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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. Precipitation of Trichoderma reesei commercial cellulase preparations under standard enzymatic hydrolysis conditions for lignocelluloses.

    PubMed

    Chylenski, Piotr; Felby, Claus; Østergaard Haven, Mai; Gama, Miguel; Selig, Michael J

    2012-08-01

    Comparative studies between commercial Trichoderma reesei cellulase preparations show that, depending on the preparation and loading, total protein precipitation can be as high as 30 % under standard hydrolysis conditions used for lignocellulosic materials. ATR-IR and SDS-PAGE data verify precipitates are protein-based and contain key cell wall hydrolyzing enzymes. Precipitation increased considerably with incubation temperature; roughly 50-150 % increase from 40 to 50 °C and 800 % greater at 60 °C. All of the reported protein losses translated into significant, and often drastic, losses in activity on related 4-nitrophenyl substrates. In addition, supplementation with the non-ionic surfactant PEG 6,000 decreased precipitation up to 80 % in 24 h precipitation levels. Protein precipitation is potentially substantial during enzymatic hydrolysis of lignocelluloses and should be accounted for during lignocellulose conversion process design, particularly when enzyme recycling is considered.

  3. 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.

  4. 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.

  5. Kinetics of cellobiose hydrolysis using cellobiase composites from Trichoderma reesei and Aspergillus niger

    SciTech Connect

    Grous, W.; Converse, A.; Grethlein, H.; Lynd, L.

    1985-01-01

    The enzymatic hydrolysis of cellulose to glucose involves the formation of cellobiose as an intermediate. It has been found necessary to add cellobiase from Aspergillus niger (NOVO) to the cellobiase component of Trichoderma reesei mutant Rut C-30 (Natick) cellulase enzymes in order to obtain after 48 h complete conversion of the cellobiose formed in the enzymatic hydrolysis of biomass. This study of the cellobiase activity of these two enzyme sources was undertaken as a first step in the formation of a kinetic model for cellulose hydrolysis that can be used in process design. In order to cover the full range of cellobiose concentrations, it was necessary to develop separate kinetic parameters for high- and low-concentration ranges of cellobiose for the enzymes from each organism. Competitive glucose inhibition was observed with the enzymes from both organisms. Substrate inhibition was observed only with the A. niger enzymes.

  6. Enzymatic hydrolysis of cellulose and various pretreated wood fractions

    SciTech Connect

    Saddler, J.N.; Brownell, H.H.; Clermont, L.P.; Levitin, N.

    1982-06-01

    Three strains of Trichoderma-Trichoderma reesei C30, Trichoderma reesei QM9414, and Trichoderma species E58-were used to study the enzymatic hydrolysis of pretreated wood substrates. Each of the culture filtrates was incubated with a variety of commercially prepared cellulose substrates and pretreated wood substrates. Solka floc was the most easily degraded commercial cellulose. The enzyme accessibility of steam-exploded samples which has been alkali extracted and then stored wet decreased with the duration of the steam treatment. Air drying reduced the extent of hydrolysis of all the samples but had a greater effect on the samples which had previously shown the greatest hydrolysis. Mild pulping using 2% chlorite increased the enzymatic hydrolysis of all the samples. Steam explosion was shown to be an excellent pretreatment method for aspen wood and was much superior to dilute nitric acid pretreatment. The results indicate that the distribution of the lignin as well as the surface area of the cellulosic substrate are important features in enzymatic hydrolysis. (Refs 17).

  7. 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.

  8. Enzymatic hydrolysis of cellulose to glucose: a report on the Natick Program. Final report

    SciTech Connect

    Not Available

    1981-09-01

    Contributions of Natick to enzymatic hydrolysis and the development of cellulase technologies are summarized. Their impact on the present economics of a commercial process is highlighted. Discussion is presented under the headings: microbial strains; growth and enzyme production in Trichoderma reesei; enzyme properties, analysis and assay; hydrolysis substrates; pretreatment; hydrolysis; process development and economics; and outlook and future developments. 99 references, 11 figures, 4 tables. (DMC)

  9. Partial acid hydrolysis of poplar wood as a pretreatment for enzymatic hydrolysis

    SciTech Connect

    Knappert, D.; Grethlein, H.; Converse, A.

    1981-01-01

    Partial acid hydrolysis was studied as a pretreatment to enhance glucose yields from enzymatic hydrolysis of poplar. The pretreatments were carried out in a continuous flow reactor at temperatures ranging from 162 to 222/sup 0/C, acid concentrations ranging from 0 to 1.5%, and treatment times from 3.6 to 12.7 s. The pretreated slurries were hydrolyzed with Trichoderma reesei C30 cellulase at 50/sup 0/C and a pH of 4.8 for 48 h. Increased yields of glucose were achieved when poplar was pretreated at temperatures higher than 180/sup 0/C. By increasing the cellobiase activity of the cellulase with the addition of NOVO cellobiase, in some cases 100% of the potential glucose content of the substrate was converted to glucose after only 24 h of enzymatic hydrolysis.

  10. 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

  11. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.

    PubMed

    Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte

    2014-07-14

    An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Kinetic study of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

    SciTech Connect

    Lee, Y.H.; Fan, L.T.

    1982-01-01

    A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein) adsorption, either apparent or intrinsic, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the hydrolysis depends on the initial crystallinity index. Several semiempirical equations have been derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present results appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose. (Refs. 31).

  18. Enzymatic hydrolysis of poly(ethylene furanoate).

    PubMed

    Pellis, Alessandro; Haernvall, Karolina; Pichler, Christian M; Ghazaryan, Gagik; Breinbauer, Rolf; Guebitz, Georg M

    2016-10-10

    The urgency of producing new environmentally-friendly polyesters strongly enhanced the development of bio-based poly(ethylene furanoate) (PEF) as an alternative to plastics like poly(ethylene terephthalate) (PET) for applications that include food packaging, personal and home care containers and thermoforming equipment. In this study, PEF powders of various molecular weights (6, 10 and 40kDa) were synthetized and their susceptibility to enzymatic hydrolysis was investigated for the first time. According to LC/TOF-MS analysis, cutinase 1 from Thermobifida cellulosilytica liberated both 2,5-furandicarboxylic acid and oligomers of up to DP4. The enzyme preferentially hydrolyzed PEF with higher molecular weights but was active on all tested substrates. Mild enzymatic hydrolysis of PEF has a potential both for surface functionalization and monomers recycling. PMID:26854948

  19. Reactor optimization for enzymatic hydrolysis of cellulose

    SciTech Connect

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

    1982-01-01

    Enzymatic hydrolysis of cellulose furnishes sugar which can be subsequently fermented to ethanol. The production of such sugar at relatively low cost is essential for commercially viable production of ethanol. Many processes have been developed for converting cellulosic materials to sugar, and their economic feasibility has been analyzed; however, relatively little has been done to optimize such processes. A comprehensive mechanistic kinetic model for enzymatic degradation was established previously; it takes into account the structure of cellulose, mode of action of celluloytic enzyme, and mode of interaction between the enzyme and the cellulosic substrate. In the present work this model has been applied to the optimal design of cellulose hydrloysis reactors. Both batch and continously stirred reactors have been considered for this purpose. The fractional contributions of various cost parameters to the production cost have been estimated. The sensitivity of sugar cost to the important cost parameters, such as raw material and enzyme costs, have been examined. 8 figures, 7 tables.

  20. 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

  1. 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

  2. Enzymatic hydrolysis of corn stover process development and evaluation

    SciTech Connect

    Perez, J.; Wilke, C.R.; Blanch, H.W.

    1981-12-01

    The hydrolysis of acid treated corn stover with cellulase from T. reesei Rut-C-30 was evaluated. Experiments were conducted with substrate concentrations of 5 to 25% by weight, enzyme activities of 0.5 to 7 IU/ml and residence times of 24 to 48 hours. Maximum conversion was 55% for specific cellulase activity of 25 to 30 IU/g. Optimum cellobiase activity for minimum cellobiose production was found to be approximately 1.8 cellobiase units to 1 FPA unit. Hydrolysis experiments with steam exploded corn stover led to a maximum conversion of 80%, significantly higher than the results obtained for acid treated substrate. Steam exploded corn stover was demonstrated as a suitable carbon source for growth of T. reesei in submerged cultures.

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

    PubMed

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

    2015-01-01

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

  4. Effects of substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw.

    PubMed

    Rosgaard, Lisa; Andric, Pavle; Dam-Johansen, Kim; Pedersen, Sven; Meyer, Anne S

    2007-10-01

    In this study, the applicability of a "fed-batch" strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low viscosity during hydrolysis, and high glucose concentrations. An enzyme system comprising Celluclast and Novozyme 188, a commercial cellulase product derived from Trichoderma reesei and a beta-glucosidase derived from Aspergillus niger, respectively, was used for the enzymatic hydrolysis. The highest final glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates of conversion varied in the different reactions. Rapid cellulose degradation was accompanied by rapid decreases in viscosity before addition of extra substrate, but when extra substrate or substrate plus enzymes were added, the viscosities of the slurries increased and the hydrolytic efficiencies decreased temporarily.

  5. 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.

  6. 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

  7. Real-time observation of the swelling and hydrolysis of a single crystalline cellulose fiber catalyzed by cellulase 7B from Trichoderma reesei.

    PubMed

    Wang, Jingpeng; Quirk, Amanda; Lipkowski, Jacek; Dutcher, John R; Hill, Christopher; Mark, Adam; Clarke, Anthony J

    2012-06-26

    The biodegradation of cellulose involves the enzymatic action of cellulases (endoglucanases), cellobiohydrolases (exoglucanases), and β-glucosidases that act synergistically. The rate and efficiency of enzymatic hydrolysis of crystalline cellulose in vitro decline markedly with time, limiting the large-scale, cost-effective production of cellulosic biofuels. Several factors have been suggested to contribute to this phenomenon, but there is considerable disagreement regarding the relative importance of each. These earlier investigations were hampered by the inability to observe the disruption of crystalline cellulose and its subsequent hydrolysis directly. Here, we show the application of high-resolution atomic force microscopy to observe the swelling of a single crystalline cellulose fiber and its-hydrolysis in real time directly as catalyzed by a single cellulase, the industrially important cellulase 7B from Trichoderma reesei. Volume changes, the root-mean-square roughness, and rates of hydrolysis of the surfaces of single fibers were determined directly from the images acquired over time. Hydrolysis dominated the early stage of the experiment, and swelling dominated the later stage. The high-resolution images revealed that the combined action of initial hydrolysis followed by swelling exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of the fiber structure and the exposure of microfibrils at the fiber surface. Both the hydrolysis and swelling were catalyzed by the native cellulase; under the same conditions, its isolated carbohydrate-binding module did not cause changes to crystalline cellulose. We anticipate that the application of our AFM-based analysis on other cellulolytic enzymes, alone and in combination, will provide significant insight into the process of cellulose biodegradation and greatly facilitate its application for the efficient and economical production of cellulosic ethanol.

  8. 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

  9. 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%.

  10. Expression of Trichoderma reesei β-mannanase in tobacco chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis.

    PubMed

    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

  11. 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

  12. 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

  13. Adsorption and synergism of cellobiohydrolase I and II of Trichoderma reesei during hydrolysis of microcrystalline cellulose

    SciTech Connect

    Medve, J.; Tjerneld, F. . Dept. of Biochemistry); Staahlberg, J. . Dept. of Molecular Biology)

    1994-11-05

    Hydrolysis of microcrystalline cellulose (Avicel) by cellobiohydrolase I and II (CBH I and II) from Trichoderma reesei has been studied. Adsorption and synergism of the enzymes were investigated. Experiments were performed at different temperatures and enzyme/substrate ratios using CBH I and CBH II alone and in reconstituted equimolar mixtures. Fast protein liquid chromatography (FPLC) analysis was found to be an accurate and reproducible method to follow the enzyme adsorption. A linear correlation was found between the conversion and the amount of adsorbed enzyme when Avicel was hydrolyzed by increasing amounts of CBH I and/or CBH II. CBH I had lower specific activity compared to CBH II although, over a wide concentration range, more CBH I was adsorbed than CBH II. Synergism between the cellobiohydrolases during hydrolysis of the amorphous fraction of Avicel showed a maximum as a function of total enzyme concentration. Synergism measured as a function of bound enzyme showed a continuous increase, which indicates that by decreasing the distance between the two enzymes the synergism is enhanced. The adsorption process for both enzymes was slow. Depending on the enzyme/substrate ratio it took 30--90 min to reach 95% of the equilibrium binding. The amount of bound enzyme decreased with increasing temperature. The two enzymes compete for the adsorption sites but also bind to specific sites. Stronger competition for adsorption sites was shown by CBH I.

  14. Effect of Ultrasonic Frequency on Enzymatic Hydrolysis of Cellulose

    NASA Astrophysics Data System (ADS)

    Yasuda, Keiji; Kato, Daiki; Xu, Zheng; Sakka, Makiko; Sakka, Kazuo

    2010-07-01

    The effect of ultrasonic frequency on the enzymatic hydrolysis of cellulose was examined. As the cellulose and enzyme, needle unbleached kraft pulp and cellulase were used. In the cases of the horn-type transducer at 20 kHz and the plate-type transducer at 28 kHz, the enzymatic hydrolysis was accelerated by ultrasonic irradiation. Total sugar concentration linearly increased with ultrasonic intensity. On the other hand, in the case of the plate-type transducer at 500 kHz, the enzymatic hydrolysis was inhibited. Total sugar concentration decreased with increasing ultrasonic intensity.

  15. 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

  16. 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

  17. 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.

  18. Comparison of different pretreatment strategies for enzymatic hydrolysis of wheat and barley straw.

    PubMed

    Rosgaard, Lisa; Pedersen, Sven; Meyer, Anne S

    2007-12-01

    In biomass-to-ethanol processes a physico-chemical pretreatment of the lignocellulosic biomass is a critical requirement for enhancing the accessibility of the cellulose substrate to enzymatic attack. This report evaluates the efficacy on barley and wheat straw of three different pretreatment procedures: acid or water impregnation followed by steam explosion versus hot water extraction. The pretreatments were compared after enzyme treatment using a cellulase enzyme system, Celluclast 1.5 L from Trichoderma reesei, and a beta-glucosidase, Novozyme 188 from Aspergillus niger. Barley straw generally produced higher glucose concentrations after enzymatic hydrolysis than wheat straw. Acid or water impregnation followed by steam explosion of barley straw was the best pretreatment in terms of resulting glucose concentration in the liquid hydrolysate after enzymatic hydrolysis. When the glucose concentrations obtained after enzymatic hydrolyses were related to the potential glucose present in the pretreated residues, the highest yield, approximately 48% (g g-1), was obtained with hot water extraction pretreatment of barley straw; this pretreatment also produced highest yields for wheat straw, producing a glucose yield of approximately 39% (g g-1). Addition of extra enzyme (Celluclast 1.5 L+Novozyme 188) during enzymatic hydrolysis resulted in the highest total glucose concentrations from barley straw, 32-39 g L-1, but the relative increases in glucose yields were higher on wheat straw than on barley straw. Maldi-TOF MS analyses of supernatants of pretreated barley and wheat straw samples subjected to acid and water impregnation, respectively, and steam explosion, revealed that the water impregnated + steam-exploded samples gave a wider range of pentose oligomers than the corresponding acid-impregnated samples.

  19. Ultrasound enhanced enzymatic hydrolysis of Parthenium hysterophorus: A mechanistic investigation.

    PubMed

    Singh, Shuchi; Agarwal, Mayank; Bhatt, Aditya; Goyal, Arun; Moholkar, Vijayanand S

    2015-09-01

    This study has attempted to establish the mechanism of the ultrasound-induced enhancement of enzymatic hydrolysis of pretreated and delignified biomass of Parthenium hysterophorus. A dual approach of statistical optimization of hydrolysis followed by application of sonication at optimum conditions has been adopted. The kinetics of hydrolysis shows a marked 6× increase with sonication, while net sugar yield shows marginal rise of ∼ 20%. The statistical experimental design reveals the hydrolysis process to be enzyme limited. Profile of sugar yield in ultrasound-assisted enzymatic hydrolysis has been analyzed using HCH-1 model coupled with Genetic Algorithm optimization. The trends in the kinetic and physiological parameters of HCH-1 model reveal that sonication enhances enzyme/substrate affinity and reaction velocity of hydrolysis. The product inhibition of enzyme in all forms (free, adsorbed, complexed) also reduces with ultrasound. These effects are attributed to intense micro-convection induced by ultrasound and cavitation in the liquid medium.

  20. Preparation and enzymatic hydrolysis of nanoparticles made from single xyloglucan polysaccharide chain.

    PubMed

    Mkedder, Ilham; Travelet, Christophe; Durand-Terrasson, Amandine; Halila, Sami; Dubreuil, Frédéric; Borsali, Redouane

    2013-05-15

    In this work, polysaccharide nanoparticles based on tamarind seeds xyloglucan are prepared, analyzed in term of characteristic sizes and morphology, and degraded by the action of a glycoside-hydrolase. Obtained in an aqueous NaNO2 solution (0.1M), these unaggregated nanoparticles have a characteristic diameter of ca. 60 nm (DLS, AFM and TEM measures). They are not compact, but highly swollen and look like hyperbranched and dendrimer-like (soft sphere model) structures. This observation is coherent with the native structure of the xyloglucan macromolecules which are themselves branched. The enzymatic hydrolysis by cellulase of Trichoderma reesei of the xyloglucan nanoparticles is investigated. In particular, the apparent mass molecular weight drastically decreases meaning that the xyloglucan nanoparticles are effectively fully hydrolyzed by the endo-β-(1,4)-glucanase. Furthermore, we observe that the enzyme has to uncoil the nanoparticles before cutting the β-(1→4) bonds and digesting the xyloglucan. PMID:23544652

  1. 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...

  2. Enzymatic hydrolysis of plant polysaccharides: substrates for fermentation.

    PubMed

    Dekker, R F

    1989-01-01

    The enzymatic hydrolysis of plant carbohydrate polymers is discussed with particular emphasis on lignocellulose. The polysaccharides include starch, inulin, cellulose and the hemicelluloses, i.e., the heteroxylans and glucomannans. Commercial operations exist for the enzymatic hydrolysis of starch and its fermentation into chemicals such as ethanol. Enzymatic hydrolysis of lignocellulose is more complex and the enzymes are rather expensive to produce, which currently precludes the commercial processing of lignocellulosic materials. The bioconversion of lignocellulose consists of 4 process steps: pretreatment, enzyme production, enzymatic saccharification and fermentation. Except for the last step, each of these process steps is discussed. The discussion is highlighted with examples of lignocellulosic waste materials (e.g., sugar cane and a hardwood and softwood sawdust) which are of potential use in a bioconversion process for providing sugar hydrolysates that can serve as fermentation substrates.

  3. 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.

  4. 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

  5. Epidemic based modeling of enzymatic hydrolysis of lignocellulosic biomass.

    PubMed

    Tai, Chao; Arellano, Maria G; Keshwani, Deepak R

    2014-01-01

    An epidemic based model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, dilute sulfuric acid pretreated corn stover. The process of substrate getting adsorbed and digested by enzyme was simulated as susceptibles getting infected by viruses and becoming removed and recovered. This model simplified the dynamic enzyme "infection" process and the catalysis of cellulose into a two-parameter controlled, enzyme behavior guided mechanism. Furthermore, the model incorporates the adsorption block by lignin and inhibition effects on cellulose catalysis. The model satisfactorily predicted the enzyme adsorption and hydrolysis, negative role of lignin, and inhibition effects over hydrolysis for a broad range of substrate and enzyme loadings. Sensitivity analysis was performed to evaluate the incorporation of lignin and other inhibition effects. Our model will be a useful tool for evaluating the effects of parameters during hydrolysis and guide a design strategy for continuous hydrolysis and the associated process control.

  6. 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…

  7. 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...

  8. 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.)

  9. Enzymatic saccharification of pretreated wheat straw: comparison of solids-recycling, sequential hydrolysis and batch hydrolysis.

    PubMed

    Pihlajaniemi, Ville; Sipponen, Satu; Sipponen, Mika H; Pastinen, Ossi; Laakso, Simo

    2014-02-01

    In the enzymatic hydrolysis of lignocellulose materials, the recycling of the solid residue has previously been considered within the context of enzyme recycling. In this study, a steady state investigation of a solids-recycling process was made with pretreated wheat straw and compared to sequential and batch hydrolysis at constant reaction times, substrate feed and liquid and enzyme consumption. Compared to batch hydrolysis, the recycling and sequential processes showed roughly equal hydrolysis yields, while the volumetric productivity was significantly increased. In the 72h process the improvement was 90% due to an increased reaction consistency, while the solids feed was 16% of the total process constituents. The improvement resulted primarily from product removal, which was equally efficient in solids-recycling and sequential hydrolysis processes. No evidence of accumulation of enzymes beyond the accumulation of the substrate was found in recycling. A mathematical model of solids-recycling was constructed, based on a geometrical series.

  10. 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.

  11. Membrane reactor for enzymatic hydrolysis of cellobiose

    SciTech Connect

    Hong, J.; Tsao, G.T.; Wankat, P.C.

    1981-07-01

    A pressurized, stirred vessel attached with an ultrafiltration membrane was used as a membrane reactor. Cellobiose hydrolysis by cellobiase was carried out and theoretically analyzed in terms of steady-state conversion and flow rate through the membrane. When the flow rate exceeds a critical value, a significant fraction of the enzyme inside the reactor is localized in the concentration polarization layer where shear from stirring is high. Consequently, enzyme deactivation inside the concentration polarization layer is accelerated and the conversion decreased due to an exchange of active enzyme in bulk with deactivated enzyme in the polarization layer via convection and back diffusion. Successful operation can be obtained at flow rates lower than the critical point to avoid the polarization and thus the deactivation. It is shown that 6.5 L of 2mg/mL of cellobiose solution is hydrolyzed to glucose with a conversion of 91% in 20 hours with 1.617 mg of cellobiase enzyme in a reactor attached with a PM 10 membrane of an effective surface area of 39.2 sq.cm. (Refs. 17).

  12. Enzymatic hydrolysis of cellulosic materials: a kinetic study

    SciTech Connect

    Beltrame, P.L.; Carniti, P.; Focher, B.; Marzetti, A.; Sarto, V.

    1984-01-01

    A kinetic study of the enzymatic hydrolysis of two celluloses with different structural features was performed at various temperatures (26-50/sup 0/C). The enzymatic system consisted of three types of enzymes: E/sub 1/-..beta..-1,4-glucan glucanohydrolase; E/sub 2/-..beta..-1,4-glucan cellobiohydrolase; and E/sub 3/-..beta..-glucosidase. A mathematical model for the mechanism of the hydrolysis of cellulosic materials catalyzed by a multienzymatic system was checked and a good rationalization of the experimental results was achieved. Uncompetitive and competitive glucose inhibition on E/sub 1/ and E/sub 2/, respectively, appeared to occur for both substrates. Inhibition by cellobiose was checked at 34/sup 0/C on one substrate. The V/sub max/, K/sub m/, and glucose inhibition constants were optimized and their dependence on temperature determined.

  13. 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

  14. Determining yields in high solids enzymatic hydrolysis of biomass.

    PubMed

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

    2009-05-01

    As technologies for utilizing biomass for fuel and chemical production continue to improve, enzymatic hydrolysis can be run at still higher solids concentrations. For hydrolyses that initially contain little or no free water (10-40% total solids, w/w), the saccharification of insoluble polymers into soluble sugars involves changes of volume, density, and proportion of insoluble solids. This poses a new challenge when determining the degree of hydrolysis (conversion yield). Experiments have shown that calculating the yield from the resulting sugar concentration in the supernatant of the slurry and using the assumed initial volume leads to significant overestimations of the yield. By measuring the proportion of insoluble solids in the slurry as well as the sugar concentration and specific gravity of the aqueous phase, it is possible to precisely calculate the degree of conversion. The discrepancies between the different ways of calculating yields are demonstrated along with a nonlaborious method for approximating yields in high solids hydrolysis. PMID:18836690

  15. 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

  16. Mechanism of the positive effect of poly(ethylene glycol) addition in enzymatic hydrolysis of steam pretreated lignocelluloses.

    PubMed

    Sipos, Bálint; Szilágyi, Mátyás; Sebestyén, Zoltán; Perazzini, Raffaella; Dienes, Dóra; Jakab, Emma; Crestini, Claudia; Réczey, Kati

    2011-11-01

    The efficiency of enzymatic hydrolysis of lignocellulses can be increased by addition of surfactants and polymers, such as poly(ethylene glycol) (PEG). The effect of PEG addition on the cellulase adsorption was tested on various steam pretreated lignocellulose substrates (spruce, willow, hemp, corn stover, wheat straw, sweet sorghum bagasse). A positive effect of PEG addition was observed, as protein adsorption has decreased and free enzyme activities (FP, β-glucosidase) have increased due to the additive. However, the degree of enhancement differed among the substrates, being highest on steam pretreated spruce. Results of lignin analysis (pyrolysis-GC/MS, (31)P NMR) suggest that the effect of PEG addition is in connection with the amount of unsubstituted phenolic hydroxyl groups of lignin in the substrate. Adsorption experiments using two commercial enzyme preparations, Celluclast 1.5L (Trichoderma reesei cellulase) and Novozym 188 (Aspergillus niger β-glucosidase) suggested that enzyme origins affected on the adsorptivity of β-glucosidases.

  17. Enzymatic hydrolysis: a method in alleviating legume allergenicity.

    PubMed

    Kasera, Ramkrashan; Singh, A B; Lavasa, S; Prasad, Komarla Nagendra; Arora, Naveen

    2015-02-01

    Legumes are involved in IgE mediated food allergy in many countries. Avoidance of allergenic food is the only way to avoid symptomatic reaction. The present study investigated the effect of enzymatic hydrolysis on the allergenicity of three legumes - kidney bean (Phaseolus vulgaris), black gram (Vigna mungo) and peanut (Arachis hypogaea). Soluble protein extracts of the study legumes were sequentially treated by Alcalase(®) and Flavourzyme(®). Allergenicity of hydrolysates was then determined by ELISA, immunoblot, stripped basophil histamine release and skin prick test (SPT). Hydrolysis resulted in the loss of all IgE binding fractions determined by immunoblot in the three legumes. Specific IgE binding in ELISA was reduced by 62.2 ± 7.7%, 87.1 ± 9.6% and 91.8 ± 7.2% in the hydrolysates of kidney bean, black gram and peanut, respectively (p < 0.01). The release of histamine was decreased significantly when sensitized basophils were challenged with hydrolysates as compared to raw extracts. Significant reduction in the biopotency of hydrolysates was also observed in SPT where only 1/10 kidney bean-sensitive individuals, 2/6 black gram-sensitive individuals and 1/7 peanut-sensitive individuals were found positive to their respective hydrolysates. In conclusion, enzymatic hydrolysis is effective in attenuating allergenicity of legume proteins and may be employed for preparing hypoallergenic food extracts.

  18. 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).

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

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

    PubMed

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

    2015-09-01

    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

  7. 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

  8. 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

  9. The effect of crystallinity of cellulose on the rate of reducing sugars production by heterogeneous enzymatic hydrolysis.

    PubMed

    Al-Zuhair, Sulaiman

    2008-07-01

    A kinetic model is devised, from the reaction mechanism steps, to predict the rate of reducing sugar production by hydrolysis of two types of cellulose, namely, amorphous carboxymethylcellulose (CMC) and highly crystalline wood shavings, using Aspergillus niger cellulase. Experimental results in a stirred batch reactor at 40 degrees C show that the production of reducing sugar reduced at much shorter times for wood shavings in comparison to CMC at the same initial substrate concentration. The experimental results are used to determine the kinetic parameters of the model equations. The significance of crystallinity was determined using inert fraction coefficient, which is assumed to be constant and equals 0.05 and 0.98 for CMC and wood shavings, respectively. It is shown there is a good agreement between the experimental results and proposed kinetic model predictions. The effect of the inert fraction coefficient on the production of reducing sugar by the enzymatic hydrolysis of cellulose is also determined. It is found that the cellulase used extracted from A. niger is much more sensitive towards the substrate structure in comparison to that extracted from Trichoderma reesei.

  10. Non-ionic surfactants do not consistently improve the enzymatic hydrolysis of pure cellulose.

    PubMed

    Zhou, Yan; Chen, Hongmei; Qi, Feng; Zhao, Xuebing; Liu, Dehua

    2015-04-01

    Non-ionic surfactants have been frequently reported to improve the enzymatic hydrolysis of pretreated lignocellulosic biomass and pure cellulose. However, how the hydrolysis condition, substrate structure and cellulase formulation affect the beneficial action of surfactants has not been well elucidated. In this work, it was found that the enzymatic hydrolysis of pure cellulose was not consistently improved by surfactants. Contrarily, high surfactant concentration, e.g. 5 g/L, which greatly improved the hydrolysis of dilute acid pretreated substrates, actually showed notable inhibition to pure cellulose conversion in the late phase of hydrolysis. Under an optimal hydrolysis condition, the improvement by surfactant was limited, but under harsh conditions surfactant indeed could enhance cellulose conversion. It was proposed that non-ionic surfactants could interact with substrates and cellulases to impact the adsorption behaviors of cellulases. Therefore, the beneficial action of surfactants on pure cellulose hydrolysis is influenced by hydrolysis condition, cellulose structural features and cellulase formulation.

