Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase

The gene encoding the family 6 carbohydrate-binding module (CtCBM6A) from Clostridium thermocellum, cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant CtCBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of CtCBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (K _a of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (K _a of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, CtCBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant K _a of 230 M^?1 and binding capacity (N ₀) of 11 μmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58°C in the presence of Ca²⁺, indicating that Ca²⁺ imparts thermal stability to the CtCBM6A structure. Homology modeling of CtCBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of CtCBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that CtCBM6A has a stable conformation.     

Mannan specific family 35 carbohydrate-binding module (CtCBM35) of Clostridium thermocellum: structure analysis and ligand binding

The three-dimensional model of the CtCBM35 (Cthe 2811), i.e. the family 35 carbohydrate binding module (CBM) from the Clostridium thermocellum family 26 glycoside hydrolase (GH) β-mannanase, generated by Modeller9v8 displayed predominance of β-sheets arranged as β-sandwich fold. Multiple sequence alignment of CtCBM35 with other CBM35s showed a conserved signature sequence motif Trp-Gly-Tyr, which is probably a specific determinant for mannan binding. Cloned CtCBM35 from Clostridium thermocellum ATCC 27405 was a homogenous, soluble 16 kDa protein. Ligand binding analysis of CtCBM35 by affinity electrophoresis displayed higher binding affinity against konjac glucomannan (K _a = 2.5 × 10⁵ M^?1) than carob galactomannan (K _a = 1.4 × 10⁵ M^?1). The presence of Ca²⁺ ions imparted slightly higher binding affinity of CtCBM35 against carob galactomannan and konjac glucomannan than without Ca²⁺ ion additive. However, CtCBM35 exhibited a low ligand-binding affinity K _a = 2.5 × 10^?5 M^?1 with insoluble ivory nut mannan. Ligand binding study by fluorescence spectroscopy showed K _a against konjac glucomannan and carob galactomannan, 2.4 × 10⁵ M^?1 and 1.44 × 10⁵ M^?1, and ΔG of binding ?27.0 and ?25.0 kJ/mol, respectively, substantiating the findings of affinity electrophoresis. Ca²⁺ ions escalated the thermostability of CtCBM35 and its melting temperature was shifted to 70°C from initial 55°C. Therefore thermostable CtCBM35 targets more β-(1,4)-manno-configured ligands from plant cell wall hemicellulosic reservoir. Thus a non-catalytic CtCBM35 of multienzyme cellulosomal enzymes may gain interest in the biofuel and food industry in the form of released sugars by targeting plant cell wall polysaccharides.     

Structure and functional investigation of ligand binding by a family 35 carbohydrate binding module (CtCBM35) of β-mannanase of family 26 glycoside hydrolase from Clostridium thermocellum

Functional attributes of recombinant CtCBM35 (family 35 carbohydrate binding module) of β-mannanase of family 26 Glycoside Hydrolase from Clostridium thermocellum were deduced by biochemical and in silico approaches. Ligand-binding analysis of expressed CtCBM35 analyzed by affinity-gel electrophoresis and fluorescence spectroscopy exhibited association constants K _a ～ 1.2·10⁵ and 3.0·10⁵ M^?1 with locust bean galactomannan and mannotriose, respectively. However, CtCBM35 showed low ligand-binding affinity with insoluble ivory nut mannan with K _a of 5.0·10^?5 M^?1. Unfolding transition analysis by fluorescence spectroscopy explained the conformational changes of CtCBM35 in the presence of guanidine hydrochloride (5 M) and urea (6.25 M). This explained that CtCBM35 has good conformational stability and requires higher free energy of denaturation to invoke unfolding. The three-dimensional (3-D) model of CtCBM35 from C. thermocellum generated by Modeller9v8 displayed predominance of β-sheets arranged as β-jelly-roll fold. The secondary structure of CtCBM35 by PredictProtein showed the presence of two α-helices (3%), 12 β-sheets (45%), and 15 random coils (52%). Secondary structural element analysis of cloned, expressed, and purified recombinant CtCBM35 by circular dichroism also corroborated the in silico predicted secondary structure. Multiple sequence alignment of CtCBM35 showed conserved residues (Tyr123, Gly124, and Phe125), which are commonly observed in mannan specific CBMs. Docking analysis of CtCBM35 with manno-oligosaccharide displayed the involvement of Tyr26, Gln29, Asn43, Trp66, Tyr68, Leu69, Arg76, and Leu127 residues, making polar contact with the ligand molecules. Ligand docking analysis of CtCBM35 exhibiting higher binding affinity with mannotriose and galactomannan (Man-Gal-Man moiety) substantiated the affinity binding and fluorescence results, displaying similar values of K _a.     

