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1.
We investigated the enzymatic complex produced by selected fungi strains isolated from the environment using the agro-industrial residues rice husk, soybean hull, and spent malt as substrates. Microbial growth was carried out in solid-state cultivation (SSC) and in submerged cultivations (SC) and the enzymatic activities of xylanase, cellulase, β-xylosidase, and β-glucosidase were determined. All substrates were effective in inducing enzymatic activities, with one strain of Aspergillus brasiliensis BLf1 showing maximum activities for all enzymes, except for cellulases. Using this fungus, the enzymatic activities of xylanase, cellulase, and β-glucosidase were generally higher in SSC compared to SC, producing maxima activities of 120.5, 25.3 and 47.4 U g?1 of dry substrate, respectively. β-xylosidase activity of 28.1 U g?1 of dry substrate was highest in SC. Experimental design was carried out to optimize xylanase activity by A. brasiliensis BLf1 in SSC using rice husk as substrate, producing maximum xylanase activity 183.5 U g?1 dry substrate, and xylooligosaccharides were produced and characterized. These results suggest A. brasiliensis BLf1 can be used to produce important lytic enzymes to be applied in the preparation of xylooligosaccharides.  相似文献   

2.
A novel bacterial isolate, capable of producing extracellular highly thermostable, halo-alkali-stable and cellulase-free xylanase, was isolated from soil and identified as Bacillus halodurans TSPV1 by polyphasic approach. The Plackett–Burman design identified wheat bran, lactose, tryptone and NaCl as the factors that significantly affect xylanase production, and thus, these were optimized by response surface methodology. The data analysis suggested that optimum levels of wheat bran (15–20 g L?1), lactose (1.0–1.5 g L?1), tryptone (2–2.5 g L?1) and NaCl (7.0–8.0 g L?1) support 6.75-fold higher xylanase production than that in the un-optimized medium. The xylanase is optimally active at 90 °C and pH 10, and stable for 4 h at 90 °C (T 1/2 60 h) over a broad range of NaCl concentrations (0–29 %). This is the first report on the isolation of polyextremophilic B. halodurans strain that produces thermo-halo-alkali-stable xylanase in submerged fermentation. This enzyme efficiently saccharifies agro residues like wheat bran and corncobs. Fifty-six percent of hemicellulose of wheat bran could be hydrolyzed by xylanase (100 U g?1 substrate) along with cellulase (22 U FPase and 50 U CMCase g?1). The xylanase, being thermo-alkali stable and cellulase free, can find applications in pre-bleaching of paper pulps and hydrolysis of xylan in agricultural residues.  相似文献   

3.
Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and β-glucosidase activities of 1,270, 146, 34, and 15 U mL?1, respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k cat/K m?=?1,440 mg?mL?1?s?1) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.  相似文献   

4.
Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L?1 for rice straw, wheat bran, peptone, and CaCO3, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL?1 were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.  相似文献   

5.
In the conversion of lignocellulose into high-value products, including fuels and chemicals, the production of cellulase and the enzymatic hydrolysis for producing fermentable sugar are the largest contributors to the cost of production of the final products. The marine bacterium Saccharophagus degradans 2-40T can degrade more than ten different complex polysaccharides found in the ocean, including cellulose and xylan. Accordingly, S. degradans has been actively considered as a practical source of crude enzymes needed for the saccharification of lignocellulose to produce ethanol by others including a leading commercial company. However, the overall enzyme system of S. degradans for hydrolyzing cellulose and hemicellulose has not been quantitatively evaluated yet in comparison with commercial enzymes. In this study, the inductions and activities of cellulase and xylanase of cell-free lysate of S. degradans were investigated. The growth of S. degradans cells and the activities of cellulase and xylanase were promoted by adding 2 % of cellulose and xylan mixture (cellulose:xylan = 4:3 in mass ratio) to the aquarium salt medium supplemented with 0.2 % glucose. The specific cellulase activity of the cell-free lysate of S. degradans, as determined by the filter paper activity assay, was approximately 70 times lower than those of commercial cellulases, including Celluclast 1.5 L and Accellerase 1000. These results imply that significant improvement in the cellulase activity of S. degradans is needed for the industrial uses of S. degradans as the enzyme source.  相似文献   

