanofiltration is applied to purify the glucose from molecular intermediates, such as cellobiose originating from the hydrolysis reaction. Finally, the ionic liquid is removed from the hydrolysate via electrodialysis. Technically, these process steps are feasible. An economic analysis of the process reveals that the selling price of glucose from this production process is about 2.75 €/kg which is too high as compared to the current market price. PMID:24084205

  11. Cellulase deactivation based kinetic modeling of enzymatic hydrolysis of steam-exploded wheat straw.

    PubMed

    Zhang, Yu; Xu, Jing-Liang; Xu, Hui-Juan; Yuan, Zhen-Hong; Guo, Ying

    2010-11-01

    Applying mass action law and quasi-steady-state theory, two cellulase kinetic models namely Eqs. (5) and (8) were developed on the basis of the first and second order reactions of enzyme deactivation, respectively. The two models are compared according to analysis of experimental data from enzymatic hydrolysis steam-exploded wheat straw. Both simulation and prediction results show Eq. (8) has much higher accuracy than Eq. (5). Analysis of initial hydrolysis rate is also in accordance with Eq. (8) and against Eq. (5). Fitted values of k(2) (the rate constant of product formation), k(de2) (the rate constant of enzyme deactivation) and K(e) (the equilibrium constant) determined from Eq. (8) are 0.4732 h(-1), 0.4011 L/(hg), and 16.8597 g/L, respectively. The higher the enzyme concentration is, the larger the deactivation rate. PMID:20594825

  12. Does densification influence the steam pretreatment and enzymatic hydrolysis of softwoods to sugars?

    PubMed

    Kumar, Linoj; Tooyserkani, Zahra; Sokhansanj, Shahab; Saddler, Jack N

    2012-10-01

    The global trade in wood pellets continues to grow. However, their potential as a feedstock for large scale cellulosic ethanol production has not been evaluated. We anticipated that the reduced moisture content and pressure exerted on the wood biomass during the pelletisation process would result in some carbohydrate loss as well as making the biomass more recalcitrant to pretreatment and subsequent hydrolysis. However, when softwood chips and pellets were steam pretreated at medium severity, little hemicellulose loss occurred while more than two-thirds of the cellulose present in the cellulose rich water insoluble fractions were hydrolysed (at 20 FPU cellulase/g cellulose). In addition, prior steaming substantially reduced the particle size of the wood chips enabling direct pelletisation without the need for grinding. Surprisingly, it was also possible to apply a single steam pretreatment to facilitate both pelletisation and subsequent enzymatic hydrolysis without the need for a further pretreatment step. PMID:22858485

  13. Model-Based Fed-Batch for High-Solids Enzymatic Cellulose Hydrolysis

    SciTech Connect

    Hodge, D. B.; Karim, M. N.; Schell, D. J.; McMillan, J. D.

    2008-01-01

    While many kinetic models have been developed for the enzymatic hydrolysis of cellulose, few have been extensively applied for process design, optimization, or control. High-solids operation of the enzymatic hydrolysis of lignocellulose is motivated by both its operation decreasing capital costs and increasing product concentration and hence separation costs. This work utilizes both insights obtained from experimental work and kinetic modeling to develop an optimization strategy for cellulose saccharification at insoluble solids levels greater than 15% (w/w), where mixing in stirred tank reactors (STRs) becomes problematic. A previously developed model for batch enzymatic hydrolysis of cellulose was modified to consider the effects of feeding in the context of fed-batch operation. By solving the set of model differential equations, a feeding profile was developed to maintain the insoluble solids concentration at a constant or manageable level throughout the course of the reaction. Using this approach, a stream of relatively concentrated solids (and cellulase enzymes) can be used to increase the final sugar concentration within the reactor without requiring the high initial levels of insoluble solids that would be required if the operation were performed in batch mode. Experimental application in bench-scale STRs using a feed stream of dilute acid-pretreated corn stover solids and cellulase enzymes resulted in similar cellulose conversion profiles to those achieved in batch shake-flask reactors where temperature control issues are mitigated. Final cellulose conversions reached approximately 80% of theoretical for fed-batch STRs fed to reach a cumulative solids level of 25% (w/w) initial insoluble solids.

  14. Assessment of taste attributes of peanut meal enzymatic-hydrolysis hydrolysates using an electronic tongue.

    PubMed

    Wang, Li; Niu, Qunfeng; Hui, Yanbo; Jin, Huali; Chen, Shengsheng

    2015-01-01

    Peanut meal is the byproduct of high-temperature peanut oil extraction; it is mainly composed of proteins, which have complex tastes after enzymatic hydrolysis to free amino acids and small peptides. The enzymatic hydrolysis method was adopted by using two compound proteases of trypsin and flavorzyme to hydrolyze peanut meal aiming to provide a flavor base. Hence, it is necessary to assess the taste attributes and assign definite taste scores of peanut meal double enzymatic hydrolysis hydrolysates (DEH). Conventionally, sensory analysis is used to assess taste intensity in DEH. However, it has disadvantages because it is expensive and laborious. Hence, in this study, both taste attributes and taste scores of peanut meal DEH were evaluated using an electronic tongue. In this regard, the response characteristics of the electronic tongue to the DEH samples and standard five taste samples were researched to qualitatively assess the taste attributes using PCA and DFA. PLS and RBF neural network (RBFNN) quantitative prediction models were employed to compare predictive abilities and to correlate results obtained from the electronic tongue and sensory analysis, respectively. The results showed that all prediction models had good correlations between the predicted scores from electronic tongue and those obtained from sensory analysis. The PLS and RBFNN prediction models constructed using the voltage response values from the sensors exhibited higher correlation and prediction ability than that of principal components. As compared with the taste performance by PLS model, that of RBFNN models was better. This study exhibits potential advantages and a concise objective taste assessment tool using the electronic tongue in the assessment of DEH taste attributes in the food industry. PMID:25985162

  15. Assessment of Taste Attributes of Peanut Meal Enzymatic-Hydrolysis Hydrolysates Using an Electronic Tongue

    PubMed Central

    Wang, Li; Niu, Qunfeng; Hui, Yanbo; Jin, Huali; Chen, Shengsheng

    2015-01-01

    Peanut meal is the byproduct of high-temperature peanut oil extraction; it is mainly composed of proteins, which have complex tastes after enzymatic hydrolysis to free amino acids and small peptides. The enzymatic hydrolysis method was adopted by using two compound proteases of trypsin and flavorzyme to hydrolyze peanut meal aiming to provide a flavor base. Hence, it is necessary to assess the taste attributes and assign definite taste scores of peanut meal double enzymatic hydrolysis hydrolysates (DEH). Conventionally, sensory analysis is used to assess taste intensity in DEH. However, it has disadvantages because it is expensive and laborious. Hence, in this study, both taste attributes and taste scores of peanut meal DEH were evaluated using an electronic tongue. In this regard, the response characteristics of the electronic tongue to the DEH samples and standard five taste samples were researched to qualitatively assess the taste attributes using PCA and DFA. PLS and RBF neural network (RBFNN) quantitative prediction models were employed to compare predictive abilities and to correlate results obtained from the electronic tongue and sensory analysis, respectively. The results showed that all prediction models had good correlations between the predicted scores from electronic tongue and those obtained from sensory analysis. The PLS and RBFNN prediction models constructed using the voltage response values from the sensors exhibited higher correlation and prediction ability than that of principal components. As compared with the taste performance by PLS model, that of RBFNN models was better. This study exhibits potential advantages and a concise objective taste assessment tool using the electronic tongue in the assessment of DEH taste attributes in the food industry. PMID:25985162

  16. Modeling enzymatic hydrolysis of lignocellulosic substrates using fluorescent confocal microscopy II: pretreated biomass.

    PubMed

    Luterbacher, Jeremy S; Moran-Mirabal, Jose M; Burkholder, Eric W; Walker, Larry P

    2015-01-01

    In this study, we extend imaging and modeling work that was done in Part I of this report for a pure cellulose substrate (filter paper) to more industrially relevant substrates (untreated and pretreated hardwood and switchgrass). Using confocal fluorescence microscopy, we are able to track both the structure of the biomass particle via its autofluorescence, and bound enzyme from a commercial cellulase cocktail supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A. Imaging was performed throughout hydrolysis at temperatures relevant to industrial processing (50°C). Enzyme bound predominantly to areas with low autofluorescence, where structure loss and lignin removal had occurred during pretreatment; this confirms the importance of these processes for successful hydrolysis. The overall shape of both untreated and pretreated hardwood and switchgrass particles showed little change during enzymatic hydrolysis beyond a drop in autofluorescence intensity. The permanence of shape along with a relatively constant bound enzyme signal throughout hydrolysis was similar to observations previously made for filter paper, and was consistent with a modeling geometry of a hollowing out cylinder with widening pores represented as infinite slits. Modeling estimates of available surface areas for pretreated biomass were consistent with previously reported experimental results. PMID:25042048

  17. Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry.

    PubMed

    van den Heuvel, Robert H H; Gato, Sara; Versluis, Cees; Gerbaux, Pascal; Kleanthous, Colin; Heck, Albert J R

    2005-01-01

    A fast and direct method for the monitoring of enzymatic DNA hydrolysis was developed using electrospray ionization mass spectrometry. We incorporated the use of a robotic chip-based electrospray ionization source for increased reproducibility and throughput. The mass spectrometry method allows the detection of DNA fragments and intact non-covalent protein-DNA complexes in a single experiment. We used the method to monitor in real-time single-stranded (ss) DNA hydrolysis by colicin E9 DNase and to characterize transient non-covalent E9 DNase-DNA complexes present during the hydrolysis reaction. The mass spectra showed that E9 DNase interacts with ssDNA in the absence of a divalent metal ion, but is strictly dependent on Ni2+ or Co2+ for ssDNA hydrolysis. We demonstrated that the sequence selectivity of E9 DNase is dependent on the ratio protein:ssDNA or the ssDNA concentration and that only 3'-hydroxy and 5'-phosphate termini are produced. It was also shown that the homologous E7 DNase is reactive with Zn2+ as transition metal ion and that this DNase displays a different sequence selectivity. The method described is of general use to analyze the reactivity and specificity of nucleases. PMID:15956101

  18. [Effects of hot-NaOH pretreatment on Jerusalem artichoke stalk composition and subsequent enzymatic hydrolysis].

    PubMed

    Wang, Qing; Qiu, Jingwen; Li, Yang; Shen, Fei

    2015-10-01

    In order to explore the possibility of Jerusalem artichoke stalk for bioenergy conversion, we analyzed the main composition of whole stalk, pitch, and core of the stalk. Meanwhile, these parts were pretreated with different NaOH concentrations at 121 degrees C. Afterwards, enzymatic hydrolysis was performed to evaluate the pretreatment efficiency. Jerusalem artichoke stalk was characterized by relatively high lignin content (32.0%) compared with traditional crop stalks. The total carbohydrate content was close to that of crop stalks, but with higher cellulose content (40.5%) and lower hemicellulose (19.6%) than those of traditional crop stalks. After pretreatment, the lignin content in the whole stalk, pitch, and core decreased by 13.1%-13.4%, 8.3%-13.5%, and 19.9%-27.2%, respectively, compared with the unpretreated substrates. The hemicellulose content in the whole stalk, pitch, and core decreased 87.8%-96.9%, 87.6%-95.0%, and 74.0%-90.2%, respectively. Correspondingly, the cellulose content in the pretreated whole stalk, pitch, and core increased by 56.5%-60.2%, 52.2%-55.4%, and 62.7%-73.2%, respectively. Moreover, increase of NaOH concentration for pretreatment could improve the enzymatic hydrolysis of the whole stalk and pitch by 2.3-2.6 folds and 10.3-18.5 folds, respectively. The hydrolysis of pretreated stalk core decreased significantly as 2.0 mol/L NaOH was employed, although the increased NaOH concentration can also improve its hydrolysis performance. Based on these results, hot-NaOH can be regarded as an option for Jerusalem artichoke stalk pretreatment. Increasing NaOH concentration was beneficial to hemicellulose and lignin removal, and consequently improved sugar conversion. However, the potential decrease of sugar conversion of the pretreated core by higher NaOH concentration suggested further optimization on the pretreatment conditions should be performed. PMID:26964335

  19. Simultaneous enzymatic hydrolysis and anaerobic biodegradation of lipid-rich wastewater from poultry industry

    NASA Astrophysics Data System (ADS)

    Dors, Gisanara; Mendes, Adriano A.; Pereira, Ernandes B.; de Castro, Heizir F.; Furigo, Agenor

    2013-03-01

    Simultaneous enzymatic hydrolysis and anaerobic biodegradation of lipid-rich wastewater from poultry industry with porcine pancreatic lipase at different concentrations (from 1.0 to 3.0 g L-1) were performed. The efficiency of the enzymatic pretreatment was measured by the Chemical Oxygen Demand (COD) removal and formation of methane. All samples pretreated with lipase showed a positive effect on the COD removal and formation of methane. After 30 days of anaerobic biodegradation the methane production varied from 569 ± 95 to 1,101 ± 10 mL for crude wastewater and pretreated at 3.0 g L-1 enzyme, respectively. COD removal of wastewater supplemented at different enzyme concentrations was found to be threefold higher than crude wastewater. The use of lipases seems to be a promising alternative for treating lipid-rich wastewaters such as those from the poultry industry.

  20. Enzymatic hydrolysis of organic-core microcapsules to produce aqueous-core microcapsules.

    PubMed

    Breguet, Veronique; Vojinovic, Vojislav; Von Stockar, Urs; Marison, Ian W

    2008-05-01

    This paper describes the development of a new method to obtain aqueous-core microcapsules from organic-core capsules. The direct production of microcapsules, using tripropionin as organic material, followed by the hydrolysis of the core by a lipase was investigated. The enzymatic study showed that the enzyme obeyed a Michaelis-Menten mechanism and conditions for optimal activity were pH 7.5, 25-37 degrees C and 0% NaCl. Under these conditions, incubation of tripropionin-alginate microcapsules in a buffer containing the enzyme successfully produced aqueous-core capsules with reduced accumulation of alginate in the core in approximately 3 h. PMID:18382924

  1. Improved enzymatic hydrolysis of lignocellulosic biomass through pretreatment with plasma electrolysis.

    PubMed

    Gao, Jing; Chen, Li; Zhang, Jian; Yan, Zongcheng

    2014-11-01

    A comprehensive research on plasma electrolysis as pretreatment method for water hyacinth (WH) was performed based on lignin content, crystalline structure, surface property, and enzymatic hydrolysis. A large number of active particles, such as HO and H2O2, generated by plasma electrolysis could decompose the lignin of the biomass samples and reduce the crystalline index. An efficient pretreatment process made use of WH pretreated at a load of 48 wt% (0.15-0.18 mm) in FeCl3 solution for 30 min at 450 V. After the pretreatment, the sugar yield of WH was increased by 126.5% as compared with unpretreated samples. PMID:25205055

  2. Pretreatment of microcrystalline cellulose in organic electrolyte solutions for enzymatic hydrolysis

    PubMed Central

    2011-01-01

    Background Previous studies have shown that the crystalline structure of cellulose is negatively correlated with enzymatic digestibility, therefore, pretreatment is required to break down the highly ordered crystalline structure in cellulose, and to increase the porosity of its surface. In the present study, an organic electrolyte solution (OES) composed of an ionic liquid (1-allyl-3-methylimidazolium chloride ([AMIM]Cl)) and an organic solvent (dimethyl sulfoxide; DMSO) was prepared, and used to pretreat microcrystalline cellulose for subsequent enzymatic hydrolysis; to our knowledge, this is the first time that this method has been used. Results Microcrystalline cellulose (5 wt%) rapidly dispersed and then completely dissolved in an OES with a molar fraction of [AMIM]Cl per OES (χ [AMIM]Cl) of greater than or equal to 0.2 at 110°C within 10 minutes. The cellulose was regenerated from the OES by precipitation with hot water, and enzymatically hydrolyzed. As the χ [AMIM]Cl of the OES increased from 0.1 to 0.9, both the hydrolysis yield and initial hydrolysis rate of the regenerated cellulose also increased gradually. After treatment using OES with χ [AMIM]Cl of 0.7, the glucose yield (54.1%) was 7.2 times that of untreated cellulose. This promotion of hydrolysis yield was mainly due to the decrease in the degree of crystallinity (that is, the crystallinity index of cellulose I). Conclusions An OES of [AMIM]Cl and DMSO with χ [AMIM]Cl of 0.7 was chosen for cellulose pretreatment because it dissolved cellulose rapidly to achieve a high glucose yield (54.1%), which was only slightly lower than the value (59.6%) obtained using pure [AMIM]Cl. OES pretreatment is a cost-effective and environmentally friendly technique for hydrolysis, because it 1) uses the less expensive OES instead of pure ionic liquids, 2) shortens dissolution time, 3) requires lower energy for stirring and transporting, and 4) is recyclable. PMID:22099703

  3. Comparative study of different alcoholate pretreatments for enhanced enzymatic hydrolysis of sugarcane bagasse.

    PubMed

    Huang, Qing; Yan, Qiuli; Fu, Jing; Lv, Xiaojing; Xiong, Chunjiang; Lin, Jianghai; Liu, Zehuan

    2016-07-01

    Pretreatment of sugarcane bagasse (SCB) with alcoholates, sodium methoxide (CH3ONa), potassium methoxide (CH3OK) and sodium ethoxide (C2H5ONa), was investigated. Analyses of lignocellulose composition and enzymatic saccharification indicated that C2H5ONa showed the highest enzymatic efficiency of 102.1%. The response surface optimization of C2H5ONa pretreatment showed that under optimal conditions (4% of C2H5ONa, 121°C, 1h), 65.4% of lignin was removed and the enzymatic efficiency reached 105.2%. Hydrolysis of SCB with cellulases and xylanase at a ratio of 4:1 showed the strongest synergism with reducing sugar production of 21g/L and conversion rates of cellulose and xylan reaching 110.4% and 94.5%, respectively. These results indicated that C2H5ONa is a promising alkali to pretreat SCB and the synergism between cellulases and xylanase has a significant effect on enzymatic saccharification of the pretreated SCB. PMID:27035479

  4. Extraction of cellulose nano-crystals from old corrugated container fiber using phosphoric acid and enzymatic hydrolysis followed by sonication.

    PubMed

    Tang, Yanjun; Shen, Xiaochuang; Zhang, Junhua; Guo, Daliang; Kong, Fangong; Zhang, Nan

    2015-07-10

    Due to its amazing physicochemical properties and high environmental compatibility, cellulose nano-crystals (CNC) hold great promise for serving as a strategic platform for sustainable development. Now, there has been growing interest in the development of processes using waste or residual biomass as CNC source for addressing economic and environmental concerns. In the present work, a combined process involving phosphoric acid hydrolysis, enzymatic hydrolysis and sonication was proposed aiming to efficiently exact CNC from low-cost old corrugated container (OCC) pulp fiber. The effect of enzymatic hydrolysis on the yield and microstructure of resulting CNC was highlighted. Results showed that the enzymatic hydrolysis was effective in enhancing CNC yield after phosphoric acid hydrolysis. CNC was obtained with a yield of 23.98 wt% via the combined process with phosphoric acid concentration of 60 wt%, cellulase dosage of 2 mL (84 EGU) per 2g fiber and sonication intensity of 200 W. Moreover, the presence of enzymatic hydrolysis imparted the obtained CNC with improved dispersion, increased crystallinity and thermal stability. PMID:25857993

  5. A process for reduction in viscosity of coffee extract by enzymatic hydrolysis of mannan.

    PubMed

    Chauhan, Prakram Singh; Sharma, Prince; Puri, Neena; Gupta, Naveen

    2014-07-01

    Mannan is the main polysaccharide component of coffee extract and is responsible for its high viscosity, which in turn negatively affects the technological processing involved in making instant coffee. In our study, we isolated mannan from coffee beans and extract of commercial coffee and it was enzymatically hydrolyzed using alkali-thermostable mannanase obtained from Bacillus nealsonii PN-11. As mannan is found to be more soluble under alkaline conditions, an alkali-thermostable mannanase is well suited for its hydrolysis. The process of enzymatic hydrolysis was optimized by response surface methodology. Under the following optimized conditions viz enzyme dose of 11.50 U mannanase g(-1) coffee extract, temperature of 44.50 °C and time of 35.80 min, significant twofold decrease in viscosity (50 mPas to 26.00 ± 1.56 mPas) was achieved. The application of this process in large-scale industrial production of coffee will help in reduction of energy consumption used during freeze-drying. It will also make technological processing involved in making coffee more economical. PMID:24390577

  6. Enzymatic hydrolysis of beer brewers' spent grain and the influence of pretreatments

    SciTech Connect

    Beldman, G.; Hennekam, J.; Voragen, A.G.J.

    1987-01-01

    The enzymatic saccharification of plant material has been shown to be of interest in various fields, such as the production of fruit juices and the utilization of biomass. A combination of cellulase, pectinase, and hemicellulases is usually used because of the chemical composition of the matrix of plant cell walls. For apples, beet pulp, and potato fiber, almost a complete hydrolysis of polysaccharides is obtained by combining cellulase and pectinase. For nonparenchymatic tissue, the situation is somewhat different: pectin is a minor component and the hemicellulose content is much higher. Enzyme action is restricted by the lignin barrier and by the high crystallinity of cellulose in this material. For such materials, mechanical, thermal, or chemical pretreatments are necessary to achieve efficient hydrolysis. This communication describes various enzymatic treatments and chemical and physical pretreatment, using brewers' spent grain as substrate. Spent grain is the residue of malt and grain which remains in the mash-kettle after the liquefied and saccharified starch has been removed by filtration. (Refs. 15).

  7. Strategies to achieve high-solids enzymatic hydrolysis of dilute-acid pretreated corn stover.

    PubMed

    Geng, Wenhui; Jin, Yongcan; Jameel, Hasan; Park, Sunkyu

    2015-01-01

    Three strategies were presented to achieve high solids loading while maximizing carbohydrate conversion, which are fed-batch, splitting/thickening, and clarifier processes. Enzymatic hydrolysis was performed at water insoluble solids (WIS) of 15% using washed dilute-acid pretreated corn stover. The carbohydrate concentration increased from 31.8 to 99.3g/L when the insoluble solids content increased from 5% to 15% WIS, while the final carbohydrate conversion was decreased from 78.4% to 73.2%. For the fed-batch process, a carbohydrate conversion efficiency of 76.8% was achieved when solid was split into 60:20:20 ratio, with all enzymes added first. For the splitting/thickening process, a carbohydrate conversion of 76.5% was realized when the filtrate was recycled to simulate a steady-state process. Lastly, the clarifier process was evaluated and the highest carbohydrate conversion of 81.4% was achieved. All of these results suggests the possibility of enzymatic hydrolysis at high solids to make the overall conversion cost-competitive. PMID:25836373

  8. Correlation between lignin physicochemical properties and inhibition to enzymatic hydrolysis of cellulose.

    PubMed

    Yang, Qiang; Pan, Xuejun

    2016-06-01

    Using isolated organosolv lignins from hardwood poplar and softwood lodgepole pine with varied physicochemical properties (molecular weight, aliphatic hydroxyl, phenolic hydroxyl, and hydrophobicity), the inhibitory effect of the lignins on enzymatic hydrolysis of cellulose was investigated and the relationship between lignin properties and the inhibitory effect was elucidated. The results indicated that the lignin inhibition to enzymatic hydrolysis of cellulose was closely related to the hydrophobicity and the phenolic hydroxyl groups of the lignin. The overall hydrophobicity of the lignin quantified by contact angle could serve as a predictor of the inhibitory effect of lignin. Hydrophilic modification of the lignin by carboxylation and sulfonation reduced the hydrophobicity by 22-30% and thereby removed the lignin inhibition by 76-96%. Phenolic hydroxyl group was a crucial factor affecting the inhibitory effect of lignin. Blocking free phenolic hydroxyl group by chemical reaction such as hydroxypropylation significantly (65-91%) reduced the inhibitory effect of lignin. Biotechnol. Bioeng. 2016;113: 1213-1224. © 2015 Wiley Periodicals, Inc. PMID:26666388

  9. Topochemical pretreatment of wood biomass to enhance enzymatic hydrolysis of polysaccharides to sugars.

    PubMed

    Mou, Hong-Yan; Orblin, Elina; Kruus, Kristiina; Fardim, Pedro

    2013-08-01

    The surface chemistry of milled birch and pine wood pretreated by ionic liquid, hydrothermal and hydrotropic methods, followed by enzymatic hydrolysis was studied in this work. Surface coverage by lignin was measured by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to describe the surface chemical composition after pretreatment in detail, and the morphology after pretreatment was investigated by FE-SEM. Ionic liquid (1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride) pretreatment at room temperature made the samples swell but did not dissolve the wood. Comparing the surface coverage by lignin, both in the case of birch and pine wood, hydrotropic worked best to remove the lignin hampering enzymatic hydrolysis. ToF-SIMS supported this finding, and showed that in birch, the carbohydrates were degraded more than in pine after hydrotropic pretreatment. The glucose yield of birch was improved by hydrotropic pretreatment from 5.1% to 83.9%, more significantly than in case of pine. PMID:23774220

  10. Power consumption evaluation of different fed-batch strategies for enzymatic hydrolysis of sugarcane bagasse.

    PubMed

    Corrêa, Luciano Jacob; Badino, Alberto Colli; Cruz, Antonio José Gonçalves

    2016-05-01

    The minimization of costs in the distillation step of lignocellulosic ethanol production requires the use of a high solids loading during the enzymatic hydrolysis to obtain a more concentrated glucose liquor. However, this increase in biomass can lead to problems including increased mass and heat transfer resistance, decreased cellulose conversion, and increased apparent viscosity with the associated increase in power consumption. The use of fed-batch operation offers a promising way to circumvent these problems. In this study, one batch and four fed-batch strategies for solids and/or enzyme feeding during the enzymatic hydrolysis of sugarcane bagasse were evaluated. Determinations of glucose concentration, power consumption, and apparent viscosity were made throughout the experiments, and the different strategies were compared in terms of energy efficiency (mass of glucose produced according to the energy consumed). The best energy efficiency was obtained for the strategy in which substrate and enzyme were added simultaneously (0.35 kg(glucose) kWh⁻¹). This value was 52% higher than obtained in batch operation. PMID:26899602

  11. Dilute acid pretreatment and enzymatic hydrolysis of sorghum biomass for sugar recovery--a statistical approach.

    PubMed

    Akanksha, Karthik; Prasad, Arjun; Sukumaran, Rajeev K; Nampoothiri, Madhavan; Pandey, Ashok; Rao, S S; Parameswaran, Binod

    2014-11-01

    Sorghum is one of the commercially feasible lignocellulosic biomass and has a great potential of being sustainable feedstock for renewable energy. As with any lignocellulosic biomass, sorghum also requires pretreatment which increases its susceptibility to hydrolysis by enzymes for generating sugars which can be further fermented to alcohol. In the present study, sorghum biomass was evaluated for deriving maximum fermentable sugars by optimizing various pretreatment parameters using statistical optimization methods. Pretreatment studies were done with H2SO4, followed by enzymatic saccharification. The efficiency of the process was evaluated on the basis of production of the total reducing sugars released during the process. Compositional analysis was done for native as well as pretreated biomass and compared. The biomass pretreated with the optimized conditions could yield 0.408 g of reducing sugars /g of pretreated biomass upon enzymatic hydrolysis. The cellulose content in the solid portion obtained after pretreatment using optimised conditions was found to be increased by 43.37% with lesser production of inhibitors in acid pretreated liquor. PMID:25434103

  12. The effect of natural antioxidants on haemoglobin-mediated lipid oxidation during enzymatic hydrolysis of cod protein.

    PubMed

    Halldorsdottir, Sigrun M; Kristinsson, Hordur G; Sveinsdottir, Holmfridur; Thorkelsson, Gudjon; Hamaguchi, Patricia Y

    2013-11-15

    Heating and changes in pH often practised during fish protein hydrolysis can cause lipid oxidation. The effect of natural antioxidants towards haemoglobin-mediated lipid oxidation during enzymatic hydrolysis of cod proteins was investigated. Different variants of a washed cod model system, containing different combinations of haemoglobin and natural antioxidants (l-ascorbic acid and Fuscus vesiculosus extract), were hydrolysed using Protease P "Amano" 6 at pH 8 and 36°C to achieve 20% degree of hydrolysis. Lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS) were analysed periodically during the hydrolysis process. The in vitro antioxidant activity of the final products was investigated. Results indicate that oxidation can develop rapidly during hydrolysis and antioxidant strategies are preferable to produce good quality products. Oxidation products did not have an impact on the in vitro antioxidant activity of the hydrolysates. The natural antioxidants inhibited oxidation during hydrolysis and contributed to the antioxidant activity of the final product. PMID:23790867

  13. The enzymatic hydrolysis of pretreated pulp fibers predominantly involves “peeling/erosion” modes of action

    PubMed Central

    2014-01-01

    Background There is still considerable debate regarding the actual mechanism by which a “cellulase mixture” deconstructs cellulosic materials, with accessibility to the substrate at the microscopic level being one of the major restrictions that limits fast, complete cellulose hydrolysis. In the work reported here we tried to determine the predominant mode of action, at the fiber level, of how a cellulase mixture deconstructs pretreated softwood and hardwood pulp fibers. Quantitative changes in the pulp fibers derived from different pretreated biomass substrates were monitored throughout the course of enzymatic hydrolysis to see if the dominant mechanisms involved either the fragmentation/cutting of longer fibers to shorter fibers or their “peeling/delamination/erosion,” or if both cutting and peeling mechanisms occurred simultaneously. Results Regardless of the source of biomass, the type of pretreatment and the chemical composition of the substrate, under typical hydrolysis conditions (50°C, pH 4.8, mixing) longer pulp fibers (fiber length >200 μm) were rapidly broken down until a relatively constant fiber length of 130 to 160 μm was reached. In contrast, shorter fibers with an initial average fiber length of 130 to 160 μm showed no significant change in length despite their substantial hydrolysis. The fragmentation/cutting mode of deconstruction was only observed on longer fibers at early stages of hydrolysis. Although the fiber fragmentation mode of deconstruction was not greatly influenced by enzyme loading, it was significantly inhibited by glucose and was mainly observed during initial mixing of the enzyme and substrate. In contrast, significant changes in the fiber width occurred throughout the course of hydrolysis for all of the substrates, suggesting that fiber width may limit the rate and extent of cellulose hydrolysis. Conclusion It appears that, at the fiber level, pretreated pulp fibers are hydrolyzed through a two-step mode of action

  14. Optimisation of the Enzymatic Hydrolysis of Blood Cells with a Neutral Protease

    PubMed Central

    Zheng, Yanbin; Chen, Qiushi; Shan, Anshan; Zhang, Hao

    2013-01-01

    For utilizing the blood cells (BCs) effectively, enzymatic hydrolysis was applied to produce the enzymatically hydrolyzed blood cells (EHBCs) by using a neutral protease as a catalyst. The results of the single-factor experiments showed optimal substrate concentration, enzyme to substrate ratio (E/S), pH, temperature, and incubation period were 1.00%, 0.10, 7.00, 50.00°C, and 12.00 h, respectively. The optimized hydrolysis conditions from response surface methodology (RSM) were pH 6.50, E/S 0.11, temperature 45.00°C, and incubation period 12.00 h. Under these conditions (substrate concentration 1.00%), the degree of hydrolysis (DH) was 35.06%. The free amino acids (FAAs) content of the EHBCs (35.24%) was 40.46 times higher than BCs while the total amino acids (TAAs) content was lower than BCs. The scores of lysine (human 0.87; pig 0.97), valine (human 1.42; pig 1.38), leucine (human 1.50; pig 1.90), tyrosine (human 0.84; pig 1.09), and histidine (human 2.17; pig 2.50) indicated that the EHBCs basically fulfilled the adult human and pig nutritional requirements. The calculated protein efficiency ratios (C-PERs) of the EHBCs were 3.94, 6.19, 21.73, and 2.04. In summary, the EHBCs were produced successfully with optimized conditions and could be a novel protein source for humans and pigs. PMID:23484101

  15. Improvement of enzymatic hydrolysis and ethanol production from corn stalk by alkali and N-methylmorpholine-N-oxide pretreatments.

    PubMed

    Cai, Ling-Yan; Ma, Yu-Long; Ma, Xiao-Xia; Lv, Jun-Min

    2016-07-01

    A combinative technology of alkali and N-methylmorpholine-N-oxide (NMMO) was used to pretreat corn stalk (CS) for improving the efficiencies of subsequent enzymatic hydrolysis and ethanol fermentation. The results showed that this strategy could not only remove hemicellulose and lignin but also decrease the crystallinity of cellulose. About 98.0% of enzymatic hydrolysis yield was obtained from the pretreated CS as compared with 46.9% from the untreated sample. The yield for corresponding ethanol yield was 64.6% while untreated CS was only 18.8%. Besides, xylose yield obtained from the untreated CS was only 11.1%, while this value was 93.8% for alkali with NMMO pretreated sample. These results suggest that a combination of alkali with 50% (wt/wt) NMMO solution may be a promising alternative for pretreatment of lignocellulose, which can increase the productions of subsequent enzymatic hydrolysis and ethanol fermentation. PMID:27078206

  16. Utilization of waste cellulose. VI. Pretreatment of lignocellulosic materials with sodium hypochlorite and enzymatic hydrolysis by Trichoderma viride

    SciTech Connect

    David, C.; Fornasier, R.; Thiry, P.

    1985-10-01

    A pretreatment of lignocellulosic materials with sodium hypochlorite-hypochlorous acid at controlled pH (between 7 and 9) considerably increases the accessibility of the cellulosic part of the substrate to chemical and biochemical reactants. As a consequence, the yield and rate of the enzymatic hydrolysis to glucose is largely increased. Wheat straw and spruce sawdust have been investigated. The increase in accessibility is assigned to degradation and (or) detachment of the lignin network. The loss in cellulose and hemicellulose is not important, lignin being preferentially degraded under carefully controlled pH conditions. When applied to pure cellulose, the pretreatment decreases the yield of enzymatic hydrolysis; in the absence of lignin, oxidation of the anhydroglucose units is important and results in the inhibition of the enzymatic hydrolysis. 12 references.

  17. Enzymatic cellulose hydrolysis: enzyme reusability and visualization of β-glucosidase immobilized in calcium alginate.

    PubMed

    Tsai, Chien-Tai; Meyer, Anne S

    2014-01-01

    The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde cross-linked BG was entrapped in calcium alginate gel particles. More than 60% of the enzyme activity could be recovered under optimized conditions, and glutaraldehyde cross-linking decreased leakage of BG from the calcium alginate particles. The immobilized BG aggregates were visualized by confocal laser scanning microscopy (CLSM). The CLSM images, which we believe are the first to be published, corroborate that more BG aggregates were entrapped in the matrix when the enzymes were cross-linked by glutaraldehyde as opposed to when they are not cross-linked. The particles with the immobilized BG were recycled for cellulase catalyzed hydrolysis of Avicel. No significant loss in BG activity was observed for up to 20 rounds of reaction recycle steps of the BG particles of 48 h each, verifying a significant stabilization of the BG by immobilization. Similar high glucose yields were obtained by one round of enzymatic hydrolysis of hydrothermally pretreated barley straw during a 72 h reaction with immobilized BG and free BG. PMID:25429563

  18. Improving enzymatic hydrolysis of corn stover pretreated by ethylene glycol-perchloric acid-water mixture.

    PubMed

    He, Yu-Cai; Liu, Feng; Gong, Lei; Lu, Ting; Ding, Yun; Zhang, Dan-Ping; Qing, Qing; Zhang, Yue

    2015-02-01

    To improve the enzymatic saccharification of lignocellulosic biomass, a mixture of ethylene glycol-HClO4-water (88.8:1.2:10, w/w/w) was used for pretreating corn stover in this study. After the optimization in oil-bath system, the optimum pretreatment temperature and time were 130 °C and 30 min, respectively. After the saccharification of 10 g/L pretreated corn stover for 48 h, the saccharification rate was obtained in the yield of 77.4 %. To decrease pretreatment temperature and shorten pretreatment time, ethylene glycol-HClO4-water (88.8:1.2:10, w/w/w) media under microwave irradiation was employed to pretreat corn stover effectively at 100 °C and 200 W for 5 min. Finally, the recovered hydrolyzates containing glucose obtained from the enzymatic hydrolysis of pretreated corn stovers could be fermented into ethanol efficiently. These results would be helpful for developing a cost-effective pretreatment combined with enzymatic saccharification of cellulosic materials for the production of lignocellulosic ethanol. PMID:25384544

  19. Phenols and lignin: Key players in reducing enzymatic hydrolysis yields of steam-pretreated biomass in presence of laccase.

    PubMed

    Oliva-Taravilla, Alfredo; Tomás-Pejó, Elia; Demuez, Marie; González-Fernández, Cristina; Ballesteros, Mercedes

    2016-01-20

    Phenols are known as inhibitors for cellulases and fermentative microorganisms in bioethanol production processes. The addition of laccases removes the phenolic compounds and subsequently reduces the lag phase of the fermentative microorganism. However, the application of laccases diminishes glucose release during the enzymatic hydrolysis. In this study a model cellulosic substrate (Sigmacell) together with lignin extract, whole steam-pretreated wheat straw (slurry) and its water insoluble solid fraction (WIS) were subjected to enzymatic hydrolysis to evaluate the effects of laccase treatment in presence of lignin and phenols. The presence of laccase in enzymatic hydrolysis of Sigmacell with lignin extract reduced glucose yield by 37% compared with assays without laccase. Furthermore, this reduction was even more marked in presence of phenols (55% reduction). Interestingly, when hydrolyzing WIS, the addition of phenols coupled with laccase treatment did not show a reduction when compared with only laccase addition. This fact suggests the key role of lignin in the hydrolysis inhibition since in WIS the ratio cellulase per gram of lignin was much lower than in Sigmacell experiments. Finally, the lower cellobiose and xylose recoveries point out that phenolic oligomers formed by laccase oxidation play important roles in the inhibition of endoglucanases, cellobiohydrolases and xylanases. To conclude, the proportion of lignin and the composition of phenols are key players in the inhibition of cellulases when the enzymatic hydrolysis is combined with laccases detoxification. PMID:26684987

  20. Mixing design for enzymatic hydrolysis of sugarcane bagasse: methodology for selection of impeller configuration.

    PubMed

    Corrêa, Luciano Jacob; Badino, Alberto Colli; Cruz, Antonio José Gonçalves

    2016-02-01

    One of the major process bottlenecks for viable industrial production of second generation ethanol is related with technical-economic difficulties in the hydrolysis step. The development of a methodology to choose the best configuration of impellers towards improving mass transfer and hydrolysis yield together with a low power consumption is important to make the process cost-effective. In this work, four dual impeller configurations (DICs) were evaluated during hydrolysis of sugarcane bagasse (SCB) experiments in a stirred tank reactor (3 L). The systems tested were dual Rushton turbine impellers (DIC1), Rushton and elephant ear (down-pumping) turbines (DIC2), Rushton and elephant ear (up-pumping) turbines (DIC3), and down-pumping and up-pumping elephant ear turbines (DIC4). The experiments were conducted during 96 h, using 10 % (m/v) SCB, pH 4.8, 50 °C, 10 FPU/g biomass, 470 rpm. The mixing time was successfully used as the characteristic parameter to select the best impeller configuration. Rheological parameters were determined using a rotational rheometer, and the power consumptions of the four DICs were on-line measured with a dynamometer. The values obtained for the energetic efficiency (the ratio between the cellulose to glucose conversion and the total energy) showed that the proposed methodology was successful in choosing a suitable configuration of impellers, wherein the DIC4 obtained approximately three times higher energetic efficiency than DIC1. Furthermore a scale-up protocol (factor scale-up 1000) for the enzymatic hydrolysis reactor was proposed. PMID:26650719

  1. Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulase.

    PubMed

    Kim, In Jung; Lee, Hee Jin; Choi, In-Geol; Kim, Kyoung Heon

    2014-10-01

    Reducing the enzyme loadings for enzymatic saccharification of lignocellulose is required for economically feasible production of biofuels and biochemicals. One strategy is addition of small amounts of synergistic proteins to cellulase mixtures. Synergistic proteins increase the activity of cellulase without causing significant hydrolysis of cellulose. Synergistic proteins exert their activity by inducing structural modifications in cellulose. Recently, synergistic proteins from various biological sources, including bacteria, fungi, and plants, were identified based on genomic data, and their synergistic activities were investigated. Currently, an up-to-date overview of several aspects of synergistic proteins, such as their functions, action mechanisms and synergistic activity, are important for future industrial application. In this review, we summarize the current state of research on four synergistic proteins: carbohydrate-binding modules, plant expansins, expansin-like proteins, and Auxiliary Activity family 9 (formerly GH61) proteins. This review provides critical information to aid in promoting research on the development of efficient and industrially feasible synergistic proteins. PMID:25129610

  2. Characterization of sugar beet pectic-derived oligosaccharides obtained by enzymatic hydrolysis.

    PubMed

    Combo, Agnan Marie Michel; Aguedo, Mario; Quiévy, Nicolas; Danthine, Sabine; Goffin, Dorothée; Jacquet, Nicolas; Blecker, Christophe; Devaux, Jacques; Paquot, Michel

    2013-01-01

    Three pectic oligosaccharides (POS) obtained by enzymatic hydrolysis of sugar beet pectin by combining endopolygalacturonase and pectinmethylesterase, were characterized using high performance liquid chromatography, thermogravimetric analysis, Fourier transform infrared spectroscopy, differential scanning calorimetry and X-ray diffraction. According to chromatographic analyses, POS are composed of mixture of polymers with different molecular weights and different galacturonic acid contents. The thermal analysis showed no major variation in thermal behavior regarding POS composition but showed that POS were more sensitive to thermal degradation than the parent pectin as well as the deesterified pectin. No change in composition of the gaseous products was obtained through TGA-FTIR analysis. The X-ray pattern of POS clearly indicated a considerable decrease in crystallinity when compared to the native pectin. Thus, thermal characterization of POS may have practical repercussions if the formulation in which POS is incorporated is submitted to a high temperature treatment. PMID:22986181

  3. Hydrothermal pretreatment enhanced enzymatic hydrolysis and glucose production from oil palm biomass.

    PubMed

    Zakaria, Mohd Rafein; Hirata, Satoshi; Hassan, Mohd Ali

    2015-01-01

    The present works investigate hydrothermal pretreatment of oil palm empty fruit bunch and oil palm frond fiber in a batch tube reactor system with temperature and time range from 170 to 250°C and 10 to 20min, respectively. The behavior of soluble sugars, acids, furans, and phenols dramatically changed over treatment severities as determined by HPLC. The cellulose-rich treated solids were analyzed by SEM, WAXD, and BET surface area. Enzymatic hydrolysis was performed from both pretreated slurries and washed solid, and data obtained suggested that tannic acid derived from lignin degradation was a potential cellulase inhibitor. Both partial removal of hemicellulose and migration of lignin during hydrothermal pretreatment caused structural changes on the cellulose-hemicellulose-lignin matrix, resulting in the opening and expansion of specific surface area and pore volume. The current results provided important factors that maximize conversion of cellulose to glucose from oil palm biomass by hydrothermal process. PMID:25460995

  4. Process for enzymatic hydrolysis of fatty acid triglycerides with oat caryopses

    SciTech Connect

    Hammond, E.G.; Lee, I.

    1992-02-18

    This patent describes the process for enzymatic hydrolysis of fatty acid triglycerides to obtain free fatty acids and glycerol. It comprises: increasing the water content of dehulled whole oat caryopses to a total water content of 17 to 44% the thus moistened caryopses having active oat lipase associated with the outer surfaces thereof; contacting the moistened whole caryopses with a liquid medium, continuing the contacting until at least 20% by volume of the triglyceride reactant has been hydrolyzed to free fatty acids and glycerol, most of the free fatty acids dissolving in the oil phase external to the caryopses and most of the glycerol being absorbed into the water within the caryopses; and separating the glycerol-containing caryopses from the fatty acid-containing oil phase.

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

    PubMed

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

    2016-04-01

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

  6. Preparation and characterization of phenol-formaldehyde adhesives modified with enzymatic hydrolysis lignin.

    PubMed

    Jin, Yanqiao; Cheng, Xiansu; Zheng, Zuanbin

    2010-03-01

    Phenol-formaldehyde (PF) adhesives modified with enzymatic hydrolysis lignin (EHL) were synthesized by a one-step process. The phenol component of the PF adhesives was partially substituted by EHL extracted from the residues of cornstalks used to produce bio-ethanol. The EHL-PF adhesives were used to prepare plywoods by hot-pressing. The pH value, viscosity, solid content, free phenol content, free formaldehyde content and brominable substance content of EHL-PF resins were investigated. The bonding strengths of the plywoods were determined, and the influences of the replacement percentage of phenol by EHL (a) and the NaOH content (b) on the properties of the adhesives were investigated. The results showed that the performance of the modified adhesives and the plywoods glued with them almost met the Chinese National Standard (GB/T 14732-2006) for first grade plywood when 20 wt% of the phenol was replaced by EHL. PMID:19854642

  7. The role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials.

    PubMed

    Sun, Shaoni; Sun, Shaolong; Cao, Xuefei; Sun, Runcang

    2016-01-01

    Lignocellulosic materials are among the most promising alternative energy resources that can be utilized to produce cellulosic ethanol. However, the physical and chemical structure of lignocellulosic materials forms strong native recalcitrance and results in relatively low yield of ethanol from raw lignocellulosic materials. An appropriate pretreatment method is required to overcome this recalcitrance. For decades various pretreatment processes have been developed to improve the digestibility of lignocellulosic biomass. Each pretreatment process has a different specificity on altering the physical and chemical structure of lignocellulosic materials. In this paper, the chemical structure of lignocellulosic biomass and factors likely affect the digestibility of lignocellulosic materials are discussed, and then an overview about the most important pretreatment processes available are provided. In particular, the combined pretreatment strategies are reviewed for improving the enzymatic hydrolysis of lignocellulose and realizing the comprehensive utilization of lignocellulosic materials. PMID:26321216

  8. Developmental Transition from Enzymatic to Acid Hydrolysis of Sucrose in Acid Limes (Citrus aurantifolia) 1

    PubMed Central

    Echeverria, Ed

    1990-01-01

    The sucrose breakdown mechanisms in juice sacs of acid lime (Citrus aurantifolia [Christm.] Swing.) were investigated throughout fruit development. All three enzymes of sucrose catabolism (sucrose synthase, acid, and alkaline invertase) are present during the initial stages. The activities of these enzymes declined rapidly and disappeared by stage 5 (80% development) but not before vacuolar pH had decreased to approximately 2.5. At this stage, sucrose breakdown occurs by acid hydrolysis. By attaining a vacuolar pH of 2.5 prior to enzyme disappearance, the cell maintains a continuous ability to break down sucrose throughout ontogeny. Thus, acid limes possess a unique and coordinated system for sucrose breakdown that involves both enzymatic and nonenzymatic pathways. PMID:16667241

  9. Enhancement of enzymatic hydrolysis of wheat straw by gamma irradiation-alkaline pretreatment

    NASA Astrophysics Data System (ADS)

    Yin, Yanan; Wang, Jianlong

    2016-06-01

    Pretreatment of wheat straw with gamma irradiation and NaOH was performed to enhance the enzymatic hydrolysis of wheat straw for production of reducing sugar. The results showed that the irradiation of wheat straw at 50 kGy decreased the yield of reducing sugar, however, the reducing sugar yield increased with increasing dose from 50 kGy to 400 kGy. The irradiation of wheat straw at 100 kGy can significantly decrease NaOH consumption and treatment time. The reducing sugar yield could reach 72.67% after irradiation at 100 kGy and 2% NaOH treatment for 1 h. The combined pretreatment of wheat straw by gamma radiation and NaOH immersion can increase the solubilization of hemicellulose and lignin as well as the accessible surface area for enzyme molecules.

  10. Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose

    PubMed Central

    Kristensen, Jan B; Felby, Claus; Jørgensen, Henning

    2009-01-01

    Background Working at high solids (substrate) concentrations is advantageous in enzymatic conversion of lignocellulosic biomass as it increases product concentrations and plant productivity while lowering energy and water input. However, for a number of lignocellulosic substrates it has been shown that at increasing substrate concentration, the corresponding yield decreases in a fashion which can not be explained by current models and knowledge of enzyme-substrate interactions. This decrease in yield is undesirable as it offsets the advantages of working at high solids levels. The cause of the 'solids effect' has so far remained unknown. Results The decreasing conversion at increasing solids concentrations was found to be a generic or intrinsic effect, describing a linear correlation from 5 to 30% initial total solids content (w/w). Insufficient mixing has previously been shown not to be involved in the effect. Hydrolysis experiments with filter paper showed that neither lignin content nor hemicellulose-derived inhibitors appear to be responsible for the decrease in yields. Product inhibition by glucose and in particular cellobiose (and ethanol in simultaneous saccharification and fermentation) at the increased concentrations at high solids loading plays a role but could not completely account for the decreasing conversion. Adsorption of cellulases was found to decrease at increasing solids concentrations. There was a strong correlation between the decreasing adsorption and conversion, indicating that the inhibition of cellulase adsorption to cellulose is causing the decrease in yield. Conclusion Inhibition of enzyme adsorption by hydrolysis products appear to be the main cause of the decreasing yields at increasing substrate concentrations in the enzymatic decomposition of cellulosic biomass. In order to facilitate high conversions at high solids concentrations, understanding of the mechanisms involved in high-solids product inhibition and adsorption inhibition

  11. Efficient sugar release by the cellulose solvent-based lignocellulose fractionation technology and enzymatic cellulose hydrolysis.

    PubMed

    Moxley, Geoffrey; Zhu, Zhiguang; Zhang, Y-H Percival

    2008-09-10

    Efficient liberation of fermentable soluble sugars from lignocellulosic biomass waste not only decreases solid waste handling but also produces value-added biofuels and biobased products. Industrial hemp, a special economic crop, is cultivated for its high-quality fibers and high-value seed oil, but its hollow stalk cords (hurds) are a cellulosic waste. The cellulose-solvent-based lignocellulose fractionation (CSLF) technology has been developed to separate lignocellulose components under modest reaction conditions (Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Biotechnol. Bioeng. 2007, 97 (2), 214- 223). Three pretreatment conditions (acid concentration, reaction temperature, and reaction time) were investigated to treat industrial hemp hurds for a maximal sugar release: a combinatorial result of a maximal retention of solid cellulose and a maximal enzymatic cellulose hydrolysis. At the best treatment condition (84.0% H3PO4 at 50 degrees C for 60 min), the glucan digestibility was 96% at hour 24 at a cellulase loading of 15 filter paper units of cellulase per gram of glucan. The scanning electron microscopic images were presented for the CSLF-pretreated biomass for the first time, suggesting that CSLF can completely destruct the plant cell-wall structure, in a good agreement with the highest enzymatic cellulose digestibility and fastest hydrolysis rate. It was found that phosphoric acid only above a critical concentration (83%) with a sufficient reaction time can efficiently disrupt recalcitrant lignocellulose structures. PMID:18702466

  12. Torque measurements reveal large process differences between materials during high solid enzymatic hydrolysis of pretreated lignocellulose

    PubMed Central

    2012-01-01

    Background A common trend in the research on 2nd generation bioethanol is the focus on intensifying the process and increasing the concentration of water insoluble solids (WIS) throughout the process. However, increasing the WIS content is not without problems. For example, the viscosity of pretreated lignocellulosic materials is known to increase drastically with increasing WIS content. Further, at elevated viscosities, problems arise related to poor mixing of the material, such as poor distribution of the enzymes and/or difficulties with temperature and pH control, which results in possible yield reduction. Achieving good mixing is unfortunately not without cost, since the power requirements needed to operate the impeller at high viscosities can be substantial. This highly important scale-up problem can easily be overlooked. Results In this work, we monitor the impeller torque (and hence power input) in a stirred tank reactor throughout high solid enzymatic hydrolysis (< 20% WIS) of steam-pretreated Arundo donax and spruce. Two different process modes were evaluated, where either the impeller speed or the impeller power input was kept constant. Results from hydrolysis experiments at a fixed impeller speed of 10 rpm show that a very rapid decrease in impeller torque is experienced during hydrolysis of pretreated arundo (i.e. it loses its fiber network strength), whereas the fiber strength is retained for a longer time within the spruce material. This translates into a relatively low, rather WIS independent, energy input for arundo whereas the stirring power demand for spruce is substantially larger and quite WIS dependent. By operating the impeller at a constant power input (instead of a constant impeller speed) it is shown that power input greatly affects the glucose yield of pretreated spruce whereas the hydrolysis of arundo seems unaffected. Conclusions The results clearly highlight the large differences between the arundo and spruce materials, both in terms of

  13. Acceleration of the Enzymatic Hydrolysis of Corn Stover and Sugar Cane Bagasse Celluloses by Low Intensity Uniform Ultrasound

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cost-competitive production of bio-ethanol and other biofuels is currently impeded, mostly by high cost and low efficiency of enzymatic hydrolysis of feedstock biomass and especially plant celluloses. Despite substantial reduction in the cost of production of cellulolytic enzymes in recent times...

