短小芽孢杆菌碱性β-葡萄糖苷酶在毕赤酵母中的表达及酶学性质研究  

Expression and characterization of alkaline β-glucosidase in Pichia pastoris from Bacillus pumilus

作　　者：阚劲松[1] 张敏[1] 徐涛[1] KAN Jin-song;ZHANG Min;XU Tao(Department of Biological and Environmental Engineering,Hefei University,Hefei,Anhui 230601,China)

机构地区：[1]合肥学院生物与环境工程系

出　　处：《食品与机械》2019年第9期39-44,共6页Food and Machinery

基　　金：安徽高校自然科学研究重点项目（编号：KJ2016A878）;安徽省教育厅质量工程项目（编号：2016sxzx036）;安徽省自然科学基金青年项目（编号：1308085QC48）

摘　　要：以毕赤酵母密码子为基准,对短小芽孢杆菌β-葡萄糖苷酶基因密码子进行优化,设计合成全基因序列,构建表达载体转入毕赤酵母中。结果表明,短小芽孢杆菌β-葡萄糖苷酶基因已成功转入酵母菌中并分泌表达,诱导培养72 h发酵液的酶活力可达25.39 U/mL。重组酶最适反应温度45℃,最适pH 9.0,重组酶的K m值1.26 mmol/L,V max为32.15μmol/(min·mg)。重组β-葡萄糖苷酶对大豆苷水解活性相对活力是pNPG的248%,转糖苷活性催化50%底物葡萄糖合成龙胆二糖,达34.25 g/L。Engineered Pichia pastoris strain secreting and expressingβ-glucosidase gene of Bacillus pumilus was constructed and enzymatic properties of recombinant enzyme were investigated.Based on the codon usage of Pichia pastoris,the codon of theβ-glucosidase gene of Bacillus pumilus was optimized,the whole gene sequence was designed and synthesized,and the expression vector was constructed and transferred into Pichia pastoris.The results showed that theβ-glucosidase gene of Bacillus pumilus had been successfully transferred into the yeast and secreted and expressed.The activity of the fermentation broth after 72 h induction could reach 25.39 U/mL.The optimum temperature and pH for the recombinantβ-glucosidase were 45℃and 9.0,and the K m and V max of recombinant enzyme were 1.26 mmol/L and 32.15μmol/(min·mg),respectively.The relative activity of recombinantβ-glucosidase to soybean glycoside hydrolysis was 248%of that of pNPG,and the transglycosylation activity catalyzed the enzymatic synthesis of gentiobiose yield of 34.25 g/L using 50%glucose as the substrate.

关 键 词：碱性β-葡萄糖苷酶 短小芽孢杆菌 毕赤酵母 龙胆二糖 表达 

分 类 号：TS2[轻工技术与工程—食品科学与工程]