Identification and characterization of a novel xylanase-AMB

BIOTECHNOLOGICALLY RELEV ANT ENZYMES AND PROTEINSIdentification and characterization of a novel xylanase derived from a rice straw degrading enrichment cultureXin-chun Mo &Chun-lan Chen &Hao Pang &Yi Feng &Jia-xun FengReceived:4January 2010/Revised:2June 2010/Accepted:3June 2010/Published online:22June 2010#Springer-Verlag 2010Abstract A metagenomic library containing ca.3.06×108bp insert DNA was constructed from a rice straw degrading enrichment culture.A xylanase gene,umxyn 10A,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 10catalytic 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 mand V max of Umxyn10A toward oat spelt xylan were 3.2mg ml −1and 0.22mmol min −1mg −1and were 2.7mg ml −1and 1.0mmol min −1mg −1against 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.Keywords Rice straw degrading enrichment culture .Metagenomic library .Xylanase .Cloning .CharacterizationIntroductionXylan is the major hemicellulosic component in lignocel-lulose.It is a complex polysaccharide composed of β-1,4-linked D -xylopyranoside residues modified with a variety of substituents,such as acetyl,arabinosyl,and uronyl side chains.Xylan can be completely biodegraded to xylose by the synergistic action of a series of xylanases,including endo-β-1,4-xylanase (EC 3.2.1.8),β-xylosidase (EC 3.2.1.37),and several accessory enzymes,such as α-L -arabinofuranosidase (EC 3.2.1.55),α-glucuronidase (EC 3.2.1.139),acetylxylan esterase (EC 3.1.1.72),ferulic acid esterase (EC 3.1.1.73),and ρ-coumaric acid esterase (EC 3.1.1.–)(Saha 2003).Xylanase is attracting increasing attention for its potential applications in a number of biotechnological processes,such as bread-making,gluten –starch separation,improving nutritional properties of animal feed,bleaching of cellulose pulp in paper manufac-turing,and pretreatment of lignocellulose in the utilization of biomass (Belien et al.2006).Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2712-2)contains supplementary material,which is available to authorized users.X.-c.Mo :C.-l.Chen :H.Pang :Y .Feng :J.-x.FengGuangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization,Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering,College of Life Science and Technology,Guangxi University,100Daxue Road,Nanning,Guangxi 530004,People ’s Republic of China H.Pang :J.-x.FengCellulose Processing Laboratory,National Engineering Research Center for Non-food Biorefinery,and Guangxi Key Laboratory of Bioindustry Technology,Guangxi Academy of Sciences,98Daling Road,Nanning,Guangxi 530003,People ’s Republic of China J.-x.Feng (*)College of Life Science and Technology,Guangxi University,100Daxue Road,Nanning,Guangxi 530004,People ’s Republic of China e-mail:jiaxunfeng@Appl Microbiol Biotechnol (2010)87:2137–2146DOI 10.1007/s00253-010-2712-2Endo-β-1,4-xylanases(hereafter termed endoxylanases) play a key role in the degradation of xylan by attacking the internalβ-1,4-glycosidic bonds within the polymer back-bone and catalyzing the initial breakdown of xylan (Shallom and Shoham2003).Xylanases mainly belong to the glycoside hydrolase family10(GH10)and family11 (GH11)(Henrissat1991;Collins et al.2005).GH10 endoxylanases typically have a high molecular weight (≥30kDa)and a low p I(Subramaniyan and Prema2002), whereas GH11endoxylanases usually have a low molecular mass(typically around22kDa)and a high p I(Torronen and Rouvinen1997).Endoxylanases have been identified from a broad range of organisms,including microbes,plants,and animals(Sunna and Antranikian1997;Ahmed et al.2009). Some xylanases isolated from cultivated microorganisms exhibit high-performance activity and thermophilic stability (Kulkarni et al.1999;Polizeli et al.2005).In addition,some xylanase genes have been identified from a variety of environmental samples by polymerase