Birchwood Xylan Research Articles

Synergistic hydrolysis of xylan using novel xylanases, β-xylosidases, and an α-L-arabinofuranosidase from Geobacillus thermodenitrificans NG80-2.

Lignocellulosic biomass from various types of wood has become a renewable resource for production of biofuels and biobased chemicals. Because xylan is the major component of wood hemicelluloses, highly efficient enzymes to enhance xylan hydrolysis can improve the use of lignocellulosic biomass. In this study, a xylanolytic gene cluster was identified from the crude oil-degrading thermophilic strain Geobacillus thermodenitrificans NG80-2. The enzymes involved in xylan hydrolysis, which include two xylanases (XynA1, XynA2), three β-xylosidases (XynB1, XynB2, XynB3), and one α-L-arabinofuranosidase (AbfA), have many unique features, such as high pH tolerance, high thermostability, and a broad substrate range. The three β-xylosidases were highly resistant to inhibition by product (xylose) accumulation. Moreover, the combination of xylanase, β-xylosidase, and α-L-arabinofuranosidase exhibited the largest synergistic action on xylan degradation (XynA2, XynB1, and AbfA on oat spelt or beechwood xylan; XynA2, XynB3, and AbfA on birchwood xylan). We have demonstrated that the proposed enzymatic cocktail almost completely converts complex xylan to xylose and arabinofuranose and has great potential for use in the conversion of plant biomass into biofuels and biochemicals.

Biochemical characterization of the xylan hydrolysis profile of the extracellular endo-xylanase from Geobacillus thermodenitrificans T12

BackgroundEndo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates. Hemicellulose degradation by Geobacillus spp. is facilitated by the hemicellulose utilization (HUS) locus that is present in most strains belonging to this genus. As part of the HUS locus, the xynA gene encoding an extracellular endo-xylanase is one of the few secreted enzymes and considered to be the key enzyme to initiate hemicellulose degradation. Several Geobacillus endo-xylanases have been characterized for their optimum temperature, optimum pH and generation of degradation products. However, these analyses provide limited details on the mode of action of the enzymes towards various substrates resulting in a lack of understanding about their hydrolytic potential.ResultsA HUS-locus associated gene (GtxynA1) from the thermophile Geobacillus thermodenitrificans T12 encodes an extracellular endo-xylanase that belongs to the family 10 glycoside hydrolases (GH10). The GtxynA1 gene was cloned and expressed in Escherichia coli. The resulting endo-xylanase (termed GtXynA1) was purified to homogeneity and showed activity between 40 °C and 80 °C, with an optimum activity at 60 °C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60 °C. Highest activity was towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 is able to degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endo-xylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides.ConclusionsThe G. thermodenitrificans T12 endo-xylanase, GtXynA1, shows temperature tolerance up to 80 °C and high activity to a variety of xylans. The mode of action of GtXynA1 reveals that arabinose substituents do not hamper substrate degradation by GtXynA1. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates.

Prebiotic effect of xylooligosaccharides produced from birchwood xylan by a novel fungal GH11 xylanase

A fungal endoxylanase belonging to the glycoside hydrolase gene family 11 (GH11) was obtained from the ascomycete Talaromyces amestolkiae. The enzyme was purified, characterized and used to produce a mixture of xylooligosaccharides (XOS) from birchwood xylan. A notable yield of neutral XOS was obtained (28.8%) upon enzyme treatment and the mixture contained a negligible amount of xylose, having xylobiose, xylotriose and xylotetraose as its main components. The prebiotic potential of this mixture was demonstrated upon analyzing the variations in microorganisms’ composition and organic acids profile in breast-fed child faeces fermentations. The strong production of acetic and lactic acid, the decrease of potentially pathogenic bacteria and the increase of bifidobacteria, and possible beneficial commensals, confirmed the prebiotic value of these xylooligosaccharides.

Recombinant Thermostable Thermomonospora fusca TF Endo-xylanase A and Its Immobilization on Modified Mesoporous SiO2 Microspheres for Manufacturing Xylooligosaccharides