  14. Performance of coupled enzymatic hydrolysis and membrane separation bioreactor for antihypertensive peptides production from Porphyra yezoensis protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To explore more efficient production methods of antihypertensive peptides from Porphyra yezoensis protein, three methods of coupling of enzymatic hydrolysis and membrane separation (CEH-MS) were studied and compared with the traditional EH and offline MS method. The results showed that the conversio...

  15. Hydrolysis of Miscanthus for bioethanol production using dilute acid presoaking combined with wet explosion pre-treatment and enzymatic treatment.

    PubMed

    Sørensen, Annette; Teller, Philip J; Hilstrøm, Troels; Ahring, Birgitte K

    2008-09-01

    Miscanthus is a high yielding bioenergy crop. In this study we used acid presoaking, wet explosion, and enzymatic hydrolysis to evaluate the combination of the different pre-treatment methods for bioethanol production with Miscanthus. Acid presoaking is primarily carried out in order to remove xylose prior to wet explosion. The acid presoaking extracted 63.2% xylose and 5.2% glucose. Direct enzymatic hydrolysis of the presoaked biomass was found to give only low sugar yields of 24-26% glucose. Wet explosion is a pre-treatment method that combines wet-oxidation and steam explosion. The effect of wet explosion on non-presoaked and presoaked Miscanthus was investigated using both atmospheric air and hydrogen peroxide as the oxidizing agent. All wet explosion pre-treatments showed to have a disrupting effect on the lignocellulosic biomass, making the sugars accessible for enzymatic hydrolysis. The combination of presoaking, wet explosion, and enzymatic hydrolysis was found to give the highest sugar yields. The use of atmospheric air gave the highest xylose yield (94.9% xylose, 61.3% glucose), while hydrogen peroxide gave the highest glucose yield (82.4% xylose, 63.7% glucose). PMID:18164954

  16. Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of β-Keto Esters

    PubMed Central

    Dold, S. -M.; Zimmermann, S.; Hamacher, K.; Schmitz, K.; Rudat, J.

    2016-01-01

    β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods—namely, the classical Z’-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening. PMID:26730596

  17. Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of β-Keto Esters.

    PubMed

    Buß, O; Jager, S; Dold, S-M; Zimmermann, S; Hamacher, K; Schmitz, K; Rudat, J

    2016-01-01

    β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods-namely, the classical Z'-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening. PMID:26730596

  18. Sulfuric Acid Pretreatment and Enzymatic Hydrolysis of Photoperiod Sensitvie Sorghum for Ethanol Production

    SciTech Connect

    F Xu; Y Shi; X Wu

    2011-12-31

    Photoperiod sensitive (PS) sorghum, with high soluble sugar content, high mass yield and high drought tolerance in dryland environments, has great potential for bioethanol production. The effect of diluted sulfuric acid pretreatment on enzymatic hydrolysis was investigated. Hydrolysis efficiency increased from 78.9 to 94.4% as the acid concentration increased from 0.5 to 1.5%. However, the highest total glucose yield (80.3%) occurred at the 1.0% acid condition because of the significant cellulose degradation at the 1.5% concentration. Synchrotron wide-angle X-ray diffraction was used to study changes of the degree of crystallinity. With comparison of cellulosic crystallinity and adjusted cellulosic crystallinity, the crystalline cellulose decreased after low acidic concentration (0.5%) applied, but did not change significantly, as the acid concentration increased. Scanning electron microscopy was also employed to understand how the morphological structure of PS sorghum changed after pretreatment. Under current processing conditions, the total ethanol yield is 74.5% (about 0.2 g ethanol from 1 g PS sorghum). A detail mass balance was also provided.

  19. Kinetic analysis of two-phase enzymatic hydrolysis of hemicellulose of xylan type.

    PubMed

    Dutta, Sajal Kanti; Chakraborty, Saikat

    2015-12-01

    We present a coupled experimental and theoretical framework for quantifying the kinetics of two-phase enzymatic hydrolysis of hemicellulose. For xylan loading of 1-5mg/ml, the nature of inhibition by the product xylose (non-competitive), the kinetic constants (Km=3.93 mg/ml, Vmax=0.0252 mg/ml/min) and the xylose inhibition constant (Kx=0.122 mg/ml) are experimentally determined. Our multi-step two-phase kinetic model incorporating enzyme adsorption to the solid substrate and non-competitive product inhibition is simulated using our kinetic data and validated against our experimentally measured temporal dynamics of xylose and reducing sugars. Further experiments show that higher substrate loading reduces the specific adsorption of the endoxylanase to the solid xylan and the enzyme's solid-liquid distribution ratio, which decelerates the solid hydrolysis and accelerates the liquid phase reactions. Thus, the xylose yield increases with substrate loading, which increases product inhibition and decreases reducing sugar yields. An operating cost analysis gives 3mg/ml as the optimal substrate loading. PMID:26433789

  20. Enhancing the Antioxidant Ability of Trametes versicolor Polysaccharopeptides by an Enzymatic Hydrolysis Process.

    PubMed

    Jhan, Mei-Hsin; Yeh, Ching-Hua; Tsai, Chia-Chun; Kao, Ching-Tian; Chang, Chao-Kai; Hsieh, Chang-Wei

    2016-01-01

    Polysaccharopeptides (PSPs) are among the main bioactive constituents of Trametes versicolor (T. versicolor). The purpose of this research was to investigate the antioxidant activities of enzymatic hydrolysates obtained from T. versicolor polysaccharopeptides by 80 U/mL β-1,3-glucanase (PSPs-EH80). The half-inhibitory concentration (IC50) of PSPs-EH80 in metal chelating assay, ABTS and DPPH radical scavenging test results were 0.83 mg/mL, 0.14 mg/mL and 0.52 mg/mL, respectively, which were lower than that of PSPs-EH 20 U/mL. The molecular weights of the PSPs-EH80 hydrolysates were 300, 190, 140 and 50 kDa, respectively, and the hydrolysis of polysaccharides by β-1,3-glucanase did not change the original functional group. PSPs-EH80 reduced the reactive oxygen species (ROS) content at least twice that of treatment without PSPs-EH80. In addition, an oxidative damage test showed that PSPs-EH80 can improve HaCaT cell survival. According to our results, PSP demonstrates the potential of anti-oxidative damage; besides, enzyme hydrolysis can improve the ability of the PSP. PMID:27626400

  1. Ammonia fiber expansion pretreatment and enzymatic hydrolysis on two different growth stages of reed canarygrass.

    PubMed

    Bradshaw, Tamika C; Alizadeh, Hasan; Teymouri, Farzaneh; Balan, Venkatesh; Dale, Bruce E

    2007-04-01

    Plant materials from the vegetative growth stage of reed canarygrass and the seed stage of reed canarygrass are pretreated by ammonia fiber expansion (AFEX) and enzymatically hydrolyzed using 15 filter paper units (FPU) cellulase/g glucan to evaluate glucose and xylose yields. Percent conversions of glucose and xylose, effects of temperature and ammonia loading, and hydrolysis profiles are analyzed to determine the most effective AFEX treatment condition for each of the selected materials. The controls used in this study were untreated samples of each biomass material. All pretreatment conditions tested enhanced enzyme digestibility and improved sugar conversions for reed canarygrass compared with their untreated counterparts. Based on 168 h hydrolysis results using 15 FPU Spezyme CP cellulase/g glucan the most effective AFEX treatment conditions were determined as: vegetative growth stage of reed canarygrass--100 degrees C, 60% moisture content, 1.2:1 kg ammonia/kg of dry matter (86% glucose and 78% xylose) and seed stage of reed canarygrass--100 degrees C, 60% moisture content, 0.8:1 kg ammonia/kg of dry matter (89% glucose and 81% xylose). Supplementation by commercial Multifect 720 xylanase along with cellulase further increased both glucose and xylose yields by 10-12% at the most effective AFEX conditions. PMID:18478404

  2. Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover.

    PubMed

    Ohgren, Karin; Bura, Renata; Saddler, Jack; Zacchi, Guido

    2007-09-01

    Ethanol can be produced from lignocellulosic biomass using steam pretreatment followed by enzymatic hydrolysis and fermentation. The sugar yields, from both hemicellulose and cellulose are critical parameters for an economically-feasible ethanol production process. This study shows that a near-theoretical glucose yield (96-104%) from acid-catalysed steam pretreated corn stover can be obtained if xylanases are used to supplement cellulases during hydrolysis. Xylanases hydrolyse residual hemicellulose, thereby improving the access of enzymes to cellulose. Under these conditions, xylose yields reached 70-74%. When pre-treatment severity was reduced by using autocatalysis instead of acid-catalysed steam pretreatment, xylose yields were increased to 80-86%. Partial delignification of pretreated material was also evaluated as a way to increase the overall sugar yield. The overall glucose yield increased slightly due to delignification but the overall xylose yield decreased due to hemicellulose loss in the delignification step. The data also demonstrate that steam pretreatment is a robust process: corn stover from Europe and North America showed only minor differences in behaviour. PMID:17113771

  3. Enzymatic neutralization of the chemical warfare agent VX: evolution of phosphotriesterase for phosphorothiolate hydrolysis.

    PubMed

    Bigley, Andrew N; Xu, Chengfu; Henderson, Terry J; Harvey, Steven P; Raushel, Frank M

    2013-07-17

    The V-type nerve agents (VX and VR) are among the most toxic substances known. The high toxicity and environmental persistence of VX make the development of novel decontamination methods particularly important. The enzyme phosphotriesterase (PTE) is capable of hydrolyzing VX but with an enzymatic efficiency more than 5 orders of magnitude lower than with its best substrate, paraoxon. PTE has previously proven amenable to directed evolution for the improvement of catalytic activity against selected compounds through the manipulation of active-site residues. Here, a series of sequential two-site mutational libraries encompassing 12 active-site residues of PTE was created. The libraries were screened for catalytic activity against a new VX analogue, DEVX, which contains the same thiolate leaving group of VX coupled to a diethoxyphosphate core rather than the ethoxymethylphosphonate core of VX. The evolved catalytic activity with DEVX was enhanced 26-fold relative to wild-type PTE. Further improvements were facilitated by targeted error-prone PCR mutagenesis of loop-7, and additional PTE variants were identified with up to a 78-fold increase in the rate of DEVX hydrolysis. The best mutant hydrolyzed the racemic nerve agent VX with a value of kcat/Km = 7 × 10(4) M(-1) s(-1), a 230-fold improvement relative to wild-type PTE. The highest turnover number achieved by the mutants created for this investigation was 137 s(-1), an enhancement of 152-fold relative to wild-type PTE. The stereoselectivity for the hydrolysis of the two enantiomers of VX was relatively low. These engineered mutants of PTE are the best catalysts ever reported for the hydrolysis of nerve agent VX. PMID:23789980

  4. High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass.

    PubMed

    Chundawat, Shishir P S; Balan, Venkatesh; Dale, Bruce E

    2008-04-15

    Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover. PMID:18306256

  5. Enzymatic hydrolysis of recovered protein from frozen small croaker and functional properties of its hydrolysates.

    PubMed

    Choi, Yeung Joon; Hur, Sungik; Choi, Byeong-Dae; Konno, Kunihiko; Park, Jae W

    2009-01-01

    Fish protein isolate were recovered from frozen small croaker using pH shift. The partial enzymatic hydrolysates were fractionated as soluble and insoluble parts. They were dried using the drum dryer and their functional properties were examined. The total nitrogen content of the enzymatic hydrolysates ranged from 12.9% to 13.7%. The degree of hydrolysis of precipitates was 18.2% and 12.2% for croaker hydrolysates treated with Protamex 1.5 MG (Bacilllus protease complex) and Flavourzyme 500 MG (endoproteases and exoproteases, Aspergillus oryzae), respectively. The TCA supernatant, after centrifugation of hydrolysates, contained numerous peptides ranging from 100 to 4000 daltons. The solubility of the supernatants was higher than that of the precipitates at 0% to 3% NaCl and pH 2 to 10. The precipitate of Flavourzyme- and Protamex-treated hydrolysates showed a high emulsion activity index value compared to egg white and bovine plasma protein. In addition, the highest emulsion stability was observed for Protamex-treated precipitate hydrolysates. Emulsion stability of Protamex-treated precipitate hydrolysates was comparable to those of protein additives (egg white, bovine plasma protein, and soy protein concentrate). Water and fat binding capacity of precipitates were higher than those of supernatant. The results indicate that precipitate hydrolysate from undersized croaker can be used in processed muscle foods as a functional and nutritional ingredient. PMID:19200081

  6. Effect of sulfuric and phosphoric acid pretreatments on enzymatic hydrolysis of corn stover.

    PubMed

    Um, Byung-Hwan; Karim, M; Henk, Linda

    2003-01-01

    The pretreatment of corn stover with H2SO4 and H3PO4 was investigated. Pretreatments were carried out from 30 to 120 min in a batch reactor at 121 degrees C, with acid concentrations ranging from 0 to 2% (w/v) at a solid concentration of 5% (w/v). Pretreated corn stover was washed with distilled water until the filtrate was adjusted to pH 7.0, followed by surfactant swelling of the cellulosic fraction in a 0-10% (w/v) solution of Tween-80 at room temperature for 12 h. The dilute acid treatment proved to be a very effective method in terms of hemicellulose recovery and cellulose digestibility. Hemicellulose recovery was 62-90%, and enzymatic digestibility of the cellulose that remained in the solid was >80% with 2% (w/v) acid. In all cases studied, the performance of H2SO4 pretreatment (hemicellulose recovery and cellulose digestibility) was significantly better than obtained with H3PO4. Enzymatic hydrolysis was more effective using surfactant than without it, producing 10-20% more sugar. Furthermore, digestibility was investigated as a function of hemicellulose removal. It was found that digestibility was more directly related to hemicellulose removal than to delignification. PMID:12721479

  7. Solid acid catalysts pretreatment and enzymatic hydrolysis of macroalgae cellulosic residue for the production of bioethanol.

    PubMed

    Tan, Inn Shi; Lee, Keat Teong

    2015-06-25

    The aim of this study is to investigate the technical feasibility of converting macroalgae cellulosic residue (MCR) into bioethanol. An attempt was made to present a novel, environmental friendly and economical pretreatment process that enhances enzymatic conversion of MCR to sugars using Dowex (TM) Dr-G8 as catalyst. The optimum yield of glucose reached 99.8% under the optimal condition for solid acid pretreatment (10%, w/v biomass loading, 4%, w/v catalyst loading, 30min, 120°C) followed by enzymatic hydrolysis (45FPU/g of cellulase, 52CBU/g of β-glucosidase, 50°C, pH 4.8, 30h). The yield of sugar obtained was found more superior than conventional pretreatment process using H2SO4 and NaOH. Biomass loading for the subsequent simultaneous saccharification and fermentation (SSF) of the pretreated MCR was then optimized, giving an optimum bioethanol yield of 81.5%. The catalyst was separated and reused for six times, with only a slight drop in glucose yield. PMID:25839825

  8. Profiling the substitution pattern of xyloglucan derivatives by integrated enzymatic hydrolysis, hydrophilic-interaction liquid chromatography and mass spectrometry.

    PubMed

    Liu, Jun; Kisonen, Victor; Willför, Stefan; Xu, Chunlin; Vilaplana, Francisco

    2016-09-01

    Plant polysaccharides constitute arguably the most complex family of biomacromolecules in terms of the stereochemistry and regiochemistry of their intramolecular linkages. The chemical modification of such polysaccharides introduces an additional level of complexity for structural determinations. We have developed an integrated analytical procedure combining selective enzymatic hydrolysis, hydrophilic interaction liquid chromatography (HILIC), and mass spectrometry (MS) to describe the substitution pattern of xyloglucan (XyG) and its chemo-enzymatic derivatives (cationic, anionic, and benzyl aminated). Enzymatic hydrolysis of XyG derivatives by a xyloglucan-specific endoglucanase (XEG) generates oligosaccharides amenable for mass spectrometric identification with distinct structures, based on enzymatic substrate recognition and hydrolytic pattern. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) and electrospray ionisation mass spectrometry (ESI-MS) offer qualitative mass profiling of the chemical derivatives. Separation and identification of the complex oligosaccharide profiles released by enzymatic hydrolysis is achieved by hyphenation of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC-ESI-MS). Further fragmentation by tandem mass spectrometry (ESI-MS/MS) in positive mode enables the structural sequencing of modified XyG oligosaccharides and the identification of the substituent position without further derivatisation. This integrated approach can be used to obtain semi-quantitative information of the substitution pattern of hemicellulose derivatives, with fundamental implications for their modification mechanisms and performance. PMID:27524300

  9. Enzymatic Hydrolysis of Polyester Thin Films: Real-Time Analysis of Film Mass Changes and Dissipation Dynamics.

    PubMed

    Zumstein, Michael Thomas; Kohler, Hans-Peter E; McNeill, Kristopher; Sander, Michael

    2016-01-01

    Cleavage of ester bonds by extracellular microbial hydrolases is considered a key step during the breakdown of biodegradable polyester materials in natural and engineered systems. Here we present a novel analytical approach for simultaneous detection of changes in the masses and rigidities of polyester thin films during enzymatic hydrolysis using a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). In experiments with poly(butylene succinate) (PBS) and the lipase of Rhizopus oryzae (RoL), we detected complete hydrolysis of PBS thin films at pH 5 and 40 °C that proceeded through soft and water-rich film intermediates. Increasing the temperature from 20 to 40 °C resulted in a larger increase of the enzymatic hydrolysis rate of PBS than of nonpolymeric dibutyl adipate. This finding was ascribed to elevated accessibility of ester bonds to the catalytic site of RoL due to increasing polyester chain mobility. When the pH of the solution was changed from 5 to 7, initial hydrolysis rates were little affected, while a softer film intermediate that lead to incomplete film hydrolysis was formed. Hydrolysis dynamics of PBS, poly(butylene adipate), poly(lactic acid), and poly(ethylene terephthalate) in assays with RoL showed distinct differences that we attribute to differences in the polyester structure. PMID:26599203

  10. Optimization of covalent immobilization of Trichoderma reesei cellulase onto modified ReliZyme HA403 and Sepabeads EC-EP supports for cellulose hydrolysis, in buffer and ionic liquids/buffer media.

    PubMed

    Bilgin, Ramazan; Yalcin, M Serkan; Yildirim, Deniz

    2016-08-01

    The covalent immobilization of Trichoderma reesei cellulase onto modified ReliZyme HA403 and Sepabeads EC-EP supports were carried out. The optimal immobilization conditions were determined using response surface methodology. The hydrolysis of cellulose using the free and immobilized cellulase preparations in ionic liquids (IL) using cosolvents was investigated. The hydrolytic activities in buffer medium containing 25% (v/v) of 1-butyl-3-methylimidazolium hexafluorophosphate were around 2.6-, 1.6-, and 5.5-fold higher than the activities in buffer medium. The retained initial activities were 32% and 57%, respectively for cellulase preparations immobilized onto Sepabeads EC-EP support and onto modified ReliZyme HA403 support after 5 reuses. PMID:25811997

  11. Enzymatic Hydrolysis and Ethanol Fermentation of High Dry Matter Wet-Exploded Wheat Straw at Low Enzyme Loading

    NASA Astrophysics Data System (ADS)

    Georgieva, Tania I.; Hou, Xiaoru; Hilstrøm, Troels; Ahring, Birgitte K.

    Wheat straw was pretreated by wet explosion using three different oxidizing agents (H2O2, O2, and air). The effect of the pretreatment was evaluated based on glucose and xylose liberated during enzymatic hydrolysis. The results showed that pretreatment with the use of O2 as oxidizing agent was the most efficient in enhancing overall convertibility of the raw material to sugars and minimizing generation of furfural as a by-product. For scale-up of the process, high dry matter (DM) concentrations of 15-20% will be necessary. However, high DM hydrolysis and fermentation are limited by high viscosity of the material, higher inhibition of the enzymes, and fermenting microorganism. The wet-explosion pretreatment method enabled relatively high yields from both enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) to be obtained when performed on unwashed slurry with 14% DM and a low enzyme loading of 10 FPU/g cellulose in an industrial acceptable time frame of 96 h. Cellulose and hemicellulose conversion from enzymatic hydrolysis were 70 and 68%, respectively, and an overall ethanol yield from SSF was 68%.

  12. Improving enzymatic hydrolysis of lignocellulosic substrates with pre-hydrolysates by adding cetyltrimethylammonium bromide to neutralize lignosulfonate.

    PubMed

    Cai, Cheng; Qiu, Xueqing; Lin, Xuliang; Lou, Hongming; Pang, Yuxia; Yang, Dongjie; Chen, Siwei; Cai, Kaifan

    2016-09-01

    Two pretreatment methods to overcome recalcitrance of lignocelluloses, sulfite pretreatment (SPORL) and dilute acid (DA), were conducted to pretreat softwood masson pine and hardwood eucalyptus for enzymatic hydrolysis. In the presence of corresponding pre-hydrolysates, adding moderate cetyltrimethylammonium bromide (CTAB) could enhance the enzymatic hydrolysis of the SPORL-pretreated substrates, but had no enhancement for the DA-pretreated substrates. The results showed that sodium lignosulfonate (SL) in pre-hydrolysates and CTAB together had a strong enhancement on the enzymatic hydrolysis of lignocelluloses. The compound of commercial lignosulfonate SXSL and CTAB (SXSL-CTAB) could enhance the substrate enzymatic digestibility (SED) of SPORL-pretreated masson pine from 27.1% to 71.0%, and that of DA-pretreated eucalyptus from 37.6% to 67.9%. The mechanism that CTAB increased the adsorption of SL on lignin to form more effective steric hindrance and reduced the non-productive adsorption of cellulase on lignin by neutralizing the negative charge of SL was proposed. PMID:27343448

  13. Combination of enzymatic hydrolysis and ethanol organosolv pretreatments: effect on lignin structures, delignification yields and cellulose-to-glucose conversion.

    PubMed

    Obama, Patrick; Ricochon, Guillaume; Muniglia, Lionel; Brosse, Nicolas

    2012-05-01

    Enzymatic pre-hydrolysis using the industrial enzymatic cocktail Cellulyve® was assessed as a first step in a pretreatment process of Miscanthus biomass involving an aqueous-ethanol organosolv treatment. (13)C and (31)P Nuclear Magnetic Resonance and size exclusion chromatography were used to analyze the cellulose and lignin before and after treatment. It was demonstrated that despite a very low impact on the fibre structure (observed by Scanning Electron Microscopy) and composition (in terms of sugars and polyphenolics content), the enzymatic pre-treatment disrupted the lignocellulosic matrix to a considerable extend. This weakening permitted enhanced removal of lignin during organosolv pulping and increased hydrolysability of the residual cellulosic pulp for the production of monomeric glucose. Using this combined treatment, a delignification yield of 93% and an enzymatic cellulose-to-glucose conversion of 75% were obtained. PMID:22424922

  14. Enzymatic Hydrolysis of Pretreated Sugarcane Straw: Kinetic Study and Semi-Mechanistic Modeling.

    PubMed

    Pratto, Bruna; de Souza, Renata Beraldo Alencar; Sousa, Ruy; da Cruz, Antonio Jose Gonçalves

    2016-04-01

    Although there are already commercial-scale productions of second generation (2G) ethanol, focusing efforts on process optimization can be of key importance to make the production cost-effective in large scale. In this scenario, mathematical models may be useful in design, scale-up, optimization, and control of bioreactors. For this reason, the aim of this work was to study the kinetics of the enzymatic hydrolysis of cellulose from sugarcane straw. Experiments using hydrothermally pretreated sugarcane (HPS) straw (195 °C, 10 min, 200 rpm) with and without alkaline delignification (4 % NaOH m/v, 30 min, 121 °C) were carried out in shake flasks (50 °C, pH 5.0, 200 rpm). Solid load was varied in a range of 0.8 to 10 % (m/v), in initial velocity and long-term assays. Enzyme concentration (Cellic®CTec2) was varied from 5 to 80 filter paper unit (FPU) gcellulose (-1). It was possible to fit Michaelis-Menten (MM), modified MM, with and without competitive inhibition by glucose, and Chrastil models. Chrastil model and modified MM with inhibition (both suitable for heterogeneous system, with high resistance to internal diffusion) showed more appropriate than pseudo-homogeneous MM model. The fitted models were able to identify key features of the hydrolysis process and can be very useful within the perspective of bioreactors engineering. PMID:26701144

  15. Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis

    DOE PAGESBeta

    Xue, Saisi; Uppugundla, Nirmal; Bowman, Michael J.; Cavalier, David; Da Costa Sousa, Leonardo; Dale, Bruce E.; Balan, Venkatesh

    2015-11-26

    Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production frommore » cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results indicate that, oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the

  16. Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis

    SciTech Connect

    Xue, Saisi; Uppugundla, Nirmal; Bowman, Michael J.; Cavalier, David; Da Costa Sousa, Leonardo; Dale, Bruce E.; Balan, Venkatesh

    2015-11-26

    Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production from cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results indicate that, oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the

  17. Tylosin detection in animal feed by liquid chromatography-tandem mass spectrometry with enzymatic hydrolysis of the tylosin urea adduct.

    PubMed

    Van Poucke, Christof; Dumoulin, Fréderic; De Keyser, Kirsten; Elliott, Chris; Van Peteghem, Carlos

    2004-05-19

    When the use of tylosin as a feed additive was forbidden by Council Regulation 2821/98, the necessity of a chemical confirmation method for the monitoring of the ban was created. Recently a method was developed for the detection of tylosin in animal feed by means of LC-MS/MS. During the validation high deviating values for the decision limit, detection capability, and repeatability for tylosin in cattle feed were observed, and the presence of urea and the formation of a tylosin urea adduct (TUA) were suggested as possible explanations. In this study two hydrolysis approaches for the TUA adduct were compared, namely, a chemical hydrolysis and an enzymatic hydrolysis with urease. The latter yielded a more complete hydrolysis of urea and was used for further validation. The recovery increased by approximately 15-25% depending on the amount of urea present in the feed (0.5-2%). The decision limit and detection capability were hardly influenced by the enzymatic hydrolysis. PMID:15137817

  18. Evaluation of soluble fraction and enzymatic residual fraction of dilute dry acid, ethylenediamine, and steam explosion pretreated corn stover on the enzymatic hydrolysis of cellulose.

    PubMed

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

    2016-06-01

    This study is aimed to examine the inhibition of soluble fraction (SF) and enzymatic residual fraction (ERF) in dry dilute acid (DDA), ethylenediamine (EDA) and steam explosion (SE) pretreated corn stover (CS) on the enzymatic digestibility of cellulose. SF of DDA, EDA and SE pretreated CS has high xylose, soluble lignin and xylo-oligomer content, respectively. SF of EDA pretreated CS leads to the highest inhibition, followed by SE and DDA pretreated CS. Inhibition of ERF of DDA and SE pretreated CS is higher than that of EDA pretreated CS. The inhibition degree (A0/A) of SF is 1.76 and 1.21 times to that of ERF for EDA and SE pretreated CS, respectively. The inhibition degree of ERF is 1.05 times to that of SF in DDA pretreated CS. The quantitative analysis shows that SF of EDA pretreated CS, SF and ERF of SE pretreated CS cause significant inhibition during enzymatic hydrolysis. PMID:26970919

  19. Scale-up and evaluation of high solid ionic liquid pretreatment and enzymatic hydrolysis of switchgrass

    PubMed Central

    2013-01-01

    Background Ionic liquid (IL) pretreatment is receiving significant attention as a potential process that enables fractionation of lignocellulosic biomass and produces high yields of fermentable sugars suitable for the production of renewable fuels. However, successful optimization and scale up of IL pretreatment involves challenges, such as high solids loading, biomass handling and transfer, washing of pretreated solids and formation of inhibitors, which are not addressed during the development stages at the small scale in a laboratory environment. As a first in the research community, the Joint BioEnergy Institute, in collaboration with the Advanced Biofuels Process Demonstration Unit, a Department of Energy funded facility that supports academic and industrial entities in scaling their novel biofuels enabling technologies, have performed benchmark studies to identify key challenges associated with IL pretreatment using 1-ethyl-3-methylimidazolium acetate and subsequent enzymatic saccharification beyond bench scale. Results Using switchgrass as the model feedstock, we have successfully executed 600-fold, relative to the bench scale (6 L vs 0.01 L), scale-up of IL pretreatment at 15% (w/w) biomass loading. Results show that IL pretreatment at 15% biomass generates a product containing 87.5% of glucan, 42.6% of xylan and only 22.8% of lignin relative to the starting material. The pretreated biomass is efficiently converted into monosaccharides during subsequent enzymatic hydrolysis at 10% loading over a 150-fold scale of operations (1.5 L vs 0.01 L) with 99.8% fermentable sugar conversion. The yield of glucose and xylose in the liquid streams were 94.8% and 62.2%, respectively, and the hydrolysate generated contains high titers of fermentable sugars (62.1 g/L of glucose and 5.4 g/L cellobiose). The overall glucan and xylan balance from pretreatment and saccharification were 95.0% and 77.1%, respectively. Enzymatic inhibition by [C2mim][OAc] at high solids

  20. A novel non-hydrolytic protein from Pseudomonas oryzihabitans enhances the enzymatic hydrolysis of cellulose.

    PubMed

    Qin, Yi-Min; Tao, Heng; Liu, You-Yan; Wang, Yan-Dong; Zhang, Jing-Ru; Tang, Ai-Xing

    2013-10-10

    Several kinds of protein such as the expansin, expansin-like proteins and LPMOs (lytic polysaccharide monooxygenases) are known to exert enhancement effects on cellulase activity. In this study, a novel cellulase synergistic protein named POEP1 was purified from the culture filtrate of Pseudomonas oryzihabitans CGMCC 6169, and was homogeneous on SDS-PAGE with a molecular weight of 60kDa. Mass spectrometry analysis indicated that it was an unknown protein without sequence similarity to the expansin and expansin-like proteins. Evaluation of the enzymatic hydrolysis of filter paper revealed that POEP1 had no cellulase activity but displayed high synergistic activity of 364% at a cellulase concentration of 0.1FPU/g of filter paper. When a mixture containing 0.6FPU cellulase and 700μg POEP1 per g of cellulose was evaluated, the maximal sugar yield was achieved, which was 2.2-fold greater than that with the cellulase alone. POEP1 was found to have functional similarity to the expansin and expansin-like proteins, which could decrease both the hydrogen-bond intensity and crystallinity, and cause the filter paper disruption. This study provided evidence for the existence of novel bacterial proteins in nature serving the same function as expansin and expansin-like proteins. PMID:23916949

  1. Enhancing the hydrolysis and methane production potential of mixed food waste by an effective enzymatic pretreatment.

    PubMed

    Kiran, Esra Uçkun; Trzcinski, Antoine P; Liu, Yu

    2015-05-01

    In this study, a fungal mash rich in hydrolytic enzymes was produced by solid state fermentation (SSF) of waste cake in a simple and efficient manner and was further applied for high-efficiency hydrolysis of mixed food wastes (FW). The enzymatic pretreatment of FW with this fungal mash resulted in 89.1 g/L glucose, 2.4 g/L free amino nitrogen, 165 g/L soluble chemical oxygen demand (SCOD) and 64% reduction in volatile solids within 24h. The biomethane yield and production rate from FW pretreated with the fungal mash were found to be respectively about 2.3 and 3.5-times higher than without pretreatment. After anaerobic digestion of pretreated FW, a volatile solids removal of 80.4±3.5% was achieved. The pretreatment of mixed FW with the fungal mash produced in this study is a promising option for enhancing anaerobic digestion of FW in terms of energy recovery and volume reduction. PMID:25722182

  2. Responses of biomass briquetting and pelleting to water-involved pretreatments and subsequent enzymatic hydrolysis.

    PubMed

    Li, Yang; Li, Xiaotong; Shen, Fei; Wang, Zhanghong; Yang, Gang; Lin, Lili; Zhang, Yanzong; Zeng, Yongmei; Deng, Shihuai

    2014-01-01

    Although lignocellulosic biomass has been extensively regarded as the most important resource for bioethanol, the wide application was seriously restricted by the high transportation cost of biomass. Currently, biomass densification is regarded as an acceptable solution to this issue. Herein, briquettes, pellets and their corresponding undensified biomass were pretreated by diluted-NaOH and hydrothermal method to investigate the responses of biomass densification to these typical water-involved pretreatments and subsequent enzymatic hydrolysis. The densified biomass auto-swelling was initially investigated before pretreatment. Results indicated pellets could be totally auto-swollen in an hour, while it took about 24 h for briquettes. When diluted-NaOH pretreatment was performed, biomass briquetting and pelleting improved sugar conversion rate by 20.1% and 5.5% comparing with their corresponding undensified biomass. Pelleting improved sugar conversion rate by 7.0% after hydrothermal pretreatment comparing with the undensified biomass. However, briquetting disturbed hydrothermal pretreatment resulting in the decrease of sugar conversion rate by 15.0%. PMID:24189385

  3. Enzymatic Hydrolysis of Alginate to Produce Oligosaccharides by a New Purified Endo-Type Alginate Lyase

    PubMed Central

    Zhu, Benwei; Chen, Meijuan; Yin, Heng; Du, Yuguang; Ning, Limin

    2016-01-01

    Enzymatic hydrolysis of sodium alginate to produce alginate oligosaccharides has drawn increasing attention due to its advantages of containing a wild reaction condition, excellent gel properties and specific products easy for purification. However, the efficient commercial enzyme tools are rarely available. A new alginate lyase with high activity (24,038 U/mg) has been purified from a newly isolated marine strain, Cellulophaga sp. NJ-1. The enzyme was most active at 50 °C and pH 8.0 and maintained stability at a broad pH range (6.0–10.0) and temperature below 40 °C. It had broad substrate specificity toward sodium alginate, heteropolymeric MG blocks (polyMG), homopolymeric M blocks (polyM) and homopolymeric G blocks (polyG), and possessed higher affinity toward polyG (15.63 mM) as well as polyMG (23.90 mM) than polyM (53.61 mM) and sodium alginate (27.21 mM). The TLC and MS spectroscopy analysis of degradation products suggested that it completely hydrolyzed sodium alginate into oligosaccharides of low degrees of polymerization (DPs). The excellent properties would make it a promising tool for full use of sodium alginate to produce oligosaccharides. PMID:27275826

  4. Effects of grain species and cultivar, thermal processing, and enzymatic hydrolysis on gluten quantitation.

    PubMed

    Pahlavan, Autusa; Sharma, Girdhari M; Pereira, Marion; Williams, Kristina M

    2016-10-01

    Gluten from wheat, rye, and barley can trigger IgE-mediated allergy or Celiac disease in sensitive individuals. Gluten-free labeled foods are available as a safe alternative. Immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are commonly used to quantify gluten in foods. However, various non-assay related factors can affect gluten quantitation. The effect of gluten-containing grain cultivars, thermal processing, and enzymatic hydrolysis on gluten quantitation by various ELISA kits was evaluated. The ELISA kits exhibited variations in gluten quantitation depending on the gluten-containing grain and their cultivars. Acceptable gluten recoveries were obtained in 200mg/kg wheat, rye, and barley-spiked corn flour thermally processed at various conditions. However, depending on the enzyme, gluten grain source, and ELISA kit used, measured gluten content was significantly reduced in corn flour spiked with 200mg/kg hydrolyzed wheat, rye, and barley flour. Thus, the gluten grain source and processing conditions should be considered for accurate gluten analysis. PMID:27132849

  5. Enzymatic Hydrolysis of Alginate to Produce Oligosaccharides by a New Purified Endo-Type Alginate Lyase.

    PubMed

    Zhu, Benwei; Chen, Meijuan; Yin, Heng; Du, Yuguang; Ning, Limin

    2016-01-01

    Enzymatic hydrolysis of sodium alginate to produce alginate oligosaccharides has drawn increasing attention due to its advantages of containing a wild reaction condition, excellent gel properties and specific products easy for purification. However, the efficient commercial enzyme tools are rarely available. A new alginate lyase with high activity (24,038 U/mg) has been purified from a newly isolated marine strain, Cellulophaga sp. NJ-1. The enzyme was most active at 50 °C and pH 8.0 and maintained stability at a broad pH range (6.0-10.0) and temperature below 40 °C. It had broad substrate specificity toward sodium alginate, heteropolymeric MG blocks (polyMG), homopolymeric M blocks (polyM) and homopolymeric G blocks (polyG), and possessed higher affinity toward polyG (15.63 mM) as well as polyMG (23.90 mM) than polyM (53.61 mM) and sodium alginate (27.21 mM). The TLC and MS spectroscopy analysis of degradation products suggested that it completely hydrolyzed sodium alginate into oligosaccharides of low degrees of polymerization (DPs). The excellent properties would make it a promising tool for full use of sodium alginate to produce oligosaccharides. PMID:27275826

  6. Enzymatic hydrolysis and production of bioethanol from common macrophytic green alga Ulva fasciata Delile.

    PubMed

    Trivedi, Nitin; Gupta, Vishal; Reddy, C R K; Jha, Bhavanath

    2013-12-01

    The green seaweed Ulva which proliferates fast and occurs abundantly worldwide was used as a feedstock for production of ethanol following enzymatic hydrolysis. Among the different cellulases investigated for efficient saccharification, cellulase 22119 showed the highest conversion efficiency of biomass into reducing sugars than Viscozyme L, Cellulase 22086 and 22128. Pre-heat treatment of biomass in aqueous medium at 120°C for 1h followed by incubation in 2% (v/v) enzyme for 36 h at 45°C gave a maximum yield of sugar 206.82±14.96 mg/g. The fermentation of hydrolysate gave ethanol yield of 0.45 g/g reducing sugar accounting for 88.2% conversion efficiency. These values are substantially higher than those of reported so far for both agarophytes and carrageenophytes. It was also confirmed that enzyme can be used twice without compromising on the saccharification efficiency. The findings of this study reveal that Ulva can be a potential feedstock for bioethanol production. PMID:24157682

  7. Substrate Dependency and Effect of Xylanase Supplementation on Enzymatic Hydrolysis of Ammonia-Treated Biomass

    NASA Astrophysics Data System (ADS)

    Gupta, Rajesh; Kim, Tae Hyun; Lee, Yoon Y.

    Pretreatment based on aqueous ammonia was investigated under two different modes of operation: soaking in aqueous ammonia and ammonia recycle percolation. These processes were applied to three different feedstocks with varied composition: corn stover, high lignin (HL), and low lignin (LL) hybrid poplars. One of the important features of ammonia-based pretreatment is that most of the hemicellulose is retained after treatment, which simplifies the overall bioconversion process and enhances the conversion efficiency. The pretreatment processes were optimized for these feedstocks, taking carbohydrate retention as well as sugar yield in consideration. The data indicate that hybrid poplar is more difficult to treat than corn stover, thus, requires more severe conditions. On the other hand, hybrid poplar has a beneficial property that it retains most of the hemicellulose after pretreatment. To enhance the digestibility of ammonia-treated poplars, xylanase was supplemented during enzymatic hydrolysis. Because of high retention of hemicellulose in treated hybrid poplar, xylanase supplementation significantly improved xylan as well as glucan digestibility. Of the three feedstocks, best results and highest improvement by xylanase addition was observed with LL hybrid poplar, showing 90% of overall sugar yield.

  8. EFFECT OF ANATOMICAL FRACTIONATION ON THE ENZYMATIC HYDROLYSIS OF ACID AND ALKALINE PRETREATED CORN STOVER

    SciTech Connect

    K. B. Duguid; M. D. Montross; C. W. Radtke; C. L. Crofcheck; L. M. Wendt; S. A. Shearer

    2009-11-01

    Due to concerns with biomass collection systems and soil sustainability there are opportunities to investigate the optimal plant fractions to collect for conversion. An ideal feedstock would require low severity pretreatment to release a maximum amount of sugar during enzymatic hydrolysis. Corn stover fractions were separated by hand and analyzed for glucan, xylan, acid soluble lignin, acid insoluble lignin, and ash composition. The stover fractions were also pretreated with either 0, 0.4, or 0.8% NaOH for 2 hours at room temperature, washed, autoclaved and saccharified. In addition, acid pretreated samples underwent simultaneous saccharification and fermentation (SSF) to ethanol. In general, the two pretreatments produced similar trends with cobs, husks, and leaves responding best to the pretreatments, the tops of stalks responding slightly less, and the bottom of the stalks responding the least. For example, corn husks pretreated with 0.8% NaOH released over 90% (standard error of 3.8%) of the available glucan, while only 45% (standard error of 1.1%) of the glucan was produced from identically treated stalk bottoms. Estimates of the theoretical ethanol yield using acid pretreatment followed by SSF were 65% (standard error of 15.9%) for husks and 29% (standard error of 1.8%) for stalk bottoms. This suggests that integration of biomass collection systems to remove sustainable feedstocks could be integrated with the processes within a biorefinery to minimize overall ethanol production costs.

  9. Determination of amino acids in two Polysiphonia species and study of enzymatic hydrolysis method

    NASA Astrophysics Data System (ADS)

    Zhang, Li-Xin; Fan, Xiao; Wei, Yu-Xi

    2002-09-01

    The total content of the rich amino acids in two common red algae, Polysiphonia urceolata and Polysiphonia japonica growing in the Qingdao seashore were determined. The algae powder was hydrolyzed by 6 mol/L HCl at 110°C for 48 h and determined by amino acid analyzer. The content was 25.35% and 24.16%, respectively, much higher than that of some other species. In addition, a nutritive liquid with abundant amino acids was prepared (by the enzymatic hydrolysis method using Polysiphonia urceolata) as raw material for a kind of health beverage. The dried seaweed was decolored by 0.25% KMnO4 and 0.5% active carbon, then enzymalized. In the selection of enzymalizing condition, the orthogonal experimental design was used. Four factors including kinds of enzyme, quantity of enzyme, temperature and time were studied at 3 levels. According to the orthogonal design results, we can choose an optimal condition: hydrolyzing at 45°C by neutral proteinase (0.25%, w/w) for 2h, adjusting pH to 8.5, then adding trypsin (0.25%, w/w) and hydrolyzing for 2 h. Finally the above solution was alkalized by NaOH and neutralized by casein. After the hydrolyzed liquid was filtered and concentrated, suitable additives were added. The final products contain rich amino acids.

  10. A dynamic supramolecular polymer with stimuli-responsive handedness for in situ probing of enzymatic ATP hydrolysis

    NASA Astrophysics Data System (ADS)

    Kumar, Mohit; Brocorens, Patrick; Tonnelé, Claire; Beljonne, David; Surin, Mathieu; George, Subi J.

    2014-12-01

    Design of artificial systems, which can respond to fluctuations in concentration of adenosine phosphates (APs), can be useful in understanding various biological processes. Helical assemblies of chromophores, which dynamically respond to such changes, can provide real-time chiroptical readout of various chemical transformations. Towards this concept, here we present a supramolecular helix of achiral chromophores, which shows chiral APs responsive tunable handedness along with dynamically switchable helicity. This system, composing of naphthalenediimides with phosphate recognition unit, shows opposite handedness on binding with ATP compared with ADP or AMP, which is comprehensively analysed with molecular dynamic simulations. Such differential signalling along with stimuli-dependent fast stereomutations have been capitalized to probe the reaction kinetics of enzymatic ATP hydrolysis. Detailed chiroptical analyses provide mechanistic insights into the enzymatic hydrolysis and various intermediate steps. Thus, a unique dynamic helical assembly to monitor the real-time reaction processes via its stimuli-responsive chiroptical signalling is conceptualized.

  11. A dynamic supramolecular polymer with stimuli-responsive handedness for in situ probing of enzymatic ATP hydrolysis.

    PubMed

    Kumar, Mohit; Brocorens, Patrick; Tonnelé, Claire; Beljonne, David; Surin, Mathieu; George, Subi J

    2014-01-01

    Design of artificial systems, which can respond to fluctuations in concentration of adenosine phosphates (APs), can be useful in understanding various biological processes. Helical assemblies of chromophores, which dynamically respond to such changes, can provide real-time chiroptical readout of various chemical transformations. Towards this concept, here we present a supramolecular helix of achiral chromophores, which shows chiral APs responsive tunable handedness along with dynamically switchable helicity. This system, composing of naphthalenediimides with phosphate recognition unit, shows opposite handedness on binding with ATP compared with ADP or AMP, which is comprehensively analysed with molecular dynamic simulations. Such differential signalling along with stimuli-dependent fast stereomutations have been capitalized to probe the reaction kinetics of enzymatic ATP hydrolysis. Detailed chiroptical analyses provide mechanistic insights into the enzymatic hydrolysis and various intermediate steps. Thus, a unique dynamic helical assembly to monitor the real-time reaction processes via its stimuli-responsive chiroptical signalling is conceptualized. PMID:25511998

  12. Potential of phosphoric acid-catalyzed pretreatment and subsequent enzymatic hydrolysis for biosugar production from Gracilaria verrucosa.

    PubMed

    Kwon, Oh-Min; Kim, Sung-Koo; Jeong, Gwi-Taek

    2016-07-01

    This study combined phosphoric acid-catalyzed pretreatment and enzymatic hydrolysis to produce biosugars from Gracilaria verrucosa as a potential renewable resource for bioenergy applications. We optimized phosphoric acid-catalyzed pretreatment conditions to 1:10 solid-to-liquid ratio, 1.5 % phosphoric acid, 140 °C, and 60 min reaction time, producing a 32.52 ± 0.06 % total reducing sugar (TRS) yield. By subsequent enzymatic hydrolysis, a 68.61 ± 0.90 % TRS yield was achieved. These results demonstrate the potential of phosphoric acid to produce biosugars for biofuel and biochemical production applications. PMID:27003825

  13. High selective delignification using oxidative ionic liquid pretreatment at mild conditions for efficient enzymatic hydrolysis of lignocellulose.

    PubMed

    Pang, Zhiqiang; Lyu, Wenkang; Dong, Cuihua; Li, Hongxing; Yang, Guihua

    2016-08-01

    Herein, the oxidative ionic liquid (IL) pretreatment for overcoming recalcitrance of lignocellulose with selective delignification was investigated, and the subsequent enzymatic hydrolysis was evaluated. IL pretreatment incorporating oxygen delignification could enhance lignin extraction with high selectivity at low carbohydrate loss. The dual-action of oxidative decomposition and dissolution by 1-butyl-3-methlimidazolium chloride (BmimCl) on biomass were synergistically acted, accounting for efficient recalcitrance removal. In addition, the mild oxidative IL treatment only slightly converted crystalline cellulose into amorphous structure, and the extensive extraction of the amorphous lignin and carbohydrate resulted to the expose of cellulose with high susceptibility. Correspondingly, the enzymatic hydrolysis of the pretreated lignocellulose was greatly enhanced. The oxidative IL treatment at mild conditions, collaborating BmimCl treatment with oxygen delignification is a promising and effective system for overcoming the robust structure of lignocellulose. PMID:27128194

  14. [The use of enzymatic hydrolysis for isolation of barbituric acid derivatives from blood (as exemplified by phenobarbital and barbamyl)].

    PubMed

    Chuvina, N A; Kolupaeva, A S; Strelova, O Iu; Zabolotskaia, I V; Gorbacheva, T V

    2010-01-01

    Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented. PMID:21265178

  15. Structural features of dilute acid, steam exploded, and alkali pretreated mustard stalk and their impact on enzymatic hydrolysis.

    PubMed

    Kapoor, Manali; Raj, Tirath; Vijayaraj, M; Chopra, Anju; Gupta, Ravi P; Tuli, Deepak K; Kumar, Ravindra

    2015-06-25

    To overcome the recalcitrant nature of biomass several pretreatment methodologies have been explored to make it amenable to enzymatic hydrolysis. These methodologies alter cell wall structure primarily by removing/altering hemicelluloses and lignin. In this work, alkali, dilute acid, steam explosion pretreatment are systematically studied for mustard stalk. To assess the structural variability after pretreatment, chemical analysis, surface area, crystallinity index, accessibility of cellulose, FT-IR and thermal analysis are conducted. Although the extent of enzymatic hydrolysis varies upon the methodologies used, nevertheless, cellulose conversion increases from <10% to 81% after pretreatment. Glucose yield at 2 and 72h are well correlated with surface area and maximum adsorption capacity. However, no such relationship is observed for xylose yield. Mass balance of the process is also studied. Dilute acid pretreatment is the best methodology in terms of maximum sugar yield at lower enzyme loading. PMID:25839820

  16. Preparation of fluorinated RNA nucleotide analogs potentially stable to enzymatic hydrolysis in RNA and DNA polymerase assays

    PubMed Central

    Shakhmin, Anton; Jones, John-Paul; Bychinskaya, Inessa; Zibinsky, Mikhail; Oertell, Keriann; Goodman, Myron F.; Prakash, G.K. Surya

    2015-01-01

    Analogs of ribonucleotides (RNA) stable to enzymatic hydrolysis were prepared and characterized. Computational investigations revealed that this class of compounds with a modified triphosphate exhibits the correct polarity and minimal steric effects compared to the natural molecule. Non-hydrolysable properties as well as the ability of the modified nucleotide to be recognized by enzymes were probed by performing single-turnover gap filling assays with T7 RNA polymerase and DNA polymerase β. PMID:26279588

  17. Effect of anion structures on cholinium ionic liquids pretreatment of rice straw and the subsequent enzymatic hydrolysis.

    PubMed

    Hou, Xue-Dan; Xu, Jie; Li, Ning; Zong, Min-Hua

    2015-01-01

    In this work, 28 cholinium ionic liquids (ILs), most of which are good solvents for dissolving lignin, were used for rice straw pretreatment to improve subsequent enzymatic hydrolysis. The anion exerted a significant effect on the pretreatment effectiveness of the IL as well as the subsequent enzymatic hydrolysis efficiency of rice straw residues. The presence of the basic group(s) in the anion significantly enhanced the IL pretreatment effectiveness, while the carboxyl, hydroxyl and aromatic groups had a negative impact on IL delignification. Except for amino acid-based ILs, the delignification abilities of the ILs are linearly and positively correlated with the pKa values of the conjugate acids of the anions. Of the ILs tested, amino acid-based ILs, especially basic amino acid-based ILs, are the most effective pretreatment solvents. Satisfactory reducing sugar yields (81% for glucose and 26% for xylose) were obtained in the enzymatic hydrolysis of rice straw pretreated by cholinium argininate ([Ch][Arg]) under a pretty mild pretreatment severity (60°C, 6 h). The results presented in this work may be useful for rational design of novel and green ILs for delignification of lignocellulose. PMID:25067792

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

    PubMed

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

    2016-01-01

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

  19. Recycling cellulases by pH-triggered adsorption-desorption during the enzymatic hydrolysis of lignocellulosic biomass.