chain reaction (PCR)-based cloning.However,it is difficult to find novel diverse xylanase genes by this approach.Only a few xylanases have been cloned from a metagenomic library of uncultured microorganisms in environmental samples.For example,a xylanase(XynH)was isolated from a soil-derived metagenomic library showing high activity against xylan under optimal conditions of pH7.8and40°C,respectively(Hu et al.2008).Nevertheless,few reports have focused on the pre-enrichment of xylolytic environmental samples,which can expeditiously increase the population of xylan-degrading microbes and facilitate the cloning of the target genes.In this study,we describe the cloning and identification of a xylanase gene(umxyn10A)from a rice straw degrading enrichment culture by a metagenomic approach and biochemical characterization of the translated product. Materials and methodsConstruction and screening of the metagenomic DNA library of the rice straw degrading enrichment cultureSoil samples were collected from different sites around Nanning City,China.To construct a rice straw degrading enrichment culture,a mixture of different soil samples in a basal medium(Widdel and Bak1992)with1%(w/v)rice straw powder as a carbon source was cultivated at60°C. When degradation of the rice straw was observed,the culture was differentially centrifuged to harvest the micro-bial cells,and the cell pellet was collected for metagenomic DNA isolation according to the method of Zhou et al. (1996)and modifications of Feng et al.(2007).Crude DNA was purified by affinity chromatography using a Sephadex G200column and polyvinylpolypyrrolidone(PVPP)as previously described(Kuske et al.1998).The metagenomic library was constructed in E.coli strain EPI100using the pWEB::TNC Cosmid Cloning Kit(Epicentre)according to the manufacturer’s instructions.Library colonies were replica plated into Luria–Bertani (LB)agar plates containing0.5%(w/v)oat spelt xylan (Sigma)(Gibbs et al.1995)or0.04%(w/v)4-methylumbelliferyl-β-D-cellobioside(4-MUC,Sigma) (Reinhold-Hurek et al.1993)to screen for enzyme activity.Sequence analyses of the xylanase gene umxyn10AThe active clone,GXN3621,harbored25.3-kb insert DNA and showed a clear hydrolyzing zone on the plate containing0.5%oat spelt xylan as substrate and also exhibited activity towards4-MUC.In order to localize the gene(s)responsible for the enzyme activity,this clone was analyzed by subcloning.Gene activity was first localized in a5.5-kb Bam HI DNA fragment and further localized in a 3.5-kb Pst I DNA fragment.The3.5-kb DNA fragment was sequenced.Putative open reading frames(ORFs)were identified with the National Center for Biotechnology Information(NCBI)ORF finder(http://www.ncbi.nlm.nih. gov).The sequence of the cloned xylanase gene,named umxyn10A,was analyzed by BlastN,BlastX,and BlastP searches on the NCBI website.Phylogenetic analysis, predicted structurally conserved regions(PSCR)analysis (Depiereux and Feytmans1992),and homology modeling of Umxyn10A are described in the“Electronic supplemen-tary material”.Expression of umxyn10A and purificationof the recombinant Umxyn10AThe partial sequence of umxyn10A encoding the catalyt-ic domain(from136to1185nt of the ORF)was amplified from clone GXN3621using pfu DNA polymer-ase(Stratagene,La Jolla,CA,USA)by PCR with the primers5′-AGTGAATTCGGGACGGCCGCCGGCGCG -3′(Eco RI restriction site is underlined)and5′-ACT AAGCTTGCGGCGGCGGGCCTTCGC-3′(Hin dIII re-striction site is underlined).The PCR protocol was:5min at95°C,followed by30cycles of30s at95°C,1min and 30s at72°C,and a final elongation of10min at72°C. The amplified DNA fragment of1,068bp in length was digested with Eco RI/Hin dIII and the recovered DNA fragment was cloned into the pGEM-3zf(+)vector (Promega,Madison,WI,USA)for sequencing.After sequence identification,the Eco RI/Hin dIII fragment from the pGEM-3zf(+)vector