Improving the catalytic activity and thermostability of Aspergillus niger xylanase through computational design.

Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported.

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library.

Xylanases are key biocatalysts in the degradation of the ß-1,4-glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-ß-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. KEY POINTS: • A GH10 endo-1,4-ß-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB's in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis.

Purification and characterization of a highly-stable fungal xylanase from Aspergillus tubingensis cultivated on palm wastes through combined solid-state and submerged fermentation.

Fungal xylanase was produced from lignocellulosic palm wastes through combined solid-state fermentation (SSF) and submerged fermentation (SmF) by Aspergillus tubingensis TSIP9 in a helical-impeller equipped bioreactor. The combined SSF-SmF promoted the xylanase production by 15 and 70% higher than SSF and SmF, respectively. Sequential purification yielded 7.4-fold purified xylanase with 9.07% recovery. The maximum activities of crude and purified xylanase were observed at the same pH of 5.0 and the same temperature of 50 °C while purified xylanase is more active and highly stable at a wider pH range of 3-8 and temperature of 30-60 °C. The half-life of purified xylanase at various temperatures was also much improved by 2-8 folds compared to crude xylanase. Michaelis-Menten constants, Vmax and Km, for purified xylanase are 2,602.8 U/mg and 32.4 mg/mL, respectively. Purified xylanase activity was most enhanced with Ca2+ followed by Zn2+ and Fe2+ at 10 mM while significantly inhibited by Co2+, Cu2+, Pb2+, and Ag+. This study has shown the effectiveness of combined SSF-SmF for xylanase production and superior properties of purified xylanase for industrial processes.

Biochemical characterization of xylanase GH11 isolated from Aspergillus niger BCC14405 (XylB) and its application in xylooligosaccharide production.

OBJECTIVE: To develop an endo-ß-1,4-xylanase with high specificity for production of prebiotic xylooligosaccharides that optimally works at moderate temperature desirable to reduce the energy cost in the production process. RESULTS: The xylB gene, encoding for a glycosyl hydrolase family 11 xylanase from a thermoresistant fungus, Aspergillus niger BCC14405 was expressed in a methylotrophic yeast P. pastoris KM71 in a secreted form. The recombinant XylB showed a high specific activity of 3852 and 169 U mg-1 protein on beechwood xylan and arabinoxylan, respectively with no detectable side activities against different forms of cellulose (Avicel Ò PH101 microcrystalline cellulose, phosphoric acid swollen cellulose and carboxymethylcellulose). The enzyme worked optimally at 45 °C, pH 6.0. It showed a specific cleavage pattern by releasing xylobiose (X2) as the major product from xylooligosaccharides (X3 to X6) substrates. The highest XOS yield of 708 mg g-1 substrate comprising X2, X3 and X6 was obtained from beechwood xylan hydrolysis. CONCLUSION: The enzyme is potent for XOS production and for saccharification of lignocellulosic biomass.

Optimization of ß-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production.

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0-9.0 and between 30-40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (Km = 26.06 mg·mL-1 and Vmax = 5.647 U·mg-1). Wheat straw xylan hydrolysis with the purified ß-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.

Cloning, expression and characterization of C. crescentus xynA2 gene and application of Xylanase II in the deconstruction of plant biomass.

Biotechnology offers innovative alternatives for industrial bioprocesses mainly because it uses enzymes that biodegrade the hemicellulose releasing fermentable sugars. Caulobacter crescentus (C. crescentus) has seven genes responsible for xylanolytic cleavage, 5 to ß-xylosidases (EC 3.2.1.37) and 2 for endoxylanases, like xynA2 (CCNA_03137) that encodes Xylanase II (EC 3.2.1.8) of the glycohydrolases-GH10 group. The xynA2 gene was amplified by PCR, cloned into the pTrcHisA vector e efficiently overexpressed in E. coli providing a His-tag fusion protein. Recombinant xylanase (XynA2) was purified by affinity chromatography using a nickel sepharose column and exhibited a single 43 kDa band on SDS-PAGE gel. XynA2 showed an optimum alkaline pH (8) and stability at alkaline pH for 24 h. Although C. crescentus is mesophilic, XynA2 has optimum temperature of 60 °C and is thermo-resistance at 65 °C. XynA maintains 66% of the enzymatic activity at high temperatures (90 °C) without being denatured.The enzyme displayed a xylanolitic activity free of cellulase to xylan from beechwood and it was not inhibited in the presence of 50 µmol mL-1 of xylose. In addition, dithiothreitol (DTT) induced XynA2 activity, as it improved its kinetic parameters by lowering the KM (5.78 µmol mL-1) and increasing the KCat/KM ratio (1.63 U s-1). Finally, C. crescentus XynA2 efficiently hydrolyzed corn straw with high release of reducing sugars that can be applied in different branches of the industry.