    PubMed

    Shang, Yaping; Su, Rongxin; Huang, Renliang; Yang, Yang; Qi, Wei; Li, Qiujin; He, Zhimin

    2014-06-01

    Recycling of cellulases is an effective way to reduce the cost of enzymatic hydrolysis for the production of cellulosic ethanol. In this study, we examined the adsorption and desorption behaviors of cellulase at different pH values and temperatures. Furthermore, we developed a promising way to recover both free and bound cellulases by pH-triggered adsorption-desorption. The results show that acidic pH (e.g., pH 4.8) was found to favor adsorption, whereas alkaline pH (e.g., pH 10) and low temperature (4-37 °C) favored desorption. The adsorption of cellulases reached an equilibrium within 60 min at pH 4.8 and 25 °C, leading to approximately 50 % of the added cellulases bound to the substrate. By controlling the pH of eluent (citrate buffer, 25 °C), we were able to increase the desorption efficiency of bound cellulases from 15 % at pH 4.8 to 85 % at pH 10. To recover cellulases after enzymatic hydrolysis, we employed adsorption by fresh substrate and desorption at pH 10 to recover the free cellulases in supernatant and the bound cellulases in residue, respectively. The recycling performance (based on the glucose yield) of this simple strategy could reach near 80 %. Our results provided a simple, low-cost, and effective approach for cellulase recycling during the enzymatic hydrolysis of lignocellulosic biomass. PMID:24752845

  20. Isolation and purification of arctigenin from Fructus Arctii by enzymatic hydrolysis combined with high-speed counter-current chromatography.

    PubMed

    Liu, Feng; Xi, Xingjun; Wang, Mei; Fan, Li; Geng, Yanling; Wang, Xiao

    2014-02-01

    Enzymatic hydrolysis pretreatment combined with high-speed counter-current chromatography for the transformation and isolation of arctigenin from Fructus Arctii was successfully developed. In the first step, the extract solution of Fructus Arctii was enzymatic hydrolyzed by β-glucosidase. The optimal hydrolysis conditions were 40°C, pH 5.0, 24 h of hydrolysis time, and 1.25 mg/mL β-glucosidase concentration. Under these conditions, the content of arctigenin was transformed from 2.60 to 12.59 mg/g. In the second step, arctigenin in the hydrolysis products was separated and purified by high-speed counter-current chromatography with a two-phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (10:25:15:20, v/v), and the fraction was analyzed by HPLC, ESI-MS, and (1)H NMR spectroscopy. Finally, 102 mg of arctigenin with a purity of 98.9% was obtained in a one-step separation from 200 mg of hydrolyzed sample. PMID:24311558

  1. Recovery of Whey Proteins and Enzymatic Hydrolysis of Lactose Derived from Casein Whey Using a Tangential Flow Ultrafiltration Module

    NASA Astrophysics Data System (ADS)

    Das, Bipasha; Bhattacharjee, Sangita; Bhattacharjee, Chiranjib

    2013-09-01

    In this study, ultrafiltration (UF) of pretreated casein whey was carried out in a cross-flow module fitted with 5 kDa molecular weight cut-off polyethersulfone membrane to recover whey proteins in the retentate and lactose in the permeate. Effects of processing conditions, like transmembrane pressure and pH on permeate flux and rejection were investigated and reported. The polarised layer resistance was found to increase with time during UF even in this high shear device. The lactose concentration in the permeate was measured using dinitro salicylic acid method. Enzymatic kinetic study for lactose hydrolysis was carried out at three different temperatures ranging from 30 to 50 °C using β-galactosidase enzyme. The glucose formed during lactose hydrolysis was analyzed using glucose oxidase-peroxidase method. Kinetics of enzymatic hydrolysis of lactose solution was found to follow Michaelis-Menten model and the model parameters were estimated by Lineweaver-Burk plot. The hydrolysis rate was found to be maximum (with Vmax = 5.5091 mmol/L/min) at 30 °C.

  2. Rapid selection and identification of Miscanthus genotypes with enhanced glucan and xylan yields from hydrothermal pretreatment followed by enzymatic hydrolysis

    PubMed Central

    2012-01-01

    Background Because many Miscanthus genotypes can be cultivated with relatively high productivity and carbohydrate content, Miscanthus has great potential as an energy crop that can support large scale biological production of biofuels. Results In this study, batch hydrothermal pretreatment at 180°C for 35 min followed by enzymatic hydrolysis was shown to give the highest total sugar yields for Miscanthus x giganteus cv. Illinois planted in Illinois. High throughput pretreatment at 180°C for 35 min and 17.5 min followed by co-hydrolysis in a multi-well batch reactor identified two varieties out of 80 that had significantly higher sugar yields from pretreatment and enzymatic hydrolysis than others. The differences in performance were then related to compositions of the 80 varieties to provide insights into desirable traits for Miscanthus that enhance sugar yields. Conclusions High throughput pretreatment and co-hydrolysis (HTPH) rapidly identified promising genotypes from a wide range of Miscanthus genotypes, including hybrids of Miscanthus sacchariflorus/M. sinensis and Miscanthus lutarioriparius, differentiating the more commercially promising species from the rest. The total glucan plus xylan content in Miscanthus appeared to influence both mass and theoretical yields, while lignin and ash contents did not have a predictable influence on performance. PMID:22863302

  3. Simultaneous enzymatic hydrolysis and extraction of endocrine-disrupting chemicals in fish bile using polyethersulfone polymer.

    PubMed

    Ros, Oihana; Aguirre, Josu; Prieto, Ailette; Olivares, Maitane; Etxebarria, Nestor; Vallejo, Asier

    2015-09-01

    This study describes a new method for the simultaneous extraction and enzymatic hydrolysis of alkylphenols, estrogens, bisphenol-A and phthalate metabolite (mono-2-ethylhexyl ester, MEHP) in fish bile using polyethersulfone (PES) polymer as sorptive material. Parameters affecting the hydrolysis (enzyme amount) and extraction (nature of polymeric material, PES desorption solvent nature and time, PES amount and time profile) were optimised. The optimum conditions were fixed as: 5 PES tubes (1.5 cm length × 0.7 mm o.d.) were added to a vessel with 100 μL of sample, 800 μL of ultrapure water, 1.5 mL phosphate buffer (0.1 mol L(-1), pH 6) and 200 μL of β-glucuronidase (1000 U mL(-1)) enzyme and the mixture was stirred at 37 °C and 550 rpm for 3 h. Quantitative results were obtained after desorption of PES material using 500 μL of ethyl acetate. The extracts were reconstituted in 250 μL of methanol and analysed by liquid chromatography-tandem mass spectrometry, obtaining apparent recoveries in the range of 73-134 % using deuterated compounds surrogates corrections. Relative standard deviations below 27 % were obtained for all target analytes and the method detection limits (MDLs) were in low nanograms per mililliter level for all the studied compounds, except in the case of MEHP which was detected at higher concentration levels (ng μL(-1)) in bile samples that do not allow its MDL determination. Bisphenol A (MDL-10.8 ng mL(-1)), diethylstilbestrol (MDL-1.4 ng mL(-1)) and MEHP (975-2604 ng mL(-1)) were detected in grey mullets captured nearby the wastewater treatment plant of Gernika (Biosphere Reserve of Urdaibai). PMID:26229028

  4. Utilization of recombinant Trichoderma reesei expressing Aspergillus aculeatus β-glucosidase I (JN11) for a more economical production of ethanol from lignocellulosic biomass.

    PubMed

    Treebupachatsakul, Treesukon; Shioya, Koki; Nakazawa, Hikaru; Kawaguchi, Takashi; Morikawa, Yasushi; Shida, Yosuke; Ogasawara, Wataru; Okada, Hirofumi

    2015-12-01

    The capacity of Trichoderma reesei cellulase to degrade lignocellulosic biomass has been enhanced by the construction of a recombinant T. reesei strain expressing Aspergillus aculeatus β-glucosidase I. We have confirmed highly efficient ethanol production from converge-milled Japanese cedar by recombinant T. reesei expressing A. aculeatus β-glucosidase I (JN11). We investigated the ethanol productivity of JN11 and compared it with the cocktail enzyme T. reesei PC-3-7 with reinforced cellobiase activity by the commercial Novozyme 188. Results showed that the ethanol production efficiency under enzymatic hydrolysis of JN11 was comparable to the cocktail enzyme both on simultaneous saccharification and fermentation (SSF) or separate hydrolysis and fermentation (SHF) processes. Moreover, the cocktail enzyme required more protein loading for attaining similar levels of ethanol conversion as JN11. We propose that JN11 is an intrinsically economical enzyme that can eliminate the supplementation of BGL for PC-3-7, thereby reducing the cost of industrial ethanol production from lignocellulosic biomass. PMID:26026380

  5. Pilot scale production of cellulolytic enzymes by Trichoderma reesei

    SciTech Connect

    Warzywoda, M.; Chevron, F.; Ferre, V.; Pourquie, J.

    1983-01-01

    The French substitute fuels program aims at the substitution of part of gasoline by methanol. In order to avoid phase separation of the gasoline-methanol blend, a cosolvant has to be added; one of the most efficient cosolvants is the mixture of acetone and butanol produced by anaerobic acetone-butanol fermentation. The Institut Francais du Petrole is thus implementing a research and development program on the production of acetone butanol from biomass, either sugar crops (fodder beets and Jerusalem artichoke) or lignocellulosic (corn stover and wheat straw). Production of sugars from lignocellulosics is a major part of this program. The enzymatic hydrolysis route, based on Trichoderma reesei cellulolytic enzymes, has been chosen since it does not cause any degradation of C/sub 5/ sugars which are good substrates of the acetone butanol fermentation. Efficient and cheap large-scale production of cellulolytic enzymes is thus a key step in this process. This paper reports on production of cellulases by Trichoderma reesei in a 3-m/sup 3/ pilot fermentor under conditions which should facilitate the scaling-up of the process. 7 references, 2 figures, 2 tables.

  6. Protein extraction and enzymatic hydrolysis of ammonia-treated cassava leaves (Manihot esculenta Crantz).

    PubMed

    Urribarrí, Lauris; Chacón, David; González, Orlaidy; Ferrer, Alexis

    2009-05-01

    In the present work, cassava leaves were treated with 0.5 kg ammonia/kg dry matter at 78 degrees C and 30% moisture content in a 2-kg reactor. Protein extraction was carried out with a calcium hydroxide solution (pH 10) for 30 min at several temperatures (30 degrees C, 45 degrees C, 60 degrees C, 75 degrees C, and 90 degrees C) and solid/liquid ratios (1:10 and 1:15) in a thermostatized bath. Soluble protein content of the extracts was determined by Lowry's method. Dry substrate concentrations of 5%, 7.5%, and 10% and enzyme doses of 2 and 5 IU/g dry matter were used for the enzymatic hydrolysis in an orbital incubator at 50 degrees C and 100 rpm. Both cellulase and xylanase were used. Reducing sugars produced were determined with the dinitrosalicylic acid method. The highest protein extraction yield for the ammonia-treated leaves was 29.10%, which was 50% higher than with the untreated leaves (20%), and was obtained at 90 degrees C with a 1:10 solid/liquid ratio. The concentrate had a protein content of 36.35% and the amino acid profile was suitable for swine and poultry. The highest sugar yield was 54.72% with respect to theoretical and was obtained with 5% solids and an enzyme dose of 5 IU/g dry matter. This yield was 3.4 times higher than the yield of the untreated leaves (16.13%). These results indicate that cassava leaves have a great potential for animal feeding and ethanol production. Both protein extraction and sugar yields may be enhanced by optimizing the ammonia treatment. PMID:19067247

  7. Enzymatic Hydrolysis of Trilactone Siderophores: Where Chiral Recognition Occurs in Enterobactin and Bacillibactin Iron Transport1

    PubMed Central

    Abergel, Rebecca J.; Zawadzka, Anna M.; Hoette, Trisha M.; Raymond, Kenneth N.

    2009-01-01

    Bacillibactin and enterobactin are hexadentate catecholate siderophores produced by bacteria upon iron limitation to scavenge ferric ion and seem to be the ultimate siderophores of their two respective domains: Gram-positive and Gram-negative. Iron acquisition mediated by these trilactone-based ligands necessitates enzymatic hydrolysis of the scaffold for successful intracellular iron delivery. The esterases BesA and Fes hydrolyze bacillibactin and enterobactin, respectively, as well as the corresponding iron complexes. Bacillibactin binds iron through three 2,3-catecholamide moieties linked to a tri-threonine scaffold via glycine spacers, whereas in enterobactin the iron-binding moieties are directly attached to a tri-l-serine backbone; although apparently minor, these structural differences result in markedly different iron coordination properties and iron transport behavior. Comparison of the solution thermodynamic and circular dichroism properties of bacillibactin, enterobactin and the synthetic analogs d-enterobactin, SERGlyCAM and d-SERGlyCAM has determined the role of each different feature in the siderophores' molecular structures in ferric complex stability and metal chirality. While opposite metal chiralities in the different complexes did not affect transport and incorporation in Bacillus subtilis, ferric complexes formed with the various siderophores did not systematically promote growth of the bacteria. The bacillibactin esterase BesA is less specific than the enterobactin esterase Fes; BesA can hydrolyze the trilactones of both siderophores, while only the tri-l-serine trilactone is a substrate of Fes. Both enzymes are stereospecific and cannot cleave tri-d-serine lactones. These data provide a complete picture of the microbial iron transport mediated by these two siderophores, from initial recognition and transport to intracellular iron release. PMID:19673474

  8. The pretreatment of corn stover with Gloeophyllum trabeum KU-41 for enzymatic hydrolysis

    PubMed Central

    2012-01-01

    Background Pretreatment is an essential step in the enzymatic hydrolysis of biomass for bio-ethanol production. The dominant concern in this step is how to decrease the high cost of pretreatment while achieving a high sugar yield. Fungal pretreatment of biomass was previously reported to be effective, with the advantage of having a low energy requirement and requiring no application of additional chemicals. In this work, Gloeophyllum trabeum KU-41 was chosen for corn stover pretreatment through screening with 40 strains of wood-rot fungi. The objective of the current work is to find out which characteristics of corn stover pretreated with G. trabeum KU-41 determine the pretreatment method to be successful and worthwhile to apply. This will be done by determining the lignin content, structural carbohydrate, cellulose crystallinity, initial adsorption capacity of cellulase and specific surface area of pretreated corn stover. Results The content of xylan in pretreated corn stover was decreased by 43% in comparison to the untreated corn stover. The initial cellulase adsorption capacity and the specific surface area of corn stover pretreated with G. trabeum were increased by 7.0- and 2.5-fold, respectively. Also there was little increase in the cellulose crystallinity of pretreated corn stover. Conclusion G. trabeum has an efficient degradation system, and the results indicated that the conversion of cellulose to glucose increases as the accessibility of cellulose increases due to the partial removal of xylan and the structure breakage of the cell wall. This pretreatment method can be further explored as an alternative to the thermochemical pretreatment method. PMID:22559172

  9. Alkaline-sulfite pretreatment and use of surfactants during enzymatic hydrolysis to enhance ethanol production from sugarcane bagasse.

    PubMed

    Mesquita, Jéssica Faria; Ferraz, André; Aguiar, André

    2016-03-01

    Sugarcane bagasse is a by-product from the sugar and ethanol industry which contains approximately 70 % of its dry mass composed by polysaccharides. To convert these polysaccharides into fuel ethanol it is necessary a pretreatment step to increase the enzymatic digestibility of the recalcitrant raw material. In this work, sugarcane bagasse was pretreated by an alkaline-sulfite chemithermomechanical process for increasing its enzymatic digestibility. Na2SO3 and NaOH ratios were fixed at 2:1, and three increasing chemical loads, varying from 4 to 8 % m/m Na2SO3, were used to prepare the pretreated materials. The increase in the alkaline-sulfite load decreased the lignin content in the pretreated material up to 35.5 % at the highest chemical load. The pretreated samples presented enhanced glucose yields during enzymatic hydrolysis as a function of the pretreatment severity. The maximum glucose yield (64 %) was observed for the samples pretreated with the highest chemical load. The use of 2.5 g l(-1) Tween 20 in the hydrolysis step further increased the glucose yield to 75 %. Semi-simultaneous hydrolysis and fermentation of the pretreated materials indicated that the ethanol yield was also enhanced as a function of the pretreatment severity. The maximum ethanol yield was 56 ± 2 % for the sample pretreated with the highest chemical load. For the sample pretreated with the lowest chemical load (2 % m/m NaOH and 4 % m/m Na2SO3), adding Tween 20 during the hydrolysis process increased the ethanol yield from 25 ± 3 to 39.5 ± 1 %. PMID:26718203

  10. Enzymatic hydrolysis of various pretreated lignocellulosic substrates and the fermentation of the liberated sugars to ethanol and butanediol

    SciTech Connect

    Saddler, J.N.; Mes-Hartree, M.; Yu, E.K.C.; Brownell, H.H.

    1983-01-01

    Aspen wood and wheat straw were pretreated by exposure to steam at elevated temperatures. Chemical analysis of the substrates revealed that steam explosion differentially decomposed the pentosan component while leaving the glucan portion relatively unchanged. The pretreated residues could be used as substrates for growth of Trichoderma reesei C30 and T. harzianum E58. The cellulase activities detected were in some cases three times as high as those found when Solka Floc was used as the substrate. Culture filtrates of T. harzianum E58 could efficiently hydrolyze the hemicellulose-rich water-soluble fractions. This material was fermented by Klebsiella pneumoniae with 0.4-0.5 g of 2,3-butanediol produced per gram of sugar utilized. Once the steam-exploded residues had been water and alkali extracted, the enzymatically hydrolyzed substrates were readily fermented by Saccharomyces cerevisiae or Zymononas mobilis with values as high as 2% (w/v) ethanol obtained from 5% steam-exploded wood fractions. 30 references, 2 figures, 8 tables.

  11. Reducing non-productive adsorption of cellulase and enhancing enzymatic hydrolysis of lignocelluloses by noncovalent modification of lignin with lignosulfonate.

    PubMed

    Lou, Hongming; Wang, Mengxia; Lai, Huanran; Lin, Xuliang; Zhou, Mingsong; Yang, Dongjie; Qiu, Xueqing

    2013-10-01

    Four fractions of one commercial sodium lignosulfonate (SXP) with different molecular weight (MW) and anionic polymers were studied to reduce non-productive adsorption of cellulase on bound lignin in a lignocellulosic substrate. SXP with higher MW had stronger blocking effect on non-productive adsorption of a commercial Trichoderma reesi cellulase cocktail (CTec2) on lignin measured by quartz crystal microgravimetry with dissipation monitoring. Linear anionic aromatic polymers have strong blocking effect, but they would also reduce CTec2 adsorption on cellulose to decrease the enzymatic activity. The copolymer of lignin and polyethylene glycol (AL-PEG1000) has strong enhancement in enzymatic hydrolysis of lignocelluloses, because it not only improves the cellulase activity to cellulose, but also blocks the non-productive cellulase adsorption on lignin. Apart from improving the cellulase activity to cellulose, the enhancements of enzymatic hydrolysis of lignocellulose by adding AL-PEG1000 and SXPs are the result of the decreased cellulase non-productive adsorption on lignin. PMID:23958680

  12. Improving rheology and enzymatic hydrolysis of high-solid corncob slurries by adding lignosulfonate and long-chain fatty alcohols.

    PubMed

    Lou, Hongming; Wu, Shun; Li, Xiuli; Lan, Tianqing; Yang, Dongjie; Pang, Yuxia; Qiu, Xueqing; Li, Xuehui; Huang, Jinhao

    2014-08-20

    The effects of lignosulfonate (SXSL) and long-chain fatty alcohols (LFAs) on the rheology and enzymatic hydrolysis of high-solid corncob slurries were investigated. The application of 2.5% (w/w) SXSL increased the substrate enzymatic digestibility (SED) of high-solid corncob slurries at 72 h from 31.7 to 54.0%, but meanwhile it increased the slurry's yield stress and complex viscosity to make the slurry difficult to stir and pump. The smallest molecular weight (MW) SXSL fraction had the strongest enhancement on SED. The SXSL fraction with large MW had a negative effect on rheology. n-Octanol (C8) and n-decanol (C10) improved the rheological properties of high-solid slurry and are strong enough to counteract the negative effect of SXSL. Furthermore, C8 and C10 clearly enhanced the enzymatic hydrolysis of high-solid corncob slurries with and without SXSL. A mechanism was proposed to explain the observed negative effect of SXSL and the positive effect of LFAs on the rheological properties. PMID:25111907

  13. Use of an electrodialytic reactor for the simultaneous β-lactoglobulin enzymatic hydrolysis and fractionation of generated bioactive peptides.

    PubMed

    Doyen, Alain; Husson, Eric; Bazinet, Laurent

    2013-02-15

    The enzymatic hydrolysis of β-lactoglobulin and the fractionation of peptides were performed in one step in an electrodialysis cell with ultrafiltration membranes stacked. After 240 min of treatment, 15 anionic and 4 cationic peptides were detected in the anionic and cationic peptide recovery compartments. Amongst these 15 anionic peptides, 2 hypocholesterolemic, 3 antihypertensive and 1 antibacterial peptides were recovered and concentrated with migration rates ranging from 5.5% and 81.7%. Amongst the 4 cationic peptides, the peptide sequence ALPMHIR, identified as lactokinin and known to exert an important antihypertensive effect, was recovered with an estimated 66% migration rate. To our knowledge, it was the first attempt to perform hydrolysis under an electric field and to simultaneously separate anionic and cationic peptides produced. PMID:23194514

  14. Fish peptone development using enzymatic hydrolysis of silver carp by-products as a nitrogen source in Staphylococcus aureus media.

    PubMed

    Fallah, Meysam; Bahram, Somayeh; Javadian, Seyed Roholla

    2015-03-01

    Fish peptone was produced using enzymatic hydrolysis of silver carp filleting by-products by alcalase and trypsin. Also, the efficiency of the hydrolysates as a nitrogen source in Staphylococcus aureus medium was compared with commercial TSB. The results indicated that the protein hydrolysate from alcalase and trypsin had high protein content (92.92%, 91.53 respectively), and degree of hydrolysis (4.94%, 4.6% respectively).The results showed that silver carp filleting waste can be an efficient source for fish peptone production as a nitrogen source for S. aureus medium. However, the type of the used proteolytic enzyme considerably affected the performance of the resulting peptone despite the same DH. Fish peptone produced by alcalese performed significantly (P < 0.05) better than commercial TSB as a media for the bacteria while the performance of the trypsin peptone was not as good as the commercial medium. PMID:25838893

  15. Enzymatic hydrolysis of hardwood and softwood harvest residue fibers released by sulfur dioxide-ethanol-water fractionation.

    PubMed

    Yamamoto, Minna; Iakovlev, Mikhail; Bankar, Sandip; Tunc, Mehmet Sefik; van Heiningen, Adriaan

    2014-09-01

    The enzymatic hydrolysis of hardwood and softwood harvest residues treated by SO2-ethanol-water (SEW) fractionation was studied. The target was to convert these fibers with high yield into glucose monomers which could be further converted into biofuel by a subsequent fermentation stage. Hardwood biomass residues were efficiently digested at low enzyme dosage (5 FPU/g cellulose) whereas the softwood residues required notably higher enzyme dosage (20 FPU) for sufficient conversion. However, cellulase dosage of softwood could be reduced mannanase supplementation. Especially the high lignin content of softwood biomass pulps impairs the digestibility and thereby, improved delignification could notably enhance the hydrolysis yields. It was shown that inferior delignification of SW biomass is due to persistent polyphenolic acids present in coniferous bark, whereas no evidence of the negative effect of inorganics and acetone extractives was observed. Additionally, SW hydrolyzate was successfully converted into a mixture of butanol, acetone and ethanol through ABE fermentation. PMID:25022728

  16. An advanced understanding of the specific effects of xylan and surface lignin contents on enzymatic hydrolysis of lignocellulosic biomass

    SciTech Connect

    Ju, Xiaohui; Engelhard, Mark H.; Zhang, Xiao

    2013-01-17

    A deep understanding of biomass recalcitrance has been hampered by the intricate and heterogeneous nature of pretreated biomass substrates obtained from random deconstruction methods. In this study, we established a unique methodology based on chemical pulping principles to create "reference substrates" with intact cellulose fibers and controlled morphological and chemical properties that enable us to investigate the individual effect of xylan, bulk, and surface lignin content on enzymatic hydrolysis. We also developed and demonstrated an X-ray photoelectron spectroscopy (XPS) technique for quantifying surface lignin content on biomass substrates. The results from this study show that, apart from its hindrance effect, xylan can facilitate cellulose fibril swelling and thus create more accessible surface area, which improves enzyme and substrate interactions. Surface lignin has a significant impact on enzyme adsorption kinetics and hydrolysis rate. Advanced understanding of xylan, bulk, and surface lignin effects provides critical information for an effective biomass conversion process.

  17. Enzymatic hydrolysis of chitin pretreated by rapid depressurization from supercritical 1,1,1,2-tetrafluoroethane toward highly acetylated oligosaccharides.

    PubMed

    Villa-Lerma, Guadalupe; González-Márquez, Humberto; Gimeno, Miquel; Trombotto, Stéphane; David, Laurent; Ifuku, Shinsuke; Shirai, Keiko

    2016-06-01

    The hydrolysis of chitin treated under supercritical conditions was successfully carried out using chitinases obtained by an optimized fermentation of the fungus Lecanicillium lecanii. The biopolymer was subjected to a pretreatment based on suspension in supercritical 1,1,1,2-tetrafluoroethane (scR134a), which possesses a critical temperature and pressure of 101°C and 40bar, respectively, followed by rapid depressurization to atmospheric pressure and further fibrillation. This methodology was compared to control untreated chitins and chitin subjected to steam explosion showing improved production of reducing sugars (0.18mg/mL), enzymatic hydrolysis and high acetylation (FA of 0.45) in products with degrees of polymerization between 2 and 5. PMID:26970920

  18. Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation.

    PubMed

    Kim, Kyoung Hyoun; Choi, In Seong; Kim, Ho Myeong; Wi, Seung Gon; Bae, Hyeun-Jong

    2014-02-01

    The microalga Chlorella vulgaris is a potential feedstock for bioenergy due to its rapid growth, carbon dioxide fixation efficiency, and high accumulation of lipids and carbohydrates. In particular, the carbohydrates in microalgae make them a candidate for bioethanol feedstock. In this study, nutrient stress cultivation was employed to enhance the carbohydrate content of C. vulgaris. Nitrogen limitation increased the carbohydrate content to 22.4% from the normal content of 16.0% on dry weight basis. In addition, several pretreatment methods and enzymes were investigated to increase saccharification yields. Bead-beating pretreatment increased hydrolysis by 25% compared with the processes lacking pretreatment. In the enzymatic hydrolysis process, the pectinase enzyme group was superior for releasing fermentable sugars from carbohydrates in microalgae. In particular, pectinase from Aspergillus aculeatus displayed a 79% saccharification yield after 72h at 50°C. Using continuous immobilized yeast fermentation, microalgal hydrolysate was converted into ethanol at a yield of 89%. PMID:24333701

  19. Fish peptone development using enzymatic hydrolysis of silver carp by-products as a nitrogen source in Staphylococcus aureus media

    PubMed Central

    Fallah, Meysam; Bahram, Somayeh; Javadian, Seyed Roholla

    2015-01-01

    Fish peptone was produced using enzymatic hydrolysis of silver carp filleting by-products by alcalase and trypsin. Also, the efficiency of the hydrolysates as a nitrogen source in Staphylococcus aureus medium was compared with commercial TSB. The results indicated that the protein hydrolysate from alcalase and trypsin had high protein content (92.92%, 91.53 respectively), and degree of hydrolysis (4.94%, 4.6% respectively).The results showed that silver carp filleting waste can be an efficient source for fish peptone production as a nitrogen source for S. aureus medium. However, the type of the used proteolytic enzyme considerably affected the performance of the resulting peptone despite the same DH. Fish peptone produced by alcalese performed significantly (P < 0.05) better than commercial TSB as a media for the bacteria while the performance of the trypsin peptone was not as good as the commercial medium. PMID:25838893

  20. Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

    NASA Astrophysics Data System (ADS)

    Zheng, Yi; Pan, Zhongli; Zhang, Ruihong; Wang, Donghai; Jenkins, Bryan

    Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.

  1. Seafood-like flavour obtained from the enzymatic hydrolysis of the protein by-products of seaweed (Gracilaria sp.).

    PubMed

    Laohakunjit, Natta; Selamassakul, Orrapun; Kerdchoechuen, Orapin

    2014-09-01

    An enzymatic bromelain seaweed protein hydrolysate (eb-SWPH) was characterised as the precursor for thermally processed seafood flavour. Seaweed (Gracilaria fisheri) protein after agar extraction was hydrolysed using bromelain (enzyme activity=119,325 U/g) at 0-20% (w/w) for 0.5-24 h. Optimal hydrolysis conditions were determined using response surface methodology. The proposed model took into account the interaction effect of the enzyme concentration and hydrolysis time on the physicochemical properties and volatile components of eb-SWPH. The optimal hydrolysis conditions for the production of eb-SWPH were 10% bromelain for 3h, which resulted in a 38.15% yield and a 62.91% degree of hydrolysis value. Three free amino acids, arginine, lysine, and leucine, were abundant in the best hydrolysate. Ten volatile flavours of the best eb-SWPH were identified using gas chromatography/mass spectrometry. The predominant odourants were hexanal, hexanoic acid, nonanoic acid, and dihydroactinidiolide. The thermally processed seafood flavour produced from eb-SWPH exhibited a roasted seafood-like flavouring. PMID:24731327

  2. Lignin enrichment and enzyme deactivation as the root cause of enzymatic hydrolysis slowdown of steam pretreated sugarcane bagasse.

    PubMed

    Wallace, Joshua; Brienzo, Michel; García-Aparicio, María P; Görgens, Johann F

    2016-05-25

    The enzymatic hydrolysis (EH) rate normally decreases during the hydrolysis, leaving unhydrolyzed material as residue. This phenomenon occurs during the hydrolysis of both cellulose (avicel) and lignocellulosic material, in nature or even pretreated. The progression of EH of steam pretreated sugarcane bagasse was associated with an initial (fast), intermediate (slower) and recalcitrant (slowest) phases, at glucan to glucose conversion yields of 61.7, 81.6 and 86%, respectively. Even though the EH of avicel as a simpler material than steam pretreated sugarcane bagasse, EH slowdown was present. The less thermo-stable endo-xylanase lost 58% of initial enzyme activity, followed by β-glucosidase that lost 16%, culminating in FPase activity loss of 30% in the first 24hours. After 72hours of EH the total loss of FPase activity was 40% compared to the initial activity. Analysis of the solid residue from EH showed that lignin content, phenolic compounds and ash increased while glucan decreased as hydrolysis progressed. During the initial fast phase of EH, the total solid residue surface area consisted predominantly of internal surface area. Thereafter, in the intermediate and recalcitrant phases of EH, the ratio of external:internal surface area increased. The proposed fiber damage and decrease in internal surface area, probably by EH action, was visualized by scanning electron microscopy imagery. The higher lignin/glucan ratio as EH progressed and enzyme deactivation by thermo instability were the main effects observed, respectively to substrate and enzyme. PMID:26820122

  3. Limitations of the NNS assay for reducing sugars from saccharified lignocellulosics. [Trichoderma reesei

    SciTech Connect

    Rivers, D.B.; Gracheck, S.J.; Woodford, L.C.; Emert, G.H.

    1984-07-01

    An evaluation is presented of two DNS (2,4-dinitrosalicylic acid) assay procedures as well as high-performance liquid chromatography (HPLC) and YSI Glucose Analyzer analyses of sugars resulting from enzymatic saccharification of lignocellulosics. Trichoderma reesei was used to produce the cellulase system containing endoglucanase, cellobiohydrolase and cellobiase. Data suggest that the DNS assay can be used as an accurate analytical method for the evaluation of reducing sugars in pure solution as well as in supernatants from enzymatic saccharification if glucose is the sole product. However, only specific assay methods such as HPLC and YSI-type glucose analyzers should be used for the analysis of saccharides produced from the hydrolysis of native or pretreated lignocellulosics since the DNS assay is susceptible to interferences and therefore results in inaccurate analyses.

  4. Effect of dimethyl sulfoxide on ionic liquid 1-ethyl-3-methylimidazolium acetate pretreatment of eucalyptus wood for enzymatic hydrolysis.

    PubMed

    Wu, Long; Lee, Seung-Hwan; Endo, Takashi

    2013-07-01

    Ground eucalyptus wood was pretreated with 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc)-dimethyl sulfoxide (DMSO) solutions with different mixing ratios under various conditions. The changes in the composition and structure of the biomass were investigated; and the enzymatic hydrolysis performance of the pretreated biomass was evaluated. [EMIM]OAc-DMSO pretreatment had a relatively mild effect on the composition of the biomass, but excessively high pretreatment temperatures led to massive loss of xylan after pretreatment. The enzymatic digestibility of the biomass was significantly improved with increased pretreatment temperature. X-ray diffraction analysis revealed that the disruption of cellulose crystal structure by [EMIM]OAc at a sufficiently high temperature was primarily responsible for the remarkable improvement in the digestibility. Appropriate addition of DMSO could help minimize the consumption of [EMIM]OAc without impairing the performance of the ionic liquid, and contribute to the improvement in pretreatment efficiency due to the viscosity reduction effect on the pretreatment liquor. PMID:23685645

  5. Physicochemical and sensory characterization of refined and deodorized tuna (Thunnus albacares) by-product oil obtained by enzymatic hydrolysis.

    PubMed

    de Oliveira, Dayse A S B; Minozzo, Marcelo G; Licodiedoff, Silvana; Waszczynskyj, Nina

    2016-09-15

    In this study, the effects of chemical refining and deodorization on fatty acid profiles and physicochemical and sensory characteristics of the tuna by-product oil obtained by enzymatic hydrolysis were evaluated. Enzymatic extraction was conducted for 120 min at 60 °C and pH 6.5 using Alcalase at an enzyme-substrate ratio of 1:200 w/w. The chemical refining of crude oil consisted of degumming, neutralization, washing, drying, bleaching, and deodorization; deodorization was conducted at different temperatures and processing times. Although chemical refining was successful, temperature and chemical reagents favored the removal of polyunsaturated fatty acids (PUFA) from the oil. Aroma attributes of fishy odor, frying odor, and rancid odor predominantly contributed to the sensory evaluation of the product. Deodorization conditions of 160 °C for 1h and 200 °C for 1h were recommended for the tuna by-product oil, which is rich in PUFA. PMID:27080896

  6. Utilization of waste cellulose. IV. Comparative study of the reactivity of different substrates in the enzymatic hydrolysis with Trichoderma viride

    SciTech Connect

    David, C.; Thiry, P.

    1982-07-01

    Different cellulosic substrates are compared. Cellulose II (grinded Cellophane) was demonstrated to be a very reactive substrate without any other pretreatment. Indeed, Cellophane, even coated one side with PVDC or nitrocellulose, can be hydrolyzed to a very high yield of glucose in a reasonable time. Suspensions with an initial high content of substrate can be used. Different pretreatments (ball milling, ..gamma..-irradiation, and Fe/sup 2 +//H/sub 2/O/sub 2/) were found to modify the reactivity of cellulose I. The pretreatments which result in the chemical transformation of the glucose units of the initial cellulosic substrate are shown to inhibit the enzymatic hydrolysis.

  7. Modeling and simulation of an enzymatic reactor for hydrolysis of palm oil.

    PubMed

    Bhatia, S; Naidu, A D; Kamaruddin, A H

    1999-01-01

    Hydrolysis of palm oil has become an important process in Oleochemical industries. Therefore, an investigation was carried out for hydrolysis of palm oil to fatty acid and glycerol using immobilized lipase in packed bed reactor. The conversion vs. residence time data were used in Michaelis-Menten rate equation to evaluate the kinetic parameters. A mathematical model for the rate of palm oil hydrolysis was proposed incorporating role of external mass transfer and pore diffusion. The model was simulated for steady-state isothermal operation of immobilized lipase packed bed reactor. The experimental data were compared with the simulated results. External mass transfer was found to affect the rate of palm oil hydrolysis at higher residence time. PMID:10595445

  8. Influence of surfactant-free ionic liquid microemulsions pretreatment on the composition, structure and enzymatic hydrolysis of water hyacinth.

    PubMed

    Xu, Fan; Chen, Li; Wang, Aili; Yan, Zongcheng

    2016-05-01

    This study investigated the pretreatment performance of surfactant-free ionic liquid microemulsions (ILMs) on water hyacinth. Pretreatment effects were evaluated in terms of lignocellulosic composition, structure and enzymatic hydrolysis. Analysis of the regenerated water hyacinth indicated that the content of the lignocellulosic composition changed, and the surface became more porous. After being pretreated with ILM(a) (mass ratio of toluene: ethanol: 1-ethyl-3-methylimidazolium acetate ([Emim]Ac)=0.35:0.3:0.35) at 70°C for 12h, the maximum delignification of 63.6% was observed. The cellulose of the water hyacinth was well protected and retained during the pretreatment process. After being enzymatically hydrolyzed for 48 h, the reducing sugar yield of the water hyacinth pretreated with ILM(a) at 70°C for 6 h was 563.7 mg/g, and its hydrolysis yield (86.1%) was nearly four and a half times of that of the untreated one (20.2%). In conclusion, the designed surfactant-free ILMs exhibit promising potential application in biomass pretreatment. PMID:26913644

  9. Effects of Impurities in Alkali-Extracted Xylan on Its Enzymatic Hydrolysis to Produce Xylo-Oligosaccharides.

    PubMed

    Shen, Rui; Li, Hong-Qiang; Zhang, Jie; Xu, Jian

    2016-07-01

    As the second abundant natural carbohydrate, xylan is normally prepared through alkaline extraction and then used for xylo-oligosaccharides (XOS) production. However, the extracted xylan inevitably contains salt, ethanol, and pigment. In order to investigate the effects of these impurities on XOS production, the alkaline-extracted xylan with different kinds and concentrations of impurities was made and then hydrolyzed using alkaline xylanase (EC 3.2.1.8) to produce XOS. The results showed that a certain concentration of salt (NaCl) promoted the XOS production, while ethanol and pigment inhibited the enzymatic hydrolysis process significantly. The color value mainly ascribed to the phenolic compounds binding to xylan was a key restriction factor in the enzymatic hydrolysis later stage. Using optimal xylan sample (with 10 mg/mL NaCl, color value of 4.6 × 10(5), without ethanol) as substrate, the highest XOS yield of 58.58 % was obtained. As the substrate of XOS production, prepared xylan should contain colored materials and ethanol as less as possible, however, retains appropriate salt. PMID:26922729

  10. Fuel ethanol production from corn stover under optimized dilute phosphoric acid pretreatment and enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethanol is a renewable oxygenated fuel. Dilute acid pretreatment is a promising pretreatment technology for conversion of lignocellulosic biomass to fuel ethanol. Generation of fermentable sugars from corn stover involves pretreatment and enzymatic saccharification. Pretreatment is crucial as nat...

  11. Single cell oil production by Mortierella isabellina from steam exploded corn stover degraded by three-stage enzymatic hydrolysis in the context of on-site enzyme production.

    PubMed

    Fang, Hao; Zhao, Chen; Chen, Shaolin

    2016-09-01

    Single cell oil (SCO), promising as alternative oil source, was produced from steam exploded corn stover (SECS) by Mortierella isabellina. Different bioprocesses from SECS to SCO were compared and the bioprocess C using the three-stage enzymatic hydrolysis was found to be the most efficient one. The bioprocess C used the lowest enzyme input 20FPIU cellulase/g glucan and the shortest time 222h, but produced 44.94g dry cell biomass and 25.77g lipid from 327.63g dry SECS. It had the highest lipid content 57.34%, and its productivities and yields were much higher than those of the bioprocess B and comparable to the bioprocess A, indicating that the three-stage enzymatic hydrolysis could greatly improve the efficiency of the bioprocess from high solid loading SECS to SCO by Mortierella isabellina. This work testified the application value of three-stage enzymatic hydrolysis in lignocellulose-based bioprocesses. PMID:27343451

  12. Ethanol production from sugars obtained during enzymatic hydrolysis of elephant grass (Pennisetum purpureum, Schum.) pretreated by steam explosion.

    PubMed

    Scholl, Angélica Luisi; Menegol, Daiane; Pitarelo, Ana Paula; Fontana, Roselei Claudete; Zandoná Filho, Arion; Ramos, Luiz Pereira; Dillon, Aldo José Pinheiro; Camassola, Marli

    2015-09-01

    In this work, steam explosion was used a pretreatment method to improve the conversion of elephant grass (Pennisetum purpureum) to cellulosic ethanol. This way, enzymatic hydrolysis of vaccum-drained and water-washed steam-treated substrates was carried out with Penicillium echinulatum enzymes while Saccharomyces cerevisiae CAT-1 was used for fermentation. After 48 h of hydrolysis, the highest yield of reducing sugars was obtained from vaccum-drained steam-treated substrates that were produced after 10 min at 200 °C (863.42 ± 62.52 mg/g). However, the highest glucose yield was derived from water-washed steam-treated substrates that were produced after 10 min at 190 °C (248.34 ± 6.27 mg/g) and 200 °C (246.00 ± 9.60 mg/g). Nevertheless, the highest ethanol production was obtained from water-washed steam-treated substrates that were produced after 6 min at 200 °C. These data revealed that water washing is a critical step for ethanol production from steam-treated elephant grass and that pretreatment generates a great deal of water soluble inhibitory compounds for hydrolysis and fermentation, which were partly characterized as part of this study. PMID:26038327

  13. pH-stat vs. free-fall pH techniques in the enzymatic hydrolysis of whey proteins.

    PubMed

    Fernández, Ayoa; Kelly, Phil

    2016-05-15

    Enzymatic hydrolysis of a commercial whey protein isolate (WPI) using either trypsin or Protamex® was compared using controlled (pH-stat) and uncontrolled (free-fall) pH conditions. pH-stat control at the enzyme's optimum value led to a more rapid rate of WPI hydrolysis by trypsin, while the opposite was the case when Protamex® was used. Furthermore, the choice of alkaline solution used to maintain constant pH during pH-stat experiments appeared to affect the reaction rate, being higher when KOH is added to the reaction mixture instead of NaOH. It would appear that potassium may play a role as co-factor or activator for the activity of this particular protease preparation. Although pH-stat techniques are usually considered to yield better hydrolysis kinetics, these findings suggest that the response of proteolytic enzyme preparations to static or free-fall pH control should be checked in advance, particularly when undertaking large scale production of WPI hydrolysates. PMID:26775989

  14. Extracellular Enzymatic Hydrolysis of High Molecular Weight Organic Carbon in Eastern Mediterranean Sapropelic and Non-Sapropelic Subsurface Sediments

    NASA Astrophysics Data System (ADS)

    Hoarfrost, A.; Couper, L.; Arnosti, C.

    2014-12-01

    Organic carbon availability is an important constraint on microbial activity in the subsurface. Since most sedimentary organic matter is likely high molecular weight and complex, bioavailability of organic carbon is closely tied to activities of extracellular enzymes that hydrolyze organic macromolecules into transportable sizes. In part due to methodological difficulties, few measurements of extracellular enzymatic activities have been made in marine sediments below ca. 20cm depth. We measured extracellular hydrolysis of specific polysaccharides in deep sediments from sapropel and non-sapropel sections of a single core from the Eastern Mediterranean. In order to counteract adsorption of the substrate onto sediment particles, we developed an extraction protocol utilizing competitive desorption and mild heating. This treatment improved substrate recovery from incubation subsamples 5- to 10-fold, and enabled us to detect enzymatic activity in deep subsurface sediments. The wide variation in TOC between proximal sediment layers in this core provided an excellent opportunity to investigate (i) the rate at which subsurface microbial communities can hydrolyze a diversity of organic substrates, and (ii) rates and ranges of enzymatic capabilities as a function of sediment depth, organic carbon load and microbial community composition. Our experiments were carried out in long-term incubations (3-6 weeks), in which substrates were readily hydrolyzed, but hydrolysis rates differed among substrates and among sediment sections. Activity was not correlated with depth, but was highest in sections with highest organic carbon content. Isolation of strains able to grow directly on the substrates of interest are underway, and provide a promising path forward to illuminate mechanisms driving potential hydrolytic activity in the subsurface.

  15. Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production.

    PubMed

    Avci, Ayse; Saha, Badal C; Dien, Bruce S; Kennedy, Gregory J; Cotta, Michael A

    2013-02-01

    Dilute H(3)PO(4) (0.0-2.0%, v/v) was used to pretreat corn stover (10%, w/w) for conversion to ethanol. Pretreatment conditions were optimized for temperature, acid loading, and time using central composite design. Optimal pretreatment conditions were chosen to promote sugar yields following enzymatic digestion while minimizing formation of furans, which are potent inhibitors of fermentation. The maximum glucose yield (85%) was obtained after enzymatic hydrolysis of corn stover pretreated with 0.5% (v/v) acid at 180°C for 15min while highest yield for xylose (91.4%) was observed from corn stover pretreated with 1% (v/v) acid at 160°C for 10min. About 26.4±0.1g ethanol was produced per L by recombinant Escherichia coli strain FBR5 from 55.1±1.0g sugars generated from enzymatically hydrolyzed corn stover (10%, w/w) pretreated under a balanced optimized condition (161.81°C, 0.78% acid, 9.78min) where only 0.4±0.0g furfural and 0.1±0.0 hydroxylmethyl furfural were produced. PMID:23334017

  16. Fractionation of wheat straw by prehydrolysis, organosolv delignification and enzymatic hydrolysis for production of sugars and lignin.

    PubMed

    Huijgen, W J J; Smit, A T; de Wild, P J; den Uil, H

    2012-06-01

    Wheat straw was fractionated using a three-step biorefining approach: (1) aqueous pretreatment for hemicellulose prehydrolysis into sugars, (2) organosolv delignification, and (3) enzymatic cellulose hydrolysis into glucose. Prehydrolysis was applied to avoid degradation of hemicellulose sugars during organosolv delignification. Maximum xylose yield obtained was 67% or 0.17 kg/kg straw (prehydrolysis: 175 °C, 30 min, 20mM H(2)SO(4)) compared to 4% in case of organosolv without prehydrolysis (organosolv: 200 °C, 60 min, 60% w/w aqueous ethanol). Prehydrolysis was found to reduce the lignin yield by organosolv delignification due to the formation of 'pseudo-lignin' and lignin recondensation during prehydrolysis. This reduction could partly be compensated by increasing the temperature of the organosolv delignification step. Prehydrolysis substantially improved the enzymatic cellulose digestibility from 49% after organosolv without prehydrolysis to 80% (20 FPU/g substrate). Increasing the organosolv delignification temperature to 220 °C resulted in a maximum enzymatic glucose yield of 93% or 0.36 kg/kg straw. PMID:22446052

  17. Hydrolyzability of xylan after adsorption on cellulose: Exploration of xylan limitation on enzymatic hydrolysis of cellulose.

    PubMed

    Wang, Xiao; Li, Kena; Yang, Ming; Zhang, Junhua

    2016-09-01

    During pretreatment of lignocellulosic materials, the dissolved xylan would re-adsorb on cellulose, and then inhibits the cellulose hydrolysis by cellulases. However, the hydrolyzability of xylan adsorbed on cellulose is not clear. In this work, the adsorption behavior of xylans on celluloses and the hydrolysis of adsorbed xylan by xylanase (XYL) were investigated. The results indicated that the adsorption of beechwood xylan (BWX) and oat spelt xylan (OSX) on Avicel was conformed to Langmuir-type adsorption isotherm. Higher ion strength increased the adsorption of BWX on Avicel, but not that of OSX. Both BWX and OSX adsorbed on Avicel and corn stover after dilute acid pretreatment (CS-DA) could be hydrolyzed by XYL. Compared to OSX, BWX adsorbed on cellulosic materials could be more easily hydrolyzed by XYL. Thus, supplementation of XYL could hydrolyze the xylan adsorbed on cellulose and potentially improved hydrolysis efficiency of lignocelluloses. PMID:27185150

  18. Enzymatic hydrolysis of penicillin and in situ product separation in thermally induced reversible phase-separation of ionic liquids/water mixture.

    PubMed

    Mai, Ngoc Lan; Koo, Yoon-Mo

    2014-09-01

    Enzymatic hydrolysis of penicillin G to produce 6-aminopenicillanic acid, key intermediate for the production of semisynthetic β-lactam antibiotics, is one of the most relevant example of industrial implementation of biocatalysts. The hydrolysis reaction is traditionally carried out in aqueous buffer at pH 7.5-8. However, the aqueous rout exhibits several drawbacks in enzyme stability and product recovery. In this study, several ionic liquids (ILs) have been used as media for enzymatic hydrolysis of penicillin G. The results indicated that hydrophobic ILs/water two-phase system were good media for the reaction. In addition, a novel aqueous two-phase system based on the lower critical solution temperature type phase changes of amino acid based ILs/water mixture was developed for in situ penicillin G hydrolysis and product separation. For instance, hydrolysis yield of 87.13% was obtained in system containing 30 wt% [TBP][Tf-ILe] with pH control (pH 7.6). Since the phase-separation of this medium system can be reversible switched from single to two phases by slightly changing the solution temperature, enzymatic hydrolytic reaction and product recovery were more efficient than those of aqueous system. In addition, the ILs could be reused for at least 5 cycles without significant loss in hydrolysis efficiency. PMID:25039057

  19. Enantioselective enzymatic hydrolysis of racemic drugs by encapsulation in sol-gel magnetic sporopollenin.

    PubMed

    Yilmaz, Elif

    2012-05-01

    Candida rugosa lipase was encapsulated within a sol-gel procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of octyltriethoxysilane and tetraethoxysilane in the presence of magnetic sporopollenin. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e., the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of racemic naproxen methyl ester, mandelic acid methyl ester or 2-phenoxypropionic acid methyl ester that were studied in aqueous buffer solution/isooctane reaction system. The encapsulated magnetic sporopollenin (Spo-M-E) was found to give 319 U/g of support with 342% activity yield. It has been observed that the percent activity yields and enantioselectivity of the magnetic sporopollenin encapsulated lipase were higher than that of the encapsulated lipase without support. The substrate specificity of the encapsulated lipase revealed more efficient hydrolysis of the racemic naproxen methyl ester and 2-phenoxypropionic acid methyl ester than racemic mandelic acid methyl ester. It was observed that excellent enantioselectivity (E > 400) was obtained for encapsulated lipase with magnetic sporopollenin with an ee value of S-Naproxen and R-2 phenoxypropionic acid about 98%. PMID:21932062

  20. ENZYMATIC HYDROLYSIS OF ORGANIC PHOSPHORUS IN EXTRACTS IN RESUSPENSIONS OF SWINE MANURE AND CATTLE MANURE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Animal manure can be a valuable resource of P for plant growth. Organic phosphates (Po) are considered bioavailable if they can be hydrolyzed to inorganic P. Therefore, investigation of the susceptibility of manure Po to hydrolysis may increase our understanding of manure Po bioavailability. In this...