was subcloned into the expres-sion vector pET30a(+)to form the expression plasmid pET30a(+)-umxyn10A,which was transformed into hostE.coli BL21(DE3)pLysS(Novagen,Madison,WI,USA)for heterologous expression.The expression cellswere cultivated at37°C in LB medium containing15μg ml–1kanamycin and34μg ml–1chloramphenicol.When theOD600value of the culture reached0.5,a final concentrationof0.5mM isopropyl-β-D-thiogalactopyranoside was addedto induce the expression of umxyn10A.The expression cellswere centrifuged and ultrasonicated to obtain the cytoplas-matic extract.The translated product Umxyn10A was purifiedby affinity chromatography with nickel–nitrilotriacetic acidagarose resin(Ni–NTA,Qiagen).The purified enzyme waschecked by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis(SDS-PAGE)and used for the biochemicalcharacterization.Enzyme assaysTo measure endo-β-1,4-xylanase activity,10μl enzymediluted in190μl buffer was added to300μl1%(w/v)xylan from oat spelt or birchwood in the same buffer.Thereactions were carried out at the specified temperature for30min,and the released reducing sugar was measured as D-xylose equivalents according to Miller(1959).One unit (U)of endoxylanase activity was defined as the amount ofenzyme releasing1μmol reducing sugar per minute.Determination of optimal pH and temperatureof Umxyn10A toward oat spelt xylanTo test the optimal pH of Umxyn10A,enzyme assays were carried out at37°C in different buffers(100mM citrate-phosphate buffer,pH3.0–7.0;100mM sodium-phosphate buffer,pH6.0–8.0;100mM Tris-HCl buffer, pH7.0–9.0;and100mM glycine-NaOH buffer,pH8.5–10.0).To determine the optimal temperature,enzyme assays were performed in sodium-phosphate buffer (100mM,pH6.5)at different temperatures from25°C to95°C.To determine the thermostability of Umxyn10A, the purified enzyme was incubated at different temper-atures(30to80°C)for1h in the absence of substrate before performing the enzyme assays under the optimal condition.The pH stability was investigated by incubat-ing the purified enzyme in different buffers(pH 3.0–10.0)at4°C for24h,followed by enzyme assays under the optimal condition.Substrate specificity of Umxyn10A and analysisof hydrolysis products of xylooligosaccharidesand birchwood xylan digested by Umxyn10AActivities of recombinant Umxyn10A on1%(w/v)polysac-charides,such as birchwood xylan,carboxymethylcellulose (CMC),avicel,lichenan,laminarin,methyl cellulose,and 2-hydroxyethyl cellulose(all from Sigma),were tested under the optimal condition to investigate the substrate specificity.To detect the hydrolysis products of xylooligosaccharide and birchwood xylan digested by Umxyn10A, 1.17μg (468μg ml–1,2.5μl)of the enzyme was incubated with one of the following xylooligosaccharides(all from Mega-zyme,Wicklow,Ireland)or birchwood xylan at a concen-tration of1%(w/v)in0.1M citrate-phosphate buffer(pH 6.5):xylobiose,xylotriose,xylotetraose,xylopentaose,and xylohexaose.After incubation at50°C for12h,the hydrolysis products from each reaction were detected by high-performance liquid chromatography(HPLC) (Waters™600,Waters,USA),a Alltima™Amino5u column(Grace,USA),and an evaporative light scattering detector(ELSD2000ES)(Alltech,USA).Elution was performed with a solvent system of CH3CN:H2O(70:30), a pressure of1,058psi,and a flow rate of1.0ml min–1at room temperature.Effects of metals and reagents on recombinant Umxyn10A activityThe effects of metal ions(5mM)on Umxyn10A activity were determined by pre-incubating the enzyme with the individual reagents in100mM sodium-phosphate buffer, pH6.5,at75°C for30min.The effects of the chelating agent[ethylenediamine tetraacetic acid(EDTA)at5,10, and50mM,respectively]and surfactants(SDS and Triton X-100)at0.25%(w/v)were also tested.Activities were then measured at the optimal condition in the presence of the metal ions or reagents with oat spelt xylan