Fusion of a Xylan-Binding Module to Gluco-Oligosaccharide Oxidase Increases Activity and Promotes Stable Immobilization

The xylan-binding module Clostridium thermocellum CBM22A was successfully fused to a gluco-oligosaccharide oxidase, GOOX-VN, from Sarocladium strictum via a short TP linker, allowing the fused protein to effectively bind different xylans. The presence of the CtCBM22A at the N-terminal of GOOX-VN increased catalytic activity on mono- and oligo-saccharides by 2-3 fold while not affecting binding affinity to these substrates. Notably, both GOOX-VN and its CBM fusion also showed oxidation of xylo-oligosaccharides with degrees of polymerization greater than six. Whereas fusion to CtCBM22A did not alter the thermostability of GOOX-VN or reduce substrate inhibition, CtCBM22A_GOOX-VN could be immobilized to insoluble oat spelt xylan while retaining wild-type activity. QCM-D analysis showed that the fused enzyme remained bound during oxidation. These features could be harnessed to generate hemicellulose-based biosensors that detect and quantify the presence of different oligosaccharides.     

Purification of the alkaliphilic xylanases from Myceliophthora sp. IMI 387099 using cellulose-binding domain as an affinity tag

Ten xylanase isoforms produced by Myceliophthora sp. were characterized for their ability to bind to avicel. Three of the xylanases showing differential affinity for avicel were purified by column chromatography. The purified xylanase Xyl IIa, IIb and IIc showed molecular mass of 47, 41 and 30 kDa and pI of ∼3.5, 4.8 and 5.2, respectively. Xyl IIa was optimally active at pH 8.0 and temperature 70 °C, while Xyl IIb and IIc were optimally active at pH 9.0 and 60 °C and 7.0 and 80 °C, respectively. Xyl IIa and Xyl IIb showed higher stability under alkaline conditions (pH 9.0) and retained 80% of the original activity upto 1 h and 3 h respectively, at 50 °C. All three purified iso-xylanases showed enhanced activities in presence of Na⁺, Mg²⁺, Mn²⁺ and K⁺ ions, whereas, Zn²⁺ and Cu²⁺ showed negative effect on Xyl IIa. The activity of Xyl IIa increased in presence of reducing agents DTT and mercaptoethanol, however, SDS showed inhibitory effect. Kinetic studies showed that Xyl IIb and IIc degrade rye arabinoxylan, much more efficiently than oat spelt xylan, whereas, Xyl IIa showed much higher Kcat/Km value for birch wood xylan as compared to oat spelt xylan. The purified xylanases were apparently classified in family 10.     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.     

Purification and characterization of a high molecular weight endoxylanase from the solid-state culture of an alkali-tolerant Aspergillus fumigatus MKU1

Summary A high molecular weight endoxylanase (XylF2) from the solid state culture of Aspergillus fumigatus MKU1 was purified to homogeneity by a combination of tube gel electrophoresis and electroelution methods. The purity was demonstrated by SDS-PAGE and the molecular mass of the XylF2 was found to be 66 kDa. The optimal pH and temperature for activity were 5.0 and 90 °C, respectively. The apparent K _m and V _max values of XylF2 with oat spelt xylan as substrate were 1.6 mg/ml and 3.25 mmol/min/mg protein respectively. The enzyme showed high activity towards oat spelt xylan while negligible activity was observed on carboxymethylcellulose. The activity of XylF2 was strongly inhibited by Hg²⁺, Ni²⁺, Zn²⁺, SDS and N-bromosuccinimide and stimulated by l-cysteine and iodoacetamide. The hydrolysis of oat spelt xylan by XylF2 released only xylo-oligosaccharides.     

