酸性木聚糖酶基因的克隆及其在毕赤酵母中的分泌表达

Cloning of Acidic Xylanase Gene and Its Secretion Expression in Pichia pastoris

运用“鸟枪法”克隆构建了环境微生物的基因组文库,并从中筛选得到一个酸性木聚糖酶基因,命名为xyl3,其在GenBank中的登录号为gb:AY300805。BLAST分析表明,该基因的序列同源性很低,其中仅存在很短的木聚糖酶基因的同源片段,其编码的木聚糖酶属于G lycosyl hydrolases fam ily 10,与来源于Geobacillus stearotherm ophilus的intra-cellu larxylanase在氨基酸水平具77%同源性。该基因经T4 DNA polym erase处理后,克隆至经限制性内切酶CpoⅠ和NotⅠ双酶切后的毕赤酵母表达载体pHBM905,获得重组质粒pHBM706。此重组质粒转化毕赤酵母GS115,经含有交联木聚糖的选择性培养平板和PCR扩增鉴定筛选得到重组毕赤酵母GS115(pHBM706)。以0.5%甲醇于28℃诱导产酶,测得重组毕赤酵母GS115(pHBM706)在诱导的第36h产酶达最高值,所产粗酶液酶活为0.177 IU/mL。该酶的最适反应pH为5.5,最适反应温度为50℃。

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 gb： AY300805 . 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.