  11. Enzymatic hydrolysis preparation of mono-O-lauroylsucrose via a mono-O-lauroylraffinose intermediate.

    PubMed

    Lu, Yuyun; Yan, Rian; Ma, Xiang; Wang, Yong; Sun, Yuankui; Luo, Zhongming

    2013-10-01

    1'-O-Lauroylsucrose and 6'-O-lauroylsucrose were formed through hydrolysis of the C-6″ galactose group of 1'-O-lauroylraffinose and 6'-O-lauroylraffinose, respectively, in the presence of α-galactosidase. The enzymatic hydrolysis of 1'-O-lauroylraffinose and 6'-O-lauroylraffinose is discussed in detail. Acetic acid-sodium acetate was chosen as the buffer solution of the enzymatic hydrolysis reaction. The optimum conditions for the enzymatic hydrolysis reaction were as follows: buffer solution, pH 3.8; enzymatic time, 48 h; and enzymatic temperature, 37 °C. Under the optimal process conditions, the efficiency of α-galactosidase was ca. 82.6%. The isomers were fully compared in solubility, hydrophile-lipophile balance (HLB) values, critical micelle concentration (CMC), and thermal stability. The results showed that all lauroylsucrose isomers have similar solubilities in polar solvent, HLB values, CMC values, and thermal stabilities.

  12. 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

  13. 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%).

  14. Lignin-based polyoxyethylene ether enhanced enzymatic hydrolysis of lignocelluloses by dispersing cellulase aggregates.

    PubMed

    Lin, Xuliang; Qiu, Xueqing; Yuan, Long; Li, Zihao; Lou, Hongming; Zhou, Mingsong; Yang, Dongjie

    2015-06-01

    Water-soluble lignin-based polyoxyethylene ether (EHL-PEG), prepared from enzymatic hydrolysis lignin (EHL) and polyethylene glycol (PEG1000), was used to improve enzymatic hydrolysis efficiency of corn stover. The glucose yield of corn stover at 72h was increased from 16.7% to 70.1% by EHL-PEG, while increase in yield with PEG4600 alone was 52.3%. With the increase of lignin content, EHL-PEG improved enzymatic hydrolysis of microcrystalline cellulose more obvious than PEG4600. EHL-PEG could reduce at least 88% of the adsorption of cellulase on the lignin film measured by quartz crystal microbalance with dissipation monitoring (QCM-D), while reduction with PEG4600 was 43%. Cellulase aggregated at 1220nm in acetate buffer analyzed by dynamic light scattering. EHL-PEG dispersed cellulase aggregates and formed smaller aggregates with cellulase, thereby, reduced significantly nonproductive adsorption of cellulase on lignin and enhanced enzymatic hydrolysis of lignocelluloses.

  15. 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.

  16. Combined steam pretreatment and enzymatic hydrolysis of starch-free wheat fibers.

    PubMed

    Palmarola-Adrados, Beatriz; Galbe, Mats; Zacchi, Guido

    2004-01-01

    Steam treatment of an industrial process stream, denoted starch-free wheat fiber, was investigated to improve the formation of monomeric sugars in subsequent enzymatic hydrolysis for further bioconversion into ethanol. The solid fraction in the process stream, derived from a combined starch and ethanol factory, was rich in arabinose (21.1%), xylose (30.1%), and glucose (18.6%), in the form of polysaccharides. Various conditions of steam pretreatment (170-220 degrees C for 5-30 min) were evaluated, and their effect was assessed by enzymatic hydrolysis with 2 g of Celluclast + Ultraflo mixture/100 g of starch-free fiber (SFF) slurry at 5% dry matter (DM). The highest overall sugar yield for the combined steam pretreatment and enzymatic hydrolysis, 52 g/100 g of DM of SFF, corresponding to 74% of the theoretical, was achieved with pretreatment at 190 degrees C for 10 min followed by enzymatic hydrolysis.

  17. Quantification of bound and free enzymes during enzymatic hydrolysis and their reactivities on cellulose and lignocellulose.

    PubMed

    Yu, Zhiying; Jameel, Hasan; Chang, Hou-min; Philips, Richard; Park, Sunkyu

    2013-11-01

    Enzymatic hydrolysis of insoluble biomass is a surface reaction. Part of the enzyme adsorb on the surface of biomass, whereas the others stay in the liquid phase. In this study, three substrates (Avicel cellulose, bleached hardwood pulp, and green-liquor pretreated hardwood pulp) were used to study the reactivity of bound and free enzyme. In a continuous enzymatic hydrolysis, 35-65% initially added enzymes became bound enzymes, which were primarily responsible for enzymatic hydrolysis. The contribution from free enzymes became insignificant after a certain period of reaction time. SDS-PAGE analysis showed that CBH I was significantly decreased in the free enzyme, which might be the reason for the low digestibility of free enzymes due to the loss of synergistic effect. When Tween 80 was added during enzymatic hydrolysis, the digestibility of free enzyme on Avicel was greatly enhanced. However, the benefit of surfactant was not noticeable for lignocellulosic pulps, comparing to Avicel.

  18. Sugars from pine bark by enzymatic hydrolysis. Effect of sodium chlorite treatments

    SciTech Connect

    Vazquez, G.; Parajo, J.C.; Antorrena, G.; Thonart, Ph.; Paquot, M.

    1987-01-01

    For the utilization of polysaccharides from Pinus pinaster bark, both untreated and NaOH-treated samples were delignified to different stages using NaClO/sub 2/ and submitted to enzyme hydrolysis with Trichoderma reesei cellulase and Aspergillus niger beta-glucosidase. In the best conditions samples treated with 1% NaOH for 15 minutes and NaClO/sub 2/ for 7 hours the conversion of polysaccharides into sugars accounts for the 75% of the potential yield, and the glucose obtained amounts to 87% of the theoretical value. 37 references.

  19. 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.

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

    SciTech Connect

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

    1985-01-01

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

  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. 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.

  3. 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...

  4. Influence of major structural features of cellulose on rate of enzymatic hydrolysis

    SciTech Connect

    Fan, L.T.; Lee, Y.H.; Beardmore, D.R.

    1981-02-01

    The rate of enzymatic hydrolysis of cellulose is profoundly affected by the structural features of cellulose, especially by the degree of crystallinity and the specific surface area. This is a report on the results of experiments conducted on cellulosic samples to examine the relationship among their rates of hydrolysis and the two structural parameters.

  5. 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, ...

  6. 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...

  7. Production of xylooligosaccharide from wheat bran by microwave assisted enzymatic hydrolysis.

    PubMed

    Wang, Tseng-Hsing; Lu, Shin

    2013-06-01

    The effective production of xylooligosaccharides (XOS) from wheat bran was investigated. Wheat bran contains rich hemicellulose which can be hydrolyzed by enzyme; the XOS were obtained by microwave assisted enzymatic hydrolysis. To improve the productivity of XOS, repeated microwave assisted enzymatic hydrolysis and activated carbon adsorption method was chosen to eliminate macromolecules in the XOS. On the basis of experimental data, an industrial XOS production process consisting of pretreatment, repeated microwave assisted enzymatic treatment and purification was designed. Using the designed process, 3.2g dry of purified XOS was produced from 50 g dry wheat bran powder.

  8. 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.

  9. 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.

  10. Enhanced enzymatic hydrolysis of rapeseed straw by popping pretreatment for bioethanol production.

    PubMed

    Wi, Seung Gon; Chung, Byung Yeoup; Lee, Yoon Gyo; Yang, Duck Joo; Bae, Hyeun-Jong

    2011-05-01

    The objective of this study was to find a pretreatment process that enhances enzymatic conversion of biomass to sugars. Rapeseed straw was pretreated by two processes: a wet process involving wet milling plus a popping treatment, and a dry process involving popping plus dry milling. The effects of the pretreatments were studied both in terms of structural and compositional changes and change in susceptibility to enzymatic hydrolysis. After application of the wet and dry processes, the amounts of cellulose and xylose in the straw were 37-38% and 14-15%, respectively, compared to 31% and 12% in untreated counterparts. In enzymatic hydrolysis performance, the wet process presented the best glucose yield, with a 93.1% conversion, while the dry process yielded 69.6%, and the un-pretreated process yielded <20%. Electron microscopic studies of the straw also showed a relative increase in susceptibility to enzymatic hydrolysis with pretreatment.

  11. Hydrogel coated monoliths for enzymatic hydrolysis of penicillin G.

    PubMed

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

    2008-08-01

    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(-3) (monolith). 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(-3) (catalyst)), 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 reactor

  12. The pattern of cell wall deterioration in lignocellulose fibers throughout enzymatic cellulose hydrolysis.

    PubMed

    Li, Xinping; Clarke, Kimberley; Li, Kecheng; Chen, Aicheng

    2012-01-01

    Cell wall deterioration throughout enzymatic hydrolysis of cellulosic biomass is greatly affected by the chemical composition and the ultrastructure of the fiber cell wall. The resulting pattern of cell wall deterioration will reveal information on cellulose activity throughout enzymatic hydrolysis. This study investigates the progression and morphological changes in lignocellulose fibers throughout enzymatic hydrolysis, using (transmission electron microscopy) TEM and field emission scanning electron microscopy (FE-SEM). Softwood thermo-mechanical pulp (STMP) and softwood bleached kraft pulp (SBKP), lignocellulose substrates containing almost all the original fiber composition, and with lignin and some hemicellulose removed, respectively, was compared for morphology changes throughout hydrolysis. The difference of conversion between STMP and SBKP after 48 h of enzymatic hydrolysis is 11 and 88%, respectively. TEM images revealed an even fiber cell wall cross section density, with uneven middle lamella coverage in STMP fibers. SKBP fibers exhibited some spaces between cell wall and lamella layers due to the removal of lignin and some hemicellulose. After 1 h hydrolysis in SBKP fibers, there were more changes in the fiber cross-sectional area than after 10 h hydrolysis in STMP fibers. Cell wall degradation was uneven, and originated in accessible cellulose throughout the fiber cell wall. FE-SEM images illustrated more morphology changes in SBKP fibers than STMP fibers. Enzymatic action of STMP fiber resulted in a smoother fiber surface, along with fiber peeling and the formation of ribbon-disjunction layers. SBKP fibers exhibited structural changes such as fiber erosion, fiber cutting, and fiber splitting throughout enzymatic hydrolysis.

  13. 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.

  14. 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...

  15. 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.

  16. Enzymatic hydrolysis of pretreated waste paper--source of raw material for production of liquid biofuels.

    PubMed

    Brummer, Vladimir; Jurena, Tomas; Hlavacek, Viliam; Omelkova, Jirina; Bebar, Ladislav; Gabriel, Petr; Stehlik, Petr

    2014-01-01

    Enzymatic hydrolysis of waste paper is becoming a perspective way to obtain raw material for production of liquid biofuels. Reducing sugars solutions that arise from the process of saccharification are a precursors for following or simultaneous fermentation to ethanol. Different types of waste paper were evaluated, in terms of composition and usability, in order to select the appropriate type of the waste paper for the enzymatic hydrolysis process. Novozymes® enzymes NS50013 and NS50010 were used in a laboratory scale trials. Technological conditions, which seem to be the most suitable for hydrolysis after testing on cellulose pulp and filter paper, were applied to hydrolysis of widely available waste papers - offset paper, cardboard, recycled paper in two qualities, matte MYsol offset paper and for comparison again on model materials. The highest yields were achieved for the cardboard, which was further tested using various pretreatment combinations in purpose of increasing the hydrolysis yields.

  17. Effects of different pretreatment methods on chemical composition of sugarcane bagasse and enzymatic hydrolysis.

    PubMed

    Gao, Yueshu; Xu, Jingliang; Zhang, Yu; Yu, Qiang; Yuan, Zhenhong; Liu, Yunyun

    2013-09-01

    Different pretreatment processes, including liquid hot water (LHW) pretreatment, sodium hydroxide (NaOH) pretreatment, and their combinative pretreatments, were conducted to improve the enzymatic digestibility and sugar recovery from sugarcane bagasse (SCB). LHW pretreatment solubilized over 82% of xylan and 42% of lignin, after which the SCB presented the poorest enzymatic digestibility. NaOH pretreatment could remove 78% of lignin and retain most of glucan. For combinative pretreatments, the sequence of two procedures had a significant effect on the chemical composition, substrate characteristic and the subsequent enzymatic hydrolysis process. LHW-NaOH pretreatment could solubilize over 92% of xylan and remove 76% of lignin, and the highest total sugar recovery of 73% was achieved after 72 h enzymatic hydrolysis. While NaOH-LHW pretreatment, which could remove nearly 84% of lignin, but only solubilize 71% of xylan, showed the highest enzymatic digestibility. The pretreatment efficiency was: NaOH-LHW>NaOH>LHW-NaOH>LHW.

  18. 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.

  19. 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

  20. 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

  1. New miniature stirred-tank bioreactors for parallel study of enzymatic biomass hydrolysis.

    PubMed

    Riedlberger, Peter; Weuster-Botz, Dirk

    2012-02-01

    Many factors strongly influence the enzymatic hydrolysis of biomass to fermentable sugars (feedstock composition, pretreatment, enzymes and enzyme loading). In order to optimize the reaction conditions for the hydrolysis of biomass, an accurate high-throughput bioprocess development tool is mandatory, which enables a parallelization and an easy scale-up. New S-shaped impellers were developed for magnetically inductive driven stirred-tank bioreactors at a 10mL-scale. An efficient and reproducible homogenization was shown at 20% w/w solids loading of microcrystalline cellulose and at, 4-10% with wheat straw in 48 parallel operated stirred-tank bioreactors. The scale-up was successfully validated for the enzymatic hydrolysis of wheat straw suspensions and microcrystalline cellulose mixtures by application of a cellulase complex at a milliliter- and liter-scale. As an example, the parallel stirred-tank bioreactor system was applied for the evaluation of enzymatic batch hydrolyses of plant materials with varying pretreatments.

  2. Screw extrude steam explosion: a promising pretreatment of corn stover to enhance enzymatic hydrolysis.

    PubMed

    Chen, Jingwen; Zhang, Wengui; Zhang, Hongman; Zhang, Qiuxiang; Huang, He

    2014-06-01

    A screw extrude steam explosion (SESE) apparatus was designed and introduced to pretreat corn stover continuously for its following enzymatic hydrolysis. SESE parameters temperature (100, 120, 150°C) and residence time (1, 2, 3min) were investigated. The enzymatic hydrolysis of corn stover pretreated by SESE and steam explosion (SE) process was carried out and analyzed systematically. A serial of analysis methods were established, and the corn stover before/after the pretreatment were characterized by scanning electron microscope (SEM), X-ray Diffraction (XRD) and Thermal Gravity/Derivative Thermal Gravity Analysis (TG/DTG). After treated by SESE pretreatment at the optimum condition (150°C, 2min), the pretreated corn stover exhibited highest enzymatic hydrolysis yield (89%), and rare fermentation inhibitors formed. Characterization results indicated that the highest yield could be attributed to the effective removal of lignin/hemicellulose and destruction of cellulose structure by SESE pretreatment.

  3. 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.

  4. 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.

  5. Enzymatic hydrolysis of ammonia-treated sugar beet pulp.

    PubMed

    Foster, B L; Dale, B E; Doran-Peterson, J B

    2001-01-01

    Sugar beet pulp is a carbohydrate-rich coproduct generated by the table sugar industry. Beet pulp has shown promise as a feedstock for ethanol production using enzymes to hydrolyze polymeric carbohydrates and engineered bacteria to ferment sugars to ethanol. In this study, sugar beet pulp underwent an ammonia pressurization depressurization (APD) pretreatment in which the pulp was exploded by the sudden evaporation of ammonia in a reactor vessel. APD was found to substantially increase hydrolysis efficiency of the cellulose component, but when hemicellulose- and pectin-degrading enzymes were added, treated pulp hydrolysis was no better than the untreated control.

  6. 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...

  7. 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

  8. 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.

  9. Effect of bovine serum albumin (BSA) on enzymatic cellulose hydrolysis.

    PubMed

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

    2013-06-01

    Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ≥90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.

  10. 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...

  11. [Mechanisms and regulation of enzymatic hydrolysis of cellulose in filamentous fungi: classical cases and new models].

    PubMed

    Gutiérrez-Rojas, Ivonne; Moreno-Sarmiento, Nubia; Montoya, Dolly

    2015-01-01

    Cellulose is the most abundant renewable carbon source on earth. However, this polymer structure comprises a physical and chemical barrier for carbon access, which has limited its exploitation. In nature, only a few percentage of microorganisms may degrade this polymer by cellulase expression. Filamentous fungi are one of the most active and efficient groups among these microorganisms. This review describes similarities and differences between cellulase activity mechanisms and regulatory mechanisms controlling gene expression for 3 of the most studied cellulolytic filamentous fungi models: Trichoderma reesei, Aspergillus niger and Aspergillus nidulans, and the recently described model Neurospora crassa. Unlike gene expression mechanisms, it was found that enzymatic activity mechanisms are similar for all the studied models. Understanding the distinctive elements of each system is essential for the development of strategies for the improvement of cellulase production, either by providing the optimum environment (fermentation conditions) or increasing gene expression in these microorganisms by genetic engineering.

  12. Recycle of enzymes and substrate following enzymatic hydrolysis of steam-pretreated aspenwood.

    PubMed

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

    1987-09-01

    The commercial production of chemicals and fuels from lignocellulosic residues by enzymatic means still requires considerable research on both the technical and economic aspects. Two technical problems that have been identified as requiring further research are the recycle of the enzymes used in hydrolysis and the reuse of the re calcitrant cellulose remaining after incomplete hydrolysis. Enzyme recycle is required to lower the cost of the enzymes, while the reuse of the spent cellulose will lower the feedstock cost. The conversion process studied was a combined enzymatic hydrolysis and fermentation (CHF) procedure that utilized the cellulolytic enzymes derived from the fungus Trichoderma harzianum E58 and the yeast Saccharomyces cerevisiae. The rate and extent of hydrolysis and ethanol production was monitored as was the activity and hydrolytic potential of the enzymes remaining in the filtrate after the hydrolysis period. When a commercial cellulose was used as the substrate for a routine 2-day CHF process, 60% of the original treated, water-extracted aspenwood was used as the substrate, only 13% of the original filter paper activity was detected after a similar procedure. The combination of 60% spent enzymes with 40% fresh enzymes resulted in the production of 30% less reducing sugars than the original enzyme mixture. Since 100% hydrolysis of the cellulose portion is seldom accomplished in an enzymatic hydrolysis pro cess, the residual cellulose was used as a substrate for the growth of T. harzianum E58 and production of celulolytic enzymes. The residue remaining after the CHF process was used as a substrate for the production of the cellulolytic enzymes. The production of enzymes from the residue of the Solka Floc hydrolysis was greater than the production of enzymes from the original Solka Floc.

  13. Steam pretreatment of spruce forest residues: optimal conditions for biogas production and enzymatic hydrolysis.

    PubMed

    Janzon, Ron; Schütt, Fokko; Oldenburg, Saskia; Fischer, Elmar; Körner, Ina; Saake, Bodo

    2014-01-16

    Steam refining of non-debarked spruce forest residues was investigated as pretreatment for enzymatic hydrolysis as well as for biogas production. Pretreatment conditions were varied in the range of 190-220 °C, 5-10 min and 0-3.7% SO₂ according to a statistical design. For both applications highest product yields were predicted at 220 °C and 2.4% SO₂, whereas the reaction time had only a minor influence. The conformity of the model results allows the conclusion that enzymatic hydrolysis is a suitable test method to evaluate the degradability of lignocellulosic biomass in the biogas process. In control experiments under optimal conditions the results of the model were verified. The yield of total monomeric carbohydrates after enzymatic hydrolysis was equivalent to 55% of all theoretically available polysaccharides. The corresponding biogas yield from the pretreated wood amounted to 304 mL/gODM. Furthermore, furans produced under optimal process conditions showed no inhibitory effect on biogas production. It can be concluded that steam refining opens the structure of wood, thus improving the enzymatic hydrolysis of the polysaccharides to fermentable monomeric sugars and subsequently enabling a higher and faster production of biogas. Anaerobic fermentation of pretreated wood is a serious alternative to alcoholic fermentation especially when low quality wood grades and residues are used. Anaerobic digestion should be further investigated in order to diversify the biorefinery options for lignocellulosic materials.

  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. 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.

  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. 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...

  18. 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...

  19. 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...

  20. 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.

  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.

  2. Analysis of particle size reduction on overall surface area and enzymatic hydrolysis yield of corn stover.

    PubMed

    Li, Hanjie; Ye, Chenlin; Liu, Ke; Gu, Hanqi; Du, Weitao; Bao, Jie

    2015-01-01

    Particle size of lignocellulose materials is an important factor for enzymatic hydrolysis efficiency. In this study, corn stover was milled and sieved into different size fractions from 1.42, 0.69, 0.34, to 0.21 mm, and the corresponding enzymatic hydrolysis yields were 24.69, 23.96, 25.34, and 26.97 %, respectively. The results indicate that the hydrolysis yield is approximately constant with changing corn stover particle sizes in the experimental range. The overall surface area and the inner pore size measurement show that the overall specific surface area was less than 2 % with the half reduction of particle size due to the greater inner pore surface area. The scanning electron microscope photographs gave direct evidence of the much greater inner pore surface area of corn stover particles. This result provided a reference when a proper size reduction of lignocellulose materials is considered in biorefining operations.

  3. Enhanced enzymatic cellulose hydrolysis by subcritical carbon dioxide pretreatment of sugarcane bagasse.

    PubMed

    Zhang, Hongdan; Wu, Shubin

    2014-04-01

    Most biomass pretreatment processes for sugar production are run at low-solid concentration (<10 wt.%). Subcritical carbon dioxide (CO2) could provide a more sustainable pretreatment medium while using relative high-solid contents (15 wt.%). The effects of subcritical CO2 pretreatment of sugarcane bagasse to the solid and glucan recoveries at different pretreatment conditions were investigated. Subsequently, enzymatic hydrolysis at different hydrolysis time was applied to obtain maximal glucose yield, which can be used for ethanol fermentation. The maximum glucose yield in enzyme hydrolyzate reached 38.5 g based on 100g raw material after 72 h of enzymatic hydrolysis, representing 93.0% glucose in sugarcane bagasse. The enhanced digestibilities of subcritical CO2 pretreated sugarcane bagasse were due to the removal of hemicellulose, which were confirmed by XRD, FTIR, SEM, and TGA analyses.

  4. The improvement of enzymatic hydrolysis efficiency of rape straw and Miscanthus giganteus polysaccharides.

    PubMed

    Swiątek, Karolina; Lewandowska, Małgorzata; Swiątek, Magdalena; Bednarski, Włodzimierz; Brzozowski, Bartosz

    2014-01-01

    The research was carried out with the aim to determine the impact of various combinations of cellulase and hemicellulase preparations on the effectiveness of enzymatic hydrolysis of polysaccharides of rape straw and Miscanthus giganteus after alkaline pretreatment. Their effectiveness was evaluated based on the quantity of saccharides released during enzymatic reaction and yield calculated in respect of the sum of polysaccharides present in native substrates. The complex of preparations produced from Trichoderma longibrachiatum fungi turned out to be the most effective. The study demonstrated a significant effect of xylanases from T. longibrachiatum, the presence of which evoked a 27-45% increase in the effectiveness of polysaccharides hydrolysis compared to the enzymatic complexes without their addition. In addition, results achieved in this study confirmed the necessity of applying the pretreatment in lignocellulose substrates conversion into bioethanol.

  5. 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.)

  6. The comparison of obtaining fermentable sugars from cellulose by enzymatic hydrolysis and fast pyrolysis.

    PubMed

    Jiang, Liqun; Zheng, Anqing; Zhao, Zengli; He, Fang; Li, Haibin; Wu, Nannan

    2016-01-01

    Sugars are one of intermediates in the biological and chemical conversion of biomass. The objective of this study was to make comparison of obtaining fermentable sugars by enzymatic hydrolysis and fast pyrolysis of ball milling pretreated cellulose. After ball milling pretreatment for 0-18h, with the accumulation of alkali and alkali earth metals (from 50.8 to 276.4ppm) and decrease of the crystalline structure (from 89.8% to 10.1%), the hydrolysis yields increased from 23.6% to 56.0% in enzymatic saccharification, while the yields of levoglucosan diminished from 61.5% to 45.6% gradually in fast pyrolysis. Both enzymatic saccharification and fast pyrolysis had unique attractive features and unfavorable limitations. The present research provided a concept for considering choices among the technologies and feedstocks currently available. PMID:26476158

  7. Subcritical CO2 pretreatment of sugarcane bagasse and its enzymatic hydrolysis for sugar production.

    PubMed

    Zhang, Hongdan; Wu, Shubin

    2013-12-01

    The present work investigated the effects of subcritical CO2 pretreatment of sugarcane bagasse at different CO2 pressure, pretreatment time, and temperature with relative high-solid concentration (15% w/v) to the composition of prehydrolyzate and the enzymatic hydrolysis. The results indicated that the maximum xylose yields in prehydrolyzate liquid were 15.78 g (combined 3.16 g xylose and 12.62 g xylo-oligosaccharides per 100g raw material). Due to the effective removal of hemicellulose, the maximum glucose yield in enzyme hydrolyzate reached 37.99 g per 100g raw material, representing 91.87% of glucose in the sugarcane bagasse. The maximal total sugars yield (combined xylose and glucose both in prehydrolyzate and enzymatic hydrolyzate) were 52.95 g based on 100g raw material. These results indicated that subcritical CO2 pretreatment can effectively improve the enzymatic hydrolysis, so it could be successfully applied to sugarcane bagasse.

  8. 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.

  9. 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.

  10. 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

  11. Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli.

    PubMed

    Abdeljabbar, Diya M; Song, Hank J; Link, A James

    2012-01-01

    Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.

  12. 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

    2015-02-11

    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.

  13. Alternatives to Trichoderma reesei in biofuel production.

    PubMed

    Gusakov, Alexander V

    2011-09-01

    Mutant strains of Trichoderma reesei are considered indisputable champions in cellulase production among biomass-degrading fungi. So, it is not surprising that most R&D projects on bioethanol production from lignocellulosics have been based on using T. reesei cellulases. The present review focuses on whether any serious alternatives to T. reesei enzymes in cellulose hydrolysis exist. Although not widely accepted, more and more data have been accumulated that demonstrate that fungi belonging to the genera Penicillium, Acremonium and Chrysosporium might represent such alternatives because they are competitive to T. reesei on some important parameters, such as protein production level, cellulase hydrolytic performance per unit of activity or milligram of protein.

  14. Kinetic study on enzymatic hydrolysis of cellulose by cellulase from Trichoderma viride

    SciTech Connect

    Ohmine, K.; Ooshima, H.; Harano, Y.

    1983-08-01

    Pure cellulose (Avicel) was hydrolyzed batchwise at 50 degrees C and pH 4.8 by cellulase from Trichoderma viride (Meicelase CEP). Then the effects of the crystallinity of cellulose as well as the thermal deactivation and product (cellobiose and glucose) inhibition to cellulase on the hydrolysis rate were quantitatively investigated. While these factors had evidently retarded the enzymatic hydrolysis of cellulose to a significant extent, the hydrolysis rates observed could not be explained. For practical purposes, an empirical, simple rate expression was developed which included only one parameter: an overall rate retardation constant. This empirical rate expression held for the hydrolysis of at least two kinds of cellulosic materials: Avicel and tissue paper. (Refs. 10)

  15. Substrate reactivity as a function of the extent of reaction in the enzymatic hydrolysis of lignocellulose

    SciTech Connect

    Desai, S.G.; Converse, A.O.

    1997-12-20

    In an effort to better understand the role of the substrate in the rapid fall off in the rate of enzymatic hydrolysis of cellulose with conversion, substrate reactivity was measured as a function of conversion. These measurements were made by interrupting the hydrolysis of pretreated wood at various degrees of conversion; and, after boiling and washing, restarting the hydrolysis in fresh butter with fresh enzyme. The comparison of the restart rate per enzyme adsorbed with the initial rate per enzyme adsorbed, both extrapolated back to zero conversion, provides a measurement of the substrate reactivity without the complications of product inhibition or cellulase inactivation. The results indicate that the substrate reactivity falls only modestly as conversion increases. However, the restart rate is still higher than the rate of the uninterrupted hydrolysis, particularly at high conversion. Hence the authors conclude that the loss of substrate reactivity is not the principal cause for the long residence time required for complete conversion.

  16. 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.