6.
The purified extracellular xylanase of polyextremophilic Bacillus halodurans TSEV1 has been visualized as a single band on SDS-PAGE and eluted as single peak by gel filtration, with a molecular mass of 40 kDa. The peptide finger print and cloned xylanase gene sequence analyses indicate that this enzyme belongs to GH family 10. The active site carboxyl residues are mainly involved in catalysis, while tryptophan residues are involved in substrate binding. The enzyme is optimally active at 80 °C and pH 9.0, and stable in the pH range of 7.0–12.0 with T 1/2 of 35 min at 80 °C (pH 9.0). Activation energy for birch wood xylan hydrolysis is 30.51 kJ mol?1. The K m, V max and k cat (birchwood xylan) are 2.05 mg ml?1, 333.33 μmol mg?1 min?1 and 3.33 × 104 min?1, respectively. The pKa1 and pKa2 of ionizable groups of the active site that influence V max are 8.51 and 11.0. The analysis of thermodynamic parameters for xylan hydrolysis suggests this as a spontaneous process. The enzyme is resistant to chemical denaturants like urea and guanidinium-HCl. The site-directed mutagenesis of catalytic glutamic acid residues (E196 and E301) resulted in a complete loss of activity. The birch wood xylan hydrolyzate contained xylobiose and xylotriose as the main products without any trace of xylose, and the enzyme hydrolyzes xylotetraose and xylopentaose rapidly to xylobiose. Thermo-alkali-stability, resistance to various chemical denaturants and mode of action make it a useful biocatalyst for generating xylo-oligosaccharides from agro-residues and bleaching of pulp in paper industries.  相似文献   

7.
Cel5 from marine Hahella chejuensis is composed of glycoside hydrolase family-5 (GH5) catalytic domain (CD) and two carbohydrate binding modules (CBM6-2). The enzyme was expressed in Escherichia coli and purified to homogeneity. The optimum endoglucanase and xylanase activities of recombinant Cel5 were observed at 65 °C, pH 6.5 and 55 °C, pH 5.5, respectively. It exhibited K m of 1.8 and 7.1 mg/ml for carboxymethyl cellulose and birchwood xylan, respectively. The addition of Ca2+ greatly improved thermostability and endoglucanase activity of Cel5. The Cel5 retained 90 % of its endoglucanase activity after 24 h incubation in presence of 5 M concentration of NaCl. Recombinant Cel5 showed production of cellobiose after hydrolysis of cellulosic substrates (soluble/insoluble) and methylglucuronic acid substituted xylooligosaccharides after hydrolysis of glucuronoxylans by endo-wise cleavage. These results indicated that Cel5 as bifunctional enzyme having both processive endoglucanase and xylanase activities. The multidomain structure of Cel5 is clearly distinguished from the GH5 bifunctional glycoside hydrolases characterized to date, which are single domain enzymes. Sequence analysis and homology modeling suggested presence of two conserved binding sites with different substrate specificities in CBM6-2 and a single catalytic site in CD. Residues Glu132 and Glu219 were identified as key catalytic amino acids by sequence alignment and further verified by using site directed mutagenesis. CBM6-2 plays vital role in catalytic activity and thermostability of Cel5. The bifunctional activities and multiple substrate specificities of Cel5 can be utilized for efficient hydrolysis of cellulose and hemicellulose into soluble sugars.  相似文献   

8.
Xanthomonas axonopodis pv. punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28 °C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55 °C while xylanase had highest activity at 45 °C. Heat treatment of enzyme extract at 75 °C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.  相似文献   