tfxa (GenBank: U01242), which encoded the mature peptide of Thermomonospora fusca TF xylanase A (TfxA), was high-level expressed in Escherichia coli BL21 (DE3). The recombinant xylanase, reETfxA, was both secreted into culture medium and in the cytoplasm. reETfxA showed high xylanase activity and was purified to homogeneity by Ni-affinity resin (182.8 and 681.4 U mg−1 for crude and purified enzyme, respectively). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blot analysis revealed that the molecular mass of reETfxA was approximately 42.7 kDa. The reETfxA was immobilized on a novel mesoporous SiO2 microsphere (MSM)-coated chitosan via covalent bonds, which formed from Schiff base reaction between supports and enzyme. The binding capacity of the prepared MSM-coated chitosan particles to reETfxA was approximately 272.6 mg g−1-particles. The optimum temperature values of the free and immobilized reETfxA were 65 and 70 °C, respectively. Moreover, the optimum pH values of the free and immobilized reETfxA were pH 6.0 and 5.0, respectively. The reETfxA showed relatively high thermostability. When treated at 70 °C and pH 6.0 for 15 min, the residual activities of free and immobilized reETfxA were 84.2 and 100.2%, respectively. The results obtained from the HPLC analysis showed that immobilized reETfxA released xylooligosaccharides from oat spelt, beechwood and birchwood xylans, with xylotetraose, xylotriose, and xylopentaose as the major products, respectively. Additionally, the immobilized reETfxA could directly hydrolyze the wheat bran insoluble xylan.

Agrowastes as Feedstock for the Production of Endo-β-Xylanase from Cohnella sp. Strain AR92

Members of Cohnella sp. isolated from a variety of environments have been shown to be glycoside hydrolase producers. Nevertheless, most evaluations of members of this genus are limited to their taxonomic description. The strain AR92, previously identified as belonging to the genus Cohnella, formed a well-supported cluster with C. thailandensis and C. formosensis (>80% bootstrap confidence). Its growth and xylanase production were approached by using a mineral-based medium containing alkali-pretreated sugarcane bagasse as the main carbon source, which was assayed as a convenient source to produce biocatalysts potentially fitting its degradation. By means of a two-step statistical approach, the production of endoxylanase was moderately improved (20%). However, a far more significant improvement was observed (145%), by increasing the inoculum size and lowering the fermentation temperature to 25°C, which is below the optimal growth temperature of the strain AR92 (37°C). The xylanolytic preparation produced by Cohnella sp. AR92 contained mild temperature-active endoxylanase (identified as redundant GH10 family) for the main activity which resulted in xylobiose and xylo-oligosaccharides as the main products from birchwood xylan.

Improving special hydrolysis characterization into Talaromyces thermophilus F1208 xylanase by engineering of N-terminal extension and site-directed mutagenesis in C-terminal

The purpose of this study was to gain insights into the hydrolysis characteristics of xylanase in producing xylo-oligosaccharides and to improve these by protein engineering. In this study, a hybrid enzyme (T-XynFM) was created from Talaromyces thermophilus F1208 xylanase (T-Xyn) by replacement of the N-terminal extension Phe1-Pro16 with Ala1-Gln8 and substitution of amino acid Phe193 (185) with Ser in the C-terminal. The enzyme showed optimum activity at 55°C and pH 6.5. Its residual activity was more than 55% after treatment at 50°C, pH 6.5 for 12h Km values of T-XynFM for beechwood xylan, birchwood xylan, and oat-spelt xylan were 10.31, 10.03, and 8.90mgmL−1, respectively. The enzyme displayed special hydrolysis characteristics: almost no xylose was produced on using xylotriose (X3), xylotetraose (X4), and xylopentaose as substrates. Moreover, over 60% X4 existed in hydrolyzed products of X3, indicating that T-XynFM possesses formidable transglycosylation properties.

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1,4-xylanase with exo- and endo-activity.

A hyperthermophilic and thermostable xylanase of 82kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85°C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1days at 65°C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

Characterization of a neutral recombinant xylanase from Thermoactinospora rubra YIM 77501T.

A xylanase gene (TrXyn10) from Thermoactinospora rubra YIM 77501T was cloned and expressed in Escherichia coli. The amino acid sequence displayed 78% homology with Microbispora mesophila xylanase (WP_062413927.1). The recombinant xylanase (TrXyn10), with MW 46.1kDa, could hydrolyse beechwood, birchwood and oatspelt xylan. Based on the sequence, enzymatic properties and tertiary structure of the protein, TrXyn10 belongs to glycoside hydrolase family 10 (GH10). The optimal pH and temperature for the recombinant enzyme were determined to be 7.0 and 55°C, respectively. TrXyn10 was stable over a wide pH range, and it retained more than 45% of the total activity at pH 6.0-12.0 for 12h. In addition, the activity was greatly promoted, by approximately 200% of the initial activity, after incubation at pH 6.0 and 7.0 for 12h. Based on enzymatic properties and product analysis, we showed that TrXyn10 is a neutral endoxylanase.