  17. Enzymatic hydrolysis of esters containing a tetrazole ring.

    PubMed

    Łukowska-Chojnacka, Edyta; Mierzejewska, Jolanta

    2014-12-01

    The lipase-catalyzed enantioselective hydrolysis of acetates containing tetrazole moiety was studied. Among all tested lipases, Novozyme SP 435 allowed to obtain optically active 4-(5-aryl-2H-tetrazol-2yl)butan-2-ol and 1-(5-aryl-2H-tetrazol-2yl)-propan-2-ol and their acetates with the highest optical purities (ee = 95%-99%) and excellent enantioselectivity (E>100). Some of the synthesized tetrazole derivatives were screened for their antifungal activity. Racemic mixtures of 4-[5-(4-chlorophenyl)-2H-tetrazol-2-yl)butan-2-ol as well as pure enantiomers of this compound showed promising antifungal activity against F. sambucinum, F. oxysporum, C. coccodes, and A. niger.

  18. Pseudo-lignin formation and its impact on enzymatic hydrolysis.

    PubMed

    Hu, Fan; Jung, Seokwon; Ragauskas, Arthur

    2012-08-01

    Pseudo-lignin, which can be broadly defined as aromatic material that yields a positive Klason lignin value and is not derived from native lignin, has been recently reported to form during the dilute acid pretreatment of poplar holocellulose. To investigate the chemistry of pseudo-lignin formation, GPC, FT-IR and 13C NMR were utilized to characterize pseudo-lignin extracted from dilute-acid pretreated α-cellulose and holocellulose. The results showed that pseudo-lignin consisting of carbonyl, carboxylic, aromatic and aliphatic structures was produced from dilute acid pretreated cellulose and hemicellulose. Pseudo-lignin extracted from holocellulose pretreated at different conditions had similar molecular weights (Mn∼1000 g/mol; Mw∼5000 g/mol) and structural features (carbonyl, carboxylic, aromatic and methoxy structures). These characterizations have provided the pseudo-lignin formation mechanisms during pretreatment. The presence and structure of pseudo-lignin is important since pseudo-lignin decreases the enzymatic conversion. PMID:22609707

  19. 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

  20. Evaluation of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of rice straw.

    PubMed

    Taniguchi, Masayuki; Suzuki, Hiroyuki; Watanabe, Daisuke; Sakai, Kenji; Hoshino, Kazuhiro; Tanaka, Takaaki

    2005-12-01

    The effects of biological pretreatment of rice straw using four white-rot fungi (Phanerochaete chrysosporium, Trametes versicolor, Ceriporiopsis subvermispora, and Pleurotus ostreatus) were evaluated on the basis of quantitative and structural changes in the components of the pretreated rice straw as well as susceptibility to enzymatic hydrolysis. Of these white-rot fungi, P. ostreatus selectively degraded the lignin fraction of rice straw rather than the holocellulose component. When rice straw (water content of 60%) was pretreated with P. ostreatus for 60 d, the total weight loss and the degree of Klason lignin degraded were 25% and 41%, respectively. After the pretreatment, the residual amounts of cellulose and hemicellulose were 83% and 52% of those in untreated rice straw, respectively. By enzymatic hydrolysis with a commercial cellulase preparation for 48 h, 52% holocellulose and 44% cellulose in the pretreated rice straw were solubilized. The net sugar yields based on the amounts of holocellulose and cellulose of untreated rice straw were 33% for total soluble sugar from holocellulose and 32% for glucose from cellulose. The SEM observations showed that the increase in susceptibility of rice straw to enzymatic hydrolysis by pretreatment with P. ostreatus is caused by partial degradation of the lignin seal. When the content of Klason lignin was less than 15% of the total weight of the pretreated straw, enhanced degrees of enzymatic solubilization of holocellulose and cellulose fractions were observed as the content of Klason lignin decreased.

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

    PubMed

    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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

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

    SciTech Connect

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

    1980-01-01

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

  9. Pulp properties resulting from different pretreatments of wheat straw and their influence on enzymatic hydrolysis rate.

    PubMed

    Rossberg, Christine; Steffien, Doreen; Bremer, Martina; Koenig, Swetlana; Carvalheiro, Florbela; Duarte, Luís C; Moniz, Patrícia; Hoernicke, Max; Bertau, Martin; Fischer, Steffen

    2014-10-01

    Wheat straw was subjected to three different processes prior to saccharification, namely alkaline pulping, natural pulping and autohydrolysis, in order to study their effect on the rate of enzymatic hydrolysis. Parameters like medium concentration, temperature and time have been varied in order to optimize each method. Milling the raw material to a length of 4mm beforehand showed the best cost-value-ratio compared to other grinding methods studied. Before saccharification the pulp can be stored in dried form, leading to a high yield of glucose. Furthermore the relation of pulp properties (i.e. intrinsic viscosity, Klason-lignin and hemicelluloses content, crystallinity, morphology) to cellulose hydrolysis is discussed.

  10. Fungal cellulase/xylanase production and corresponding hydrolysis using pretreated corn stover as substrates.

    PubMed

    Zhang, Liang; Wang, Xiaoqing; Ruan, Zhenhua; Liu, Ying; Niu, Xiaorui; Yue, Zhengbo; Li, Zhimin; Liao, Wei; Liu, Yan

    2014-01-01

    Three pretreated corn stover (ammonia fiber expansion, dilute acid, and dilute alkali) were used as carbon source to culture Trichoderma reesei Rut C-30 for cellulase and xylanase production. The results indicated that the cultures on ammonia fiber expansion and alkali pretreated corn stover had better enzyme production than the acid pretreated ones. The consequent enzymatic hydrolysis was performed applying fungal enzymes on pretreated corn stover samples. Tukey's statistical comparisons exhibited that there were significant differences on enzymatic hydrolysis among different combination of fungal enzymes and pretreated corn stover. The higher sugar yields were achieved by the enzymatic hydrolysis of dilute alkali pretreated corn stover.

  11. 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.

  12. High temperature aqueous ammonia pretreatment and post-washing enhance the high solids enzymatic hydrolysis of corn stover.

    PubMed

    Qin, Lei; Liu, Zhi-Hua; Jin, Mingjie; Li, Bing-Zhi; Yuan, Ying-Jin

    2013-10-01

    Aqueous ammonia pretreatment was optimized and the limiting factors in high solids enzymatic hydrolysis were assessed. The recommended pretreatment condition to achieve high enzymatic yield was: 180 °C, 20% (w/w) ammonia, 30 min, and 20% solids content. FT-IR and GC-MS results indicated that most of the lignin was degraded to soluble fragments after pretreatment. The pretreated solids after post-washing showed higher enzymatic digestibility at high solids loading than that without washing. The washed solids required lower cellulase and xylanase dosage than unwashed solids to achieve high sugar yield. Enzymatic conversions were declined with the increased solids loading of pretreated solids, pretreated-washed solids, and filter papers. The results indicated that solids loading in enzymatic hydrolysis was an important factor affecting sugar yield. The increasing concentration of glucose and ligno-phenolics mainly inhibited the enzymatic hydrolysis of aqueous ammonia pretreated corn stover.

  13. Branched poly(lactide) synthesized by enzymatic polymerization: effects of molecular branches and stereochemistry on enzymatic degradation and alkaline hydrolysis.

    PubMed

    Numata, Keiji; Srivastava, Rajiv K; Finne-Wistrand, Anna; Albertsson, Ann-Christine; Doi, Yoshiharu; Abe, Hideki

    2007-10-01

    In this article the effects of the number of molecular branches (chain ends) and the stereochemistry of poly(lactide)s (PLAs) on the enzymatic degradation and alkaline hydrolysis are studied. Various linear and branched PLAs were synthesized using lipase PS (Pseudomonas fluorescens)-catalyzed ring-opening polymerization (ROP) of lactide monomers having different stereochemistries (L-lactide, D-lactide, and D,L-lactide). Five different alcohols were used as initiators for the ROP, and the monomer-to-initiator molar feed ratio was varied from 10 to 100 and 1000 for each branch in the polymer architecture. The properties of branched PLAs that would affect the enzymatic and alkaline degradations, i.e., the glass transition temperature, the melting temperature, the melting enthalpy, and the advancing contact angle, were determined. The PLA films were degraded using proteinase K or 1.0 M NaOH solution, and the weight loss and changes in the number average molecular weight (Mn) of the polymer were studied during 12 h of degradation. The results suggest that an increase in the number of molecular branches of branched PLAs enhances its enzymatic degradability and alkali hydrolyzability. Moreover, the change in Mn of the branched poly(L-lactide) (PLLA) by alkaline hydrolysis indicated that the decrease in Mn was in the first place dependent on the number of molecular branches and thereafter on the length of the molecular branch of branched PLA. The branched PLLA, poly(D-lactide) (PDLA), and poly(D,L-lactide) (PDLLA) differed in weight loss and change in Mn of the PLA segment during the enzymatic degradation. It is suggested that the branched PDLLA was degraded preferentially by proteinase K.

  14. Surfactant-assisted pretreatment and enzymatic hydrolysis of spent mushroom compost for the production of sugars.

    PubMed

    Kapu, N U S; Manning, M; Hurley, T B; Voigt, J; Cosgrove, D J; Romaine, C P

    2012-06-01

    Spent mushroom compost (SMC), a byproduct of commercial mushroom cultivation, poses serious environmental problems that have hampered the growth of this important agro-industry. In an effort to develop new applications for SMC, we explored its use as a feedstock for bioethanol production. SMC constitutes approximately 30%w/w polysaccharides, 66% of which is glucan. Following dilute-acid pretreatment and enzymatic hydrolysis, both in the presence of PEG 6000, 97% of glucan and 44% of xylan in SMC were converted into the corresponding monosaccharides. Incorporation of PEG 6000 reduced the cellulase requirement by 77%. Zwittergent 3-12 and 3-14 also significantly increased the efficacy of acid pretreatment and enzymatic hydrolysis. The use of SMC in bioethanol production represents a potential mitigation solution for the critical environmental issues associated with the stockpiling of the major byproduct of the mushroom industry.

  15. Combination of biological pretreatment with liquid hot water pretreatment to enhance enzymatic hydrolysis of Populus tomentosa.

    PubMed

    Wang, Wei; Yuan, Tongqi; Wang, Kun; Cui, Baokai; Dai, Yucheng

    2012-03-01

    A novel stepwise pretreatment of combination of fungal treatment with liquid hot water (LHW) treatment was conducted to enhance the enzymatic hydrolysis of Populus tomentosa. The results showed that lignin and cellulose increased with the elevating temperature, while significant amount of hemicellulose was degraded during the LHW pretreatment. A highest hemicellulose removal of 92.33% was observed by combination of Lenzites betulina C5617 with LHW treatment at 200°C, which was almost 2 times higher than that of sole LHW treatment at the same level. Saccharification of poplar co-treated with L. betulina C5617 and LHW at 200°C resulted in a 2.66-fold increase of glucose yield than that of sole LHW treatment, and an increase (2.25-fold) of glucose yield was obtained by the combination of Trametes ochracea C6888 with LHW. The combination pretreatment performed well at accelerating the enzymatic hydrolysis of poplar wood.

  16. 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

  17. 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.

  18. 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.

  19. Pretreatment of rice straw by a hot-compressed water process for enzymatic hydrolysis.

    PubMed

    Yu, Guoce; Yano, Shinichi; Inoue, Hiroyuki; Inoue, Seiichi; Endo, Takashi; Sawayama, Shigeki

    2010-01-01

    Hot-compressed water (HCW) is among several cost-effective pretreatment processes of lignocellulosic biomass for enzymatic hydrolysis. The present work investigated the characteristics of HCW pretreatment of rice straw including sugar production and inhibitor formation in the liquid fraction and enzymatic hydrolysis of pretreated material. Pretreatment was carried out at a temperature ranging from 140 to 240 degrees C for 10 or 30 min. Soluble oligosaccharides were found to constitute almost all the components of total sugars in the liquid fraction. The maximal production of total glucose at 180 degrees C and below accounted for 4.4-4.9% of glucan in raw material. Total xylose production peaked at 180 degrees C, accounting for 43.3% of xylan in raw material for 10-min pretreatment and 29.8% for 30-min pretreatment. The production of acetic acid increased at higher temperatures and longer treatment time, indicating more significant disruption of lignocellulosic structure, and furfural production achieved the maximum (2.8 mg/ml) at 200 degrees C for both 10-min and 30-min processes. The glucose yield by enzymatic hydrolysis of pretreated rice straw was no less than 85% at 180 degrees C and above for 30-min pretreatment and at 200 degrees C and above for 10-min pretreatment. Considering sugar recovery, inhibitor formation, and process severity, it is recommended that a temperature of 180 degrees C for a time of 30 min can be the most efficient process for HCW pretreatment of rice straw.

  20. 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

  1. Biodegradable cellulose diacetate-graft-poly(L-lactide)s: enzymatic hydrolysis behavior and surface morphological characterization.

    PubMed

    Teramoto, Yoshikuni; Nishio, Yoshiyuki

    2004-01-01

    Enzymatic hydrolysis of selected copolymers of cellulose diacetate-graft-poly(L-lactide)s (CDA-g-PLLAs) were conducted with proteinase K for film specimens, which were solely quenched from the molten state or, further, annealed at temperatures below or above their glass transition temperatures. The hydrolysis rates depended seriously on the thermal history, as well as on the graft modification. Especially, the heat treatment, followed by physical aging or crystallization of the originally amorphous materials, was a key factor to control subtly their enzymatic degradation behavior. Atomic force microscopy revealed that the enzymatic hydrolysis transformed the surface of the respective films into a more undulated one with a number of fine protuberances, for example, of several hundred nanometers in height and a few micrometers in width. Attenuated total reflection FTIR spectroscopy ensured selective release of lactyl units from the surface region. In visual appearance, some degraded films exhibited even an iridescent color due to an effect of interference of visible light reflected on the surface. These observations suggest a conception of "spatiotemporally controlled degradation", leading to a new method not only for regulation of the overall rate of degradation but also for fine surface abrasion of polymer materials.

  2. 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

  3. 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

  4. 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

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

    PubMed

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

    2015-01-01

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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. Effects of a steam explosion pretreatment on sugar production by enzymatic hydrolysis and structural properties of reed straw.

    PubMed

    Hu, Qiulong; Su, Xiaojun; Tan, Lin; Liu, Xianghua; Wu, Anjun; Su, Dingding; Tian, Kaizhong; Xiong, Xingyao

    2013-01-01

    Reed lignocellulose was subjected to a steam explosion pretreatment to obtain a high conversion rate of sugar after subsequent enzymatic hydrolysis using a commercial cellulase mixture. Under conditions of differing temperature (200 °C, 220 °C and 240 °C) and residence time (2, 5, and 8 min), the effect of the pretreatment on the sugar yield from enzymatic hydrolysis was studied. The highest respective reducing sugar and glucose yields were 36.14% and 15.35% after 60-h enzymatic hydrolysis of reed straw that had been pretreated with a steam explosion at 220 °C for 5 min. Fourier transform infrared spectrophotometry (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used in this study to comprehensively investigate the steam explosion-induced changes in the organizational structure and morphological properties of reed straw to analyze the reason for the increased sugar yield from enzymatic hydrolysis after the steam explosion.

  11. 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

  12. 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.

  13. Rapid near infrared spectroscopy for prediction of enzymatic hydrolysis of corn bran after various pretreatments.

    PubMed

    Baum, Andreas; Agger, Jane; Meyer, Anne S; Egebo, Max; Mikkelsen, Jørn Dalgaard

    2012-02-15

    Efficient generation of a fermentable hydrolysate is a primary requirement in the utilization of fibrous plant biomass as feedstocks in bioethanol processes. The first biomass conversion step usually involves a hydrothermal pretreatment before enzymatic hydrolysis. The purpose of the pretreatment step is to increase the responsivity of the substrate to enzymatic attack and the type of pretreatment affects the enzymatic conversion efficiency. Destarched corn bran is a fibrous, heteroxylan-rich side-stream from the starch industry which may be used as a feedstock for bioethanol production or as a source of xylose for other purposes. In the present study we demonstrate the use of diffuse reflectance near infrared spectroscopy (NIR) as a rapid and non-destructive analytical tool for evaluation of pretreatment effects on destarched corn bran. NIR was used to achieve classification between 43 differently pretreated corn bran samples using principal component analysis (PCA) and hierarchal clustering algorithms. Quantification of the enzymatically released monosaccharides by HPLC was used to design multivariate calibration models (biPLS) on the NIR spectra. The models could predict the enzymatic release of different levels of arabinose, xylose and glucose from all the differently pretreated destarched corn bran samples. The present study also demonstrates a generic, non-destructive solution to determine the enzymatic monosaccharide release from polymers in biomass side-streams, thereby potentially replacing the cumbersome HPLC analysis.

  14. 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.

  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.

  16. 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.

  17. 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.

  18. 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.

  19. Particle size distribution of rice flour affecting the starch enzymatic hydrolysis and hydration properties.

    PubMed

    de la Hera, Esther; Gomez, Manuel; Rosell, Cristina M

    2013-10-15

    Rice flour is becoming very attractive as raw material, but there is lack of information about the influence of particle size on its functional properties and starch digestibility. This study evaluates the degree of dependence of the rice flour functional properties, mainly derived from starch behavior, with the particle size distribution. Hydration properties of flours and gels and starch enzymatic hydrolysis of individual fractions were assessed. Particle size heterogeneity on rice flour significantly affected functional properties and starch features, at room temperature and also after gelatinization; and the extent of that effect was grain type dependent. Particle size heterogeneity on rice flour induces different pattern in starch enzymatic hydrolysis, with the long grain having slower hydrolysis as indicated the rate constant (k). No correlation between starch digestibility and hydration properties or the protein content was observed. It seems that in intact granules interactions with other grain components must be taken into account. Overall, particle size fractionation of rice flour might be advisable for selecting specific physico-chemical properties.

  20. Economic impact of total solids loading on enzymatic hydrolysis of dilute acid pretreated corn stover.

    PubMed

    Humbird, David; Mohagheghi, Ali; Dowe, Nancy; Schell, Daniel J

    2010-01-01

    In process integration studies of the biomass-to-ethanol conversion process, it is necessary to understand how cellulose conversion yields vary as a function of solids and enzyme loading and other key operating variables. The impact of solids loading on enzymatic cellulose hydrolysis of dilute acid pretreated corn stover slurry was determined using an experimental response surface design methodology. From the experimental work, an empirical correlation was obtained that expresses monomeric glucose yield from enzymatic cellulose hydrolysis as a function of solids loading, enzyme loading, and temperature. This correlation was used in a technoeconomic model to study the impact of solids loading on ethanol production economics. The empirical correlation was used to provide a more realistic assessment of process cost by accounting for changes in cellulose conversion yields at different solids and enzyme loadings as well as enzyme cost. As long as enzymatic cellulose conversion drops off at higher total solids loading (due to end-product inhibition or other factors), there is an optimum value for the total solids loading that minimizes the ethanol production cost. The optimum total solids loading shifts to higher values as enzyme cost decreases.

  1. Small angle neutron scattering reveals pH-dependent conformational changes in Trichoderma reesei cellobiohydrolase I: implications for enzymatic activity.

    PubMed

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

    2011-09-16

    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.

  2. Changes on structural properties of biomass pretreated by combined deacetylation with liquid hot water and its effect on enzymatic hydrolysis.

    PubMed

    Jiang, Wei; Chang, Senlin; Qu, Yongshui; Zhang, Zhiguo; Xu, Jian

    2016-11-01

    A novel combined pretreatment of deacetylation and liquid hot water (LHW) was invented which has been proved to be effective in increasing enzymatic hydrolysis yield of biomass. In order to further understand the effect of this new pretreatment process on biomass, the variation on structural properties including cellulose crystallinity index (CrI), specific surface area (SSA) and degree of polymerization (DP) before/after pretreatment and how these properties affected the enzymatic hydrolysis of biomass were explored. The improvement of pretreatment severity (PS) could increase CrI, SSA and reduce DP. Whereas the enhancement of degree of deacetylation could decrease SSA and DP. An optimal formula (E12Y=0.347(100-CrI)(-0.375)×(SSA)(0.203)×(1700-DP)(0.281)) was achieved to express the correlation between structural properties and enzymatic hydrolysis after 12h. The enzymatic yield was more sensitive to CrI than to SSA and DP.

  3. Hydrolysis of phosphotriesters: a theoretical analysis of the enzymatic and solution mechanisms.

    PubMed

    López-Canut, Violeta; Ruiz-Pernía, J Javier; Castillo, Raquel; Moliner, Vicent; Tuñón, Iñaki

    2012-07-27

    A theoretical study on the alkaline hydrolysis of paraoxon, one of the most popular organophosphorus pesticides, in aqueous solution and in the active site of Pseudomonas diminuta phosphotriesterase (PTE) is presented. Simulations by means of hybrid quantum mechanics/molecular mechanics (QM/MM) potentials show that the hydrolysis of paraoxon takes place through an A(N)D(N) or associative mechanism both in solution and in the active site of PTE. The results correctly reproduce the magnitude of the activation free energies and can be used to rationalize the observed kinetic isotope effects (KIEs) for the hydrolysis of paraoxon in both media. Enzymatic hydrolysis of O,O-diethyl p-chlorophenyl phosphate, a phosphotriester having a leaving group with higher pK(a) than paraoxon, was also simulated. Hydrolysis of this phosphotriester by PTE follows a A(N)+D(N) mechanism with a pentacoordinate intermediate. Moreover, the leaving group of this new substrate coordinates to one of the zinc ions of the bimetallic active site in order to stabilize the large negative charge developed on the oxygen atom of the leaving group when the P-O bond is broken in the products state. To accommodate this new ligand in the coordination shell, carbamylated Lys169 must be displaced from one zinc ion to the other, which in turn affects the acidity of Asp301, a residue originally bound to the second zinc ion. This ability to displace some of the ligands of the coordination shell of the zinc centers would explain the promiscuity of this enzyme, which is capable of catalyzing hydrolysis of different substrate by means of different mechanisms. PMID:22745111

  4. 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

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

    PubMed Central

    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

  6. Enzymatic hydrolysis in an aqueous organic two-phase system using centrifugal partition chromatography.

    PubMed

    Krause, J; Oeldorf, T; Schembecker, G; Merz, J

    2015-04-24

    Multi-phase reaction systems, mostly aqueous organic systems, are used in enzyme catalysis to convert hydrophobic substrates which are almost insoluble in aqueous media. In this study, a Centrifugal Partition Chromatograph is used as a compact device for enzymatic multi-phase reaction that combines efficient substrate supply to the aqueous phase and separation of both phases in one apparatus. A process design procedure to systematically select the aqueous and organic phase to achieve stable and efficient reaction rates and operation conditions in Centrifugal Partition Chromatography for efficient mixing and separation of the phases is presented. The procedure is applied to the hydrolysis of 4-nitrophenyl palmitate with a lipase derived from Candida rugosa. It was found that the hydrolysis rate of 4-nitrophenyl palmitate was two times higher in Centrifugal Partition Chromatography than in comparable stirred tank reactor experiments. PMID:25773726

  7. Enzymatic hydrolysis in an aqueous organic two-phase system using centrifugal partition chromatography.

    PubMed

    Krause, J; Oeldorf, T; Schembecker, G; Merz, J

    2015-04-24

    Multi-phase reaction systems, mostly aqueous organic systems, are used in enzyme catalysis to convert hydrophobic substrates which are almost insoluble in aqueous media. In this study, a Centrifugal Partition Chromatograph is used as a compact device for enzymatic multi-phase reaction that combines efficient substrate supply to the aqueous phase and separation of both phases in one apparatus. A process design procedure to systematically select the aqueous and organic phase to achieve stable and efficient reaction rates and operation conditions in Centrifugal Partition Chromatography for efficient mixing and separation of the phases is presented. The procedure is applied to the hydrolysis of 4-nitrophenyl palmitate with a lipase derived from Candida rugosa. It was found that the hydrolysis rate of 4-nitrophenyl palmitate was two times higher in Centrifugal Partition Chromatography than in comparable stirred tank reactor experiments.

  8. Biological pretreatment of corn stover with ligninolytic enzyme for high efficient enzymatic hydrolysis.

    PubMed

    Wang, Feng-Qin; Xie, Hui; Chen, Wei; Wang, En-Tao; Du, Feng-Guang; Song, An-Dong

    2013-09-01

    Aiming at increasing the efficiency of transferring corn stover into sugars, a biological pretreatment was developed and investigated in this study. The protocol was characterized by the pretreatment with crude ligninolytic enzymes from Phanerochete chrysosporium and Coridus versicolor to break the lignin structure in corn stover, followed by a washing procedure to eliminate the inhibition of ligninolytic enzyme on cellulase. By a 2 d-pretreatment, sugar yield from corn stover hydrolysis could be increased by 50.2% (up to 323 mg/g) compared with that of the control. X-ray diffractometry and FT-IR analysis revealed that biological pretreatment could partially remove the lignin of corn stover, and consequently enhance the enzymatic hydrolysis efficiency of cellulose and hemeicellulose. In addition, the amount of microbial inhibitors, such as acetic acid and furfural, were much lower in biological pretreatment than that in acid pretreatment. This study provided a promising pretreatment method for biotransformation of corn stovers.

  9. Enzymatic hydrolysis of pelletized AFEX™-treated corn stover at high solid loadings.

    PubMed

    Bals, Bryan D; Gunawan, Christa; Moore, Janette; Teymouri, Farzaneh; Dale, Bruce E

    2014-02-01

    Ammonia fiber expansion (AFEX™) pretreatment can be performed at small depots, and the pretreated biomass can then be pelletized and shipped to a centralized refinery. To determine the feasibility of this approach, pelletized AFEX-treated corn stover was hydrolyzed at high (18-36%) solid loadings. Water absorption and retention by the pellets was low compared to unpelletized stover, which allowed enzymatic hydrolysis slurries to remain well mixed without the need for fed-batch addition. Glucose yields of 68% and xylose yields of 65% were obtained with 20 mg enzyme/g glucan and 18% solid loading after 72 h, compared to 61% and 59% for unpelletized corn stover. Pelletization also slightly increased the initial rate of hydrolysis compared to unpelletized biomass. The ease of mixing and high yields obtained suggests that pelletization after AFEX pretreatment could have additional advantages beyond improved logistical handling of biomass.

  10. 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.

  11. Model-based fed-batch for high-solids enzymatic cellulose hydrolysis.

    PubMed

    Hodge, David B; Karim, M Nazmul; Schell, Daniel J; McMillan, James D

    2009-01-01

    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.

  12. 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

  13. [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

  14. 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

  15. 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.

  16. Microalgae pretreatment with liquid hot water to enhance enzymatic hydrolysis efficiency.

    PubMed

    Yuan, Tao; Li, Xiekun; Xiao, Shiyuan; Guo, Ying; Zhou, Weizheng; Xu, Jingliang; Yuan, Zhenhong

    2016-11-01

    Nowadays, microalgae are being considered as promising raw material for bioethanol production. In this work, three process variables during liquid hot water (LHW) pretreatment prior to enzymatic hydrolysis by response surface methodology on Scenedesmus sp. WZKMT were investigated to enhance glucose recovery. Results indicated that the order of significance for three parameters was temperature>solid-to-liquid ratio>time. The optimal condition was 1:13 (w/v), 147°C and 40min. The concentration and recovery of glucose under this condition were 14.223g·L(-1) and 89.32%, respectively, which were up to 5-fold higher than the samples without LHW pretreatment. In addition, the surface morphologies of microalgae cells before and after LHW pretreatment were also verified using scanning electron microscopy (SEM). LHW pretreatment can greatly enhance the enzymatic efficiency, and can be regarded as an ideal pretreatment method for glucose recovery from microalgae.

  17. 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.

  18. Microalgae pretreatment with liquid hot water to enhance enzymatic hydrolysis efficiency.

    PubMed

    Yuan, Tao; Li, Xiekun; Xiao, Shiyuan; Guo, Ying; Zhou, Weizheng; Xu, Jingliang; Yuan, Zhenhong

    2016-11-01

    Nowadays, microalgae are being considered as promising raw material for bioethanol production. In this work, three process variables during liquid hot water (LHW) pretreatment prior to enzymatic hydrolysis by response surface methodology on Scenedesmus sp. WZKMT were investigated to enhance glucose recovery. Results indicated that the order of significance for three parameters was temperature>solid-to-liquid ratio>time. The optimal condition was 1:13 (w/v), 147°C and 40min. The concentration and recovery of glucose under this condition were 14.223g·L(-1) and 89.32%, respectively, which were up to 5-fold higher than the samples without LHW pretreatment. In addition, the surface morphologies of microalgae cells before and after LHW pretreatment were also verified using scanning electron microscopy (SEM). LHW pretreatment can greatly enhance the enzymatic efficiency, and can be regarded as an ideal pretreatment method for glucose recovery from microalgae. PMID:27614155

  19. 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

  20. Effect of the molecular structure of lignin-based polyoxyethylene ether on enzymatic hydrolysis efficiency and kinetics of lignocelluloses.