  1. Recovery and reuse of cellulase catalyst in an enzymatic cellulose hydrolysis process

    DOEpatents

    Woodward, J.

    1987-09-18

    A process for recovering cellulase from the hydrolysis of cellulose, and reusing it in subsequent hydrolyois procedures. The process utilizes a commercial adsorbent that efficiently removes cellulase from reaction products which can be easily removed by simple decantation. 1 fig., 4 tabs.

  2. Effects of different enzymatic hydrolysis methods on the bioactivity of peptidoglycan in Litopenaeus vannamei

    NASA Astrophysics Data System (ADS)

    Song, Xiaoling; Zhang, Yue; Wei, Song; Huang, Jie

    2013-03-01

    The effects of different hydrolysis methods on peptidoglycan (PG) were assessed in terms of their impact on the innate immunity and disease resistance of Pacific white shrimp, Litop enaeus vannamei. PG derived from Bifidobacterium thermophilum was prepared in the laboratory and processed with lysozyme and protease under varying conditions to produce several different PG preparations. A standard shrimp feed was mixed with 0.05% PG preparations to produce a number of experimental diets for shrimp. The composition, concentration, and molecular weight ranges of the soluble PG were analyzed. Serum phenoloxidase and acid phosphatase activity in the shrimp were determined on Days 6—31 of the experiment. The protective activity of the PG preparations was evaluated by exposing shrimp to white spot syndrome virus (WSSV). Data on the composition of the PG preparations indicated that preparations hydrolyzed with lysozyme for 72 h had more low-molecular-weight PG than those treated for 24 h, and hydrolysis by protease enhanced efficiency of hydrolysis compared to lysozyme. SDS-PAGE showed changes in the molecular weight of the soluble PG produced by the different hydrolysis methods. Measurements of serum phenoloxidase and acid phosphatase activity levels in the shrimp indicated that the PG preparations processed with enzymes were superior to the preparation which had not undergone hydrolysis in enhancing the activity of the two serum enzymes. In addition, the preparation containing more low-molecular-weight PG enhanced the resistance of the shrimp to WSSV, whereas no increased resistance was observed for preparations containing less low-molecular-weight PG. These findings suggest that the immunity-enhancing activity of PG is related to its molecular weight and that increasing the quantity of low-molecular-weight PG can fortify the effect of immunity enhancement.

  3. Fungal secretomes enhance sugar beet pulp hydrolysis

    PubMed Central

    Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland

    2014-01-01

    The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g–1 protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1–17.5 mg g–1 SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. PMID:24677771

  4. Fungal secretomes enhance sugar beet pulp hydrolysis.

    PubMed

    Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland

    2014-04-01

    The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. PMID:24677771

  5. ENZYMATIC HYDROLYSIS AND FERMENTATION OF LIME PRETREATED WHEAT STRAW TO ETHANOL

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat straw used in this study contained 44.24 +/- 0.28% cellulose and 25.23 +/- 0.11% hemicellulose. Lime pretreatment and enzymatic saccharification methods were evaluated for conversion of wheat straw cellulose and hemicellulose to fermentable sugars. The maximum yield of monomeric sugars from ...

  6. Influence of enzymatic hydrolysis on the allergenicity of roasted peanut protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peanut allergy is recognized as one of the most severe food allergies. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family; such as, soybean, chickpea, and lentil. There are only a few studies carried out w...

  7. Effects of algal hydrolysate as reaction medium on enzymatic hydrolysis of lignocelluloses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Algal biomass has been proposed as a source of lipids and sugars for biofuel productions. However, a substantial portion of potentially valuable algal material remains as a liquid hydrolysate after sugar and lipid extractions. This study examined the effects of an algal hydrolysate on the enzymatic...

  8. Difference analysis of the enzymatic hydrolysis performance of acid-catalyzed steam-exploded corn stover before and after washing with water.

    PubMed

    Zhu, Junjun; Shi, Linli; Zhang, Lingling; Xu, Yong; Yong, Qiang; Ouyang, Jia; Yu, Shiyuan

    2016-10-01

    The difference in the enzymatic hydrolysis yield of acid-catalyzed steam-exploded corn stover (ASC) before and after washing with water reached approximately 15 % under the same conditions. The reasons for the difference in the yield between ASC and washed ASC (wASC) were determined through the analysis of the composition of ASC prehydrolyzate and sugar concentration of enzymatic hydrolyzate. Salts produced by neutralization (CaSO4, Na2SO4, K2SO4, and (NH4)2SO4), sugars (polysaccharides, oligosaccharides, and monosaccharides), sugar-degradation products (weak acids and furans), and lignin-degradation products (ethyl acetate extracts and nine main lignin-degradation products) were back-added to wASC. Results showed that these products, except furans, exerted negative effect on enzymatic hydrolysis. According to the characteristics of acid-catalyzed steam explosion pretreatment, the five sugar-degradation products' mixture and salts [Na2SO4, (NH4)2SO4] showed minimal negative inhibition effect on enzymatic hydrolysis. By contrast, furans demonstrated a promotion effect. Moreover, soluble sugars, such as 13 g/L xylose (decreased by 6.38 %), 5 g/L cellobiose (5.36 %), 10 g/L glucose (3.67 %), as well as lignin-degradation products, and ethyl acetate extracts (4.87 %), exhibited evident inhibition effect on enzymatic hydrolysis. Therefore, removal of soluble sugars and lignin-degradation products could effectively promote the enzymatic hydrolysis performance. PMID:27277746

  9. Optimization of nitrogen recovery in the enzymatic hydrolysis of dogfish (Squalus acanthias) protein. Composition of the hydrolysates.

    PubMed

    Diniz, F M; Martin, A M

    1997-05-01

    The recovery of nitrogen in the enzymatic hydrolysis of dogfish shark muscle was optimized by the use of response surface methodology. The optimum values for enzyme/substrate ratio, temperature and pH were found to be 3.7% (w/w), 55.3 degrees C, and 8.3, respectively. The dogfish protein hydrolysate produced under these conditions contained a high crude protein concentration (> 85%), and its high nutritional value was indicated by the presence of all essential amino acids, and by high PER values. These results indicate the potential for dogfish protein hydrolysate to be used in lieu of vegetable proteins as a protein supplement in foods. PMID:9205594

  10. Mechanical property of different corn stover morphological fractions and its correlations with high solids enzymatic hydrolysis by periodic peristalsis.

    PubMed

    Liu, Zhi-Hua; Chen, Hong-Zhang

    2016-08-01

    Selective structure fractionation combined with periodic peristalsis was exploited to improve the conversion performance of corn stover. The increase of glucan and lignin content and the decrease of xylan content in stem pith were highest after SE, whereas they were lowest in stem node. Glucan conversion increased in this order: steam nodeenzymatic hydrolysis efficiency of different corn stover morphological fractions. PMID:27140819

  11. Enhanced enzymatic hydrolysis of poplar bark by combined use of gamma ray and dilute acid for bioethanol production

    NASA Astrophysics Data System (ADS)

    Chung, Byung Yeoup; Lee, Jae Taek; Bai, Hyoung-Woo; Kim, Ung-Jin; Bae, Hyeun-Jong; Gon Wi, Seung; Cho, Jae-Young

    2012-08-01

    Pretreatment of poplar bark with a combination of sulfuric acid (3%, w/w, H2SO4) and gamma irradiation (0-1000 kGy) was performed in an attempt to enhance enzymatic hydrolysis for bioethanol production. The yields of reducing sugar were slightly increased with an increasing irradiation dose, ranging from 35.4% to 51.5%, with a 56.1% reducing sugar yield observed after dilute acid pretreatment. These results clearly showed that soluble sugars were released faster and to a greater extent in dilute acid-pretreated poplar bark than in gamma irradiation-pretreated bark. When combined pretreatment was carried out, a drastic increase in reducing sugar yield (83.1%) was found compared with individual pretreatment, indicating the possibility of increasing the convertibility of poplar bark following combined pretreatment. These findings are likely associated with cellulose crystallinity, lignin modification, and removal of hemicelluloses.

  12. Optimisation of cheese whey enzymatic hydrolysis and further continuous production of antimicrobial extracts by Lactobacillus plantarum CECT-221.

    PubMed

    Rodríguez-Pazo, Noelia; da Silva Sabo, Sabrina; Salgado-Seara, José Manuel; Arni, Saleh Al; de Souza Oliveira, Ricardo Pinheiro; Domínguez, José Manuel

    2016-08-01

    The enzymatic hydrolysis of cheese whey was optimised using the enzymes iZyme, Alcalase or Flavourzyme under different conditions. Hydrolysates supplemented with commercial nutrients were evaluated as fermentation broths to produce DL-3-Phenyllactic acid (PLA) from phenylalanine (Phe) by Lactobacillus plantarum CECT-221. Optimised hydrolysates were obtained using Flavourzyme at 50 °C and 100 rpm during 12 h, and assayed in 250 ml Erlenemyer flasks using different proportions of vinasses as economic nutrient. The process was then scaled up using a 2 litres Bioreactor working under the continuous modality. Under the intermediate dilution rate of 0·0207 h-1 0·81 ± 0·026 mM of PLA and 38·8 ± 3·253 g/l of lactic acid were produced. A final evaluation revealed that lactic acid, and bacteriocins exerted the highest inhibitory effect among the extracted components of cell-free supernatants. PMID:27600978

  13. Small-Angle Neutron Scattering Reveals pH-Dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I: Implications for Enzymatic Activity

    SciTech Connect

    Pingali, Sai Venkatesh; O'Neill, Hugh Michael; McGaughey, Joseph; Urban, Volker S; Rempe, Caroline S; Petridis, Loukas; Smith, Jeremy C; Evans, Barbara R; Heller, William T

    2011-01-01

    Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4 5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solutions having pH values of 7.0, 6.0, 5.3, and 4.2. As the pH decreases from 7.0 to 5.3, the enzyme structure remains well defined, possessing a spatial differentiation between the cellulose binding domain and the catalytic core that only changes subtly. At pH 4.2, the solution conformation of the enzyme changes to a structure that is intermediate between a properly folded enzyme and a denatured, unfolded state, yet the secondary structure of the enzyme is essentially unaltered. The results indicate that at the pH of optimal activity, the catalytic core of the enzyme adopts a structure in which the compact packing typical of a fully folded polypeptide chain is disrupted and suggest that the increased range of structures afforded by this disordered state plays an important role in the increased activity of Cel7A through conformational selection.

  14. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review.

    PubMed

    Alvira, P; Tomás-Pejó, E; Ballesteros, M; Negro, M J

    2010-07-01

    Biofuel produced from lignocellulosic materials, so-called second generation bioethanol shows energetic, economic and environmental advantages in comparison to bioethanol from starch or sugar. However, physical and chemical barriers caused by the close association of the main components of lignocellulosic biomass, hinder the hydrolysis of cellulose and hemicellulose to fermentable sugars. The main goal of pretreatment is to increase the enzyme accessibility improving digestibility of cellulose. Each pretreatment has a specific effect on the cellulose, hemicellulose and lignin fraction thus, different pretreatment methods and conditions should be chosen according to the process configuration selected for the subsequent hydrolysis and fermentation steps. This paper reviews the most interesting technologies for ethanol production from lignocellulose and it points out several key properties that should be targeted for low-cost and advanced pretreatment processes. PMID:20042329

  15. Impact of enzymatic and alkaline hydrolysis on CBD concentration in urine.

    PubMed

    Bergamaschi, Mateus M; Barnes, Allan; Queiroz, Regina H C; Hurd, Yasmin L; Huestis, Marilyn A

    2013-05-01

    A sensitive and specific analytical method for cannabidiol (CBD) in urine was needed to define urinary CBD pharmacokinetics after controlled CBD administration, and to confirm compliance with CBD medications including Sativex-a cannabis plant extract containing 1:1 ∆(9)-tetrahydrocannabinol (THC) and CBD. Non-psychoactive CBD has a wide range of therapeutic applications and may also influence psychotropic smoked cannabis effects. Few methods exist for the quantification of CBD excretion in urine, and no data are available for phase II metabolism of CBD to CBD-glucuronide or CBD-sulfate. We optimized the hydrolysis of CBD-glucuronide and/or -sulfate, and developed and validated a GC-MS method for urinary CBD quantification. Solid-phase extraction isolated and concentrated analytes prior to GC-MS. Method validation included overnight hydrolysis (16 h) at 37 °C with 2,500 units β-glucuronidase from Red Abalone. Calibration curves were fit by linear least squares regression with 1/x (2) weighting with linear ranges (r(2) > 0.990) of 2.5-100 ng/mL for non-hydrolyzed CBD and 2.5-500 ng/mL for enzyme-hydrolyzed CBD. Bias was 88.7-105.3 %, imprecision 1.4-6.4 % CV and extraction efficiency 82.5-92.7 % (no hydrolysis) and 34.3-47.0 % (enzyme hydrolysis). Enzyme-hydrolyzed urine specimens exhibited more than a 250-fold CBD concentration increase compared to alkaline and non-hydrolyzed specimens. This method can be applied for urinary CBD quantification and further pharmacokinetics characterization following controlled CBD administration. PMID:23494274

  16. The Simultineous Enzymatic Hydrolysis of Tapioca Starch for Instant Formation of Glucose

    NASA Astrophysics Data System (ADS)

    Sarbatly, Rosalam

    This study investigated the possibility of simultaneous reactions of the gelatinization, liquefaction and saccharification (SGLS) carried out at two reaction temperatures of saccharification 55 and 60°C for instant glucose production as well as controlling low viscosity of solute over the hydrolysis period. At 55°C, 10% (w/w) of the tapioca starch and 0.9 mL L-1 of a blending mixture of α-amylase and amyloglocosidase, the viscosity was kept low below 2.2x10-3 pa-s throughout the hydrolysis process. The conversion of the tapioca starch to glucose was as high as 65% (w/w) over 28 h of the hydrolysis time. Increasing the temperature to 60°C did not increase the conversion but, (1) increased the maximum rate of reaction from 8.89g L-1 h-1 to 13.3 g L-1h-1 (2) reduced the time to reach a half of the final glucose concentration from 6.1 to 5 h and also (3) slightly increased the earlier stage of solute viscosity without affecting the entire process.

  17. Enzymatic hydrolysis of starry triggerfish (Abalistes stellaris) muscle using liver proteinase from albacore tuna (Thunnus alalunga).

    PubMed

    Sripokar, P; Chaijan, M; Benjakul, S; Kishimura, H; Klomklao, S

    2016-02-01

    Proteinases from liver extract from albacore tuna (Thunnus alalunga) were used to produce protein hydrolysate from starry triggerfish (Abalistes stellaris) muscle. Hydrolysis conditions for preparing protein hydrolysate from starry triggerfish muscle were optimized. Enzyme level, reaction time and fish muscle/buffer ratio significantly affected the hydrolysis (p < 0.05). Optimum conditions for triggerfish muscle hydrolysis were 5.5 % liver extract, 40 min reaction time and fish muscle/buffer ratio of 1:3 (w/v). The freeze-dried protein hydrolysate was characterized with respect to chemical composition, amino acid composition and color. The product contained 91.73 % protein, 2.04 % lipid and 6.48 % ash. The protein hydrolysate exhibited high amount of essential amino acids (45.62 %). It was light yellow in color (L (*) = 82.94, a (*) = 0.84, b (*) = 22.83). The results indicate that the extract from liver of albacore tuna could be used to produce fish protein hydrolysate and protein hydrolysate from starry triggerfish muscle may potentially serve as a good source of desirable peptide and amino acids. PMID:27162384

  18. Comparison of Phosphorus Forms in Wet and Dried Animal Manures by Solution Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy and Enzymatic Hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Both enzymatic hydrolysis and solution 31P nuclear magnetic resonance (NMR) spectroscopy have been used to characterize P compounds in animal manures. However, no comparison of the two methods has been reported in the literature. In this study, we compared P compounds in dairy and poultry manures i...

  19. Pretreatment of dried distillers grains with solubles by soaking in aqueous ammonia and subsequent enzymatic/dilute acid hydrolysis to produce fermentable sugars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dried distillers grains with solubles (DDGS), a co-product of corn ethanol production in the dry-grind process, was pretreated by soaking in aqueous ammonia (SAA) using a 15% w/w NH4OH solution at a solid:liquid ratio of 1:10. The effect of pretreatment on subsequent enzymatic hydrolysis was studied...

  20. Effect of enzymatic hydrolysis of pancreatin and alcalase enzyme on some properties of edible bird's nest hydrolysate

    NASA Astrophysics Data System (ADS)

    Khushairay, Etty Syarmila Ibrahim; Ayub, Mohd Khan; Babji, Abdul Salam

    2014-09-01

    Edible bird nest (EBN) is a dried glutinous secretion from the salivary glands of several different swiftlet species. It is widely consumed as a health food due to its high beneficial effects to human health and has been considered to be one of the most precious food items by the Chinese for thousands of years. The aim of this study was to evaluate the effect of enzymatic hydrolysis on protein solubility (μg/g), the degree of hydrolysis (DH%), and peptide content (μg/g) of edible bird's nest hydrolysate. Initial protein solubility of boiled EBN was 25.5mg/g EBN. Treating the solubilized EBN with pancreatin 4NF for 1.0 - 1.5hours increased EBN protein solubility to 163.9mg/g and produced hydrolysate with DH% of 86.5% and 109.5mg/g peptide. EBN hydrolyzed with alcalase for 1.5 hours produced hydrolysate with protein solubility of 86.7mg/g, 82.7 DH% and had 104.1mg/g peptide content.

  1. Enzymatic hydrolysis and physical characterization of commercial celluloses and cellulose-based ion-exchange powdered mixed resins.

    PubMed

    Clarkin, S D; Clesceri, L S

    2002-12-01

    Commercial celluloses (BH20, Epicote, FC+) and their cellulose-containing powdered mixed resins (PMR) were evaluated using enzymatic and physical methods. Samples were hydrolyzed with purified Trichoderma viride cellulase extract and measured for released reducing sugar using the dinitrosalicylic acid method. Physical characterization was performed with gross specific surface areas (GSSA) and relative crystalline indices (RCI). In addition, FC+ was exposed to physical and chemical processing commonly encountered in spent PMR processing to determine potential effects on reducing sugar release in high intensity containers. Reducing sugar released from the celluloses by T. viride cellulase ranged from 135.37 to 244.48 mg day(-1); the celluloses were highly crystalline, ranging from 82.47 to 84.57%; and the GSSA medians for the celluloses ranged from 1,298.60 cm(2) g(-1) to 2,493.20 cm(2) g(-1). Most processing treatments on the FC+ reduced the amount of reducing sugar released and increased RCI. Cellulose hydrolysis rates did not show a strong correlation with the physical characterization. These results suggest that (1) celluloses and PMR can serve as abundant sources of bioavailable carbon in water treatment systems, and (2) the use of correlative physical characteristics to evaluate a cellulose-based commercial product may not accurately predict microbial activity; a complementary microbial test such as cellulose hydrolysis with cellulase may prove useful. PMID:12466892

  2. Molecular Investigation of the Mechanism of Non-Enzymatic Hydrolysis of Proteins and the Predictive Algorithm for Susceptibility.

    PubMed

    Lauer, Timothy M; Wood, Geoffrey P F; Farkas, David; Sathish, Hasige A; Samra, Hardeep S; Trout, Bernhardt L

    2016-06-14

    A number of potential degradation routes can limit the shelf life of a biotherapeutic. While these are experimentally measurable, the tests to do so require a substantial investment in both time and material, resources rarely available early in the drug development process. To address the potential degradation route of non-enzymatic hydrolysis, we performed a molecular modeling analysis, together with an experimental study, to gain detailed insight into the reaction. On the basis of the mechanism, an algorithm for predicting the likely cleavage sites of a protein has been created. This algorithm measures four key properties during a molecular dynamics simulation, which relate to the key steps of the hydrolysis mechanism, in particular the rate-determining step (which can vary depending on the local environment). The first two properties include the secondary structure and the surface exposure of the amide bond, both of which help detect if the addition of the proton to the amide bond is possible. The second two properties relate to whether the side chain can cyclize and form a furane ring. These two properties are the orientation of the side chain relative to the amide bond and the number of hydrogen bonds between the side chain and the surrounding protein. Overall, the algorithm performs well at identifying reactive versus nonreactive bonds. The algorithm correctly classifies nearly 90% of all amide bonds following an aspartic or glutamic acid residue as reactive or nonreactive. PMID:27194363

  3. A novel combined pretreatment of ball milling and microwave irradiation for enhancing enzymatic hydrolysis of microcrystalline cellulose.

    PubMed

    Peng, Huadong; Li, Hongqiang; Luo, Hao; Xu, Jian

    2013-02-01

    Microcrystalline cellulose (MCC) was performed as a mode substrate to investigate its potential ability of bioconversion in a novel combined pretreatment of ball milling (BM) and/or microwave irradiation (MWI). The variation of structure characteristics of MCC before/after pretreatment were investigated, including crystallinity index (CrI), size of crystal (S(C)), specific surface area (SSA) and degree of polymerization (DP). Their correlation with the rate of enzymatic hydrolysis was differentiated by an optimized equation which indicated the rate of hydrolysis was much more sensitive to CrI than SSA and DP. To achieve the same or higher glucose yield of BM for 3h and 6h, BM for 1h with MWI for 20min could save 54.8% and 77.40% energy consumption, respectively. Moreover, chemicals were not required in this process. It is concluded that the combination of BM and short time MWI is an environment-friendly, economical and effective approach to treat biomass. PMID:23306114

  4. Saccharification of microalgae biomass obtained from wastewater treatment by enzymatic hydrolysis. Effect of alkaline-peroxide pretreatment.

    PubMed

    Martín Juárez, Judit; Lorenzo Hernando, Ana; Muñoz Torre, Raúl; Blanco Lanza, Saúl; Bolado Rodríguez, Silvia

    2016-10-01

    An enzymatic method for the carbohydrate hydrolysis of different microalgae biomass cultivated in domestic (DWB) and pig manure (PMWB) wastewaters, at different storage conditions (fresh, freeze-dried and reconstituted), was evaluated. The DWB provided sugars yields between 40 and 63%, although low xylose yields (< 23.5%). Approximately 2% of this biomass was converted to byproducts as succinic, acetic and formic acids. For PMWB, a high fraction of the sugars (up to 87%) was extracted, but mainly converted into acetic, butyric and formic acids, which was attributed to the bacterial action. In addition, the performance of an alkaline-peroxide pretreatment, conducted for 1h, 50°C and H2O2 concentrations from 1 to 7.5% (w/w), was essayed. The hydrolysis of pretreated microalgae supported a wide range of sugars extraction for DWB (55-90%), and 100% for PMWB. Nevertheless, a large fraction of these sugars (∼30% for DWB and 100% for PMWB) was transformed to byproducts. PMID:27372005

  5. Modulating lipophilicity of rohitukine via prodrug approach: Preparation, characterization, and in vitro enzymatic hydrolysis in biorelevant media.

    PubMed

    Kumar, Vikas; Bharate, Sonali S; Vishwakarma, Ram A

    2016-09-20

    Rohitukine is a medicinally important natural product which has inspired the discovery of two anticancer clinical candidates. Rohitukine is highly hydrophilic in nature which hampers its oral bioavailability. Thus, herein our objective was to improve the drug-like properties of rohitukine via prodrug-strategy. Various ester prodrugs were synthesized and studied for solubility, lipophilicity, chemical stability and enzymatic hydrolysis in plasma/esterase. All prodrugs displayed lower aqueous solubility and improved lipophilicity compared with rohitukine, which was in accordance with the criteria of compounds in drug-discovery. The stability of synthesized prodrugs was evaluated in buffers at different pH, SGF, SIF, rat plasma and in esterase enzyme. The rate of hydrolysis in all incubation media was dependent primarily on the acyl promoieties. Hexanoyl ester prodrug of rohitukine, 3d, was stable under chemical conditions; however it was completely hydrolyzed to rohitukine, in plasma and in esterase in 4h. Hexanoate ester 3d appeared to be the most promising prodrug as it remained intact at gastric/intestinal pH and was completely transformed to the parent compound in plasma as desired for an ideal prodrug. The data presented herein, will help in designing prodrugs with desired physicochemical properties in future in structurally similar chemotypes. PMID:27422078

  6. Feasibility of enzymatic hydrolysis and alcoholic fermentation of starch contained in buffalo gourd (Cucurbita foetidissima) roots

    SciTech Connect

    Scheerens, J.C.; Kopplin, M.J.; Abbas, I.R.; Nelson, J.M.; Gathman, A.C.; Berry, J.W.

    1987-03-01

    The suitability of using annually grown, carrot-sized buffalo gourd (Cucurbita foetidissima) roots as a feedstock for alcoholic fermentation was explored. Roots grown in 1982 and 1983 were slurried, dextrinized and saccharified using Takatherm and Diazyme (commercial enzymes manufactured by Miles Laboratories), and fermented by the action of Saccharomyces cerevisiae. These processes were monitored in detail and results were compared with those displayed by controls formulated using potato tubers. The preparation of gourd root slurries with suitable viscosity characteristics for enzymatic digestion required the addition of water (at least 50% by weight) which reduced the proportion of fermentable sugars in the resulting saccharified suspensions. The resulting slurries were well-suited to enzymatic conversion of starch to sugar. Estimates of enzymatic efficiency in gourd root suspensions did not suggest the presence of naturally occurring amylase or glucosidase inhibitors in these plant materials. Saccharified gourd root mashes supported yeast growth well and produced ethanol yields at 82.2-86.5% of the theoretically maximum efficiency. 23 references.

  7. Feruloyl esterase production by Aspergillus terreus CECT 2808 and subsequent application to enzymatic hydrolysis.

    PubMed

    Pérez-Rodríguez, N; Moreira, C D; Torrado Agrasar, A; Domínguez, J M

    2016-09-01

    Ferulic acid esterases (FAE) were produced by Aspergillus terreus CECT 2808 from vine trimming shoots (VTS) and corn cob. Later, the fungal extracts thus obtained were used to enzymatically release ferulic acid (FA) from both substrates. Our findings showed a higher FAE activity in the enzymatic extracts produced on corn cob (0.070±0.004U/mL). Nevertheless, the enzymatic extracts produced on VTS demonstrated a better performance for FA release from both corn cob (2.05±0.01mg/g) and VTS (0.19±0.003mg/g). This result was probably because of the higher xylanase/FAE ratio determined in VTS extract. Therefore, an additional assay was carried out by supplementing corn cob extract with a commercial xylanase to test the influence of FAE/xylanase ratio in FA release. The results revealed the relevance of the FAE/xylanase ratio for an optimal FA release. PMID:27444329

  8. Ammonia fiber expansion (AFEX) pretreatment, enzymatic hydrolysis, and fermentation on empty palm fruit bunch fiber (EPFBF) for cellulosic ethanol production.

    PubMed

    Lau, Ming J; Lau, Ming W; Gunawan, Christa; Dale, Bruce E

    2010-11-01

    Empty palm fruit bunch fiber (EPFBF), a readily available cellulosic biomass from palm processing facilities, is investigated as a potential carbohydrate source for cellulosic ethanol production. This feedstock was pretreated using ammonia fiber expansion (AFEX) and enzymatically hydrolyzed. The best tested AFEX conditions were at 135 °C, 45 min retention time, water to dry biomass loading of 1:1 (weight ratio), and ammonia to dry biomass loading of 1:1 (weight ratio). The particle size of the pretreated biomass was reduced post-AFEX. The optimized enzyme formulation consists of Accellerase (84 μL/g biomass), Multifect Xylanase (31 μL/g biomass), and Multifect Pectinase (24 μL/g biomass). This mixture achieved close to 90% of the total maximum yield within 72 h of enzymatic hydrolysis. Fermentation on the water extract of this biomass affirms that nutrients solely from the pretreated EPFBF can support yeast growth for complete glucose fermentation. These results suggest that AFEX-treated EPFBF can be used for cellulosic biofuels production because biomass recalcitrance has been overcome without reducing the fermentability of the pretreated materials. PMID:20419480

  9. Combination of liquid hot water pretreatment and wet disk milling to improve the efficiency of the enzymatic hydrolysis of eucalyptus.

    PubMed

    Weiqi, Wei; Shubin, Wu; Liguo, Liu

    2013-01-01

    Combination of liquid hot water pretreatment (LHWP) and wet disk milling (WDM) was investigated in this study to enhance the sugar recovery yield both in prehydrolyzate and enzymatic hydrolyzate. The results show that WDM with LHWP at 180 °C for 20 min produced maximum xylose and glucose yields of 91.62% and 88.12%, respectively, which are higher than that of dilute acid pretreatment or individual LHWP. Corresponding concentration of fermentation inhibitors such as acetic acid, HMF, and furfural in the prehydrolyzate are about 0.98, 0.07 and 0.78 g/L, respectively, which indicated that the detoxification may be not required in the next fermentation step. The acid-insoluble lignin recovery in the insoluble solid resulting from enzymatic hydrolysis, was 25.67/100g raw material, representing 90.7% of acid-insoluble lignin in the eucalyptus biomass. It can be concluded that liquid hot water pretreatment combined with wet disk milling can be successfully applied to eucalyptus. PMID:23260273

  10. An on-line method for pressurized hot water extraction and enzymatic hydrolysis of quercetin glucosides from onions.

    PubMed

    Lindahl, Sofia; Liu, Jiayin; Khan, Samiullah; Karlsson, Eva Nordberg; Turner, Charlotta

    2013-06-27

    A novel environmentally sound continuous-flow hot water extraction and enzymatic hydrolysis method for determination of quercetin in onion raw materials was successfully constructed using a stepwise optimization approach. In the first step, enzymatic hydrolysis of quercetin-3,4'-diglucoside to quercetin was optimized using a three level central composite design considering temperature (75-95°C), pH (3-6) and volume concentration of ethanol (5-15%). The enzyme used was a thermostable β-glucosidase variant (termed TnBgl1A_N221S/P342L) covalently immobilized on either of two acrylic support-materials (Eupergit(®) C 250L or monolithic cryogel). Optimal reaction conditions were irrespective of support 84°C, 5% ethanol and pH 5.5, and at these conditions, no significant loss of enzyme activity was observed during 72 h of use. In a second step, hot water extractions from chopped yellow onions, run at the optimal temperature for hydrolysis, were optimized in a two level design with respect to pH (2.6 and 5.5), ethanol concentration (0 and 5%) and flow rate (1 and 3 mL min(-1)) Obtained results showed that the total quercetin extraction yield was 1.7 times higher using a flow rate of 3 mL min(-1) (extraction time 90 min), compared to a flow rate of 1 mL min(-1) (extraction time 240 min). Presence of 5% ethanol was favorable for the extraction yield, while a further decrease in pH was not, not even for the extraction step alone. Finally, the complete continuous flow method (84°C, 5% ethanol, pH 5.5, 3 mL min(-1)) was used to extract quercetin from yellow, red and shallot onions and resulted in higher or similar yield (e.g. 8.4±0.7 μmol g(-1) fresh weight yellow onion) compared to a conventional batch extraction method using methanol as extraction solvent. PMID:23764443

  11. Enzymatic synthesis of S-phenyl-L-cysteine from keratin hydrolysis industries wastewater with tryptophan synthase.

    PubMed

    Xu, Lisheng; Wang, Zhiyuan; Mao, Pingting; Liu, Junzhong; Zhang, Hongjuan; Liu, Qian; Jiao, Qing-Cai

    2013-04-01

    An economical method for production of S-phenyl-L-cysteine from keratin acid hydrolysis wastewater (KHW) containing L-serine was developed by recombinant tryptophan synthase. This study provides us with an alternative KHW utilization strategy to synthesize S-phenyl-L-cysteine. Tryptophan synthase could efficiently convert L-serine contained in KHW to S-phenyl-L-cysteine at pH 9.0, 40°C and Trion X-100 of 0.02%. In a scale up study, L-serine conversion rate reach 97.1% with a final S-phenyl-L-cysteine concentration of 38.6 g l(-1). PMID:23478091

  12. Bioconversion of wheat straw to ethanol: chemical modification, enzymatic hydrolysis, and fermentation

    SciTech Connect

    Detroy, R.W.; Lindenfelser, L.A.; Sommer, S.; Orton, W.L.

    1981-07-01

    The current studies involved a series of chemical treatment combinations upon wheat straw (WS), and subsequent hydrolysis of released cellulose to fermentable sugars. Primary chemical treatment of WS was followed by additional, secondary treatment of residues with either acids or alkali. Following primary and secondary treatment the WS residues were hydrolyzed with cellulase, and final glucose yields were determined. Samples of residues and effluents taken after each reaction also were analyzed. Large-scale experiments for both saccharification and alcohol production were investigated in a glass-column reactor. The results showed that conversion of the cellulosic component to sugar varied with the chemical modification steps. 23 refs.

  13. Building blocks for the solution phase synthesis of oligonucleotides: regioselective hydrolysis of 3',5'-Di-O-levulinylnucleosides using an enzymatic approach.

    PubMed

    García, Javier; Fernández, Susana; Ferrero, Miguel; Sanghvi, Yogesh S; Gotor, Vicente

    2002-06-28

    A short and convenient synthesis of 3'- and 5'-O-levulinyl-2'-deoxynucleosides has been developed from the corresponding 3',5'-di-O-levulinyl derivatives by regioselective enzymatic hydrolysis, avoiding several tedious chemical protection/deprotection steps. Thus, Candida antartica lipase B (CAL-B) was found to selectively hydrolyze the 5'-levulinate esters, furnishing 3'-O-levulinyl-2'-deoxynucleosides 3 in >80% isolated yields. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) and Candida antarctica lipase A (CAL-A) exhibit the opposite selectivity toward the hydrolysis at the 3'-position, affording 5'-O-levulinyl derivatives 4 in >70% yields. A similar hydrolysis procedure was successfully extended to the synthesis of 3'- and 5'-O-levulinyl-protected 2'-O-alkylribonucleosides 7 and 8. This work demonstrates for the first time application of commercial CAL-B and PSL-C toward regioselective hydrolysis of levulinyl esters with excellent selectivity and yields. It is noteworthy that protected cytidine and adenosine base derivatives were not adequate substrates for the enzymatic hydrolysis with CAL-B, whereas PSL-C was able to accommodate protected bases during selective hydrolysis. In addition, we report an improved synthesis of dilevulinyl esters using a polymer-bound carbodiimide as a replacement for dicyclohexylcarbodiimide (DCC), thus considerably simplifying the workup for esterification reactions. PMID:12076150

  14. Morphine-3-D glucuronide stability in postmortem specimens exposed to bacterial enzymatic hydrolysis.

    PubMed

    Carroll, F T; Marraccini, J V; Lewis, S; Wright, W

    2000-12-01

    Medical examiners frequently rely on the finding of free morphine present in postmortem specimens to assist in certifying deaths associated with narcotics. In vitro hydrolysis of morphine-3-D glucuronide (M3DG) to free morphine was studied using variable specimen pH, initial degree of specimen putrefaction, storage temperature and time, and the effectiveness of sodium fluoride (NaF) preservation. Reagent M3DG was added to opiate-free fresh blood and urine and to autopsy-derived blood specimens. Reagent bovine glucuronidase was also added to certain specimens. Freshly collected and refrigerated NaF-preserved blood produced minimal free morphine, whereas four of five autopsy blood specimens produced free morphine from M3DG. Increased storage time, temperature, and initial degree of putrefaction resulted in greater free morphine generation despite the absence of viable bacteria. Hydrolysis occurring during specimen storage can generate free morphine from M3DG and may result in erroneous conclusions in certifying narcotic deaths. PMID:11111790

  15. Optimization of enzymatic hydrolysis of shrimp waste for recovery of antioxidant activity rich protein isolate.

    PubMed

    Sowmya, R; Ravikumar, T M; Vivek, R; Rathinaraj, K; Sachindra, N M

    2014-11-01

    Shrimp waste is an important source of astaxanthin, which occur as a complex with proteins, and protein isolates as well as carotenoids are known to possess antioxidant activity. Investigations were carried out to optimize hydrolysis of shrimp waste using a bacterial protease to obtain antioxidant activity rich protein isolate. The effect of three process variables namely enzyme concentration to waste, incubation temperature and time on carotenoid recovery, protein content, trichloro acetic acid (TCA) soluble peptide content and DiPhenyl Picryl Hydrazylchloride (DPPH) scavenging activity was evaluated using a fractionally factorial design. A high correlation coefficient (>0.90) between the observed and the predicted values indicated the appropriateness of the design employed. Maximum carotenoid recovery was obtained by hydrolysing the shrimp waste with 0.3 % enzyme for 4 h. DPPH radical scavenging activity of carotenoprotein isolate was markedly affected by enzyme concentration, temperature and time of hydrolysis. The study indicated that in order to obtain the carotenoprotein from shrimp waste with higher carotenoid content hydrolysing with an enzyme concentration of 0.2-0.4 %, at lower temperature of 25-30° upto 4 h is ideal. However, in order to obtain the protein isolate with increased antioxidant activity hydrolysing at higher temperature of 50 °C, with higher enzyme concentration of 0.5 % for shorter duration is more ideal. PMID:26396312

  16. Kinetics of enzymatic hydrolysis of olive oil in batch and fed-batch systems.

    PubMed

    Cabral, Paloma Souza; Filho, Arion Zandoná; Voll, Fernando Augusto Pedersen; Corazza, Marcos Lúcio

    2014-07-01

    This work reports experimental data, kinetic modeling, and simulations of enzyme-catalyzed hydrolysis of olive oil. This reaction was performed in batch system and an ordered-sequential Bi Bi model was used to model the kinetic mechanism. A fed-batch system was proposed and experimental data were obtained and compared to the simulated values. The kinetic model used was able to correlate the experimental data, in which a satisfactory agreement between the experimental data and modeling results was obtained under different enzyme concentration and initial free water content. Therefore, the modeling allowed a better understanding of the reaction kinetics and affords a fed-batch simulation for this system. From the results obtained, it was observed that the fed-batch approach showed to be more advantageous when compared to the conventional batch system. PMID:24793196

  17. Effects of laccase on lignin depolymerization and enzymatic hydrolysis of ensiled corn stover.

    PubMed

    Chen, Qin; Marshall, Megan N; Geib, Scott M; Tien, Ming; Richard, Tom L

    2012-08-01

    The aim of this study was to explore the synergies of laccase, a ligninolytic enzyme, with cellulose and hemicellulase amendments on ensiled corn stover. Molecular signals of lignin decomposition were observed by tetramethylammonium hydroxide thermochemolysis and gas chromatography-mass spectroscopy (TMAH-GC-MS) analysis. The significant findings suggest that ensilage might provide a platform for biological pretreatment. By partially hydrolyzing cellulose and hemicellulose into soluble sugars, ensilage facilitates laccase penetration into the lignocellulose complex to enhance lignin degradation. Downstream cellulose hydrolysis was improved 7% with increasing laccase loading rate. These results demonstrate the potential of enzymes, either directly amended or expressed by microbes during ensilage, to maximize utilization of corn stover for cellulosic biofuels and other downstream fermentations. PMID:22613895

  18. Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

    PubMed Central

    Kopera, Edyta; Belczyk-Ciesielska, Agnieszka; Bal, Wojciech

    2012-01-01

    In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques. PMID:22574150

  19. Fungal pretreatment of non-sterile miscanthus for enhanced enzymatic hydrolysis.

    PubMed

    Vasco-Correa, Juliana; Ge, Xumeng; Li, Yebo

    2016-03-01

    Miscanthus was pretreated with the fungus Ceriporiopsis subvermispora under non-sterile conditions, using sterile miscanthus that had been previously colonized with the fungus as the inoculum. Inoculum ratios equal to or greater than 30% yielded a successful pretreatment, enhancing the enzymatic digestibility of miscanthus by 3- to 4-fold over that of raw miscanthus, which was comparable with the fungal pretreatment under sterile conditions. This enhanced digestibility was linearly correlated with lignin degradation. Although cellulose loss of up to 13% was observed for the successful non-sterile pretreatments, the final glucose yield was 3-4 times higher than that of raw miscanthus and comparable to that of the sterile pretreated miscanthus. A time course study showed that maximum glucose yield can be achieved with a pretreatment time of 21 days. PMID:26722811

  20. Pretreatment of cane bagasse with alkaline hydrogen peroxide for enzymatic hydrolysis of cellulose and ethanol fermentation

    SciTech Connect

    Azzam, A.M. )

    1989-01-01

    Pretreatment of the agrocellulosic waste, cane bagasse with alkaline hydrogen peroxide greatly enhances its susceptibility to enzymatic cellulolysis and thus the ethanol production from it. Various process conditions have been studied to optimize the enzymate effectiveness. These conditions include the contact time, the hydrogen peroxide concentration and the pretreatment temperature. Results obtained show, that about 50% of lignin and most of hemicellulose content of can bagasse was solubilized, by 2% alkaline hydrogen peroxide at 30{sup 0}C within 8 h. The cellulose content was consequently increased from 42% in the original cane bagasse to 75% in the oxidized pulp. Saccharification of this pulp residue with cellulase from Trichorderma viride at 45{sup 0}C for 24 h, yielded glucose with 95% efficiency. The efficiency of ethanol production from the insoluble fraction with S. cervisiae was 90% compared to about 50% for untreated cane bagasse.

  1. Dilute oxalic acid pretreatment for high total sugar recovery in pretreatment and subsequent enzymatic hydrolysis.

    PubMed

    Qing, Qing; Huang, Meizi; He, Yucai; Wang, Liqun; Zhang, Yue

    2015-12-01

    Oxalic acid was evaluated as an alternative reagent to mineral inorganic acid in pretreatment of corncob to achieve high xylose yield in addition to highly digestible solid residue. A quadratic polynomial model of xylose formation was developed for optimization of pretreatment process by the response surface methodology based on the impact factors of pretreatment temperature, reaction time, acid concentration, and solid-to-liquid ratio. The highest xylose yield was 94.3 % that was obtained under the pretreatment condition of 140 °C for 40 min with 0.5 wt% oxalic acid at a solid loading of 7.5 %. Under these conditions, the xylose yield results of verification experiments were very close to the model prediction, which indicated that the model was applicable. The solid residue generated under this condition also demonstrated a satisfactory enzymatic digestibility and fermentability. PMID:26494137

  2. Evaluation of the effect of hot-compressed water treatment on enzymatic hydrolysis of lignocellulosic nanofibrils with different lignin content using a quartz crystal microbalance.

    PubMed

    Kumagai, Akio; Lee, Seung-Hwan; Endo, Takashi

    2016-07-01

    Hot-compressed water (HCW) treatment is known to not only improve enzymatic hydrolysis efficiency of lignocellulosic biomass but to also generate insoluble lignin droplets, which retard enzymatic hydrolysis. In this study, the inhibitory effect of the lignin droplets was evaluated by monitoring the initial enzyme adsorption and degradation of lignocellulosic nanofibrils (LCNFs) using a quartz crystal microbalance (QCM). Lignin content was adjusted by the sodium chlorite-acetic acid method and divided into samples with high (24.9 wt%) and low (5.6 wt%) lignin content, which were then subjected to HCW treatment at various temperatures. The changes in lignin content were small with increasing HCW temperature, whereas hemicellulose content decreased, regardless of the initial lignin content. The formation of lignin droplets and pseudo-lignin-like products was confirmed in both LCNFs by atomic force microscopy (AFM) and was predominant in LCNFs with high lignin content treated at 200°C. QCM data showed that the enzyme adsorption amount in both LCNFs after HCW treatment was increased and was greater in LCNFs with low lignin content. Initial enzymatic degradation was substantially slowed in LCNFs with high lignin content, particularly after HCW treatment at temperatures higher than 180°C. These QCM results suggest that the steric hindrance of the deposited lignin is the primary mechanism by which the initial enzymatic hydrolysis is delayed. Biotechnol. Bioeng. 2016;113: 1441-1447. © 2015 Wiley Periodicals, Inc. PMID:26694223

  3. Identification of the Major ACE-Inhibitory Peptides Produced by Enzymatic Hydrolysis of a Protein Concentrate from Cuttlefish Wastewater

    PubMed Central

    Rodríguez Amado, Isabel; Vázquez, José Antonio; González, Pilar; Esteban-Fernández, Diego; Carrera, Mónica; Piñeiro, Carmen

    2014-01-01

    The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC50 = 76.8 ± 15.2 μg mL−1) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC50 value of 58.4 ± 4.6 μg mL−1. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A–D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC50 values of the fractions ranged from 1.92 to 8.83 μg mL−1, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions. PMID:24619242

  4. The effect of enzymatic pre-hydrolysis of dairy wastewater on the granular and immobilized microbial community in anaerobic bioreactors.

    PubMed

    Cammarota, Magali C; Rosa, Daniela R; Duarte, Iolanda C S; Saavedra, Nora K; Varesche, Maria B A; Zaiat, Marcelo; Freire, Denise M G

    2013-01-01

    The effect of a lipase-rich enzyme preparation produced by the fungus Penicillium sp. on solid-state fermentation was evaluated in two anaerobic bioreactors (up-flow anaerobic sludge blanket (UASB) and horizontal-flow anaerobic immobilized biomass (HAIB)) treating dairy wastewater with 1200 mg oil and grease/L. The oil and grease hydrolysis step was carried out with 0.1% (w/v) of the solid enzymatic preparation at 30 degrees C for 24 h. This resulted in a final concentration of free acids eight times higher than the initial value. The bioreactors operated at 30 degrees C with hydraulic retention times of 12 h (HAIB) and 20 h (UASB) for a period of 430 days, and had high chemical oxygen demand (COD) removal efficiencies (around 90%) when fed with pre-hydrolyzed wastewater. There was, however, an increase in the effluent oil and grease concentration (from values as low as 17 mg/L to values above 150 mg/L in the UASB bioreactor, and from 38-242 mg/L in the HAIB bioreactor), and oil and grease accumulation in the biomass throughout the operational period (the oil and grease content reached 1.7 times that found in the inoculum of the UASB bioreactor). The HAIB bioreactor gave better results because the support for biomass immobilization acted as a filter, retaining oil and grease at the entry of the bioreactor. The molecular analysis of the Bacteria and Archaea domains revealed significant differences in the microbial profiles in experiments conducted with and without the pre-hydrolysis step. The differences observed in the overall parameters could be related to the microbial diversity of the anaerobic sludge. PMID:23530355

  5. The role of residue stability in transient protein-protein interactions involved in enzymatic phosphate hydrolysis. A computational study.

    PubMed

    Bonet, Jaume; Caltabiano, Gianluigi; Khan, Abdul Kareem; Johnston, Michael A; Corbí, Carles; Gómez, Alex; Rovira, Xavier; Teyra, Joan; Villà-Freixa, Jordi

    2006-04-01

    Finding why protein-protein interactions (PPIs) are so specific can provide a valuable tool in a variety of fields. Statistical surveys of so-called transient complexes (like those relevant for signal transduction mechanisms) have shown a tendency of polar residues to participate in the interaction region. Following this scheme, residues in the unbound partners have to compete between interacting with water or interacting with other residues of the protein. On the other hand, several works have shown that the notion of active site electrostatic preorganization can be used to interpret the high efficiency in enzyme reactions. This preorganization can be related to the instability of the residues important for catalysis. In some enzymes, in addition, conformational changes upon binding to other proteins lead to an increase in the activity of the enzymatic partner. In this article the linear response approximation version of the semimacroscopic protein dipoles Langevin dipoles (PDLD/S-LRA) model is used to evaluate the stability of several residues in two phosphate hydrolysis enzymes upon complexation with their activating partners. In particular, the residues relevant for PPI and for phosphate hydrolysis in the CDK2/Cyclin A and Ras/GAP complexes are analyzed. We find that the evaluation of the stability of residues in these systems can be used to identify not only active site regions but it can also be used as a guide to locate "hot spots" for PPIs. We also show that conformational changes play a major role in positioning interfacing residues in a proper "energetic" orientation, ready to interact with the residues in the partner protein surface. Thus, we extend the preorganization theory to PPIs, extrapolating the results we obtained from the above-mentioned complexes to a more general case. We conclude that the correlation between stability of a residue in the surface and the likelihood that it participates in the interaction can be a general fact for transient

  6. Two Novel Class II Hydrophobins from Trichoderma spp. Stimulate Enzymatic Hydrolysis of Poly(Ethylene Terephthalate) when Expressed as Fusion Proteins

    PubMed Central

    Espino-Rammer, Liliana; Ribitsch, Doris; Przylucka, Agnieszka; Marold, Annemarie; Greimel, Katrin J.; Herrero Acero, Enrique; Guebitz, Georg M.; Kubicek, Christian P.

    2013-01-01

    Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET. PMID:23645195

  7. The effect of photoisomerization on the enzymatic hydrolysis of polymeric micelles bearing photo-responsive azobenzene groups at their cores.

    PubMed

    Harnoy, Assaf J; Slor, Gadi; Tirosh, Einat; Amir, Roey J

    2016-06-28

    The design of stable polymeric micelles that can respond to specific stimuli is crucial for the development of smart micellar nanocarriers that can release their active cargo selectively at the target site, thus diminishing the therapeutic limitations due to non-selective damage to healthy tissues. Here we report the design and synthesis of photo- and enzyme-responsive amphiphilic PEG-dendron hybrids bearing one, two or four enzymatically cleavable azobenzene end-groups. These dual-responsive hybrids can respond to light through the reversible isomerization of the azobenzene end-groups from the non-polar trans isomer to the highly polar cis isomer and vice versa, upon UV and visible irradiation, respectively. The high structural precision of these hybrids, which emerges from the dendritic architecture, enabled a detailed study of the photoisomerization of the azobenzene end-groups with high molecular resolution. Remarkably, although the transition from trans-to-cis led to a significant increase in the polarity of the micellar cores, the micelles remained stable. Our kinetic studies show that although the trans isomer is a better substrate for the activating enzyme, the UV induced formation of the cis azobenzene end-groups led to significant acceleration of the enzymatic hydrolysis of the end-groups. These results provide strong indication that the enzyme cannot reach the core of the micelles and instead the end-groups have to leave the hydrophobic core in order to be exposed on the micelle's surface or even leave the micelle in order to allow their cleavage by the activating enzymes. PMID:27093537

  8. Electron beam pretreatment of switchgrass to enhance enzymatic hydrolysis to produce sugars for biofuels.

    PubMed

    Sundar, Smith; Bergey, N Scott; Salamanca-Cardona, Lucia; Stipanovic, Arthur; Driscoll, Mark

    2014-01-16

    Conversion of lignocellulosic biomass to value added products such as ethanol and other platform chemicals is enhanced by pretreatment, which reduces the crystallinity and molecular weight of cell wall polymers, thus increasing the available reaction sites. In this study, switchgrass (Panicum virgatum L.) was pretreated with high energy electron beam (EB) irradiation to reduce its recalcitrance and achieve higher sugar conversion rates during treatment with cellulases and β-glucosidase. Conversion rates to sugars were compared before and after hot water (HW) extraction of EB-treated and control samples of switchgrass. Thermogravimetric analysis (TGA) was employed to determine peak degradation temperature of these EB-treated biomass samples before and after HW extraction, and near infrared spectroscopy (NIR) was used as a rapid technique to determine cellulose, hemicellulose, and lignin contents in the samples. TGA data confirm previously reported results that EB pretreatment reduces the molecular weight and crystallinity of cellulose and hemicellulose. This leaves hemicellulose more amenable to HW extraction and creates more cellulase-accessible sites, as shown by NIR and glucose yield data, respectively. Hemicellulose content was reduced from 30.2 to 16.9% after HW extraction and 1000 kGy EB treatment, and ultimate glucose yield after cellulase hydrolysis increased more than 4-fold. This study provides evidence that when EB pretreatment is utilized in combination with HW extraction, higher conversion rates and yields of glucose can be obtained from the cellulosic fraction of switchgrass. PMID:24188854

  9. Synthesis, DNA Polymerase Incorporation, and Enzymatic Phosphate Hydrolysis of Formamidopyrimidine Nucleoside Triphosphates

    PubMed Central

    Imoto, Shuhei; Patro, Jennifer N.; Jiang, Yu Lin; Oka, Natsuhisa; Greenberg, Marc M.