Characterization of a novel xylanase from an extreme temperature hot spring metagenome for xylooligosaccharide production.

In this study, the metagenomic resource generated from an aquatic habitat of extreme temperature was screened for the identification of a novel xylanase, XynM1. Gene sequence analysis designated it as a member of glycoside hydrolase (GH) family 10. The metagenomic DNA fragment was cloned, expressed in Escherichia coli, and the purified protein was biochemically characterized. The optimum temperature and pH for the XynM1 xylanase were found to be at 80 °C and 7, respectively. It exhibited worthwhile pH stability by retaining about 70% activity in the range of pH 6 to 9 after the exposure for 12 h at 25 °C. Thermostability analysis established considerable heat tolerance in XynM1 protein at elevated temperatures, displaying about 50% residual activity after the exposure of 40 °C, 50 °C, 60 °C, and 70 °C for 20 h, 12 h, 6 h, and 1.5 h, respectively. The effects of additives such as metals, surfactants, and organic solvents were evaluated on the activity of XynM1. It was able to retain about 50% of its initial activity in the presence of NaCl concentration of 1 to 5 M. The novel xylanase was capable of hydrolyzing the hemicellulosic polymer, derived from diverse biomass sources, e.g., beechwood xylan, wheat arabinoxylan, corncob xylan, and sweet sorghum xylan. The XynM1-treated beechwood xylan manifested catalytic release of xylooligosaccharides (XOS) of 2-6 DP. The novel GH10 xylanase is a promising biocatalyst that could be ascribed for biomass conversion and production of prebiotic XOS biomolecules.

Isolation of four xylanases capable of hydrolyzing corn fiber xylan from Paenibacillus sp. H2C.

Corn fibre xylan (CX) shows high resistance to enzymatic hydrolysis due to its densely decorated side chains. To find enzymes capable of hydrolyzing CX, we isolated a bacterial strain (named H2C) from soil, by enrichment culture using non-starch polysaccharides of corn as the sole carbon source. Analysis based on the 16S rRNA sequence placed strain H2C within genus Paenibacillus. Enzymes were purified from supernatant of culture broth of strain H2C based on solubilizing activities toward CX. Four enzymes, Xyn5A, Xyn10B, Xyn11A, and Xyn30A, were successfully identified, which belong to glycoside hydrolase (GH) families, 5, 10, 11, and 30, respectively. Phylogenetic analysis classified Xyn5A in subfamily 35 of GH family 5, a subfamily of unknown function. Their activities toward beechwood xylan and/or wheat arabinoxylan indicated that these enzymes are ß-1,4-xylanases. They showed high solubilizing activities toward a feed material, corn dried distiller's grains with solubles, compared to five previously characterized xylanases.Abbreviations : CX: corn fibre xylan; DDGS: corn dried distiller's grains with solubles.

Isolation and Characterization of a Novel Cold-Active, Halotolerant Endoxylanase from Echinicola rosea sp. Nov. JL3085T.