    PubMed

    Lin, Xuliang; Qiu, Xueqing; Zhu, Duming; Li, Zihao; Zhan, Ningxin; Zheng, Jieyi; Lou, Hongming; Zhou, Mingsong; Yang, Dongjie

    2015-10-01

    Effect of the molecular structure of lignin-based polyoxyethylene ether (EHL-PEG) on enzymatic hydrolysis of Avicel and corn stover was investigated. With the increase of PEG contents and molecular weight of EHL-PEG, glucose yield of corn stover increased. EHL-PEG enhanced enzymatic hydrolysis of corn stover significantly at buffer pH 4.8-5.5. Glucose yield of corn stover at 20% solid content increased from 32.8% to 63.8% by adding EHL-PEG, while that with PEG4600 was 54.2%. Effect of EHL-PEG on enzymatic hydrolysis kinetics of cellulose film was studied by quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). An enhancing mechanism of EHL-PEG on enzymatic hydrolysis kinetics of cellulose was proposed. Cellulase aggregates dispersed by EHL-PEG excavated extensive cavities into the surface of cellulose film, making the film become more loose and exposed. After the maximum enzymatic hydrolysis rate, the film was mainly peeled off layer by layer until equilibrium.

  1. Suitable technological conditions for enzymatic hydrolysis of waste paper by Novozymes® enzymes NS50013 and NS50010.

    PubMed

    Brummer, Vladimir; Skryja, Pavel; Jurena, Tomas; Hlavacek, Viliam; Stehlik, Petr

    2014-10-01

    Waste paper belongs to a group of quantitatively the most produced waste types. Enzymatic hydrolysis is becoming a suitable way to treat this type of waste and at the same time, to produce a valuable liquid biofuel, because reducing sugars solutions that are formed during the process of saccharification can be a precursor for following or simultaneous fermentation. If it will be possible to make the enzymatic hydrolysis of the waste paper economically viable, it could serve as one of the new ways to lower the dependence of the transport sector on oil in the future. Only several studies comparing the enzymatic hydrolysis of different waste papers were performed in the past; they are summarized in this manuscript. In our experimental trials, suitable technological conditions for waste paper enzymatic hydrolysis using enzymes from Novozymes® biomass kit: enzymes NS50013 and NS50010 were investigated. The following enzymatic hydrolysis parameters in laboratory scale trials were verified on high cellulose content substrates-filter paper and cellulose pulp: type of buffer, pH, temperature, concentration of the substrate, loading of the enzyme and rate of stirring.

  2. 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.

  3. Optimization of enzymatic hydrolysis conditions for extraction of pectin from rapeseed cake (Brassica napus L.) using commercial enzymes.

    PubMed

    Jeong, Han-Seob; Kim, Ho-Yong; Ahn, Sye Hee; Oh, Sei Chang; Yang, In; Choi, In-Gyu

    2014-08-15

    The aims of this study were to extract pectin from rapeseed cake (RSC) by enzymatic hydrolysis using commercial enzymes (Celluclast and Alcalase) and to investigate the effects of different reaction conditions, such as enzymatic hydrolysis time, enzyme-RSC ratio, and Celluclast-Alcalase ratio, on the degradation of RSC and pectin yield. RSC was treated using a combined extraction process that consisted of a fat removal process, enzymatic hydrolysis, and isopropanol/ethanol precipitation. After the fat removal process and enzymatic hydrolysis, defatted-RSC was suitably decomposed, and the loss of liberated reducing sugars was minimized when the hydrolysis condition reached a hydrolysis time of 270 min or an enzyme-RSC ratio of 1:50. Based on these results, various Celluclast-Alcalase ratios were applied. Alcalase led to the destruction of protein-carbohydrate complex in defatted-RSC, whereas Celluclast cleaved some linkages of carbohydrate slightly. As a result, the highest pectin yield was 6.85% at the Celluclast-Alcalase ratio of 1:4.

  4. 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.

  5. 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.

  6. Enzymatic Hydrolysis Optimization to Ethanol Production by Simultaneous Saccharification and Fermentation

    NASA Astrophysics Data System (ADS)

    Vásquez, Mariana Peñuela; da Silva, Juliana Nascimento C.; de Souza, Maurício Bezerra; Pereira, Nei

    There is tremendous interest in using agro-industrial wastes, such as cellulignin, as starting materials for the production of fuels and chemicals. Cellulignin are the solids, which result from the acid hydrolysis of the sugarcane bagasse. The objective of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cellulignin, and to study its fermentation to ethanol using Saccharomyces cerevisiae. Cellulose conversion was optimized using response surface methods with pH, enzyme loading, solid percentage, and temperature as factor variables. The optimum conditions that maximized the conversion of cellulose to glucose, calculated from the initial dried weight of pretreated cellulignin, (43°C, 2%, and 24.4 FPU/g of pretreated cellulignin) such as the glucose concentration (47°C, 10%, and 25.6 FPU/g of pretreated cellulignin) were found. The desirability function was used to find conditions that optimize both, conversion to glucose and glucose concentration (47°C, 10%, and 25.9 FPU/g of pretreated cellulignin). The resulting enzymatic hydrolyzate was fermented yielding a final ethanol concentration of 30.0 g/L, in only 10 h, and reaching a volumetric productivity of 3.0 g/L·h, which is close to the values obtained in the conventional ethanol fermentation of sugar cane juice (5.0-8.0 g/L·h) in Brazil.

  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.

  8. Effect of high dry solids loading on enzymatic hydrolysis of acid bisulfite pretreated Eastern redcedar.

    PubMed

    Ramachandriya, Karthikeyan D; Wilkins, Mark; Atiyeh, Hasan K; Dunford, Nurhan T; Hiziroglu, Salim

    2013-11-01

    This study investigates hydrolysis of cellulose from Eastern redcedar to glucose at high solids loading. Enzymatic hydrolysis of pretreated redcedar was performed with 0.5 ml Accelerase® 1500/g glucan (46 FPU/g glucan) using dry solids loading from 2% to 20% (w/w). Rheological challenges observed at high solids loading were overcome by adding stainless steel balls to shake flask reactors. The highest glucose concentration, 126 g/L (84% glucan-to-glucose yield), was obtained using 20% solids loading with stainless steel balls as a mixing aid. This enzymatic hydrolyzate was fermented into ethanol using Saccharomyces cerevisiae D5A to produce 52 g/L of ethanol (corresponding to 166 L/dry Mg of redcedar). Reducing enzyme dosage at 16% solids loading from 46 to 11.5 FPU/g glucan reduced glucan-to-glucose yields. This study has demonstrated the possibility of extracting sugars from the invasive species of Eastern redcedar with high solid loadings and their conversion into ethanol.

  9. Sea buckthorn (Hippophae rhamnoides) proanthocyanidins inhibit in vitro enzymatic hydrolysis of protein.

    PubMed

    Arimboor, Ranjith; Arumughan, C

    2011-08-01

    Interactions of phenolics with other food constituents and digestive enzymes are likely to have interference with the digestion and bioavailability of food and phenolics. In this study the effect of sea buckthorn proanthocyanidins on in vitro digestion of protein was evaluated. Optimization of the extraction conditions showed that maximum recovery of sea buckthorn proanthocyanidins was obtained with acidified acetone; water mixture (60% to 70%, v/v). Crude proanthocyanidin extracts thus prepared were purified using sephadex gel column chromatography and their average degree of polymerization and the effects on enzymatic hydrolysis of bovine serum albumin as influenced by their protein precipitation capacities were studied. Average degree of polymerization of proanthocyanidins in berry pulp, kernel, seed coat, and leaves was 7.4, 5.6, 8.2, and 10.6, respectively. The EC50 values for the protein precipitation by the PA of berry pulp, kernel seed coat, and leaves were 44.2, 44.1, 65.8, and 39.8 μg, respectively. Relative enzymatic hydrolysis of the protein-proanthocyanidin complexes was 44.1% to 60.3% for pepsin and 57.5% to 67.7% for trypsin. Interactions of sea buckthorn proanthocyanidins with food proteins and digestive enzymes might alter the protein digestibility and phenolic bioavailabilty. PMID:22417524

  10. 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

  11. 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).

  12. Microbial pretreatment of corn stover with Ceriporiopsis subvermispora for enzymatic hydrolysis and ethanol production.

    PubMed

    Wan, Caixia; Li, Yebo

    2010-08-01

    The feasibility of concurrent wet storage and microbial pretreatment of corn stover with Ceriporiopsis subvermispora for ethanol production was investigated in this study. The effects of particle size (5-15 mm), moisture content (45-85%), pretreatment time (18-35 d), and temperature (4-37 degrees C) on lignin degradation and enzymatic hydrolysis yield were studied. The results showed that C. subvermispora selectively degraded lignin up to 31.59% with a limited cellulose loss of less than 6% during an 18-d pretreatment. When 5mm corn stover was pretreated at 28 degrees C with 75% moisture content, overall glucose yields of 57.67%, 62.21%, and 66.61% were obtained with 18-, 28-, and 35-d microbial pretreated corn stover, respectively. For the above conditions, the highest overall ethanol yield of 57.80% was obtained with 35-d-pretreated corn stover. Enzymatic hydrolysis yield was highly related to the lignin removal during microbial pretreatment.

  13. [Role of genistein in enzymatic albumin hydrolysis in the presence of nitrates (III) and (V)].

    PubMed

    Tokarz, Andrzej; Pokorska-Lis, Grazyna; Popiel, Elzbieta

    2008-01-01

    Polyphenols and nitrates are essential ingredients of human diet. Harm caused by nitrates is well know and studied. Positive role of polyphenols is investigated. The aim of the study was to analyze interactions between nitrates (III) and (V) and genistein in systems of enzymatic protein (albumin) hydrolysis. In vitro model of enzymatic acidic-alkaline albumine hydrolysis in the presence of nitrates, polyphenols and vitamin C in different concentrations was used. Content of nitrates was measured in dialysation fluid spectrophotometrically according to Griess' method. The study revealed inhibiting influence of genistein on nitrares(III) concentration in external compartment. The influence depended on polyphenol dose (for nitrates (III) between 11.21% and 7.27%, for nitrates (V) between 95.64% and 79.64% of dialysis). When genistein was introduced in too high concentrations--over 2,4 mg/system--it did not improve the effect, but inhibited it. The influence of genistein was synergic with resveratrol and vitamin C.

  14. Production of fermentable sugars from sugarcane bagasse by enzymatic hydrolysis after autohydrolysis and mechanical refining.

    PubMed

    Batalha, Larisse Aparecida Ribas; Han, Qiang; Jameel, Hasan; Chang, Hou-Min; Colodette, Jorge Luiz; Borges Gomes, Fernando José

    2015-03-01

    The autohydrolysis process has been considered a simple, low-cost and environmental friendly technology for generation of sugars from biomass. In order to improve accessibility of enzymes during enzymatic hydrolysis as well as to allow the recovery of hemicellulose in the filtrate, the sugarcane bagasse was pretreated using autohydrolysis followed by a mechanical refining process. The autohydrolysis was carried out in three different conditions. Autohydrolysis at 190°C for 10min provided the highest overall sugar (19.2/100g raw bagasse) in prehydrolyzate. The enzymatic hydrolysis step was performed for all the post-treated solids with and without refining at enzyme loadings of 5 and 10FPU/g for 96h. A total of 84.4% of sugar can be recovered from sugarcane bagasse at 180°C for 20min with 5 FPU/g enzyme charge. The economic analysis for the proposed method showed that the bioethanol production can have a financial return larger than 12%.

  15. Optimization of thermo-chemical pretreatment and enzymatic hydrolysis of kitchen wastes.

    PubMed

    Vavouraki, Aikaterini I; Volioti, Vassiliki; Kornaros, Michael E

    2014-01-01

    The use of abundant waste materials with high carbohydrate content may contribute substantially to reduction of biofuels production cost. The present study aimed at optimizing the combined effect of thermo-chemical pretreatment and enzymatic hydrolysis of kitchen wastes (KW) for maximizing the production of fermentable soluble sugars. To this end, acid pretreatment of KW samples was performed with hydrochloric acid (0-3% HCl) at 30-100 °C for 0-120 min treatment time. Alternatively, alkaline pretreatment of KW samples was performed with potassium hydroxide solution (0-11%) at constant temperature and time (0 °C and 20 min, respectively). KOH pretreatment at such conditions targets to degrade the resistant starch of KW samples. Both acid and alkaline pretreatments were followed by addition of variable levels of enzyme dosage (0-3.6% v/v α-amylase and 0-3.2% v/v amyloglucosidase-AMG) at constant pH, temperature and time (pH = 5, T = 50 °C and t = 30 min, respectively). Based on our results, glucose concentration increased by ~300% after pretreatment with either acid or KOH in combination with enzymatic hydrolysis (2% HCl, 85 °C, 80 min, 0.1% α-amylase, AMG, and 1% KOH, 0 °C, 20 min, 1.1% α-amylase, 0.4% AMG) compared to raw (untreated) KW. Estimating the different YG yields at KW loading of 5%, an increase of 192% and 121% for total soluble monosugars and total soluble sugars, respectively, was succeeded compared to untreated KW. The effect of solids loading on the obtained sugar yields using the optimum conditions for thermo-chemical pretreatment followed by enzymatic hydrolysis was also tested resulting to 27.5% increase of the soluble glucose yield when half of the solids loading (2.5%) was used. A decrease of total soluble sugars yield by 32.2% was observed when solely acid hydrolysis at optimum conditions from our previous study was applied at 30% solids loading.

  16. Feasibility of pressurization to speed up enzymatic hydrolysis of biological materials for multielement determinations.

    PubMed

    Moreda-Piñeiro, Antonio; Bermejo-Barrera, Adela; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Jorge; Alonso-Rodríguez, Elia; Muniategui-Lorenzo, Soledad; López-Mahía, Purificación; Prada-Rodríguez, Darío

    2007-03-01

    The feasibility of pressurized solvents (liquids at a high pressure and/or high temperature without the subcritical point being reached) has been newly investigated to accelerate enzymatic hydrolysis processes of mussel tissue for multielement determinations. The target elements (Al, As, Cd, Co, Cu, Fe, Hg, Li, Mn, Pb, Se, Sr, V, and Zn) were released from dried mussel tissue by action of two proteases (pepsin and pancreatin), and they have been evaluated by inductively coupled plasma optical emission spectrometry (ICP-OES). Variables inherent to the enzymatic activity (pH, ionic strength, temperature, and enzyme mass) and factors affecting pressurization (static time, pressure, and number of cycles) were simultaneously studied by applying a Plackett-Burman design (PBD) as the screening method. Results showed that pH, ionic strength, and temperature were the most statistically significant factors (confidence interval of 95%) under pressurized conditions for pepsin, while pH and ionic strength affected pancreatin activity. This means that metal extraction is mostly attributed to enzymatic activity. The static time (enzymatic hydrolysis time) was found statistically nonsignificant for most of the elements, meaning that the hydrolysis procedure can be finished within a 2-15 min range. For pepsin, optimized conditions (pH 1.0, temperature 40 degrees C, pressure 1500 psi, static time 2 min, and number of cycles 3) gave quantitative extractions for As, Cd, Co, Cu, Hg, Li, Mn, Pb, Se, Sr, V, and Zn. The pepsin mass was 0.05 g, and the solution was Milli-Q water at pH 1.0 (adjusted with hydrochloric acid). For pancreatin, quantitative recoveries were only reached for As, Cd, Cu, Li, Pb, and Sr at room temperature, at a pressure of 1500 psi, for a static time of 2 min and a number of cycles of 3. The extraction solution was a 0.3 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer at a pH of 7.5 working at room temperature. Around 0.5 g of diatomaceous

  17. 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

  18. Ethanol production from cashew apple bagasse: improvement of enzymatic hydrolysis by microwave-assisted alkali pretreatment.

    PubMed

    Rodrigues, Tigressa Helena Soares; Rocha, Maria Valderez Ponte; de Macedo, Gorete Ribeiro; Gonçalves, Luciana R B

    2011-07-01

    In this work, the potential of microwave-assisted alkali pretreatment in order to improve the rupture of the recalcitrant structures of the cashew able bagasse (CAB), lignocellulosic by-product in Brazil with no commercial value, is obtained from cashew apple process to juice production, was studied. First, biomass composition of CAB was determined, and the percentage of glucan and lignin was 20.54 ± 0.70% and 33.80 ± 1.30%, respectively. CAB content in terms of cellulose, hemicelluloses, and lignin, 19.21 ± 0.35%, 12.05 ± 0.37%, and 38.11 ± 0.08%, respectively, was also determined. Results showed that, after enzymatic hydrolysis, alkali concentration exerted influence on glucose formation, after pretreatment with 0.2 and 1.0 mo L(-1) of NaOH (372 ± 12 and 355 ± 37 mg g(glucan)(-1) ) when 2% (w/v) of cashew apple bagasse pretreated by microwave-assisted alkali pretreatment (CAB-M) was used. On the other hand, pretreatment time (15-30 min) and microwave power (600-900 W) exerted no significant effect on hydrolysis. On enzymatic hydrolysis step, improvement on solid percentage (16% w/v) and enzyme load (30 FPU g (CAB-M) (-1) ) increased glucose concentration to 15 g L(-1). The fermentation of the hydrolyzate by Saccharomyces cerevesiae resulted in ethanol concentration and productivity of 5.6 g L(-1) and 1.41 g L(-1) h(-1), respectively.

  19. 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

  20. 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

  1. 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.

  2. Enhanced enzymatic hydrolysis and ethanol production from cashew apple bagasse pretreated with alkaline hydrogen peroxide.

    PubMed

    da Costa, Jessyca Aline; Marques, José Edvan; Gonçalves, Luciana Rocha Barros; Rocha, Maria Valderez Ponte

    2015-03-01

    The effect of combinations and ratios between different enzymes has been investigated in order to assess the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen peroxide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic hydrolysis conducted with cellulase complex and β-glucosidase in a ratio of 0.61:0.39, enzyme loading of 30FPU/g(CAB-AHP) and 66CBU/g(CAB-AHP), respectively, using 4% cellulose from CAB-AHP, turned out to be the most effective conditions, with glucose and xylose yields of 511.68 mg/g(CAB-AHP) and 237.8 mg/g(CAB-AHP), respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an ethanol yield of 61.8kg/ton(CAB), corresponding to 15 g/L ethanol and productivity of 3.75 g/( Lh). The ethanol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80% yield and 74.2kg/ton(CAB).

  3. Step enzymatic hydrolysis of sodium hydroxide-pretreated Chinese liquor distillers' grains for ethanol production.

    PubMed

    Liu, Yue-Hong; Wu, Zheng-Yun; Yang, Jian; Yuan, Yu-Ju; Zhang, Wen-Xue

    2014-01-01

    Distillers' grains are a co-product of ethanol production. In China, only a small portion of distillers' grains have been used to feed the livestock because the amount was so huge. Nowadays, it has been reported that the distillers' grains have the potential for fuel ethanol production because they are composed of lignocelluloses and residual starch. In order to effectively convert distillers' grains to fuel ethanol and other valuable production, sodium hydroxide pretreatment, step-by-step enzymatic hydrolysis, and simultaneous saccharification and fermentation (SSF) were investigated. The residual starch was first recycled from wet distillers' grains (WDG) with glucoamylase to obtain glucose-rich liquid. The total sugar concentration was 21.3 g/L, and 111.9% theoretical starch was hydrolyzed. Then the removed-starch dry distillers' grains (RDDG) were pretreated with NaOH under optimal conditions and the pretreated dry distillers' grains (PDDG) were used for xylanase hydrolysis. The xylose concentration was 19.4 g/L and 68.6% theoretical xylose was hydrolyzed. The cellulose-enriched dry distillers' grains (CDDG) obtained from xylanase hydrolysis were used in SSF for ethanol production. The ethanol concentration was 42.1 g/L and the ethanol productivity was 28.7 g/100 g CDDG. After the experiment, approximately 80.6% of the fermentable sugars in WDG was converted to ethanol.

  4. Enhanced enzymatic hydrolysis and ethanol production from cashew apple bagasse pretreated with alkaline hydrogen peroxide.

    PubMed

    da Costa, Jessyca Aline; Marques, José Edvan; Gonçalves, Luciana Rocha Barros; Rocha, Maria Valderez Ponte

    2015-03-01

    The effect of combinations and ratios between different enzymes has been investigated in order to assess the optimal conditions for hydrolysis of cashew apple bagasse pretreated with alkaline hydrogen peroxide (the solids named CAB-AHP). The separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes were evaluated in the ethanol production. The enzymatic hydrolysis conducted with cellulase complex and β-glucosidase in a ratio of 0.61:0.39, enzyme loading of 30FPU/g(CAB-AHP) and 66CBU/g(CAB-AHP), respectively, using 4% cellulose from CAB-AHP, turned out to be the most effective conditions, with glucose and xylose yields of 511.68 mg/g(CAB-AHP) and 237.8 mg/g(CAB-AHP), respectively. Fermentation of the pure hydrolysate by Kluyveromyces marxianus ATCC 36907 led to an ethanol yield of 61.8kg/ton(CAB), corresponding to 15 g/L ethanol and productivity of 3.75 g/( Lh). The ethanol production obtained for SSF process using K. marxianus ATCC 36907 was 18 g/L corresponding to 80% yield and 74.2kg/ton(CAB). PMID:25545094

  5. Step enzymatic hydrolysis of sodium hydroxide-pretreated Chinese liquor distillers' grains for ethanol production.

    PubMed

    Liu, Yue-Hong; Wu, Zheng-Yun; Yang, Jian; Yuan, Yu-Ju; Zhang, Wen-Xue

    2014-01-01

    Distillers' grains are a co-product of ethanol production. In China, only a small portion of distillers' grains have been used to feed the livestock because the amount was so huge. Nowadays, it has been reported that the distillers' grains have the potential for fuel ethanol production because they are composed of lignocelluloses and residual starch. In order to effectively convert distillers' grains to fuel ethanol and other valuable production, sodium hydroxide pretreatment, step-by-step enzymatic hydrolysis, and simultaneous saccharification and fermentation (SSF) were investigated. The residual starch was first recycled from wet distillers' grains (WDG) with glucoamylase to obtain glucose-rich liquid. The total sugar concentration was 21.3 g/L, and 111.9% theoretical starch was hydrolyzed. Then the removed-starch dry distillers' grains (RDDG) were pretreated with NaOH under optimal conditions and the pretreated dry distillers' grains (PDDG) were used for xylanase hydrolysis. The xylose concentration was 19.4 g/L and 68.6% theoretical xylose was hydrolyzed. The cellulose-enriched dry distillers' grains (CDDG) obtained from xylanase hydrolysis were used in SSF for ethanol production. The ethanol concentration was 42.1 g/L and the ethanol productivity was 28.7 g/100 g CDDG. After the experiment, approximately 80.6% of the fermentable sugars in WDG was converted to ethanol. PMID:24397718

  6. 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

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

    PubMed

    Tsai, Chien-Tai; Meyer, Anne S

    2014-11-25

    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.

  8. 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.

  9. 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

  10. Increase in ethanol production from sugarcane bagasse based on combined pretreatments and fed-batch enzymatic hydrolysis.

    PubMed

    Wanderley, Maria Carolina de Albuquerque; Martín, Carlos; Rocha, George Jackson de Moraes; Gouveia, Ester Ribeiro

    2013-01-01

    Enzymatic hydrolysis of pretreated sugarcane bagasse was performed to investigate the production of ethanol. The sugarcane bagasse was pretreated in a process combining steam explosion and alkaline delignification. The lignin content decreased to 83%. Fed-batch enzymatic hydrolyses was initiated with 8% (w/v) solids loading, and 10 FPU/g cellulose. Then, 1% solids were fed at 12, 24 or 48 h intervals. After 120 h, the hydrolysates were fermented with Saccharomyces cerevisiae UFPEDA 1238, and a fourfold increase in ethanol production was reached when fed-batch hydrolysis with a 12-h addition period was used for the steam pretreated and delignified bagasse.

  11. 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

  12. 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.

  13. Mechanism of the discrepancy in the enzymatic hydrolysis efficiency between defatted peanut flour and peanut protein isolate by Flavorzyme.

    PubMed

    Zheng, Lin; Zhao, Yijun; Xiao, Chuqiao; Sun-Waterhouse, Dongxiao; Zhao, Mouming; Su, Guowan

    2015-02-01

    Both defatted peanut flour (DPF) and peanut protein isolate (PPI) are widely used to prepare peanut protein hydrolysates. To compare their enzymatic hydrolysis efficiencies, DPF and PPI were hydrolysed by Alcalase, Neutrase, Papain, Protamex and Flavorzyme. Alcalase and Flavorzyme were found to be the most efficient proteases to hydrolyse both DPF and PPI. The efficiency was comparable to each other when using Alcalase, while PPI was hydrolysed less efficiently than DPF when using Flavorzyme. Analysis of changes in the protein solubility, subunit and conformation, and amino acid composition of DPF, PPI and their Flavorzyme hydrolysis residues indicated that the PPI preparation process had minimal effect on it, but peptide aggregation via non-covalent bonding (including hydrophobic interactions and hydrogen bonds) during hydrolysis and/or thermal treatment after hydrolysis were likely responsible for the reduced hydrolysis efficiency of PPI by Flavorzyme.

  14. 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.

  15. 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.

  16. 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.

  17. Effects of NIPAm polymer additives on the enzymatic hydrolysis of Avicel and pretreated Miscanthus.

    PubMed

    Mackenzie, Katherine J; Francis, Matthew B

    2014-09-01

    There is currently much interest in the economic use of cellulosic biomass as a source of renewable fuels. This process typically involves the enzymatic hydrolysis of plant matter to afford soluble sugars for subsequent fermentation steps. The cost of cellulase enzymes presents a critical barrier to the commercialization of these processes. In this work, we demonstrate that a new family of polymer additives based on NIPAm can increase enzyme performance substantially. When applied to an industrially relevant combination of enzymes and lignin-containing biomass, polymer additives allow a 60% reduction in enzyme loading to achieve the same level of saccharification. Evidence presented herein suggests that these polymers function through multiple mechanisms, including (1) preventing enzyme denaturation through shear and interfacial interactions, (2) preventing non-productive adsorption to lignin, and (3) altering the cellulose structure. An advantage of these polymers over other additives is their thermoresponsive behavior, enabling their recovery and reuse. PMID:24729018

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

    PubMed

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

    2003-09-01

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

  19. 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.

  20. 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

  1. 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...

  2. 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...

  3. Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars.

    PubMed

    Murnen, Hannah K; Balan, Venkatesh; Chundawat, Shishir P S; Bals, Bryan; Sousa, Leonardo da Costa; Dale, Bruce E

    2007-01-01

    Miscanthus x giganteus is a tall perennial grass whose suitability as an energy crop is presently being appraised. There is very little information on the effect of pretreatment and enzymatic saccharification of Miscanthus to produce fermentable sugars. This paper reports sugar yields during enzymatic hydrolysis from ammonia fiber expansion (AFEX) pretreated Miscanthus. Pretreatment conditions including temperature, moisture, ammonia loading, residence time, and enzyme loadings are varied to maximize hydrolysis yields. In addition, further treatments such as soaking the biomass prior to AFEX as well as washing the pretreated material were also attempted to improve sugar yields. The optimal AFEX conditions determined were 160 degrees C, 2:1 (w/w) ammonia to biomass loading, 233% moisture (dry weight basis), and 5 min reaction time for water-soaked Miscanthus. Approximately 96% glucan and 81% xylan conversions were achieved after 168 h enzymatic hydrolysis at 1% glucan loading using 15 FPU/(g of glucan) of cellulase and 64 p-NPGU/(g of glucan) of beta-glucosidase along with xylanase and tween-80 supplementation. A mass balance for the AFEX pretreatment and enzymatic hydrolysis process is presented.

  4. 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

  5. Kinetic modeling of enzymatic hydrolysis of pretreated kitchen wastes for enhancing bioethanol production.

    PubMed

    Cekmecelioglu, Deniz; Uncu, Oya N

    2013-03-01

    It is well known that use of low cost and abundant waste materials in microbial fermentations can reduce product costs. Kitchen wastes disposed of in large amounts from cafeterias, restaurants, dining halls, food processing plants, and household kitchens contain high amounts of carbohydrate components such as glucose, starch, and cellulose. Efficient utilization of these sugars is another opportunity to reduce ethanol costs. In this study, the effect of pretreatment methods (hot water, acid solutions, and a control) on enzymatic hydrolysis of kitchen wastes was evaluated using a kinetic modeling approach. Fermentation experiments conducted with and without traditional fermentation nutrients were assessed at constant conditions of pH 4.5 and temperature of 30°C for 48h using commercial dry baker's yeast, Saccharomyces cerevisiae. The control, which involved no treatment, and hot water treated samples gave close glucose concentrations after 6h. The highest and lowest rates of glucose production were found as 0.644 and 0.128 (h(-1)) for the control (or no-pretreated (NPT)) and 1% acid solutions, respectively. The fermentation results indicated that final ethanol concentrations are not significantly improved by adding nutrients (17.2-23.3g/L). Thus, it was concluded that product cost can be lowered to a large extent if (1) kitchen wastes are used as a substrate, (2) no fermentation nutrient is used, and (3) hydrolysis time is applied for about 6h. Further optimization study is needed to increase the yield to higher levels.