as substrate. The activity assayed in the absence of the reagents was taken as100%.umxyn10A nucleotide sequence accession numberThe umxyn10A nucleotide sequence was deposited in the GenBank database(accession number EF118907). ResultsConstruction and screening of the metagenomic DNA library from the rice straw degrading enrichment culture A cosmid library containing about12,000clones was constructed with the metagenomic DNA isolated from the rice straw degrading enrichment culture.Restriction analy-ses of the plasmids of16randomly chosen clones showed that the inserted DNA fragments ranged from14to37kb with an average size of25.5kb.The total capacity of the library was estimated to be3.06×108bp.One clone of thelibrary showed activity on plates containing oat spelt xylan and 4-MUC and was chosen for further study.Sequence analyses of the xylanase gene umxyn10A The sequenced DNA fragment contained an ORF (umxyn 10A)encoding a putative endoxylanase (Umxyn10A)with 395amino acids (aa)and predicted molecular mass of 44kDa.The ORF sequence of umxyn 10A showed the most similarity to the endo-β-1,4-xylanase precursor gene (accession number FJ458449)with 76%identity (595/782bases),which was recently cloned from the deep-sea microbe Kocuria sp.Mn22(Li et al.2009).The aa sequence of Umxyn10A showed the highest similarity to the xylanase from T.fusca YX (accession number AAZ56824.1)with 58%identity and 73%similarity and to the endo-β-1,4-xylanase precursor from Kocuria sp.Mn22(accession number ACJ73932.1)with 58%identity and 72%similarity.Multi-alignment with bacterial and fungal GH10xylanases (Table S 1)showed that two putative catalytic Glu residues (Glu199and Glu307)were present in the catalytic domain of Umxyn10A (Fig.1).The multi-alignment also identified nine conserved aa residues in Umxyn10A,which were H35,W39,N81,E82,N124,H160,E188,D190,and W236,respectively,as highlighted in yellow in Fig.1[the numbers were calculated from the aa sequence of Xyn10A from Cellulomonas fimiA TCC 484(accession number AAA56792.1)].Six PSCRs were found in those aligned sequences,designated as Box1(–GMxVRGHTLVWHSQ –),Box2(–KIxAWDVVNEAIxD –),Box3(–xKLYYNDYNLE –),Box4(–IDGIGMQSHLxI –),Box5(–VEVxITELDI –)and Box6(–xVTVWGVxDxxSWL –)(Fig.1).The phylogenetic relationships of Umxyn10A are illustrated in Fig.2.Umxyn10A showed the closest genetic relationship with xylanases from Kocuria sp.Mn22,Thermobifida fusca YX,and Thermotoga maritima MSB8(accession number AAD35164.1).Homology mod-eling revealed that folded Umxyn10A has a (β/α)8parallel structure (Fig.S 1a ).Six PSCRs are located in the protein model at different αhelixes and βsheets,such as Box1at α3helix,Box2at α4helix,Box3at β6sheet,Box4at β7sheet,Box5at α7sheet,and Box6at α8helix,parison of the predicted 3D structure of Umxyn10A with that of T.maritima MSB8xylanase 10B (PDB 1vbu)revealed that the nine conserved aa residues were located at the same position (Fig.S 1b ).These results demonstrate that Umxyn10A is a typical GH10xylanase.Expression of umxyn10A and purification of the recombinant Umxyn10AE.coli BL21(DE3)harboring pET30a (+)-umxyn10A showed xylanase activity on a LB plate with oatspeltFig.1Conserved amino acid residues and structurally con-served regions of GH10xyla-nases predicted by the ClustalW software and MATCH-BOX server.The protein codes corre-spond to those listed in Table S 1in the “Electronic supplementary material ”.Only the PSCRs are shown in the figure according to the results obtained from the MATCH-BOX server.The aa sequences highlighted in green represent the block of similar aa residues in all aligned sequen-ces,the aa residues highlighted in red and yellow represent the identical aa sequences,and the aa sequences highlighted in blue represent the conservative aa sequences.The predicted con-served putative catalytic Glu residues (Glu199and Glu307)are indicated