Optimization of Cellulase and Xylanase Production by Micrococcus Species under Submerged Fermentation

This paper reports on the optimization of culture conditions for cellulase and xylanase production by bacterial isolate from lignocellulosic biomass. The bacterial isolate was screened for cellulase and xylanase production on carboxyl methyl cellulose (CMC) and birch wood xylan as substrates, respectively. One bacterial isolate showing the highest halo zone diameter (isolate PLY1) was selected for detailed studies. The analysis of the 16S ribosomal ribonucleic acid (rRNA) gene nucleotide sequence of PLY1 revealed it to have 98% similarity to Micrococcus luteus strain Fse9 and the sequence was deposited in the GenBank as Micrococcus luteus strain SAMRC-UFH3 with accession number KU171371. Cellulase production was achieved in the presence of CMC (1% w/v) under an incubation temperature of 25 °C (198 U/mL), pH 5 (173 U/mL), agitation speed 50 rpm (173 U/mL) and incubation period of 96 h (102 U/mL). Xylanase was produced maximally when birch wood xylan (1% w/v) was used as the substrate at 25 °C (1007 U/mL), pH 10 (2487 U/mL), 200 rpm (1814 U/mL), and under an incubation period of 84 h (1296 U/mL). Our findings showed that Micrococcus sp. SAMRC-UFH3 appears to be a potentially important candidate for lignocellulosic waste degradation and other relevant industrial applications.

Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, β-d-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities.

The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited β-xylosidase activity toward a chromogenic substrate, p-nitrophenyl-β-d-xylopyranoside, and xylobiose, while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose, α-l-Araf-(1→2)-d-Xylp or α-l-Araf-(1→3)-d-Xylp and α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp Moreover, the combination of PcAxy43A and P. curdlanolyticus B-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for the saccharification of arabinoxylan into simple sugars for many applications. IMPORTANCE In this study, the glycoside hydrolase 43 (GH43) intracellular multifunctional endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase (AXH) from P. curdlanolyticus B-6 were characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)-2 and C(O)-3 positions of xylose residues doubly substituted with arabinosyl, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxy43A and endo-xylanase Xyn10C from strain B-6, providing novel metabolic potential for processing arabinoxylans into xylose and arabinose.

Characterization of a Salt-Tolerant and Cold-Adapted Xylanase from Bacillus cellulosilyticus

A xylanase (Xyn10A) gene from the saline-alkali-tolerant microorganism Bacillus cellulosilyticus DSM 2522 was cloned and expressed in Escherichia coli BL21 (DE3). The open reading frame was composed of 1008 base pairs, and it encoded 335 amino acid residues belonging to glycosyl hydrolase family 10. The optimal temperature and pH of the purified Xyn10A were 40 °C and 8.0, respectively. The Xyn10A was sensitive to heat and showed obvious cold-adapted activity, retaining 38.3%, 55.7%, and 82.9% of the optimal activity at 4, 20, and 30 °C, respectively. Xyn10A also showed a high level of NaCl tolerance. The highest activity was observed with 1.5 M NaCl. The specific enzyme activity of Xyn10A was as much as 163.8 U/mg. Kinetic assays showed that Km, Vmax, and Kcat were 2.56 mg/mL, 202.5 μM/min/mg, and 132.6 /s, respectively. Additionally, the main hydrolysis products using birchwood xylan as substrate were xylobiose, xylotriose, and xylotetraose, as determined by thin layer chromatography analysis. As a cold-adapted and salt-tolerant enzyme, Xyn10A is an ideal candidate for further research and biotechnological applications.

Functional characterization of the native swollenin from Trichoderma reesei: study of its possible role as C1 factor of enzymatic lignocellulose conversion.