  6. 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

  7. 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

  8. Kinetic modeling of enzymatic hydrolysis of pretreated kitchen wastes for enhancing bioethanol production.

    PubMed

    Cekmecelioglu, Deniz; Uncu, Oya N

    2013-03-01

    It is well known that use of low cost and abundant waste materials in microbial fermentations can reduce product costs. Kitchen wastes disposed of in large amounts from cafeterias, restaurants, dining halls, food processing plants, and household kitchens contain high amounts of carbohydrate components such as glucose, starch, and cellulose. Efficient utilization of these sugars is another opportunity to reduce ethanol costs. In this study, the effect of pretreatment methods (hot water, acid solutions, and a control) on enzymatic hydrolysis of kitchen wastes was evaluated using a kinetic modeling approach. Fermentation experiments conducted with and without traditional fermentation nutrients were assessed at constant conditions of pH 4.5 and temperature of 30°C for 48h using commercial dry baker's yeast, Saccharomyces cerevisiae. The control, which involved no treatment, and hot water treated samples gave close glucose concentrations after 6h. The highest and lowest rates of glucose production were found as 0.644 and 0.128 (h(-1)) for the control (or no-pretreated (NPT)) and 1% acid solutions, respectively. The fermentation results indicated that final ethanol concentrations are not significantly improved by adding nutrients (17.2-23.3g/L). Thus, it was concluded that product cost can be lowered to a large extent if (1) kitchen wastes are used as a substrate, (2) no fermentation nutrient is used, and (3) hydrolysis time is applied for about 6h. Further optimization study is needed to increase the yield to higher levels. PMID:22959156

  9. 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.

  10. Effect of liquid hot water pretreatment severity on properties of hardwood lignin and enzymatic hydrolysis of cellulose.

    PubMed

    Ko, Ja Kyong; Kim, Youngmi; Ximenes, Eduardo; Ladisch, Michael R

    2015-02-01

    Lignin, one of the major components of lignocellulosic biomass, plays an inhibitory role on the enzymatic hydrolysis of cellulose. This work examines the role of lignin in pretreated hardwood, where extents of cellulose hydrolysis decrease, rather than increase with increasing severity of liquid hot water pretreatment. Hardwood pretreated with liquid hot water at severities ranging from log Ro  = 8.25 to 12.51 resulted in 80-90% recovery of the initial lignin in the residual solids. The ratio of acid insoluble lignin (AIL) to acid soluble lignin (ASL) increased and the formation of spherical lignin droplets on the cell wall surface was observed as previously reported in the literature. When lignins were isolated from hardwoods pretreated at increasing severities and characterized based on glass transition temperature (Tg ), the Tg of isolated lignins was found to increase from 171 to 180°C as the severity increased from log Ro  = 10.44 to 12.51. The increase in Tg suggested that the condensation reactions of lignin molecules occurred during pretreatment and altered the lignin structure. The contribution of the changes in lignin properties to enzymatic hydrolysis were examined by carrying out Avicel hydrolysis in the presence of isolated lignins. Lignins derived from more severely pretreated hardwoods had higher Tg values and showed more pronounced inhibition of enzymatic hydrolysis.

  11. 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.

  12. 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

  13. 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

  14. Effects of dilute acid pretreatment conditions on enzymatic hydrolysis monomer and oligomer sugar yields for aspen, balsam, and switchgrass.

    PubMed

    Jensen, Jill R; Morinelly, Juan E; Gossen, Kelsey R; Brodeur-Campbell, Michael J; Shonnard, David R

    2010-04-01

    The effects of dilute acid hydrolysis conditions were investigated on total sugar (glucose and xylose) yields after enzymatic hydrolysis with additional analyses on glucose and xylose monomer and oligomer yields from the individual hydrolysis steps for aspen (a hardwood), balsam (a softwood), and switchgrass (a herbaceous energy crop). The results of this study, in the form of measured versus theoretical yields and a severity analysis, show that for aspen and balsam, high dilute acid hydrolysis xylose yields were obtainable at all acid concentrations (0.25-0.75 wt.%) and temperatures (150-175 degrees C) studied as long as reaction time was optimized. Switchgrass shows a relatively stronger dependence on dilute acid hydrolysis acid concentration due to its higher neutralizing mineral content. Maximum total sugar (xylose and glucose; monomer plus oligomer) yields post-enzymatic hydrolysis for aspen, balsam, and switchgrass, were 88.3%, 21.2%, and 97.6%, respectively. In general, highest yields of total sugars (xylose and glucose; monomer plus oligomer) were achieved at combined severity parameter values (log CS) between 2.20 and 2.40 for the biomass species studied.

  15. 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.

  16. The family II carbohydrate-binding module of xylanase CflXyn11A from Cellulomonas flavigena increases the synergy with cellulase TrCel7B from Trichoderma reesei during the hydrolysis of sugar cane bagasse.

    PubMed

    Pavón-Orozco, Patricia; Santiago-Hernández, Alejandro; Rosengren, Anna; Hidalgo-Lara, María Eugenia; Stålbrand, Henrik

    2012-01-01

    Synergy between Cellulomonas flavigena xylanase CflXyn11A and Trichoderma reesei endoglucanase TrCel7B was assessed during hydrolysis of alkaline pretreated sugar cane bagasse (SCB) after 12-48 h, applying the individual enzymes and mixtures of the enzymes. A high degree of synergy (6.3) between CflXyn11A and TrCel7B in hydrolysis of SCB was observed after 12h in the equimolar mixture. A threefold decrease in the degree of synergy was observed with TrCel7B and the catalytic module of CflXyn11A; suggesting an important role played by the carbohydrate-binding module of CflXyn11A (CflXyn11A-CBM) in the observed synergy. Affinity electrophoresis and binding assays showed that CflXyn11A-CBM binds to xylans and to a lesser extent to cellulose. Our results suggest that synergy is more pronounced at early stages of hydrolysis. Furthermore, for the first time it is described that a CBM carried by a xylanase significantly enhances the synergy with a cellulase (threefold increase in synergy).

  17. An efficient process for the saccharification of wood chips by combined ionic liquid pretreatment and enzymatic hydrolysis.

    PubMed

    Viell, Jörn; Wulfhorst, Helene; Schmidt, Thomas; Commandeur, Ulrich; Fischer, Rainer; Spiess, Antje; Marquardt, Wolfgang

    2013-10-01

    A process concept combining pretreatment of wood in ionic liquids and subsequent enzymatic hydrolysis to sugars is herein investigated to identify operating conditions which allow for (i) the processing of larger wood chips of 10 mm length, (ii) low temperature, (iii) high sugar yield, and (iv) short processing time. A careful quantitative study of the interaction of pretreatment and hydrolysis reveals that hydrolysis is most effective if beech chips are first disintegrated in [EMIM][Ac] at 115 °C for 1.5 h. The cellulose conversion varies between 70.5 wt% and 90.2wt% for hydrolysis times between 5 h and 72 h. A complete recovery of cellulose and xylan resulting in a total saccharification of 65 wt% of the wood chips could be demonstrated. It is shown that short pretreatment times are required to enable high sugar yield as well as to limit product degradation.

  18. Improvement of the enzymatic hydrolysis of furfural residues by pretreatment with combined green liquor and ethanol organosolv.

    PubMed

    Yu, Hailong; Xing, Yang; Lei, Fuhou; Liu, Zhiping; Liu, Zuguang; Jiang, Jianxin

    2014-09-01

    Furfural residues (FRs) were pretreated with ethanol and a green liquor (GL) catalyst to produce fermentable sugar. Anthraquinone (AQ) was used as an auxiliary reagent to improve delignification and reduce cellulose decomposition. The results showed that 42.7% of lignin was removed and 96.5% of cellulose was recovered from substrates pretreated with 1.0 mL GL/g of dry substrate and 0.4% (w/w) AQ at 140°C for 1h. Compared with raw material, ethanol-GL pretreatment of FRs increased the glucose yield from 69.0% to 85.9% after 96 h hydrolysis with 18 FPU/g-cellulose for cellulase, 27 CBU/g-cellulose for β-glucosidase. The Brauner-Emmett-Teller surface area was reduced during pretreatment, which did not inhibit the enzymatic hydrolysis. Owing to the reduced surface area, the unproductive binding of cellulase to lignin was decreased, thus improving the enzymatic hydrolysis. The degree of polymerization of cellulose from FRs was too low to be a key factor for improving enzymatic hydrolysis.

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

    NASA Astrophysics Data System (ADS)

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

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

  20. 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

  1. Changes on structural properties of biomass pretreated by combined deacetylation with liquid hot water and its effect on enzymatic hydrolysis.

    PubMed

    Jiang, Wei; Chang, Senlin; Qu, Yongshui; Zhang, Zhiguo; Xu, Jian

    2016-11-01

    A novel combined pretreatment of deacetylation and liquid hot water (LHW) was invented which has been proved to be effective in increasing enzymatic hydrolysis yield of biomass. In order to further understand the effect of this new pretreatment process on biomass, the variation on structural properties including cellulose crystallinity index (CrI), specific surface area (SSA) and degree of polymerization (DP) before/after pretreatment and how these properties affected the enzymatic hydrolysis of biomass were explored. The improvement of pretreatment severity (PS) could increase CrI, SSA and reduce DP. Whereas the enhancement of degree of deacetylation could decrease SSA and DP. An optimal formula (E12Y=0.347(100-CrI)(-0.375)×(SSA)(0.203)×(1700-DP)(0.281)) was achieved to express the correlation between structural properties and enzymatic hydrolysis after 12h. The enzymatic yield was more sensitive to CrI than to SSA and DP. PMID:27611028

  2. 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

  3. 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

  4. Enzymatic hydrolysis of gelatin layers on used lith film using thermostable alkaline protease for recovery of silver and PET film.

    PubMed

    Masui, Akihiko; Yasuda, Masahiro; Fujiwara, Nobuaki; Ishikawa, Haruo

    2004-01-01

    To develop a new efficient and potential industrial enzymatic process for the recovery of silver and poly(ethylene terephthalate) (PET) from used lith film for printing, which has not been recycled at all, enzymatic hydrolysis of gelatin layers on lith film was investigated using the thermostabilized mutant enzyme of the alkaline protease from alkaliphilic Bacillus sp. B21-2. The rate of gelatin hydrolysis of lith film in a stirred-tank reactor increased with the temperature and enzyme concentration. The time required to complete the hydrolysis of gelatin on lith film was longer than that on X-ray film because of the tightly cross-linked structure of the gelatin layers of lith film. The time required to complete the hydrolysis by using the mutant enzyme was less than that using the wild-type enzyme. The gelatin hydrolysis of lith film was well explained by a model that took into consideration a number of physical processes in addition to the chemical process. PMID:15296460

  5. Enzymatic hydrolysis of penicillin in mixed ionic liquids/water two-phase system.

    PubMed

    Jiang, Yangyang; Xia, Hansong; Guo, Chen; Mahmood, Iram; Liu, Huizhou

    2007-01-01

    In this paper, an integrated process involving the mixed ionic liquids/water two-phase system (MILWS) is proposed to improve the efficiency for enzymatic hydrolysis of penicillin G. First, hydrophilic [C4mim]BF4 (1-butyl-3-methylimidazolium tetrafluoraborate) and NaH2PO4 salt form an ionic liquids aqueous two-phase system (ILATPS), which could extract penicillin from its fermentation broth efficiently. Second, a hydrophobic [C4mim]PF6 (1-butyl-3-methylimidazolium hexafluoraphosphate) is introduced into the ionic liquids-rich phase of ILATPS containing penicillin and converses it into MILWS. Penicillin is hydrolyzed by penicillin acylase in the water phase of MILWS at pH 5. The byproduct phenylacetic acid (PAA) is partitioned into the ionic liquids mixture phase, while the intended product 6-aminopenicillanic acid (6-APA) is precipitated at this pH. In comparison with a similar butyl acetate/water system (BAWS) at pH 4, MILWS exhibits two advantages. (1) The selectivity between PAA and penicillin is greatly optimized at pH 5 by varying the mole ratio of [C4mim]PF6/[C4mim]BF4 in MILWS, whereas in BAWS the unalterable nature of the organic solvent restricts the optimized pH for maximum selectivity between PAA and penicillin at pH 4. (2) The pH for 6-APA precipitation in BAWS is 4, whereas it shifts to pH 5 in MILWS due to the complexation between negatively charged 6-APA and the cationic surface of the ionic liquids micelle. As a result, the removal of the two products from the enzyme sphere at relatively high pH is permitted in MILWS, which is beneficial for enzymatic activity and stability in comparison with the acidic pH 4 environment in BAWS.

  6. 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

  7. 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.

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

    PubMed

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

    2012-01-01

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

  9. 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.

  10. Enzymatic hydrolysis of inulin to fructose by glutaraldehyde fixed yeast cells.

    PubMed

    Workman, W E; Day, D F

    1984-08-01

    Inulin, a polyfruction, is found as the reserve carbohydrate in the roots and tubers of various plants (i.e. Jerusalem artichoke, chicory, and dahlia tubers). The beta-fructofuranosidase (inulase) from the yeast Kluyveromyces fragilis is of interest because of its industrial potential in fructose syrup and alcohol production from inulin containing plants. We have found that the inulase of K. fragilis can be immobilized in the yeast cells by glutaraldehyde treatment. These cells are resistant to physical and enzymatic destruction. Although the exact nature of the immobilization is not fully understood, the kinetic parameters of the immobilized enzyme are similar to those of the soluble enzyme. No reduction of enzyme activity was observed after glutaraldehyde treatment and glutaraldehyde concentration did not affect enzyme activity. A 96% hydrolysis of dahlia inulin was achieved in 10.5 h with a 9.5% (w/v) fixed enzyme suspension. A Jerusalem artichoke extract containing 16.8%polyfructan was completely hydrolyzed in 3.5 h with a 0.24% (w/v)fixed enzyme suspension. This is a time frame feasible for industrial consideration. PMID:18553476

  11. 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%.

  12. 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

  13. 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

  14. 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.

  15. 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.

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

    PubMed

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

    2012-01-01

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

  17. 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.

  18. 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

  19. Fermentable sugar release from Jatropha seed cakes following lime pretreatment and enzymatic hydrolysis.

    PubMed

    Liang, Yanna; Siddaramu, Thara; Yesuf, Jemil; Sarkany, Nicolas

    2010-08-01

    Composition change of Jatropha seed cake samples was evaluated upon lime pretreatment at 100 degrees C with different parameters. With a lime dose of 0.2 g and a water content of 10 ml per gram of cake and a treatment period of 1 h, 38.2+/-0.6% of lignin was removed. However, 65+/-16% of hemicellulose was also lost under this condition. For all the treatments tested, cellulose content was not affected by lime supplementation. Through further examining total reducing sugar (TRS) release by enzymatic hydrolysis after lime pretreatment, we have found that 0.1 g of lime and 9 ml of water per gram of cake and 3 h pretreatment produced the maximal 68.9% conversion of cellulose. Without lime pretreatment, the highest cellulose conversion was 33.3%. One microalgal species, Schizochytrium limacinum SR21 was able to grow on the hydrolyzates and generate a biomass density of 3.2 g/l in 4 days.

  20. Effects of enzymatic hydrolysis of buckwheat protein on antigenicity and allergenicity

    PubMed Central

    Sung, Dong-Eun; Lee, Jeongok; Han, Youngshin; Shon, Dong-Hwa; Ahn, Kangmo

    2014-01-01

    BACKGROUND/OBJECTIVES Due to its beneficial health effects, use of buckwheat has shown a continuous increase, and concerns regarding the allergic property of buckwheat have also increased. This study was conducted for evaluation of the hydrolytic effects of seven commercial proteases on buckwheat allergens and its allergenicity. MATERIALS/METHODS Extracted buckwheat protein was hydrolyzed by seven proteolytic enzymes at individual optimum temperature and pH for four hours. Analysis was then performed using SDS-PAGE, immunoblotting, and competitive inhibition ELISA (ciELISA) with rabbit antiserum to buckwheat protein, and direct ELISA with pooled serum of 21 buckwheat-sensitive patients. RESULTS Alkaline protease, classified as serine peptidase, was most effective in reducing allergenicity of buckwheat protein. It caused decomposition of the whole buckwheat protein, as shown on SDS-PAGE, and results of immunoblotting showed that the rabbit antiserum to buckwheat protein no longer recognized it as an antigen. Allergenicity showed a decrease of more than 50% when pooled serum of patients was used in ELISA. Two proteolytic enzymes from Aspergillus sp. could not hydrolyze buckwheat allergens effectively, and the allergenicity even appeared to increase. CONCLUSIONS Serine-type peptidases appeared to show a relatively effective reduction of buckwheat allergenicity. However, the antigenicity measured using rabbit antiserum did not correspond to the allergenicity measured using sera from human patients. Production of less allergenic buckwheat protein may be possible using enzymatic hydrolysis. PMID:24944772

  1. Enhancing the enzymatic hydrolysis of corn stover by an integrated wet-milling and alkali pretreatment.

    PubMed

    He, Xun; Miao, Yelian; Jiang, Xuejian; Xu, Zidong; Ouyang, Pingkai

    2010-04-01

    An integrated wet-milling and alkali pretreatment was applied to corn stover prior to enzymatic hydrolysis. The effects of NaOH concentration in the pretreatment on crystalline structure, chemical composition, and reducing-sugar yield of corn stover were investigated, and the mechanism of increasing reducing-sugar yield by the pretreatment was discussed. The experimental results showed that the crystalline structure of corn stover was disrupted, and lignin was removed, while cellulose and hemicellulose were retained in corn stover by the pretreatment with 1% NaOH in 1 h. The reducing-sugar yield from the pretreated corn stovers increased from 20.2% to 46.7% when the NaOH concentration increased from 0% to 1%. The 1% NaOH pretreated corn stover had a holocellulose conversion of 55.1%. The increase in reducing-sugar yield was related to the crystalline structure disruption and delignification of corn stover. It was clarified that the pretreatment significantly enhanced the conversion of cellulose and hemicellulose in the corn stover to sugars.

  2. 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.

  3. A novel surfactant-assisted ionic liquid pretreatment of sugarcane bagasse for enhanced enzymatic hydrolysis.

    PubMed

    Nasirpour, N; Mousavi, S M; Shojaosadati, S A

    2014-10-01

    This study investigated a novel pretreatment method, as an essential step, for production of second generation bioethanol from sugarcane bagasse (SCB). Effect of tween 80 (TW) and polyethylene glycol 4000 (PEG) on SCB pretreatment was assessed using 1-butyl-3-methyl imidazolium chloride ([BMIM]Cl) as an ionic liquid (IL). Different concentrations of TW and PEG were used to determine the optimum concentration of surfactant for the highest percentage of cellulose conversion. TW and PEG increased lignin removal by 12.5% over the IL-only pretreated sample. The 3% (w/w) PEG showed a significant increase in enzymatic digestibility with an efficiency of 96.2% after 12h of hydrolysis; this was 23% higher than the efficiency of SCB pretreated with IL. The increase in digestibility of surfactant assisted IL pretreatment method can be attributed to the decrease in cellulose crystallinity, changes in the cellulose lattice, and delignification; which was confirmed by FT-IR, XRD and FE-SEM analysis.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. The promoting effects of manganese on biological pretreatment with Irpex lacteus and enzymatic hydrolysis of corn stover.

    PubMed

    Song, Lili; Ma, Fuying; Zeng, Yelin; Zhang, Xiaoyu; Yu, Hongbo

    2013-05-01

    The effect of metal ions on biological pretreatment was evaluated for improving subsequent enzymatic hydrolysis. Results showed that the efficiency of fungal pretreatment was greatly improved with manganese supplement in biomass. After enzymatic hydrolysis of 28-d pretreated corn stover, maximum glucose yield was 308.98 mg/g corn stover with manganese supplement, which increased by 61.39% as compared to the conventional fungal pretreatment. Furthermore, manganese also enhanced the production of ethanol, corresponding to a high ethanol conversion (83.39%). Manganese greatly improved the delignification of Irpex lacteus specially. Correspondingly, the efficiency of saccharification and fermentation was closely related to the removal of lignin. This study showed a promising effect of manganese on fungal pretreatment and the production of biofuels.

  11. 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

  12. Temperature induced decoupling of enzymatic hydrolysis and carbon remineralization in long-term incubations of Arctic and temperate sediments

    NASA Astrophysics Data System (ADS)

    Robador, Alberto; Brüchert, Volker; Steen, Andrew D.; Arnosti, Carol

    2010-04-01

    Extracellular enzymatic hydrolysis of high-molecular weight organic matter is the initial step in sedimentary organic carbon degradation and is often regarded as the rate-limiting step. Temperature effects on enzyme activities may therefore exert an indirect control on carbon mineralization. We explored the temperature sensitivity of enzymatic hydrolysis and its connection to subsequent steps in anoxic organic carbon degradation in long-term incubations of sediments from the Arctic and the North Sea. These sediments were incubated under anaerobic conditions for 24 months at temperatures of 0, 10, and 20 °C. The short-term temperature response of the active microbial community was tested in temperature gradient block incubations. The temperature optimum of extracellular enzymatic hydrolysis, as measured with a polysaccharide (chondroitin sulfate), differed between Arctic and temperate habitats by about 8-13 °C in fresh sediments and in sediments incubated for 24 months. In both Arctic and temperate sediments, the temperature response of chondroitin sulfate hydrolysis was initially similar to that of sulfate reduction. After 24 months, however, hydrolysis outpaced sulfate reduction rates, as demonstrated by increased concentrations of dissolved organic carbon (DOC) and total dissolved carbohydrates. This effect was stronger at higher incubation temperatures, particularly in the Arctic sediments. In all experiments, concentrations of volatile fatty acids (VFA) were low, indicating tight coupling between VFA production and consumption. Together, these data indicate that long-term incubation at elevated temperatures led to increased decoupling of hydrolytic DOC production relative to fermentation. Temperature increases in marine sedimentary environments may thus significantly affect the downstream carbon mineralization and lead to the increased formation of refractory DOC.

  13. 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.

  14. High glucose recovery from direct enzymatic hydrolysis of bisulfite-pretreatment on non-detoxified furfural residues.

    PubMed

    Xing, Yang; Bu, Lingxi; Sun, Dafeng; Liu, Zhiping; Liu, Shijie; Jiang, Jianxin

    2015-10-01

    This study reports four schemes to pretreat wet furfural residues (FRs) with sodium bisulfite for production of fermentable sugar. The results showed that non-detoxified FRs (pH 2-3) had great potential to lower the cost of bioconversion. The optimal process was that unwashed FRs were first pretreated with bisulfite, and the whole slurry was then directly used for enzymatic hydrolysis. A maximum glucose yield of 99.4% was achieved from substrates pretreated with 0.1 g NaHSO3/g dry substrate (DS), at a relatively low temperature of 100 °C for 3 h. Compared with raw material, enzymatic hydrolysis at a high-solid of 16.5% (w/w) specifically showed more excellent performance with bisulfite treated FRs. Direct bisulfite pretreatment improved the accessibility of substrates and the total glucose recovery. Lignosulfonate in the non-detoxified slurry decreased the non-productive adsorption of cellulase on the substrate, thus improving enzymatic hydrolysis.

  15. High glucose recovery from direct enzymatic hydrolysis of bisulfite-pretreatment on non-detoxified furfural residues.

    PubMed

    Xing, Yang; Bu, Lingxi; Sun, Dafeng; Liu, Zhiping; Liu, Shijie; Jiang, Jianxin

    2015-10-01

    This study reports four schemes to pretreat wet furfural residues (FRs) with sodium bisulfite for production of fermentable sugar. The results showed that non-detoxified FRs (pH 2-3) had great potential to lower the cost of bioconversion. The optimal process was that unwashed FRs were first pretreated with bisulfite, and the whole slurry was then directly used for enzymatic hydrolysis. A maximum glucose yield of 99.4% was achieved from substrates pretreated with 0.1 g NaHSO3/g dry substrate (DS), at a relatively low temperature of 100 °C for 3 h. Compared with raw material, enzymatic hydrolysis at a high-solid of 16.5% (w/w) specifically showed more excellent performance with bisulfite treated FRs. Direct bisulfite pretreatment improved the accessibility of substrates and the total glucose recovery. Lignosulfonate in the non-detoxified slurry decreased the non-productive adsorption of cellulase on the substrate, thus improving enzymatic hydrolysis. PMID:26150072

  16. 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.

  17. Investigation of the pellets produced from sugarcane bagasse during liquid hot water pretreatment and their impact on the enzymatic hydrolysis.

    PubMed

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

    2015-08-01

    In the process of liquid hot water (LHW) pretreatment, there are numbers of pellets formed on the lignocellulosic surface. The characteristics and effect of pellets on the enzymatic hydrolysis of LHW-treated sugarcane bagasse (SCB) were investigated. After SCB was treated with LHW at 180°C, the pellets deposited on the surface of solid residues were extracted gently with 1% sodium hydroxide (NaOH) solution. They were composed of 81.0% lignin, 7.0% glucan, and 3.2% xylan. The LHW pretreatment solution (PS) was sprayed to the filter paper, and the pellets were observed on its surface. Fourier transform infrared spectroscopy (FTIR) data showed that lignin was also the main component of the PS pellets. The effect of the pellets on enzymatic hydrolysis was chiefly attributed to the steric hindrance, not the cellulase adsorption. The structural characteristics of LHW-treated SCB might play a more important role in influencing the enzymatic hydrolysis than the pellets.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

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

    PubMed

    Sangkharak, Kanokphorn

    2011-11-01

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

  7. 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.

  8. 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.

  9. Rapid enzymatic hydrolysis using a novel recombinant β-glucuronidase in benzodiazepine urinalysis.

    PubMed

    Morris, Ayodele A; Chester, Scot A; Strickland, Erin C; McIntire, Gregory L

    2014-10-01

    Only trace amounts of parent benzodiazepines are present in urine following extensive metabolism and conjugation. Thus, hydrolysis of glucuronides is necessary for improved detection. Enzyme hydrolysis is preferred to retain identification specificity, but can be costly and time-consuming. The assessment of a novel recombinant β-glucuronidase for rapid hydrolysis in benzodiazepine urinalysis is presented. Glucuronide controls for oxazepam, lorazepam and temazepam were treated with IMCSzyme™ recombinant β-glucuronidase. Hydrolysis efficiency was assessed at 55°C and at room temperature (RT) using the recommended optimum pH. Hydrolysis efficiency for four other benzodiazepines was evaluated solely with positive patient samples. Maximum hydrolysis of glucuronide controls at 5 min at RT (mean analyte recovery ≥ 94% for oxazepam and lorazepam and ≥ 80% for temazepam) was observed. This was considerably faster than the optimized 30 min incubation time for the abalone β-glucuronidase at 65°C. Mean analyte recovery increased at longer incubation times at 55°C for temazepam only. Total analyte in patient samples compared well to targets from abalone hydrolysis after recombinant β-glucuronidase hydrolysis at RT with no incubation. Some matrix effect, differential reactivity, conjugation variability and transformation impacting total analyte recovery were indicated. The unique potential of the IMCSzyme™ recombinant β-glucuronidase was demonstrated with fast benzodiazepine hydrolysis at RT leading to decreased processing time without the need for heat activation. PMID:25217555

  10. Pectinase from Trichoderma reesei QM 9414

    SciTech Connect

    Haltmeier, T.; Leisola, M.; Ulmer, D.; Waldner, R.; Fiechter, A.

    1983-06-01

    The present study was undertaken to determine whether T. reesei produces pectinolytic enzymes and, if so, to determine their action pattern and specificity. The aim was also to find out the practical importance of pectinolytic activity in the hydrolysis of cellulosic materials. It was found that the presence of pectinase does not appear to facilitate the hydrolysis of cellulose in plant material through any cell-separating mechanism. It is concluded that cellulase and xylanase activities are the important activities in the practical hydrolysis of cellulosic materials and that lignin is the real limiting factor. (Refs. 12).

  11. 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.

  12. 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.

  13. Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites.

    PubMed

    Wang, Ping; Stone, Judith A; Chen, Katherine H; Gross, Susan F; Haller, Christine A; Wu, Alan H B

    2006-10-01

    Confirmation of opioids in urine samples of clinical patients requires liberation of opioids from their glucuronide conjugates. Both acid hydrolysis and enzyme hydrolysis using beta-glucuronidase from various sources have been reported, with the latter approach prevailing in most clinical toxicology laboratories. The goal of this study was to compare the efficiency of acid versus different enzyme hydrolysis methods in recovering morphine and common semisynthetic opioids from glucuronide standards and 78 patient urine samples that were screened positive for opioids as a class. Specimens were analyzed with a validated gas chromatography-mass spectrometry (GC-MS) procedure. With the exception of oxycodone, the results indicated that the majority of opioids tested were extensively glucuronide-conjugated in urine. Significantly, acid hydrolysis liberated > 90% of morphine and hydromorphone from their glucuronide standards but enzyme hydrolysis had lower and variable efficiency, depending on the opiate type and the enzyme source. In patient specimens, much higher concentrations of free codeine, morphine, hydromorphone, and oxymorphone were obtained with acid hydrolysis than with various enzyme methods. Incomplete hydrolysis using beta-glucuronidase could lead to false-negative results for many opioids when urine is tested for drugs of abuse. We conclude that acid hydrolysis is the method of choice for GC-MS confirmation of urine opioids.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. A water molecule identified as a substrate of enzymatic hydrolysis of cellulose: A statistical-mechanics study

    NASA Astrophysics Data System (ADS)

    Ikuta, Yasuhiro; Karita, Shuichi; Kitago, Yu; Watanabe, Nobuhisa; Hirata, Fumio

    2008-11-01

    We calculated three-dimensional (3D) distribution of water molecules around and inside a complex of a cellulase, Cel44A, with a cellohexaose, based on the 3D-RISM theory. A distinct peak is observed in the 3D-distribution of water at the position within the hydrogen-bond distance from the two residues Glu186 and Glu359 in the enzyme. We identified the water molecule as a substrate of the enzymatic hydrolysis reaction. The finding provides strong support to one of the proposed mechanisms concerning the reaction, that is the retention process.