    2007-01-01

    The nucleoside triphosphates of N6-(2-deoxy-α,β-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy·dGTP) and its C-nucleoside analogue (β-C-Fapy·dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy·dGTP and β-C-Fapy·dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3′-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy·dG observed in cells if 1% of the dGTP pool is converted to Fapy·dGTP. Similarly, polymerase-mediated introduction of β-C-Fapy·dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy·dGTP and β-C-Fapy·dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates. PMID:17090045

  10. Sweet sorghum as feedstock for ethanol production: enzymatic hydrolysis of steam-pretreated bagasse.

    PubMed

    Sipos, Bálint; Réczey, Jutka; Somorai, Zsolt; Kádár, Zsófia; Dienes, Dóra; Réczey, Kati

    2009-05-01

    Sweet sorghum is an attractive feedstock for ethanol production. The juice extracted from the fresh stem is composed of sucrose, glucose, and fructose and can therefore be readily fermented to alcohol. The solid fraction left behind, the so-called bagasse, is a lignocellulosic residue which can also be processed to ethanol. The objective of our work was to test sweet sorghum, the whole crop, as a potential raw material of ethanol production, i.e., both the extracted sugar juice and the residual bagasse were tested. The juice was investigated at different harvesting dates for sugar content. Fermentability of juices extracted from the stem with and without leaves was compared. Sweet sorghum bagasse was steam-pretreated using various pretreatment conditions (temperatures and residence times). Efficiency of pretreatments was characterized by the degree of cellulose hydrolysis of the whole pretreated slurry and the separated fiber fraction. Two settings of the studied conditions (190 degrees C, 10 min and 200 degrees C, 5 min) were found to be efficient to reach conversion of 85-90%. PMID:19015818

  11. Mathematical modeling and optimization of cellulase protein production using Trichoderma reesei RL-P37

    SciTech Connect

    Tholudur, A.; Ramirez, W.F.; McMillan, J.D.

    1999-07-01

    The enzyme cellulase, a multienzyme complex made up of several proteins, catalyzes the conversion of cellulose to glucose in an enzymatic hydrolysis-based biomass-to-ethanol process. Production of cellulase enzyme proteins in large quantities using the fungus Trichoderma reesei requires understanding the dynamics of growth and enzyme production. The method of neural network parameter function modeling, which combines the approximation capabilities of neural networks with fundamental process knowledge, is utilized to develop a mathematical model of this dynamic system. In addition, kinetic models are also developed. Laboratory data from bench-scale fermentations involving growth and protein production by T. reesei on lactose and xylose are used to estimate the parameters in these models. The relative performance of the various models and the results of optimizing these models on two different performance measures are presented. An approximately 33% lower root-mean-squared error (RMSE) in protein predictions and about 40% lower total RMSE is obtained with the neural network-based model, the RMSE in predicting optimal conditions for two performance indices, is about 67% and 40% lower, respectively, when compared with the kinetic models. Thus, both model predictions and optimization results from the neural network-based model are found to be closer to the experimental data than the kinetic models developed in this work. It is shown that the neural network parameter function modeling method can be useful as a macromodeling technique to rapidly develop dynamic models of a process.

  12. Chemical characterization and hydrothermal pretreatment of Salicornia bigelovii straw for enhanced enzymatic hydrolysis and bioethanol potential.

    PubMed

    Cybulska, Iwona; Chaturvedi, Tanmay; Brudecki, Grzegorz P; Kádár, Zsófia; Meyer, Anne S; Baldwin, Robert M; Thomsen, Mette Hedegaard

    2014-02-01

    Salicornia bigelovii straw was characterized and evaluated as a potential lignocellulosic bioethanol feedstock. S. bigelovii used in the study was grown in the United Arab Emirates using saltwater (40ppt) for irrigation. Salt removal was performed prior to pretreatment to protect the processing equipment and avoid inhibition of enzymes and yeast. Composition of the washed biomass was comparable to traditional lignocellulosic biomasses with relatively high glucan and xylan content (26 and 22g/100gDM, respectively) but with lower lignin content (7g/100gDM). The washed feedstock was subjected to hydrothermal pretreatment, producing highly digestible (up to 92% glucan-to-glucose conversion) and fermentable (up to 100% glucose-to-ethanol conversion) fiber fractions. Liquid fractions obtained in the pretreatment did not show inhibition towards Saccharomyces cerevisiae. No significant differences among the enzymatic convertibility and microbial fermentability of the fibers as well as low xylose recoveries suggest that lower severity pretreatment conditions could be exploited for S. bigelovii. PMID:24362358

  13. Enzymatic hydrolysis of steryl glucosides, major contaminants of vegetable oil-derived biodiesel.

    PubMed

    Aguirre, Andres; Peiru, Salvador; Eberhardt, Florencia; Vetcher, Leandro; Cabrera, Rodolfo; Menzella, Hugo G

    2014-05-01

    Biodiesels are mostly produced from lipid transesterification of vegetable oils, including those from soybean, jatropha, palm, rapeseed, sunflower, and others. Unfortunately, transesterification of oil produces various unwanted side products, including steryl glucosides (SG), which precipitate and need to be removed to avoid clogging of filters and engine failures. So far, efficient and cost-effective methods to remove SGs from biodiesel are not available. Here we describe for the first time the identification, characterization and heterologous production of an enzyme capable of hydrolyzing SGs. A synthetic codon-optimized version of the lacS gene from Sulfolobus solfataricus was efficiently expressed and purified from Escherichia coli, and used to treat soybean derived biodiesel containing 100 ppm of SGs. After optimizing different variables, we found that at pH 5.5 and 87 °C, and in the presence of 0.9 % of the emulsifier polyglycerol polyricinoleate, 81 % of the total amount of SGs present in biodiesel were hydrolyzed by the enzyme. This remarkable reduction in SGs suggests a path for the removal of these contaminants from biodiesel on industrial scale using an environmentally friendly enzymatic process. PMID:24265025

  14. Angiotensin I-Converting Enzyme Inhibitory Peptides of Chia (Salvia hispanica) Produced by Enzymatic Hydrolysis

    PubMed Central

    Segura Campos, Maira Rubi; Peralta González, Fanny; Chel Guerrero, Luis

    2013-01-01

    Synthetic angiotensin I-converting enzyme (ACE-I) inhibitors can have undesirable side effects, while natural inhibitors have no side effects and are potential nutraceuticals. A protein-rich fraction from chia (Salvia hispanica L.) seed was hydrolyzed with an Alcalase-Flavourzyme sequential system and the hydrolysate ultrafiltered through four molecular weight cut-off membranes (1 kDa, 3 kDa, 5 kDa, and 10 kDa). ACE-I inhibitory activity was quantified in the hydrolysate and ultrafiltered fractions. The hydrolysate was extensive (DH = 51.64%) and had 58.46% ACE-inhibitory activity. Inhibition ranged from 53.84% to 69.31% in the five ultrafiltered fractions and was highest in the <1 kDa fraction (69.31%). This fraction's amino acid composition was identified and then it was purified by gel filtration chromatography and ACE-I inhibition measured in the purified fractions. Amino acid composition suggested that hydrophobic residues contributed substantially to chia peptide ACE-I inhibitory strength, probably by blocking angiotensin II production. Inhibitory activity ranged from 48.41% to 62.58% in the purified fractions, but fraction F1 (1.5–2.5 kDa) exhibited the highest inhibition (IC50 = 3.97 μg/mL; 427–455 mL elution volume). The results point out the possibility of obtaining bioactive peptides from chia proteins by means of a controlled protein hydrolysis using Alcalase-Flavourzyme sequentional system. PMID:26904588

  15. Angiotensin I-Converting Enzyme Inhibitory Peptides of Chia (Salvia hispanica) Produced by Enzymatic Hydrolysis.

    PubMed

    Segura Campos, Maira Rubi; Peralta González, Fanny; Chel Guerrero, Luis; Betancur Ancona, David

    2013-01-01

    Synthetic angiotensin I-converting enzyme (ACE-I) inhibitors can have undesirable side effects, while natural inhibitors have no side effects and are potential nutraceuticals. A protein-rich fraction from chia (Salvia hispanica L.) seed was hydrolyzed with an Alcalase-Flavourzyme sequential system and the hydrolysate ultrafiltered through four molecular weight cut-off membranes (1 kDa, 3 kDa, 5 kDa, and 10 kDa). ACE-I inhibitory activity was quantified in the hydrolysate and ultrafiltered fractions. The hydrolysate was extensive (DH = 51.64%) and had 58.46% ACE-inhibitory activity. Inhibition ranged from 53.84% to 69.31% in the five ultrafiltered fractions and was highest in the <1 kDa fraction (69.31%). This fraction's amino acid composition was identified and then it was purified by gel filtration chromatography and ACE-I inhibition measured in the purified fractions. Amino acid composition suggested that hydrophobic residues contributed substantially to chia peptide ACE-I inhibitory strength, probably by blocking angiotensin II production. Inhibitory activity ranged from 48.41% to 62.58% in the purified fractions, but fraction F1 (1.5-2.5 kDa) exhibited the highest inhibition (IC50 = 3.97 μg/mL; 427-455 mL elution volume). The results point out the possibility of obtaining bioactive peptides from chia proteins by means of a controlled protein hydrolysis using Alcalase-Flavourzyme sequentional system. PMID:26904588

  16. Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize (Zea mays L.) lines

    PubMed Central

    Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D.; Williams, Daniel L.; Magee, Timothy D.; Kaeppler, Shawn M.; de Leon, Natalia; Hodge, David B.

    2015-01-01

    A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA) content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. This indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment. PMID:25871649

  17. Chemical synthesis and enzymatic, stereoselective hydrolysis of a functionalized dihydropyrimidine for the synthesis of β-amino acids.

    PubMed

    Slomka, Christin; Zhong, Sabilla; Fellinger, Anna; Engel, Ulrike; Syldatk, Christoph; Bräse, Stefan; Rudat, Jens

    2015-12-01

    A novel substrate, 6-(4-nitrophenyl)dihydropyrimidine-2,4(1H,3H)-dione (pNO2PheDU), was chemically synthesized and analytically verified for the potential biocatalytic synthesis of enantiopure β-amino acids. The hydantoinase (EC 3.5.2.2) from Arthrobacter crystallopoietes DSM20117 was chosen to prove the enzymatic hydrolysis of this substrate, since previous investigations showed activities of this enzyme toward 6-monosubstituted dihydrouracils. Whole cell biotransformations with recombinant Escherichia coli expressing the hydantoinase showed degradation of pNO2PheDU. Additionally, the corresponding N-carbamoyl-β-amino acid (NCarbpNO2 βPhe) was chemically synthesized, an HPLC-method with chiral stationary phases for detection of this product was established and thus (S)-enantioselectivity toward pNO2PheDU has been shown. Consequently this novel substrate is a potential precursor for the enantiopure β-amino acid para-nitro-β-phenylalanine (pNO2 βPhe). PMID:26705241

  18. Direct lactic acid fermentation of Jerusalem artichoke tuber extract using Lactobacillus paracasei without acidic or enzymatic inulin hydrolysis.

    PubMed

    Choi, Hwa-Young; Ryu, Hee-Kyoung; Park, Kyung-Min; Lee, Eun Gyo; Lee, Hongweon; Kim, Seon-Won; Choi, Eui-Sung

    2012-06-01

    Lactic acid fermentation of Jerusalem artichoke tuber was performed with strains of Lactobacillus paracasei without acidic or enzymatic inulin hydrolysis prior to fermentation. Some strains of L. paracasei, notably KCTC13090 and KCTC13169, could ferment hot-water extract of Jerusalem artichoke tuber more efficiently compared with other Lactobacillus spp. such as L. casei type strain KCTC3109. The L. paracasei strains could utilize almost completely the fructo-oligosaccharides present in Jerusalem artichoke. Inulin-fermenting L. paracasei strains produced c.a. six times more lactic acid compared with L. casei KCTC3109. Direct lactic fermentation of Jerusalem artichoke tuber extract at 111.6g/L of sugar content with a supplement of 5 g/L of yeast extract by L. paracasei KCTC13169 in a 5L jar fermentor produced 92.5 ce:hsp sp="0.25"/>g/L of lactic acid with 16.8 g/L fructose equivalent remained unutilized in 72 h. The conversion efficiency of inulin-type sugars to lactic acid was 98% of the theoretical yield. PMID:22516247

  19. Production of cellulosic ethanol from cotton processing residues after pretreatment with dilute sodium hydroxide and enzymatic hydrolysis.

    PubMed

    Fockink, Douglas Henrique; Maceno, Marcelo Adriano Corrêa; Ramos, Luiz Pereira

    2015-01-01

    In this study, production of cellulosic ethanol from two cotton processing residues was investigated after pretreatment with dilute sodium hydroxide. Pretreatment performance was investigated using a 2(2) factorial design and the highest glucan conversion was achieved at the most severe alkaline conditions (0.4g NaOH g(-1) of dry biomass and 120°C), reaching 51.6% and 38.8% for cotton gin waste (CGW) and cotton gin dust (CGD), respectively. The susceptibility of pretreated substrates to enzymatic hydrolysis was also investigated and the best condition was achieved at the lowest total solids (5wt%) and the highest enzyme loading (85mg of Cellic CTec2 g(-1) of dry substrate). However, the highest concentration of fermentable sugars - 47.8 and 42.5gL(-1) for CGD and CGW, respectively - was obtained at 15wt% total solids using this same enzyme loading. Substrate hydrolysates had no inhibitory effects on the fermenting microorganism. PMID:25841187

  20. Preparation of Yeast Hydrolysate Enriched in Cyclo-His-Pro (CHP) by Enzymatic Hydrolysis and Evaluation of Its Functionality

    PubMed Central

    Lee, Hyun Jung; Son, Heung Soo; Park, Chung; Suh, Hyung Joo

    2015-01-01

    In this study, we attempted to enrich cyclo-His-Pro (CHP) using enzymatic hydrolysis of yeast and to evaluate the functionality of yeast hydrolysate (YH)-enriched CHP. Flavourzyme offered a better performance in enhancing CHP content than other proteases. The CHP enrichment conditions were optimized as follows: addition of 1% Flavourzyme, 48-h incubation at 60°C, and pH 6.0. The CHP content significantly increased by 20-fold after ultra-filtration (UF). Maximal CHP translation was obtained after heating for 8 h at 50°C and pH 7.0. YH showed poor foaming capacity between pH 3.0 to 9.0. The emulsifying activities of YHs were slightly higher at near acidic pH. Increase in heating temperature and time resulted in decreased CHP content. The results indicate that YH is more heat stable after UF. Therefore, the CHP in YH after UF can be used as a food additive with physiological CHP activity and high heat stability. PMID:26770916

  1. Integrated chemical and multi-scale structural analyses for the processes of acid pretreatment and enzymatic hydrolysis of corn stover.

    PubMed

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

    2016-05-01

    Corn stover was pretreated with acid under moderate conditions (1.5%, w/w, 121°C, 60min), and kinetic enzymolysis experiments were performed on the pretreated substrate using a mixture of Celluclast 1.5L (20FPU/g dry substrate) and Novozyme 188 (40CBU/g dry substrate). Integrated chemical and multi-scale structural methods were then used to characterize both processes. Chemical analysis showed that acid pretreatment removed considerable hemicellulose (from 19.7% in native substrate to 9.28% in acid-pretreated substrate) and achieved a reasonably high conversion efficiency (58.63% of glucose yield) in the subsequent enzymatic hydrolysis. Multi-scale structural analysis indicated that acid pretreatment caused structural changes via cleaving acetyl linkages, solubilizing hemicellulose, relocating cell wall surfaces and enlarging substrate porosity (pore volume increased from 0.0067cm(3)/g in native substrate to 0.019cm(3)/g in acid-pretreated substrate), thereby improving the polysaccharide digestibility. PMID:26876990

  2. Non-severe thermochemical hydrolysis of stover from white corn and sequential enzymatic saccharification and fermentation to ethanol.

    PubMed

    Vargas-Tah, Alejandra; Moss-Acosta, Cessna L; Trujillo-Martinez, Berenice; Tiessen, Axel; Lozoya-Gloria, Edmundo; Orencio-Trejo, Montserrat; Gosset, Guillermo; Martinez, Alfredo

    2015-12-01

    A parametric study, with an initial load of 15%w/w of dry stover from white corn, was conducted to evaluate the sequential thermochemical hydrolysis (TH), enzymatic saccharification (ES) and fermentation of the whole slurry with ethanologenic Escherichia coli. The TH was designed to release the maximum amount of xylose with a concomitant formation of minimal amounts of furans. It was found that 29.0% or 93.2% of the xylan was recovered as free xylose at 130°C after 8 min in the presence of 1% or 2%w/w H2SO4 and produced only 0.06 or 0.44 g/L of total furans, respectively. After 24h of ES, 76.14-77.18 g/L of monosaccharides (pentoses and hexoses) were obtained. These slurries, which contained 0.03-0.26 g/L of total furans and 5.14-5.91 g/L of acetate, were fermented with 3.7 g/L of ethanologenic E. coli to produce 24.5-23.5 g/L of ethanol. PMID:26433785

  3. Bisulfite pretreatment changes the structure and properties of oil palm empty fruit bunch to improve enzymatic hydrolysis and bioethanol production.

    PubMed

    Tan, Liping; Sun, Wan; Li, Xuezhi; Zhao, Jian; Qu, Yinbo; Choo, Yuen May; Loh, Soh Kheang

    2015-06-01

    Bisulfite pretreatment is a proven effective method for improving the enzymatic hydrolysis of empty fruit bunch (EFB) from oil palm for bioethanol production. In this study, we set out to determine the changes that occur in the structure and properties of EFB materials and fractions of hemicellulose and lignin during the bisulfite pretreatment process. The results showed that the crystallinity of cellulose in EFB increased after bisulfite pretreatment, whereas the EFB surface was damaged to various degrees. The orderly structure of EFB, which was maintained by hydrogen bonds, was destroyed by bisulfite pretreatment. Bisulfite pretreatment also hydrolyzed the glycosidic bonds of the xylan backbone of hemicellulose, thereby decreasing the molecular weight and shortening the xylan chains. The lignin fractions obtained from EFB and pretreated EFB were typically G-S lignin, and with low content of H units. Meanwhile, de-etherification occurred at the β-O-4 linkage, which was accompanied by polymerization and demethoxylation as a result of bisulfite pretreatment. The adsorption ability of cellulase differed for the various lignin fractions, and the water-soluble lignin fractions had higher adsorption capacity on cellulase than the milled wood lignin. In general, the changes in the structure and properties of EFB provided insight into the benefits of bisulfite pretreatment. PMID:25866127

  4. Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization.

    PubMed

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-08-01

    Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool. PMID:27177457

  5. Enzymatic hydrolysis of blue whiting (Micromesistius poutassou); functional and bioactive properties.

    PubMed

    Geirsdottir, Margret; Sigurgisladottir, Sjofn; Hamaguchi, Patricia Y; Thorkelsson, Gudjon; Johannsson, Ragnar; Kristinsson, Hordur G; Kristjansson, Magnus M

    2011-01-01

    Functional and biochemical properties of fish protein hydrolysates (FPH) from blue whiting (BW) were studied. FPH (2.5%, 5%, 10%, and 15% degree of hydrolysis [DH]) were made from isolated proteins from headed and gutted BW with Alcalase 2.4 L. The properties of dried BW mince and protein isolate compared to 4 reference proteins (soy and milk protein) were studied: color, solubility, water-holding capacity (WHC), oil-binding capacity (OBC), emulsion capacity (EC), and emulsion stability (ES). The angiotensin I-converting enzyme (ACE) inhibitory activities of the soluble fraction of BW powders were also investigated. Furthermore, the products were characterized by analyzing their chemical composition. Chemical composition, solubility, OBC, and EC of the BW powders was significantly (P < 0.05) different with different DH, while color, ES, and WHC were not significantly (P > 0.05) different. Salt content of the FPH was high (4% to 19%) and increased with increased DH. Protein solubility varied from 10% to 70% and increased with increased DH. WHC of the FPH was around 97% and was higher than that of all the reference proteins tested. OBC decreased with increased DH (from 3.5 to 2.1 g oil/g protein) and was higher than OBC of the soy and milk proteins (1.6 to 1.9 g oil/g protein). EC of FPH was similar or lower than the reference proteins. ES of FPH (60% to 90%) was similar to or lower than soy and whey proteins (60% to 98%) but higher than casein (20%). ACE inhibition activity increased as DH was increased. Practical Application: The results from this study demonstrate that a functional bioactive hydrolysate can be produced from BW, which is an underutilized fish species, and may aid the industry in better utilizing this raw material. The novelty of this research was the use of BW as a raw material where the protein has been isolated with the pH shift method. Furthermore, it was novel that bioactivity and functionality was measured in the same samples. PMID:21535642

  6. A novel stepwise pretreatment on corn stalk by alkali deacetylation and liquid hot water for enhancing enzymatic hydrolysis and energy utilization efficiency.

    PubMed

    Jiang, Wei; Xu, Jian

    2016-06-01

    A novel stepwise pretreatment on corn stalk (CS) by alkali deacetylation combined with liquid hot water (LHW) was investigated to enhance enzymatic hydrolysis. After deacetylated treatment, strength of alkali deacetylation of CS was from 1.79% to 91.34% which was subsequently pretreated by LHW with severity from 3.27 to 4.27. It was found that higher strength of alkali deacetylation could reduce both the degradation of hemicellulose and inhibitors formation in liquid hot water pretreatment (LHWP). Enzymatic hydrolysis efficiency was confirmed to be affected by LHW pretreatment severity (PS) and strength of alkali treatment. This combined pretreatment of alkali deacetylation and LHW could not only increase glucose yield, but also enhance energy utilization efficiency. The maximum enzymatic hydrolysis of 87.55%±3.64 with the ratio of glucose yield to energy input at 50.39gglucosekJ(-1) was obtained when strength of alkali deacetylation at 84.96% with PS at 3.97 were used. PMID:26967334

  7. Effect of pretreatment and enzymatic hydrolysis on the physical-chemical composition and morphologic structure of sugarcane bagasse and sugarcane straw.

    PubMed

    Moretti, Marcia Maria de Souza; Perrone, Olavo Micali; Nunes, Christiane da Costa Carreira; Taboga, Sebastião; Boscolo, Maurício; da Silva, Roberto; Gomes, Eleni

    2016-11-01

    The present work aimed to study the effect of the pretreatment of sugarcane bagasse and straw with microwave irradiation in aqueous and acid glycerol solutions on their chemical composition, fiber structure and the efficiency of subsequent enzymatic hydrolysis. Thermogravimetric analysis showed that the pretreatment acted mainly on the lignin and hemicellulose fractions of the bagasse, whereas, in the straw, lesser structural and chemical changes were observed. The images from transmission electron microscopy (TEM) revealed that treating bagasse and straw with acid glycerol solution loosened the cell walls and there was a breakdown in the pit membrane. The treated material was submitted to hydrolysis for 72h and higher yields of reducing sugars were observed compared to the untreated material (250.9mg/g from straw and 197.4mg/g from bagasse). TEM images after hydrolysis confirmed the possible points of access of the enzymes to the secondary cell wall region of the pretreated biomass. PMID:27578061

  8. Strategy for biotechnological process design applied to the enzymatic hydrolysis of agave fructo-oligosaccharides to obtain fructose-rich syrups.

    PubMed

    García-Aguirre, Mauricio; Sáenz-Alvaro, Victor A; Rodríguez-Soto, Mayra A; Vicente-Magueyal, Francisco J; Botello-Alvarez, Enrique; Jimenez-Islas, Hugo; Cárdenas-Manríquez, Marcela; Rico-Martínez, Ramiro; Navarrete-Bolaños, Jose L

    2009-11-11

    A strategy to optimize biotechnological process design is illustrated for the production of fructose-rich syrups via enzymatic hydrolysis of agave fructo-oligosaccharides. The optimization process includes ecological studies from natural fermentations leading to the selection of a strain with capacity for inulinase synthesis, and variable optimization for the synthesis, and enzymatic hydrolysis using the response surface methodology. The results lead to the selection of Kluyveromyces marxianus , endogenous strains isolated from aguamiel (natural fermented sugary sap from agave plants), as the main strain with high capacity for enzyme synthesis with inulinase activity. Production optimization at bioreactor level revealed that operation at 30.6 degrees C, 152 rpm, 1.3 VVM of aeration, and pH 6.3 leads to maximum inulinase synthesis, whereas 31 degrees C, 50 rpm, and pH 6.2 leads to maximum hydrolysis of agave fructo-oligosaccharides. HPLC analysis of the fructose-rich syrups obtained at these optimal conditions showed an average composition of 95% of fructose and 5% of glucose and the absence of sucrose. The analysis also revealed that the syrups are free of residues and toxic compounds, an undesirable occurrence often present when traditional methods based on thermal or acid hydrolysis are applied for their obtainment. Therefore, the product may be suitable for use as additive in many applications in the food and beverage industries. PMID:19827764

  9. ENZYMATIC HYDROLYSIS OF YEAST CELL WALLS. I. ISOLATION OF WALL-DECOMPOSING ORGANISMS AND SEPARATION AND PURIFICATION OF LYTIC ENZYMES.

    PubMed

    TANAKA, H; PHAFF, H J

    1965-06-01

    Tanaka, Hirosato (University of California, Davis), and Herman J. Phaff. Enzymatic hydrolysis of yeast cell walls. I. Isolation of wall-decomposing organisms and separation and purification of lytic enzymes. J. Bacteriol. 89:1570-1580. 1965.-A number of microorganisms, able to decompose and grow on yeast cell walls, were isolated from soil. These isolates demonstrated various types of attack on yeast walls. A bacterium, identified as Bacillus circulans, and a species of Streptomyces produced clear, lysed zones when grown on an agar medium containing baker's yeast cell walls. The streptomycete formed glucanase, mannanase, and protease, but B. circulans produced only glucanases. Purified mannan could be prepared from the culture fluid of B. circulans grown on baker's yeast cell walls. In a liquid, mineral medium, extracellular lytic enzyme production by B. circulans was optimal after 3 days of aerobic growth at 30 C with 0.5% baker's yeast cell walls as the carbon source. Twelve other carbon sources were ineffective as inducers. Among a number of polysaccharides tested, the crude enzymes of B. circulans hydrolyzed only beta-1-->3 glucan (laminarin) and beta-1-->6 glucan (pustulan), both by a random mechanism, to a mixture of dimer and glucose. The beta-1-->3 and beta-1-->6 glucanases were separated from each other by diethylaminoethyl cellulose column chromatography. Water-soluble oat glucan, which contains in the linear chain both beta-1-->3 and beta-1-->4 bonds, was also hydrolyzed by the bacterial beta-1-->3 glucanase. The products of this reaction indicated that this enzyme hydrolyzes beta-1-->3 or beta-1-->4 glucosidic linkages, provided the beta-glucopyranosyl units composing these bonds are substituted in the 3 position by another glucose unit. PMID:14291597

  10. Matrix solid phase dispersion assisted enzymatic hydrolysis as a novel approach for cocaine and opiates isolation from human hair.

    PubMed

    Míguez-Framil, Martha; Cabarcos, Pamela; Tabernero, María Jesús; Bermejo, Ana María; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

    2013-11-01

    The possibility of assisting enzymatic hydrolysis (EH) procedures by sample disruption mechanisms inherent to matrix solid phase dispersion (MSPD) has been explored in the current study. EH of hair specimens from poly-drug abusers was assisted by dispersing/blending the sample (0.05 g) with alumina (2.25 g) before loading the dissolved enzyme (6 mL of 1 mg mL(-1) Pronase E in 1.4 M/1.4 M Tris/HCl, pH 7.3) through the hair-alumina solid phase packaged inside a disposable MSPD syringe. The MSPD-EH method was developed, and it proved to offer quantitative results when isolating cocaine, benzoylecgonine (BZE), codeine, morphine and 6-monoacethylmorphine (6-MAM) from human hair samples. The procedure allows an on column clean-up/pre-concentration procedure of the isolated targets by attaching a previously conditioned Oasis HLB cartridge to the end of the MSPD syringe. The EH procedure of human hair with Pronase E can therefore be shortened to approximately 30 min. Within this time, sample blending/dispersion, MSPD syringe package, elution (EH when dissolved Pronase E is passing through the sample-dispersant bed), and extract clean-up and target pre-concentration stages are achieved. Gas chromatography-mass spectrometry (GC-MS) was used for determining each target after elution from the Oasis HLB cartridges with 2 mL of 2% (v/v) acetic acid in methanol, concentration by N2 stream evaporation, and dried extract derivatization with N-methyl-tert-butylsilyltrifluoroacetamide (BSTFA) and chlorotrimethylsilane (TMCS). The method was validated according to the guidance for bioanalytical method validation of the US Department of Health and Human Services, Food and Drug Administration. The simplicity of the proposed approach makes it a useful procedure for screening/quantifying drugs of abuse in hair specimens from poly-drug abusers. PMID:24112826

  11. 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

  12. Monitoring of enzymatic hydrolysis of starch by microdialysis sampling coupled on-line to anion exchange chromatography and integrated pulsed electrochemical detection using post-column switching

    SciTech Connect

    Torto, N.; Gorton, L.; Emneus, J.; Laurell, T.; Marko-Varga, G.; Akerberg, C.; Zacchi, G. |

    1997-12-05

    A quantitative evaluation of the hydrolysis of wheat starch using Termamyl, a thermostable {alpha}-amylase, is reported. Data from the monitoring of the hydrolysis of wheat starch indicated that, after 1 h, glucose and maltooligosaccharides up to DP 7 were the main hydrolysis products and thus enabled optimization of a liquefaction step during the production of L-lactic acid. The monitoring system used, both in the on- and off-line mode, was based on continuous flow microdialysis sampling (CFMS) coupled to anion exchange chromatography and integrated pulsed electrochemical detection (IPED). A microdialysis probe equipped with a 5-mm polysulfone (SPS 4005) membrane, with a molecular-weight cut-off of 5 kDa, was used to sample the hydrolysis products of native wheat starch at 90 C. Characteristic fingerpoint separations were achieved by anion exchange chromatography after enzymatic hydrolysis. Post-column switching improved the detection and, consequently, also quantification of the hydrolysates as fouling of the electrode could be reduced. Maltooligosaccharide standards were used for quantification and to verify the elution of the hydrolysates by spiking the off-line samples.

  13. Pretreatment of Dried Distiller Grains with Solubles by Soaking in Aqueous Ammonia and Subsequent Enzymatic/Dilute Acid Hydrolysis to Produce Fermentable Sugars.

    PubMed

    Nghiem, Nhuan P; Montanti, Justin; Kim, Tae Hyun

    2016-05-01

    Dried distillers grains with solubles (DDGS), a co-product of corn ethanol production in the dry-grind process, was pretreated by soaking in aqueous ammonia (SAA) using a 15 % w/w NH4OH solution at a solid/liquid ratio of 1:10. The effect of pretreatment on subsequent enzymatic hydrolysis was studied at two temperatures (40 and 60 °C) and four reaction times (6, 12, 24, and 48 h). Highest glucose yield of 91 % theoretical was obtained for the DDGS pretreated at 60 °C and 24 h. The solubilized hemicellulose in the liquid fraction was further hydrolyzed with dilute H2SO4 to generate fermentable monomeric sugars. The conditions of acid hydrolysis included 1 and 4 wt% acid, 60 and 120 °C, and 0.5 and 1 h. Highest yields of xylose and arabinose were obtained at 4 wt% acid, 120 °C, and 1 h. The fermentability of the hydrolysate obtained by enzymatic hydrolysis of the SAA-pretreated DDGS was demonstrated in ethanol fermentation by Saccharomyces cerevisiae. The fermentability of the hydrolysate obtained by consecutive enzymatic and dilute acid hydrolysis was demonstrated using a succinic acid-producing microorganism, strain Escherichia coli AFP184. Under the fermentation conditions, complete utilization of glucose and arabinose was observed, whereas only 47 % of xylose was used. The succinic acid yield was 0.60 g/g total sugar consumed. PMID:26797927

  14. Enzymatic hydrolysis of cuttlefish (Sepia officinalis) and sardine (Sardina pilchardus) viscera using commercial proteases: effects on lipid distribution and amino acid composition.

    PubMed

    Kechaou, Emna Soufi; Dumay, Justine; Donnay-Moreno, Claire; Jaouen, Pascal; Gouygou, Jean-Paul; Bergé, Jean-Pascal; Amar, Raja Ben

    2009-02-01

    Total lipid and phospholipid recovery as well as amino acid quality and composition from cuttlefish (Sepia officinalis) and sardine (Sardina pilchardus) were compared. Enzymatic hydrolyses were performed using the three proteases Protamex, Alcalase, and Flavourzyme by the pH-stat method (24 h, pH 8, 50 degrees C). Three fractions were generated: an insoluble sludge, a soluble aqueous phase, and an oily phase. For each fraction, lipids, phospholipids, and proteins were quantified. Quantitative and qualitative analyses of the raw material and hydrolysates were performed. The degree of hydrolysis (DH) for cuttlefish viscera was 3.2% using Protamex, 6.8% using Flavourzyme, and 7% using Alcalase. DH for sardine viscera was 1.9% (using Flavourzyme), 3.1% (using Protamex) and 3.3% (using Alcalase). Dry matter yields of all hydrolysis reactions increased in the aqueous phases. Protein recovery following hydrolysis ranged from 57.2% to 64.3% for cuttlefish and 57.4% to 61.2% for sardine. Tissue disruption following protease treatment increased lipid extractability, leading to higher total lipid content after hydrolysis. At least 80% of the lipids quantified in the raw material were distributed in the liquid phases for both substrates. The hydrolysed lipids were richer in phospholipids than in the lipids extracted by classical chemical extraction, especially after Flavourzyme hydrolysis for cuttlefish and Alcalase hydrolysis for sardine. The total amino acid content differed according to the substrate and the enzyme used. However, regardless of the raw material or the protease used, hydrolysis increased the level of essential amino acids in the hydrolysates, thereby increasing their potential nutritional value for feed products. PMID:19217554

  15. Comparison the effect of three commercial enzymes for enzymatic hydrolysis of two substrates (rice bran protein concentrate and soy-been protein) with SDS-PAGE.

    PubMed

    Ahmadifard, Nasrollah; Murueta, Julio Humberto Cordova; Abedian-Kenari, Abdolmohammad; Motamedzadegan, Ali; Jamali, Hadi

    2016-02-01

    In this research enzymatic hydrolysis of rice bran protein concentrate (RBPC) and soybean Protein (SBP) as control were studied with 3 commercial enzymes (Alcalase®, Papain and acommercial 3-enzyme cocktail containing of 1.6 mg ml(-1) Trypsin, 3.1 mg ml(-1) Chymotrypsin, 1.3 mg ml(-1)Aminopeptidase (SIGMA P7500) and 7.95 mg ml(-1)pronase type XIV (SIGMA P5147) by the pH stat method. The hydrolysis was carried out at temperature of 28 C, 60 min and pH 8.00. Results were showed that RBPC, and SBP had higher Degree hydrolysis (DH %) with Alcalase® enzyme. Alcalase®had stronger capability for hydrolysis compared to the other tested enzymes. After 60 minute of hydrolysis time, the DH% of Alcalase® for RBPC and SBP was 12.69 and 12.50 %, respectively. In contrast, papain enzyme was showed lowest DH% in three substrates that 1.56 and 1.24 % were for SBP and RBPC, respectively.The hydrolysis of the protein fraction performed the three enzymes on the two substrates was followed in SDS-PAGE. RBPC and SBP showed almost complete digestion with Alcalase® enzyme after 60 minutes. 3-enzyme cocktail enzyme hydrolyzed better the RBPC than the SBP. Papain enzyme had less effect on the two substrates than other 2 enzymes. It was found that Alcalase® has highest capability for hydrolysis compared to other enzyme preparations. The high value protein hydrolysates prepared by Alcalase® can be used as value added ingredients in many food formulations. They are also suitable for a broad range of industrial food applications and also for cosmetic and personal care products. PMID:27162408

  16. Performance and molecular evaluation of an anaerobic system with suspended biomass for treating wastewater with high fat content after enzymatic hydrolysis.

    PubMed

    Rosa, Daniela R; Duarte, Iolanda C S; Saavedra, N Katia; Varesche, Maria B; Zaiat, Marcelo; Cammarota, Magali C; Freire, Denise M G

    2009-12-01

    The effect of a lipase-rich fungal enzymatic preparation, produced by a Penicillium sp. during solid-state fermentation, was evaluated in an anaerobic digester treating dairy wastewater with 1200 mg of oil and grease/L. The oil and grease hydrolysis step was carried out with 0.1% (w/v) of solid enzymatic preparation at 30 degrees C for 24 h, and resulted in a final free acid concentration eight times higher than the initial value. The digester operated in sequential batches of 48 h at 30 degrees C for 245 days, and had high chemical oxygen demand (COD) removal efficiencies (around 90%) when fed with pre-hydrolyzed wastewater. However, when the pre-hydrolysis step was removed, the anaerobic digester performed poorly (with an average COD removal of 32%), as the oil and grease accumulated in the biomass and effluent oil and grease concentration increased throughout the operational period. PCR-DGGE analysis of the Bacteria and Archaea domains revealed remarkable differences in the microbial profiles in trials conducted with and without the pre-hydrolysis step, indicating that differences observed in overall parameters were intrinsically related to the microbial diversity of the anaerobic sludge. PMID:19656674

  17. Enhanced enzymatic hydrolysis of mild alkali pre-treated rice straw at high-solid loadings using in-house cellulases in a bench scale system.

    PubMed

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

    2016-06-01

    In the present study, scale-up systems for cellulase production and enzymatic hydrolysis of pre-treated rice straw at high-solid loadings were designed, fabricated and tested in the laboratory. Cellulase production was carried out using tray fermentation at 45 °C by Aspergillus terreus in a temperature-controlled humidity chamber. Enzymatic hydrolysis studies were performed in a horizontal rotary drum reactor at 50 °C with 25 % (w/v) solid loading and 9 FPU g(-1) substrate enzyme load using in-house as well commercial cellulases. Highly concentrated fermentable sugars up to 20 % were obtained at 40 h with an increased saccharification efficiency of 76 % compared to laboratory findings (69.2 %). These findings demonstrate that we developed a simple and less energy intensive bench scale system for efficient high-solid saccharification. External supplementation of commercial β-glucosidase and hemicellulase ensured better hydrolysis and further increased the saccharification efficiency by 14.5 and 20 %, respectively. An attempt was also made to recover cellulolytic enzymes using ultrafiltration module and nearly 79-84 % of the cellulases and more than 90 % of the sugars were recovered from the saccharification mixture. PMID:26941245

  18. Monitoring real-time enzymatic hydrolysis of Distillers Dried Grains with Solubles (DDGS) by dielectric spectroscopy following hydrothermal pre-treatment by steam explosion.

    PubMed

    Bryant, David N; Firth, Elliot; Kaderbhai, Naheed; Taylor, Stephen; Morris, Stephen M; Logan, Daniel; Garcia, Naroa; Ellis, Andrew; Martin, Steven M; Gallagher, Joe A

    2013-01-01

    Dielectric spectroscopy (DS) has been used to monitor the simultaneous saccharification and fermentation of lignocellulosic biomass by measuring its dielectric state. However, it is unknown whether following steam explosion (SE) pre-treatment, lignocellulose would still maintain a dielectric state, and, if maintained, whether the dissipation during enzymatic hydrolysis could be monitored. Distillers Dried Grains with Solubles (DDGS), pre-treated by SE, was found to have a capacitance (C = 580 kHz) of approximately 24 pF cm(-1). Following addition of full-strength cellulolytic cocktail A (CC-A; R(2) = 0.97) and 1/3 strength cocktail B (CC-B; R(2) = 0.96), a natural logarithmic decay in capacitance was determined. Furthermore, the DS biomass probes quantified the initial linear rate of dissipation in capacitance during hydrolysis. The rate of CC-B was 34% that of CC-A. These data extend scope and utility of DS biomass probes for monitoring the enzymatic hydrolysis of SE-pre-treated lignocellulosic substrates in real-time. PMID:23228453

  19. Fermentative l-lactic acid production from pretreated whole slurry of oil palm trunk treated by hydrothermolysis and subsequent enzymatic hydrolysis.

    PubMed

    Eom, In-Yong; Oh, Young-Hoon; Park, Si Jae; Lee, Seung-Hwan; Yu, Ju-Hyun

    2015-06-01

    A simple and cost-effective biochemical conversion process consisting of hydrothermal treatment, enzymatic hydrolysis and fermentation of pretreated whole slurry (PWS) was developed for producing l-lactic acid (L-LA) from oil palm trunk (OPT). When OPT was hydrothermally treated at optimal condition capable of achieving maximum yield of hemicellulosic sugars after enzymatic hydrolysis, the enzymatic digestibility of the PWS afforded a yield of 81.4% of the theoretical glucose yield (TGY). However, glucose yield from washed pretreated solid (WPS) was only 43.5% of TGY. The use of two hydrolysates from PWS and WPS for fermentation by Lactobacillus paracasei engineered to selectively produce L-LA afforded yields of 89.5% and 45.8% of the theoretical LA yield (TLY), respectively. This study confirmed the inevitable extensive sugar loss during washing of pretreated slurry due to loss of soluble starch. Alternatively, the proposed design process is considered suitable for converting OPT to L-LA without such starch loss. PMID:25768416

  20. Characterisation of legumes by enzymatic hydrolysis, microdialysis sampling, and micro-high-performance anion-exchange chromatography with electrospray ionisation mass spectrometry.

    PubMed

    Okatch, Harriet; Torto, Nelson; Armateifio, Joan

    2003-04-11

    An assay based on enzymatic hydrolysisand microdialysis sampling, micro-high-performance anion-exchange chromatography (micro-HPAEC) with electrospray ionisation mass spectrometry (ESI-MS) for the characterisation of legumes is presented. Characterisation of two bean varieties; Phaseolus mungo and P. acutifilous was based upon enzymatic hydrolysis using an endo-beta-mannanase from Aspergillus niger with subsequent analysis of the hydrolysates with HPAEC-MS. The hydrolysates were detected in the positive ionisation mode after desalting the chromatographic effluent, employing a cation-exchange membrane desalting device with water as the regenerating liquid. Mass chromatograms, acquiredafter hydrolysis of both bean samples for 12 h, showed two different profiles of hydrolysates. The P. mungo bean hydrolysate showed the presence of saccharides with a degree of polymerisation (DP) in the range of 2-6, whereas that of P. acutifilous showed only DPs of 2-5. Both bean samples had one type of DP 2, but showed different types of DPs 3, 4 and 5. Only the P. mungo sample showed the presence of DP 6. The most abundant fraction for P. mungo was DP 4, whereas that for P. acutifilous was DP 5. Tandem MS of the hydrolysates showed that the DP 2 hydrolysates observed for the samples were of the same type, having a 1,6 linkage. Also tandem MS data for DPs 3, 4, and 5 showed that similar hydrolysates were present within the same sample as well as among the two samples. The data also showed the existence of 1,6 linkages for DP 3, 4, and 5 hydrolysates. The single enzymatic hydrolysis in combination with microdialysis and HPAEC with ESI-MS proved to be sufficient and reproducible for profiling and showing the difference between the two bean samples. PMID:12735463

  1. Microwave-assisted enzymatic hydrolysis followed by extraction with restricted access nanocomposites for rapid analysis of glucocorticoids residues in liver tissue.

    PubMed

    Feng, Jianan; Liu, Xiaodan; Li, Yan; Duan, Gengli

    2016-10-01

    We developed a novel, simple and fast method for the determination of glucocorticoids residues in liver tissue by combining microwave-assisted enzymatic hydrolysis and restricted access matrix dispersive solid phase extraction (RAM-dSPE) followed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Firstly, microwave-assisted enzymatic hydrolysis was introduced in order to obtain a maximum amount of unconjugated parent drug in a short time (8s), while the traditional method commonly needs 4-12h. Secondly, further cleanup was carried out by RAM-dSPE based on the graphene@mSiO2-C8 nanomaterials which were synthesized by coating mesoporous silica onto hydrophilic graphene nano-sheets through a surfactant-mediated co-condensation sol-gel process. The enzymatic hydrolysis influencing factors (pH of the buffer, microwave radiation power, incubation time) and the experimental conditions of RAM-dSPE (sorbents amount, type and volume of the elution solvent, adsorption and desorption time) were optimized. Three glucocorticoids (prednisolone (PREL), betamethasone (BE) and dexamethasone (DE)) were selected as models to evaluate the feasibility of the method. According to the results, the developed method provided low detection limit (S/N=3) of 0.01-0.05μgkg(-1) and good linearity range of 0.25-800μgkg(-1) (R(2)>0.996) for glucocorticoids. The limit of quantification (S/N=10) range from 0.03 to 0.19μgkg(-1). Compared with other traditional methods, the developed method could provide similar or even better results in a greatly reduced analysis time. PMID:27474293

  2. Enzymatic hydrolysis of beech wood lignocellulose at high solid contents and its utilization as substrate for the production of biobutanol and dicarboxylic acids.

    PubMed

    Tippkötter, Nils; Duwe, Anna-Maria; Wiesen, Sebastian; Sieker, Tim; Ulber, Roland

    2014-09-01

    The development of a cost-effective hydrolysis for crude cellulose is an essential part of biorefinery developments. To establish such high solid hydrolysis, a new solid state reactor with static mixing is used. However, concentrations >10% (w/w) cause a rate and yield reduction of enzymatic hydrolysis. By optimizing the synergetic activity of cellulolytic enzymes at solid concentrations of 9%, 17% and 23% (w/w) of crude Organosolv cellulose, glucose concentrations of 57, 113 and 152 g L(-1) are reached. However, the glucose yield decreases from 0.81 to 0.72 g g(-1) at 17% (w/w). Optimal conditions for hydrolysis scale-up under minimal enzyme addition are identified. As result, at 23% (w/w) crude cellulose the glucose yield increases from 0.29 to 0.49 g g(-1). As proof of its applicability, biobutanol, succinic and itaconic acid are produced with the crude hydrolysate. The potential of the substrate is proven e.g. by a high butanol yield of 0.33 g g(-1). PMID:25006020

  3. Comparison of cell wall polysaccharide hydrolysis by a dilute acid/enzymatic saccharification process and rumen microorganisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance of biomass in the bioenergy conversion process. Cell wall polysaccharide hydrolysis by dilute sulfuric acid pretreatment at 121 degrees C followed by cellulase hydrolysis for 72 h (CONV) and in v...

  4. Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei

    PubMed Central

    Chen, Ji-Hong; Li, Wen-Jian; Liu, Jing; Hu, Wei; Xiao, Guo-Qing; Dong, Miao-Yin; Wang, Yu-Chen

    2015-01-01

    The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and β-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme. PMID:26656155

  5. Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei.

    PubMed

    Wang, Min; Lu, Xuefeng

    2016-01-01

    Recent demands for the production of lignocellulose biofuels boosted research on cellulase. Hydrolysis efficiency and production cost of cellulase are two bottlenecks in "biomass to biofuels" process. The Trichoderma cellulase mixture is one of the most commonly used enzymes for cellulosic hydrolysis. During hydrolytic process cellobiose accumulation causes feedback inhibition against most cellobiohydrolases and endoglucanases. In this study, we demonstrated the synergism effects between cellobiose dehydrogenase (CDH) and cellulase both in vitro and in vivo. The CDH from Phanerochaete chrysosporium was heterologously expressed in Pichia pastoris. Supplementation of the purified CDH in Trichoderma cellulase increased the cellulase activities. Especially β-glucosidase activity was increased by 30-100% varying at different time points. On the other hand, the cdh gene was heterologously expressed in Trichoderma reesei to explore the synergism between CDH and cellulases in vivo. The analyses of gene expression and enzymatic profiles of filter paper activity, carboxymethylcellulase (CMCase) and β-glucosidase show the increased cellulase activity and the enhanced cellulase production in the cdh-expressing strains. The results elucidate a possible mechanism for diminishing the cellobiose inhibition of cellulase by CDH. These findings provide a novel perspective to make more economic enzyme cocktails for commercial application or explore alternative strategies for generating cellulase-producing strains with higher efficiency. PMID:27199949

  6. Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei

    PubMed Central

    Wang, Min; Lu, Xuefeng

    2016-01-01

    Recent demands for the production of lignocellulose biofuels boosted research on cellulase. Hydrolysis efficiency and production cost of cellulase are two bottlenecks in “biomass to biofuels” process. The Trichoderma cellulase mixture is one of the most commonly used enzymes for cellulosic hydrolysis. During hydrolytic process cellobiose accumulation causes feedback inhibition against most cellobiohydrolases and endoglucanases. In this study, we demonstrated the synergism effects between cellobiose dehydrogenase (CDH) and cellulase both in vitro and in vivo. The CDH from Phanerochaete chrysosporium was heterologously expressed in Pichia pastoris. Supplementation of the purified CDH in Trichoderma cellulase increased the cellulase activities. Especially β-glucosidase activity was increased by 30–100% varying at different time points. On the other hand, the cdh gene was heterologously expressed in Trichoderma reesei to explore the synergism between CDH and cellulases in vivo. The analyses of gene expression and enzymatic profiles of filter paper activity, carboxymethylcellulase (CMCase) and β-glucosidase show the increased cellulase activity and the enhanced cellulase production in the cdh-expressing strains. The results elucidate a possible mechanism for diminishing the cellobiose inhibition of cellulase by CDH. These findings provide a novel perspective to make more economic enzyme cocktails for commercial application or explore alternative strategies for generating cellulase-producing strains with higher efficiency. PMID:27199949

  7. Solar assisted alkali pretreatment of garden biomass: Effects on lignocellulose degradation, enzymatic hydrolysis, crystallinity and ultra-structural changes in lignocellulose

    SciTech Connect

    Gabhane, Jagdish; William, S.P.M. Prince; Vaidya, Atul N.; Das, Sera; Wate, Satish R.

    2015-06-15

    Highlights: • SAAP is an efficient and economic means of pretreatment. • SAAP was found to be efficient in lignin and hemicellulose removal. • SAAP enhanced the enzymatic hydrolysis. • FTIR, XRD and SEM provided vivid understanding about the mode of action of SAAP. • Mass balance closer of 98% for pretreated GB confirmed the reliability of SAAP. - Abstract: A comprehensive study was carried out to assess the effectiveness of solar assisted alkali pretreatment (SAAP) on garden biomass (GB). The pretreatment efficiency was assessed based on lignocellulose degradation, conversion of cellulose into reducing sugars, changes in the ultra-structure and functional groups of lignocellulose and impact on the crystallinity of cellulose, etc. SAAP was found to be efficient for the removal of lignin and hemicellulose that facilitated enzymatic hydrolysis of cellulose. FTIR and XRD studies provided details on the effectiveness of SAAP on lignocellulosic moiety and crystallinity of cellulose. Scanning electron microscopic analysis showed ultra-structural disturbances in the microfibrils of GB as a result of pretreatment. The mass balance closer of 97.87% after pretreatment confirmed the reliability of SAAP pretreatment. Based on the results, it is concluded that SAAP is not only an efficient means of pretreatment but also economical as it involved no energy expenditure for heat generation during pretreatment.

