Enzymatic Hydrolysis Production Research Articles

A Novel Biochemical Route for Fuels and Chemicals Production from Cellulosic Biomass

The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing β-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate—glucose and gluconate—can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of β-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.

Construction and characterization of chimeric cellulases with enhanced catalytic activity towards insoluble cellulosic substrates

The chimeric proteins viz. CBM3–Cel9A, CBM4–Cel9A and CBM30–Cel9A, are constructed by fusion of family 3, 4, and 30 cellulose binding modules (CBMs) to N-terminus of family 9 endoglucanase (Cel9A) from Alicyclobacillus acidocaldrious. The chimeric enzymes were successfully expressed in Escherichia coli and purified to homogeneity. The chimeric enzymes showed significant increase in Avicel (8–12 folds) and filter paper (7–10 folds) degradation activities compared to Cel9A endoglucanase. Computational protein modeling and simulation on the chimeric enzymes were applied to analyze the fused CBMs effect on the increased insoluble cellulosic substrates degradation activity. Thin layer chromatography analysis of the enzymatic hydrolysis products and distribution of reducing sugars between soluble and insoluble fractions indicated processive cleavage of insoluble cellulosic substrates by the chimeras. The fused CBMs played a critical accessory role for the Cel9A catalytic domain and changed its character to facilitate the processive cleavage of insoluble cellulosic substrates.

The effect of refrigerated storage of raw milk on the physicochemical and microbiological quality of Tunisian semihard Gouda‐type cheese during ripening

A Tunisian semihard Gouda‐type cheese made from milk kept at 4 °C for 24, 48, 72 and 96 h was monitored during 45 days of ripening. The effect of milk refrigeration on the evolution of physicochemical parameters in relation to the quantitative variation of the microbial population during ripening of Gouda‐type cheese was investigated. Microbiological and physicochemical analyses were performed on raw milk and cheese samples after curding, 2, 9, 16, 23, 30, 37 and 45 days of ripening time. The raw milk kept under refrigeration at 4 °C for 96 h showed the highest microbial count and proteolysis level. The duration of storage significantly reduced the cheese yield as a result of important solubilisation casein in proteoses‐peptones. Results of different nitrogenous fractions by Kjeldahl method showed enzymatic hydrolysis products of casein whose intensity depended on the maturing stage as well as the refrigeration time. Besides the evident action of the plasmin, original milk protease, on the hydrolysis of casein in soluble fractions, the proteolysis of cheese caseins is also initiated by proteolytic action of the chymosin and extracellular heat‐resistant proteases notably produced by the same psychrotrophic microflora. Lactic acid bacteria starters that constitute the dominant microflora of this type of cheese are also considered as aroma precursors.

Sugar Purification from Enzymatic Rice Straw Hydrolysis Products using Cross-Flow Diafiltration

The sugars produced by enzymatic hydrolysis of rice straw are separated using cross-flow diafiltration in this study. The effects of membrane type, membrane pore size, cross-flow velocity and transmembrane pressure on the filtration flux, sugar rejection, and sugar mass flux transported to the filtrate are discussed. The filtration flux increases with increasing cross-flow velocity or transmembrane pressure. When the membrane made of mixed cellulose ester (MCE) is used, over 70% filtration resistances are caused by the membrane fouling; while the resistance due to virgin membrane is dominant when regenerated cellulose (RC) membranes are used. A force balance model is applied to relate the filtration flux and filtration resistance to operating conditions. The calculated data of filtration flux based on this model agree fairly well with experimental data. In addition, a theoretical model is used to explain the sugar transmission through the cake and membrane pores. The sugar rejection coefficient decreases with increasing cross-flow velocity because of the effect of cake reduction. This effect is more significant when the MCE membrane is used. Comparing the sugar mass flux transported into the filtrate, it is more effective for sugar purification by using 10 kD RC membrane and under higher transmembrane pressures.

Comparative studies of acidic and enzymatic hydrolysis for production of soyasapogenols from soybean saponin

Soyasapogenols, aglycones of soyasaponins, can be produced from crude soybean saponin extract by acid or enzymatic hydrolysis. Soyasapogenol B is known to have hepatoprotective, antimutagenic, antivirus, and anti-inflammatory activities. Hydrolysis of soyabean saponin extract for 72 h with 2 M HCl in methanol gave three soyasapogenols, namely: soyasapogenol D, soyasapogenol B1 and soyasapogenol A. However, the microbial hydrolysis of soybean saponin extract by Aspergillus terreus led to isolation of soyasapogenol B as a major product. A systematic evaluation of the effect of key operational parameters on the microbial transformation was performed. Maximum production of soyasapogenol B (about 152.3 mg/50ml) was observed using 1.5% (w/v) soybean saponin and 1.5% (w/v) glucose, 32°C after 72 h at pH 7 using phosphate buffer. Under these optimal conditions, the cells’ bioconversion efficiency increased from 20.5 to 85.3%. The isolation of soyasapogenols was performed using chromatographic methods and their structures identified on the basis of spectroscopic tools.