  19. 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.

  20. Effect of four pretreatments on enzymatic hydrolysis and ethanol fermentation of wheat straw. Influence of inhibitors and washing.

    PubMed

    Toquero, Cristina; Bolado, Silvia

    2014-04-01

    Pretreatment is essential in the production of alcohol from lignocellulosic material. In order to increase enzymatic sugar release and bioethanol production, thermal, dilute acid, dilute basic and alkaline peroxide pretreatments were applied to wheat straw. Compositional changes in pretreated solid fractions and sugars and possible inhibitory compounds released in liquid fractions were analysed. SEM analysis showed structural changes after pretreatments. Enzymatic hydrolysis and fermentation by Pichia stipitis of unwashed and washed samples from each pretreatment were performed so as to compare sugar and ethanol yields. The effect of the main inhibitors found in hydrolysates (formic acid, acetic acid, 5-hydroxymethylfurfural and furfural) was first studied through ethanol fermentations of model media and then compared to real hydrolysates. Hydrolysates of washed alkaline peroxide pretreated biomass provided the highest sugar concentrations, 31.82g/L glucose, and 13.75g/L xylose, their fermentation yielding promising results, with ethanol concentrations reaching 17.37g/L.

  1. Susceptibility of a polycaprolactone-based root canal filling material to degradation. II. Gravimetric evaluation of enzymatic hydrolysis.

    PubMed

    Tay, Franklin R; Pashley, David H; Yiu, Cynthia K Y; Yau, Joyce Y Y; Yiu-fai, Mak; Loushine, Robert J; Weller, R Norman; Kimbrough, W Frank; King, Nigel M

    2005-10-01

    Polycaprolactone is susceptible to enzymatic biodegradation via ester bond cleavage. This study examined the susceptibility of Resilon, a polycaprolactone-based root filling material to enzymatic hydrolysis. Resilon, gutta-percha, and polycaprolactone disks, prepared by compression molding, were incubated in phosphate-buffered saline, lipase PS or cholesterol esterase at 37 degrees C for 96 h. They were retrieved at different time intervals for gravimetric analysis and scanning electron microscopy. The materials exhibited slight weight gains when incubated in phosphate-buffered saline that can be attributed to water sorption. Gutta-percha showed similar weight gains in the two enzymes. Conversely, Resilon and polycaprolactone exhibited extensive surface thinning and weight losses after incubation in lipase PS and cholesterol esterase. Glass filler particles in Resilon were exposed following surface dissolution of the polymer matrix, creating rough surface topography. Biodegradation of Resilon by bacterial and salivary enzymes warrants further investigation of their activities using cultures of endodontically relevant microbes and human saliva extracts.

  2. 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

  3. 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

  4. Effect of a milling pre-treatment on the enzymatic hydrolysis of carbohydrates in brewer's spent grain.

    PubMed

    Niemi, Piritta; Faulds, Craig B; Sibakov, Juhani; Holopainen, Ulla; Poutanen, Kaisa; Buchert, Johanna

    2012-07-01

    Millions of tonnes of brewer's spent grain (BSG) are annually produced worldwide as a by-product of the brewing industry. BSG has the potential to be a valuable source of food, chemicals and energy if cost-efficient fractionation methods can be developed. A 2-fold improvement in carbohydrate solubilisation could be achieved through the introduction of a milling step prior to enzymatic hydrolysis. Course and fine milled fractions were characterized by particle size distribution and light microscopy. Fine milling decreased particle size down to the micron level and this in turn improved the carbohydrate solubility yield by a multi-enzyme mixture from 23% up to 45%. Carbohydrate solubilisation could be further increased through the supplementation of this enzyme preparation with additional cellulases. The physical degradation caused by the milling also liberated soluble carbohydrates without the requirement of any enzymatic treatment.

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

    PubMed

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

    2014-08-01

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

  6. Enzymatic hydrolysis of cellulose: evaluation of cellulase culture filtrates under use conditions

    SciTech Connect

    Mandels, M.; Medeiros, J.E.; Andreotti, R.E.; Bissett, F.H.

    1981-09-01

    Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 hours. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given time was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration. (Refs. 28).

  7. Enzymatic hydrolysis of cellulose: evaluation of cellulase culture filtrates under use conditions

    SciTech Connect

    Mandels, M.; Medeiros, J.E.; Andreotti, R.E.; Bissett, F.H.

    1981-09-01

    Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 h. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given time was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme.

  8. 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.

  9. Effects of glycerol on enzymatic hydrolysis and ethanol production using sugarcane bagasse pretreated by acidified glycerol solution.

    PubMed

    Zhang, Zhanying; Wong, Heng H; Albertson, Peter L; Harrison, Mark D; Doherty, William O S; O'Hara, Ian M

    2015-09-01

    In this study, for the first time the effects of glycerol on enzymatic hydrolysis and ethanol fermentation were investigated. Enzymatic hydrolysis was inhibited slightly with 2.0 wt% glycerol, leading to reduction in glucan digestibility from 84.9% without glycerol to 82.9% (72 h). With 5.0 wt% and 10.0 wt% glycerol, glucan digestibility was reduced by 4.5% and 11.0%, respectively. However, glycerol did not irreversibly inhibit cellulase enzymes. Ethanol fermentation was not affected by glycerol up to 5.0 wt%, but was inhibited slightly at 10.0 wt% glycerol, resulting in reduction in ethanol yield from 86.0% in the absence of glycerol to 83.7% (20 h). Based on the results of laboratory and pilot-scale experiments, it was estimated that 0.142 kg ethanol can be produced from 1.0 kg dry bagasse (a glucan content of 38.0%) after pretreatment with acidified glycerol solution. PMID:26056778

  10. 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

  11. Combined deacetylation and PFI refining pretreatment of corn cob for the improvement of a two-stage enzymatic hydrolysis.

    PubMed

    Zhang, Yuedong; Mu, Xindong; Wang, Haisong; Li, Bin; Peng, Hui

    2014-05-21

    A combined deacetylation and PFI refining pretreatment was applied to corn cob for the improvement of a two-stage enzymatic hydrolysis. In stage 1, the pretreated corn cob was first hydrolyzed by xylanase to produce xylo-oligosaccharides (XOS). In stage 2, the solid residue isolated from stage 1 was further hydrolyzed by cellulase and β-glucosidase. NaOH, Na2CO3, and Ca(OH)2 were tested to remove acetyl groups in the process of deacetylation, and it was found that Ca(OH)2 could be the most suitable alkali for deacetylation in this work. After deacetylation using 0.8 mmol of Ca(OH)2/g of substrate and PFI refining, 50.5% xylan in the raw material could be hydrolyzed into XOS. The corresponding xylan yield of stage 1, the glucan yield of stage 2, and the total sugar yield (all sugars released in the hydrolyzate) after the two-stage enzymatic hydrolysis were 0.306, 0.305, and 0.661 g/g of corn cob, respectively. PMID:24810587

  12. Effects of glycerol on enzymatic hydrolysis and ethanol production using sugarcane bagasse pretreated by acidified glycerol solution.

    PubMed

    Zhang, Zhanying; Wong, Heng H; Albertson, Peter L; Harrison, Mark D; Doherty, William O S; O'Hara, Ian M

    2015-09-01

    In this study, for the first time the effects of glycerol on enzymatic hydrolysis and ethanol fermentation were investigated. Enzymatic hydrolysis was inhibited slightly with 2.0 wt% glycerol, leading to reduction in glucan digestibility from 84.9% without glycerol to 82.9% (72 h). With 5.0 wt% and 10.0 wt% glycerol, glucan digestibility was reduced by 4.5% and 11.0%, respectively. However, glycerol did not irreversibly inhibit cellulase enzymes. Ethanol fermentation was not affected by glycerol up to 5.0 wt%, but was inhibited slightly at 10.0 wt% glycerol, resulting in reduction in ethanol yield from 86.0% in the absence of glycerol to 83.7% (20 h). Based on the results of laboratory and pilot-scale experiments, it was estimated that 0.142 kg ethanol can be produced from 1.0 kg dry bagasse (a glucan content of 38.0%) after pretreatment with acidified glycerol solution.

  13. 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...

  14. Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: II. Quantification of inhibition and suitability of membrane reactors.

    PubMed

    Andrić, Pavle; Meyer, Anne S; Jensen, Peter A; Dam-Johansen, Kim

    2010-01-01

    Product inhibition of cellulolytic enzymes affects the efficiency of the biocatalytic conversion of lignocellulosic biomass to ethanol and other valuable products. New strategies that focus on reactor designs encompassing product removal, notably glucose removal, during enzymatic cellulose conversion are required for alleviation of glucose product inhibition. Supported by numerous calculations this review assesses the quantitative aspects of glucose product inhibition on enzyme-catalyzed cellulose degradation rates. The significance of glucose product inhibition on dimensioning of different ideal reactor types, i.e. batch, continuous stirred, and plug-flow, is illustrated quantitatively by modeling different extents of cellulose conversion at different reaction conditions. The main operational challenges of membrane reactors for lignocellulose conversion are highlighted. Key membrane reactor features, including system set-up, dilution rate, glucose output profile, and the problem of cellobiose are examined to illustrate the quantitative significance of the glucose product inhibition and the total glucose concentration on the cellulolytic conversion rate. Comprehensive overviews of the available literature data for glucose removal by membranes and for cellulose enzyme stability in membrane reactors are given. The treatise clearly shows that membrane reactors allowing continuous, complete, glucose removal during enzymatic cellulose hydrolysis, can provide for both higher cellulose hydrolysis rates and higher enzyme usage efficiency (kg(product)/kg(enzyme)). Current membrane reactor designs are however not feasible for large scale operations. The report emphasizes that the industrial realization of cellulosic ethanol requires more focus on the operational feasibility within the different hydrolysis reactor designs, notably for membrane reactors, to achieve efficient enzyme-catalyzed cellulose degradation.

  15. 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

  16. 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.

  17. 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.

  18. Constitutively cellulose-producing mutant of trichoderma reesei

    SciTech Connect

    Mishra, S.; Gopalkrishnan, K.S.; Ghose, T.K.

    1982-01-01

    The single major factor limiting the economy of enzymatic hydrolysis of cellulose is the cost of the cellulase enzymes. Most of the work to date in reducing this cost has centered around selection of hyperproducing strains using the standard cellulose medium. Cellulose is an insoluble substrate with obvious design disadvantages in bioreactors. It is often indicated that another reasonable approach to the problem would be the isolation of constitutive mutants which require no inducer to form cellulase. In fact, the goal of several laboratories around the world working on the hydrolysis of cellulosic substances has been to isolate a constitutive mutant. Reported here is the isolation of a constitutively cellulase producing mutant in T. reesei that produces high levels of all of the component enzymes in the cellulase complex in the presence of glucose or sucrose. With this constitutive mutant, it is hoped that a major hurdle of working with an insoluble substrate will be overcome. The mutant will allow easier operation and control of cellulose bioreactors and can be used for continuous enzyme production, as opposed to the present batch production system. Also, a number of other unusable susbtrates like whey and molasses can be used for commercial preparations of cellulases. (Refs. 2).

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. High solids enzymatic hydrolysis of pretreated lignocellulosic materials with a powerful stirrer concept.

    PubMed

    Ludwig, Daniel; Michael, Buchmann; Hirth, Thomas; Rupp, Steffen; Zibek, Susanne

    2014-02-01

    In this study, we present a powerful stirred tank reactor system that can efficiently hydrolyse lignocellulosic material at high solid content to produce hydrolysates with glucose concentration > 100 g/kg. As lignocellulosic substrates alkaline-pretreated wheat straw and organosolv-pretreated beech wood were used. The developed vertical reactor was equipped with a segmented helical stirrer, which was specially designed for high biomass hydrolysis. The stirrer was characterised according to mixing behaviour and power input. To minimise the cellulase dosage, a response surface plan was used. With the empirical relationship between glucose yield, cellulase loading and solid content, the minimal cellulase dosage was calculated to reach at least 70% yield at high glucose and high substrate concentrations within 48 h. The optimisation resulted in a minimal enzyme dosage of 30 FPU/g dry matter (DM) for the hydrolysis of wheat straw and 20 FPU/g DM for the hydrolysis of beech wood. By transferring the hydrolysis reaction from shaking flasks to the stirred tank reactor, the glucose yields could be increased. Using the developed stirred tank reactor system, alkaline-pretreated wheat straw could be converted to 110 g/kg glucose (76%) at a solid content of 20% (w/w) after 48 h. Organosolv-pretreated beech wood could be efficiently hydrolysed even at 30% (w/w) DM, giving 150 g/kg glucose (72%). PMID:24242162

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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...

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. Double enzymatic hydrolysis preparation of heme from goose blood and microencapsulation to promote its stability and absorption.

    PubMed

    Wang, Baowei; Cheng, Fansheng; Gao, Shun; Ge, Wenhua; Zhang, Mingai

    2017-02-15

    Iron deficiency anemia (IDA) is the most common nutritional deficiency worldwide. This deficiency could be solved by preparing stable, edible, and absorbable iron food ingredients using environmentally friendly methods. This study investigated enzymatic hydrolysis and microencapsulation process of goose blood. The physicochemical properties, stabilities of the microencapsulated goose blood hydrolysate (MGBH) and a supplement for rats with IDA were also evaluated. The results showed that the synergetic hydrolytic action of neutrase and alkaline protease significantly increased the heme-releasing efficiency. The heme was then microencapsulated using sodium caseinate, maltodextrin and carboxymethyl cellulose (CMC) as the edible wall material, and the encapsulation efficiency of the product reached 98.64%. Meanwhile, favorable thermal, storage and light stabilities were observed for the microencapsulation. It was found that MGBH can significantly improve the body weight and hematological parameters of IDA Wistar rat. PMID:27664688

  16. Double enzymatic hydrolysis preparation of heme from goose blood and microencapsulation to promote its stability and absorption.

    PubMed

    Wang, Baowei; Cheng, Fansheng; Gao, Shun; Ge, Wenhua; Zhang, Mingai

    2017-02-15

    Iron deficiency anemia (IDA) is the most common nutritional deficiency worldwide. This deficiency could be solved by preparing stable, edible, and absorbable iron food ingredients using environmentally friendly methods. This study investigated enzymatic hydrolysis and microencapsulation process of goose blood. The physicochemical properties, stabilities of the microencapsulated goose blood hydrolysate (MGBH) and a supplement for rats with IDA were also evaluated. The results showed that the synergetic hydrolytic action of neutrase and alkaline protease significantly increased the heme-releasing efficiency. The heme was then microencapsulated using sodium caseinate, maltodextrin and carboxymethyl cellulose (CMC) as the edible wall material, and the encapsulation efficiency of the product reached 98.64%. Meanwhile, favorable thermal, storage and light stabilities were observed for the microencapsulation. It was found that MGBH can significantly improve the body weight and hematological parameters of IDA Wistar rat.

  17. Effect of endoxylanase and α-L-arabinofuranosidase supplementation on the enzymatic hydrolysis of steam exploded wheat straw.

    PubMed

    Alvira, P; Negro, M J; Ballesteros, M

    2011-03-01

    The cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of the bioethanol production process from lignocellulosic biomass. Hemicellulases and other accessory enzymes are becoming crucial to increase enzymatic hydrolysis (EH) yields at low cellulase dosages. The aim of this work was to evaluate the effect of two recombinant hemicellulolytic enzymes on the EH of steam pretreated wheat straw. Pretreatments at two severity conditions were performed and the whole slurry obtained after steam explosion pretreatment was employed as substrate. An endoxylanase (Xln C) from Aspergillus nidulans and an α-L-arabinofuranosidase (AF) from Aspergillus niger, have been applied in combination with cellulase enzymes. A degree of synergism of 29.5% and increases up to 10% in the EH yields were obtained, showing the potential of accessory activities to improve the EH step and make the whole process more effective.

  18. Enzymatic hydrolysis of aspen biomass into fermentable sugars by using lignocellulases from Armillaria gemina.

    PubMed

    Jagtap, Sujit Sadashiv; Dhiman, Saurabh Sudha; Kim, Tae-Su; Li, Jinglin; Lee, Jung-Kul; Kang, Yun Chan

    2013-04-01

    A white rot fungus, identified as Armillaria gemina SKU2114 on the basis of morphological and phylogenetic analyses, was found to secrete efficient lignocellulose-degrading enzymes. The strain showed maximum endoglucanase, cellobiohydrolase, and β-glucosidase activities of 146, 34, and 15 U/mL, respectively, and also secreted xylanase, laccase, mannanase, and lignin peroxidase with activities of 1270, 0.16, 57, and 0.31 U/mL, respectively, when grown with rice straw as a carbon source. Among various plant biomasses tested for saccharification, aspen biomass produced the maximum amount of reducing sugar. Response surface methodology was used to optimize the hydrolysis of aspen biomass to achieve the highest level of sugar production. A maximum saccharification yield of 62% (429 mg/g-substrate) was obtained using Populus tomentiglandulosa biomass after 48 h of hydrolysis. A. gemina was shown to be a good option for use in the production of reducing sugars from lignocellulosic biomass.

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

    PubMed Central

    Bergamaschi, Mateus M.; Barnes, Allan; Queiroz, Regina H. C.; Hurd, Yasmin L.

    2013-01-01

    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 Δ9-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/x2 weighting with linear ranges (r2>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

  20. 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

  1. Focused ultrasound-assisted acceleration of enzymatic hydrolysis of alkylphenols and 17β-oestradiol glucuronide in fish bile.

    PubMed

    Vallejo, Asier; Usobiaga, Aresatz; Ortiz-Zarragoitia, Maren; Cajaraville, Miren P; Fernández, Luis A; Zuloaga, Olatz

    2010-11-01

    According to the European Water Framework Directive (WFD), alkylphenols, such as octylphenols and nonylphenols, and 17β-oestradiol are considered as priority or emerging pollutants, respectively, mainly due to their possible properties as endocrine-disrupting compounds (EDCs). EDCs are accumulated in liver, fat, kidney and bile in the glucuronide form. In order to determine the concentration of these compounds in bile, an enzymatic hydrolysis step is necessary. This step is usually long (~16 h), and in this sense, ultrasound probes were studied as a possible alternative energy source to accelerate this process. Enzymatic hydrolysis was reduced to 20 min using an ultrasound probe at one cycle and 10% of amplitude. For validation of analytical procedure, nonylphenol glucuronide (4NP-G), 4-tert-octylphenol glucuronide (4tOP-G) and 4-n-octylphenol glucuronide (4nOP-G) were synthesised while 17β-oestradiol glucuronide (E2-G) was commercially available. Bile from thick-lip grey mullets (Chelon labrosus) was spiked with known amounts of 4NP-G, 4tOP-G, 4nOP-G and E2-G and submitted to the optimised procedure. Good recoveries (77-122%), precision in the 5% to 12% range and limits of detection, ranging from the low nanogramme per gramme level for 4tOP, 4nOP and E2 to the low microgramme per gramme level for nonylphenols, were obtained. The optimised method was applied for the determination of alkylphenol in the bile of thick-lip grey mullets fish bile from the Urdaibai estuary (UNESCO reserve of the Biosphere, Bay of Biscay), and high concentrations (2.3-14.2 μg/g), such as those obtained in polluted areas, were measured. E2 was determined in the bile of thick-lip grey mullets, intraperitoneally injected with E2.

  2. Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF.

    PubMed

    Cantarella, Maria; Cantarella, Laura; Gallifuoco, Alberto; Spera, Agata; Alfani, Francesco

    2004-01-01

    Steam-exploded (SE) poplar wood biomass was hydrolyzed by means of a blend of Celluclast and Novozym cellulase complexes in the presence of the inhibiting compounds produced during the preceding steam-explosion pretreatment process. The SE temperature and time conditions were 214 degrees C and 6 min, resulting in a log R(0) of 4.13. In enzymatic hydrolysis tests at 45 degrees C, the biomass loading in the bioreactor was 100 g(DW)/L (dry weight) and the enzyme-to-biomass ratio 0.06 g/g(DW). The enzyme activities for endo-glucanase, exo-glucanase, and beta-glucosidase were 5.76, 0.55, and 5.98 U/mg, respectively. The inhibiting effects of components released during SE (formic, acetic, and levulinic acids, furfural, 5-hydroxymethyl furfural (5-HMF), syringaldehyde, 4-hydroxy benzaldehyde, and vanillin) were studied at different concentrations in hydrolysis runs performed with rinsed SE biomass as model substrate. Acetic acid (2 g/L), furfural, 5-HMF, syringaldehyde, 4-hydroxybenzaldehyde, and vanillin (0.5 g/L) did not significantly effect the enzyme activity, whereas formic acid (11.5 g/L) inactivated the enzymes and levulinic acid (29.0 g/L) partially affected the cellulase. Synergism and cumulative concentration effects of these compounds were not detected. SSF experiments show that untreated SE biomass during the enzymatic attack gives rise to a nonfermentable hydrolysate, which becomes fermentable when rinsed SE biomass is used. The presence of acetic acid, vanillin, and 5-HMF (0.5 g/L) in SSF of 100 g(DW) /L biomass gave rise to ethanol yields of 84.0%, 73.5%, and 91.0% respectively, with respective lag phases of 42, 39, and 58 h.

  3. 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

  4. 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.

  5. 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...

  6. Enzymatic Hydrolysis and Simultaneous Extraction for Preparation of Genipin from Bark of Eucommia ulmoides after Ultrasound, Microwave Pretreatment.

    PubMed

    Li, Lili; Guo, Yupin; Zhao, Lianfei; Zu, Yuangang; Gu, Huiyan; Yang, Lei

    2015-01-01

    A continuous process based on the combination of ultrasounds and/or microwaves pretreatments followed by enzymatic hydrolysis and simultaneous extraction (EHSE) has been proposed to recover genipin from Eucommia ulmoides bark. At first, in the pretreatment step, the mixture of 1.0 g dried bark powder and 10 mL deionized water were irradiated by microwave under 500 W for 10 min. Then, in hydrolysis step, the optimal conditions were as follows: 0.5 mg/mL of cellulase concentration, 4.0 pH of enzyme solution, 24 h of incubation time and 40 °C of incubation temperature. After incubation, 10 mL ethanol was added to extract genipin for 30 min by ultrasound. After EHSE treatment, the yield of genipin could reach 1.71 μmol/g. Moreover, scanning electron micrographs illustrated that severe structural disruption of plant was obtained by EHSE. The results indicated that the EHSE method provided a good alternative for the preparation of genipin from Eucommia ulmoides bark as well as other herbs.

  7. 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

  8. 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

  9. 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

  10. Enzymatic hydrolysis of nylons: quantification of the reaction rate of nylon hydrolase for thin-layered nylons.

    PubMed

    Nagai, Keisuke; Iida, Kazuki; Shimizu, Kimiaki; Kinugasa, Ryo; Izumi, Motoki; Kato, Dai-Ichiro; Takeo, Masahiro; Mochiji, Kozo; Negoro, Seiji

    2014-10-01

    Nylon hydrolase degrades various aliphatic nylons, including nylon-6 and nylon-66. We synthesized a nylon-66 copolymer (M w = 22,900, M n = 7,400), in which a part of an adipoyl unit (32 % molar ratio) of nylon-66 was replaced with a succinyl unit by interfacial polymerization. To quantify the reaction rate of the enzymatic hydrolysis of nylons at the surface of solid polymers, we prepared a thin layer of nylons on the bottom surface of each well in a polystyrene-based micro-assay plate. The thickness of the nylon layer was monitored by imaging analysis of the photographic data. More than 99 % of the copolymer with thicknesses of 260 nm (approximately 600 layers of polymer strands) were converted to water-soluble oligomers by nylon hydrolase (3 mg enzyme ml(-1)) at 30 °C within 60 h. These results were further confirmed by TLC analysis of the reaction products and by assay of liberated amino groups in the soluble fractions. The degradation rate of the thin-layered nylon-6 was similarly analyzed. We demonstrate that this assay enables a quantitative evaluation of the reaction rate of hydrolysis at the interface between the solid and aqueous phases and a quantitative comparison of the degradability for various polyamides.

  11. 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.

  12. 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.

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

    PubMed

    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 Takathermtrade mark and Diazymetrade mark (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.

  14. 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

  15. Effect of ionic liquid pretreatment on the chemical composition, structure and enzymatic hydrolysis of energy cane bagasse.

    PubMed

    Qiu, Zenghui; Aita, Giovanna M; Walker, Michelle S

    2012-08-01

    Ionic liquids (ILs) are promising solvents for the pretreatment of lignocellulose as they are thermally stable, environmentally friendly, recyclable, and have low volatility. This study evaluated the effect of 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]) for the pretreatment of energy cane bagasse in terms of biomass composition, structural changes and enzymatic digestibility. Energy cane bagasse was pretreated with [EMIM][OAc] (5% (w/w)) at 120 °C for 30 min followed by hydrolysis with commercially available enzymes, Spezyme CP and Novozyme 188. IL-treated energy cane bagasse resulted in significant lignin removal (32.0%) with slight glucan and xylan losses (8.8% and 14.0%, respectively), and exhibited a much higher enzymatic digestibility (87.0% and 64.3%) than untreated (5.5% and 2.8%) or water-treated (4.0% and 2.1%) energy cane bagasse in terms of both cellulose and hemicellulose digestibilities, respectively. The enhanced digestibilities of IL-treated biomass can be attributed to delignification and reduction of cellulose crystallinity as confirmed by FTIR and XRD analyses.

  16. 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

  17. [Use of Endoglucanase IV from Trichoderma reesei to Enhance the Hydrolytic Activity of a Cellulase Complex from the Fungus Penicillium verruculosum].

    PubMed

    Proskurina, O V; Korotkova, O G; Rozhkova, A M; Kondrat'eva, E G; Matys, V Yu; Zorov, I N; Koshelev, A V; Okunev, O N; Nemashkalov, V A; Bubnova, T V; Sinitsyn, A P

    2015-01-01

    The effect of polysaccharide monooxygenase (endoglucanase IV) from the fungus Trichoderma reesei on the hydrolysis of polysaccharide substrates by cellulases secreted by the fungus Penicillium verruculosum has been investigated. Supplementation of the enzyme complex from P. verruculosum by endoglucanase IV from T. reesei has been shown to elevate the efficiency of cellulose hydrolysis by 45%.

  18. 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.

  19. Comparison of microbial inhibition and enzymatic hydrolysis rates of liquid and solid fractions produced from pretreatment of biomass with carbonic acid and liquid hot water.

    PubMed

    Yourchisin, Damon M; Van Walsum, G Peter

    2004-01-01

    This research quantified the enzymatic digestibility of the solid component and the microbial inhibition of the liquid component of pretreated aspen wood and corn stover hydrolysates. Products of liquid hot water and carbonic acid pretreatment were compared. Pretreatment temperatures tested ranged from 180 to 220 degrees C, and reaction times were varied between 4 and 64 min. Both microbial inhibition rates and enzymatic hydrolysis rates showed no difference between pretreatments containing carbonic acid and those not containing no carbonic acid. Microbial inhibition increased as the reaction severity increased, but only above a midpoint severity parameter of 200 degrees C for 16 min. Both the rates and yields of enzymatic hydrolysis displayed an increase from the lowest tested reaction severity to the highest tested reaction severity.

  20. 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

  1. 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.

  2. Effect of alkali pretreatment on the structural properties and enzymatic hydrolysis of corn cob.

    PubMed

    Sahare, Padmavati; Singh, Rajkumar; Laxman, R Seeta; Rao, Mala

    2012-12-01

    An effective alkali pretreatment which affects the structural properties of cellulose (corn cob) has been studied. The pretreatment of corn cob was carried out with different combinations of alkali at varying temperatures. The most effective pretreatment of corn cob was achieved with 1 % alkali at 50 °C in 4 h. The crystallinity index (CrI) and specific surface area (SSA) of untreated corn cob was 39 % and 0.52 m(2)/g wherein after alkali pretreatment CrI decreased to 15 % and SSA increased to 3.32 m(2)/g. The fungal organism was identified as Penicillium pinophilum on the basis of ITS sequence. At 5 % substrate concentration using a complete cellulase from Penicillium pinophilum the hydrolysis of untreated corn cob with 5, 10 and 20 FPU/g enzyme loadings were 11 %, 13 % and 16 %, whereas after alkali treatment the hydrolysis increased to 78 %, 90 % and 100 %, respectively. Further hydrolytic potential of commercial cellulases viz. Accellerase™ 1,000, Palkofeel-30 and Palkocel-40 were investigated under similar conditions. PMID:22983744

  3. 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

  4. 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.

