Alkaline Xylanase Research Articles

A Calcium-Dependent Xylan-Binding Domain of Alkaline Xylanase from AlkaliphilicBacillussp. Strain 41M-1

Xylanase J of alkaliphilic Bacillus sp. strain 41M-1 contains a carbohydrate-binding module family 36 xylan-binding domain (XBD). Mutational analysis of the XBD revealed that Tyr237, Asp313, Trp317, and Asp318 were involved in Ca(2+)-dependent xylan-binding, and that Asp313 and Asp318 were especially important.

Microbiome of Fungus-Growing Termites: a New Reservoir for Lignocellulase Genes

Fungus-growing termites play an important role in lignocellulose degradation and carbon mineralization in tropical and subtropical regions, but the degradation potentiality of their gut microbiota has long been neglected. The high quality and quantity of intestinal microbial DNA are indispensable for exploring new cellulose genes from termites by function-based screening. Here, using a refined intestinal microbial DNA extraction method followed by multiple-displacement amplification (MDA), a fosmid library was constructed from the total microbial DNA isolated from the gut of a termite growing in fungi. Functional screening for endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase resulted in 12 β-glucosidase-positive clones and one xylanase-positive clone. The sequencing result of the xylanase-positive clone revealed an 1,818-bp open reading frame (ORF) encoding a 64.5-kDa multidomain endo-1,4-β-xylanase, designated Xyl6E7, which consisted of an N-terminal GH11 family catalytic domain, a CBM_4_9 domain, and a Listeria-Bacteroides repeat domain. Xyl6E7 was a highly active, substrate-specific, and endo-acting alkaline xylanase with considerably wide pH tolerance and stability but extremely low thermostability.

A novel <i>Bacillus pumilus</i> strain for alkaline xylanase production at and above 40°C

The objective of the study was to select a bacterial strain which can produce xylanase at alkaline pH and temperatures above 40°C and to characterize the strain. Among the 45 bacterial strains, isolated from different sources, five strains, which were expected to produce alkaline xylanase, above 40°C, isolated from open media containing xylan as carbon source (GS7, GS15, GS17, GS20 & GS*), were selected for the study. Xylanase production by the strains GS17 and GS*, was less affected by pH changes between pH 8.0 and 9.5 than the strains GS7, GS15 and GS20. Strains GS17 and GS* produced xylanase at pH 10.0 while the others did not produce. Therefore strains GS17 and GS* were selected. Xylanase production, by GS17 at 39 hours was 1.2, 1.3, 1.2 and 3.2 times higher at 40, 45, 50 and 55°C than by GS*. At 60°C, GS17 produced 4.25 UmL-1 (30h) of xylanase activity while GS* did not. As GS17 produced xylanase at and above pH 9.0 and 40 to 60°C, it was selected. The xylanase of GS17 showed activity in the temperature range from 40 to 95°C showing highest activity at 60°C. Strain GS17 is non-branching, gram-positive, sporulating, motile, aerobic, catalase positive, β hemolytic, oxidase positive long rods. Hence, it belongs to the genus Bacillus. Slightly oval shaped terminal and subterminal endospores were observed in 24h old cultures. When the culture became old, formation of spores was very much reduced. Further, based on the shape and arrangement of spores, ability to produce acid from glucose, xylose & mannose; growth temperature; inability to hydrolyze starch and tyrosine; inability to produce urease and indole; inability to reduce nitrate; ability to grow in NaCl and ability to grow in different carbohydrate sources, the strain, GS17 was identified as Bacillus pumilus. Thus, Bacillus pumilus which was locally isolated can grow and produce xylanase at and above 40°C and pH 9.0. Key words: alkaline xylanase; Bacillus pumilus; carbon source. DOI: 10.4038/cjsbs.v39i1.2354Cey. J. Sci. (Bio. Sci.) 39 (1): 61-70, 2010

Cloning and characterization of a xylanase, KRICT PX1 from the strain Paenibacillus sp. HPL-001