The isotopic exchange of oxygen as a tool for detection of the glycation sites in proteins

A nonenzymatic reaction of reducing sugars with the free amino group located at the N terminus of the polypeptide chain or in the lysine side chain results in glycation of proteins. The fragments of glycated proteins obtained by enzymatic hydrolysis could be considered as the biomarkers of both the aging process and diabetes mellitus. Here we propose a new method for the identification of peptide-derived Amadori products in the enzymatic digest of glycated proteins. The products of enzymatic hydrolysis of the model protein ubiquitin were incubated with H 2 18O under microwave activation. We observed that at these conditions the Amadori compounds selectively exchange one oxygen atom in the hexose moiety. The characteristic isotopic pattern of Amadori products treated with H 2 18O allows fast and convenient identification of this group of compounds, whereas nonglycated peptides are not susceptible to isotopic exchange.

Extracellular production of Streptomyces exfoliatus poly(3-hydroxybutyrate) depolymerase in Rhodococcus sp. T104: determination of optimal biocatalyst conditions

The phaZ ( Sex ) gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T (m)) of 55.8°C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine-histidine-carboxylic acid in the active site. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40°C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.

Biological pretreatment of lignocelluloses with white-rot fungi and its applications: A review

Lignocellulosic carbohydrates, i.e. cellulose and hemicellulose, have abundant potential as feedstock for production of biofuels and chemicals. However, these carbohydrates are generally infiltrated by lignin. Breakdown of the lignin barrier will alter lignocelluloses structures and make the carbohydrates accessible for more efficient bioconversion. White-rot fungi produce ligninolytic enzymes (lignin peroxidase, manganese peroxidase, and laccase) and efficiently mineralise lignin into CO2 and H2O. Biological pretreatment of lignocelluloses using white-rot fungi has been used for decades for ruminant feed, enzymatic hydrolysis, and biopulping. Application of white-rot fungi capabilities can offer environmentally friendly processes for utilising lignocelluloses over physical or chemical pretreatment. This paper reviews white-rot fungi, ligninolytic enzymes, the effect of biological pretreatment on biomass characteristics, and factors affecting biological pretreatment. Application of biological pretreatment for enzymatic hydrolysis, biofuels (bioethanol, biogas and pyrolysis), biopulping, biobleaching, animal feed, and enzymes production are also discussed.

Biological pretreatment of lignocelluloses with white-rot fungi and its applications: A review

Lignocellulosic carbohydrates, i.e. cellulose and hemicellulose, have abundant potential as feedstock for production of biofuels and chemicals. However, these carbohydrates are generally infiltrated by lignin. Breakdown of the lignin barrier will alter lignocelluloses structures and make the carbohydrates accessible for more efficient bioconversion. White-rot fungi produce ligninolytic enzymes (lignin peroxidase, manganese peroxidase, and laccase) and efficiently mineralise lignin into CO2 and H2O. Biological pretreatment of lignocelluloses using white-rot fungi has been used for decades for ruminant feed, enzymatic hydrolysis, and biopulping. Application of white-rot fungi capabilities can offer environmentally friendly processes for utilising lignocelluloses over physical or chemical pretreatment. This paper reviews white-rot fungi, ligninolytic enzymes, the effect of biological pretreatment on biomass characteristics, and factors affecting biological pretreatment. Application of biological pretreatment for enzymatic hydrolysis, biofuels (bioethanol, biogas and pyrolysis), biopulping, biobleaching, animal feed, and enzymes production are also discussed.

Evaluation of steam pretreatment on sweet sorghum bagasse for enzymatic hydrolysis and bioethanol production

Sweet sorghum bagasse (SSB) was steam-pretreated at various temperatures, residence times and SO2 impregnation dosages. A 23 factorial experimental design was employed to investigate the effects of temperature, residence time and SO2 impregnation and their interactions on enzymatic hydrolysis and ethanol production. The results showed that temperature, residence time, SO2 impregnation and the interaction of residence time and SO2 impregnation significantly affected enzymatic hydrolysis in an order of residence time>temperature>SO2 impregnation>the interaction of residence time and SO2 impregnation. Both of water-soluble and water-insoluble fraction derived from steam pretreatment were used for ethanol production, respectively. The results indicated that the effects of mentioned factors and their interactions on total ethanol yield were not significant. However, 200°C, 7.5min and 2.5% SO2 impregnation could be determined as a group of optimal pretreatment conditions, in which the highest ethanol yield of 15.3g/100g SSB (dry basis) could be obtained without pentose fermentation.

