具蛋白酶抗性的Armillariella tabescens β-甘露聚糖酶MAN47的分子定向改造  

Directional Molecular Rebuilding of β-mannanase MAN47 with Trypsin-resistance from Armillariella tabescens

作　　者：胡凤娟[1] 王旭曼[1] 刘大岭[1,2] 姚冬生[1,3] 

机构地区：[1]暨南大学微生物技术研究所,广州510632 [2]广东省生物工程药物重点实验室,广州510632 [3]基因药物国家工程研究中心,广州510632

出　　处：《中国生物工程杂志》2011年第10期75-82,共8页China Biotechnology

基　　金：广东省科技攻关资助项目(2005B20601004)

摘　　要：利用定点突变的方法提高Armillariella tabescensβ-甘露聚糖酶MAN47的胰蛋白酶抗性。首先根据其氨基酸序列,找到胰蛋白酶的水解位点—赖氨酸(Lys,K)和精氨酸(Arg,R),再利用生物信息学软件获得酶分子结构中K和R与周围溶剂的接触程度,选定暴露程度最大的K280为候选突变位点,进行模拟突变,并分析突变前后的氢键键长和整体结构的变化。根据氢键键长的变化,确定突变体为K280N。对K280N设计突变引物,用重叠延伸PCR技术对MAN47野生型man基因进行突变,PCR产物与大肠杆菌-酿酒酵母穿梭表达载体PYCα连接,在大肠杆菌DH5α中扩增后转入酿酒酵母Saccharomyces cerevisiae,经人工肠液(pH 6.8 10mg/ml胰蛋白酶溶液)筛选,得到抗胰蛋白酶的最佳突变株。结果表明突变酶在用人工肠液处理180min后,其半衰期为173min,而野生型酶为99min,其他酶学性质与野生型酶基本一致。The trypsin-resistance of β-mannanase MAN47 from Armillariella tabescens was improved by site-directed mutagenesis method.In order to determine the amino acid for mutation,homology modeling were used.Firstly,the structure of β-mannanase MAN47 was constructed by homology modeling and the contact of intramolecular trypsin cleavage sites（K and R） with the surrounding solvent（exposure extent） was analyzed on the basic principles of the catalytic characteristics of trypsin.Among those trypsin cleavage sites,K280 with most exposure extent to the surrounding solvent was chosen as the candidate mutation site.Then K280 was subject to simulating mutation.And K280N was determined as the suitable mutant after calculating the changes in H-bond length and the structure of enzymes before and after simulating mutation.Then the gene of the mutant K280N by site-directed mutagenesis were obtained,and it was cloned into secreted expression vector pYCα.The amplified recombinant plasmids in DH5α cells were transformed into Saccharomyces cerevisiae BJ5465 for the mutant enzyme expression.Subsequently,transformants were screened out by incubation with simulated intestinal fluid（pH 6.8 10mg/ml trypsin solution）.And both the wild-type and mutated enzymes were characterized and evaluated on trypsin-resistance.Results showed that the mutant enzyme K280N had a half-life of 173 min after incubation with trypsin at 40°C,which was 74 min longer than that of the wild-type enzyme（99 min）.But the optima pH and temperature of both the two enyzmes were similar.In conclusion,the trypsin-resistance of the β-mannanase MAN47 was successfully improved after site-directed mutagenesis.

关 键 词：定点突变 Β-甘露聚糖酶 重叠延伸PCR 同源建模 分子计算 

分 类 号：Q819[生物学—生物工程]