嗜热子囊菌光孢变种β-葡萄糖苷酶的基因克隆、表达及酶学性质分析  

Cloning,Expression and Enzymic Property Anylysis of β-glucosidase from A Thermophilic Fungus Thermoascus aurantiacus var.levisporus

作　　者：陈卫红[1] 李多川[1] 

机构地区：[1]山东农业大学植物保护学院环境生物系,泰安271018

出　　处：《应用与环境生物学报》2009年第4期549-553,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家"863"计划资助项目(Nos.2008AA05Z403;2006AA10Z304)~~

摘　　要：从嗜热子囊菌光孢变种(Thermoascus aurantiacus var.levisporus)RNA中通过RT-PCR克隆出β-葡萄糖苷酶基因bglⅠ的全长序列,cDNA序列为2672bp,Genbank登录号为EU269025,将该片段插入巴斯德毕赤酵母Pichia pastoris分泌型表达载体pPIC9K中,获得重组质粒pPIC9K/bgl,经线性化后用电穿孔法导入毕赤酵母GS115中,在醇氧化酶AOX1基因启动子作用下,获得高效表达β-葡萄糖苷酶的毕赤酵母工程菌株.经DEAE-Sepharose Fast Flow阴离子层析纯化了该重组表达蛋白.SDS-PAGE测得该重组蛋白相对分子质量(Mr)约为120×103.经甲醇诱导,培养基中β-葡萄糖苷酶的活力可达1.2U/mg,小规模发酵量达0.45mg/mL.该酶的最适反应温度为60℃,最适反应pH为5.0.于70℃保温30min仍保持80%的酶活力,具有较高的热稳定性,在pH3.0～9.0的条件下酸碱耐受性强.β-glucosidase bgl I gene was isolated from thermophilic fungus Thermoascus aurantiacus var. levisporus RNA through RT-PCR. The length of the gene was 2 672 bp, which was registered in GenBank with accession number EU269025, and the gene was ligated with the Pichia pastoris expression vector pPIC9K, resulting in recombinant plasmid pPIC9K/bgl. pPIC9K/bgl was then linearized and transformed into P. pastoris GS115 by electroporation, and highly efficient recombinant strains were obtained under control of the AOX1 promotor. The recombinant β-glucosidase was purified using DEAE- Sepharose Fast Flow chromatography. The molecular mass of the purified enzyme was 120 ×10^3 determined by SDS-PAGE. With methanol induction, the recombinant strain expressed and secreted β-glucosidase into the culture with activity of 1.2 U/rag, and the expression level was 0.45 mg/mL by small-scale culturing. The expression of β-glucosidase exhibited the optimum catalytic activity at 60 ℃ and pH 5.0, remained 80% of its original activity after 30 rain at 70℃, possessed high thermostability, and had high acid and alkali tolerance under the condition ofpH 3.0-9.0. Fig 6, Tab l, Ref22

关 键 词：嗜热子囊菌光孢变种 表达 纯化 毕赤酵母 Β-葡萄糖苷酶 

分 类 号：Q93[生物学—微生物学] Q78