Structure and Cytotoxic Activity of Enzymatic Hydrolysis Products of Secoiridoid Glucosides, Isoligustroside and Isooleuropein

Hydrolysis of isoligustroside (1) and isooleuropein (2), secoiridoid glucosides, in the presence of β-glucosidase provided 2-(4-hydroxyphenyl)methyl (2R,3S,4S)-3-formyl-3,4-dihydro-4-(2-methoxy-2-oxoethyl)-2-methyl-2H-pyran-5-carboxylate (3) and 2-(3,4-dihydroxyphenyl)methyl (2R,3S,4S)-3-formyl-3,4-dihydro-4-(2-methoxy-2-oxoethyl)-2-methyl-2H-pyran-5-carboxylate (4), respectively. The structures of 3 and 4 were elucidated on the basis of extensive spectral analyses, including 2D-NMR experiments. Compounds 3 and 4 were found to be new rearrangement products of the aglycones of 1 and 2. The cytotoxic activities of 3 and 4 were evaluated using a disease-oriented panel of 39 human cancer cell lines and showed moderate cytotoxic activity for 4, while 3 exhibited weaker activity compared to that of 4.

Microwave-assisted alkali pretreatment of rice straw to promote enzymatic hydrolysis and hydrogen production in dark- and photo-fermentation

Rice straw was pretreated by microwave-assisted alkali to improve saccharification in enzymatic hydrolysis and hydrogen yield in combined dark- and photo-fermentation in this paper. A maximum reducing sugar yield of 69.3g/100gTVS was obtained when 50g/l rice straw was pretreated by microwave heating for 15min at 140°C in 0.5% NaOH solution and then enzymatically hydrolyzed for 96h. When hydrolyzed rice straw was used for hydrogen production by anaerobic bacteria in dark-fermentation, a maximum hydrogen yield of 155ml/gTVS was obtained. The residual solution (mainly acetate and butyrate) from dark-fermentation was reutilized for hydrogen production by immobilized photosynthetic bacteria in photo-fermentation. By combination of dark- and photo-fermentation, the maximum hydrogen yield was greatly enhanced to 463ml/gTVS, which is 43.2% of the theoretical hydrogen yield.

Ultra-sensitive biosensor based on mesocellular silica foam for organophosphorous pesticide detection

A sensitive amperometric acetylcholinesterase (AChE) biosensor was fabricated based on mesocellular silica foam (MSF), which functioned as both an enzyme immobilization matrix and a solid phase extraction (SPE) material for the preconcentration of target molecules. The hydrophilic interface, the good mechanical/chemical stability, and the suitable pore dimension of MSF provided the entrapped AChE a good environment to well maintain its bioactivity at basic condition. The AChE immobilized in MSF showed improved catalytic ability for the hydrolysis of acetylthiocholine, as evidenced by the increasing of the oxidation current of thiocholine, the enzymatic catalytic hydrolysis production of acetylthiocholine. In addition, the MSF with large surface area showed a modest adsorption capacity for monocrotophos, a model organophosphate used in this study, via the hydrogen bond or physical adsorption interaction. The combination of the SPE and the good enzyme immobilization ability in MSF significantly promoted the sensitivity of the biosensor, and the limit of detection has lowered to 0.05ng/mL. The biosensor exhibited accuracy, good reproducibility, and acceptable stability when used for garlic samples analysis. The strategy may provide a new method to fabricate highly sensitive biosensors for the detection of ultra-trace organophosphorous pesticide infield.

Effect of degree of polymerization of lignocellulosic biomass on characteristics of enzymatic hydrolysis products for L-lactic acid production

Effect of degree of polymerization of lignocellulosic biomass on characteristics of enzymatic hydrolysis products for L-lactic acid production

Optimization by orthogonal array design of ion-pair HPLC separation of the enzymatic hydrolysis products of yeast RNA

The separation of enzymatic hydrolysis products of yeast RNA by ion-pair HPLC was studied. A modified chromatographic response function (MCRF) was proposed to appraise the effectiveness of chromatographic separation. This function takes the number of peaks, resolution and the retention time of the last peak into consideration. It shows advantages for optimization of HPLC separation of complex mixtures. An orthogonal array design was used to separate the hydrolysate of yeast RNA and the optimal chromatographic conditions were obtained.

