【作者】 吴喆瑜； 于越； 巩建业； 李利君； 倪辉；

【Author】 Wu Zheyu;Yu Yue;Gong Jianye;Li Lijun;Ni Hui;College of Food and Biological Engineering,Jimei University;Key Laboratory of Food Microbiology and Enzyme Engineering of Fujian Province;

【机构】 集美大学食品与生物工程学院； 福建省食品微生物与酶工程重点实验室；

【摘要】 α-L-鼠李糖苷酶(EC 3.2.1.40),是一种在工业上有着重要应用的食品酶,属于糖苷水解酶类,能够水解天然及合成的具有糖苷类的物质末端的鼠李糖基。本实验以实验室前期构建成功的α-L-鼠李糖苷酶重组质粒(pPIC9K-rha)为模板,采用易错PCR的方法,建立库容量为2×10~5,阳性克隆率为92%的突变库。基于酵母异源表达产生胞外酶的原理,构建了96孔板筛选法,最终从3000个转化子中筛选得到了5株热稳定性改变的突变体。通过热稳定性研究发现,突变酶D80N/V529A在65℃条件下,热稳定性比原始型α-L-鼠李糖苷酶(WT)提高了10分钟。另外四个突变酶的热稳定性显著降低,且均在600位氨基酸附近存在氨基酸突变,由此推测600位氨基酸附近区域为影响热稳定性的有效区域。更多还原

【Abstract】 Hydrolytic enzymes a-L-rhamnosidases(EC 3.2.1.40),which can specifically hydrolyze the terminal a-l-rhamnoside groups from polysaccharides and glycosides,are the important enzymes of food industry.In the previous study,the recombinant plasmid(pPIC9K-rha) containing the α-L-rhamnosidase gene of Aspergillus niger TS528 was constructed.A random mutant library based on this α-L-rhamnosidase was generated using error-prone PCR.The colony positive rate of the library was 92%,and the library had approximately 2 × 10 random mutants.Since Pichia pastoris can extracellularly express heterologous proteins,a screening method by using 96-well plates was established according to this principle.Through the screening of 3000 mutant colonies,five mutants were observed to display different thermostability compared towild-type α-L-rhamnosidase(WT).Among these mutants,the double mutants D80N/V529 Ashowed 10 min longer half-life than WT,at65℃ and the other four mutants decreased the thermostability.According to the sequence analysis,most thermostability changed mutants had amino acids alteration around 600 position.Therefore,this domain was predicted to be important for the thermostability of α-L-rhamnosidase.更多还原