  5. Enzymatic hydrolysis of cellulose coupled with electricity generation in a microbial fuel cell.

    PubMed

    Rezaei, Farzaneh; Richard, Tom L; Logan, Bruce E

    2008-12-15

    Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from a variety of biodegradable substrates, including cellulose. Particulate materials have not been extensively examined for power generation in MFCs, but in general power densities are lower than those produced with soluble substrates under similar conditions likely as a result of slow hydrolysis rates of the particles. Cellulases are used to achieve rapid conversion of cellulose to sugar for ethanol production, but these enzymes have not been previously tested for their effectiveness in MFCs. It was not known if cellulases would remain active in an MFC in the presence of exoelectrogenic bacteria or if enzymes might hinder power production by adversely affecting the bacteria. Electricity generation from cellulose was therefore examined in two-chamber MFCs in the presence and absence of cellulases. The maximum power density with enzymes and cellulose was 100 +/- 7 mW/m(2) (0.6 +/- 0.04 W/m(3)), compared to only 12 +/- 0.6 mW/m(2) (0.06 +/- 0.003 W/m(3)) in the absence of the enzymes. This power density was comparable to that achieved in the same system using glucose (102 +/- 7 mW/m(2), 0.56 +/- 0.038 W/m(3)) suggesting that the enzyme successfully hydrolyzed cellulose and did not otherwise inhibit electricity production by the bacteria. The addition of the enzyme doubled the Coulombic efficiency (CE) to CE = 51% and increased COD removal to 73%, likely as a result of rapid hydrolysis of cellulose in the reactor and biodegradation of the enzyme. These results demonstrate that cellulases do not adversely affect exoelectrogenic bacteria that produce power in an MFC, and that the use of these enzymes can increase power densities and reactor performance.

  6. Hydrolysis of fungal and plant cell walls by enzymatic complexes from cultures of Fusarium isolates with different aggressiveness to rye (Secale cereale).

    PubMed

    Jaroszuk-Ściseł, Jolanta; Kurek, Ewa

    2012-08-01

    The efficiency of hydrolysis of fungal (Fusarium spp.) cell wall and rye root cell wall by crude enzymatic complexes from (42-day-old) cultures of three F. culmorum isolates, a plant growth-promoting rhizosphere isolate (PGPF) DEMFc2, a deleterious rhizosphere isolate (DRMO) DEMFc5, and a pathogenic isolate DEMFc37, as well as two other, pathogenic isolates belonging to F. oxysporum and F. graminearum species was studied. In the enzymatic complexes originating from the Fusarium spp. cultures, the activities of the following cell wall-degrading enzymes were identified: glucanases, chitinases, xylanases, endocellulases, exocellulases, pectinases, and polygalacturonases. The preparation originating from a culture of the PGPF isolate was the least efficient in plant cell wall (PCW) hydrolysis. There were no significant differences in the efficiency of PCW hydrolysis between preparations from cultures of the DRMO and the pathogenic isolates. PGPF was the most efficient in liberating reducing sugars and N-acetylglucosamine (GlcNAc) from fungal cell walls (FCW). Xylanase activities of the enzymatic complexes were strongly positively (R > +0.9) correlated with their efficiency in hydrolyzing PCW, whereas chitinase activities were correlated with the efficiency in FCW hydrolysis.

  7. 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

  8. 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.

  9. Processing surface sizing starch using oxidation, enzymatic hydrolysis and ultrasonic treatment methods--Preparation and application.

    PubMed

    Brenner, Tobias; Kiessler, Birgit; Radosta, Sylvia; Arndt, Tiemo

    2016-03-15

    The surface application of starch is a well-established method for increasing paper strength. In surface sizing, a solution of degraded starch is applied to the paper. Two procedures have proved valuable for starch degradation in the paper mill: enzymatic and thermo-oxidative degradation. The objective of this study was to determine achievable efficiencies of cavitation in preparing degraded starch for surface application on paper. It was found that ultrasonic-assisted starch degradation can provide a starch solution that is suitable for surface sizing. The molecular composition of starch solutions prepared by ultrasonic treatment differed from that of starch solutions degraded by enzymes or by thermo-oxidation. Compared to commercial degradation processes, this resulted in intensified film formation and in greater penetration during surface sizing and ultimately in a higher starch content of the paper. Paper sized with ultrasonically treated starch solutions show the same strength properties compared to commercially sized paper.

  10. 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.

  11. 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.

  12. Enzymatic hydrolysis and fermentation of pretreated cashew apple bagasse with alkali and diluted sulfuric Acid for bioethanol production.

    PubMed

    Rocha, Maria Valderez Ponte; Rodrigues, Tigressa Helena Soares; de Macedo, Gorete Ribeiro; Gonçalves, Luciana R B

    2009-05-01

    The aim of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cashew apple bagasse (CAB) after diluted acid (CAB-H) and alkali pretreatment (CAB-OH), and to evaluate its fermentation to ethanol using Saccharomyces cerevisiae. Glucose conversion of 82 +/- 2 mg/g CAB-H and 730 +/- 20 mg/g CAB-OH was obtained when 2% (w/v) of solid and 30 FPU/g bagasse was used during hydrolysis at 45 degrees C, 2-fold higher than when using 15 FPU/g bagasse, 44 +/- 2 mg/g CAB-H, and 450 +/- 50 mg/g CAB-OH, respectively. Ethanol concentration and productivity, achieved after 6 h of fermentation, were 20.0 +/- 0.2 g L(-1) and 3.33 g L(-1) h(-1), respectively, when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g L(-1)). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g L(-1)), ethanol concentration and productivity were 8.2 +/- 0.1 g L(-1) and 2.7 g L(-1) h(-1) in 3 h, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 and 0.47 g/g glucose with pretreated CAB-OH and CAB-H, respectively. Ethanol concentration and productivity, obtained using CAB-OH hydrolyzate, were close to the values obtained in the conventional ethanol fermentation of cashew apple juice or sugar cane juice.

  13. 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

  14. 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

  15. Sequential acid and enzymatic hydrolysis in situ and bioethanol production from Gracilaria biomass.

    PubMed

    Wu, Fang-Chen; Wu, Jane-Yii; Liao, Yi-Jyun; Wang, Man-Ying; Shih, Ing-Lung

    2014-03-01

    Gracilaria sp., a red alga, was used as a feedstock for the production of bioethanol. Saccharification of Gracilaria sp. by sequential acid and enzyme hydrolysis in situ produced a high quality hydrolysate that ensured its fermentability to produce ethanol. The optimal saccharification process resulted in total 11.85g/L (59.26%) of glucose and galactose, Saccharomyces cerevisiae Wu-Y2 showed a good performance on co-fermentability of glucose and galactose released in the hydrolysate from Gracilaria sp. The final ethanol concentrations of 4.72g/L (0.48g/g sugar consumed; 94% conversion efficiency) and the ethanol productivity 4.93g/L/d were achieved. 1g of dry Gracilaria can be converted to 0.236g (23.6%) of bioethanol via the processes developed. Efficient alcohol production by immobilized S. cerevisiae Wu-Y2 in batch and repeated batch fermentation was also demonstrated. The findings of this study revealed that Gracilaria sp. can be a potential feedstock in biorefinery for ethanol production.

  16. Enzymatic hydrolysis of steam-exploded sugarcane bagasse using high total solids and low enzyme loadings.

    PubMed

    Ramos, Luiz Pereira; da Silva, Larissa; Ballem, Annielly Comelli; Pitarelo, Ana Paula; Chiarello, Luana Marcele; Silveira, Marcos Henrique Luciano

    2015-01-01

    Hydrolysis of phosphoric acid-impregnated steam-treated sugarcane bagasse was pre-optimized using a face-centered central composite design in which the process variables were the substrate total solids (TS, %), agitation intensity (AI, rpm) and enzyme loading (EL, gg(-1)). Pretreatment was carried out at 180°C for 10min using cane bagasse with 50wt% moisture content containing 9.5mg of H3PO4 per gram of dry biomass. Hydrolyses were performed for 96h at 50°C using Cellic CTec2® and water-washed steam-treated substrates. The highest amount of fermentable sugars was obtained with 20wt% TS, producing 76.8gL(-1) of glucose equivalents, which corresponded to a total glucan conversion of 69.2wt% and to a theoretical net increase of 39% in ethanol production from the same sugarcane tonnage without considering the use of leaves, tops and the additional yields from C5 sugars.

  17. Enzymatic hydrolysis of starch in the presence of cereal soluble fibre polysaccharides.

    PubMed

    Dhital, Sushil; Dolan, Grace; Stokes, Jason R; Gidley, Michael J

    2014-03-01

    The in vitro amylolysis of both granular and cooked maize starch and the diffusion of glucose in the presence of 1% and 2% cereal soluble fibre polysaccharides (arabinoxylan and mixed linkage beta-glucan) were studied at various levels of shear mixing in order to identify potential molecular mechanisms underlying observed glycemia-reducing effects of soluble fibres in vivo. The presence of soluble fibres increased viscosity by ca. 10× and 100× for 1% and 2% concentrations respectively. Despite this large difference in viscosity, measured digestion and mass transfer coefficients were only reduced by a factor of 1.5 to 2.5 at the same mixing speed. In contrast, introduction of mixing in the digesting and diffusing medium significantly increased the rate of amylolytic starch digestion and mass transfer of glucose. This effect is such that mixing at high speeds negates the hindering effect of the 100× increased viscosity imparted by the presence of 2% soluble fibre; this is essentially captured by the Reynolds number (the ratio of inertial and viscous forces) that defines the flow kinematics. The modest reduction of in vitro starch hydrolysis and glucose diffusion at increased viscosity suggests that the established benefits of soluble fibres on post-prandial glycaemia, in terms of attenuation of the overall rate and extent of dietary starch conversion to blood glucose, are not primarily due to a direct effect of viscosity. Alternative hypotheses are proposed based on gastric emptying, restriction of turbulent flow, and/or stimulation of mucus turnover.

  18. 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%.

  19. 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.

  20. 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

  1. Process optimization for the production of diosgenin with Trichoderma reesei.

    PubMed

    Zhu, Yuling; Ni, Jinren; Huang, Wen

    2010-06-01

    Based on the response surface methodology, an effective microbial system for diosgenin production from enzymatic pretreated Dioscorea zingiberensis tubers with Trichoderma reesei was studied. The fermentation medium was optimized with central composite design (3(5)) depended on Plackett-Burmann design which identified significant impacts of peptone, K(2)HPO(4) and Tween 80 on diosgenin yield. The effects of different fermentation conditions on diosgenin production were also studied. Four parameters, i.e. incubation period, temperature, initial pH and substrate concentration were optimized using 4(5) central composite design. The highest diosgenin yield of 90.57% was achieved with 2.67% (w/v) of peptone, 0.29% (w/v) of K(2)HPO(4), 0.73% (w/v) of Tween 80 and 9.77% (w/v) of substrate, under the condition of pH 5.8, temperature 30 degrees C. The idealized incubation time was 6.5 days. After optimization, the product yield increased by 33.70% as compared to 67.74 +/- 1.54% of diosgenin yield in not optimized condition. Scale-up fermentation was carried out in a 5.0 l bioreactor, maximum diosgenin yield of 90.17 +/- 3.12% was obtained at an aeration of 0.80 vvm and an agitation rate of 300 rpm. The proposed microbial system is clean and effective for diosgenin production and thus more environmentally acceptable than the traditional acid hydrolysis.

  2. Ionic liquid-mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis.

    PubMed

    Lee, Sang Hyun; Doherty, Thomas V; Linhardt, Robert J; Dordick, Jonathan S

    2009-04-01

    Lignocellulose represents a key sustainable source of biomass for transformation into biofuels and bio-based products. Unfortunately, lignocellulosic biomass is highly recalcitrant to biotransformation, both microbial and enzymatic, which limits its use and prevents economically viable conversion into value-added products. As a result, effective pretreatment strategies are necessary, which invariably involves high energy processing or results in the degradation of key components of lignocellulose. In this work, the ionic liquid, 1-ethyl-3-methylimidazolium acetate ([Emim][CH3COO]), was used as a pretreatment solvent to extract lignin from wood flour. The cellulose in the pretreated wood flour becomes far less crystalline without undergoing solubilization. When 40% of the lignin was removed, the cellulose crystallinity index dropped below 45, resulting in > 90% of the cellulose in wood flour to be hydrolyzed by Trichoderma viride cellulase. [Emim] [CH3COO] was easily reused, thereby resulting in a highly concentrated solution of chemically unmodified lignin, which may serve as a valuable source of a polyaromatic material as a value-added product.

  3. 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.

  4. 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

  5. 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

  6. Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize (Zea mays L.) lines.

    PubMed

    Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D; Williams, Daniel L; Magee, Timothy D; Kaeppler, Shawn M; de Leon, Natalia; Hodge, David B

    2015-07-01

    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.

  7. 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.

  8. Preparation of Yeast Hydrolysate Enriched in Cyclo-His-Pro (CHP) by Enzymatic Hydrolysis and Evaluation of Its Functionality.

    PubMed

    Lee, Hyun Jung; Son, Heung Soo; Park, Chung; Suh, Hyung Joo

    2015-12-01

    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.

  9. Evaluation of ammonia fibre expansion (AFEX) pretreatment for enzymatic hydrolysis of switchgrass harvested in different seasons and locations

    PubMed Central

    2010-01-01

    Background When producing biofuels from dedicated feedstock, agronomic factors such as harvest time and location can impact the downstream production. Thus, this paper studies the effectiveness of ammonia fibre expansion (AFEX) pretreatment on two harvest times (July and October) and ecotypes/locations (Cave-in-Rock (CIR) harvested in Michigan and Alamo harvested in Alabama) for switchgrass (Panicum virgatum). Results Both harvest date and ecotype/location determine the pretreatment conditions that produce maximum sugar yields. There was a high degree of correlation between glucose and xylose released regardless of the harvest, pretreatment conditions, or enzyme formulation. Enzyme formulation that produced maximum sugar yields was the same across all harvests except for the CIR October harvest. The least mature sample, the July harvest of CIR switchgrass, released the most sugars (520 g/kg biomass) during enzymatic hydrolysis while requiring the least severe pretreatment conditions. In contrast, the most mature harvest released the least amount of sugars (410 g/kg biomass). All hydrolysates were highly fermentable, although xylose utilisation in the July CIR hydrolysate was poor. Conclusions Each harvest type and location responded differently to AFEX pretreatment, although all harvests successfully produced fermentable sugars. Thus, it is necessary to consider an integrated approach between agricultural production and biochemical processing in order to insure optimal productivity. PMID:20047650

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. New insights into enzymatic hydrolysis of heterogeneous cellulose by using carbohydrate-binding module 3 containing GFP and carbohydrate-binding module 17 containing CFP

    PubMed Central

    2014-01-01

    Background The in-depth understanding of the enzymatic hydrolysis of cellulose with heterogeneous morphology (that is, crystalline versus amorphous) may help develop better cellulase cocktail mixtures and biomass pretreatment, wherein cost-effective release of soluble sugars from solid cellulosic materials remains the largest obstacle to the economic viability of second generation biorefineries. Results In addition to the previously developed non-hydrolytic fusion protein, GC3, containing a green fluorescent protein (GFP) and a family 3 carbohydrate-binding module (CBM3) that can bind both surfaces of amorphous and crystalline celluloses, we developed a new protein probe, CC17, which contained a mono-cherry fluorescent protein (CFP) and a family 17 carbohydrate-binding module (CBM17) that can bind only amorphous cellulose surfaces. Via these two probes, the surface accessibilities of amorphous and crystalline celluloses were determined quantitatively. Our results for the enzymatic hydrolysis of microcrystalline cellulose (Avicel) suggested that: 1) easily accessible amorphous cellulose on the surface of Avicel is preferentially hydrolyzed at the very early period of hydrolysis (that is, several minutes with a cellulose conversion of 2.8%); 2) further hydrolysis of Avicel is a typical layer-by-layer mechanism, that is, amorphous and crystalline cellulose regions were hydrolyzed simultaneously; and 3) most amorphous cellulose within the interior of the Avicel particles cannot be accessed by cellulase. Conclusions The crystallinity index (CrI), reflecting a mass-average (three-dimensional) cellulose characteristic, did not represent the key substrate surface (two-dimensional) characteristic related to enzymatic hydrolysis. PMID:24552554

  16. 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.

  17. 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

  18. 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.

  19. Effect of enzymatic hydrolysis with subsequent mild thermal oxidation of tallow on precursor formation and sensory profiles of beef flavours assessed by partial least squares regression.

    PubMed

    Song, Shiqing; Tang, Qi; Hayat, Khizar; Karangwa, Eric; Zhang, Xiaoming; Xiao, Zuobing

    2014-03-01

    Effects of different pretreatments of tallow on flavour precursor development and flavour profiles of beef flavours (BFs) were evaluated. Analysis of free fatty acids and volatiles of tallow by GC and GC-MS indicated that the enzymatic hydrolyzed-thermally oxidized tallow formed the most characteristic flavour precursors compared with others. The results of descriptive sensory analysis confirmed that beef flavour 4 from enzymatic hydrolyzed-thermally oxidized tallow had the strongest beefy, meaty and odour characteristics, followed by beef flavour 2 from oxidized tallow. Electronic nose data confirmed the accuracy of the sensory analysis results. The correlation analysis of 51 volatile compounds in tallow and sensory attributes of BFs showed that some compounds, especially aldehydes, made a significant contribution to sensory attributes. Correlation analysis of free fatty acids and sensory attributes through partial least squares regression (PLSR) confirmed that the moderate enzymatic hydrolysis-thermal oxidation pretreatment of tallow was necessary to achieve the characteristic beef flavour. PMID:24334040

  20. Effect of enzymatic hydrolysis with subsequent mild thermal oxidation of tallow on precursor formation and sensory profiles of beef flavours assessed by partial least squares regression.

    PubMed

    Song, Shiqing; Tang, Qi; Hayat, Khizar; Karangwa, Eric; Zhang, Xiaoming; Xiao, Zuobing

    2014-03-01

    Effects of different pretreatments of tallow on flavour precursor development and flavour profiles of beef flavours (BFs) were evaluated. Analysis of free fatty acids and volatiles of tallow by GC and GC-MS indicated that the enzymatic hydrolyzed-thermally oxidized tallow formed the most characteristic flavour precursors compared with others. The results of descriptive sensory analysis confirmed that beef flavour 4 from enzymatic hydrolyzed-thermally oxidized tallow had the strongest beefy, meaty and odour characteristics, followed by beef flavour 2 from oxidized tallow. Electronic nose data confirmed the accuracy of the sensory analysis results. The correlation analysis of 51 volatile compounds in tallow and sensory attributes of BFs showed that some compounds, especially aldehydes, made a significant contribution to sensory attributes. Correlation analysis of free fatty acids and sensory attributes through partial least squares regression (PLSR) confirmed that the moderate enzymatic hydrolysis-thermal oxidation pretreatment of tallow was necessary to achieve the characteristic beef flavour.

  1. 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.

  2. Screening of cellulases for biofuel production: online monitoring of the enzymatic hydrolysis of insoluble cellulose using high-throughput scattered light detection.

    PubMed

    Jäger, Gernot; Wulfhorst, Helene; Zeithammel, Erik U; Elinidou, Efthimia; Spiess, Antje C; Büchs, Jochen

    2011-01-01

    A new prospective cellulase assay simultaneously combining high-throughput, online analysis and insoluble cellulosic substrates is described. The hydrolysis of three different insoluble cellulosic substrates, catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast), was monitored using the BioLector - allowing online monitoring of scattered light intensities in a continuously shaken microtiter plate. Cellulase activities could be quantitatively assayed using the BioLector. At low cellulase/cellulose ratios, the Michaelis-Menten parameters of the cellulase mixture were mainly affected by the crystallinity index of the cellulose. Here, the apparent maximum cellulase activities inversely correlated with the crystallinity index of the cellulose. At high cellulase/cellulose ratios the particle size of the cellulose, defining the external surface area accessible to the cellulases, was the key determining factor for cellulase activity. The developed technique was also successfully applied to evaluate the pH optimum of cellulases. Moreover, the non-hydrolytic deagglomeration of cellulose particles was investigated, for the first time, using high-throughput scattered light detection. In conclusion, this cellulase assay ideally links high-throughput, online analysis and realistic insoluble cellulosic substrates in one simple system. It will considerably simplify and accelerate fundamental research on cellulase screening.

  3. 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

  4. Enzymatic degradation of monolayer for poly(lactide) revealed by real-time atomic force microscopy: effects of stereochemical structure, molecular weight, and molecular branches on hydrolysis rates.

    PubMed

    Numata, Keiji; Finne-Wistrand, Anna; Albertsson, Ann-Christine; Doi, Yoshiharu; Abe, Hideki

    2008-08-01

    The influences of the stereochemical structure, the molecular weight, and the number of molecular branches for poly(lactide) (PLA) on enzymatic hydrolysis rates of PLA monolayers were studied by atomic force microscopy (AFM) and the Langmuir-Blodgett (LB) technique. Monolayers of six kinds of PLA with different molecular weights, stereochemical structure, and numbers of molecular branches were prepared by LB techniques and then characterized by AFM in air. The PLA molecules covered homogeneously with a silicon substrate and did not form lamellar crystals in the monolayer. We determined the initial hydrolysis rate of PLA monolayers in presence of proteinase K by volumetric analysis from the continuous AFM height images. The presence of D-lactyl unit reduced the hydrolysis rate of the monolayer. The hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. In contrast, the rates of erosion for branched PLLA monolayers were independent of the molecular weight of samples. The erosion rate of branched PLLA monolayers was found to be dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. From these results, furthermore, the hydrolysis mode of PLAs by proteinase K is discussed.

  5. 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.

  6. 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

  7. 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

  8. Industrial-scale steam explosion pretreatment of sugarcane straw for enzymatic hydrolysis of cellulose for production of second generation ethanol and value-added products.

    PubMed

    Oliveira, Fernando M V; Pinheiro, Irapuan O; Souto-Maior, Ana M; Martin, Carlos; Gonçalves, Adilson R; Rocha, George J M

    2013-02-01

    Steam explosion at 180, 190 and 200°C for 15min was applied to sugarcane straw in an industrial sugar/ethanol reactor (2.5m(3)). The pretreated straw was delignificated by sodium hydroxide and hydrolyzed with cellulases, or submitted directly to enzymatic hydrolysis after the pretreatment. The pretreatments led to remarkable hemicellulose solubilization, with the maximum (92.7%) for pretreatment performed at 200°C. Alkaline treatment of the pretreated materials led to lignin solubilization of 86.7% at 180°C, and only to 81.3% in the material pretreated at 200°C. All pretreatment conditions led to high hydrolysis conversion of cellulose, with the maximum (80.0%) achieved at 200°C. Delignification increase the enzymatic conversion (from 58.8% in the cellulignin to 85.1% in the delignificated pulp) of the material pretreated at 180°C, but for the material pretreated at 190°C, the improvement was less remarkable, while for the pretreated at 200°C the hydrolysis conversion decreased after the alkaline treatment.

  9. A NaBH₄ Coupled Ninhydrin-Based Assay for the Quantification of Protein/Enzymes During the Enzymatic Hydrolysis of Pretreated Lignocellulosic Biomass.

    PubMed

    Mok, Yiu Ki; Arantes, Valdeir; Saddler, Jack N

    2015-07-01

    Accurate protein quantification is necessary in many of the steps during the enzymatic hydrolysis of pretreated lignocellulosic biomass, from the fundamental determination of enzyme kinetics to techno-economic assessments, such as the use of enzyme recycling strategies, evaluation of enzyme costs, and the optimization of various process steps. In the work described here, a modified, more rapid ninhydrin-based protein quantification assay was developed to better quantify enzyme levels in the presence of lignocellulosic biomass derived compounds. The addition of sodium borohydride followed by acid hydrolysis at 130 °C greatly reduced interference from monosaccharides and oligosaccharides and decreased the assay time 6-fold. The modified ninhydrin assay was shown to be more accurate as compared to various traditional colorimetric protein assays when commercial cellulase enzyme mixtures were quantified under typical pretreated lignocellulosic biomass enzymatic hydrolysis conditions. The relatively short assay time and microplate-reading capability of the modified assay indicated that the method could likely be used for high-throughput protein determination. PMID:25987134

  10. 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.

  11. 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...

  12. 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

  13. Simultaneous pressurized enzymatic hydrolysis extraction and clean up for arsenic speciation in seafood samples before high performance liquid chromatography-inductively coupled plasma-mass spectrometry determination.

    PubMed

    Moreda-Piñeiro, Jorge; Alonso-Rodríguez, Elia; Moreda-Piñeiro, Antonio; Moscoso-Pérez, Carmen; Muniategui-Lorenzo, Soledad; López-Mahía, Purificación; Prada-Rodríguez, Darío; Bermejo-Barrera, Pilar

    2010-10-29

    The feasibility of pressurized conditions to assist enzymatic hydrolysis of seafood tissues for arsenic speciation was novelty studied. A simultaneous in situ (in cell) clean-up procedure was also optimized, which speeds up the whole sample treatment. Arsenic species (As(III), MMA, DMA, As(V), AsB and AsC) were released from dried seafood tissues using pepsin as a protease, and the arsenic species were separated/quantified by anion exchange high performance liquid chromatography (HPLC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). Variables inherent to the enzymatic activity (pH, temperature and ionic strength), the amount of enzyme (pepsin), and factors affecting pressurization (pressure, static time, number of cycles and amount of dispersing agent, C-18) were fully evaluated. Pressurized assisted enzymatic hydrolysis (PAEH) with pepsin can be finished after few minutes (two cycles of 2 min each one plus 3 min to reach the hydrolysis temperature of 50 °C). A total sample solubilisation is not achieved after the procedure, however it is efficient enough for breaking down certain bonds of bio-molecules and for releasing arsenic species. The developed method has been found to be precise (RSDs lower than 6% for As(III), DMA and As(V); and 3% for AsB) and sensitive (LOQs of 18.1, 36.2, 35.7, 28.6, 20.6 and 22.5 ng/g for As(III), MMA, DMA, As(V), AsB and AsC, respectively). The optimized methodology was successfully applied to different certified reference materials (DORM-2 and BCR 627) which offer certified AsB and DMA contents, and also to different seafood products (mollusks, white fishes and cold water fishes).

  14. 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

  15. Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

    PubMed

    Wang, Shu-Yang; Jiang, Bo-Ling; Zhou, Xiang; 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.

  16. Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

    PubMed

    Wang, Shu-Yang; Jiang, Bo-Ling; Zhou, Xiang; 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. Mutational effects on the catalytic mechanism of cellobiohydrolase I from Trichoderma reesei.

    PubMed

    Yan, Shihai; Li, Tong; Yao, Lishan

    2011-05-01

    QM/MD simulations are performed to study mutational effects on the glycosylation step of the oligosaccharide hydrolysis catalyzed by Trichoderma reesei cellobiohydrolase I. The potential of mean force along the reaction pathway is determined by the umbrella sampling method. A detailed mechanism is developed to illustrate the decrease in activity of the mutants. Our calculations demonstrate that (1) the E212Q mutation increases the overall activation barrier by ~4.0 kcal/mol, while the D214N mutation causes ~0.4 kcal/mol increase of the barrier, and (2) there is only one transition state identified in the wild type (WT) and D214N mutant, while two transition states exist in the E212Q mutant for the glycosylation process. The results explain the experimental observation that the E212Q mutant loses most of its hydrolysis capability, while the D214N mutant only reduces it slightly compared to the WT. Further analysis suggests that the proton transfer from Glu(217) to O(4) and the glycosidic bond cleavage between subsites +1 and -1 are concerted, facilitating the subsequent nucleophilic attack of Glu(212) on C(1)' in subsite -1. Our QM/MD study illustrates the importance of the prearrangement of the active site and provides atomic details of the enzymatic catalytic mechanism.

  2. Spectrophotometric assays for the enzymatic hydrolysis of the active metabolites of chlorpyrifos and parathion by plasma paraoxonase/arylesterase.