Purification and characterization of a high-thermostable β-xylanase from newly isolated Thermomyces lanuginosus THKU-49

Highly thermostable β-xylanase produced by newly isolated Thermomyces lanuginosus THKU-49 strain was purified in a four-step procedure involving ammonium sulfate precipitation and subsequent separation on a DEAE-Sepharose fast flow column, hydroxylapatite column, and Sephadex G-100 column, respectively. The enzyme purified to homogeneity had a specific activity of 552 U/mg protein and a molecular weight of 24.9 kDa. The optimal temperature of the purified xylanase was 70°C, and it was stable at temperatures up to 60°C at pH 6.0; the optimal pH was 5.0–7.0, and it was stable in the pH range 3.5–8.0 at 4°C. Xylanase activity was inhibited by Mn²⁺, Sn²⁺, and ethylenediaminetetraacetic acid. The xylanase showed a high activity towards soluble oat spelt xylan, but it exhibited low activity towards insoluble oat spelt xylan; no activity was found to carboxymethylcellulose, avicel, filter paper, locust bean gum, cassava starch, and p-nitrophenyl β-d-xylopyranoside. The apparent K _m value of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was 7.3 ± 0.236 and 60.2 ± 6.788 mg/ml, respectively. Thin-layer chromatography analysis showed that the xylanase hydrolyzed oat spelt xylan to yield mainly xylobiose and xylose as end products, but that it could not release xylose from the substrate xylobiose, suggesting that it is an endo-xylanase.     

Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module

The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE–CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE–CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.     

Effect of Carbon Source on Production of α-L-Arabinofuranosidase by Aureobasidium pullulans

A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced α-L-arabinofuranosidase (α-L-AFase) when grown in liquid culture on sugar beet arabinan, wheat arabinoxylan, L-arabinose, L-arabitol, xylose, xylitol, oat spelt xylan, corn fiber, or arabinogalactan. L-Arabinose was most effective for production of both whole-broth and extracellular α-L-AFase activity, followed by L-arabitol. Oat spelt xylan, sugar beet arabinan, xylose, xylitol, and wheat arabinoxylan were intermediate in their ability to support α-L-AFase production. Lower amounts of enzyme activity were detected in corn fiber- and arabinogalactan-grown cultures. Received: 16 April 1998 / Accepted: 17 June 1998     

Production and initial characterisation of the xylan-degrading system of Phanerochaete chrysosporium

We report the optimum conditions for the degradation of oat spelt arabinoxylan and a preliminary characterisation of the inducible xylan-degrading system of the lignin-degrading white-rot fungus Phanerochaete chrysosporium. Xylanase activity was optimal at pH 5.0 and 50°C; see attached sheet the maximum reaction velocity (V_max) of the system was 3.86 units (U) mg^–1 protein with arabinoxylan as substrate and the substrate concentration giving half V_max (S_0.5) was 0.52 mg ml^–1. At concentrations of arabinoxylan greater than 15 mg ml^–1 excess substrate inhibition was observed. Xylose at 0.9 mm inhibited activity to the extent of 50%. Xylanase activity increased as a function of the dilution of the enzyme preparation prior to assay. It was resolved into four peaks by using a DEAE-Biogel column; the material in these peaks differed with respect to xylan solubilisation and the formation of reducing sugars. Electrofocusing gels allowed visualisation of several bands of activity corresponding to each peak. The arabinoxylan degradation system of P. chrysosporium is therefore composed of multiple components. Correspondence to: P. Broda     

Activity and thermostability increase of xylanase following transplantation with modules sub-divided from hyper-thermophilic CBM9_1-2

Transplantation is useful for elucidating the functions of structural modules and for engineering enzyme properties. Unexpectedly, transplanting a hyper-thermophilic carbohydrate-binding module, CBM9_1-2, into the mesophilic Aspergillus niger GH11 xylanase (Xyn) slightly decreased the thermal inactivation half-life of Xyn. This effect was further investigated by dividing the CBM9_1-2 module into two smaller parts, C1 and C2, which were transplanted into Xyn to create the chimeras Xyn-C1 and Xyn-C2. Both chimeras exhibited higher catalytic activities on xylan than native Xyn. Xyn-C2 exhibited higher binding affinities for both oat spelt and birch wood xylans, and its thermal inactivation half-life (69.3 min) was 4 or 5 times longer than that of Xyn (17.6 min), Xyn-C1 (13.4 min), and the original chimera containing CBM9_1-2 (13.8 min). In contrast, Xyn-C1 exhibited higher binding affinity for oat spelt xylan, but not for birch wood xylan. Through this rational engineering of the fungal xylanase, the C2 sub-module was shown to have a different thermostabilizing effect than the C1 sub-module. The different functions of the smaller parts of a large module can play pivotal roles in transplantation.     