9.
A neutral xylanase (XynII) from Volvariella volvacea was identified and characterized. Unlike other modular xylanases, it consists of only a single GH10 catalytic domain with a unique C-terminal sequence (W-R-W-F) and a phenylalanine and proline-rich motif (T-P-F-P-P-F) at N-terminus, indicating that it is a novel GH10 xylanase. XynII exhibited optimal activity at pH 7 and 60 °C and stability over a broad range of pH 4.0–10.0. XynII displayed extreme highly SDS resistance retaining 101.98, 92.99, and 69.84 % activity at the presence of 300 mM SDS on birchwood, soluble oat spelt, and beechwood xylan, respectively. It remained largely intact after 24 h of incubation with proteinase K at a protease to protein ratio of 1:50 at 37 °C. The kinetic constants K m value towards beechwood xylan was 0.548 mg ml?1, and the k cat/K m ratio, reflecting the catalytic efficiency of the enzyme, was 126.42 ml mg?1 s?1 at 60 °C. XynII was a true endo-acting xylanase lacking cellulase activity. It has weak activity towards xylotriose but efficiently hydrolyzed xylans and xylooligosaccharides larger than xylotriose mainly to xylobiose. Synergistic action with acetyl xylan esterase (AXEI) from V. volvacea was observed for de-starched wheat bran. The highest degree of synergy (DS 1.42) was obtained in sequential reactions with AXEI digestion preceding XynII. The high SDS resistance and intrinsic stability suggested XynII may have potential applications in various industrial processes especially for the detergent and textile industries and animal feed industries.  相似文献   

10.
This study aimed to assess the variability in respect of titer and properties of xylanase from Trichoderma reesei SAF3 under both solid-state and submerged fermentation. SSF was initially optimized with different agro-residues and among them wheat bran was found to be the best substrate that favored maximum xylanase production of 219 U (gws)?1 at 96 h of incubation. The mycelial stage of the fungi and intracellular accumulation of Ca++ and Mg++ induced maximum enzyme synthesis. Inoculum level of 10 × 106 spores 5 g?1 of dry solid substrate and water activity of 0.6 were found to be optimum for xylanase production under SSF. Further optimization was made using a Box-Behnken design under response surface methodology. The optimal cultivation conditions predicted from canonical analysis of this model were incubation time (A) = 96–99 h, inoculum concentration (B) = 10 × 106 spores 5 g?1 of dry substrate, solid substrate concentration (C) = 10–12 g flask?1, initial moisture level (D) = 10 mL flask?1 (equivalent to a w  = 0.55) and the level of xylanase was 299.7 U (gws)?1. Subsequent verification of these levels agreed (97 % similar) with model predictions. Maximum amount of xylanase was recovered with water (6:1, v/w) and under shaking condition (125 rpm). Purified xylanase from SSF showed better stability in salt and pH, was catalytically and thermodynamically more efficient over enzyme from SmF, though molecular weight of both enzymes was identical (53.8 kDa).  相似文献   

11.
Tissue was cultured and protoplasts isolated from the carrageenophyte Chondracanthus acicularis with the aim of developing micropropagation as an alternative to harvesting raw material from natural beds. Both adventitious shoots and filamentous calluses were induced by tissue culture on medium solidified with 0.4–1 % (w/v) agar. Adventitious shoots were mainly produced from discoid bases while filamentous calluses were mainly induced from basal zones and sub-apical explants. A gradient of the regeneration ability was observed from the top to the bottom of the thallus. The discoid base was the most reactive explant and produced the highest number of adventitious shoots compared to basal zones and sub-apical explants, irrespective of the concentration of agar. Protoplasts were isolated enzymatically from the whole thallus using a combination of cellulase R-10 Onozuka, macerozyme R-10, and crude extract of the gland gut of algivorous molluscs. The highest mean yield of protoplasts (1.2?×?106 protoplasts g?1 fresh weight) was obtained after 16 h of digestion with an enzyme mixture containing 2 % (w/v) cellulase R-10, 1 % (w/v) macerozyme R-10 Onozuka, 4 % (v/v) crude extract of gut gland of Haliotis, 0.8 M mannitol, 50 mM sodium citrate, 0.3 % (w/v) bovine serum albumin. Depending on the conditions, mean protoplast yields ranged from 3.14?×?105 to 1.2?×?106 protoplasts g?1 fresh weight. Different factors (storage duration, mannitol, sodium citrate, crude extract of the gland gut of algivorous molluscs) were tested to improve the yield of protoplasts but none has a significantly effect.  相似文献   

12.
To gain better knowledge of the variety of digestive enzymes in phytophagous coleopteran pests, a sequencing screen of 76 random cDNAs from a gut library from Phaedon cochleariae larvae was performed. The screen yielded 21 cDNAs encoding amino-acid sequences homologous to known digestive enzymes, most of them were cell wall-hydrolysing enzymes. The deduced protein sequences of 7 cDNAs encoding putative α-amylase, cysteine proteinase, trypsin, chymotrypsin, cellulase, pectinase and xylanase display all the structural features that characterize these enzymes in other eukaryotic organisms. Except the α-amylase and chymotrypsin cDNAs, the other cDNAs probably derive from multigene families. The distribution of the corresponding enzymatic activities at various developmental stages of P. cochleariae was examined. α-amylase activity is present in guts of larvae and adults, proteinases are abundant in guts of larvae and adults, but scarce in eggs and larval carcasses, xylanases are present in the guts of larvae and adults, as well as in carcasses of larvae, whereas cellulase and pectinase activities are distributed in larval and adult guts, larval carcasses, and eggs. Only a minor fraction of the cellulases is secreted by microorganisms, suggesting that P. cochleariae synthesizes most of its own cell-wall hydrolysing enzymes. The physiological role of the enzymes is discussed, as well as the significance of these results for pest management strategies involving transgenic plants expressing enzyme inhibitors.  相似文献   

13.
Xylanase III (Xyn III), a specific endoxylanase that belongs to family 10 of the glycoside hydrolases, was overexpressed in Trichoderma reesei QM9414 using a constitutive strong promoter of the gene encoding pyruvate decarboxylase (pdc). The maximum recombinant xylanase activity achieved was 817.2?±?65.2 U/mL in the transformant fermentation liquid. The productivities of Xyn III accounted for approximately 53 % of the total protein secreted by the recombinant. The enzyme was optimally active at 60 °C and pH 6. The recombinant Xyn III was stable at pH 5–8. This is the first report on the homologous expression of xyn3 in T. reesei QM9414. The properties of Xyn III make it promising in a variety of industrial use.  相似文献   

14.
15.
Sixty fungal cultures were isolated from agricultural soil, industrial soil, forest canopy soil having decomposed leaf litter and compost samples collected from different regions of India. Fifteen fungal cultures were selected qualitatively for the production of xylanase and cellulases and were identified employing ITS, NS and MNS primers. The enzyme cocktail consisting of 3811 IU g?1 of xylanase and 9.9 IU g?1 of cellulase from Trichoderma longibrachiatum MDU-6 was selected quantitatively for the deinking of diverse paper wastes. The enzyme production increased two fold when produced at tray level in comparison with flasks. The enzyme cocktail was effective in the deinking of old newspaper samples with significant removal of chromophores, phenolics and hydrophobic compounds and less sugar loss. While in case of examination papers and laser printed papers, ink removal was not very significant. Moreover, the sugar loss was significantly high in case of examination papers. The deinking results were further confirmed with FTIR analysis. Deinked newspaper pulp sample shows brightness of 52 %, which was 9.6 % high than its control sample. The ERIC value for deinked newspaper pulp was found to be 655.9 ppm. Thereafter, the deinked newspaper pulp was examined under light microscope after differential staining with safranin and malachite green and also examined under scanning and transmission electron microscope, which revealed fibrillation and perforation.  相似文献   

16.
The thermostable bifunctional CMCase and xylanase encoding gene (rBhcell-xyl) from Bacillus halodurans TSLV1 has been expressed in Escherichia coli. The recombinant E. coli produced rBhcell-xyl (CMCase 2272 and 910 U L?1 xylanase). The rBhcell-xyl is a ~62-kDa monomeric protein with temperature and pH optima of 60 °C and 6.0 with T1/2 of 7.0 and 3.5 h at 80 °C for CMCase and xylanase, respectively. The apparent K m values (CMC and Birchwood xylan) are 3.8 and 3.2 mg mL?1. The catalytic efficiency (k cat/K m ) values of xylanase and CMCase are 657 and 171 mL mg?1 min?1, respectively. End-product analysis confirmed that rBhcell-xyl is a unique endo-acting enzyme with exoglucanase activity. The rBhcell-xyl is a GH5 family enzyme possessing single catalytic module and carbohydrate binding module. The action of rBhcell-xyl on corn cobs and wheat bran liberated reducing sugars, which can be fermented to bioethanol and fine biochemicals.  相似文献   

17.
The delta-endotoxin of Bacillus thuringiensis. II. On the mode of action   总被引:1,自引:0,他引:1  
Uptake of glucose-14C by guts of the silkworm Bombyx mori was stimulated within 1 min after administration of Bacillus thuringiensis δ-endotoxin. Uptake of amino acids or carbonate ions was not similarly stimulated. General metabolic breakdown of the gut epithelium appears to occur between 10 and 20 min after administration of the toxin.  相似文献   

18.
The aim of this study was to develop simple and efficient method for immobilization of Candida antarctica lipase B onto hydrophobic anion exchange resin Purolite® MN102 and to apply immobilized catalyst for the enzymatic synthesis of two valuable esters—isoamyl acetate and l-ascorbyl oleate. At optimized conditions (1 M phosphate buffer pH = 7, 7 h at 25 °C, and 18.75 mg of offered proteins g?1 of support), immobilized lipase with hydrolytic activity of 888.4 p-nitrophenyl butyrate units g?1 was obtained. Afterwards, preparation was applied for the solvent-free synthesis of isoamyl acetate from triacetin and isoamyl alcohol. At 75 °C, 1 M of isoamyl alcohol, and 6 mg ml?1 of enzyme 100 % yield was achieved in 6 h, while at prolonged reaction times, complete conversion was enabled even at lower temperatures, lower lipase loadings, and higher substrate concentrations. After 15 consecutive reuses (60 h), activity of catalyst dropped to 50 % of its initial value and total amount of 1.31 mol (170.55 g) of ester with 1 g of biocatalyst was produced. Even higher operational stability of lipase (25 % loss of activity in 200 h) was observed in the synthesis of l-ascorbyl oleate performed in organic solvent (t-butanol). Multiple use of one batch of immobilized biocatalyst in both cases led to a significant process cost reduction and substantial increment of corresponding productivities.  相似文献   

19.
A cutinase gene (ScCut1) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N- or O-glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris, and the recombinantly produced ScCut1 enzyme was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K m and V max values determined for pNP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg?1, respectively. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with pNP-butyrate and tritiated cutin as the substrates, respectively. With both substrates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p-nitrophenyl butyrate (relative activity of 25 %) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p-nitrophenol than towards longer ones. Circular dichroism analysis suggested that the denaturation of ScCut1 by heating the protein sample to 80 °C was to a great extent reversible.  相似文献   

20.
A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20 g L?1 and 80 % dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53 U mL?1 was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13 U mL?1) was measured at a C/N ratio of 50.08. These conditions led to a 45.8 % increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500 g L?1 glucose led to a 6.2-fold increase (465.07 U mL?1) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.  相似文献   

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