BackgroundThrough binding to cellulose, expansin-like proteins are thought to loosen the structural order of crystalline surface material, thus making it more accessible for degradation by hydrolytic enzymes. Swollenin SWO1 is the major expansin-like protein from the fungus Trichoderma reesei. Here, we have performed a detailed characterization of a recombinant native form of SWO1 with respect to its possible auxiliary role in the enzymatic saccharification of lignocellulosic substrates.ResultsThe swo1 gene was overexpressed in T. reesei QM9414 Δxyr1 mutant, featuring downregulated cellulase production, and the protein was purified from culture supernatant. SWO1 was N-glycosylated and its circular dichroism spectrum suggested a folded protein. Adsorption isotherms (25 °C, pH 5.0, 1.0 mg substrate/mL) revealed SWO1 to be 120- and 20-fold more specific for binding to birchwood xylan and kraft lignin, respectively, than for binding to Avicel PH-101. The SWO1 binding capacity on lignin (25 µmol/g) exceeded 12-fold that on Avicel PH-101 (2.1 µmol/g). On xylan, not only the binding capacity (22 µmol/g) but also the affinity of SWO1 (Kd = 0.08 µM) was enhanced compared to Avicel PH-101 (Kd = 0.89 µM). SWO1 caused rapid release of a tiny amount of reducing sugars (<1 % of total) from different substrates (Avicel PH-101, nanocrystalline cellulose, steam-pretreated wheat straw, barley β-glucan, cellotetraose) but did not promote continued saccharification. Atomic force microscopy revealed that amorphous cellulose films were not affected by SWO1. Also with AFM, binding of SWO1 to cellulose nanocrystallites was demonstrated at the single-molecule level, but adsorption did not affect this cellulose. SWO1 exhibited no synergy with T. reesei cellulases in the hydrolysis of the different celluloses. However, SWO1 boosted slightly (1.5-fold) the reducing sugar release from a native grass substrate.ConclusionsSWO1 is a strongly glycosylated protein, which has implications for producing it in heterologous hosts. Although SWO1 binds to crystalline cellulose, its adsorption to xylan is much stronger. SWO1 is not an auxiliary factor of the enzymatic degradation of a variety of cellulosic substrates. Effect of SWO1 on sugar release from intact plant cell walls might be exploitable with certain (e.g., mildly pretreated) lignocellulosic feedstocks.

Bifunctional recombinant cellulase-xylanase (rBhcell-xyl) from the polyextremophilic bacterium Bacillus halodurans TSLV1 and its utility in valorization of renewable agro-residues.

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 910UL-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 3.5h at 80°C for CMCase and xylanase, respectively. The apparent K m values (CMC and Birchwood xylan) are 3.8 and 3.2mgmL-1. The catalytic efficiency (k cat/K m ) values of xylanase and CMCase are 657 and 171mLmg-1min-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.

Purification, characterization, gene cloning and expression of GH-10 xylanase (Penicillium citrinum isolate HZN13).

An extracellular thermostable xylanase (Xyl-IIb) produced by Penicillium citrinum isolate HZN13 was purified to homogeneity using DEAE-Sepharose, Sephadex G-100 and Bio-Gel P-60 chromatography with specific activity of 6272.7 U/mg and 19.6-fold purification. The purification revealed the occurrence of multiple forms of xylanases (Xyl-I, Xyl-IIa, Xyl-IIb and Xyl-III). The molecular mass of highly purified Xyl-IIb was ~31 kDa with SDS-PAGE. The enzyme was cellulase-free, thermostable (55–75 °C) and acidophilic (3.5–5.0). It was activated by Ca2+, Ba2+, DTT and β-mercaptoethanol, whereas inhibited by Hg2+, Pb2+, Ni2+ and p-CMB. Purified Xyl-IIb exhibited highest specificity toward birchwood and oat spelts xylan. Kinetics of Xyl-IIb revealed a K m of 10 mg/ml and 16.7 mg/ml and V max of 9523g and 15,873 U/mg with birchwood and oat spelts xylan, respectively, indicating high affinity toward birchwood xylan. The xylanase (Xyl-IIb) belongs to glycosyl hydrolase (GH) family 10 based on conserved regions. Xylanase-encoding gene (xynB) consists of 1501 bp with an open reading frame of 264 bp which was predicted to encode a protein having 87 amino acids and shared homology with endo-1,4-beta-xylanase (xynB) gene from Penicillium citrinum. Cloned xynB gene was expressed in E. coli BL21 (DE3) with xylanase activity (80 U/mg) and confirmed to be GH-10 Xyl-IIa based on molecular mass (~40 kDa). These properties of xylanase make it promising for their applications in biofuel industries.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0489-4) contains supplementary material, which is available to authorized users.

Purification and characterization of low molecular weight alkaline xylanase from Neosartorya tatenoi KKU-CLB-3-2-4-1

A low molecular weight alkaline xylanase from Neosartorya tatenoi KKU-CLB-3-2-4-1 was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-100 and DEAE-cellulose column chromatography. The xylanase was purified 32.09 fold, with the specific activity of 16.07 U mg−1 protein and a recovery yield of 2.67%. The purified xylanase was estimated by SDS-PAGE with a molecular weight of 20 kDa. The purified xylanase was active at pH 10.0 and retained over 75% of the original activity in pH range 7.0–11.0 after incubation at 4 °C for 24 h. The optimum temperature of the purified xylanase was 45 °C. As for thermo-stability, more than 80% of original xylanase activity was retained after heating at 45 °C for 120 min. Xylanase activity was stimulated by Ag+ and inhibited by Hg2+. The purified xylanase was specific to beechwood xylan, birchwood xylan and oat spelt xylan. The Km and Vmax values for beechwood xylan were 10.34 mg/mL and 45.66 μmol/min/mg, respectively. In addition, the xylanase hydrolyzed beechwood xylan yielded mainly xylotriose and xylotetraose as end products, suggesting it was an endo-xylanase. Thus, its properties may make it attractive for application in the pulp and paper industry.