Purification and Characterization of a Low-Molecular-Weight Xylanase Produced by Acrophialophora nainiana

A low-molecular-weight xylanase activity (XynI) was isolated from the fungus Acrophialophora nainiana after growth in a solid medium containing wheat bran. XynI was purified to apparent homogeneity by ultrafiltration and gel filtration chromatography. The purified enzyme had a molecular weight value of approx. 17 kDa, as determined by SDS-PAGE. This enzyme was most active at 50°C and pH 6.0. At 50°C the half-life was 150 min. The apparent K _m value for birchwood xylan was much lower than the K _m value for oat spelt xylan. XynI was activated by L-cysteine, DTE, β-mercaptoethanol, and L-tryptophan. XynI did not show significant sequence homology with other xylanases. The analysis of hydrolysis products of xylans and wood pulps showed that XynI was able to release xylooligomers ranging from X2 to X3 and X2 to X6, respectively. The enzyme was not active against acetylated xylan. A small amount of xylose was released from deacetylated, birchwood, and oat spelt xylans. The results obtained with enzymatic treatment of Kraft pulp indicated a reduction in the amount of chlorine compounds required for the process and enhanced brightness gain. Received: 6 May 1998 / Accepted: 29 July 1998     

Characterization of a family 54 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

A glycosyl hydrolase family 54 (GH54) α-l-arabinofuranosidase gene (abfA) of Aureobasidium pullulans was amplified by polymerase chain reaction from genomic DNA and a 498-amino-acid open reading frame deduced from the DNA sequence. Modeling of the highly conserved A. pullulans AbfA protein sequence on the crystal structure of Aspergillus kawachii AkabfB showed that the catalytic amino acid arrangement and overall structure were highly similar including the N-terminal catalytic and C-terminal arabinose binding domains. The abfA gene was expressed in Saccharomyces cerevisiae, and the heterologous enzyme was purified. The protein was monomeric, migrating at 49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and eluting at 36 kDa upon gel filtration. AbfA showed maximal activity at 55°C and between pH 3.5 and pH 4. The enzyme had a K _m value for p-nitrophenyl-α-l-arabinofuranoside of 3.7 mM and a V _max of 34.8 μmol min⁻¹ mg protein⁻¹. Arabinose acted as a noncompetitive inhibitor with a K _i of 38.4 mM. The enzyme released arabinose from maize fiber, oat spelt arabinoxylan, and wheat arabinoxylan, but not from larch wood arabinogalactan or α-1,5-debranched arabinan. AbfA displayed low activity against α-1,5-l-arabino-oligosaccharides. The enzyme acted synergistically with endo-β-1,4-xylanase in the breakdown of wheat arabinoxylan. Binding of AbfA to xylan from several sources confirmed the presence of a functional carbohydrate-binding module. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and characterization of a novel xylanase derived from a rice straw degrading enrichment culture

A metagenomic library containing ca. 3.06 × 10⁸ bp insert DNA was constructed from a rice straw degrading enrichment culture. A xylanase gene, umxyn10A, was cloned by screening the library for xylanase activity. The encoded enzyme Umxyn10A showed 58% identity and 73% similarity with a xylanase from Thermobifida fusca YX. Sequence analyses showed that Umxyn10A contained a glycosyl hydrolase family 10 catalytic domain. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically. Recombinant Umxyn10A was highly active toward xylan. However, the purified enzyme could slightly hydrolyze β-1,3/4-glucan and β-1,3/6-glucan. Umxyn10A displayed maximal activity toward oat spelt xylan at a high temperature (75°C) and weak acidity (pH 6.5). The K _m and V _max of Umxyn10A toward oat spelt xylan were 3.2 mg ml⁻¹ and 0.22 mmol min⁻¹ mg⁻¹ and were 2.7 mg ml⁻¹ and 1.0 mmol min⁻¹ mg⁻¹ against birchwood xylan, respectively. Metal ions did not appear to be required for the catalytic activity of this enzyme. The enzyme Umxyn10A could efficiently hydrolyze birchwood xylan to release xylobiose as the major product and a negligible amount of xylose. The xylanase identified in this work may have potential application in producing xylobiose from xylan.     