We cloned a xylanase gene (xynT) from marine bacterium Echinicola rosea sp. nov. JL3085T and recombinantly expressed it in Escherichia coli BL21. This gene encoded a polypeptide with 379 amino acid residues and a molecular weight of ~43 kDa. Its amino acid sequence shared 45.3% similarity with an endoxylanase from Cellvibrio mixtus that belongs to glycoside hydrolases family 10 (GH10). The XynT showed maximum activity at 40 °C and pH 7.0, and a maximum velocity of 62 µmoL min-1 mg-1. The XynT retained its maximum activity by more than 69%, 51%, and 26% at 10 °C, 5 °C, and 0 °C, respectively. It also exhibited the highest activity of 135% in the presence of 4 M NaCl and retained 76% of its activity after 24 h incubation with 4 M NaCl. This novel xylanase, XynT, is a cold-active and halotolerant enzyme that may have promising applications in drug, food, feed, and bioremediation industries.

A novel acid-tolerant ß-xylanase from Scytalidium candidum 3C for the synthesis of o-nitrophenyl xylooligosaccharides.

Endo-ß-xylanases are hemicellulases involved in the conversion of xylans in plant biomass. Here, we report a novel acidophilic ß-xylanase (ScXynA) with high transglycosylation abilities that was isolated from the filamentous fungus Scytalidium candidum 3C. ScXynA was identified as a glycoside hydrolase family 10 (GH10) dimeric protein, with a molecular weight of 38 ± 5 kDa per subunit. The enzyme catalyzed the hydrolysis of different xylans under acidic conditions and was stable in the pH range 2.6-4.5. The kinetic parameters of ScXynA were determined in hydrolysis reactions with p-nitrophenyl-ß-d-cellobioside (pNP-ß-Cel) and p-nitrophenyl-ß-d-xylobioside (pNP-ß-Xyl2 ), and kcat /Km was found to be 0.43 ± 0.02 (s·mM)-1 and 57 ± 3 (s·mM)-1 , respectively. In the catalysis of the transglycosylation o-nitrophenyl-ß-d-xylobioside (oNP-ß-Xyl2 ) acted both as a donor and an acceptor, resulting in the efficient production of o-nitrophenyl xylooligosaccharides, with a degree of polymerization of 3-10 and o-nitrophenyl-ß-d-xylotetraose (oNP-ß-Xyl4 ) as the major product (18.5% yield). The modeled ScXynA structure showed a favorable position for ligand entry and o-nitrophenyl group accommodation in the relatively open -3 subsite, while the cleavage site was covered with an extended loop. These structural features provide favorable conditions for transglycosylation with oNP-ß-Xyl2 . The acidophilic properties and high transglycosylation activity make ScXynA a suitable choice for various biotechnological applications, including the synthesis of valuable xylooligosaccharides.

Purification, Identification, and Characterization of an Endo-1,4-ß-Xylanase from Wheat Malt.

In this study, an endo-1,4-ß-xylanase was purified from wheat malt following the procedures of ammonium sulfate precipitation, cation-exchange chromatography, and two-step anion-exchange chromatography. The purified endo-1,4-ß-xylanase had a specific activity of 3.94 u/mg, demonstrating a weight average molecular weight (Mw) of approximately 58,000 Da. After LC-MS/MS (Liquid chromatography-tandem mass spectrometry) identification, the purified enzyme had the highest matching degree with a GH10 (Glycoside Hydrolase 10) domain-containing protein from wheat, there were 23 match peptides with a score above the threshold and the prot-cover was 45.5%. The resulting purified enzyme was used to investigate its degradation ability on high viscosity wheat-derived water-extractable arabinoxylan (WEAX). Degradation experiments confirmed that the purified enzyme was a true endo-acting enzyme, which could degrade large WEAX into smaller WEAX. The average degree of polymerization (avDP) and the viscosity of WEAX decreased with the increasing reaction time. The enzyme could degrade a small amount of WEAX into arabinoxylan-oligosaccharides (AXOS) with a degree of polymerization of 2-6, but no monosaccharide was produced. The degradation occurred rapidly in the first 3.5 h and decreased with the further prolongation of reaction time.

Purification and characterization of a new xylanase with excellent stability from Aspergillus flavus and its application in hydrolyzing pretreated corncobs.

