耐碱性甘露聚糖酶基因的克隆及其在毕赤酵母中的表达

Cloning and expression in Pichia pastoris of an alkaline mannanase gene

通过功能平板从土壤中筛选得到含甘露聚糖酶基因的耐碱菌株。构建其基因组文库,从中筛选到甘露聚糖酶基因TM1并测序分析,用BLAST分析表明,TM1的氨基酸序列与其他在GenBank发表的甘露聚糖酶的氨基酸序列的同源性均低于60%,故确定其为一个新的甘露聚糖酶基因(GenBank登录号为AY623903)。将此基因去除信号肽后的编码序列克隆到表达载体pHBM905C上,得到重组质粒pHBM1201。经SalⅠ酶切后分别转化毕赤酵母(Pichiapastoris)KM71、GS115、SMD1168,得到分泌表达的重组毕赤酵母。挑选相对表达量最高的重组毕赤酵母SMD1168-3在摇瓶中诱导产酶,对该酶的粗酶进行酶学性质分析表明,其最适反应温度为55℃,最适PH值为7.5,以魔芋粉为底物所测得的最高酶活为41.8U,半衰期为1h,在80℃保温5min其酶活由最初酶活的77%下降到11%,温度下降到55℃后活性可恢复到最初酶活的60%以上。

A strain containing alkaline mannanase gene was isolated from soil by functional plates and the genome library was constructed. From it a mannanase gene TM1 was acquired and was sequenced. The BLAST analysis showed a lower-than-60% similarity of the amino acid sequence to those in GenBank and proved TM1 to be a new mannanase gene (GenBank accession number AY623903). The new gene without signal peptide was cloned into the Pichia pastoris expression vector pHBM905C. The recombinant plasmid pHBM1201 was digested by SalⅠ and transformed into Pichia pastoris KM71, GS115, SMD1168, respectively. All of the recombinant Pichia pastroris strains containing pHBM1201 secreted functional β-mannanase. Because of its high mass of expression, the recombinant Pichia pastoris SMD1168-3 containing pHBM1201 was induced at shake flasks. The optimal temperature and pH of the β-mannanase produced by the recombinant strains were 55℃and 7.5, respectively. The enzymatic activity for konjak powder reached 41.8 with a half life of one hour. After keeping at 80℃ for 5min, the enzymatic activity declined from 77% to 11% and the enzymatic activity could recover up to more than 60% when the temperature descended to 55℃.