Comparative characterization of deletion derivatives of the modular xylanase XynA of Thermotoga maritima

The modular Xylanase XynA from Thermotoga maritima consists of five domains (A1-A2-B-C1-C2). Two similar N-terminal domains (A1-A2-) are family 22 carbohydrate-binding modules (CBMs), followed by the catalytic domain (-B-) belonging to glycoside hydrolase family 10, and the C-terminal domains (-C1-C2), which are members of family 9 of CBMs. The gradual deletion of the non-catalytic domains resulted in deletion derivatives (XynAΔC; XynAΔA1C and XynAΔNC) with increased maximum activities (V _max) at 75°C, pH 6.2. Furthermore, these deletions led to a shift of the optimal NaCl concentration for xylan hydrolysis from 0.25 (XynA) to 0.5 M (XynAΔNC). In the presence of the family 22 CBMs, the catalytic domain retained more activity in the acidic range of the pH spectrum than without these domains. In addition to the deletion derivatives of XynA, the N-terminal domains A1 and A2 were produced recombinantly, purified, and investigated in binding studies. For soluble xylan preparations, linear β-1,4-glucans and mixed-linkage β-1,3-1,4-glucans, only the A2 domain mediated binding, not the A1 domain, in accordance with previous observations. The XynA deletion enzymes lacking the C domains displayed low affinity also to hydroxyethylcellulose and carboxymethylcellulose. With insoluble oat spelt xylan and birchwood xylan as the binding substrates, the highest affinity was observed with XynAΔC and the lowest affinity with XynAΔNC. Although the domain A1 did not bind to soluble xylan preparations, the insoluble oat spelt xylan-binding data suggest that this domain does play a role in substrate binding in that it improves the binding to insoluble xylans.     

Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities

Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as α, β and γ. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the γ sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of β and γ sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities.     

Constructing arabinofuranosidases for dual arabinoxylan debranching activity

Enzymatic conversion of arabinoxylan requires α‐L‐arabinofuranosidases able to remove α‐L‐arabinofuranosyl residues (α‐L‐Araf) from both mono‐ and double‐substituted D‐xylopyranosyl residues (Xylp) in xylan (i.e., AXH‐m and AXH‐d activity). Herein, SthAbf62A (a family GH62 α‐L‐arabinofuranosidase with AXH‐m activity) and BadAbf43A (a family GH43 α‐L‐arabinofuranosidase with AXH‐d3 activity), were fused to create SthAbf62A_BadAbf43A and BadAbf43A_SthAbf62A. Both fusion enzymes displayed dual AXH‐m,d and synergistic activity toward native, highly branched wheat arabinoxylan (WAX). When using a customized arabinoxylan substrate comprising mainly α‐(1 → 3)‐L‐Araf and α‐(1 → 2)‐L‐Araf substituents attached to disubstituted Xylp (d‐2,3‐WAX), the specific activity of the fusion enzymes was twice that of enzymes added as separate proteins. Moreover, the SthAbf62A_BadAbf43A fusion removed 83% of all α‐L‐Araf from WAX after a 20 hr treatment. ¹H NMR analyses further revealed differences in SthAbf62A_BadAbf43 rate of removal of specific α‐L‐Araf substituents from WAX, where 9.4 times higher activity was observed toward d‐α‐(1 → 3)‐L‐Araf compared to m‐α‐(1 → 3)‐L‐Araf positions.     

Cloning,purification and characterization of an alkali-stable endoxylanase from thermophilic <Emphasis Type="Italic">Geobacillus</Emphasis> sp. 71

The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially inhibited by Al³⁺ and Cu²⁺ but strongly inhibited by Hg²⁺. The enzyme follows Michaelis–Menten kinetics, with K_m and V_max values of 0.425 mg xylan/ml and 500 μmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose.