Cloning,Expression,and Characterization Of A Novel Xylanase Gene Been Cloned From Strain Isolated From Special Environments

Endo-1,4-?-D-xylanase is the crucial enzyme of xylan degrading enzymes.Xylanases have great applications in baking,animal feed,paper and other industries.Nowadays,screening enzyme-producing strains from special environments and obtaining enzymes with superior properties has been one of the important researching methods about xylanase.In this study,xylanase-producing strains were screened from various special environments and 13 GH10 xylanase gene fragments were cloned,2 full-length xylanase genes were obtained by using thermal asymmetric interlaced-PCR(TAIL-PCR)procedure.The heterologous expression and recombinant enzyme's biochemical characterizations of an xylanase XynSL4 were reported.The results are as follows:1.With beechwood xylan chosen as the only carbon source of primary screening medium,totally 20 strains which showed transparent zones and different morphology were screened from 5 special environments of soda Lake Dasubu,Guangxi mangrove,Yunnan hot spring,compost from Changfu ranch and mud sample of the south China sea.After amplify objective gene fragments from these strains,8 sequences were confirmed to be GH10 xylanase gene fragments,and 5 belong to GH11.Of them,6 xylanase gene fragments which had similarities of 70-90%with known xylanases and 3 novel xylanase gene fragments which had similarities of below 80%with known xylanases were successful obtained.2.Two full-length xylanase genes(xynSL3 and xynSL4),which were cloned from strains isolated from soda lake Dabusu,were obtained by using thermal asymmetric interlaced-PCR(TAIL-PCR)procedure.Their nucleotide sequences and deduced amino acid sequences were analyzed:?xynSL3 was 2259 bp,encoded a polypeptide of 752 amino acid residues,including a putative signal peptide,a catalytic domain and two carbohydrate-binding module(CBM)domains,which had similarities of 55%with known xylanases;?xynSL4 was 1143 bp,encoded a polypeptide of 380 amino acid residues,including a putative signal peptide and a catalytic domain,which had similarities of 77%with known xylanases.xynSL4 was selected for further expression?purified and characterized.The multiple sequence alignment,three-dimensional structure and phylogenetic analysis of xylanase XynSL4 were also reported.3.The novel xylanase gene xynSL4 was successful heterologously expressed in E.coli.The crude enzyme was purified to electrophoretic homogeneity by ammonium sulfate precipitation,ultrafiltration and Ni-affinity chromatography.Enzymatic properties of purified XynSL4 are as follows:?It had an apparent pH and temperature optimum of pH 7 and 70?,it was stable at pH 11(retained more than 60%activity),which was characterized to be thermophilic and alkaline-tolerant xylanase;?Its thermosability at 70? is worse,XynSL4 was far more thermostable at high temperature in the presence of beechwood xylan which confirmed that substrate can protect enzyme against heat;?XynSL4 has great tolerance to high concentration of NaCl up to 4 mol/L.The enzyme activity was significantly enhanced by ?-mercaptoethanol and Ca2,but strongly inhibited by heavy metal ions and SDS;?Using beechwood xylan as the substrate,the Km,Vmax and Kcat value were 1.45±0.08mg/mL?362.74±8.55 ?mol/(mg·min)and 262.62?6.19 s-1;?The enzyme degraded xylan,producing xylose and xylobiose as the predominant products,indicating that it was endoxylanase.In this study,xylanase-producing strains from various special environments were screened and several xylanase genes were cloned.An xylanase XynSL4 cloned from soda lake were reported.This thermophilic and alkaline and salt-tolerant enzyme has great value for theoretical research and application.