  8. Characterization of a Novel Dye-Decolorizing Peroxidase (DyP)-Type Enzyme from Irpex lacteus and Its Application in Enzymatic Hydrolysis of Wheat Straw

    PubMed Central

    Salvachúa, Davinia; Prieto, Alicia

    2013-01-01

    Irpex lacteus is a white rot basidiomycete proposed for a wide spectrum of biotechnological applications which presents an interesting, but still scarcely known, enzymatic oxidative system. Among these enzymes, the production, purification, and identification of a new dye-decolorizing peroxidase (DyP)-type enzyme, as well as its physico-chemical, spectroscopic, and catalytic properties, are described in the current work. According to its N-terminal sequence and peptide mass fingerprinting analyses, I. lacteus DyP showed high homology (>95%) with the hypothetical (not isolated or characterized) protein cpop21 from an unidentified species of the family Polyporaceae. The enzyme had a low optimal pH, was very stable to acid pH and temperature, and showed improved activity and stability at high H2O2 concentrations compared to other peroxidases. Other attractive features of I. lacteus DyP were its high catalytic efficiency oxidizing the recalcitrant anthraquinone and azo dyes assayed (kcat/Km of 1.6 × 106 s-1 M-1) and its ability to oxidize nonphenolic aromatic compounds like veratryl alcohol. In addition, the effect of this DyP during the enzymatic hydrolysis of wheat straw was checked. The results suggest that I. lacteus DyP displayed a synergistic action with cellulases during the hydrolysis of wheat straw, increasing significantly the fermentable glucose recoveries from this substrate. These data show a promising biotechnological potential for this enzyme. PMID:23666335

  9. Comparative study of lignin characteristics from wheat straw obtained by soda-AQ and kraft pretreatment and effect on the following enzymatic hydrolysis process.

    PubMed

    Yang, Haitao; Xie, Yimin; Zheng, Xing; Pu, Yunqiao; Huang, Fang; Meng, Xianzhi; Wu, Weibing; Ragauskas, Arthur; Yao, Lan

    2016-05-01

    To understand the structural changes of lignin after soda-AQ and kraft pretreatment, milled straw lignin, black liquor lignin and residual lignin extracted from wheat straw were characterized by FT-IR, UV, GPC and NMR. The results showed that the main lignin linkages were β-aryl ether substructures (β-O-4'), followed by phenylcoumaran (β-5') and resinol (β-β') substructures, while minor content of spirodienone (β-1'), dibenzodioxocin (5-5') and α,β-diaryl ether linkages were detected as well. After pretreatment, most lignin inter-units and lignin-carbohydrate complex (LCC) linkages were degraded and dissolved in black liquor, with minor amount left in residual pretreated biomass. In addition, through quantitative (13)C and 2D-HSQC NMR spectral analysis, lignin and LCC were found to be more degraded after kraft pretreatment than soda-AQ pretreatment. Furthermore, the subsequent enzymatic hydrolysis results showed that more cellulose in wheat straw was converted to glucose after kraft pretreatment, indicating that LCC linkages were important in the enzymatic hydrolysis process. PMID:26897415

  10. Novel DDR Processing of Corn Stover Achieves High Monomeric Sugar Concentrations from Enzymatic Hydrolysis (230 g/L) and High Ethanol Concentration (10% v/v) During Fermentation

    SciTech Connect

    Chen, Xiaowen; Jennings, Ed; Shekiro, Joe; Kuhn, Erik M.; O'Brien, Marykate; Wang, Wei; Schell, Daniel J.; Himmel, Mike; Elander, Richard T.; Tucker, Melvin P.

    2015-04-03

    Distilling and purifying ethanol, butanol, and other products from second and later generation lignocellulosic biorefineries adds significant capital and operating cost for biofuels production. The energy costs associated with distillation affects plant gate and life cycle analysis costs. Lower titers in fermentation due to lower sugar concentrations from pretreatment increase both energy and production costs. In addition, higher titers decrease the volumes required for enzymatic hydrolysis and fermentation vessels. Therefore, increasing biofuels titers has been a research focus in renewable biofuels production for several decades. In this work, we achieved over 200 g/L of monomeric sugars after high solids enzymatic hydrolysis using the novel deacetylation and disc refining (DDR) process on corn stover. The high sugar concentrations and low chemical inhibitor concentrations from the DDR process allowed ethanol titers as high as 82 g/L in 22 hours, which translates into approximately 10 vol% ethanol. To our knowledge, this is the first time that 10 vol% ethanol in fermentation derived from corn stover without any sugar concentration or purification steps has been reported. Techno-economic analysis shows the higher titer ethanol achieved from the DDR process could significantly reduce the minimum ethanol selling price from cellulosic biomass.

  11. Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

    SciTech Connect

    Anderson, Lindsey N.; Culley, David E.; Hofstad, Beth A.; Chauvigne-Hines, Lacie M.; Zink, Erika M.; Purvine, Samuel O.; Smith, Richard D.; Callister, Stephen J.; Magnuson, Jon M.; Wright, Aaron T.

    2013-12-01

    Development of alternative, non-petroleum based sources of bioenergy that can be applied in the short-term find great promise in the use of highly abundant and renewable lignocellulosic plant biomass.1 This material obtained from different feedstocks, such as forest litter or agricultural residues, can yield liquid fuels and other chemical products through biorefinery processes.2 Biofuels are obtained from lignocellulosic materials by chemical pretreatment of the biomass, followed by enzymatic decomposition of cellulosic and hemicellulosic compounds into soluble sugars that are converted to desired chemical products via microbial metabolism and fermentation.3, 4 To release soluble sugars from polymeric cellulose multiple enzymes are required, including endoglucanase, exoglucanase, and β-glucosidase.5, 6 However, the enzymatic hydrolysis of cellulose into soluble sugars remains a significant limiting factor to the efficient and economically viable utilization of lignocellulosic biomass for transport fuels.7, 8 The primary industrial source of cellulose and hemicellulases is the mesophilic soft-rot fungus Trichoderma reesei,9 having widespread applications in food, feed, textile, pulp, and paper industries.10 The genome encodes 200 glycoside hydrolases, including 10 cellulolytic and 16 hemicellulolytic enzymes.11 The hypercellulolytic catabolite derepressed strain RUT-C30 was obtained through a three-step UV and chemical mutagenesis of the original T. reesei strain QM6a,12, 13 in which strains M7 and NG14 were intermediate, having higher cellulolytic activity than the parent strain but less activity and higher catabolite repression than RUT-C30.14 Numerous methods have been employed to optimize the secreted enzyme cocktail of T. reesei including cultivation conditions, operational parameters, and mutagenesis.3 However, creating an optimal and economical enzyme mixture for production-scale biofuels synthesis may take thousands of experiments to identify.

  12. Development of a novel method for hot-pressure extraction of protein from chicken bone and the effect of enzymatic hydrolysis on the extracts.

    PubMed

    Dong, Xian-bing; Li, Xia; Zhang, Chun-hui; Wang, Jin-zhi; Tang, Chun-hong; Sun, Hong-mei; Jia, Wei; Li, Yin; Chen, Lin-li

    2014-08-15

    To investigate the hot-pressure extraction of protein from chicken bone (CB), chicken bone extracts (CBE) was prepared from CB by heating at 130±0.5 °C for 120 min, followed by filtration, standing, defatting, and concentration. Effects of enzymatic hydrolysis on the properties of hydrolysates were examined. Results showed CBE contained 25.59% of protein, and showed a desirable value of protein digestibility-corrected amino acid score for adult. The total amino acid (AA) content of CBE is 21.99%, among which 40.62% and 54.66% are essential and fresh AA, respectively. Forty kinds of volatile compounds were identified after 24 h of hydrolysis, with 2,3,5-trimethylpyrazine as the key flavor compound. After 8 h of hydrolysis of CBE, the content of small MW of peptides (400-1000 Da) increased by 74 times compared with that of 1 h. CBE and its hydrolysates demonstrate a new kind of potential suitable nutritional supplement in various foods. PMID:24679789

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

    PubMed Central

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

    2011-01-01

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

  14. Effects of tea saponin on glucan conversion and bonding behaviour of cellulolytic enzymes during enzymatic hydrolysis of corncob residue with high lignin content

    PubMed Central

    2013-01-01

    Background Recently, interest in the utilization of corncob residue (CCR, with high lignin of 45.1%) as a feedstock for bioethanol has been growing. Surfactants have been one of the most popular additives intended to prevent the inhibitory effect of lignin on cellulolytic enzymes, thereby improving hydrolysis. In this study, the effects of biosurfactant tea saponin (TS) on the enzymatic hydrolysis of CCR and the bonding behavior of cellulolytic enzymes to the substrate were investigated. The surface tension in the supernatant was also detected to obtain information about the characteristics and stability of TS. Results The glucose concentration was 17.15 mg/mL at 120 hours of hydrolysis with the low loading of cellulolytic enzymes (7.0 FPU/g cellulose and 10.5 BGU/g cellulose) and 5% CCR. The optimal dosage of TS was its critical micelle concentration (cmc, 1.80 mg/mL). The glucose yield was enhanced from 34.29 to 46.28 g/100 g dry matter by TS. The results indicate that TS can promote the adsorption of cellulolytic enzymes on the substrate and mediate the release of adsorbed enzymes. Meanwhile, TS improves the recovery of the cellulolytic enzymes after a hydrolysis cycle and prevents deactivation of the enzymes during the intense shaking process. The surface tension in supernatants of digested CCR with TS remained at 50.00 mN/m during the course of hydrolysis. It is interesting to note that biosurfactant TS can maintain the surface tension in supernatants, despite its digestibility by cellulolytic enzymes. Conclusions Serving as an accelerant of lignocellulose hydrolysis, TS can also be degraded by the cellulolytic enzymes and release glucose while retaining stability, which reduces the cost of both the cellulolytic enzymes and the additive. As the glucose from the TS could be utilized by yeast, further efforts will investigate the mechanism of function and the application of TS in the production of ethanol by simultaneous saccharification and fermentation

  15. Deposition of Lignin Droplets Produced During Dilute Acid Pretreatment of Maize Stems Retards Enzymatic Hydrolysis of Cellulose

    SciTech Connect

    Selig, M. J.; Viamajala, S.; Decker, S. R.; Tucker, M. P.; Himmel, M. E.; Vinzant, T. B.

    2007-01-01

    Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these lignin droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.

  16. Solar assisted alkali pretreatment of garden biomass: Effects on lignocellulose degradation, enzymatic hydrolysis, crystallinity and ultra-structural changes in lignocellulose.

    PubMed

    Gabhane, Jagdish; William, S P M Prince; Vaidya, Atul N; Das, Sera; Wate, Satish R

    2015-06-01

    A comprehensive study was carried out to assess the effectiveness of solar assisted alkali pretreatment (SAAP) on garden biomass (GB). The pretreatment efficiency was assessed based on lignocellulose degradation, conversion of cellulose into reducing sugars, changes in the ultra-structure and functional groups of lignocellulose and impact on the crystallinity of cellulose, etc. SAAP was found to be efficient for the removal of lignin and hemicellulose that facilitated enzymatic hydrolysis of cellulose. FTIR and XRD studies provided details on the effectiveness of SAAP on lignocellulosic moiety and crystallinity of cellulose. Scanning electron microscopic analysis showed ultra-structural disturbances in the microfibrils of GB as a result of pretreatment. The mass balance closer of 97.87% after pretreatment confirmed the reliability of SAAP pretreatment. Based on the results, it is concluded that SAAP is not only an efficient means of pretreatment but also economical as it involved no energy expenditure for heat generation during pretreatment. PMID:25816769

  17. Biomass-to-electricity: analysis and optimization of the complete pathway steam explosion--enzymatic hydrolysis--anaerobic digestion with ICE vs SOFC as biogas users.

    PubMed

    Santarelli, M; Barra, S; Sagnelli, F; Zitella, P

    2012-11-01

    The paper deals with the energy analysis and optimization of a complete biomass-to-electricity energy pathway, starting from raw biomass towards the production of renewable electricity. The first step (biomass-to-biogas) is based on a real pilot plant located in Environment Park S.p.A. (Torino, Italy) with three main steps ((1) impregnation; (2) steam explosion; (3) enzymatic hydrolysis), completed by a two-step anaerobic fermentation. In the second step (biogas-to-electricity), the paper considers two technologies: internal combustion engines and a stack of solid oxide fuel cells. First, the complete pathway has been modeled and validated through experimental data. After, the model has been used for an analysis and optimization of the complete thermo-chemical and biological process, with the objective function of maximization of the energy balance at minimum consumption. The comparison between ICE and SOFC shows the better performance of the integrated plants based on SOFC. PMID:22940353

  18. Effects of the pretreatment method on high solids enzymatic hydrolysis and ethanol fermentation of the cellulosic fraction of sugarcane bagasse.

    PubMed

    Martins, Luiza Helena da Silva; Rabelo, Sarita Cândida; da Costa, Aline Carvalho

    2015-09-01

    This work evaluated ethanol production from sugarcane bagasse at high solids loadings in the pretreatment (20-40% w/v) and hydrolysis (10-20% w/v) stages. The best conditions for diluted sulfuric acid, AHP and Ox-B pretreatments were determined and mass balances including pretreatment, hydrolysis and fermentation were calculated. From a technical point of view, the best pretreatment was AHP, which enabled the production of glucose concentrations near 8% with high productivity (3.27 g/Lh), as well as ethanol production from 100.9 to 135.4 kg ethanol/ton raw bagasse. However, reagent consumption for acid pretreatment was much lower. Furthermore, for processes that use pentoses and hexoses separately, this pretreatment produces the most desirable pentoses liquor, with higher xylose concentration in the monomeric form. PMID:26004382

  19. Effects of extrusion pretreatment parameters on sweet sorghum bagasse enzymatic hydrolysis and its subsequent conversion into bioethanol.

    PubMed

    Heredia-Olea, Erick; Pérez-Carrillo, Esther; Montoya-Chiw, Manuel; Serna-Saldívar, Sergio O

    2015-01-01

    Second-generation bioethanol production from sweet sorghum bagasse first extruded at different conditions and then treated with cell wall degrading enzymes and fermented with I. orientalis was determined. The twin extruder parameters tested were barrel temperature, screws speed, and feedstock moisture content using surface response methodology. The best extrusion conditions were 100°C, 200 rpm, and 30% conditioning moisture content. This nonchemical and continuous pretreatment did not generate inhibitory compounds. The extruded feedstocks were saccharified varying the biocatalysis time and solids loading. The best conditions were 20% solids loading and 72 h of enzymatic treatment. These particular conditions converted 70% of the total fibrous carbohydrates into total fermentable C5 and C6 sugars. The extruded enzymatically hydrolyzed sweet sorghum bagasse was fermented with the strain I. orientalis at 12% solids obtaining a yield of 198.1 mL of ethanol per kilogram of bagasse (dw). PMID:25866776

  20. Effects of Extrusion Pretreatment Parameters on Sweet Sorghum Bagasse Enzymatic Hydrolysis and Its Subsequent Conversion into Bioethanol

    PubMed Central

    Heredia-Olea, Erick; Pérez-Carrillo, Esther; Serna-Saldívar, Sergio O.

    2015-01-01

    Second-generation bioethanol production from sweet sorghum bagasse first extruded at different conditions and then treated with cell wall degrading enzymes and fermented with I. orientalis was determined. The twin extruder parameters tested were barrel temperature, screws speed, and feedstock moisture content using surface response methodology. The best extrusion conditions were 100°C, 200 rpm, and 30% conditioning moisture content. This nonchemical and continuous pretreatment did not generate inhibitory compounds. The extruded feedstocks were saccharified varying the biocatalysis time and solids loading. The best conditions were 20% solids loading and 72 h of enzymatic treatment. These particular conditions converted 70% of the total fibrous carbohydrates into total fermentable C5 and C6 sugars. The extruded enzymatically hydrolyzed sweet sorghum bagasse was fermented with the strain I. orientalis at 12% solids obtaining a yield of 198.1 mL of ethanol per kilogram of bagasse (dw). PMID:25866776

  1. The effect of harvest time, dry matter content and mechanical pretreatments on anaerobic digestion and enzymatic hydrolysis of miscanthus.

    PubMed

    Frydendal-Nielsen, Susanne; Hjorth, Maibritt; Baby, Sanmohan; Felby, Claus; Jørgensen, Uffe; Gislum, René

    2016-10-01

    Miscanthus x giganteus was harvested as both green and mature biomass and the dry matter content of the driest harvest was artificially decreased by adding water in two subsamples, giving a total of five dry matter contents. All five biomass types were mechanically pretreated by roller-milling, extrusion or grinding and accumulated methane production and enzymatically-accessible sugars were measured. Accumulated methane production was studied using sigmoid curves that allowed comparison among the treatments of the rate of the methane production and ultimate methane yield. The green biomass gave the highest methane yield and highest levels of enzymatically-accessible cellulose. The driest biomass gave the best effect from extrusion but with the highest energy consumption, whereas roller-milling was most efficient on wet biomass. The addition of water to the last harvest improved the effect of roller-milling and equalled extrusion of the samples in efficiency. PMID:27455125

  2. Does Enzymatic Hydrolysis of Glycosidically Bound Volatile Compounds Really Contribute to the Formation of Volatile Compounds During the Oolong Tea Manufacturing Process?

    PubMed

    Gui, Jiadong; Fu, Xiumin; Zhou, Ying; Katsuno, Tsuyoshi; Mei, Xin; Deng, Rufang; Xu, Xinlan; Zhang, Linyun; Dong, Fang; Watanabe, Naoharu; Yang, Ziyin

    2015-08-12

    It was generally thought that aroma of oolong tea resulted from hydrolysis of glycosidically bound volatiles (GBVs). In this study, most GBVs showed no reduction during the oolong tea manufacturing process. β-Glycosidases either at protein or gene level were not activated during the manufacturing process. Subcellular localization of β-primeverosidase provided evidence that β-primeverosidase was located in the leaf cell wall. The cell wall remained intact during the enzyme-active manufacturing process. After the leaf cell disruption, GBV content was reduced. These findings reveal that, during the enzyme-active process of oolong tea, nondisruption of the leaf cell walls resulted in impossibility of interaction of GBVs and β-glycosidases. Indole, jasmine lactone, and trans-nerolidol were characteristic volatiles produced from the manufacturing process. Interestingly, the contents of the three volatiles was reduced after the leaf cell disruption, suggesting that mechanical damage with the cell disruption, which is similar to black tea manufacturing, did not induce accumulation of the three volatiles. In addition, 11 volatiles with flavor dilution factor ≥4(4) were identified as relatively potent odorants in the oolong tea. These results suggest that enzymatic hydrolysis of GBVs was not involved in the formation of volatiles of oolong tea, and some characteristic volatiles with potent odorants were produced from the manufacturing process. PMID:26212085

  3. Investigation of enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle (Collichthys niveatus) and evaluation of its functional properties.

    PubMed

    Shen, Qing; Guo, Rui; Dai, Zhiyuan; Zhang, Yanping

    2012-05-23

    This study was carried out to investigate the enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle of the marine fish species Collichthys niveatus. About 160 fish samples were tested, and the analyzed fish species was found to be a lean fish with low fat (1.77 ± 0.01%) and high protein (16.76 ± 1.21%). Fish muscle of C. niveatus was carefully collected and hydrolyzed with four commercial enzymes: Alcalase, Neutrase, Protamex, and Flavourzyme under the conditions recommended by the manufacturers. Among the tested proteases, Neutrase catalyzed the hydrolysis process most effectively since the hydrolysate generated by Neutrase has the highest content of sweet and umami taste amino acids (SUA). The effect of hydrolysis conditions was further optimized using response surface methodology (RSM), and the optimum values for temperature, pH, and enzyme/substrate ratio (E/S ratio) were found to be 40.7 °C, 7.68, and 0.84%, respectively. Finally, the amino acid composition of the hydrolysate was analyzed by AccQ·Tag derivatization and HPLC-PDA determination. Major amino acids of the muscle of C. niveatus were threonine, glutamic acid, phenyalanine, tryptophan, and lysine, accounting for respectively 10.92%, 10.85%, 10.79%, 9.86%, and 9.76% of total amino acid content. The total content of essential amino acids was 970.7 ng·mL(-1), while that of nonessential amino acids was 709.1 ng·mL(-1). The results suggest that the fish muscle and its protein hydrolysate from C. niveatus provide a versatile supply of the benefits and can be incorporated as supplements in health-care foods. PMID:22530872

  4. Synthesis, Chemical and Enzymatic Hydrolysis, and Aqueous Solubility of Amino Acid Ester Prodrugs of 3-Carboranyl Thymidine Analogues for Boron Neutron Capture Therapy of Brain Tumors

    PubMed Central

    Hasabelnaby, Sherifa; Goudah, Ayman; Agarwal, Hitesh K.; Abd alla, Mosaad S. M.; Tjarks, Werner

    2012-01-01

    Various water-soluble L-valine-, L-glutamate-, and glycine ester prodrugs of two 3-Carboranyl Thymidine Analogues (3-CTAs), designated N5 and N5-2OH, were synthesized for Boron Neutron Capture Therapy (BNCT) of brain tumors since the water solubilities of the parental compounds proved to be insufficient in preclinical studies. The amino acid ester prodrugs were prepared and stored as hydrochloride salts. The water solubilities of these amino acid ester prodrugs, evaluated in phosphate buffered saline (PBS) at pH 5, pH 6 and pH 7.4, improved 48 to 6600 times compared with parental N5 and N5-2OH. The stability of the amino acid ester prodrugs was evaluated in PBS at pH 7.4, Bovine serum, and Bovine cerebrospinal fluid (CSF). The rate of the hydrolysis in all three incubation media depended primarily on the amino acid promoiety and, to a lesser extend, on the site of esterification at the deoxyribose portion of the 3-CTAs. In general, 3'-amino acid ester prodrugs were less sensitive to chemical and enzymatic hydrolysis than 5'-amino acid ester prodrugs and the stabilities of the latter decreased in the following order: 5'-valine > 5'-glutamate > 5'-glycine. The rate of the hydrolysis of the 5'-amino acid ester prodrugs in Bovine CSF was overall higher than in PBS and somewhat lower than in Bovine serum. Overall, 5'-glutamate ester prodrug of N5 and the 5'-glycine ester prodrugs of N5 and N5-2OH appeared to be the most promising candidates for preclinical BNCT studies. PMID:22889558

  5. Genome Sequencing and Analysis of the Biomass-Degrading Fungus Trichoderma reesei (syn. Hypocrea jecorina)

    SciTech Connect

    Martinez, Antonio D.; Berka, Randy; Henrissat, Bernard; Saloheimo, Markku; Arvas, Mikko; Baker, Scott E.; Chapman, Jaro d; Chertkov, Olga; Coutinho, Pedro M.; Cullen, Dan; Danchin, Etienne G.; Grigoriev, Igor V.; Harris, Paul; Jackson, Melissa ?.; kubicek, Christian P.; Han, Cliff F.; Ho, Isaac; Larrando, Luis F.; Lopez de Leon, Alfredo; Magnuson, Jon K.; Merino, Sandy; Misra, Monica; Nelson, Beth; Putnam, Nicholas; Robbertse, Barbara; Salamov, Asaf; Schmoll, Monika; Terry, Astrid ?.; Thayer, Nina; Westerholm-Parvinen, Ann; Schoch, Conrad L.; Yao, Jian ?.; Barbote, Ravi; Nelson, Mary Anne; Detter, Chris J.; Bruce, David; Kuske, Cheryl; Xie, Gary; Richardson, P. M.; Rokhsar, Daniel S.; Lucas, Susan; Rubin, Eddie M.; Dunn-Coleman, Nigel; Ward, Michael ?.; Brettin, T.

    2008-05-01

    A major thrust of the white biotechnology movement involves the development of enzyme systems which depolymerize biomass to simple sugars which are subsequently converted to sustainable biofuels (e.g., ethanol) and chemical intermediates. The fungus Trichoderma reesei (syn. Hypocrea jecorina) represents a paradigm for the industrial production of highly efficient cellulases and hemicellulases needed for hydrolysis of biomass polysaccharides. Herein we describe intriguing attributes of the T. reeseigenome in relation to the future of fuel biotechnology. The T. reesei genome sequence was derived using a whole genome shotgun approach combined with finishing work to generate an assembly comprising 89 scaffolds totaling 34 Mbp with few gaps. In total, 9,130 gene models were predicted using a combination of ab initio and sequence similarity-based methods and EST data. Considering the industrial utility and effectiveness of its enzymes, the T. reesei genome surprisingly encodes the fewest cellulases and hemicellulases of any fungus having the ability to hydrolyze plant cell wall polysaccharides and whose genome has been sequenced. Many genes encoding carbohydrate active enzymes are distributed non-randomly in groups or clusters that interestingly lie between regions of synteny with other Sordariomycetes. Additionally, the T. reesei genome contains a multitude of genes encoding biosynthetic pathways for secondary metabolites (possible antibacterial and antifungal compounds) which may promote successful competition and survival in the crowded and competitive soil habitat occupied by T. reesei. Our analysis coupled with the availability of genome sequence data provides a roadmap for construction of enhanced T. reesei strains for industrial applications.

  6. Effect of enzymatic hydrolysis of starch on pasting, rheological and viscoelastic properties of milk-barnyard millet (Echinochloa frumentacea) blends meant for spray drying.

    PubMed

    Kumar, P Arun; Pushpadass, Heartwin A; Franklin, Magdaline Eljeeva Emerald; Simha, H V Vikram; Nath, B Surendra

    2016-10-01

    The influence of enzymatic hydrolysis of starch on the pasting properties of barnyard millet was studied using a rheometer. The effects of blending hydrolyzed barnyard millet wort with milk at different ratios (0:1, 1:1, 1:1.5 and 1:2) on flow and viscoelastic behavior were investigated. From the pasting curves, it was evident that enzymatically-hydrolyzed starch did not exhibit typical pasting characteristics expected of normal starch. The Herschel-Bulkley model fitted well to the flow behaviour data, with coefficient of determination (R(2)) ranging from 0.942 to 0.988. All milk-wort blends demonstrated varying degree of shear thinning with flow behavior index (n) ranging from 0.252 to 0.647. Stress-strain data revealed that 1:1 blend of milk to wort had the highest storage modulus (7.09-20.06Pa) and an elastically-dominant behavior (phase angle <45°) over the tested frequency range. The crossover point of G' and G" shifted to higher frequencies with increasing wort content. From the flow and viscoelastic behavior, it was concluded that the 1:1 blend of milk to wort would have least phase separation and better flowability during spray drying. PMID:27296446

  7. Cellulosimicrobium cellulans strain E4-5 enzymatic hydrolysis of curdlan for production of (1 → 3)-linked β-D-glucan oligosaccharides.

    PubMed

    Fu, Yunbin; Cheng, Likun; Meng, Yanyu; Li, Shuguang; Zhao, Xiaoming; Du, Yuguang; Yin, Heng

    2015-12-10

    In order to find an efficient enzymatic tool for curdlan degradation to produce (1 → 3)-linked β-D-glucan oligosaccharides, strain E4-5 (registration number JN089883, Genbank) was isolated from seaside soil. The 16S rRNA gene sequencing classified it as Cellulosimicrobium cellulans. It was the first reported microorganism that succeeded in degrading high-set heated curdlan blocks. The ferments of strain E4-5 also showed good degradation effects on laminaran and alkali-neutralized curdlan. Due to the products with less amount of glucose, it was assumed that endo-1,3-β-glucanases of strain E4-5 had a greater hydrolyzing effect than exo-1,3-β-glucanases. This indicated that strain E4-5 was a promising microorganism to hydrolyze (1 → 3)-linked β-D-glucan. Moreover, alkali-neutralization pretreatment was effective for promoting a more diversified degree of polymerization (DP) of (1 → 3)-linked β-D-glucan oligosaccharides under enzymatic hydrolysis and will pave the way for making full use of curdlan for production of glucan oligosaccharides. PMID:26428180

  8. Enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide by α-chymotrypsin in DMSO-water/AOT/n-heptane reverse micelles. A unique interfacial effect on the enzymatic activity.

    PubMed

    Moyano, Fernando; Setien, Evangelina; Silber, Juana J; Correa, N Mariano

    2013-07-01

    The reverse micelle (RM) media are very good as nanoreactors because they can create a unique microenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is to determine the influence of different water-dimethyl sulfoxide (DMSO) mixtures encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane RMs on the enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide (Bz-Try-pNA) by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media at different DMSO-water mixture compositions and in DMSO-water/AOT/n-heptane RMs. The hydrolysis rates of Bz-Try-pNA catalyzed by α-CT were determined by UV-vis spectroscopy. The reaction follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM, and kcat/KM were evaluated under different conditions. In this homogeneous media, DMSO plays an important role in the solubilization process of the peptide which is almost insoluble in water, but it has a tremendous impact on the inactivation of α-CT. It is shown that the enzyme dissolved in a 20% molar ratio of the DMSO-water mixture does not present enzymatic activity. Dynamic light scattering has been used to assess the formation of DMSO-water/AOT/heptane RMs at different DMSO compositions. The results also show that there is preferential solvation of the AOT RM interface by water molecules. To test the use of these RMs as nanoreactors, the kinetic parameters for the enzymatic reaction in these systems have been evaluated. The parameters were determined at fixed W(S) {W(S) = ([water] + [DMSO])/[AOT] = 20} at different DMSO-water compositions. The results show that the Michaelis-Menten mechanism is valid for α-CT in all the RM systems studied and that the reaction takes place at the RM interface. Surprisingly, it was observed that the enzyme encapsulated by the RMs show catalytic effects with similar kcat/KM values at any DMSO composition investigated, which evidence that DMSO molecules are

  9. 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.

  10. Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30.

    PubMed

    Anderson, Lindsey N; Culley, David E; Hofstad, Beth A; Chauvigné-Hines, Lacie M; Zink, Erika M; Purvine, Samuel O; Smith, Richard D; Callister, Stephen J; Magnuson, Jon M; Wright, Aaron T

    2013-12-01

    Lignocellulosic biomass has great promise as a highly abundant and renewable source for the production of biofuels. However, the recalcitrant nature of lignocellulose toward hydrolysis into soluble sugars remains a significant challenge to harnessing the potential of this source of bioenergy. A primary method for deconstructing lignocellulose is via chemical treatments, high temperatures, and hydrolytic enzyme cocktails, many of which are derived from the fungus Trichoderma reesei. Herein, we use an activity-based probe for glycoside hydrolases to rapidly identify optimal conditions for maximum enzymatic lignocellulose deconstruction. We also demonstrate that subtle changes to enzyme composition and activity in various strains of T. reesei can be readily characterized by our probe approach. The approach also permits multimodal measurements, including fluorescent gel-based analysis of activity in response to varied conditions and treatments, and mass spectrometry-based quantitative identification of labelled proteins. We demonstrate the promise this probe approach holds to facilitate rapid production of enzyme cocktails for high-efficiency lignocellulose deconstruction to accommodate high-yield biofuel production. PMID:24121482

  11. Production of a chimeric enzyme tool associating the Trichoderma reesei swollenin with the Aspergillus niger feruloyl esterase A for release of ferulic acid.

    PubMed

    Levasseur, Anthony; Saloheimo, Markku; Navarro, David; Andberg, Martina; Monot, Frédéric; Nakari-Setälä, Tiina; Asther, Marcel; Record, Eric

    2006-12-01

    The main goals of this work were to produce the fusion protein of the Trichoderma reesei swollenin I (SWOI) and Aspergillus niger feruloyl esterase A (FAEA) and to study the effect of the physical association of the fusion partners on the efficiency of the enzyme. The fusion protein was produced up to 25 mg l(-1) in the T. reesei strains Rut-C30 and CL847. In parallel, FAEA alone was produced for use as a control protein in application tests. Recombinant FAEA and SWOI-FAEA were purified to homogeneity and characterized. The biochemical and kinetic characteristics of the two recombinant proteins were found to be similar to those of native FAEA, except for the temperature stability and specific activity of the SWOI-FAEA. Finally, the SWOI-FAEA protein was tested for release of ferulic acid from wheat bran. A period of 24 h of enzymatic hydrolysis with the SWOI-FAEA improved the efficiency of ferulic acid release by 50% compared with the results obtained using the free FAEA and SWOI. Ferulic acid is used as an antioxidant and flavor precursor in the food and pharmaceutical industries. This is the first report of a potential application of the SWOI protein fused with an enzyme of industrial interest. PMID:16957894

  12. Morphology and enzyme production of Trichoderma reesei Rut C-30 are affected by the physical and structural characteristics of cellulosic substrates.

    PubMed

    Peciulyte, Ausra; Anasontzis, George E; Karlström, Katarina; Larsson, Per Tomas; Olsson, Lisbeth

    2014-11-01

    The industrial production of cellulolytic enzymes is dominated by the filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina). In order to develop optimal enzymatic cocktail, it is of importance to understand the natural regulation of the enzyme profile as response to the growth substrate. The influence of the complexity of cellulose on enzyme production by the microorganisms is not understood. In the present study we attempted to understand how different physical and structural properties of cellulose-rich substrates affected the levels and profiles of extracellular enzymes produced by T. reesei. Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel® and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS (13)C-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD001304. In total 124 proteins were identified and quantified in the culture supernatants, including cellulases, hemicellulases, other glycoside hydrolases, lignin-degrading enzymes, auxiliary activity 9 (AA9) family (formerly GH61), supporting activities of proteins and enzymes acting on cellulose, proteases, intracellular proteins and several hypothetical proteins. Surprisingly, substantial differences in the enzyme profiles were found even though there were minor differences in the chemical composition between the cellulose-rich substrates

  13. Enzymatic hydrolysis of whey lactose to glucose for alcohol production. Final report, September 1, 1979-August 31, 1981

    SciTech Connect

    Hirasuna, T.J.

    1981-09-15

    This report covers the initial phase of a whey-to-glucose-to-alcohol process via an immobilized beta-galactosidase reactor in series with a fermentor. The first stage takes pure lactose and its hydrolysis with a soluble enzyme system. This stage involves the development of an assay to assess conversion in the hydrolysis reaction and the selections of the best reaction conditions for the enzyme selected. For the Embiozyme Lactase enzyme, the best conditions are 45/sup 0/C and pH 6.5. Thus, this enzyme would be more applicable to sweet whey systems rather than to acid whey. In the range of 1.0 to 3.0 g/l enzyme concentration, increased conversion is seen with increased enzyme concentration; however, the economics need to be studied to determine the optimum enzyme level taking into account cost and yield considerations. Some substrate or product inhibition is seen and is especially apparent above 50 g/l substrate concentration. Inhibition needs to be studied in more detail. Stability seems to be a problem with the Embiozyme Lactase enzyme. This may lead to problems in the future immobilization. Alternate processing conditions may have to be determined giving stability considerations the highest priority. Alternate suppliers of lactase can also be investigated. Acid denaturation seems to be more reliable than heat denaturation. Whether buffer or water is used in enzyme reconstitution makes little short-term difference in conversion. However, there may be an effect on long-term stability. 33 refs., 8 figs., 13 tabs.

  14. Combined pretreatment using alkaline hydrothermal and ball milling to enhance enzymatic hydrolysis of oil palm mesocarp fiber.

    PubMed

    Zakaria, Mohd Rafein; Hirata, Satoshi; Hassan, Mohd Ali

    2014-10-01

    Hydrothermal pretreatment of oil palm mesocarp fiber was conducted in tube reactor at treatment severity ranges of log Ro = 3.66-4.83 and partial removal of hemicellulose with migration of lignin was obtained. Concerning maximal recovery of glucose and xylose, 1.5% NaOH was impregnated in the system and subsequent ball milling treatment was employed to improve the conversion yield. The effects of combined hydrothermal and ball milling pretreatments were evaluated by chemical composition changes by using FT-IR, WAXD and morphological alterations by SEM. The successful of pretreatments were assessed by the degree of enzymatic digestibility of treated samples. The highest xylose and glucose yields obtained were 63.2% and 97.3% respectively at cellulase loadings of 10 FPU/g-substrate which is the highest conversion from OPMF ever reported. PMID:25058299

  15. Ultrasound-assisted enzymatic hydrolysis for iodinated amino acid extraction from edible seaweed before reversed-phase high performance liquid chromatography-inductively coupled plasma-mass spectrometry.

    PubMed

    Romarís-Hortas, Vanessa; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

    2013-09-27

    The combination of reverse phase high performance liquid chromatography (RP-HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) was used for the determination of monoiodotyrosine (MIT) and diiodotyrosine (DIT) in edible seaweed. A sample pre-treatment based on ultrasound assisted enzymatic hydrolysis was optimized for the extraction of these iodinated amino acids. Pancreatin was selected as the most adequate type of enzyme, and parameters affecting the extraction efficiency (pH, temperature, mass of enzyme and extraction time) were evaluated by univariate approaches. In addition, extractable inorganic iodine (iodide) was also quantified by anion exchange high performance liquid chromatography (AE-HPLC) coupled with ICP-MS. The proposed procedure offered limits of detection of 1.1 and 4.3ngg(-1) for MIT and DIT, respectively. Total iodine contents in seaweed, as well as total iodine in enzymatic digests were measured by ICP-MS after microwave assisted alkaline digestion with tetramethylamonium hydroxide (TMAH) for total iodine assessment, and also by treating the pancreatin extracts (extractable total iodine assessment). The optimized procedure was successfully applied to five different types of edible seaweed. The highest total iodine content, and also the highest iodide levels, was found in the brown seaweed Kombu (6646±45μgg(-1)). Regarding iodinated amino acids, Nori (a red seaweed) was by far the one with the highest amount of both species (42±3 and 0.41±0.024μgg(-1) for MIT and DIT, respectively). In general, MIT concentrations were much higher than the amounts of DIT, which suggests that iodine from iodinated proteins in seaweed is most likely bound in the form of MIT residues. PMID:23972456

  16. Comparative Study of Corn Stover Pretreated by Dilute Acid and Cellulose Solvent-Based Lignocellulose Fractionation: Enzymatic Hydrolysis, Supramolecular Structure, and Substrate Accessibility

    SciTech Connect

    Zhu, Z.; Sathitsuksanoh, N.; Vinzant, T.; Schell, D. J.; McMillian, J. D.; Zhang, Y. H. P.

    2009-07-01

    Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF-pretreated corn stover but reached only {approx}60% for the DA-pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were measured for the first time. The COSLIF-pretreated corn stover had a CAC of 11.57 m{sup 2}/g, nearly twice that of the DA-pretreated biomass (5.89 m{sup 2}/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA- and COSLIF-pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility.

  17. Determination and stability of divicine and isouramil produced by enzymatic hydrolysis of vicine and convicine of faba bean.

    PubMed

    Pulkkinen, Marjo; Zhou, Xiao; Lampi, Anna-Maija; Piironen, Vieno

    2016-12-01

    The aglycones of vicine and convicine, divicine and isouramil, are the causative agents of favism and, therefore, should be analysed along with vicine and convicine in research seeking to eliminate them. This study investigated the stability of the aglycones produced by hydrolysis with β-glucosidase. Reversed-phase, high-performance liquid chromatography (HPLC) with UV detection was shown to be able to observe both aglycone formation and further reactions in isolated fractions and extract made from faba bean and in faba bean suspension. Divicine and isouramil were unstable and degraded almost completely in extract in 60min and completely in fractions in 120min at a pH of 5 at 37°C. Adding sodium ascorbate delayed degradation of divicine. Divicine was more stable at 20°C than at 37°C. Being able to show formation and degradation of the aglycones, the proposed method allows monitoring of the vicine and convicine detoxification process. PMID:27374500

  18. Enhancement of enzymatic hydrolysis and Klason lignin removal of corn stover using photocatalyst-assisted ammonia pretreatment.

    PubMed

    Yoo, Chang Geun; Wang, Chao; Yu, Chenxu; Kim, Tae Hyun

    2013-03-01

    Photocatalyst-assisted ammonia pretreatment was explored to improve lignin removal of the lignocellulosic biomass for effective sugar conversion. Corn stover was treated with 5.0-12.5 wt.% ammonium hydroxide, two different photocatalysts (TiO(2) and ZnO) in the presence of molecular oxygen in a batch reactor at 60 °C. Various solid-to-liquid ratios (1:20-1:50) were also tested. Ammonia pretreatment assisted by TiO(2)-catalyzed photo-degradation removed 70 % of Klason lignin under the optimum condition (12.5 % ammonium hydroxide, 60 °C, 24 h, solid/liquid=1:20, photocatalyst/biomass=1:10 with oxygen atmosphere). The enzymatic digestibilities of pretreated corn stover were 85 % for glucan and 75 % for xylan with NH(3)-TiO(2)-treated solid and 82 % for glucan and 77 % for xylan with NH(3)-ZnO-treated solid with 15 filter paper units/g-glucan of cellulase and 30 cellobiase units/g-glucan of β-glucosidase, a 2-13 % improvement over ammonia pretreatment alone. PMID:23329141

  19. Process intensification of delignification and enzymatic hydrolysis of delignified cellulosic biomass using various process intensification techniques including cavitation.

    PubMed

    Nagula, Karuna Narsappa; Pandit, Aniruddha Bhalchandra

    2016-08-01

    Different methods of pretreatment including alkali treatment, treatment with ultrasound, biological treatment using laccase enzyme and combined treatment like ultrasound-laccase for Napier grass have been tried. With alkali pretreatment optimized conditions obtained were sodium hydroxide 0.3% w/v giving 86% delignification at temperature of 80°C, treatment time of 2h. In physical methods of treatment ultrasound, at a temperature of 45°C, treatment time of 2h, operating at frequency 24kHz and power of 100W gave 18% delignification. For laccase pretreatment, optimized conditions obtained were 300rpm impeller speed, enzyme concentration 10U/gm of Napier grass gave 50% delignification with cellulose. The optimized conditions for delignification by using combination treatment of ultrasound & enzymatic were obtained at 24kHz frequency, 100W giving 75% of delignification in 6h. An enhancement in lignin degradation by 25% and reduction in the treatment time from 12 to 6h is achieved as compared to only laccase treatment. PMID:27090406

  20. Fungal treatment followed by FeCl3 treatment to enhance enzymatic hydrolysis of poplar wood for high sugar yields.

    PubMed

    Wang, Wei; Yuan, Tong Qi; Cui, Bao Kai

    2013-12-01

    Fungal treatment followed by FeCl3 treatment was used to improve saccharification of wood from Populus tomentosa. Combined treatments accumulated lignin and slightly degraded cellulose, whereas almost all hemicelluloses were removed. The white rot fungus, Trametes orientalis, and the brown rot fungus, Fomitopsis palustris, both accompanied by FeCl3 post-treatment resulted in 98.8 and 99.7 % of hemicelluloses loss at 180 °C, respectively, which were over twice than that of hot water pretreatment at the same level. In addition, the solid residue from the T. orientalis-assisted and F. palustris-assisted FeCl3 treatment at 180 °C released 84.5 and 95.4 % of reducing sugars, respectively: 1.4- and 1.6-fold higher than that of FeCl3 treatment alone at the same temperature. Combined treatments disrupted the intact cell structure and increased accessible surface area of cellulose therefore enhancing the enzymatic digestibility, as evidenced by XRD and SEM analysis data. PMID:23907674

  1. Optimization of extraction efficiency by shear emulsifying assisted enzymatic hydrolysis and functional properties of dietary fiber from deoiled cumin (Cuminum cyminum L.).

    PubMed

    Ma, Mengmei; Mu, Taihua; Sun, Hongnan; Zhang, Miao; Chen, Jingwang; Yan, Zhibin

    2015-07-15

    This study evaluated the optimal conditions for extracting dietary fiber (DF) from deoiled cumin by shear emulsifying assisted enzymatic hydrolysis (SEAEH) using the response surface methodology. Fat adsorption capacity (FAC), glucose adsorption capacity (GAC), and bile acid retardation index (BRI) were measured to evaluate the functional properties of the extracted DF. The results revealed that the optimal extraction conditions included an enzyme to substrate ratio of 4.5%, a reaction temperature of 57 °C, a pH value of 7.7, and a reaction time of 155 min. Under these conditions, DF extraction efficiency and total dietary fiber content were 95.12% and 84.18%, respectively. The major components of deoiled cumin DF were hemicellulose (37.25%) and cellulose (33.40%). FAC and GAC increased with decreasing DF particle size (51-100 μm), but decreased with DF particle sizes <26 μm; BRI increased with decreasing DF particle size. The results revealed that SEAEH is an effective method for extracting DF. DF with particle size 26-51 μm had improved functional properties. PMID:25722165

  2. Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis

    PubMed Central

    2012-01-01

    Background Cellulose amorphogenesis, described as the non-hydrolytic “opening up” or disruption of a cellulosic substrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosic biomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play a major role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of this process. Results In this work, techniques for measuring changes in cellulose accessibility are reviewed and a new quantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to cellulose accessibility were successfully quantified using this technique. Conclusions The adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick, accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs used for adsorption studies to include those specific for such compounds as xylan or mannan should also allow for the accurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomass matrix. PMID:22828270

  3. Data on synthesis of oligomeric and polymeric poly(butylene adipate-co-butylene terephthalate) model substrates for the investigation of enzymatic hydrolysis

    PubMed Central

    Perz, Veronika; Bleymaier, Klaus; Sinkel, Carsten; Kueper, Ulf; Bonnekessel, Melanie; Ribitsch, Doris; Guebitz, Georg M.

    2016-01-01

    The aliphatic-aromatic copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT), also known as ecoflex, contains adipic acid, 1,4-butanediol and terephthalic acid and is proven to be compostable [1], [2], [3]). We describe here data for the synthesis and analysis of poly(butylene adipate-co-butylene terephthalate variants with different adipic acid:terephatalic acid ratios and 6 oligomeric PBAT model substrates. Data for the synthesis of the following oligomeric model substrates are described: mono(4-hydroxybutyl) terephthalate (BTa), bis(4-(hexanoyloxy)butyl) terephthalate (HaBTaBHa), bis(4-(decanoyloxy)butyl) terephthalate (DaBTaBDa), bis(4-(tetradecanoyloxy)butyl) terephthalate (TdaBTaBTda), bis(4-hydroxyhexyl) terephthalate (HTaH) and bis(4-(benzoyloxy)butyl) terephthalate (BaBTaBBa). Polymeric PBAT variants were synthesized with adipic acid:terephatalic acid ratios of 100:0, 90:10, 80:20, 70:30, 60:40 and 50:50. These polymeric and oligomeric substances were used as ecoflex model substrates in enzymatic hydrolysis experiments in the article “Substrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substrates” [4]. PMID:26981550

  4. NREL 2012 Achievement of Ethanol Cost Targets: Biochemical Ethanol Fermentation via Dilute-Acid Pretreatment and Enzymatic Hydrolysis of Corn Stover

    SciTech Connect

    Tao, L.; Schell, D.; Davis, R.; Tan, E.; Elander, R.; Bratis, A.

    2014-04-01

    For the DOE Bioenergy Technologies Office, the annual State of Technology (SOT) assessment is an essential activity for quantifying the benefits of biochemical platform research. This assessment has historically allowed the impact of research progress achieved through targeted Bioenergy Technologies Office funding to be quantified in terms of economic improvements within the context of a fully integrated cellulosic ethanol production process. As such, progress toward the ultimate 2012 goal of demonstrating cost-competitive cellulosic ethanol technology can be tracked. With an assumed feedstock cost for corn stover of $58.50/ton this target has historically been set at $1.41/gal ethanol for conversion costs only (exclusive of feedstock) and $2.15/gal total production cost (inclusive of feedstock) or minimum ethanol selling price (MESP). This year, fully integrated cellulosic ethanol production data generated by National Renewable Energy Laboratory (NREL) researchers in their Integrated Biorefinery Research Facility (IBRF) successfully demonstrated performance commensurate with both the FY 2012 SOT MESP target of $2.15/gal (2007$, $58.50/ton feedstock cost) and the conversion target of $1.41/gal through core research and process improvements in pretreatment, enzymatic hydrolysis, and fermentation.

  5. Data on synthesis of oligomeric and polymeric poly(butylene adipate-co-butylene terephthalate) model substrates for the investigation of enzymatic hydrolysis.

    PubMed

    Perz, Veronika; Bleymaier, Klaus; Sinkel, Carsten; Kueper, Ulf; Bonnekessel, Melanie; Ribitsch, Doris; Guebitz, Georg M

    2016-06-01

    The aliphatic-aromatic copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT), also known as ecoflex, contains adipic acid, 1,4-butanediol and terephthalic acid and is proven to be compostable [1], [2], [3]). We describe here data for the synthesis and analysis of poly(butylene adipate-co-butylene terephthalate variants with different adipic acid:terephatalic acid ratios and 6 oligomeric PBAT model substrates. Data for the synthesis of the following oligomeric model substrates are described: mono(4-hydroxybutyl) terephthalate (BTa), bis(4-(hexanoyloxy)butyl) terephthalate (HaBTaBHa), bis(4-(decanoyloxy)butyl) terephthalate (DaBTaBDa), bis(4-(tetradecanoyloxy)butyl) terephthalate (TdaBTaBTda), bis(4-hydroxyhexyl) terephthalate (HTaH) and bis(4-(benzoyloxy)butyl) terephthalate (BaBTaBBa). Polymeric PBAT variants were synthesized with adipic acid:terephatalic acid ratios of 100:0, 90:10, 80:20, 70:30, 60:40 and 50:50. These polymeric and oligomeric substances were used as ecoflex model substrates in enzymatic hydrolysis experiments in the article "Substrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substrates" [4]. PMID:26981550

  6. Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process.

    PubMed

    Welderufael, Fisseha Tesfay; Gibson, Trevor; Methven, Lisa; Jauregi, Paula

    2012-10-15

    The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of β-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC(50)=287 μg/mL) and (ii) from β-lactoglobulin (IC(50)=128 μg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC(50)). PMID:23442643

  7. DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g L-1) during enzymatic hydrolysis and high ethanol concentrations (>10% v/v) during fermentation without hydrolysate purification or concentration

    DOE PAGESBeta

    Chen, Xiaowen; Kuhn, Erik; Jennings, Edward W.; Nelson, Robert; Tao, Ling; Zhang, Min; Tucker, Melvin P.