The KRICT PX1 gene (GB: FJ380951) consisting of 996 bp encoding a protein of 332 amino acids (38.1 kDa) from the recently isolated Paenibacillus sp. strain HPL-001 (KCTC11365BP) has been cloned and expressed in Escherichia coli. The xylanase KRICT PX1 showed high activity on birchwood xylan, and was active over a pH range of 5.0 to 11.0, with two optima at pH 5.5 and 9.5 at 50 °C with K m value of 5.35 and 3.23, respectively. The xylanase activity was not affected by most salts, such as NaCl, LiCl, KCl, NH 4Cl, CaCl 2, MgCl 2, MnCl 2, and CsCl 2 at 1 mM, but affected by CuSO 4, ZnSO 4, and FeCl 3. One mM of EDTA, 2-mercaptoethanol, and PMSF did not affect the xylanase activity. TLC analysis of the catalyzed products after reaction with birchwood xylan revealed that xylobiose was the major product with smaller amounts of xylotriose and xylose. A similarity analysis of the amino acids in KRICT PX1 resulted 72% identity with xylanase from Geobacillus stearothermophilus (GB: ZP_03040360), 70% identity with intracellular xylanase from an uncultured bacterium (GB: AAP51133), 68% identity with endo-1-4-xylanse from Paenibacillus sp. (GB: ZP_02847150). In addition, the amino acid alignment of KRICT PX1 with glycosyl hydralase (GH) family 10 xylanases revealed a high degree of homology in highly conserved regions including the catalytic sites, and this was confirmed through PROSITE scan. These results imply that KRICT PX1 is a new xylanase gene, and this alkaline xylanase belongs to GH family 10.

A Highly Thermostable Alkaline Cellulase-Free Xylanase from Thermoalkalophilic Bacillus sp. JB 99 Suitable for Paper and Pulp Industry: Purification and Characterization

A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0-10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K (m) and V (max) of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min(-1) mg(-1), respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

Analysis of Functional Domains and Improvement of Alkaliphily of an Alkaline Xylanase on the Basis of Its Three-dimensional Structure

Xylanase J (XynJ) from alkaliphilic Bacillus sp. strain 41M-1 is a multi-domain enzyme consisting of a glycoside hydrolase (GH) family 11 catalytic domain and a carbohydrate-binding module family 36 xylan-binding domain (XBD). Structural comparison of the GH family 11 catalytic domains indicated that there were several specific salt bridges in the catalytic cleft of XynJ. Mutant enzymes were prepared by substituting several amino acid residues responsible for the characteristic salt bridges. Elimination of the salt bridges caused an acidophilic shift in optimum pH, suggesting that the characteristic salt bridges contributed to the alkaliphily of XynJ. On the other hand, reinforcing one of the characteristic salt bridges in the catalytic domain shifted the optimum pH of XynJ from 8.5 to 9.0. Furthermore, introducing excess Arg residues on the protein surface was also effective to improve the alkaliphily of XynJ. Xylan-binding properties of various XBD mutants were investigated as fusion proteins with glutathione-S-transferase. Furthermore, the same substitutions were introduced into the XBD region of XynJ and insoluble xylan-hydrolyzing activity was measured. The results indicated that some Asp, Trp and Tyr residues were important for xylan-binding activity of the XBD, and that xylan-hydrolyzing activity of XynJ was closely correlated to xylan-binding activity of the XBD region.

In Vitro Renaturation of Alkaline Family G/11 Xylanase via a Folding Intermediate: α-Crystallin Facilitates Refolding in an ATP-Independent Manner

In this study, alkaliphilic family G/11 xylanase from alkali-tolerant filamentous fungi Penicillium citrinum MTCC 6489 was used as a model system to gain insight into the molecular aspects of unfolding/refolding of alkaliphilic glycosyl hydrolase protein family. The intrinsic protein fluorescence suggested a putative intermediate state of protein in presence of 2 M guanidium hydrochloride (GdmCl) with an emission maximum of 353 nm. Here we studied the refolding of GdmCl-denatured alkaline xylanase in the presence and the absence of a multimeric chaperone protein alpha-crystallin to elucidate the molecular mechanism of intramolecular interactions of the alkaliphilic xylanase protein that dictates its extremophilic character. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1- naphthalene sulfonate-binding studies, suggest that alpha-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of xylanase in an ATP-independent manner. A 2 M GdmCl is sufficient to denature alkaline xylanase completely. The hydrodynamic radius (R(H)) of a native alkaline xylanase is 4.0, which becomes 5.0 in the presence of 2 M GdmCl whereas in presence of the higher concentration of GdmCl R(H) value was shifted to 100, indicating the aggregation of denatured xylanase. The alpha-crystallin.xylanase complex exhibited the recovery of functional activity with the extent of approximately 43%. Addition of ATP to the complex did not show any significant effect on activity recovery of the denatured protein.