with an arrowhead at the bottom of the figure.All protein sequences mentioned in the text are marked with an arrow to the left of the protein codexylan,whereas that of E.coli BL21(DE3)harboring plasmid pET30a(+)did not(data not shown).The cytoplasmatic extract of the expression cells presented a distinctive50-kDa protein in SDS-PAGE(Fig.3,lane3),in agreement with the predicted molecular mass of the fusion protein with a6×His tag.The recombinant enzyme was purified to apparent homogeneity by affinity chromatogra-phy(Fig.3,lane4).Zymogram analysis of the recombinant Umxyn10A indicated that only one apparent active band with the expected molecular size was present in the respective zymogram(Fig.3,lanes5and6).Enzyme properties of the recombinant Umxyn10AThe optimal pH of the purified recombinant Umxyn10A toward oat spelt xylan was approximately pH 6.0–6.5 (Fig.4a).The optimal temperature for the hydrolysis reaction was75°C(Fig.4b).The enzyme retained more than80%activity upon incubation at pH 5.0to7.0 (Fig.4c).The enzyme was stable at temperatures below 65°C with more than80%activity retained,and its activity was dramatically lost after30-min incubation at temper-atures above70°C(Fig.4d).The K m and V max of the recombinant Umxyn10A toward oat spelt xylan were 3.2mg ml–1and0.22mmol min−1mg−1,respectively. When using birchwood xylan as substrate,the K m and V max of recombinant Umxyn10A were2.7mg ml–1and1.0mmol min−1mg−1,respectively.Substrate specificity,xylan hydrolysis products,and effect of metals and reagentsThe activities of Umxyn10A on various substrates are shown in Table1.Umxyn10A showed high activity for the substrates containingβ-1,4-xylan bonds,such as xylan from birchwood and oat spelt.It showed some activity towards other substrates that consisted ofβ-1,3/4-glucan or β-1,3/6-glucan.The hydrolysates of xylooligosaccharides and birchwood xylan digested by purified Umxyn10A xylanase were analyzed by HPLC(Fig.5).The enzyme was not active on xylobiose.Umxyn10A could completely hydrolyze xylotriose to produce xylobiose and xylose.Xylotetraose and xylohexaose were completely degraded to produce solely xylobiose,whereas xylopentaose was totally digested to release xylobiose and xylose,with xylobiose as themajorproduct (Fig.5b –f ).Xylobiose was the main product of hydrolysis of birchwood xylan by Umxyn10A (Fig.5g ).A negligible amount of xylose was detected in the hydrolysate of birchwood xylan after digestion by Umxyn10A (Fig.5g ).Umxyn10A activity was not stimulated by Mg 2+,Mn 2+,Ca 2+,Cr 2+,Zn 2+,Li +,Cu 2+,K +,Ni 2+,or Fe 2+(each at a concentration of 5mM),and it was neither inhibited nor activated by EDTA at concentrations ranging from 5to 50mM.Only Co 2+slightly stimulated the activity of Umxyn10A.Metal ions do not appear to be required for the catalytic activity of Umxyn10A.Instead,Fe 3+and anionic surfactant SDS strongly inhibited the enzyme activity by 36%and 65%,respectively (Table 2).DiscussionCurrently,several genes for xylanases have been cloned from environmental DNA,by PCR-based cloning based on high homology or by screening a soil-derived metagenomic library (Sunna and Bergquist 2003;Hayashi et al.2005;HupH valueR e l a t i v e a c t i v i t y (%)T (°C)R e l a t i v e a c t i v i t y (%)pH valueR e l a t i v e a c t i v i t y (%)T (°C)R e l a t i v e a c t i v i t y (%)ab c d Fig.4Effects of pH and temperature on the activity of the purified recombinant Umxyn10A toward oat spelt xylan.a Effect of pH on enzymatic activity toward oat spelt xylan measured in 100mM citrate-phosphate buffer,pH 3.0–7.0;sodium-phosphate buffer,pH 6.0–8.0;Tris-HCl buffer,pH 7.0–9.0;and glycine-NaOH buffer,pH 8.5–10.0.b Effect of temperature on the activity of Umxyn10A toward oat spelt xylan.Experiments were conducted in the temperature range of 25to 95°C at pH 6.5in 100mM citrate-phosphate buffer.c pH stability