    2016-04-01

    Distilling and purifying ethanol and other products from second generation lignocellulosic biorefineries adds significant capital and operating costs to biofuel production. The energy usage associated with distillation negatively affects plant gate costs and causes environmental and life-cycle impacts, and the lower titers in fermentation caused by lower sugar concentrations from pretreatment and enzymatic hydrolysis increase energy and water usage and ethanol production costs. In addition, lower ethanol titers increase the volumes required for enzymatic hydrolysis and fermentation vessels increase capital expenditure (CAPEX). Therefore, increasing biofuel titers has been a research focus in renewable biofuel production for several decades. In thismore » work, we achieved approximately 230 g L-1 of monomeric sugars after high solid enzymatic hydrolysis using deacetylation and mechanical refining (DMR) processed corn stover substrates produced at the 100 kg per day scale. The high sugar concentrations and low chemical inhibitor concentrations achieved by the DMR process allowed fermentation to ethanol with titers as high as 86 g L-1, which translates into approximately 10.9% v/v ethanol. To our knowledge, this is the first time that titers greater than 10% v/v ethanol in fermentations derived from corn stover without any sugar concentration or purification steps have been reported. As a result, the potential cost savings from high sugar and ethanol titers achieved by the DMR process are also reported using TEA analysis.« less

  8. Coupling alkaline pre-extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic hydrolysis and fermentability

    PubMed Central

    2014-01-01

    Background A two-stage chemical pretreatment of corn stover is investigated comprising an NaOH pre-extraction followed by an alkaline hydrogen peroxide (AHP) post-treatment. We propose that conventional one-stage AHP pretreatment can be improved using alkaline pre-extraction, which requires significantly less H2O2 and NaOH. To better understand the potential of this approach, this study investigates several components of this process including alkaline pre-extraction, alkaline and alkaline-oxidative post-treatment, fermentation, and the composition of alkali extracts. Results Mild NaOH pre-extraction of corn stover uses less than 0.1 g NaOH per g corn stover at 80°C. The resulting substrates were highly digestible by cellulolytic enzymes at relatively low enzyme loadings and had a strong susceptibility to drying-induced hydrolysis yield losses. Alkaline pre-extraction was highly selective for lignin removal over xylan removal; xylan removal was relatively minimal (~20%). During alkaline pre-extraction, up to 0.10 g of alkali was consumed per g of corn stover. AHP post-treatment at low oxidant loading (25 mg H2O2 per g pre-extracted biomass) increased glucose hydrolysis yields by 5%, which approached near-theoretical yields. ELISA screening of alkali pre-extraction liquors and the AHP post-treatment liquors demonstrated that xyloglucan and β-glucans likely remained tightly bound in the biomass whereas the majority of the soluble polymeric xylans were glucurono (arabino) xylans and potentially homoxylans. Pectic polysaccharides were depleted in the AHP post-treatment liquor relative to the alkaline pre-extraction liquor. Because the already-low inhibitor content was further decreased in the alkaline pre-extraction, the hydrolysates generated by this two-stage pretreatment were highly fermentable by Saccharomyces cerevisiae strains that were metabolically engineered and evolved for xylose fermentation. Conclusions This work demonstrates that this two

  9. Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose

    PubMed Central

    2013-01-01

    Background The complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. But endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation. Results The glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum β-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in V max /K m in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188. Conclusions The hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the

  10. Natural deep eutectic solvent mediated pretreatment of rice straw: bioanalytical characterization of lignin extract and enzymatic hydrolysis of pretreated biomass residue.

    PubMed

    Kumar, Adepu K; Parikh, Bhumika S; Pravakar, Mohanty

    2016-05-01

    The present investigation demonstrated pretreatment of lignocellulosic biomass rice straw using natural deep eutectic solvents (NADESs), and separation of high-quality lignin and holocellulose in a single step. Qualitative analysis of the NADES extract showed that the extracted lignin was of high purity (>90 %), and quantitative analysis showed that nearly 60 ± 5 % (w/w) of total lignin was separated from the lignocellulosic biomass. Addition of 5.0 % (v/v) water during pretreatment significantly enhanced the total lignin extraction, and nearly 22 ± 3 % more lignin was released from the residual biomass into the NADES extract. X-ray diffraction studies of the untreated and pretreated rice straw biomass showed that the crystallinity index ratio was marginally decreased from 46.4 to 44.3 %, indicating subtle structural alterations in the crystalline and amorphous regions of the cellulosic fractions. Thermogravimetric analysis of the pretreated biomass residue revealed a slightly higher T dcp (295 °C) compared to the T dcp (285 °C) of untreated biomass. Among the tested NADES reagents, lactic acid/choline chloride at molar ratio of 5:1 extracted maximum lignin of 68 ± 4 mg g(-1) from the rice straw biomass, and subsequent enzymatic hydrolysis of the residual holocellulose enriched biomass showed maximum reducing sugars of 333 ± 11 mg g(-1) with a saccharification efficiency of 36.0 ± 3.2 % in 24 h at 10 % solids loading. PMID:26032452

  11. Occurrence of bound 3-monochloropropan-1,2-diol content in commonly consumed foods in Hong Kong analysed by enzymatic hydrolysis and GC-MS detection.

    PubMed

    Chung, Stephen W C; Chan, Benny T P; Chung, H Y; Xiao, Ying; Ho, Y Y

    2013-01-01

    The aim of this study was to determine the level of bound 3-monochloropropan-1,2-diol in foodstuffs commonly consumed in Hong Kong, China, by an enzymatic hydrolysis indirect method which proved to be free from interferences. A total of 290 samples were picked up randomly from the local market and analysed. About 73% of these samples were found to contain detectable amounts of bound 3-MCPD. Amongst the 73 food items, bound 3-MCPD was not detected in 13 food items, including extra virgin olive oil, beef ball/salami, beef flank, ham/Chinese ham, nuts, seeds, soy sauce, oyster sauce, butter, yoghurt, cream, cheese and milk. For those found to contain detectable bound 3-MCPD, the content ranged up to 2500 µg kg(-1). The highest mean bound 3-MCPD content among the 14 food groups was in biscuits (440 [50-860] µg kg(-1)), followed by fats and oils (390 [n.d.-2500] µg kg(-1)), snacks (270 [9-1000] µg kg(-1)), and Chinese pastry (270 [n.d.-1200] µg kg(-1)). Among the samples, the highest bound 3-MCPD content was in a grape seed oil (2500 µg kg(-1)), followed by a walnut flaky pastry (1200 µg kg(-1)) and a grilled corn (1000 µg kg(-1)). Basically, the results of this study agreed well with other published results in peer-reviewed journals, except for cheese, cream, ham, nuts and seeds. PMID:23819821

  12. Effect of feeding lipids recovered from fish processing waste by lactic acid fermentation and enzymatic hydrolysis on antioxidant and membrane bound enzymes in rats.

    PubMed

    Rai, Amit Kumar; Bhaskar, N; Baskaran, V

    2015-06-01

    Fish oil recovered from fresh water fish visceral waste (FVW-FO) through lactic acid fermentation (FO-LAF) and enzymatic hydrolysis (FO-EH) were fed to rats to study their influence on lipid peroxidation and activities of antioxidant and membrane bound enzyme in liver, heart and brain. Feeding of FO-LAF and FO-EH resulted in increase (P < 0.05) in lipid peroxides level in serum, liver, brain and heart tissues compared to ground nut oil (control). Activity of catalase (40-235 %) and superoxide dismutase (17-143 %) also increased (P < 0.05) with incremental level of EPA + DHA in diet. The increase was similar to cod liver oil fed rats at same concentration of EPA + DHA. FO-LAF and FO-EH increased (P < 0.05) the Na(+)K(+) ATPase activity in liver and brain microsomes, Ca(+)Mg(+) ATPase in heart microsome and acetylcholine esterase in brain microsomes when fed with 5 % EPA + DHA. There was also significant change in fatty acid composition and cholesterol/phospholipid ratio in microsomes of rat fed with FVW-FO. Feeding FVW-FO recovered by biotechnological approaches enhanced the activity of antioxidant enzymes in tissues, modulates the activities of membrane bound enzymes and improved the fatty acid composition in microsomes of tissues similar to CLO. Utilization of these processing wastes for the production of valuable biofunctional products can reduce the mounting economic values of fish oil and minimize the environmental pollution problems. PMID:26028754

  13. Isomerization of 1-O-indol-3-ylacetyl-beta-D-glucose. Enzymatic hydrolysis of 1-O, 4-O, and 6-O-indol-3-ylacetyl-beta-D-glucose and the enzymatic synthesis of indole-3-acetyl glycerol by a hormone metabolizing complex

    NASA Technical Reports Server (NTRS)

    Kowalczyk, S.; Bandurski, R. S.

    1990-01-01

    The first compound in the series of reactions leading to the ester conjugates of indole-3-acetic acid (IAA) in kernels of Zea mays sweet corn is the acyl alkyl acetal, 1-O-indol-3-ylacetyl-beta-D-glucose (1-O-IAGlu). The enzyme catalyzing the synthesis of this compound is UDP-glucose:indol-3-ylacetate glucosyl-transferase (IAGlu synthase). The IAA moiety of the high energy compound 1-O-IAGlu may be enzymatically transferred to myo-inositol or to glycerol or the 1-O-IAGlu may be enzymatically hydrolyzed. Alternatively, nonenzymatic acyl migration may occur to yield the 2-O, 4-O, and 6-O esters of IAA and glucose. The 4-O and 6-O esters may then be enzymatically hydrolyzed to yield free IAA and glucose. This work reports new enzymatic activities, the transfer of IAA from 1-O-IAGlu to glycerol, and the enzyme-catalyzed hydrolysis of 4-O and 6-O-IAGlu. Data is also presented on the rate of non-enzymatic acyl migration of IAA from the 1-O to the 4-O and 6-O positions of glucose. We also report that enzymes catalyzing the synthesis of 1-O-IAGlu and the hydrolysis of 1-O, 4-O, and 6-O-IAGlu fractionate as a hormone metabolizing complex. The association of synthetic and hydrolytic capabilities in enzymes which cofractionate may have physiological significance.

  14. Enzymatic hydrolysis studies of arabinogalactan-protein structure from Acacia gum: the self-similarity hypothesis of assembly from a common building block.

    PubMed

    Renard, D; Lavenant-Gourgeon, L; Lapp, A; Nigen, M; Sanchez, C

    2014-11-01

    Arabinogalactan (AG) and arabinogalactan-protein (AGP) fractions were treated enzymatically using several proteases in acidic (pH 4) and alkaline (pH 7) conditions in order to go deeper insight into the structure and conformations of the two main fractions of Acacia senegal gum. Endoproteinase Glu-C, pepsin and phosphatase acid were thus used in acidic conditions while subtilisin A, pronase, trypsin, papain and proteinase K were used in alkaline conditions to cleave protein moieties of the two fractions. Structures of AG and AGP were probed using HPSEC-MALLS, small angle neutron scattering and far-UV circular dichroism. Enzymes did not affect AG fraction structure whatever the pH conditions used, highlighting the inaccessibility of the peptide backbone and the remarkable stability of this fraction in acidic and alkaline conditions. This result was in agreement with the thin oblate ellipsoid model we previously identified for the AG fraction where the 43 amino-acid residues peptide sequence was supposed, based on spectroscopic methods, to be totally buried. Contrary to AG fraction, AGP protein component was therefore cleaved using enzymes in alkaline conditions, the absence of enzymatic efficiency in acidic conditions being probably ascribed to long range electrostatic repulsions occurring between negatively charged AGP and enzymes at pH 4. The decrease of AGP molecular weight after hydrolysis in alkaline conditions went from 1.79 × 10(6) g mol(-1) for control AGP to as low as 1.68 × 10(5) g mol(-1) for papain-treated AGP. The overall structure of the enzyme-treated AGPs was found to be surprisingly quite similar whatever the enzyme used and close, with however some subtle differences, to AG unit. A tri-axial ellipsoid conformation was found in enzyme-treated AGPs and the two main preferential distances identified in the pair distance distribution function would claim in favor of rod-like or elongated particles or alternatively would indicate the presence of two

  15. Enzymatic hydrolysis of wheat arabinoxylan by a recombinant "minimal" enzyme cocktail containing beta-xylosidase and novel endo-1,4-beta-xylanase and alpha-l-arabinofuranosidase activities.

    PubMed

    Sørensen, Hanne R; Pedersen, Sven; Jørgensen, Christel T; Meyer, Anne S

    2007-01-01

    This study describes the identification of the key enzyme activities required in a "minimal" enzyme cocktail able to catalyze hydrolysis of water-soluble and water-insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat. The optimal arabinose-releasing and xylan-depolymerizing enzyme activities were identified from data obtained when selected, recombinant enzymes were systematically supplemented to the different arabinoxylan substrates in mixtures; this examination revealed three novel alpha-l-arabinofuranosidase activities: (i) one GH51 enzyme from Meripilus giganteus and (ii) one GH51 enzyme from Humicola insolens, both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from H. insolens able to catalyze the release of arabinose from doubly substituted xylose. Treatment of water-soluble and water-insoluble wheat arabinoxylan with an enzyme cocktail containing a 20%:20%:20%:40% mixture and a 25%:25%:25%:25% mixture, respectively, of the GH43 alpha-l-arabinofuranosidase from H. insolens (Abf II), the GH51 alpha-l-arabinofuranosidase from M. giganteus (Abf III), a GH10 endo-1,4-beta-xylanase from H. insolens (Xyl III), and a GH3 beta-xylosidase from Trichoderma reesei (beta-xyl) released 322 mg of arabinose and 512 mg of xylose per gram of water-soluble wheat arabinoxylan dry matter and 150 mg of arabinose and 266 mg of xylose per gram of water-insoluble wheat arabinoxylan dry matter after 24 h at pH 5, 50 degrees C. A 10%:40%:50% mixture of Abf II, Abf III, and beta-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 degrees C. The optimal dosages of the "minimal" enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme protein per kilogram of substrate dry matter for the water-soluble wheat arabinoxylan, the water-insoluble wheat arabinoxylan, and the vinasse, respectively. These enzyme

  16. Validation of a novel sequential cultivation method for the production of enzymatic cocktails from Trichoderma strains.

    PubMed

    Florencio, C; Cunha, F M; Badino, A C; Farinas, C S

    2015-02-01

    The development of new cost-effective bioprocesses for the production of cellulolytic enzymes is needed in order to ensure that the conversion of biomass becomes economically viable. The aim of this study was to determine whether a novel sequential solid-state and submerged fermentation method (SF) could be validated for different strains of the Trichoderma genus. Cultivation of the Trichoderma reesei Rut-C30 reference strain under SF using sugarcane bagasse as substrate was shown to be favorable for endoglucanase (EGase) production, resulting in up to 4.2-fold improvement compared with conventional submerged fermentation. Characterization of the enzymes in terms of the optimum pH and temperature for EGase activity and comparison of the hydrolysis profiles obtained using a synthetic substrate did not reveal any qualitative differences among the different cultivation conditions investigated. However, the thermostability of the EGase was influenced by the type of carbon source and cultivation system. All three strains of Trichoderma tested (T. reesei Rut-C30, Trichoderma harzianum, and Trichoderma sp INPA 666) achieved higher enzymatic productivity when cultivated under SF, hence validating the proposed SF method for use with different Trichoderma strains. The results suggest that this bioprocess configuration is a very promising development for the cellulosic biofuels industry. PMID:25399068

  17. A cellular automaton model of crystalline cellulose hydrolysis by cellulases

    PubMed Central

    2011-01-01

    Background Cellulose from plant biomass is an abundant, renewable material which could be a major feedstock for low emissions transport fuels such as cellulosic ethanol. Cellulase enzymes that break down cellulose into fermentable sugars are composed of different types - cellobiohydrolases I and II, endoglucanase and β-glucosidase - with separate functions. They form a complex interacting network between themselves, soluble hydrolysis product molecules, solution and solid phase substrates and inhibitors. There have been many models proposed for enzymatic saccharification however none have yet employed a cellular automaton approach, which allows important phenomena, such as enzyme crowding on the surface of solid substrates, denaturation and substrate inhibition, to be considered in the model. Results The Cellulase 4D model was developed de novo taking into account the size and composition of the substrate and surface-acting enzymes were ascribed behaviors based on their movements, catalytic activities and rates, affinity for, and potential for crowding of, the cellulose surface, substrates and inhibitors, and denaturation rates. A basic case modeled on literature-derived parameters obtained from Trichoderma reesei cellulases resulted in cellulose hydrolysis curves that closely matched curves obtained from published experimental data. Scenarios were tested in the model, which included variation of enzyme loadings, adsorption strengths of surface acting enzymes and reaction periods, and the effect on saccharide production over time was assessed. The model simulations indicated an optimal enzyme loading of between 0.5 and 2 of the base case concentrations where a balance was obtained between enzyme crowding on the cellulose crystal, and that the affinities of enzymes for the cellulose surface had a large effect on cellulose hydrolysis. In addition, improvements to the cellobiohydrolase I activity period substantially improved overall glucose production. Conclusions

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

    PubMed Central

    Khokhar, Zia-ullah; Syed, Qurat-ul-Ain; Wu, Jing; Athar, Muhammad Amin

    2014-01-01

    Bioethanol production from lignocellulosic raw materials involves process steps like pre-treatment, enzymatic hydrolysis, fermentation and distillation. In this study, wheat straw was explored as feedstock for on-site cellulase production by T. reesei 3EMS35 mutant, and as a substrate for second generation bioethanol production from baker yeast. Scanning electron microscopy (SEM) and X-ray diffractography (XRD) of untreated wheat straw (UWS) and acid treated wheat straw (TWS) were done to understand the structural organization and changes in the cellulase accessibility and reactivity. The effect of delignification and structural modification for on-site cellulase enzyme production was comparably studied. The efficiency of crude cellulase enzyme for digestion of UWS and TWS and then production of ethanol from TWS was studied using same-vessel saccharification and fermentation (SVSF) technique, both in shaking flasks as well as in fermenters. Two different methods of operation were tested, i.e. the UWSEnz method, where UWS was used for on-site enzyme production, and TWSEnz method where TWS was applied as substrate for cellullase production. Results obtained showed structural modifications in cellulose of TWS due to delignification, removal of wax and change of crystallinity. UWS was better substrate than TWS for cellulase production due to the fact that lignin did not hinder the enzyme production by fungus but acted as a booster. On-site cellulase enzyme produced by T. reesei 3EMS35 mutant hydrolyzed most of cellulose (91 %) in TWS within first 24 hrs. Shake flasks experiments showed that ethanol titers and yields with UWSEnz were 2.9 times higher compared to those obtained with TWSEnz method respectively. Comparatively, titer of ethanol in shake flask experiments was 10 % higher than this obtained in 3 L fermenter with UWSEnz. Outcomes from this investigation clearly demonstrated the potential of on-site cellulase enzyme production and SVSF for ethanol production

  19. A green and efficient technology for the degradation of cellulosic materials: structure changes and enhanced enzymatic hydrolysis of natural cellulose pretreated by synergistic interaction of mechanical activation and metal salt.

    PubMed

    Zhang, Yanjuan; Li, Qian; Su, Jianmei; Lin, Ye; Huang, Zuqiang; Lu, Yinghua; Sun, Guosong; Yang, Mei; Huang, Aimin; Hu, Huayu; Zhu, Yuanqin

    2015-02-01

    A new technology for the pretreatment of natural cellulose was developed, which combined mechanical activation (MA) and metal salt treatments in a stirring ball mill. Different valent metal nitrates were used to investigate the changes in degree of polymerization (DP) and crystallinity index (CrI) of cellulose after MA+metal salt (MAMS) pretreatment, and Al(NO3)3 showed better pretreatment effect than NaNO3 and Zn(NO3)2. The destruction of morphological structure of cellulose was mainly resulted from intense ball milling, and the comparative studies on the changes of DP and crystal structure of MA and MA+Al(NO3)3 pretreated cellulose samples showed a synergistic interaction of MA and Al(NO3)3 treatments with more effective changes of structural characteristics of MA+Al(NO3)3 pretreated cellulose and substantial increase of reducing sugar yield in enzymatic hydrolysis of cellulose. In addition, the results indicated that the presence of Al(NO3)3 had significant enhancement for the enzymatic hydrolysis of cellulose. PMID:25490099

  20. Secretome data from Trichoderma reesei and Aspergillus niger cultivated in submerged and sequential fermentation methods.

    PubMed

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

    2016-09-01

    The cultivation procedure and the fungal strain applied for enzyme production may influence levels and profile of the proteins produced. The proteomic analysis data presented here provide critical information to compare proteins secreted by Trichoderma reesei and Aspergillus niger when cultivated through submerged and sequential fermentation processes, using steam-explosion sugarcane bagasse as inducer for enzyme production. The proteins were organized according to the families described in CAZy database as cellulases, hemicellulases, proteases/peptidases, cell-wall-protein, lipases, others (catalase, esterase, etc.), glycoside hydrolases families, predicted and hypothetical proteins. Further detailed analysis of this data is provided in "Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation process: enzyme production for sugarcane bagasse hydrolysis" C. Florencio, F.M. Cunha, A.C Badino, C.S. Farinas, E. Ximenes, M.R. Ladisch (2016) [1]. PMID:27419196

  1. Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414

    PubMed Central

    Ng, Thomas K.; Zeikus, J. G.

    1981-01-01

    The crude extracellular cellulase of Clostridium thermocellum LQRI (virgin strain) was very active and solubilized microcrystalline cellulose at one-half the rate observed for the extracellular cellulase of Trichoderma reesei QM9414 (mutant strain). C. thermocellum cellulase activity differed considerably from that of T. reesei as follows: higher endoglucanase/exoglucanase activity ratio; absence of extracellular cellobiase or β-xylosidase activity; long-chain oligosaccharides instead of short-chain oligosaccharides as initial (15-min) hydrolytic products on microcrystalline cellulose; mainly cellobiose or xylobiose as long-term (24-h) hydrolysis products of Avicel and MN300 or xylan; and high activity and stability at 60 to 70°C. Under optimized reaction conditions, the kinetic properties (Vmax, 0.4 μmol/min per mg of protein; energy of activation, 33 kJ; temperature coefficient, 1.8) of C. thermocellum cellulose-solubilizing activity were comparable to those reported for T. reesei, except that the dyed Avicel concentration at half-maximal velocity was twofold higher (182 μM). The cellulose-solubilizing activity of the two crude cellulases differed considerably in response to various enzyme inhibitors. Most notably, Ag2+ and Hg2+ effectively inhibited C. thermocellum but not T. reesei cellulase at <20 μM, whereas Ca2+, Mg2+, and Mn2+ inhibited T. reesei but not C. thermocellum cellulase at >10 mM. Both enzymes were inhibited by Cu2+ (>20 mM), Zn2+ (>1.0 mM), and ethylene glycol-bis(β-aminoethyl ether)- N,N-tetraacetic acid (>10 mM). T. reesei but not C. thermocellum cellulose-solubilizing activity was 20% inhibited by glucose (73 mM) and cellobiose (29 mM). Both cellulases preferentially cleaved the internal glycosidic bonds of cellooligosaccharides. The overall rates of cellooligosaccharide degradation were higher for T. reesei than for C. thermocellum cellulase, except that the rates of conversion of cellohexaose to cellotriose were equivalent. Images PMID

  2. Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering

    PubMed Central

    2012-01-01

    increased by 40% at 55°C and 169% at 60°C when its enzyme mixture was used in the hydrolysis. Conclusions This study shows that optimizations of the promoter and linker for hybrid genes can dramatically improve the efficiency of heterologous expression of cellulase genes in T. reesei. PMID:22314137

  3. Biomimetic Approach to Enhance Enzymatic Hydrolysis of the Synthetic Polyester Poly(1,4-butylene adipate): Fusing Binding Modules to Esterases.

    PubMed

    Perz, Veronika; Zumstein, Michael Thomas; Sander, Michael; Zitzenbacher, Sabine; Ribitsch, Doris; Guebitz, Georg M

    2015-12-14

    Mimicking a concept of nature for the hydrolysis of biopolymers, the Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) was fused to a polymer binding module (PBM) to enhance the hydrolysis of the polyester poly(1,4-butylene adipate) (PBA). Namely, the binding module of a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (Thc_Cut1_PBM) was attached to the cutinase via two different linker sequences varying in length. In order to investigate the adsorption behavior, catalytically inactive mutants both of Thc_Cut1 and Thc_Cut1_PBM were successfully constructed by site-directed mutagenesis of serine 131 to alanine. Quartz crystal microbalance with dissipation monitoring (QCM-D) analysis revealed that the initial mass increase during enzyme adsorption was larger for the inactive enzymes linked with the PBM as compared to the enzyme without the PBM. The hydrolysis rates of PBA were significantly enhanced when incubated with the active, engineered Thc_Cut1_PBM as compared to the native Thc_Cut1. Thc_Cut1_PBM completely hydrolyzed PBA thin films on QCM-D sensors within approximately 40 min, whereas twice as much time was required for the complete hydrolysis by the native Thc_Cut1. PMID:26566664

  4. A review of the enzymatic hydrolysis of mannans and synergistic interactions between β-mannanase, β-mannosidase and α-galactosidase.

    PubMed

    Malgas, Samkelo; van Dyk, J Susan; Pletschke, Brett I

    2015-08-01

    Mannan is an important polysaccharide found in softwoods and many other plant sources. Mannans from various sources display large differences in composition, structure and complexity. To hydrolyse mannan into its monomer sugars requires a number of enzymes working in synergy. This review examines mannan structure and the enzymes required for its hydrolysis. Several studies have investigated the effect of supplementing β-mannanases with β-mannosidases and α-galactosidases in binary and ternary combinations. Synergistic enhancement of hydrolysis has been found in some, but not all cases. In the case of mannosidases, they sometimes display an anti-synergistic effect with mannanases, most likely due to competition for binding sites. Most importantly, in the case of α-galactosidases, the same enzyme from different families display differences in synergistic interactions due to different specificities. An improved understanding of enzyme interactions will aid in achieving enhanced hydrolysis of mannans and higher sugar yields. This review highlights areas which require further research in order to gain a better understanding of mannan hydrolysis and utilisation. Such knowledge is very important as this can be used in the optimisation of commercial or purified enzyme mixtures to improve the economic viability of the conversion of high mannan-containing biomass such as softwoods into fermentable sugars for bioethanol production. PMID:26026279

  5. Valorization of cruor slaughterhouse by-product by enzymatic hydrolysis for the production of antibacterial peptides: focus on α 1-32 family peptides mechanism and kinetics modeling.

    PubMed

    Hedhili, K; Dimitrov, K; Vauchel, P; Sila, A; Chataigné, G; Dhulster, P; Nedjar, N

    2015-10-01

    Bovine hemoglobin is the major component of the cruor (slaughterhouse by-product) and can be considered as an important source of active peptides that could be obtained by pepsic hydrolysis. The kinetics of appearance and disappearance of several antibacterial peptides from α 1-32 family during hydrolysis of synthesized α 1-32 peptide, of purified bovine hemoglobin and of cruor was studied, and reaction scheme for the hydrolysis of α 1-32 family peptides from these three sources was determined. On this basis, a mathematical model was proposed to predict the concentration of each peptide of interest of this family depending on hydrolysis time, and also on temperature (in the range 15-37 °C), pH (in the range 3.5-5.5) and enzyme to substrate ratio (in the range 1/50-1/200 for the synthesized peptide and 1/5-1/20 for purified bovine hemoglobin and cruor). Apparent rate constants of reactions were determined by applying the model on a set of experimental data and it was shown that they depended on the temperature according to Arrhenius's law, that their dependence on the pH was linear, and that enzyme to substrate ratio influence was limited (in the studied range). PMID:26099509

  6. Comparison of the enzymatic digestibility of physically and chemically pretreated selected line of diploid-Miscanthus sinensis Shiozuka and triploid-M.×giganteus.

    PubMed

    Hideno, Akihiro; Kawashima, Ayato; Anzoua, Kossonou Guillaume; Yamada, Toshihiko

    2013-10-01

    The diploid Miscanthus sinensis "Shiozuka" which was selected as a high-biomass producing line, and the triploid M. × giganteus (M×G) were treated by ball milling (physical treatment) and alkaline hydrogen peroxide treatment (AHP; chemical treatment), and their structural sugar compositions and enzymatic digestibility were compared. The structural sugar content of Shiozuka was moderate and lower than that of M×G. The Klason lignin content of Shiozuka was also lower than that of M×G. However, Shiozuka was sensitive to ball milling and AHP treatment; ball milled and AHP-treated Shiozuka had higher enzymatic digestibility than ball milled and AHP-treated M×G. Shiozuka would be promising feedstock to obtain fermentable sugars with low energy consumption. Finally, enzymes for the hydrolysis of chemically treated Miscanthus were isolated from Trichoderma reesei ATCC 66589 and Penicillium pinophilum. The sugar yield could be increased by enzymatic hydrolysis of AHP-treated samples with NaOH and H2O2 and the isolated enzymes. PMID:23954245

  7. Glucosinolates and derived products in cruciferous vegetables: total glucosinolates by retention on anion exchange resin and enzymatic hydrolysis to measure released glucose.

    PubMed

    VanEtten, C H; Daxenbichler, M E

    1977-07-01

    Details are given for determining total glucosinolates in Cruciferae plants by a procedure measuring released glucose. The glucosinolates are separated from about 90% of other material in the plant extract by adsorption on an anion exchange resin. Then, by a selective thioglucosidase hydrolysis of the glucosinolates retained on the exchange resin, the glucose and aglucons are separated from other substances retained by the resin. Glucose is released into an aqueous medium and is equivalent to the total glucosinolates. The aglucons formed by the hydrolysis are extracted into methylene chloride and determined by gas-liquid chromatography. Based on 29 determinations of the glucose from sinigrin, analyzed under different conditions, accuracy of the total glucosinolate determination was 94.8 +/- 7.3%. The coefficient of variation, determined by duplicate analyses on extracts from 58 cabbage samples, was 4.6%. PMID:893313

  8. Genome sequencing of the Trichoderma reesei QM9136 mutant identifies a truncation of the transcriptional regulator XYR1 as the cause for its cellulase-negative phenotype

    DOE PAGESBeta

    Lichius, Alexander; Bidard, Frédérique; Buchholz, Franziska; Le Crom, Stéphane; Martin, Joel; Schackwitz, Wendy; Austerlitz, Tina; Grigoriev, Igor V; Baker, Scott E; Margeot, Antoine; et al

    2015-04-20

    Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a.

  9. Process Design Report for Wood Feedstock: Lignocellulosic Biomass to Ethanol Process Desing and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis Current and Futuristic Scenarios

    SciTech Connect

    Wooley, Robert; Ruth, Mark; Sheehan, John; Ibsen, Kelly; Majdeski, Henry; Galves, Adrian

    1999-07-01

    The National Renewable Energy Laboratory (NREL) has undertaken a complete review and update of the process design and economic model for the biomass-to-ethanol process based on co-current dilute acid prehydrolysis, along with simultaneous saccharification (enzymatic) and co-fermentation. The process design includes the core technologies being researched by the U.S. Department of Energy (DOE): prehydrolysis, simultaneous saccharification and co-fermentation, and cellulase enzyme production.

  10. Interactions of the Trichoderma reesei rho3 with the secretory pathway in yeast and T. reesei.

    PubMed

    Vasara, T; Salusjärvi, L; Raudaskoski, M; Keränen, S; Penttilä, M; Saloheimo, M

    2001-12-01

    We recently isolated from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) a gene encoding RHOIII as a multicopy suppressor of the yeast temperature-sensitive secretory mutation, sec15-1. To characterize this gene further, we tested its ability to suppress other late-acting secretory mutations. The growth defect of yeast strains with sec1-1, sec1-11, sec3-2, sec6-4 and sec8-9 mutations was suppressed. Expression of rho3 also improved the impaired actin organization of sec15-1 cells at +38 degrees C. Overproduction of yeast Rho3p using the same expression vector as T. reesei RHOIII appeared to be toxic in sec3-101, sec5-24, sec8-9, sec10-2 and sec15-1 cells. When expressed from the GAL1 promoter, RHO3 suppressed the growth defect of sec1 at the restrictive temperature and inhibited the growth of sec3-101 at the permissive temperature. Disruption of the rho3 gene in the T. reesei genome did not affect the hyphal or colony morphology nor the cellular cytoskeleton organization. Furthermore, the growth of T. reesei was not affected on glucose by the rho3 disruption. Instead, both growth and protein secretion of T. reesei in cellulose cultures was remarkably decreased in rho3 disruptant strains when compared with the parental strain. These results suggest that rho3 is involved in secretion processes in T. reesei. PMID:11886564

  11. Improving the enzymatic hydrolysis of dilute acid pretreated wheat straw by metal ion blocking of non-productive cellulase adsorption on lignin.

    PubMed

    Akimkulova, Ardak; Zhou, Yan; Zhao, Xuebing; Liu, Dehua

    2016-05-01

    Eleven salts were selected to screen the possible metal ions for blocking the non-productive adsorption of cellulase onto the lignin of dilute acid pretreated wheat straw. Mg(2+) was screened finally as the promising candidate. The optimal concentration of MgCl2 was 1mM, but the beneficial action was also dependent on pH, hydrolysis time and cellulase loading. Significant improvement of glucan conversion (19.3%) was observed at low cellulase loading (5FPU/g solid). Addition of isolated lignins, tannic acid and lignin model compounds to pure cellulose hydrolysis demonstrated that phenolic hydroxyl group (Ph-OH) was the main active site blocked by Mg(2+). The interaction between Mg(2+) and Ph-OH of lignin monomeric moieties followed an order of p-hydroxyphenyl (H)>guaiacyl (G)>syringyl (S). Mg(2+) blocking made the lignin surface less negatively charged, which might weaken the hydrogen bonding and electrostatically attractive interaction between lignin and cellulase enzymes. PMID:26930032

  12. Simultaneous Cellulase Production, Saccharification and Detoxification Using Dilute Acid Hydrolysate of S. spontaneum with Trichoderma reesei NCIM 992 and Aspergillus niger.

    PubMed

    Sateesh, Lanka; Rodhe, Adivikatla Vimala; Naseeruddin, Shaik; Yadav, Kothagauni Srilekha; Prasad, Yenumulagerard; Rao, Linga Venkateswar

    2012-06-01

    Bioethanol production from lignocellulosic materials has several limitations. One aspect is the high production cost of cellulases used for saccharification of substrate and inhibition of fermenting yeast due to inhibitors released in acid hydrolysis. In the present work we have made an attempt to achieve simultaneous cellulases production, saccharification and detoxification using dilute acid hydrolysate of Saccharum spontaneum with and without addition of nutrients, supplemented with acid hydrolyzed biomass prior to inoculation in one set and after 3 days of inoculation in another set. Organisms used were T. reesei NCIM 992, and Aspergillus niger isolated in our laboratory. Cellulase yield obtained was 0.8 IU/ml on fourth day with T. reesei. Sugars were found to increase from fourth to fifth day, when hydrolysate was supplemented with nutrients and acid hydrolyzed biomass followed by inoculation with T. reesei. Phenolics were also found to decrease by 67%. PMID:23729891

  13. The impact of heating and soaking on the in vitro enzymatic hydrolysis of protein varies in different species of tropical legumes.

    PubMed

    Torres, Julieta; Rutherfurd, Shane M; Muñoz, Luz S; Peters, Michael; Montoya, Carlos A

    2016-03-01

    The effects of different thermal (raw, autoclaving or boiling for 5 and 20min) and soaking (with or without) treatments on the degree of hydrolysis (DH) of protein were investigated for selected legumes (Canavalia brasiliensis; Lablab purpureus; pink, red and white colour hulls Vigna unguiculata). Each legume preparation underwent in vitro simulated gastrointestinal tract digestion comprising either pepsin (120min) or pepsin/pancreatin (120/240min) digestion. The DH was determined based on the amount of free amino groups released. Autoclaving for 5min increased the pepsin/pancreatin DH for all the unsoaked and soaked legumes (+20% to 46% units) except Canavalia, while boiling for 5min only increased DH for two soaked legumes (+12% to 28% units). Extending boiling from 5 to 20min increased the DH for three soaked legumes (+5% to 29% units). In conclusion, autoclaving, in general, extensively increased the sequential pepsin/pancreatin DH, while boiling only increased it for selected legumes. PMID:26471569

  14. Corn stover semi-mechanistic enzymatic hydrolysis model with tight parameter confidence intervals for model-based process design and optimization.

    PubMed

    Scott, Felipe; Li, Muyang; Williams, Daniel L; Conejeros, Raúl; Hodge, David B; Aroca, Germán

    2015-02-01

    Uncertainty associated to the estimated values of the parameters in a model is a key piece of information for decision makers and model users. However, this information is typically not reported or the confidence intervals are too large to be useful. A semi-mechanistic model for the enzymatic saccharification of dilute acid pretreated corn stover is proposed in this work, the model is a modification of an existing one providing a statistically significant improved fit towards a set of experimental data that includes varying initial solid loadings (10-25% w/w) and the use of the pretreatment liquor and washed solids with or without supplementation of key inhibitors. A subset of 8 out of 17 parameters was identified, showing sufficiently tight confidence intervals to be used in uncertainty propagation and model analysis, without requiring interval truncation via expert judgment. PMID:25496946

  15. Pretreatment of bamboo by ultra-high pressure explosion with a high-pressure homogenizer for enzymatic hydrolysis and ethanol fermentation.

    PubMed

    Jiang, Zehui; Fei, Benhua; Li, Zhiqiang

    2016-08-01

    Bamboo shoots, 2- and 5-year-old bamboo were treated by using a homogenizer in a constant suspended state, a process termed as ultra-high pressure explosion (UHPE). The bamboo powder was heated in 2% NaOH solution at 121°C, and then 100MPa UHPE-treated through a homogenizer. The results verified that UHPE changed the suspension solution of powder into a stick fluid. The contents of lignin were decreased significantly. The bamboo shoots and 2-year-old bamboo were completely hydrolyzed to glucose within 48h by enzymes loading of 15 FPU of cellulase and 30IU of β-glucosidase per gram glucan. Fermentation of enzymatic hydrolyzates with Saccharomyces cerevisiae resulted in about 89.7-95.1% of the theoretical ethanol yield after 24h. Therefore, NaOH+UHPE is argued to be a potential alternative technology for pretreatment of bamboo. PMID:27189535

  16. Cyclomaltoheptaose mixed esters of anti-inflammatory drugs and short-chain fatty acids and study of their enzymatic hydrolysis in vitro.

    PubMed

    Cao, Feng; Ren, Yong; Hua, Weiyi

    2009-03-10

    In an effort to enhance the drug-loading capacity of cyclomaltoheptaose (beta-cyclodextrin, betaCD) and to combine the function of anti-inflammatory drugs with short-chain fatty acids (SCFA), ternary esters incorporating seven copies of an anti-inflammatory drug and 14 copies of a SCFA onto a beta-cyclodextrin core were designed and prepared. Acetic, propionic, or butyric esters were introduced at secondary OH groups, and ibuprofen, flurbiprofen, or felbinac was attached to primary OH groups through ester bonds. Heptakis[2,3-di-O-butanoyl-6-O-2-(biphenyl-4-yl)-ethanoyl]-cyclomaltoheptaose was very stable in aqueous and esterase solution. It was hydrolyzed by alpha-amylase (4 units/mL) with t(1/2) value of 18h. The total released amount of biphenyl acetic acid was 38% after 24h when the esterase was added after the alpha-amylase hydrolysis. The present results suggest that these nine betaCD conjugates may release the anti-inflammatory drug in the colonic contents. PMID:19185291

  17. Systematic optimization of fed-batch simultaneous saccharification and fermentation at high-solid loading based on enzymatic hydrolysis and dynamic metabolic modeling of Saccharomyces cerevisiae.

    PubMed

    Unrean, Pornkamol; Khajeeram, Sutamat; Laoteng, Kobkul

    2016-03-01

    An integrative simultaneous saccharification and fermentation (SSF) modeling is a useful guiding tool for rapid process optimization to meet the techno-economic requirement of industrial-scale lignocellulosic ethanol production. In this work, we have developed the SSF model composing of a metabolic network of a Saccharomyces cerevisiae cell associated with fermentation kinetics and enzyme hydrolysis model to quantitatively capture dynamic responses of yeast cell growth and fermentation during SSF. By using model-based design of feeding profiles for substrate and yeast cell in the fed-batch SSF process, an efficient ethanol production with high titer of up to 65 g/L and high yield of 85 % of theoretical yield was accomplished. The ethanol titer and productivity was increased by 47 and 41 %, correspondingly, in optimized fed-batch SSF as compared to batch process. The developed integrative SSF model is, therefore, considered as a promising approach for systematic design of economical and sustainable SSF bioprocessing of lignocellulose. PMID:26610806

  18. Trichoderma reesei FS10-C enhances phytoremediation of Cd-contaminated soil by Sedum plumbizincicola and associated soil microbial activities

    PubMed Central

    Teng, Ying; Luo, Yang; Ma, Wenting; Zhu, Lingjia; Ren, Wenjie; Luo, Yongming; Christie, Peter; Li, Zhengao

    2015-01-01

    This study aimed to explore the effects of Trichoderma reesei FS10-C on the phytoremediation of Cd-contaminated soil by the hyperaccumulator Sedum plumbizincicola and on soil fertility. The Cd tolerance of T. reesei FS10-C was characterized and then a pot experiment was conducted to investigate the growth and Cd uptake of S. plumbizincicola with the addition of inoculation agents in the presence and absence of T. reesei FS10-C. The results indicated that FS10-C possessed high Cd resistance (up to 300 mg L-1). All inoculation agents investigated enhanced plant shoot biomass by 6–53% of fresh weight and 16–61% of dry weight and Cd uptake by the shoots by 10–53% compared with the control. All inoculation agents also played critical roles in increasing soil microbial biomass and microbial activities (such as biomass C, dehydrogenase activity and fluorescein diacetate hydrolysis activity). Two inoculation agents accompanied by FS10-C were also superior to the inoculation agents, indicating that T. reesei FS10-C was effective in enhancing both Cd phytoremediation by S. plumbizincicola and soil fertility. Furthermore, solid fermentation powder of FS10-C showed the greatest capacity to enhance plant growth, Cd uptake, nutrient release, microbial biomass and activities, as indicated by its superior ability to promote colonization by Trichoderma. The solid fermentation powder of FS10-C might serve as a suitable inoculation agent for T. reesei FS10-C to enhance both the phytoremediation efficiency of Cd-contaminated soil and soil fertility. PMID:26113858

  19. Trichoderma reesei FS10-C enhances phytoremediation of Cd-contaminated soil by Sedum plumbizincicola and associated soil microbial activities.

    PubMed

    Teng, Ying; Luo, Yang; Ma, Wenting; Zhu, Lingjia; Ren, Wenjie; Luo, Yongming; Christie, Peter; Li, Zhengao

    2015-01-01

    This study aimed to explore the effects of Trichoderma reesei FS10-C on the phytoremediation of Cd-contaminated soil by the hyperaccumulator Sedum plumbizincicola and on soil fertility. The Cd tolerance of T. reesei FS10-C was characterized and then a pot experiment was conducted to investigate the growth and Cd uptake of S. plumbizincicola with the addition of inoculation agents in the presence and absence of T. reesei FS10-C. The results indicated that FS10-C possessed high Cd resistance (up to 300 mg L(-1)). All inoculation agents investigated enhanced plant shoot biomass by 6-53% of fresh weight and 16-61% of dry weight and Cd uptake by the shoots by 10-53% compared with the control. All inoculation agents also played critical roles in increasing soil microbial biomass and microbial activities (such as biomass C, dehydrogenase activity and fluorescein diacetate hydrolysis activity). Two inoculation agents accompanied by FS10-C were also superior to the inoculation agents, indicating that T. reesei FS10-C was effective in enhancing both Cd phytoremediation by S. plumbizincicola and soil fertility. Furthermore, solid fermentation powder of FS10-C showed the greatest capacity to enhance plant growth, Cd uptake, nutrient release, microbial biomass and activities, as indicated by its superior ability to promote colonization by Trichoderma. The solid fermentation powder of FS10-C might serve as a suitable inoculation agent for T. reesei FS10-C to enhance both the phytoremediation efficiency of Cd-contaminated soil and soil fertility. PMID:26113858

  20. Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis Current and Futuristic Scenarios

    SciTech Connect

    Wooley, R.; Ruth, M.; Sheehan, J.; Ibsen, K.; Majdeski, H.; Galvez, A.

    1999-07-20

    The National Renewable Energy Laboratory (NREL) has undertaken a complete review and update of the process design and economic model for the biomass-to-ethanol enzymatic based process. The process design includes the core technologies being researched by the U.S. Department of Energy (DOE): prehydrolysis, simultaneous saccharification and co-fermentation, and cellulase enzyme production. In addition, all ancillary areas--feed handling, product recovery and purification, wastewater treatment lignin burner and boiler--turbogenerator, and utilities--are included. NREL engaged Delta-T Corporation to assist in the process design evaluation, equipment costing, and overall plant integration. The process design and costing for the lignin burner and boiler turbogenerator has been reviewed by Reaction Engineering Inc. and the wastewater treatment by Merrick and Company. An overview of both reviews is included here. The purpose of this update was to ensure that the process design and equipment costs were reasonable and consistent with good engineering practice for plants of this type using available technical data. This work has resulted in an economic model that can be used to predict the cost of producing ethanol from cellulosic biomass using this technology if a plant were to be built in the next few years. The model was also extended using technology improvements that are expected to be developed based on the current DOE research plan. Future process designs and cost estimates are given for the years 2005, 2010, and 2015.

  1. Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis Current and Futuristic Scenarios

    SciTech Connect

    Galvez, A.; Ibsen, K.; Majdeski, H.; Ruth, M.; Sheehan, J.; Wooley, R.

    1999-07-20

    The National Renewable Energy Laboratory (NREL) has undertaken a complete review and update of the process design and economic model for the biomass-to-ethanol enzymatic based process. The process design includes the core technologies being researched by the U.S. Department of Energy (DOE): prehydrolysis, simultaneous saccharification and co-fermentation, and cellulase enzyme production. In addition, all ancillary areasfeed handling, product recovery and purification, wastewater treatment lignin burner and boiler-turbogenerator, and utilitiesare included. NREL engaged Delta-T Corporation to assist in the process design evaluation, equipment costing, and overall plant integration. The process design and costing for the lignin burner and boiler turbogenerator has been reviewed by Reaction Engineering Inc. and the wastewater treatment by Merrick Company. An overview of both reviews is included here. The purpose of this update was to ensure that the process design and equipment costs were reasonable and consistent with good engineering practice for plants of this type using available technical data. This work has resulted in an economic model that can be used to predict the cost of producing ethanol from cellulosic biomass using this technology if a plant were to be built in the next few years. The model was also extended using technology improvements that are expected to be developed based on the current DOE research plan. Future process designs and cost estimates are given for the years 2005, 2010, and 2015.

  2. Process Design and Economics for Biochemical Conversion of Lignocellulosic Biomass to Ethanol: Dilute-Acid Pretreatment and Enzymatic Hydrolysis of Corn Stover

    SciTech Connect

    Humbird, D.; Davis, R.; Tao, L.; Kinchin, C.; Hsu, D.; Aden, A.; Schoen, P.; Lukas, J.; Olthof, B.; Worley, M.; Sexton, D.; Dudgeon, D.

    2011-03-01

    This report describes one potential biochemical ethanol conversion process, conceptually based upon core conversion and process integration research at NREL. The overarching process design converts corn stover to ethanol by dilute-acid pretreatment, enzymatic saccharification, and co-fermentation. Building on design reports published in 2002 and 1999, NREL, together with the subcontractor Harris Group Inc., performed a complete review of the process design and economic model for the biomass-to-ethanol process. This update reflects NREL's current vision of the biochemical ethanol process and includes the latest research in the conversion areas (pretreatment, conditioning, saccharification, and fermentation), optimizations in product recovery, and our latest understanding of the ethanol plant's back end (wastewater and utilities). The conceptual design presented here reports ethanol production economics as determined by 2012 conversion targets and 'nth-plant' project costs and financing. For the biorefinery described here, processing 2,205 dry ton/day at 76% theoretical ethanol yield (79 gal/dry ton), the ethanol selling price is $2.15/gal in 2007$.

  3. Effect of the addition of a nonaqueous polar solvent (glycerol) on enzymatic catalysis in reverse micelles. Hydrolysis of 2-naphthyl acetate by alpha-chymotrypsin.

    PubMed

    Falcone, R Darío; Biasutti, M Alicia; Correa, N Mariano; Silber, Juana J; Lissi, Eduardo; Abuin, Elsa

    2004-07-01

    The kinetics of hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by alpha-chymotrypsin (alpha-CT), in reverse micellar solutions formed by glycerol (GY)-water (38% v/v) mixture/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-heptane has been determined by spectroscopic measurements. To compare the efficiency of this reaction with that observed in micelles with water in the core, as well as in the corresponding homogeneous media, the reaction was also studied in water/AOT/n-heptane reverse micellar solutions and in both homogeneous media (water and GY-water, 38% v/v mixture). In every media, alpha-CT was characterized by the absorption and emission spectra, the fluorescence lifetimes, and the fluorescence anisotropy of its tryptophan residues. The effect of AOT concentration on the kinetic parameters obtained in the micellar systems was determined, at a constant molar ratio of the inner polar solvent and surfactant. Moreover, the data obtained allowed the evaluation of the 2-NA partition constant between the organic and the micellar pseudophase. It is shown that the addition of GY to the micelle interior results in an increase in the catalytic properties of alpha-CT. The fluorescence anisotropy studies in the different media show that the addition of GY increases the viscosity as compared with the aqueous systems. It seems that the GY addition to the reverse micellar aggregates results in a decrease of the conformational mobility of alpha-CT, which leads to an increase of the enzyme stability and activity. PMID:16459586

  4. Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides

    PubMed Central

    2011-01-01

    Background Lignin and hemicelluloses are the major components limiting enzyme infiltration into cell walls. Determination of the topochemical distribution of lignin and aromatics in sugar cane might provide important data on the recalcitrance of specific cells. We used cellular ultraviolet (UV) microspectrophotometry (UMSP) to topochemically detect lignin and hydroxycinnamic acids in individual fiber, vessel and parenchyma cell walls of untreated and chlorite-treated sugar cane. Internodes, presenting typical vascular bundles and sucrose-storing parenchyma cells, were divided into rind and pith fractions. Results Vascular bundles were more abundant in the rind, whereas parenchyma cells predominated in the pith region. UV measurements of untreated fiber cell walls gave absorbance spectra typical of grass lignin, with a band at 278 nm and a pronounced shoulder at 315 nm, assigned to the presence of hydroxycinnamic acids linked to lignin and/or to arabino-methylglucurono-xylans. The cell walls of vessels had the highest level of lignification, followed by those of fibers and parenchyma. Pith parenchyma cell walls were characterized by very low absorbance values at 278 nm; however, a distinct peak at 315 nm indicated that pith parenchyma cells are not extensively lignified, but contain significant amounts of hydroxycinnamic acids. Cellular UV image profiles scanned with an absorbance intensity maximum of 278 nm identified the pattern of lignin distribution in the individual cell walls, with the highest concentration occurring in the middle lamella and cell corners. Chlorite treatment caused a rapid removal of hydroxycinnamic acids from parenchyma cell walls, whereas the thicker fiber cell walls were delignified only after a long treatment duration (4 hours). Untreated pith samples were promptly hydrolyzed by cellulases, reaching 63% of cellulose conversion after 72 hours of hydrolysis, whereas untreated rind samples achieved only 20% hydrolyzation. Conclusion The low

  5. Microwave Pretreatment For Hydrolysis Of Cellulose

    NASA Technical Reports Server (NTRS)

    Cullingford, Hatice S.; George, Clifford E.; Lightsey, George R.

    1993-01-01

    Microwave pretreatment enhances enzymatic hydrolysis of cellulosic wastes into soluble saccharides used as feedstocks for foods, fuels, and other products. Low consumption of energy, high yield, and low risk of proposed hydrolysis process incorporating microwave pretreatment makes process viable alternative to composting.

  6. Expression of Barley Endopeptidase B in Trichoderma reesei

    PubMed Central

    Saarelainen, R.; Mantyla, A.; Nevalainen, H.; Suominen, P.

    1997-01-01

    The gene for barley endopeptidase B (EPB) has been expressed in the filamentous fungus Trichoderma reesei from the cbh1 promoter. The EPB signal sequence allowed secretion of over 90% of the recombinant protein. Yields reached about 500 mg of immunoreactive protein per liter and exceeded values for any other protein derived from a higher eukaryotic organism produced in T. reesei. PMID:16535756

  7. Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions

    PubMed Central

    2013-01-01

    Background On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions. Results The effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold β-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high β-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars. Conclusions The sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode

  8. Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover

    SciTech Connect

    Aden, A.; Ruth, M.; Ibsen, K.; Jechura, J.; Neeves, K.; Sheehan, J.; Wallace, B.; Montague, L.; Slayton, A.; Lukas, J.