Effect of cutinase on the degradation of cotton seed coat in bio-scouring

In this paper the effect of cutinase on the degradation of cotton seed coat is analyzed. Fourier transform infrared (FT-IR) microspectroscopy was applied to study the changes of chemical compositions in cotton seed coat epidermal layer and gas chromatography/mass spectrometry (GC/MS) was used to analyse cutinase depolymerization of cotton seed coat. Based on these arguments the ability of cutinase to degrade aliphatic components in cotton seed coat was verified. Positive effect of cutinase on degradation of cotton seed coat was observed with the combination of alkaline pectinase or xylanase. The removal of aliphatic components by cutinase enables other enzymes to penetrate into the inner of cotton seed coat. Cutinase can potentially improve the degradation of cotton seed coat during cotton fabric bio-scouring process.

Improvement of Alkaliphily ofBacillusAlkaline Xylanase by Introducing Amino Acid Substitutions Both on Catalytic Cleft and Protein Surface

Xylanase J (XynJ) from alkaliphilic Bacillus sp. 41M-1 is an alkaline xylanase. The crystal structure has been solved with XynJ. Improvement of the alkaliphily of XynJ was attempted by amino acid substitutions. Reinforcing the characteristic salt bridge in the catalytic cleft and introducing excess Arg residues on the protein surface shifted the optimum pH of the wild-type enzyme from 8.5 to 9.5.

Contribution of salt bridges to alkaliphily of Bacillusalkaline xylanase

Xylanase J (XynJ) from alkaliphilic Bacillussp. strain 41M-1 is an alkaline xylanase. Structure comparison indicated that there were several specific salt bridges in the catalytic cleft of XynJ compared with neutral xylanases. Mutant enzymes were prepared by substituting several amino acids comprising the salt bridges. Some mutants exhibited acidophilic shift in optimum pH, whereas another showed alkaliphilic shift. These results suggested that the characteristic salt bridges could contribute to the alkaliphily of XynJ.

Statistical optimization of alkaline xylanase production from Streptomyces violaceoruber under submerged fermentation using response surface methodology

Response surface methodology employing central composite design (CCD) was used to optimize fermentation medium for the production of cellulase-free, alkaline xylanase from Streptomyces violaceoruber under submerged fermentation. The design was employed by selecting wheat bran, peptone, beef extract, incubation time and agitation as model factors. A second-order quadratic model and response surface method showed that the optimum conditions for xylanase production (wheat bran 3.5 % (w/v), peptone 0.8 % (w/v), beef extract 0.8 % (w/v), incubation time 36 h and agitation 250 rpm) results in 3.0-fold improvement in alkaline xylanase production (1500.0 IUml(-1)) as compared to initial level (500.0 IUml(-1)) after 36 h of fermentation, whereas its value predicted by the quadratic model was 1347 IUml(-1). Analysis of variance (ANOVA) showed a high coefficient of determination (R(2)) value of 0.9718, ensuring a satisfactory adjustment of the quadratic model with the experimental data.The economical and cellulase-free nature of xylanase would enhance its applicability in pulp and paper industry.

Influence of combined enzymatic treatment on one-bath scouring of cotton knitted fabrics

The scouring of cotton knitted fabrics with alkaline pectinase, neutral cellulase, neutral–alkaline protease, and alkaline xylanase and mixtures thereof, in one bath was studied. The effects of enzyme combinations were studied against the criteria of wettability, burst strength loss and whiteness. The best combinations were (1) pectinase (4 g L−1) and cellulase (0.5 g L−1), (2) pectinase (4 g L−1), cellulase (0.5 g L−1) and protease (1 g L−1), (3) pectinase (4 g L−1), cellulase (0.5 g L−1) and xylanase (2 g L−1). The results showed that the scouring effects of enzyme mixtures were better than those of individual enzymes, and dyeing properties (K/S, colour parameters and colour fastness) of bioscoured cotton knits were comparable to those of conventionally alkaline scoured cotton knits.

Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris

The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644 U mL−1 and the optimal secretion time was 4 days at 25°C. SDS-PAGE showed two bands, of 32.2 kDa and 29.6 kDa, both larger than the predicted mass of 22.4 kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6–9 and 50°C, respectively.

Partial purification and properties of cellulase-free alkaline xylanase produced by Rhizopus stolonifer in solid-state fermentation

Rhizopus stolonifer was cultivated in wheat bran to produce a cellulase-free alkaline xylanase. The purified enzyme obtained after molecular exclusion chromatography in Sephacryl S-200 HR showed optimum temperature as 45º C and hydrolysis pHs optima as pH 6.0 and 9.0. Xylanase presented higher Vmax at pH 9.0 (0.87 µmol/mg protein) than at pH 6.0 and minor Km at pH 6.0 (7.42 mg/mL) than at pH 9.0.

Xylanases of marine fungi of potential use for biobleaching of paper pulp.

