Cloning of Acidic Xylanase Gene and Its Secretion Expression in Pichia pastoris / 微生物学通报

ABSTRACT

An acidic xylanase gene,named xyl3,was cloned from the genomic library of enviromental microbes constructed by using shotgun cloning strategy,and submitted to GeneBank with accession number of gbAY300805.BLAST analysis indicated that the gene xyl3 has low similarity with other xylanase genes and the encoded xylanase,sorted as Glycosyl hydrolases family 10,has 77% similarity with the intra-cellular xylanase from Geobacillus stearothermophilus at amino acid level.Treated with T4 DNA polymerase,the gene xyl3 was ligated with the linearized Pichia pastoris expression vector pHBM905 produced by digestion of restriction endonuclease CpoI and NotI to generate the recombinant plasmid pHBM706.Then the plasmid pHBM706,digested by restriction endonuclease SalI,was transformed into P.pastoris GS115 to obtain the recombinant P.pastoris GS115(pHBM706),which was induced to produce the recombinant xylanase with 0.5% methanol at 28℃.At the 36th hours of induction,the porduced crude enzyme was detected to reach the higest enzyme activity of 0.177 IU/mL.The optimal pH and temperature of the enzyme activity is 5.5 and 50℃ respectively.