Application of Enzymatic Hydrolysis on Sunflower Lecithin Using a Pancreatic PLA2

Journal of the American Oil Chemists' SocietyVolume 88, Issue 3 p. 443-446 Letter to the Editor Application of Enzymatic Hydrolysis on Sunflower Lecithin Using a Pancreatic PLA2 D. M. Cabezas, D. M. Cabezas Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CCT La Plata, CONICET, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (FCE, UNLP), 47 y 116 (1900), La Plata, ArgentinaSearch for more papers by this authorR. Madoery, R. Madoery Cátedra de Química Orgánica, Facultad de Ciencias Agrarias, Universidad Nacional de Córdoba (FCA, UNC), Ciudad Universitaria S/N, Córdoba, 5000 ArgentinaSearch for more papers by this authorB. W. K. Diehl, B. W. K. Diehl Spectral Service GmbH Laboratorium für Auftragsanalytik, Emil Hoffman Str. 33, Cologne, 50996 GermanySearch for more papers by this authorM. C. Tomás, Corresponding Author M. C. Tomás [email protected] 54-221-425485354-221-4890741 | Fax: 54-221-4249287 Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CCT La Plata, CONICET, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (FCE, UNLP), 47 y 116 (1900), La Plata, Argentina [email protected]Search for more papers by this author D. M. Cabezas, D. M. Cabezas Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CCT La Plata, CONICET, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (FCE, UNLP), 47 y 116 (1900), La Plata, ArgentinaSearch for more papers by this authorR. Madoery, R. Madoery Cátedra de Química Orgánica, Facultad de Ciencias Agrarias, Universidad Nacional de Córdoba (FCA, UNC), Ciudad Universitaria S/N, Córdoba, 5000 ArgentinaSearch for more papers by this authorB. W. K. Diehl, B. W. K. Diehl Spectral Service GmbH Laboratorium für Auftragsanalytik, Emil Hoffman Str. 33, Cologne, 50996 GermanySearch for more papers by this authorM. C. Tomás, Corresponding Author M. C. Tomás [email protected] 54-221-425485354-221-4890741 | Fax: 54-221-4249287 Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), CCT La Plata, CONICET, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (FCE, UNLP), 47 y 116 (1900), La Plata, Argentina [email protected]Search for more papers by this author First published: 01 October 2010 https://doi.org/10.1007/s11746-010-1684-9Citations: 6Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume88, Issue3March 2011Pages 443-446 RelatedInformation

Glucosinolates and their respective enzymatic hydrolysis products are not involved in the in vitro antioxidant properties of rocket salad species

Glucosinolates and their respective enzymatic hydrolysis products are not involved in the in vitro antioxidant properties of rocket salad species

Combination of biological pretreatment with mild acid pretreatment for enzymatic hydrolysis and ethanol production from water hyacinth

The mild acid pretreatment and the combination of biological pretreatment by a white rot fungus Echinodontium taxodii or a brown rot fungus Antrodia sp. 5898 with mild acid pretreatment were evaluated under different pretreatment conditions for enzymatic hydrolysis and ethanol production from water hyacinth. The combined pretreatment with E. taxodii (10 days) and 0.25% H(2)SO(4) was proved to be more effective than the sole acid pretreatment. The reducing sugar yield from enzymatic hydrolysis of co-treated water hyacinth increased 1.13-2.11 fold than that of acid-treated water hyacinth at the same conditions. The following study on separate hydrolysis and fermentation with Saccharomyces cerevisiae indicated that the ethanol yield from co-treated water hyacinth achieved 0.192 g/g of dry matter, which increased 1.34-fold than that from acid-treated water hyacinth (0.146 g/g of dry matter). This suggested that the combination of biological and mild acid pretreatment is a promising method to improve enzymatic hydrolysis and ethanol production from water hyacinth with low lignin content.

Hydrogen production from water hyacinth through dark- and photo- fermentation

This article discusses the method of producing hydrogen from water hyacinth. Water hyacinth was pretreated with microwave heating and alkali to enhance the enzymatic hydrolysis and hydrogen production in a two-step process of dark- and photo- fermentation. Water hyacinth with various concentrations of 10–40 g/l was pretreated with four methods: (1) steam heating; (2) steam heating and microwave heating/alkali pretreatment; (3) steam heating and enzymatic hydrolysis; (4) steam heating, microwave heating/alkali pretreatment and enzymatic hydrolysis. Water hyacinth (20 g/l) pretreated with method 4 gave the maximum reducing sugar yield of 30.57 g/100 g TVS, which was 45.6% of the theoretical reducing sugar yield (67.0 g/100 g TVS). The pretreated water hyacinth was used to produce hydrogen by mixed H 2-producing bacteria in dark fermentation. The maximum hydrogen yield of 76.7 ml H 2/g TVS was obtained at 20 g/l of water hyacinth. The residual solutions from dark fermentation (mainly acetate and butyrate) were used to further produce hydrogen by immobilized Rhodopseudomonas palustris in photo fermentation. The maximum hydrogen yield of 522.6 ml H 2/g TVS was obtained at 10 g/l of water hyacinth. Through a combined process of dark- and photo- fermentation, the maximum hydrogen yield from water hyacinth was dramatically enhanced from 76.7 to 596.1 ml H 2/g TVS, which was 59.6% of the theoretical hydrogen yield.

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

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 °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 5 mm corn stover was pretreated at 28 °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.