Characterisation of Klebsiella pneumoniae Xylanase and Increment of Its Activity in Heterologous Expression System

A xylanase DNA sequence with a total length of 642 bp was previously isolated from a xylanolytic Klebsiellapneumoniae. Xylanase gene primers were designed with the addition of BamH1 and EcoR1 restriction enzymesites in order get a full xylanase gene that is in-frame with pSTAG expression vector. The isolated xylanasegene was amplified using the designed primers through PCR, then cloned and expressed in E. coli BL21 (DE3).In-silico characterization showed that the recombinant xylanase has a molecular weight of 23.9 kDa and a pI of9.32. The signal peptide cleavage site for the recombinant xylanase was predicted to be between residues 61and 62. The activity of the crude recombinant xylanase was 2.015 U/mL, which was higher than the crudenative xylanase activity, with maximum at 0.642 U/mL. Staining of the birchwood xylan agar plate with Congored showed a clearing zone around E. coli BL21 (DE3) colonies with recombinant pSTAG plasmid evenwithout being induced with IPTG. This implied leaky expression of the E. coli BL21 (DE3) secretion system,which recognized the signal sequence of the recombinant xylanase, and proceeded to cleave and secreted outthe mature protein into the culture medium. MALDI-TOF analysis of a 20 kDa protein present in the culturemedium confirmed that the recombinant xylanase had been secreted into the culture medium.

Characterization of Heterologously Expressed Acetyl Xylan Esterase1 Isolated from the Anaerobic Rumen Fungus Neocallimastix frontalis PMA02.

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine α-helixes and 12 β-strands. The enzyme expressed in E.coli had the highest activity at 40°C and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at 40°C, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

A high-molecular-weight, alkaline, and thermostable β-1,4-xylanase of a subseafloor Microcella alkaliphila

An endo β-1,4-xylanase (XynE15) from a culture broth of a deep subseafloor microorganism, Microcella alkaliphila JAM-AC0309, was purified to homogeneity. The molecular mass of XynE15 was approximately 150kDa as judged by SDS-PAGE. The optimal pH and temperature for hydrolysis of xylan were pH 8 and 65°C. The enzyme was stable to incubation for 30min at up to 75°C, and the half-life at 50°C was 48h. XynE15 hydrolyzed arabinoxylan, oat spelt xylan, and birchwood xylan well, but not avicel, carboxymethylcellulose, or arabinan. Xylooligosaccharides were hydrolyzed to mainly xylobiose from higher than xylotetraose. The genome sequencing analysis of strain JAM-AC03039 revealed that XynE15 was composed of 1,319 amino acids with one catalytic domain and three carbohydrate-binding domains belonging to glycoside hydrolase (GH) family 10 and carbohydrate-binding module (CBM) family 4, respectively.

Statistical optimization of thermo-alkali stable xylanase production from Bacillus tequilensis strain ARMATI

Background: Xylanase from bacteria finds use in prebleaching process and bioconversion of lignocelluloses into feedstocks. The xylanolytic enzyme brings about the hydrolysis of complex biomolecules into simple monomer units. This study aims to optimize the cellulase-free xylanase production and cell biomass of Bacillus tequilensis strain ARMATI using response surface methodology (RSM). Results: Statistical screening of medium constituents and the physical factors affecting xylanase and biomass yield of the isolate were optimized by RSM using central composite design at N = 30, namely 30 experimental runs with 4 independent variables. The central composite design showed 3.7 fold and 1.5 fold increased xylanase production and biomass yield of the isolate respectively compared to ‘one factor at a time approach', in the presence of the basal medium containing birchwood xylan (1.5% w/v) and yeast extract (1% w/v), incubated at 40oC for 24 h. Analysis of variance (ANOVA) revealed high coefficient of determination (R2) of 0.9978 and 0.9906 for the respective responses at significant level (p Conclusions: The cellulase-free xylanase showed an alkali-tolerant and thermo-stable property with potentially applicable nature at industrial scale. This statistical approach established a major contribution in enzyme production from the isolate by optimizing independent factors and represents a first reference on the enhanced production of thermo-alkali stable cellulase-free xylanase from B. tequilensis.

An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67U/mg; k cat=178/s, K m=4.90mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley β-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70% activity towards WAX with K i of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.