A new extracellular xylanase was purified from a non-toxic mesophilic fungus Aspergillus flavus, and characterized as the ß-1, 4-endoxylanase (designated as AfXynA) that appeared in a single protein band on SDS-PAGE with a molecular mass of 20.2 kDa, which is different from all other reported xylanases from the same strain. The AfXynA exhibited a specific activity of 838.2 U/mg. Its optimal temperature and pH were determined to be 55 °C and 7.5, respectively. It was stable up to 50 °C and within pH 3.5-10.5. AfXynA also exhibited an excellent tolerance to various proteases. This new xylanase had an endohydrolytic mode of action and could hydrolyze xylotriose to xylobiose through transglycosylation. It could efficiently degrade xylan to mainly yield xylobiose, xylotriose, xylopentose and xylohexaose. In addition, the AfXynA was effective in hydrolyzing pretreated corncobs, and shows a great potential in the production of xylooligosaccharides. These unique enzymatic properties make the AfXynA attractive for more biotechnological applications.

Efficient expression and secretion of endo-1,4-ß-xylanase from Penicillium citrinum in non-conventional yeast Yarrowia lipolytica directed by the native and the preproLIP2 signal peptides.

Filamentous fungi are the most common industrial xylanase producers. In this study, the xynA gene encoding xylanase A of Penicilium citrinum was successfully synthesized and expressed in Yarrowia lipolytica under the control of the strong constitutive TEF promoter. Native and preproLIP2 secretion signals were used for comparison of the expression and secretion level. The recombinant xylanase was produced as a soluble protein, and the total activity production reached 11 and 52 times higher than the level of activity produced by the fungus P. citrinum native strain, respectively. Maximum activity was observed with the preproLIP2 secretion signal at 180 U/mL. Post translational glycosylation affected the molecular mass of the recombinant xylanase, resulting in an apparent molecular weight larger than 60 kDa, whereas after deglycosylation, the recombinant XynA displayed a molecular mass of 20 kDa. The deglycosylated xylanase was purified by ion exchange chromatography and reached 185-fold of purification. The enzyme was optimally active at 55 °C and pH 5 and stable over a broad pH range (3-9). It retained more than 80% of the original activity after 24 h. It conserved around 80% of the original activity after pre-incubation at 40 °C for 6 h. With birchwood xylan as substrate, the enzyme showed a Km of 5.2 mg/mL, and kcat of 245 per s. The high level of secretion and the stability over a wide range of pH and at moderate temperatures of the re-XynA could be useful for variety of biotechnological applications.

Heterologous expression, purification and biochemical characterization of a new endo-1,4-ß-xylanase from Rhodothermaceae bacterium RA.

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

Promiscuity of host cell proteins in the purification of histidine tagged recombinant xylanase A by IMAC procedures: A case study with a Ni2+-tacn-based IMAC system.

Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisite to validate the chromatographic purification of recombinant proteins. This study explores how different experimental conditions affect the HCP profiles generated during the immobilised metal ion affinity chromatographic (IMAC) purification with a Ni2+-1,4,7-triaza-cyclononane (tacn) Sepharose FF™ sorbent of the Bacillus halodurans N- and C-terminal His6-tagged xylanase A, expressed by Escherichia coli BL21(DE3) cells, and captured directly from cell lysates. Comparative studies were also carried out under identical loading, wash and elution conditions using nitrilotriacetic acid (NTA), also immobilised onto an agarose support and complexed with Ni2+ ions. High-resolution tandem mass spectrometry of the tryptic peptides derived from the proteins present in the IMAC flow-through, wash and elution fractions confirmed that the E. coli BL21(DE3) HCP profiles were dependent on the choice of adsorbent. With feedstocks containing the N- or C-terminal His6-tagged xylanase A, in several instances the same E. coli BL21(DE3) HCPs were found to co-elute with the tagged protein from either adsorbent, indicating a preferential ability of some HCPs to bind to both the IMAC resin and to the recombinant protein. This promiscuous behaviour has been found to be due to factors other than just the presence of histidine-rich motifs within the amino acid sequences of these HCPs. This case study demonstrates that the choice of protein expression and separation conditions impact on the levels of HCP contamination when different IMAC systems are employed.