of the enzyme.Enzyme activity was measured under the optimal condition (citrate-phosphate buffer,pH 6.5,75°C,30min)after the enzyme was incubated in the buffers specified above at 4°C for 24h.d Thermostability of Umxyn10A.Enzyme activity was measured under the optimal condition (citrate-phosphate buffer,pH 6.5,75°C,30min)after the enzyme had been incubated at the indicated temperature for 1h.The error bars represent the standard deviation of triplicate measurementsFig.3SDS-PAGE analysis of the purified protein ne 1,protein molecular weight marker (116.0,66.2,45.0,35.0,25.0,18.0,and 14.0kDa);lane 2,cytoplasmatic extracts of E.coli BL21(DE3)harboring the empty plasmid pET30a (+);lane 3,cytoplasmatic extracts of the expression cell;lane 4,recombinant Umxyn10A purified by Ni-NTA;lanes 5and 6,in-gel refolded cytoplasmatic extracts of the expression cell showing activity on 4-MUC under ultraviolet light at total protein concentrations of 30and 10mg ml −1,respectivelyFig.5HPLC chromatograms ofthe hydrolysates of xylooligo-saccharides and birchwood xy-lan digested with the purified recombinant Umxyn10A.The recombinant Umxyn10A (468μg ml−1,2.5μl)was added to100μl of each xylooligosac-charide solution[1%(w/v)in 0.1M citrate-phosphate buffer, pH6.5].The mixture was incu-bated at50°C for12h and diluted to a volume of500μl before loading to an Alltima™Amino5u column for HPLC analysis.Elution was performed with a solvent system ofCH3CN:H2O(70:30),a pressure of1,058psi,and a flow rate of 1.0ml min–1at room tempera-ture.a Mixture of authentic xylooligosaccharides of xylose (X1),xylobiose(X2),xylotriose (X3),xylotetraose(X4),xylo-pentaose(X5),and xylohexaose (X6).b Hydrolysate of X2.c Hydrolysate of X3.d Hydroly-sate of X4.e Hydrolysate of X5.f Hydrolysate of X6.g Hydro-lysate of birchwood xylanet al.2008;Yamada et al.2008).In this study,an enrichment culture of rice straw-degrading microorganisms from soils was constructed.Metagenomic approach and functional screening were employed to identify a new xylanase,Umxyn10A.The optimal temperature of Umxyn10A toward oat spelt xylan is 75°C,which is higher than that of xylanases cloned previously from a soil-derived meta-genomic library or by a PCR-based method or from cultivated microorganisms,such as 50°C for XynH cloned from a soil-derived metagenomic library (Hu et al.2008),40°C for XynZG from Plectosphaerella cucu-merina (Zhang et al.2007),40°C for XynB from Clostridium cellulovorans (Han et al.2004),and 40°C for a xylanase from Trichoderma harzianum T4(Franco etal.2004).However,it was observed that the xylanase Umxyn10A was unstable at the apparent optimum tem-perature of 75°C and at 70°C (Fig.4b,d ).Denaturation of enzymes is time dependent as well as temperature dependent,and at the apparent optimum temperature denaturation of enzymes becomes significant (Daniel et al.2001).In accordance,in the thermostability assay for Umxyn10A,more severe thermal denaturation of the enzyme occurs at 75°C and 70°C than at 65°C during the 1-h preincubation of the enzyme at the corresponding temperature in the absence of substrates (Fig.4d ).Neverthe-less,Umxyn10A was stable at temperatures below 65°C,with more than 80%activity retained.These properties of Umxyn10A may provide important advantages for the potential biotechnological application of the enzyme.Metal ion or chemical reagent Concentration Relative activity (%)a No addition –100±2.48CoCl 25mM 103±3.29CaCl 25mM 88±1.05NiCl 25mM 85±3.55CrCl 25mM90±1.34LiCl 5mM 96±1.56FeCl 25mM 97±2.05KCl 5mM 97±2.63MgCl 25mM 84±1.08FeCl 35mM 64±2.45MnCl 25mM 85±1.73ZnCl 25mM 99±1.11CuCl 25mM 85±1.91EDTA 5mM 89±1.71EDTA 10mM 84±1.89EDTA 50mM 83±3.53SDS0.25%(w /v )35±2.99Triton X-1000.25%(w /v )92±2.48Table 2Effects of metal ions and chemical reagents onUmxyn10A activity toward oat spelt xylanaThe reaction system contained 0.01mg ml −1Umxyn10A