Showing papers on "Xylanase published in 2007"

Optimization of enzyme complexes for lignocellulose hydrolysis.

Abstract: The ability of a commercial Trichoderma reesei cellulase preparation (Celluclast 1.5L), to hydrolyze the cellulose and xylan components of pretreated corn stover (PCS) was significantly improved by supplementation with three types of crude commercial enzyme preparations nominally enriched in xylanase, pectinase, and beta-glucosidase activity. Although the well-documented relief of product inhibition by beta-glucosidase contributed to the observed improvement in cellulase performance, significant benefits could also be attributed to enzymes components that hydrolyze non-cellulosic polysaccharides. It is suggested that so-called "accessory" enzymes such as xylanase and pectinase stimulate cellulose hydrolysis by removing non-cellulosic polysaccharides that coat cellulose fibers. A high-throughput microassay, in combination with response surface methodology, enabled production of an optimally supplemented enzyme mixture. This mixture allowed for a approximately twofold reduction in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis targets (99% and 88% conversion, respectively), thereby validating this approach towards enzyme improvement and process cost reduction for lignocellulose hydrolysis.

Thermostable enzymes in lignocellulose hydrolysis.

Abstract: Thermostable enzymes offer potential benefits in the hydrolysis of lignocellulosic substrates; higher specific activity decreasing the amount of enzymes, enhanced stability allowing improved hydrolysis performance and increased flexibility with respect to process configurations, all leading to improvement of the overall economy of the process. New thermostable cellulase mixtures were composed of cloned fungal enzymes for hydrolysis experiments. Three thermostable cellulases, identified as the most promising enzymes in their categories (cellobiohydrolase, endoglucanase and β-glucosidase), were cloned and produced in Trichoderma reesei and mixed to compose a novel mixture of thermostable cellulases. Thermostable xylanase was added to enzyme preparations used on substrates containing residual hemicellulose. The new optimised thermostable enzyme mixtures were evaluated in high temperature hydrolysis experiments on technical steam pretreated raw materials: spruce and corn stover. The hydrolysis temperature could be increased by about 10–15 °C, as compared with present commercial Trichoderma enzymes. The same degree of hydrolysis, about 90% of theoretical, measured as individual sugars, could be obtained with the thermostable enzymes at 60 °C as with the commercial enzymes at 45 °C. Clearly more efficient hydrolysis per assayed FPU unit or per amount of cellobiohydrolase I protein used was obtained. The maximum FPU activity of the novel enzyme mixture was about 25% higher at the optimum temperature at 65 °C, as compared with the highest activity of the commercial reference enzyme at 60 °C. The results provide a promising basis to produce and formulate improved enzyme products. These products can have high temperature stability in process conditions in the range of 55–60 °C (with present industrial products at 45–50 °C) and clearly improved specific activity, essentially decreasing the protein dosage required for an efficient hydrolysis of lignocellulosic substrates. New types of process configurations based on thermostable enzymes are discussed.

Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose.

Abstract: An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.

Enzymatic production of xylooligosaccharides from cotton stalks.

Abstract: Xylooligosaccharide (XO) production was performed from xylan, which was obtained by alkali extraction from cotton stalk, a major agricultural waste in Turkey. Enzymatic hydrolysis was selected to prevent byproduct formation such as xylose and furfural. Xylan was hydrolyzed using a commercial xylanase preparation, and the effects of pH, temperature, hydrolysis period, and substrate and enzyme concentrations on the XO yield and degree of polymerization (DP) were investigated. Cotton stalk contains about 21% xylan, the composition of which was determined as 84% xylose, 7% glucose, and 9% uronic acid after complete acid hydrolysis. XOs in the DP range of 2-7 (X6 approximately X5>X2>X3) were obtained with minor quantities of xylose in all of the hydrolysis conditions used. Although after 24 h of hydrolysis at 40 degrees C, the yield was about 53%, the XO production rate leveled off after 8-24 h of hydrolysis. XO yield was affected by all of the parameters investigated; however, none of them affected the DP of the end product significantly, except the hydrolysis period. Enzyme hydrolysis was maintained by the addition of fresh substrate after 72 h of hydrolysis, indicating the persistence of enzyme activity. The optimal hydrolysis conditions were determined as 40 degrees C, pH 5.4, and 2% xylan. The obtained product was fractionated via ultrafiltration by using 10, 3, and 1 kDa membranes. Complete removal of xylanase and unhydrolyzed xylan was achieved without losing any oligosaccharides having DP 5 or smaller by 10 kDa membrane. After a two-step membrane processing, a permeate containing mostly oligosaccharides was obtained.

Elucidating the mechanism of laccase and tyrosinase in wheat bread making.

Abstract: Cross-linking enzymes generate covalent bonds in and between food biopolymers. These enzymes are interesting tools for tailoring dough and bread structures, as the characteristics of the biopolymers significantly determine the viscoelastic and fracture properties of dough and bread. In this study, the influence of oxidative cross-linking enzymes, tyrosinase from the filamentous fungus Trichoderma reesei and laccase from the white rot fungus Trametes hirsuta, on dough and bread were examined. Oxidation of low molecular weight phenolic model compounds of flour, cross-linking of gluten proteins, dough rheology, and bread making were characterized during or after the enzymatic treatments. In the dough and bread experiments, laccase and tyrosinase were also studied in combination with xylanase. Of the model compounds tyrosine, p-coumaric acid, caffeic acid, ferulic acid, and Gly-Leu-Tyr tripeptide, tyrosinase oxidized all except ferulic acid. Laccase was able to oxidize each of the studied compounds. The pheno...

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

Abstract: The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.

Xylanase contribution to the efficiency of cellulose enzymatic hydrolysis of barley straw

Abstract: In this study, different enzyme preparations available from Novozymes were assessed for their efficiency to hydrolyze lignocellulosic materials. The enzyme mixture was evaluated on a pretreated cellulose-rich material, and steam-exploded barley straw pretreated under different temperatures (190, 200, and 210°C, respectively) in order to produce fermentable sugars. Results show that xylanase supplementation improves initial cellulose hydrolysis effectiveness of water-insoluble solid fraction from all steam-exploded barley straw samples, regardless of the xylan content of substrate. The mixture constituted by cellulase: β-glucosidase: endoxylanase of the new kit for lignocellulose conversion at a ratio 10∶1∶5% ([v/w], enzyme [E]/substrate [S]) provides the highest increment of cellulose conversion in barley straw pretreated at 210°C, for 10 min.

Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically.

Abstract: The genome of Clostridium thermocellum contains a number of genes for polysaccharide degradation-associated proteins that are not cellulosome bound. The list includes β-glucanases, glycosidases, chitinases, amylases and a xylanase. One of these ‘soluble’-enzyme genes codes for a second glycosyl hydrolase (GH)48 cellulase, Cel48Y, which was expressed in Escherichia coli and biochemically characterized. It is a cellobiohydrolyse with activity on native cellulose such as microcrystalline and bacterial cellulose, and low activity on carboxymethylcellulose. It is about 100 times as active on amorphic cellulose and mixed-linkage barley β-glucan compared with cellulase Cel9I. The enzyme Cel48Y shows a distinct synergism of 2.1 times with the noncellulosomal processive endoglucanase Cel9I on highly crystalline bacterial cellulose at a 17-fold excess of Cel48Y over Cel9I. These data show that C. thermocellum has, besides the cellulosome, the genes for a second cellulase system for the hydrolysis of crystalline cellulose that is not particle bound.

Production of xylooligosaccharides from xylans by extracellular xylanases from Thermobifida fusca.

Abstract: Xylooligosaccharides are produced for use as a valuable food sweetener or additive. They have many beneficial biomedical and health effects. In this study, a process for producing xylooligosaccharides from lignocellulolytic agricultural waste was developed. Bagasse, corncob, wheat bran, and peanut shell were used as carbon sources for production of xylanolytic enzymes from Thermobifida fusca NTU22. When using bagasse as the carbon source, the xylanolytic enzymes that simultaneously accumulated in the broth in a 500 mL Hinton flask after 72 h of cultivation at 50 degrees C were measured as xylanase (14.0 U/mL), beta-xylosidase (74.1 mU/mL), and acetyl esterase (29.1 mU/mL). The optimum pH and temperature for xylanases were 6.0-8.0 and 70 degrees C, respectively. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gel. This was followed by heat treatment at 70 degrees C for 30 min that eliminated 90% of the beta-xylosidase activity. The xylanase and acetyl esterase activities were still 100%. Two percent of xylan extracted from the bagasse was then hydrolyzed by heat-treated crude xylanase preparation at 60 degrees C, pH 7.0, for 10 h. The xylooligosaccharides that accumulated in the broth were about 23.7%. After the purification process by activated charcoal chromatography, the purity of xylooligosaccharides was 71.4%.

Immobilization of xylanase from Bacillus pumilus strain MK001 and its application in production of xylo-oligosaccharides.

Abstract: Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0-10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 degrees C higher (68.0 degrees C) than free enzyme (60.0 degrees C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 degrees C. The immobilized xylanase registered marginal increase and decrease in Km and Vmax values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.

Role of the transcriptional activator xlnR of Fusarium oxysporum in regulation of xylanase genes and virulence.

Abstract: Fungal infection of plants involves degradation of the host cell wall through the action of lytic enzymes secreted by the pathogen. The role of these enzymes in virulence is difficult to determine due to their functional redundancy and, therefore, remains controversial. Here, we have studied XlnR, a zinc-finger transcription factor from the vascular wilt pathogen Fusarium oxysporum that is orthologous to the major transcriptional activator of xylanase genes in Aspergillus spp. Transcription of the xlnR gene was activated by inducing carbon sources such as oat spelt xylan (OSX) and repressed by glucose. Targeted knockout of xlnR in F. oxysporum resulted in lack of transcriptional activation of structural xylanase genes, both in culture and during infection of tomato plants, as well as in dramatically reduced extracellular xylanase activity. By contrast, overexpression of xlnR under the control of the Aspergillus nidulans gpdA promoter did not significantly increase xylanase activity, suggesting that XlnR is regulated not only at the transcriptional but also at the post-translational level. The ΔxlnR mutants were still fully virulent on tomato plants. Thus, XlnR, the major transcriptional activator of xylanase genes, is not an essential virulence determinant in F. oxysporum.

Alkali-thermostable and cellulase-free xylanase production by an extreme thermophile Geobacillus thermoleovorans

Abstract: The optimization of cultural variables resulted in a marked enhancement in the secretion of cellulase-free and alkali-thermostable xylanase (EC 3.2.1.8) by an extreme thermophile Geobacillus thermoleovorans. The enzyme secretion was enhanced when the medium was supplemented with xylan (0.15%) and Tween-80 (0.1% v/v). In wheat bran-tryptone medium, the peak in enzyme production was attained within 42 h in a fermenter as compared to 72 h in shake flasks. Optimization of the culture conditions resulted in a 7.72-fold enhancement in enzyme production. The cellulase-free xylanase was optimally active at pH 8.5 and 80°C, and it was found to be useful in the pre-bleaching process of paper pulps.

Xylanase production by Aspergillus niger ANL 301 using agro - wastes

Abstract: Xylanase production by wild-type Aspergillus niger ANL301, newly isolated from wood-waste, was monitored at 24 h intervals for a period 168 h in media containing different carbon sources. The carbon sources were oat-spelt xylan (Fluka) and three agro-wastes (sawdust, sugarcane pulp and wheat bran). Highest xylanase activity of 6.47 units/mL was obtained at 96 h in media containing wheat bran as sole carbon source. Maximum activity value for the media containing sugarcane pulp was 0.95 units/mL obtained also at 96 h. Sawdust and oat spelt xylan gave the peak enzyme activities of 0.65 and 0.80 units/mL respectively at 120 h. High protein yield was obtained in media containing the agro-wastes, with wheat bran giving the highest value of 1.14 mg/mL at 96 h. The maximum specific xylanase activities were 3.86, 3.37, 5.69, and 9.36 units/ mg protein for sawdust, sugarcane pulp, wheat bran and oat spelt xylan, respectively. Out of the three agro-wastes used in this study, wheat bran holds greatest promise for low cost production of the xylanase enzyme.

Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina.

Abstract: A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at 25 degrees C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml(-1). The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 40 degrees C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The Km and Vmax values for birchwood xylan are 2.06 mg ml(-1) and 0.49 mmol min(-1)mg(-1), respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu2+ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity is unaffected by EDTA even at a concentration of 5 mM.

A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and characterization

Abstract: Aims: The enzymatic hydrolysis of xylan has potential economic and environment-friendly applications. Therefore, attention is focused here on the discovery of new extremophilic xylanase in order to meet the requirements of industry. Methods and Results: An extracellular xylanase was purified from the culture filtrate of P. citrinum grown on wheat bran bed in solid substrate fermentation. Single step purification was achieved using hydrophobic interaction chromatography. The purified enzyme showed a single band on SDS-PAGE with an apparent molecular weight of c. 25 kDa and pI of 3·6. Stimulation of the activity by β mercaptoethanol, dithiotheritol (DTT) and cysteine was observed. Moderately thermostable xylanase showed optimum activity at 50°C at pH 8·5. Conclusion: Xylanase purified from P. citrinum was alkaliphilic and moderately thermostable in nature. Significance and Impact of the Study: The present work reports for the first time the purification and characterization of a novel endoglucanase free alkaliphilic xylanase from the alkali tolerant fungus Penicillium citrinum. The alkaliphilicity and moderate thermostability of this xylanase may have potential implications in paper and pulp industries.

A multifunctional hybrid glycosyl hydrolase discovered in an uncultured microbial consortium from ruminant gut

Abstract: A unique multifunctional glycosyl hydrolase was discovered by screening an environmental DNA library prepared from a microbial consortium collected from cow rumen. The protein consists of two adjacent catalytic domains. Sequence analysis predicted that one domain conforms to glycosyl hydrolase family 5 and the other to family 26. The enzyme is active on several different β-linked substrates and possesses mannanase, xylanase, and glucanase activities. Site-directed mutagenesis studies on the catalytic residues confirmed the presence of two functionally independent catalytic domains. Using site-specific mutations, it was shown that one catalytic site hydrolyzes β-1,4-linked mannan substrates, while the second catalytic site hydrolyzes β-1,4-linked xylan and β-1,4-linked glucan substrates. Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE) also confirmed that the enzyme has discrete domains for binding and hydrolysis of glucan- and mannan-linked polysaccharides. Such multifunctional enzymes have many potential industrial applications in plant processing, including biomass saccharification, animal feed nutritional enhancement, textile, and pulp and paper processing.