    PubMed

    Furlong, C E; Richter, R J; Seidel, S L; Costa, L G; Motulsky, A G

    1989-08-01

    Human serum plasma paraoxonase/arylesterase exhibits a genetic polymorphism for the hydrolysis of paraoxon. One allelic form of the enzyme hydrolyzes paraoxon slowly with a low turnover number and the other(s) hydrolyzes paraoxon rapidly with a high turnover number. Chlorpyrifos-oxon, the active metabolite of the insecticide chlorpyrifos (Dursban), is also hydrolyzed by plasma arylesterase/paraoxonase. A specific assay for measuring hydrolysis of this compound is described. This assay is not subject to interference by the esterase activity of serum albumin. The Km for chlorpyrifos-oxon hydrolysis was 75 microM. Hydrolysis was inhibited by phenyl acetate, EDTA, and organic solvents. Enzyme activity required calcium ions and was stimulated by sodium chloride. Hydrolysis was optimized by using methanol instead of acetone to dissolve substrate. Unlike the multimodal distribution of paraoxonase, the distribution of chlorpyrifos-oxonase activity failed to show clear multimodality. An improvement in the assay for hydrolysis of paraoxon by plasma arylesterase/paraoxonase was achieved by elimination of organic solvents. Plotting chlorpyrifos-oxonase activity vs paraoxonase activity for a human population using the new assay conditions provided an excellent resolution of low activity homozygotes from heterozygotes for this allele. A greater than 40-fold difference in rates of chlorpyrifosoxon hydrolysis observed between rat (low activity) and rabbit sera (high activity) correlated well with the reported large differences in LD50 values for chlorpyrifos in these two animals, consistent with an important role of serum paraoxonase in detoxification of organophosphorus pesticides in vivo.

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

    PubMed

    Kataria, Rashmi; Ghosh, Sanjoy

    2011-11-01

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

  4. 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.

  5. Full enzymatic hydrolysis of commercial sucrose laurate by immobilized-stabilized derivatives of lipase from Thermomyces lanuginosa.

    PubMed

    Marciello, Marzia; Mateo, Cesar; Guisan, Jose Manuel

    2011-06-01

    Sucrose laurate is a detergent that is useful for various biochemical applications because it is a green compound and is easily degradable after hydrolysis with a lipase or esterase. One problem observed in the process of sucrose laurate degradation is that most commercial detergent preparations are impure, necessitating the hydrolysis of all of the sucrose esters present in the preparation, all of them with detergent properties. In this article, a highly active catalyst, which is able to perform the hydrolysis of commercial sucrose laurate, is presented. The use of glyoxyl agarose preparations of a previously aminated Thermomyces lanuginosa lipase (TLL) enabled complete hydrolysis, in less than 30 min, of all of the compounds that comprise the mixture. In addition, this derivative is stable in the presence of 20% ethanol, which is necessary to prevent microbial contamination.

  6. 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.

  7. Identification of characteristic flavour precursors from enzymatic hydrolysis-mild thermal oxidation tallow by descriptive sensory analysis and gas chromatography-olfactometry and partial least squares regression.

    PubMed

    Shi, Xiaoxia; Zhang, Xiaoming; Song, Shiqing; Tan, Chen; Jia, Chengsheng; Xia, Shuqin

    2013-01-15

    The "enzymatic hydrolysis-mild thermal oxidation" method was employed to obtain oxidized tallow. Nine beeflike flavours (BFs) were prepared through Maillard reaction with oxidized tallow and other ingredients. Volatile compounds of oxidized tallow and beeflike flavours were analysed by SPME/GC-MS. Six sensory attributes (meaty, beefy, tallowy, simulate, burnt and off-flavour) were selected to assess BFs. Thirty four odour-active compounds were identified to represent beef odour through GC-O analysis based on detection frequency method. GC-MS profiles of oxidized tallow were correlated with GC-O responses and sensory attributes of BFs using partial least squares regression modelling (PLSR). Twenty nine compounds were considered as the potential precursors of oxidized tallow. Among them, tetradecanoic acid, d-limonene, 1,7-heptandiol, 2-butyltetrahydrofuran, (Z)-4-undecenal, (Z)-4-decenal, (E)-4-nonenal and 5-pentyl-2(3H)-furanone were unique products generated from enzymatic hydrolysis-mild thermal oxidation of tallow, while hexanal, heptanal, octanal, nonanal, decanal, pentanal, acetic acid, butanoic acid, hexanoic acid, 1-heptanol, 1-octanol, 3-methylbutanal, 2-pentylfuran, γ-nonalactone, 2-undecenal, (E,E)-2,4-decadienal, (E,E)-2,4-nonadienal, (E)-2-nonenal, (E)-2-octenal, (E)-2-decenal and (Z)-2-heptenal were common products generated from thermal oxidation of tallow. PMID:23270941

  8. Heterogeneous Expression and Functional Characterization of Cellulose-Degrading Enzymes from Aspergillus niger for Enzymatic Hydrolysis of Alkali Pretreated Bamboo Biomass.

    PubMed

    Ali, Nasir; Ting, Zhang; Li, Hailong; Xue, Yong; Gan, Lihui; Liu, Jian; Long, Minnan

    2015-09-01

    Enzymatic hydrolysis of cellulosic biomass has caught much attention because of modest reaction conditions and environment friendly conditions. To reduce the cost and to achieve good quantity of cellulases, a heterologous expression system is highly favored. In this study, cellulose-degrading enzymes, GH3 family β-glucosidase (BGL), GH7 family-related cellobiohydrolases (CBHs), and endoglucanase (EG) from a newly isolated Aspergillus niger BE-2 are highly expressed in Pichia pastoris GS115. The strain produced EG, CBHs, and BGL enzymatic concentration of 0.56, 0.11, and 22 IU/mL, respectively. Mode of actions of the recombinant enzymes for substrate specificity and end product analysis are verified and found specific for cellulose degradation. Bamboo biomass saccharification with A. niger cellulase released a high level of fermentable sugars. Hydrolysis parameters are optimized to obtain reducing sugars level of 3.18 g/L. To obtain reducing sugars from a cellulosic biomass, A. niger could be a good candidate for enzymes resource of cellulase to produce reducing sugars from a cellulosic biomass. This study also facilitates the development of highly efficient enzyme cocktails for the bioconversion of lignocellulosic biomass into monosaccharides and oligosaccharides. PMID:26202492

  9. Comparative study of lignin characteristics from wheat straw obtained by soda-AQ and kraft pretreatment and effect on the following enzymatic hydrolysis process

    DOE PAGESBeta

    Yang, Haitao; Xie, Yimin; Zheng, Xing; Pu, Yunqiao; Huang, Fang; Meng, Xianzhi; Wu, Weibing; Ragauskas, Arthur; Yao, Lan

    2016-02-18

    With this study, 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,more » through quantitative 13C 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.« less

  10. 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.

  11. 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.

  12. Supercritical carbon dioxide combined with 1-butyl-3-methylimidazolium acetate and ethanol for the pretreatment and enzymatic hydrolysis of sugarcane bagasse.

    PubMed

    Silveira, Marcos Henrique Luciano; Vanelli, Bruno Angelo; Corazza, Marcos Lucio; Ramos, Luiz Pereira

    2015-09-01

    The use of green solvents for the partial delignification of milled sugarcane bagasse (1mm particle size) and for the enhancement of its susceptibility to enzymatic hydrolysis was demonstrated. The experiments were carried out for 2h using 40 g of supercritical carbon dioxide combined with 1-butyl-3-methylimidazolium acetate and 15.8 g of ethanol. The effects of temperature (110-180 °C), pressure (195-250 bar) and IL-to-bagasse mass ratio (0:1-1:1) were investigated through a factorial design in which the response variables were the extent of delignification and both anhydroglucose and anhydroxylose contents in the pretreated materials. The highest delignification degree (41%) led to the best substrate for hydrolysis, giving a 70.7 wt% glucose yield after 12h using 5 wt% and Cellic CTec2® (Novozymes) at 10 mg g(-1) total solids. Hence, excellent substrates for hydrolysis were produced with a minimal IL requirement, which could be recovered by ethanol washing for its downstream processing and reuse. PMID:26056781

  13. Supercritical carbon dioxide combined with 1-butyl-3-methylimidazolium acetate and ethanol for the pretreatment and enzymatic hydrolysis of sugarcane bagasse.

    PubMed

    Silveira, Marcos Henrique Luciano; Vanelli, Bruno Angelo; Corazza, Marcos Lucio; Ramos, Luiz Pereira

    2015-09-01

    The use of green solvents for the partial delignification of milled sugarcane bagasse (1mm particle size) and for the enhancement of its susceptibility to enzymatic hydrolysis was demonstrated. The experiments were carried out for 2h using 40 g of supercritical carbon dioxide combined with 1-butyl-3-methylimidazolium acetate and 15.8 g of ethanol. The effects of temperature (110-180 °C), pressure (195-250 bar) and IL-to-bagasse mass ratio (0:1-1:1) were investigated through a factorial design in which the response variables were the extent of delignification and both anhydroglucose and anhydroxylose contents in the pretreated materials. The highest delignification degree (41%) led to the best substrate for hydrolysis, giving a 70.7 wt% glucose yield after 12h using 5 wt% and Cellic CTec2® (Novozymes) at 10 mg g(-1) total solids. Hence, excellent substrates for hydrolysis were produced with a minimal IL requirement, which could be recovered by ethanol washing for its downstream processing and reuse.

  14. Enhanced sugar production from pretreated barley straw by additive xylanase and surfactants in enzymatic hydrolysis for acetone-butanol-ethanol fermentation.

    PubMed

    Yang, Ming; Zhang, Junhua; Kuittinen, Suvi; Vepsäläinen, Jouko; Soininen, Pasi; Keinänen, Markku; Pappinen, Ari

    2015-01-01

    This study aims to improve enzymatic sugar production from dilute sulfuric acid-pretreated barley straw for acetone-butanol-ethanol (ABE) fermentation. The effects of additive xylanase and surfactants (polyethylene glycol [PEG] and Tween) in an enzymatic reaction system on straw hydrolysis yields were investigated. By combined application of 2g/100g dry-matter (DM) xylanase and PEG 4000, the glucose yield was increased from 53.2% to 86.9% and the xylose yield was increased from 36.2% to 70.2%, which were considerably higher than results obtained with xylanase or surfactant alone. The ABE fermentation of enzymatic hydrolysate produced 10.8 g/L ABE, in which 7.9 g/L was butanol. The enhanced sugar production increased the ABE yield from 93.8 to 135.0 g/kg pretreated straw. The combined application of xylanase and surfactants has a large potential to improve sugar production from barley straw pretreated with a mild acid and that the hydrolysate showed good fermentability in ABE production.

  15. Hybrid model for an enzymatic reactor: hydrolysis of cheese whey proteins by alcalase immobilized in agarose gel particles.

    PubMed

    Sousa, Ruy; Resende, Mariam M; Giordano, Raquel L C; Giordano, Roberto C

    2003-01-01

    Cheese whey proteolysis, carried out by immobilized enzymes, can either change or evidence functional properties of the produced peptides, increasing the potential applications of this byproduct of the dairy industry. Optimization and scale-up of the enzymatic reactor relies on its mathematical model-a set of mass balance equations, with reaction rates usually given by Michaelis-Menten-like kinetics; no information about the distribution of peptides' molecular sizes is supplied. In this article, a hybrid model of a batch enzymatic reactor is presented, consisting of differential mass balances coupled to a "neural-kinetic model," which provides the molecular weight distributions of the resulting peptides. PMID:12721464

  16. Non-enzymatic and enzymatic hydrolysis of alkyl halides: a haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an SN2 displacement reaction.

    PubMed

    Lightstone, F C; Zheng, Y J; Maulitz, A H; Bruice, T C

    1997-08-01

    The semiempirical PM3 method, calibrated against ab initio HF/6-31+G(d) theory, has been used to elucidate the reaction of 1, 2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of Xanthobacter autothropicus. Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations. The three most significant observations of the present study are that: (i) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of AcO- with DCE; (ii) the structures of the transition states in the gas-phase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (iii) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal/mol. The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. A new sunscreen of the cinnamate class: synthesis and enzymatic hydrolysis evaluation of glyceryl esters of p-methoxycinnamic acid.

    PubMed

    de Freitas, Zaida Maria Faria; dos Santos, Elisabete Pereira; da Rocha, João Ferreira; Dellamora-Ortiz, Gisela Maria; Gonçalves, José Carlos Saraiva

    2005-05-01

    Glyceryl esters of p-methoxycinnamic acid, 1,3-dipalmitoyl-2-p-methoxycinnamoyl-1,2,3-propanetriol and 1,3-dioctanoyl-2-p-methoxycinnamoyl-1,2,3-propanetriol were synthesised in an attempt to increase substantivity and decrease eventual undesirable effects of sunscreens of this class. To assess if the glyceryl esters could present a higher stability towards hydrolysis by lipases in the stratum corneum, hydrolysis rates were determined in vitro using a commercial fungal lipase from Rhizomucor miehei. Results presented herein show that the glyceryl esters have similar lambda(max) and epsilon values to sunscreens of the cinnamate class. The ester 1,3-dipalmitoyl-2-p-methoxycinnamoyl-1,2,3-propanetriol presented a 2.8 times lower hydrolysis rate by lipase, in vitro, than the commercial sunscreen 2-ethylhexyl-p-methoxycinnamate (alkyl ester). This finding suggests that this triacylglycerol can possibly have a longer retention time in the skin and consequently promote a more intense and effective antisolar action than the commercial sunscreen.

  3. Rigorous kinetic model considering positional specificity of lipase for enzymatic stepwise hydrolysis of triolein in biphasic oil-water system.

    PubMed

    Hermansyah, Heri; Wijanarko, Anondho; Kubo, Masaki; Shibasaki-Kitakawa, Naomi; Yonemoto, Toshikuni

    2010-09-01

    A rigorous kinetic model describing the stepwise triglyceride hydrolysis at the oil-water interface, based on the Ping Pong Bi Bi mechanism using suspended lipase having positional specificity, was constructed. The preference of the enzyme to cleave to the ester bonds at the edge and the center of the glycerol backbone of the substrates (tri-, di- or monoglyceride) was incorporated in the model. This model was applied to the experimental results for triolein hydrolysis using suspended Porcine pancreatic lipase (an sn-1,3 specific lipase) and Candida rugosa lipase (a non-specific lipase) in a biphasic oil-water system under various operating conditions. In order to discuss the model's advantages, other models that do not consider the positional specificity of the lipase were also applied to our experimental results. The model considering the positional specificity of the lipase gave results which fit better with the experimental data and described the effect of the initial enzyme concentration, the interfacial area, and the initial concentrations of triolein on the entire process of the stepwise triolein hydrolysis. This model also gives a good representation of the rate for cleaving the respective ester bonds of each substrate by each type of lipase.

  4. 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.

  5. Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

    PubMed Central

    2011-01-01

    Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX) and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy. PMID:21702938

  6. 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

  7. 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

  8. 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.

  9. Synthesis, chemical and enzymatic hydrolysis, and aqueous solubility of amino acid ester prodrugs of 3-carboranyl thymidine analogs for boron neutron capture therapy of brain tumors.

    PubMed

    Hasabelnaby, Sherifa; Goudah, Ayman; Agarwal, Hitesh K; abd Alla, Mosaad S M; Tjarks, Werner

    2012-09-01

    Various water-soluble L-valine-, L-glutamate-, and glycine ester prodrugs of two 3-Carboranyl Thymidine Analogs (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 48-6600 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

  10. 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.

  11. Dilute sulfuric acid pretreatment of corn stover for enzymatic hydrolysis and efficient ethanol production by recombinant Escherichia coli FBR5 without detoxification.

    PubMed

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

    2013-08-01

    A pretreatment strategy for dilute H2SO4 pretreatment of corn stover was developed for the purpose of reducing the generation of inhibitory substances during pretreatment so that a detoxification step is not required prior to fermentation while maximizing sugar yield. The optimal conditions for pretreatment of corn stover (10%, w/v) were: 0.75% H2SO4, 160°C, and 0-5 min holding time. The conditions were chosen based on maximum glucose release after enzymatic hydrolysis, minimum loss of pentose sugars and minimum formation of sugar degradation products such as furfural and hydroxymethyl furfural. The pretreated corn stover after enzymatic saccharification generated 63.2 ± 2.2 and 63.7 ± 2.3 g total sugars per L at 0 and 5 min holding time, respectively. Furfural production was 0.45 ± 0.1 and 0.87 ± 0.4 g/L, respectively. The recombinant Escherichia coli strain FBR5 efficiently fermented non-detoxified corn stover hydrolyzate if the furfural content is <0.5 g/L.

  12. 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

  13. Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose.

    PubMed

    Szijártó, Nóra; Szengyel, Zsolt; Lidén, Gunnar; Réczey, Kati

    2004-01-01

    An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources-including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucose-grown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO2 evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO2 evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO2 evolution are analyzed and discussed herein.

  14. 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.

  15. 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.

  16. 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

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

    NASA Astrophysics Data System (ADS)

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

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

  18. Inhibition of beta-glucosidase activity in Trichoderma reesei C30 cellulase by derivatives and isomers of glucose

    SciTech Connect

    Woodward, J.; Arnold, S.L.

    1981-07-01

    The inhibition of Beta-glucosidase in Trichoderma reesei C30 cellulase by D-glucose, its isomers, and derivatives was studied using cellobiose and p-nitrophenyl-beta-glucoside (PNPG) as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose with approximate K1 values of 0.5mM and 8.7mM for cellobiose and PNPG as substrate, respectively. This inhibition by glucose was maximal at pH 4.8 and no inhibition was observed at pH 6.5 and above. The alpha anomer of glucose inhibited beta-glucosidase to a greater extent than did the beta form. Compared with D-glucose, L-glucose, D-glucose-6-phosphate, and D-glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D-glucose-L-cysteine which was almost as inhibitory as glucose itself when cellobiose was used as substrate. Fructose (2-100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by beta-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase.

  19. De-ashing treatment of corn stover improves the efficiencies of enzymatic hydrolysis and consequent ethanol fermentation.

    PubMed

    He, Yanqing; Fang, Zhenhong; Zhang, Jian; Li, Xinliang; Bao, Jie

    2014-10-01

    In this study, corn stover with different ash content was pretreated using dry dilute acid pretreatment method at high solids loading of 67% (w/w). The results indicate that the hydrolysis yield of corn stover is increased from 43.30% to 70.99%, and ethanol yield is increased from 51.74% to 73.52% when ash is removed from 9.60% to 4.98%. The pH measurement of corn stover slurry indicates that the decrease of pretreatment efficiency is due to the neutralization of sulfuric acid by alkaline compounds in the ash. The elemental analysis reveals that the ash has the similar composition with the farmland soil. This study demonstrates the importance of ash removal from lignocellulose feedstock under high solids content pretreatment.

  20. 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.

  1. Relationship between the fine structure of native cellulose and cellulose degradability by the cellulase complexes of Trichoderma reesei and Clostridium thermocellum

    SciTech Connect

    Weimer, P.J.; Weston, W.M.

    1985-11-01

    The initial rate of hydrolysis of six commercially available native (type 1) celluloses was determined for the crude cellulase complexes of the thermophilic anaerobic bacterium C. thermocellum and the mesophilic fungus T. reesei. These rates were then compared with certain physical features of the substrates in an attempt to determine the role of cellulose structure in its degradability. Within the substrate series tested, the Clostridium system showed a greater relative range in rate of enzymatic hydrolysis than did the Trichoderma system. Average correlation coefficients for the kinetic rates from bacterial and fungal cellulases, respectively, and the following physical parameters were obtained: relative crystallinity index (RCI) from acid hydrolysis, -0.61 and -0.85; RCI from x-ray diffraction, -0.75 and -0.89; accessibility to formylation at 4 degrees C, +0.49 and +0.60; nonaccessibility to formylation at 65 degrees, -0.40 and - 0.73; fiber saturation point, +0.83 and +0.85. Kinetic and pore volume distribution data suggest that the rate-limiting components of both the bacterial and fungal cellulase systems are of similar size, approximately 43 Angstroms along one axis. 32 references.

  2. 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.

  3. 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.

  4. Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility.

    PubMed

    Zhu, Zhiguang; Sathitsuksanoh, Noppadon; Vinzant, Todd; Schell, Daniel J; McMillan, James D; Zhang, Y-H Percival

    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 approximately 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(2)/g, nearly twice that of the DA-pretreated biomass (5.89 m(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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. The role of oxidation and enzymatic hydrolysis on the in vivo degradation of trimethylene carbonate based photocrosslinkable elastomers.

    PubMed

    Chapanian, Rafi; Tse, M Yat; Pang, Stephen C; Amsden, Brian G

    2009-01-01

    The in vivo degradation of trimethylene carbonate (TMC) containing elastomers was investigated, and the mechanism of degradation explored through in vitro degradation under enzymatic and oxidative conditions. The elastomers were prepared via UV initiated crosslinking of prepolymers of TMC and equimolar amounts of TMC and epsilon-caprolactone (CL). The degradation process was followed by investigating the changes in the mechanical properties, mass loss, water uptake, sol content, differential scanning calorimetry, and surface chemistry through attenuated total reflectance infrared (ATR-FTIR) spectroscopy. During in vivo degradation, TMC and TMCCL elastomers exhibited surface erosion. The tissue response was of greater intensity in the case of the TMC elastomer. Both elastomers exhibited degradation in cholesterol esterase containing solutions in vitro, but no parallels were found between the rate of in vivo degradation and the rate of in vitro degradation. Only the TMCCL elastomer degraded in lipase. Degradation in a stable superoxide anion in vitro medium was consistent with the observed in vivo degradation results, indicating a dominant role of oxidation through the secretion of this reactive oxygen species by adherent phagocytic cells in the degradation of these elastomers. PMID:18947866

  10. Mathematical model-based optimization of physico-enzymatic hydrolysis of Pinus roxburghii needles for the production of reducing sugars.

    PubMed

    Vats, Siddharth; Maurya, Devendra Prasad; Jain, Ayushi; Mall, Varija; Negi, Sangeeta

    2013-11-01

    The objective of this study was to optimize the physico-enzymatic pretreatment of P. roxburghii fallen foliage (needles) to produce reducing sugars through response surface methodology (RSM) with central composite face centered design (CCD). Under this, five parameters, i.e., concentration of laccase, cellulose and xylanase, steam explosion pressure and incubation period, at three levels with twenty six runs were taken into account. Cellulase, xylanase and laccase enzymes with activity 4.563, 38.32 and 0.05 IU/mL, respectively, were produced from locally isolated microbial strains. The analysis of variance (ANOVA) was applied for the validation of the predicted model at 95% of confidence level. This model predicted 334 mg/g release of reducing sugars on treating P. roxburghii fallen foliage with 1.18 mL of cellulose, 0.31 mL of xylanase and 0.01 mL of laccase, 14.39 psi steam explosion pressure and 24 h of incubation time. The experimental results obtained were in good agreement to predicted values, making it a reliable optimized model for five factors in combination to predict reducing sugar yield for ethanol production for bio-fuel industry.

  11. 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.

  12. 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

  13. Pretreating lignocellulosic biomass by the concentrated phosphoric acid plus hydrogen peroxide (PHP) for enzymatic hydrolysis: evaluating the pretreatment flexibility on feedstocks and particle sizes.

    PubMed

    Wang, Qing; Wang, Zhanghong; Shen, Fei; Hu, Jinguang; Sun, Fubao; Lin, Lili; Yang, Gang; Zhang, Yanzong; Deng, Shihuai

    2014-08-01

    In order to seek a high-efficient pretreatment path for converting lignocellulosic feedstocks to fermentable sugars by enzymatic hydrolysis, the concentrated H₃PO₄ plus H₂O₂ (PHP) was attempted to pretreat different lignocellulosic biomass for evaluating the pretreatment flexibility on feedstocks. Meanwhile, the responses of pretreatment to particle sizes were also evaluated. When the PHP-pretreatment was employed (final H₂O₂ and H₃PO₄ concentration of 1.77% and 80.0%), 71-96% lignin and more than 95% hemicellulose in various feedstocks (agricultural residues, hardwood, softwood, bamboo, and their mixture, and garden wastes mixture) can be removed. Consequently, more than 90% glucose conversion was uniformly achieved indicating PHP greatly improved the pretreatment flexibility to different feedstocks. Moreover, when wheat straw and oak chips were PHP-pretreated with different sizes, the average glucose conversion reached 94.9% and 100% with lower coefficient of variation (7.9% and 0.0%), which implied PHP-pretreatment can significantly weaken the negative effects of feedstock sizes on subsequent conversion.

  14. 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.

  15. Determination of opiates in whole blood and vitreous humor: a study of the matrix effect and an experimental design to optimize conditions for the enzymatic hydrolysis of glucuronides.

    PubMed

    Sanches, Livia Rentas; Seulin, Saskia Carolina; Leyton, Vilma; Paranhos, Beatriz Aparecida Passos Bismara; Pasqualucci, Carlos Augusto; Muñoz, Daniel Romero; Osselton, Michael David; Yonamine, Mauricio

    2012-04-01

    Undoubtedly, whole blood and vitreous humor have been biological samples of great importance in forensic toxicology. The determination of opiates and their metabolites has been essential for better interpretation of toxicological findings. This report describes the application of experimental design and response surface methodology to optimize conditions for enzymatic hydrolysis of morphine-3-glucuronide and morphine-6-glucuronide. The analytes (free morphine, 6-acetylmorphine and codeine) were extracted from the samples using solid-phase extraction on mixed-mode cartridges, followed by derivatization to their trimethylsilyl derivatives. The extracts were analysed by gas chromatography-mass spectrometry with electron ionization and full scan mode. The method was validated for both specimens (whole blood and vitreous humor). A significant matrix effect was found by applying the F-test. Different recovery values were also found (82% on average for whole blood and 100% on average for vitreous humor). The calibration curves were linear for all analytes in the concentration range of 10-1,500 ng/mL. The limits of detection ranged from 2.0 to 5.0 ng/mL. The method was applied to a case in which a victim presented with a previous history of opiate use.

  16. On-line supercritical fluid extraction/enzymatic hydrolysis of vitamin a esters: a new simplified approach for the determination of vitamins a and e in food.

    PubMed

    Turner, C; King, J W; Mathiasson, L

    2001-02-01

    An on-line supercritical fluid extraction (SFE)/enzymatic hydrolysis procedure using immobilized lipase has been developed for the determination of vitamin A in dairy and meat products. Several lipases were tried, of which Novozyme 435 (Candida antarctica type B) showed the highest activity toward retinyl palmitate. There was no observed activity with alpha-tocopheryl acetate. When pressure, temperature, modifiers, flow rate, extraction time, and water content were varied, high vitamin A recovery was obtained in milk powder. Collected extracts were analyzed by reversed-phase high-performance liquid chromatography with ultraviolet and fluorescence detection without additional sample cleanup. The procedure gave reliable values of vitamin A as well as of vitamin E in other food items such as infant formula, minced pork and beef meat, and low- and high-fat liver paste. The described method is faster and more automated than conventional methods based on liquid-liquid extraction, or SFE using off-line saponification, for vitamin A and E determination. Results obtained with the new method did not differ significantly from those obtained with the other two methods mentioned above.

  17. Fast screening and secure confirmation of milk powder adulteration with maltodextrin via electrospray ionization-mass spectrometry [ESI(+)-MS] and selective enzymatic hydrolysis.

    PubMed

    Sanvido, Gustavo B; Garcia, Jerusa S; Corilo, Yuri E; Vaz, Boniek G; Zacca, Jorge J; Cosso, Ricardo G; Eberlin, Marcos N; Peter, Martin G

    2010-09-01

    Direct-infusion electrospray ionization-mass spectrometry [ESI(+)-MS] of several milk powder samples, confiscated by the Brazilian Federal Police, showed ions accounting for sodiated and potassiated molecules of disaccharides (m/z 365 and 381) as well as trisaccharides (m/z 527 and 543), whereas monosaccharide ions were not detected. The trisaccharide ions were not detected in samples of genuine milk powder, raising the suspicion that their presence indicates adulteration by the addition of maltodextrin. In control samples, maltose and maltotriose were hydrolyzed by alpha-glucosidase and not beta-galactosidase, whereas lactose was resistant to alpha-glucosidase but was hydrolyzed with beta-galactosidase. Samples suspected of being adulterated behaved in the same fashion, confirming the presence of maltose and maltotriose or maltodextrin. Direct-infusion ESI-MS is shown therefore to provide rapid screening of milk powder for adulteration with maltodextrin, whereas its combination with selective enzymatic hydrolysis provides highly reliable confirmation for unambiguous results.

  18. 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

  19. Optimization of chip size and moisture content to obtain high, combined sugar recovery after sulfur dioxide-catalyzed steam pretreatment of softwood and enzymatic hydrolysis of the cellulosic component.

    PubMed

    Olsen, Colin; Arantes, Valdeir; Saddler, Jack

    2015-01-01

    The influence of chip size and moisture content on the combined sugar recovery after steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis of the cellulosic component were investigated using response surface methodology. Chip size had little influence on sugar recovery after both steam pretreatment and enzymatic hydrolysis. In contrast, the moisture of the chips greatly influenced the relative severity of steam pretreatment and, as a result, the combined sugar recovery from the hemicellulosic and cellulosic fractions. Irrespective of chip size and the pretreatment temperature, time, and SO2 loading that were used, the relative severity of pretreatment was highest at a moisture of 30-40w/w%. However, the predictive model indicated that an elevated moisture content of roughly 50w/w% (about the moisture content of a standard softwood mill chip) would result in the highest, combined sugar recovery (80%) over the widest range of steam pretreatment conditions.

  20. 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