Characterization of recombinant endo-1,4-ß-xylanase of Bacillus halodurans C-125 and rational identification of hot spot amino acid residues responsible for enhancing thermostability by an in-silico approach.

Increased demand of enzymes for industrial use has led the scientists towards protein engineering techniques. In different protein engineering strategies, rational approach has emerged as the most efficient method utilizing bioinformatics tools to produce enzymes with desired reaction kinetics; physiochemical (temperature, pH, half life, etc) and biological (selectivity, specificity, etc.) characteristics. Xylanase is one of the widely used enzymes in paper and food industry to degrade xylan component present in plant pulp. In this study endo 1,4-ß-xylanase (Xyl-11A) from Bacillus halodurans C-125 was cloned in pET-22b (+) vector and expressed in Escherichia coli BL21 (DE3) expression strain. The enzyme had Michaelis constant Km of 1.32 mg ml-1 birchwoodxylan (soluble form) and maximum reaction velocity (Vmax) 73.53 mmol min-1 mg-1 with an optimum temperature of 75 °C and pH 9.0. The thermostability analysis showed that enzyme retained more than 80% of its residual activity when incubated at 75 °C for 2 h. In addition, to increase Xyl-11A thermostability, an in-silico analysis was performedto identify the hot spot amino acid residues. Consensus-based amino acid substitution was applied to evaluate multiple sequence alignment of homologs and identified 20 amino acids positions by following Jensen-Shnnon Divergence method. 3D models of 20 selected mutants were analyzed for conformational transition in protein structures by using NMSim server. Two selected mutants T6K and I17M of Xyl-11A retained 40, 60% residual activity respectively, at 85 °C for 120 min as compared to wild type enzyme which retained 37% initial activity under same conditions, confirming the enhanced thermostability of mutants. The present study showed a good approach for the identification of promising amino acid residues responsible for enhancing the thermostability of enzymes of industrial importance.

Biochemical characterization and low-resolution SAXS shape of a novel GH11 exo-1,4-ß-xylanase identified in a microbial consortium.

Biotechnologies that aim to produce renewable fuels, chemicals, and bioproducts from residual ligno(hemi)cellulosic biomass mostly rely on enzymatic depolymerization of plant cell walls (PCW). This process requires an arsenal of diverse enzymes, including xylanases, which synergistically act on the hemicellulose, reducing the long and complex xylan chains to oligomers and simple sugars. Thus, xylanases play a crucial role in PCW depolymerization. Until recently, the largest xylanase family, glycoside hydrolase family 11 (GH11) has been exclusively represented by endo-catalytic ß-1,4- and ß-1,3-xylanases. Analysis of a metatranscriptome library from a microbial lignocellulose community resulted in the identification of an unusual exo-acting GH11 ß-1,4-xylanase (MetXyn11). Detailed characterization has been performed on recombinant MetXyn11 including determination of its low-resolution small-angle X-ray scattering (SAXS) molecular envelope in solution. Our results reveal that MetXyn11 is a monomeric globular enzyme that liberates xylobiose from heteroxylans as the only product. MetXyn11 has an optimal activity in a pH range from 6 to 9 and an optimal temperature of 50 °C. The enzyme maintained above 65% of its original activity in the pH range 5 to 6 after being incubated for 72 h at 50 °C. Addition of the enzyme to a commercial enzymatic cocktail (CelicCtec3) promoted a significant increase of enzymatic hydrolysis yields of hydrothermally pretreated sugarcane bagasse (16% after 24 h of hydrolysis).

Characterization of a novel xylanase from Aspergillus flavus with the unique properties in production of xylooligosaccharides.