    2002-06-01

    This report is an update of NREL's ongoing process design and economic analyses of processes related to developing ethanol from lignocellulosic feedstocks. The U.S. Department of Energy (DOE) is promoting the development of ethanol from lignocellulosic feedstocks as an alternative to conventional petroleum-based transportation fuels. DOE funds both fundamental and applied research in this area and needs a method for predicting cost benefits of many research proposals. To that end, the National Renewable Energy Laboratory (NREL) has modeled many potential process designs and estimated the economics of each process during the last 20 years. This report is an update of the ongoing process design and economic analyses at NREL. We envision updating this process design report at regular intervals; the purpose being to ensure that the process design incorporates all new data from NREL research, DOE funded research and other sources, and that the equipment costs are reasonable and consistent with good engineering practice for plants of this type. For the non-research areas this means using equipment and process approaches as they are currently used in industrial applications. For the last report, published in 1999, NREL performed a complete review and update of the process design and economic model for the biomass-to-ethanol process utilizing co-current dilute acid prehydrolysis with simultaneous saccharification (enzymatic) and co-fermentation. The process design included the core technologies being researched by the DOE: prehydrolysis, simultaneous saccharification and co-fermentation, and cellulase enzyme production. In addition, all ancillary areas--feed handling, product recovery and purification, wastewater treatment (WWT), lignin combustor and boiler-turbogenerator, and utilities--were included. NREL engaged Delta-T Corporation (Delta-T) to assist in the process design evaluation, the process equipment costing, and overall plant integration. The process design and

  9. Optimizing cellulase mixtures for maximum rate and extent of hydrolysis. Final report

    SciTech Connect

    Walker, L.P.; Wilson, D.B.

    1997-03-01

    Pure Thomomonospora fusca and Trichoderma reesei cellulases and their mixtures were studied to determine the optimal set of cellulases for biomass hydrolysis. The objective was to reduce the cost of cellulase in order to help lower the overall processing cost of the enzymatic conversion of biomass cellulose to sugars, which can then be fermented into fuels and other energy-intensive chemicals. No cellulase mixture was obtained that was much better than the best commercially available preparations. However, the study has greatly increased knowledge of T. fusca cellulases, synergism, and cellulose binding, and provide evidence that future work will produce cellulases with higher activity in degrading crystalline cellulose. T. fusca cellulases may have good industrial potential because: (1) they are compatible with industrial processes that operate at elevated temperatures; (2) they retain 90% of their activity under neutral or basic conditions, which provides a great deal of flexibility in reactor design and operation; and (3) tools are now available to change specific amino acid residues in their catalytic domains and to assess how these changes influence catalysis. 74 refs.

  10. Genome sequencing of the Trichoderma reesei QM9136 mutant identifies a truncation of the transcriptional regulator XYR1 as the cause for its cellulase-negative phenotype

    SciTech Connect

    Lichius, Alexander; Bidard, Frédérique; Buchholz, Franziska; Le Crom, Stéphane; Martin, Joel; Schackwitz, Wendy; Austerlitz, Tina; Grigoriev, Igor V; Baker, Scott E; Margeot, Antoine; Seiboth, Bernhard; Kubicek, Christian P

    2015-04-20

    Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a.

  11. Targets of light signalling in Trichoderma reesei

    PubMed Central

    2013-01-01

    Background The tropical ascomycete Trichoderma reesei (Hypocrea jecorina) represents one of the most efficient plant cell wall degraders. Regulation of the enzymes required for this process is affected by nutritional signals as well as other environmental signals including light. Results Our transcriptome analysis of strains lacking the photoreceptors BLR1 and BLR2 as well as ENV1 revealed a considerable increase in the number of genes showing significantly different transcript levels in light and darkness compared to wild-type. We show that members of all glycoside hydrolase families can be subject to light dependent regulation, hence confirming nutrient utilization including plant cell wall degradation as a major output pathway of light signalling. In contrast to N. crassa, photoreceptor mediated regulation of carbon metabolism in T. reesei occurs primarily by BLR1 and BLR2 via their positive effect on induction of env1 transcription, rather than by a presumed negative effect of ENV1 on the function of the BLR complex. Nevertheless, genes consistently regulated by photoreceptors in N. crassa and T. reesei are significantly enriched in carbon metabolic functions. Hence, different regulatory mechanisms are operative in these two fungi, while the light dependent regulation of plant cell wall degradation appears to be conserved. Analysis of growth on different carbon sources revealed that the oxidoreductive D-galactose and pentose catabolism is influenced by light and ENV1. Transcriptional regulation of the target enzymes in these pathways is enhanced by light and influenced by ENV1, BLR1 and/or BLR2. Additionally we detected an ENV1-regulated genomic cluster of 9 genes including the D-mannitol dehydrogenase gene lxr1, with two genes of this cluster showing consistent regulation in N. crassa. Conclusions We show that one major output pathway of light signalling in Trichoderma reesei is regulation of glycoside hydrolase genes and the degradation of hemicellulose

  12. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

    PubMed Central

    Stålbrand, H; Saloheimo, A; Vehmaanperä, J; Henrissat, B; Penttilä, M

    1995-01-01

    beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei. PMID:7793911

  13. Steady and dynamic shear characterization of cellulase-producing Trichoderma reesei suspensions

    SciTech Connect

    Marten, M.R.; Velkovska, S.; Khan, S.A.

    1995-12-31

    Suspension rheology of fungal fermentations is important in determination of mass transfer rates, as well as mixing quality. We have characterized Trichorderma reesei RUT-C30 suspension rheology during growth on xylose (soluble) and cellulose (particulate) substrates, using both steady and dynamic shear measurements. Biomass growth was monophasic on xylose and biphasic on cellulose; the latter behavior is consistent with relatively rapid, early growth on soluble sugars derived from rapidly hydrolyzed material, followed by a second, slower growth phase owing to hydrolysis of more recalcitrant cellulose by increasing cellulose concentrations. Steady shear measurements established the presence of a yield stress for fermentation broths when using a 10 (vol)% fungal inoculum. The Casson equation represented all data well. Casson parameters of viscosity and yield stress followed biomass evolution: two maxima in both parameters were observed with cellulose substrates, and a single maximum with xylose. Dynamic shear measurements on broths indicated a gel behavior at small strains and a shear thinning liquid behavior at larger displacements. These results indicate the need to include rheology and mixing considerations in the subsequent development of a full biological and physical kinetic description of T. reesei cellulose conversions.

  14. Rheological, mass transfer, and mixing characterization of cellulase-producing Trichoderma reesei suspensions

    SciTech Connect

    Marten, M.R.; Velkovska, S.; Khan, S.A.; Ollis, D.F.

    1996-09-01

    Steady and dynamic shear measurements are utilized to characterize the rheological behavior of Trichoderma reesei RUT-C30 fungal suspensions during batch growth on xylose (soluble substrate) or cellulose (particulate solid substrate) at three different fermentor impeller speeds (250, 400, and 550 rpm). Biomass concentrations versus time were unimodal on xylose and bimodal on cellulose. This behavior is consistent with relatively rapid, early growth on easily metabolized growth medium components (yeast extract), followed by a second, slower growth phase due to hydrolysis of recalcitrant cellulose by increasing cellulose concentrations. Critical dissolved oxygen (DO) concentration for T. reesei growth on cellulose was found to be 0.073 mmol/L. The DO was kept above this level by supplementing the air feed with pure oxygen, implying that mass transfer limitations were not the cause of bimodal cell growth. Steady shear rheological data showed shear thinning behavior and a yield stress for all broth samples regardless of substrate. Dynamic shear measurements on broth suspensions indicated `gel-like` behavior at low strains, with microstructural breakdown at larger displacements. Time variations of the Casson model parameters (yield stress and Casson viscosity) and dynamic moduli (elastic and viscous modulus) followed both cell mass and morphology. 45 refs., 14 figs.

  15. New-to-nature sophorose analog: a potent inducer for gene expression in Trichoderma reesei.

    PubMed

    Huang, Tom Tao; Wages, John M

    2016-04-01

    Controlled hydrolysis of lactonic sophorolipids from Starmerella bombicola yields a previously undescribed sophorose analog that potently induces cellulase in Trichoderma reesei Rut-C30. Acid treatment of natural sophorolipids results in a mixture of monoacetylated, deacetylated, and diacetylated sophorolipids in acidic and lactonic forms. Isolation of the active components of the mixture, followed by structure determination by MS and NMR, reveals a new chemical entity, in which the lactone ring has been opened at the C-1' rather than at the C-4″ position of the sophorose moiety. This sophorose ester is resistant to degradation by the host and is at least 28 times more powerful an inducer than sophorose in shake-flask culture. Even at low concentrations (0.05 mM), the chemically modified sophorolipid effectively induces cellulase. With further improvements, this highly enabling technology can potentially reduce the cost of enzymes produced in T. reesei and can facilitate the rapid deployment of enzyme plants to support the nascent cellulosic biofuels and biochemicals industries. PMID:26920480

  16. Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing

    SciTech Connect

    Le Crom, Stphane; Schackwitz, Wendy; Pennacchiod, Len; Magnuson, Jon K.; Culley, David E.; Collett, James R.; Martin, Joel X.; Druzhinina, Irina S.; Mathis, Hugues; Monot, Frdric; Seiboth, Bernhard; Cherry, Barbara; Rey, Michael; Berka, Randy; Kubicek, Christian P.; Baker, Scott E.; Margeot, Antoine

    2009-09-22

    Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels, such as ethanol, and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions and 18 larger deletions leading to the loss of more than 100 kb of genomic DNA. From these events we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild type strain QM6a. Our analysis provides the first genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus, and suggests new areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.

  17. Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing

    PubMed Central

    Le Crom, Stéphane; Schackwitz, Wendy; Pennacchio, Len; Magnuson, Jon K.; Culley, David E.; Collett, James R.; Martin, Joel; Druzhinina, Irina S.; Mathis, Hugues; Monot, Frédéric; Seiboth, Bernhard; Cherry, Barbara; Rey, Michael; Berka, Randy; Kubicek, Christian P.; Baker, Scott E.; Margeot, Antoine

    2009-01-01

    Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production. PMID:19805272

  18. Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing.

    PubMed

    Le Crom, Stéphane; Schackwitz, Wendy; Pennacchio, Len; Magnuson, Jon K; Culley, David E; Collett, James R; Martin, Joel; Druzhinina, Irina S; Mathis, Hugues; Monot, Frédéric; Seiboth, Bernhard; Cherry, Barbara; Rey, Michael; Berka, Randy; Kubicek, Christian P; Baker, Scott E; Margeot, Antoine

    2009-09-22

    Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production. PMID:19805272

  19. Production of cellulase enzymes and hydrolysis of steam-exploded wood.

    PubMed

    San Martin, R; Blanch, H W; Wilke, C R; Sciamanna, A F

    1986-04-01

    Steam-exploded aspen has been examined as a candidate feedstock for both cellulose production and enzymatic hydrolysis of wood. Batch and fed-batch cultivation methods were evaluated and compared with previous experiments using ball-milled, crystalline cellulose (Solka Floe). Batch cultivation of Trichoderma reesei Rut C-30 on 9 wt% water-washed aspen yielded enzyme productivities and activities comparable to those obtained on Solka Floe (40 FP IU/L-h; 7. 5 FP IU/mL). Fed-batch cultivation of Rut C-30 resulted in higher enzyme productivities and tilers than batch cultivation (50 FP IU/L-h; 15 FP IU/mL). However, the overall enzyme production performance was lower than on Solka Floe at comparable cellulose feeding rates and concentrations. This may be due to the accumulation of steam explosion by-products and lignin in the fermentor.The hydrolysis of water-washed steam-exploded aspen was performed at different enzyme loadings and wood concentrations. Glucose production, using 10 and 15 wt% suspension, showed that while glucose concentration increased with wood load, the yield of glucose derived from cellulose decreased. With 10 wt% suspensions, it was possible to obtain a cellulose conversion to glucose above 95%. Low cellulose levels in the hydrolyzates indicated that the filter paper activity ratios (approximately 1.5), a significant result since the fungus was grown exclusively on wood. mIt also suggested that the observed yield decrease is more likely to be caused by glucose than cellobiose inhibition of the enzymes. PMID:18555361

  20. Determination of free and total myo-inositol in infant formula and adult/pediatric nutritional formula by high- performance anion exchange chromatography with pulsed amperometric detection, including a novel total extraction using microwave-assisted acid hydrolysis and enzymatic treatment: first action 2012.12.

    PubMed

    Ellingson, David; Pritchard, Ted; Foy, Pamela; King, Kathryn; Mitchell, Barbara; Austad, John; Winters, Doug; Sullivan, Darryl; Dowell, Dawn

    2013-01-01

    After an assessment of data generated from a single-laboratory validation study published in J. AOAC Int. 95, 1469-1478 (2012), a method for determining total myo-inositol in infant formula and adult/ pediatric nutritional formula by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), including extraction by using microwave-assisted acid hydrolysis and enzymatic treatment was presented for consideration by AOAC during the AOAC Annual Meeting held in Las Vegas, NV, from September 30 to October 3, 2012. The Expert Review Panel on Infant Formula and Adult Nutritionals concluded that the method met the criteria set by the standard method performance requirements (SMPRs) for the determination of free myo-inositol and approved the method as AOAC Official First Action. The method also determines total myo-inositol, but includes bound sources that the SMPRs exclude. The method involves using HPAEC-PAD for free myo-inositol and a total myo-inositol determination by two different techniques. The first technique uses the conventional acid hydrolysis with 6 h incubation in an autoclave. The second uses a microwave-assisted acid hydrolysis with enzymatic treatment that decreases the extraction time. PMID:24282949

  1. Pretreatment and Enzymatic Hydrolysis of Sorghum Fiber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum fiber has potential to serve as a low-cost feedstock for production of fuel ethanol. Sorghum fiber from a decortication process was used for this study. The approximate chemical composition of sorghum fiber is 30% starch, 18% hemicellulose, 11% cellulose, 11% protein, 10% crude fat, and 3% ...

  2. Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose*

    PubMed Central

    Murphy, Leigh; Cruys-Bagger, Nicolaj; Damgaard, Heidi Delcomyn; Baumann, Martin J.; Olsen, Søren Nymand; Borch, Kim; Lassen, Søren Flensted; Sweeney, Matt; Tatsumi, Hirosuke; Westh, Peter

    2012-01-01

    The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s−1 at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2–300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase. PMID:22110134

  3. Hydrolysis of biomass material

    DOEpatents

    Schmidt, Andrew J.; Orth, Rick J.; Franz, James A.; Alnajjar, Mikhail

    2004-02-17

    A method for selective hydrolysis of the hemicellulose component of a biomass material. The selective hydrolysis produces water-soluble small molecules, particularly monosaccharides. One embodiment includes solubilizing at least a portion of the hemicellulose and subsequently hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. A second embodiment includes solubilizing at least a portion of the hemicellulose and subsequently enzymatically hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. A third embodiment includes solubilizing at least a portion of the hemicellulose by heating the biomass material to greater than 110.degree. C. resulting in an aqueous portion that includes the solubilized hemicellulose and a water insoluble solids portion and subsequently separating the aqueous portion from the water insoluble solids portion. A fourth embodiment is a method for making a composition that includes cellulose, at least one protein and less than about 30 weight % hemicellulose, the method including solubilizing at least a portion of hemicellulose present in a biomass material that also includes cellulose and at least one protein and subsequently separating the solubilized hemicellulose from the cellulose and at least one protein.

  4. Improving the activity of Trichoderma reesei cel7B through stabilizing the transition state.

    PubMed

    Wang, Yefei; Song, Xiangfei; Zhang, Shujun; Li, Jingwen; Shu, Zhiyu; He, Chunyan; Huang, Qingshan; Yao, Lishan

    2016-06-01

    Trichoderma reesei (Tr.) cellulases, which convert cellulose to reducing sugars, are a promising catalyst used in the lignocellulosic biofuel production. Improving Tr. cellulases activity, though very difficult, is highly desired due to the recalcitrance of lignocellulose. Meanwhile, it is preferable to enhance the cellulase's promiscuity so that substrates other than cellulose can also be hydrolyzed. In this work, an attempt is made to improve the catalytic activity of a major endogluanase Tr. Cel7B against xylan which crosslinks with cellulose in lignocellulose. By using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations, the transition state of the xylo-oligosaccharide hydrolysis is identified. Then, mutations are introduced and their effect on the transition state stabilization is ranked based on the free energy calculations. Seven top ranked mutants are evaluated experimentally. Three mutants A208Q, A222D, and G230R show a higher activity than the wild-type Tr. Cel7B in the hydrolysis of xylan (by up to 47%) as well as filter paper (by up to 50%). The combination of the single mutants can further improve the enzyme activity. Our work demonstrates that the free energy method is effective in engineering the Tr. Cel7B activity against xylan and cellulose, and thus may also be useful for improving the activity of other Tr. cellulases. Biotechnol. Bioeng. 2016;113: 1171-1177. © 2015 Wiley Periodicals, Inc. PMID:26616246

  5. Phosphatase hydrolysis of organic phosphorus compounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatases are diverse groups of enzymes that deserve special attention because of the significant roles they play in mineralizing organic phosphorus (P) into inorganic available form. For getting more insight on the enzymatically hydrolysis of organic P, in this work, we compared the catalytic pa...

  6. Cloning of genes encoding alpha-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae.

    PubMed Central

    Margolles-Clark, E; Tenkanen, M; Nakari-Setälä, T; Penttilä, M

    1996-01-01

    A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae. Two genes, abf1 and bxl1, were isolated by screening the yeast library for extracellular alpha-L-arabinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-arabinofuranoside. The genes abf1 and bxl1 encode 500 and 758 amino acids, respectively, including the signal sequences. The deduced amino acid sequence of ABFI displays high-level similarity to the alpha-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases. The deduced amino acid sequence of BXLI shows similarities to the beta-glucosidases grouped in family 3. The yeast-produced enzymes were tested for enzymatic activities against different substrates. ABFI released L-arabinose from p-nitrophenyl-alpha-L-arabinofuranoside and arabinoxylans and showed some beta-xylosidase activity toward p-nitrophenyl-beta-D-xylopyranoside. BXLI did not release L-arabinose from arabinoxylan. It showed alpha-L-arabinofuranosidase, alpha-L-arabinopyranosidase, and beta-xylosidase activities against p-nitrophenyl-alpha-L-arabinofuranosidase, p-nitrophenyl-alpha-L-arabinopyranoside, and p-nitrophenyl-beta-D- xylopyranoside, respectively, with the last activity being the highest. It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan. ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes. Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei. PMID:8837440

  7. 77 FR 35331 - Trichoderma reesei; Proposed Significant New Use Rule

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-13

    ... produced, and at what levels if the genetically modified T. reesei is grown on various plant biomass... rule (SNUR) under the Toxic Substances Control Act (TSCA) for the genetically modified microorganism.... Background A. What action is the agency taking? EPA is proposing this SNUR for the genetically...

  8. Improving Trichoderma reesei Cel7B thermostability by targeting the weak spots.

    PubMed

    Zhang, Shujun; Wang, Yefei; Song, Xiangfei; Hong, Jingbo; Zhang, Yu; Yao, Lishan

    2014-10-27

    For proteins that denature irreversibly, the denaturation is typically triggered by a partial unfolding, followed by a permanent change (e.g., aggregation). The regions that initiate the partial unfolding are named "weak spots". In this work, a molecular dynamics (MD) simulation and data analysis protocol is developed to identify the weak spots of Trichoderma reesei Cel7B, an important endoglucanase in cellulose hydrolysis, through assigning the local melting temperature (Tmp) to individual residue pairs. To test the predicted weak spots, a total of eight disulfide bonds were designed in these regions and all enhanced the enzyme thermostability. The increased stability, quantified by ΔT50 (which is the T50 difference between the mutant and the wild type enzyme), is negatively correlated with the MD-predicted Tmp, demonstrating the effectiveness of the protocol and highlighting the importance of the weak spots. Strengthening interactions in these regions proves to be a useful strategy in improving the thermostability of Tr. Cel7B. PMID:25286389

  9. A mechanistic study of Trichoderma reesei Cel7B catalyzed glycosidic bond cleavage.

    PubMed

    Zhang, Yu; Yan, Shihai; Yao, Lishan

    2013-07-25

    An ONIOM study is performed to illustrate the mechanism of Trichoderma reesei Cel7B catalyzed p-nitrophenyl lactoside hydrolysis. In both the glycosylation and deglycosylation steps, the reaction proceeds in a concerted way, meaning the nucleophilic attack and the glycosidic bond cleavage occur simultaneously. The glycosylation step is rate limiting with a barrier of 18.9 kcal/mol, comparable to the experimental value derived from the kcat measured in this work. The function of four residues R108, Y146, Y170, and D172, which form a hydrogen-bond network involving the substrate, is studied by conservative mutations. The mutants, including R108K, Y146F, Y170F, and D172N, decrease the enzyme activity by about 150-8000-fold. Molecular dynamics simulations show that the mutations disrupt the hydrogen-bond network, cause the substrate to deviate from active binding and hinder either the proton transfer from E201 to O4(+1) or the nucleophilic attack from E196 to C1(-1). PMID:23822607

  10. Enzymatic Products from Modified Soybean Oil Containing Hydrazinoester

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We use soybean oil to produce new, non-petroleum based products. The starting material is the ene reaction product of soybean oil and diethyl azodicarboxylate (DEAD), which can then be hydrolyzed chemically and enzymatically. Chemical hydrolysis gives hydrazino-fatty acids, whereas enzymatic hydro...

  11. Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps

    SciTech Connect

    Brady, Sonia K.; Sreelatha, Sarangapani; Feng, Yinnian; Chundawat, Shishir P. S.; Lang, Matthew J.

    2015-12-10

    Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor’s mechanistic action has yet to be revealed. We develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Moreover, through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A’s molecular motility mechanism.

  12. Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps

    DOE PAGESBeta

    Brady, Sonia K.; Sreelatha, Sarangapani; Feng, Yinnian; Chundawat, Shishir P. S.; Lang, Matthew J.

    2015-12-10

    Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor’s mechanistic action has yet to be revealed. We develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, whichmore » likely includes energy from glycosidic bonds and other sources. Moreover, through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A’s molecular motility mechanism.« less

  13. Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps

    NASA Astrophysics Data System (ADS)

    Brady, Sonia K.; Sreelatha, Sarangapani; Feng, Yinnian; Chundawat, Shishir P. S.; Lang, Matthew J.

    2015-12-01

    Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor's mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A's molecular motility mechanism.

  14. Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and cellulases.

    PubMed

    Oksanen, T; Pere, J; Paavilainen, L; Buchert, J; Viikari, L

    2000-02-28

    Effects of recycling ECF-bleached softwood kraft pulp on pulp properties were evaluated in the laboratory. The tensile strength, fiber flexibility and WRV lost during drying of the pulp were recovered by refining between the cycles which, however, resulted in deteriorated drainage properties. The recycled pulps were treated with purified Trichoderma reesei cellulases and hemicellulases and the changes in fiber properties due to enzymatic treatments were characterized. The endoglucanases (EG I and EG II) significantly improved pulp drainage already at low dosage levels, and EG II was found to be more effective at a given level of carbohydrate solubilization. Combining hemicellulases with the endoglucanase treatments increased the positive effects of the endoglucanases on pulp drainage. However, as a result of the endoglucanase treatments a slight loss in strength was observed. Combining mannanase with endoglucanase treatments appeared to increase this negative effect, whereas the impact of xylanase was not significant. Although the drainage properties of the pulps could be improved by selected enzymes, the water retention capacity of the dried hornified fibers could not be recovered by any of the enzymes tested. PMID:10702909

  15. Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps.

    PubMed

    Brady, Sonia K; Sreelatha, Sarangapani; Feng, Yinnian; Chundawat, Shishir P S; Lang, Matthew J

    2015-01-01

    Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor's mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A's molecular motility mechanism. PMID:26657780

  16. Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps

    PubMed Central

    Brady, Sonia K.; Sreelatha, Sarangapani; Feng, Yinnian; Chundawat, Shishir P. S.; Lang, Matthew J

    2015-01-01

    Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor's mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A's molecular motility mechanism. PMID:26657780

  17. Improvement of thermostability and activity of Trichoderma reesei endo-xylanase Xyn III on insoluble substrates.

    PubMed

    Matsuzawa, Tomohiko; Kaneko, Satoshi; Yaoi, Katsuro

    2016-09-01

    Trichoderma reesei Xyn III, an endo-β-1,4-xylanase belonging to glycoside hydrolase family 10 (GH10), is vital for the saccharification of xylans in plant biomass. However, its enzymatic thermostability and hydrolytic activity on insoluble substrates are low. To overcome these difficulties, the thermostability of Xyn III was improved using random mutagenesis and directed evolution, and its hydrolytic activity on insoluble substrates was improved by creating a chimeric protein. In the screening of thermostable Xyn III mutants from a random mutagenesis library, we identified two amino acid residues, Gln286 and Asn340, which are important for the thermostability of Xyn III. The Xyn III Gln286Ala/Asn340Tyr mutant showed xylanase activity even after heat treatment at 60 °C for 30 min or 50 °C for 96 h, indicating a dramatic enhancement in thermostability. In addition, we found that the addition of a xylan-binding domain (XBD) to the C-terminal of Xyn III improved its hydrolytic activity on insoluble xylan. PMID:27138202

  18. Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei.

    PubMed

    Bischof, Robert H; Ramoni, Jonas; Seiboth, Bernhard

    2016-01-01

    More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei. PMID:27287427

  19. Hydrolysis of olive mill waste to enhance rhamnolipids and surfactin production.

    PubMed

    Moya Ramírez, Ignacio; Altmajer Vaz, Deisi; Banat, Ibrahim M; Marchant, Roger; Jurado Alameda, Encarnación; García Román, Miguel

    2016-04-01

    The aim of this work was to demonstrate the effectiveness of hydrolysis pretreatment of olive mill (OMW) waste before use as a carbon source in biosurfactant production by fermentation. Three hydrolysis methods were assessed: enzymatic hydrolysis, acid pretreatment plus enzymatic hydrolysis, and acid hydrolysis. Fermentation was carried out using two bacterial species: Pseudomonas aeruginosa and Bacillus subtilis. Our results showed that the enzymatic hydrolysis was the best pretreatment, yielding up to 29.5 and 13.7mg/L of rhamnolipids and surfactins respectively. Glucose did not show significant differences in comparison to enzymatically hydrolysed OMW. At the best conditions found rhamnolipids and surfactins reached concentrations of 299 and 26.5mg/L; values considerably higher than those obtained with non-hydrolysed OMW. In addition, enzymatic pretreatment seemed to partially reduce the inhibitory effects of OMW on surfactin production. Therefore, enzymatic hydrolysis proved to effectively increase the productivity of these biosurfactants using OMW as the sole carbon source. PMID:26796482

  20. Synergistic action of recombinant accessory hemicellulolytic and pectinolytic enzymes to Trichoderma reesei cellulase on rice straw degradation.

    PubMed

    Laothanachareon, Thanaporn; Bunterngsook, Benjarat; Suwannarangsee, Surisa; Eurwilaichitr, Lily; Champreda, Verawat

    2015-12-01

    Synergism between core cellulases and accessory hydrolytic/non-hydrolytic enzymes is the basis of efficient hydrolysis of lignocelluloses. In this study, the synergistic action of three recombinant accessory enzymes, namely GH62 α-l-arabinofuranosidase (ARA), CE8 pectin esterase (PET), and GH10 endo-1,4-beta-xylanase (XYL) from Aspergillus aculeatus expressed in Pichia pastoris to a commercial Trichoderma reesei cellulase (Accellerase® 1500; ACR) on hydrolysis of alkaline pretreated rice straw was studied using a mixture design approach. Applying the full cubic model, the optimal ratio of quaternary enzyme mixture was predicted to be ACR:ARA:PET:XYL of 0.171:0.079:0.100:0.150, which showed a glucose releasing efficiency of 0.173 gglc/FPU, higher than the binary ACR:XYL mixture (0.122 gglc/FPU) and ACR alone (0.081 gglc/FPU) leading to a 47.3% increase in glucose yield compared with that from ACR at the same cellulase dosage. The result demonstrates the varying degree of synergism of accessory enzymes to cellulases useful for developing tailor-made enzyme systems for bio-industry. PMID:26433794

  1. Genome sequencing of the Trichoderma reesei QM9136 mutant identifies a truncation of the transcriptional regulator XYR1 as the cause for its cellulase-negative phenotype

    SciTech Connect

    Lichius, Alexander; Bidard, Frederique; Buchholz, Franziska; Le Crom, Stphane; Martin, Joel X.; Schackwitz, Wendy; Austerlitz, Tina; Grigoriev, Igor V.; Baker, Scott E.; Margeot, Antoine; Seiboth, Bernhard; Kubicek, Christian P.

    2015-12-01

    Background: Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a. Results: In QM9136, we detected a surprisingly low number of mutagenic events in the promoter and coding regions of genes, i.e. only eight indels and six single nucleotide variants. One of these indels led to a frame-shift in the Zn2Cys6 transcription factor XYR1, the general regulator of cellulase and xylanase expression, and resulted in its C-terminal truncation by 140 amino acids. Retransformation of strain QM9136 with the wild-type xyr1 allele fully recovered the ability to produce cellulases, and is thus the reason for the cellulase-negative phenotype. Introduction of an engineered xyr1 allele containing the truncating point mutation into the moderate producer T. reesei QM9414 rendered this strain also cellulase-negative. The correspondingly truncated XYR1 protein was still able to enter the nucleus, but failed to be expressed over the basal constitutive level. Conclusion: The missing 140 C-terminal amino acids of XYR1 are therefore responsible for its previously observed auto-regulation which is essential for cellulases to be expressed. Our data present a working example of the use of genome sequencing leading to a functional explanation of the QM9136 cellulase-negative phenotype.

  2. Enzymatic pretreatment for preparing starch nanocrystals.

    PubMed

    LeCorre, Déborah; Vahanian, Elina; Dufresne, Alain; Bras, Julien

    2012-01-01

    Starch nanocrystals (SNCs) are crystalline platelets resulting from the acid hydrolysis of starch. A limiting factor for their more widespread use is their preparation duration. Therefore, this study investigates the possibility of developing an enzymatic pretreatment of starch to reduce the acid hydrolysis duration. A screening of three types of enzymes, namely, α-amylase, β-amylase, and glucoamylase, is proposed, and the latter was selected for a pretreatment. Compared with the regular kinetics of hydrolysis for preparing SNC, that of pretreated starch was much faster. The extent of hydrolysis normally reached in 24 h was obtained after only 6 h, and the regular final yield (15% after 5 days) was reached in 45 h. AFM and X-ray diffraction measurements confirmed that the obtained nanoparticles were indeed SNC. PMID:22133316

  3. Characterization of a Neocallimastix patriciarum cellulase cDNA (celA) homologous to Trichoderma reesei cellobiohydrolase II.

    PubMed Central

    Denman, S; Xue, G P; Patel, B

    1996-01-01

    The nucleotide sequence of a cellulase cDNA (celA) from the rumen fungus Neocallimastix patriciarum and the primary structure of the protein which it encodes were characterized. The celA cDNA was 1.95 kb long and had an open reading frame of 1,284 bp, which encoded a polypeptide having 428 amino acid residues. A sequence alignment showed that cellulase A (CELA) exhibited substantial homology with family B cellulases (family 6 glycosyl hydrolases), particularly cellobiohydrolase II from the aerobic fungus Trichoderma reesei. In contrast to previously characterized N. patriciarum glycosyl hydrolases, CELA did not exhibit homology with any other rumen microbial cellulases described previously. Primary structure and function studies in which deletion analysis and a sequence comparison with other well-characterized cellulases were used revealed that CELA consisted of a cellulose-binding domain at the N terminus and a catalytic domain at the C terminus. These two domains were separated by an extremely Asn-rich linker. Deletion of the cellulose-binding domain resulted in a marked decrease in the cellulose-binding ability and activity toward crystalline cellulose. When CELA was expressed in Escherichia coli, it was located predominantly in the periplasmic space, indicating that the signal sequence of CELA was functional in E.coli. Enzymatic studies showed that CELA had an optimal pH of 5.0 and an optimal temperature of 40 degrees C. The specific activity of immunoaffinity-purified CELA against Avicel was 9.7 U/mg of protein, and CELA appeared to be a relatively active cellobiohydrolase compared with the specific activities reported for other cellobiohydrolases, such as T. reesei cellobiohydrolases I and II. PMID:8787388

  4. Enhanced hydrolysis of soluble cellulosic substrates by a metallocellulase with veratryl alcohol-oxidase activity

    SciTech Connect

    Evans, B.R.; Margalt, R.; Woodward, J.

    1995-12-31

    A cellulose enzyme fraction was separated from Trichoderma reesei Pulpzyme HA{trademark}, and its characteristics suggested that it was mainly composed of cellobiohydrolase II (CBH II). The covalent attachment of pentaammineruthenium (III) to this enzyme resulted in threefold and fourfold enhancements of its hydrolytic activity on carboxymethyl cellulose (CMC) and barley {beta}-glucan, respectively, as well as endowing it with veratryl alcohol-oxidase activity. Enhancement of hydrolysis was not affected by addition of tartrate or hydrogen peroxide to the reaction mixture. Both native and pentaammineruthenium modified enzymes had negligible activity on cellobiose and p-nitrophenyl {beta}-cellobioside (PNPC).

  5. Soybean bio-refinery platform: enzymatic process for production of soy protein concentrate, soy protein isolate and fermentable sugar syrup.

    PubMed

    Loman, Abdullah Al; Islam, S M Mahfuzul; Li, Qian; Ju, Lu-Kwang

    2016-10-01

    Soybean carbohydrate is often found to limit the use of protein in soy flour as food and animal feed due to its indigestibility to monogastric animal. In the current study, an enzymatic process was developed to produce not only soy protein concentrate and soy protein isolate without indigestible carbohydrate but also soluble reducing sugar as potential fermentation feedstock. For increasing protein content in the product and maximizing protein recovery, the process was optimized to include the following steps: hydrolysis of soy flour using an Aspergillus niger enzyme system; separation of the solid and liquid by centrifugation (10 min at 7500×g); an optional step of washing to remove entrapped hydrolysate from the protein-rich wet solid stream by ethanol (at an ethanol-to-wet-solid ratio (v/w) of 10, resulting in a liquid phase of approximately 60 % ethanol); and a final precipitation of residual protein from the sugar-rich liquid stream by heat treatment (30 min at 95 °C). Starting from 100 g soy flour, this process would produce approximately 54 g soy protein concentrate with 70 % protein (or, including the optional solid wash, 43 g with 80 % protein), 9 g soy protein isolate with 89 % protein, and 280 ml syrup of 60 g/l reducing sugar. The amino acid composition of the soy protein concentrate produced was comparable to that of the starting soy flour. Enzymes produced by three fungal species, A. niger, Trichoderma reesei, and Aspergillus aculeatus, were also evaluated for effectiveness to use in this process. PMID:27207010

  6. A single molecule study of cellulase hydrolysis of crystalline cellulose

    NASA Astrophysics Data System (ADS)

    Liu, Yu-San; Luo, Yonghua; Baker, John O.; Zeng, Yining; Himmel, Michael E.; Smith, Steve; Ding, Shi-You

    2010-02-01

    Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate β-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.

  7. Erythritol production on wheat straw using Trichoderma reesei

    PubMed Central

    2014-01-01

    We overexpressed the err1 gene in the Trichoderma reesei wild-type and in the cellulase hyperproducing, carbon catabolite derepressed strain Rut-C30 in order to investigate the possibility of producing erythritol with T. reesei. Two different promoters were used for err1 overexpression in both strains, a constitutive (the native pyruvat kinase (pki) promoter) and an inducible one (the native β-xylosidase (bxl1) promoter). The derived recombinant strains were precharacterized by analysis of err1 transcript formation on D-xylose and xylan. Based on this, one strain of each type was chosen for further investigation for erythritol production in shake flasks and in bioreactor experiments. For the latter, we used wheat straw pretreated by an alkaline organosolve process as lignocellulosic substrate. Shake flask experiments on D-xylose showed increased erythritol formation for both, the wild-type and the Rut-C30 overexpression strain compared to their respective parental strain. Bioreactor cultivations on wheat straw did not increase erythritol formation in the wild-type overexpression strain. However, err1 overexpression in Rut-C30 led to a clearly higher erythritol formation on wheat straw. PMID:24949268

  8. Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei.

    PubMed

    Hackbart, H C S; Machado, A R; Christ-Ribeiro, A; Prietto, L; Badiale-Furlong, E

    2014-08-01

    This study evaluated the ability of the microorganisms Rhizopus oryzae (CCT7560) and Trichoderma reesei (QM9414), producers of generally recognized as safe (GRAS) enzymes, to reduce the level of aflatoxins B1, B2, G1, G2, and M1. The variables considered to the screening were the initial number of spores in the inoculum and the culture time. The culture was conducted in contaminated 4 % potato dextrose agar (PDA) medium, and the residual mycotoxins were determined every 24 h by HPLC-FL. The fungus R. oryzae has reduced aflatoxins B1, B2, and G1 in the 96 h and aflatoxins M1 and G2 in the range of 120 h of culture by approximately 100 %. The fungus T. reesei has reduced aflatoxins B1, B2, and M1 in the 96 h and aflatoxin G1 in the range of 120 h of culture by approximately 100 %. The highest reduction occurred in the middle of R. oryzae culture. PMID:24925827

  9. Erythritol production on wheat straw using Trichoderma reesei.

    PubMed

    Jovanović, Birgit; Mach, Robert L; Mach-Aigner, Astrid R

    2014-01-01

    We overexpressed the err1 gene in the Trichoderma reesei wild-type and in the cellulase hyperproducing, carbon catabolite derepressed strain Rut-C30 in order to investigate the possibility of producing erythritol with T. reesei. Two different promoters were used for err1 overexpression in both strains, a constitutive (the native pyruvat kinase (pki) promoter) and an inducible one (the native β-xylosidase (bxl1) promoter). The derived recombinant strains were precharacterized by analysis of err1 transcript formation on D-xylose and xylan. Based on this, one strain of each type was chosen for further investigation for erythritol production in shake flasks and in bioreactor experiments. For the latter, we used wheat straw pretreated by an alkaline organosolve process as lignocellulosic substrate. Shake flask experiments on D-xylose showed increased erythritol formation for both, the wild-type and the Rut-C30 overexpression strain compared to their respective parental strain. Bioreactor cultivations on wheat straw did not increase erythritol formation in the wild-type overexpression strain. However, err1 overexpression in Rut-C30 led to a clearly higher erythritol formation on wheat straw. PMID:24949268

  10. The hydrolysis of polyimides

    NASA Technical Reports Server (NTRS)

    Hoagland, P. D.; Fox, S. W.

    1973-01-01

    Thermal polymerization of aspartic acid produces a polysuccinimide (I), a chain of aspartoyl residues. An investigation was made of the alkaline hydrolysis of the imide rings of (I) which converts the polyimide to a polypeptide. The alkaline hydrolysis of polyimides can be expected to be kinetically complex due to increasing negative charge generated by carboxylate groups. For this reason, a diimide, phthaloyl-DL-aspartoyl-beta-alanine (IIA) was synthesized for a progressive study of the hydrolysis of polyimides. In addition, this diimide (IIA) can be related to thalidomide and might be expected to exhibit similar reactivity during hydrolysis of the phthalimide ring.

  11. How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis

    PubMed Central

    2011-01-01

    Background In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis. Results After expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin

  12. The effect of extracellular polysaccharides on the goethite-surface promoted hydrolysis of organophosphates.

    NASA Astrophysics Data System (ADS)

    Kenney, J. P. L.; Olsson, R.; Giesler, R.; Persson, P.

    2012-04-01

    Organophosphate monoesters comprise a significant fraction of phosphate in soils. In order to access phosphorus needed for growth, plants and microorganisms often require the hydrolysis of large organophosphate molecules. This hydrolysis can be enzymatic or a reaction promoted by contact with an environmental surface. Because phosphorus strongly adsorbs to environmental particles, the fate and transport of phosphorus in the biosphere can be significantly impacted by reactions at the surfaces of these particles. Soil minerals, including the common Fe(III) mineral goethite, have been shown to increase the rate of hydrolysis of organophosphates by acting as catalysts. Many enzyme-secreting microbes and plants can also release extracellular polysaccharides (EPS) into their local environments. EPS is known to adsorb to environmental particles, including goethite. The adsorption of EPS may alter the physico-chemistry of the mineral-phosphate-enzyme system by impacting either the adsorption or enzymatic hydrolysis of organophosphate. Currently, there is little information available regarding the ability of EPS to enhance or inhibit the availability of essential nutrients, such as phosphate, in the environment. In this study we have investigated the hydrolysis of the phosphate monoesters, glucose phosphate (GP) and p-nitrophenyl phosphate (pNPP). To investigate the hydrolysis mechanisms and extent of hydrolysis of phosphate monoesters we studied three systems: 1) abiotic hydrolysis, where monoesters are adsorbed on goethite surfaces; 2) enzymatic hydrolysis where the monoesters are adsorbed to goethite then exposed to an enzyme; and 3) testing whether the presence of alginate, which is used as a model for EPS, can inhibit or enhance the abiotic or enzymatic hydrolysis. To investigate this we used infrared spectroscopy and the ATR sampling technique. Abiotic hydrolysis was examined using goethite as the environmental surface. Adsorption of each monoester to the goethite was

  13. Comparative hydrolysis and fermentation of sugarcane and agave bagasse.

    PubMed

    Hernández-Salas, J M; Villa-Ramírez, M S; Veloz-Rendón, J S; Rivera-Hernández, K N; González-César, R A; Plascencia-Espinosa, M A; Trejo-Estrada, S R

    2009-02-01

    Sugarcane and agave bagasse samples were hydrolyzed with either mineral acids (HCl), commercial glucanases or a combined treatment consisting of alkaline delignification followed by enzymatic hydrolysis. Acid hydrolysis of sugar cane bagasse yielded a higher level of reducing sugars (37.21% for depithed bagasse and 35.37% for pith bagasse), when compared to metzal or metzontete (agave pinecone and leaves, 5.02% and 9.91%, respectively). An optimized enzyme formulation was used to process sugar cane bagasse, which contained Celluclast, Novozyme and Viscozyme L. From alkaline-enzymatic hydrolysis of sugarcane bagasse samples, a reduced level of reducing sugar yield was obtained (11-20%) compared to agave bagasse (12-58%). Selected hydrolyzates were fermented with a non-recombinant strain of Saccharomyces cerevisiae. Maximum alcohol yield by fermentation (32.6%) was obtained from the hydrolyzate of sugarcane depithed bagasse. Hydrolyzed agave waste residues provide an increased glucose decreased xylose product useful for biotechnological conversion. PMID:19000863

  14. The intracellular galactoglycome in Trichoderma reesei during growth on lactose.

    PubMed

    Karaffa, Levente; Coulier, Leon; Fekete, Erzsébet; Overkamp, Karin M; Druzhinina, Irina S; Mikus, Marianna; Seiboth, Bernhard; Novák, Levente; Punt, Peter J; Kubicek, Christian P

    2013-06-01

    Lactose (1,4-0-β-D-galactopyranosyl-D-glucose) is used as a soluble carbon source for the production of cellulases and hemicellulases for-among other purposes-use in biofuel and biorefinery industries. The mechanism how lactose induces cellulase formation in T. reesei is enigmatic, however. Previous results from our laboratory raised the hypothesis that intermediates from the two galactose catabolic pathway may give rise to the accumulation of intracellular oligogalactosides that could act as inducer. Here we have therefore used high-performance anion-exchange chromatography-mass spectrometry to study the intracellular galactoglycome of T. reesei during growth on lactose, in T. reesei mutants impaired in galactose catabolism, and in strains with different cellulase productivities. Lactose, allo-lactose, and lactulose were detected in the highest amounts in all strains, and two trisaccharides (Gal-β-1,6-Gal-β-1,4-Glc/Fru and Gal-β-1,4-Gal-β-1,4-Glc/Fru) also accumulated to significant levels. Glucose and galactose, as well as four further oligosaccharides (Gal-β-1,3/1,4/1,6-Gal; Gal-β-1,2-Glc) were only detected in minor amounts. In addition, one unknown disaccharide (Hex-β-1,1-Hex) and four trisaccharides were also detected. The accumulation of the unknown hexose disaccharide was shown to correlate with cellulase formation in the improved mutant strains as well as the galactose pathway mutants, and Gal-β-1,4-Gal-β-1,4-Glc/Fru and two other unknown hexose trisaccharides correlated with cellulase production only in the pathway mutants, suggesting that these compounds could be involved in cellulase induction by lactose. The nature of these oligosaccharides, however, suggests their formation by transglycosylation rather than by glycosyltransferases. Based on our results, the obligate nature of both galactose catabolic pathways for this induction must have another biochemical basis than providing substrates for inducer formation. PMID:23299458

  15. Comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates.

    PubMed

    Chandra, Richard P; Ewanick, Shannon M; Chung, Pablo A; Au-Yeung, Kathy; Del Rio, Luis; Mabee, Warren; Saddler, Jack N

    2009-08-01

    Fiber size analysis, water retention value, and Simons' stain measurements were assessed for their potential to predict the susceptibility of a given substrate to enzymatic hydrolysis. Slight modifications were made to the fiber size analysis and water retention protocols to adapt these measurements to evaluate substrates for cellulolytic hydrolysis rather than pulps for papermaking. Lodgepole pine was pretreated by the steam and ethanol-organosolv processes under varying conditions. The Simons' stain procedure proved to be an effective method for indicating the potential ease of enzymatic hydrolysis of substrates pretreated by either process or when the pretreatment conditions were altered. PMID:19357812

  16. The cargo and the transport system: secreted proteins and protein secretion in Trichoderma reesei (Hypocrea jecorina).

    PubMed

    Saloheimo, Markku; Pakula, Tiina M

    2012-01-01

    Trichoderma reesei (Hypocrea jecorina) is an efficient cell factory for protein production that is exploited by the enzyme industry. Yields of over 100 g secreted protein l(-1) from industrial fermentations have been reported. In this review we discuss the spectrum of proteins secreted by T. reesei and the studies carried out on its protein secretion system. The major enzymes secreted by T. reesei under production conditions are those degrading plant polysaccharides, the most dominant ones being the major cellulases, as demonstrated by the 2D gel analysis of the secretome. According to genome analysis, T. reesei has fewer genes encoding enzymes involved in plant biomass degradation compared with other fungi with sequenced genomes. We also discuss other T. reesei secreted enzymes and proteins that have been studied, such as proteases, laccase, tyrosinase and hydrophobins. Investigation of the T. reesei secretion pathway has included molecular characterization of the pathway components functioning at different stages of the secretion process as well as analysis of the stress responses caused by impaired folding or trafficking in the pathway or by expression of heterologous proteins. Studies on the transcriptional regulation of the secretory pathway have revealed similarities, but also interesting differences, with other organisms, such as a different induction mechanism of the unfolded protein response and the repression of genes encoding secreted proteins under secretion stress conditions. PMID:22053009

  17. Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei.

    PubMed

    Geng, Alei; Zou, Gen; Yan, Xing; Wang, Qianfu; Zhang, Jun; Liu, Fanghua; Zhu, Baoli; Zhou, Zhihua

    2012-11-01

    A metagenomic fosmid library containing 1 × 10(5) clones was constructed from a biogas digester fed with pig ordure and rice straw. In total, 121 clones with activity of 4-methylumbelliferyl-cellobiosidase were screened from the metagenomic library. A novel GH5 cellulase gene exo2b was identified from a sequenced clone EXO02C10 and expressed in Escherichia coli BL21. The corresponding recombinant Exo2b protein showed high specific activity toward both carboxymethylcellulose (CMC; 260 U/mg protein) and β-D-glucan from barley (849 U/mg), with an optimal pH and temperature of 7.5 and 58 °C, respectively. Exo2b showed stable activity at a wide pH range from 5.5 to 9.0 and was highly thermostable at 60 °C in the presence of 60 mM cysteine. Residual activity was maintained at nearly 100% when Exo2b was incubated at 60 °C for 15 h. A thin-layer chromatography analysis of the hydrolysis products confirmed that Exo2b was an endo-β-1,4-glucanase and it could also produce oligosaccharide smaller than cellotetraose. The fragment encoding the Exo2b catalytic domain was then fused with the cbh1 gene from Trichoderma reesei, and the fused gene was successfully expressed in T. reesei Rut-C30. Compared to that of the parent strain, the filter paper activity and CMCase activity of the secreted proteins of a selected transformant A1 increased by 24% and 18%, respectively. Besides, the glucose concentration from the hydrolysis of pretreated corn stover by the A1 secreted proteins increased by 19.8%. The present study demonstrated the potential application of metagenome originated cellulase genes to modify cellulase producing fungi. PMID:22270237

  18. Polykaryon formation using a swollen conidium of Trichoderma reesei.

    PubMed

    Toyama, Hideo; Yano, Makiko; Hotta, Takeshi

    2004-01-01

    The cellulolytic fungus, Trichoderma has oval and mononucleate conidia. When these conidia are incubated in a liquid medium, they begin to swell and their shape becomes spherical followed by an increase in inner space. In such swollen conidia, it is possible to produce a larger autopolyploid nucleus using a mitotic arrester compared with the case of the original conidia. In this study, polykaryon formation was attempted using these swollen conidia. Dried mature green conidia of Trichoderma reesei QM6a (IFO 31326) were incubated in Mandel's medium in order to swell. The swollen conidia were treated with a mitotic arrester, colchicine, for autopolyploidization. After autopolyploidization, polykaryon formation was carried out using the swollen conidia. After the treatment, multiple smaller nuclei whose diameter was almost the same as that of the original strain were generated from an autopolyploid nucleus in a swollen conidium. A cellulase hyperproducer without decrease in growth rate could be selected using such swollen conidia. PMID:15054260

  19. Systems biological approaches towards understanding cellulase production by Trichoderma reesei.

    PubMed

    Kubicek, Christian P

    2013-01-20

    Recent progress and improvement in "-omics" technologies has made it possible to study the physiology of organisms by integrated and genome-wide approaches. This bears the advantage that the global response, rather than isolated pathways and circuits within an organism, can be investigated ("systems biology"). The sequencing of the genome of Trichoderma reesei (teleomorph Hypocrea jecorina), a fungus that serves as a major producer of biomass-degrading enzymes for the use of renewable lignocellulosic material towards production of biofuels and biorefineries, has offered the possibility to study this organism and its enzyme production on a genome wide scale. In this review, I will highlight the use of genomics, transcriptomics, proteomics and metabolomics towards an improved and novel understanding of the biochemical processes that involve in the massive overproduction of secreted proteins. PMID:22750088

  20. Production of Trichoderma reesei cellulases on glucose-containing media.

    PubMed Central

    Nakari-Setälä, T; Penttilä, M

    1995-01-01

    The filamentous fungus Trichoderma reesei was shown to secrete active cellobiohydrolase I and the endoglucanase I catalytic core domain into the culture medium when the fungus was grown on glucose-containing medium. The expression of the proteins was driven by the promoters of the elongation factor 1 alpha, tef1, and the unidentified gene for cDNA1. The cDNA1 promoter gave the best yields. The highest amounts of cellobiohydrolase I and the endoglucanase I core, being 50 to 100 mg/liter, accounted for more than half of the total protein secreted by the fungus. The levels obtained with the tef1 promoter were 20 to 50 times lower. PMID:7487002