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l(-1) xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55 degrees C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50 degrees C, with a second peak of activity at 90 degrees C. When assayed at pH 8.5, optimum activity was seen at 80 degrees C. The crude enzyme was thermostable at 55 degrees C for at least 4 h and retained about 60% activity. Gel filtration of the 50-80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)(-1). The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50-80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of beta-xylosidase and alpha- l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.

Production and Characterization of Alkaline Xylanases from Bacillus sp. Isolated from an Alkaline Soda Lake

Production and Characterization of Alkaline Xylanases from Bacillus sp. Isolated from an Alkaline Soda Lake

Compatibility of alkaline xylanases from an alkaliphilic Bacillus NCL (87-6-10) with commercial detergents and proteases.

Alkaline xylanases from alkaliphilic Bacillus strains NCL (87-6-10) and Sam III were compared with the commercial xylanases Pulpzyme HC and Biopulp for their compatibility with detergents and proteases for laundry applications. Among the four xylanases evaluated, the enzyme from the alkaliphilic Bacillus strain NCL (87-6-10) was the most compatible. The enzyme retained its full activity (40 degrees C for 1 h) in the presence of detergents, whereas Pulpzyme HC and Sam III showed only 30% and 50% of their initial activity, respectively. Biopulp, though stable to detergents, had only marginal activity (5%)at pH 10. However, all four enzymes retained significant activity (80%) for 60 min in the presence of the proteases Alcalase and Conidiobolus protease. Supplementation of the enzyme enhanced the cleaning ability of the detergents.

Characterization of alkaline thermoactive cellulase-free xylanases from alkalophilic Bacillus (NCL 87-6-10).

Two alkaline xylanases designated as "A" and "C", respectively, were isolated from the culture filtrates of the alkalophilic Bacillus grown on a wheat bran-yeast extract medium. The two xylanases occurred in the culture filtrate in a ratio of 10:90. These xylanases were purified to homogeneity on a CM-Sephadex matrix followed by further separation of Xylanase "A" on a phenyl sepharose column and preparative electrophoresis. The two xylanases differed considerably in their physico-chemical properties, kinetics and in their mode of action. Xylanase "C" had a molecular weight of 25,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was a cationic protein with a pI of 8.9. In contrast xylanase "A" had a molecular weight of 45,000 with a pI of 5.3. The two xylanases showed distinct differences in their hydrolysis pattern. Xylanase "A" produced comparatively larger amounts of small molecular weight oligosaccharides and xylose namely xylotriose (X(3)), xylobiose (X(2)) and xylose even in the initial stages of hydrolysis (2 and 5 h) while xylanase "C" produced negligible amounts of X(2) and no xylose for the same period of incubation. At 24 h only traces of xylose was produced by xylanase "C" while substantial amounts of the monomer was produced by xylanase A in 24 h. Xylanase "A" had a broad pH optimum ranging from pH 6.0-10.0 at 40-60 degrees C while xylanase "C" had an optimum pH of 8.0 at 40-60 degrees C. Xylanases "A" and "C" differed in their K(m) and V(max) values. Xylanase "A" had a K(m) of 1.67 mg/ml and a V(max) of 3.85 x 10(2) micromol/ml/min, whereas xylanase "C" had a K(m) of 10 mg/ml and a V(max) of 1.43 x 10(4) micromol/ml/min.

Studies on the key amino acid residues responsible for the alkali-tolerance of the xylanase by site-directed or random mutagenesis

Asparagine (Asn)-71 of the xylanase (XYN) from Bacillus pumilus A-30 was found highly conserved in alkaline xylanases of family G/11. The mutated gene fragments containing different substitutions of Asn-71 was obtained by site-directed mutagenesis to study its role in the alkali-tolerant mechanism of xylanase. The xylanase activity was completely lost if Asn-71 residue was replaced by alkaline arginine (Arg) or lysine (Lys) residues, but obviously depressed with a shift in the pH optimum of the enzyme from 6.7 to 6.3 if substituted by serine (Ser) or aspartate (Asp) residues. No mutant with a shift of the pH optimum to a more basic value was found. Furthermore, N71D lost its activity in the alkaline pH range completely, while N71S did not lose as much as that of N71D. Except for Asn-71, the random mutagenesis to other residues of the xylanase was also studied. The alkali-tolerant mechanism of the xylanase was analyzed by their charged character, ionized state, and the hydrogen bond network of the residues surrounding the two catalytic residues on the basis of homology modeling of the mutated xylanases.

Employing Chimeric Xylanases to Identify Regions of an Alkaline Xylanase Participating in Enzyme Activity at Basic pH.

Employing Chimeric Xylanases to Identify Regions of an Alkaline Xylanase Participating in Enzyme Activity at Basic pH.