A novel xylanase from the filamentous fungus Aspergillus flavus was purified and characterized as the ß-1, 4-endoxylanase (designed as AfXynB) with a molecular mass (32.2 kDa), which is different from all of the previously reported xylanases from the same strain. AfXynB was optimally active at pH 7.5 and 55 °C, respectively. It was stable up to 50 °C within range of pH 4.0-9.5, and displayed an excellent tolerance to various cations, reagents, and proteases. AfXynB showed specific activity toward beechwood xylan but no detected activity toward CMC and pNP-ß-D-xylopyranoside. The xylanase is a typical endo-xylanase; it could hydrolyze beechwood xylan to only yield xylobiose (X2) and xylopentaose (X5). Actually, this may be the first report for the endo-xylanases that displayed such a unique hydrolytic property. These findings in the present study have great implications for its future applications of the novel xylanase.

Purification and characterization of an endo-xylanase from Trichoderma sp., with xylobiose as the main product from xylan hydrolysis.

Fungal endo-ß-1,4-xylanases (endo-xylanases) can hydrolyze xylan into xylooligosaccharides (XOS), and have potential biotechnological applications for the exploitation of natural renewable polysaccharides. In the current study, we aimed to screen and characterize an efficient fungal endo-xylanase from 100 natural humus-rich soil samples collected in Guizhou Province, China, using extracted sugarcane bagasse xylan (SBX) as the sole carbon source. Initially, 182 fungal isolates producing xylanases were selected, among which Trichoderma sp. strain TP3-36 was identified as showing the highest xylanase activity of 295 U/mL with xylobiose (X2) as the main product when beechwood xylan was used as substrate. Subsequently, a glycoside hydrolase family 11 endo-xylanase, TXyn11A, was purified from strain TP3-36, and its optimal pH and temperature for activity against beechwood xylan were identified to be 5.0 and 55 °C, respectively. TXyn11A was stable across a broad pH range (3.0-10.0), and exhibited strict substrate specificity, including xylan from beechwood, wheat, rye, and sugarcane bagasse, with Km and Vmax values of 5 mg/mL and 1250 µmol/mg min, respectively, toward beechwood xylan. Intriguingly, the main product obtained from hydrolysis of beechwood xylan by TXyn11A was xylobiose, whereas SBX hydrolysis resulted in both X2 and xylotriose. Overall, these characteristics of the endo-xylanase TXyn11A indicate several potential industrial applications.

Delineating thermophilic xylanase from Bacillus licheniformis DM5 towards its potential application in xylooligosaccharides production.

In this present study novel endoxylanase producing Bacillus licheniformis DM5 isolated, identified based on 16S rDNA from Garampani hotspring, Assam, India and enzyme was purified. RNA secondary structure predicted the similarity of B. licheniformis DM5 with B. licheniformis ATCC14580. Highest production of xylanase from B. licheniformis DM5 was achieved in the TY medium with cell densities 12 g/l and extracellular protein concentration containing xylanase 400 mg/l. Partially purified extracellular xylanase displayed optimum pH 6.5 and temperature 50 °C. Thermostability of the xylanase at the elevated temperature showed stability between 50 and 60 °C retaining its 99% activity. Kinetic parameters of thermophilic xylanase revealed Km 1.5 ± 0.2 mg/ml, Vmax 2.7 ± 0.2 U/ml and and Kcat 1.8 ± 0.2 s-1 against beechwood xylan but ruled out any exo-acting activity against synthetic pNP-xylopyranoside substrate. Time dependent enzymatic hydrolysis of beechwood xylan and preprocessed agrowaste corncob exhibited the release of xylotriose and xylobiose oligosaccharide (XOS) significantly high. Xylobiose and xylotriose exhibited higher binding affinities with BIAXP transporter protein of probiotic bacteria explaining their easy uptake by the cells. Mixed oligosaccharides also exhibited better prebiotic activity by promoting growth of Bifidobacterium infantis and Lactobacillus delbrueckii. Mixed XOS when tested for their cytotoxicity on Hela cell lines in in vitro MTT assay displayed significant lowering of cell viability after 48 h and 24 h at 100 µg/ml to 60% and 50%, respectively. In contrast, cytotoxicity wasn't observed against normal cervical cell line (VK2/E6E7-ATCC-CRL-2616). Therefore, thermophilic endoxylanase from B. licheniformis DM5 could be attributed for the production of prebiotic and anti-inflammatory XOS from agrowaste.