新型甘露聚糖酶基因的克隆及在毕赤酵母中的表达

Cloning of Novel Mannanase Gene and Its Secreted Expression in Pichia Pastoris

构建土壤基因组文库,通过功能筛选得到一水解底物魔芋粉的阳性克隆,测序后,序列分析表明:该基因全长为1 530 bp,包含一个糖基水解家族26结构域,编码510个氨基酸,相对分子质量为56 438。此基因推导的氨基酸序列与Cellulomonas fimi的Man26A和Cellvibrio japonicus的mannanase A氨基酸序列同源性分别为64.3%、38%。将此基因的成熟肽的编码序列克隆到表达载体pHBM905上,得到重组质粒pHBM730,将pHBM730经SalI酶切线性化后转化3株毕赤酵母GS115、KM71、SMD1168,该甘露聚糖酶基因在这3种毕赤酵母中均实现分泌表达。将此3种重组毕赤酵母GS115(pHBM730)、KM71(pHBM730)、SMD1168(pHBM730)诱导产酶,对这3种重组酶进行相关的酶学性质研究。分析表明,在KM71中表达量最高,其最适反应pH值均约为6.6,最适反应温度均约为45℃。在最适反应条件下测得其酶活力为19.64 U。此结果为瓜尔豆胶降解产物的工业化应用提供了参考。

A positive clone that can hydrolyze Konjac powder was obtained when constructing genome library of soil.The sequence analysis of the clone indicated that the full-length gene was 1 530 bp and containing the 26th domain of a mannoside-hydrolyzed family,which encoded 510 amino acid residues and relative molecular mass 56 438 D.The amino acid sequence encoded by this gene was homologous to the Man26A of Cellulomonas fimi at the degree of 64.3% and mannanase A of Cellvibrio japonicus at degree 38%.Then the coding sequence of the gene was cloned into the Pichia pastoris expression vector pHBM905,the recombinant plasmid named pHBM730.Then pHBM730 was transformed into Pichia pastoris GS115,KM71,SMD1168 respectively after being linearized by SalI.The recombinant Pichia pastoris GS115（pHBM730）,KM71（pHBM730）,SMD1168（pHBM730） secreted functional mannanase and the expression in KM71（pHBM730）was the best.The analysis of the recombinant mannanase indicated that the optimum temperature and pH for the enzyme were 45 ℃ and pH 6.6 respectively.The enzyme activity reached 19.64 U using guar gum as substrate,and it could provide a reference for the guar gum degradation products.