and 10mg ml −1of oat spelt xylan in 100mM citrate-phosphate buffer (pH 6.5)with addition of the respective chemical reagent at the concentration specified.The reac-tion was performed at 75°C for 30min in triplicate.Activity assayed in the absence of the reagents is set at 100%Table 1Substrate specificity of the recombinant xylanase Umxyn10A SubstrateConcentration (%,w /v )Specific activity (U mg −1)Relative activity (%)a Xylan from oat spelt (β-1,4-xylan)(control)1144.44±2.38100±1.33Xylan from birchwood (β-1,4-xylan)1211.16±12.82148±1.29Lichenan (β-1,3/4-glucan)130.57±2.1917.7±4.35Avicel (β-1,4-glucan)129.48±1.4716.9±2.12Laminarin (β-1,3/6-glucan)128.40±0.5516.3±7.35Carboxymethylcellulose (β-1,4-glucan)123.68±0.5512.9±3.04Methyl cellulose (β-1,4-glucan)123.32±0.4212.6±2.342-Hydroxyethyl cellulose (β-1,4-glucan)122.95±0.5512.4±3.01aThe reaction system contained 0.01mg ml −1Umxyn10A and 10mg ml −1of different substrates in 100mM citrate-phosphate buffer (pH 6.5).The reaction was performed at 75°C for 30min in triplicate.One unit (U)of activity was defined as the amount of enzyme releasing 1μmol reducing sugar per minute.The enzyme activity against oat spelt xylan is set at 100%In previous reports,researchers found that the activity of xylanases is often inhibited by various metal ions,such as Mn2+,Zn2+,Co2+,Ag+,and Cu2+(Araki et al.1999;Gupta et al.2000;Fialho and Carmona2004).However,in the present study,most of these metal ions did not significantly reduce the activity of Umxyn10A.All the data suggested that Umxyn10A is a novel type of xylanase.Substrate specificity analysis revealed that Umxyn10A can efficiently digest birchwood xylan,which consists of mainly xylose(≥90%),and oat spelt xylan,which consists of xylose(≥70%),glucose(≤15%),and arabinose(≤10%) (Kim et al.2009),but showed higher specific activity toward birchwood xylan than toward oat spelt xylan.The V max value of Umxyn10A for birchwood xylan was approximately fivefold higher than that of the enzyme for oat spelt xylan.The enzyme also had a smaller K m value toward birchwood xylan than that toward oat spelt xylan. These data suggest that Umxyn10A is a birchwood xylan-selective endo-β-1,4-xylanase with broad substrate speci-ficity that can efficiently degrade heteropolymeric xylans composed of various constituents.The endo-β-1,4-xylanases from Bacillus halodurans S7(Mamo et al.2006)and Clostridium acetobutylicum ATCC824(Ali et al.2005) were reported to be birchwood xylan-selective enzymes but have different enzymatic properties compared with those of Umxyn10A.In addition,the ability of Umxyn10A to degrade polysaccharides consisting ofβ-1,3/4-glucan orβ-1,3/6-glucan or cellulose,such as Avicel and CMC,indicates that it is a multifunctional enzyme.Xylanases are subject to end-product inhibition by xylo-biose and xylan hydrolysis products(Bachmann and McCarthy 1991;Polizeli et al.2005).However,the final product, xylobiose,seems not to inhibit Umxyn10A activity since the xylooligosaccharides could be completely hydrolyzed to release xylobiose or a mixture of xylobiose and xylose. Interestingly,Umxyn10A can efficiently degrade birchwood xylan to release xylobiose as the major product and a negligible amount of xylose.Xylobiose is of interest for human health and nutrition,as it has been found to be a selective growth stimulant of human intestinal Bifidobacte-rium,which is beneficial for the maintenance of a healthy intestinal microflora(Okazaki et al.1990).The selective stimulative effect of xylobiose on Bifidobacterium was much higher than that of other oligosaccharides(Degnan and Macfarlane1991).However,the xylobiose production costs are very high(Jiang et al.2004).Nevertheless,the xylanase Umxyn10A described in this work may have potential application in the production of xylobiose from xylan. 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