基于定向进化的烷基间苯二酚合酶改造  

Application of directed evolution in modification of alkylresorcinol synthase

作　　者：包莹玲 赵威棣 吕旭浩 周靖阳 祝衢鑫 李忠 陈小龙 BAO Yingling;ZHAO Weidi;LU Xuhao;ZHOU Jingyang;ZHU Quxing;LI Zhong;CHEN Xiaolong(Institution of Fermentation Engineering, College of Biotechnology and Bioengineering, Zhejiang University of Technology,Hangzhou 310014, China;Zhejiang Tonglu Huifeng Biotechnology Co., Ltd., Hangzhou 311500, China)

机构地区：[1]浙江工业大学生物工程学院发酵工程研究所,浙江杭州310014 [2]浙江省桐庐汇丰生物科技有限公司,浙江杭州311500

出　　处：《发酵科技通讯》2018年第4期204-208,共5页Bulletin of Fermentation Science and Technology

基　　金：浙江省自然科学基金资助项目(LQ14C010001)

摘　　要：烷基间苯二酚合酶(ARS)属于研究蛋白结构与功能关系的模式分子——Ⅲ型聚酮合酶家族,其产物烷基间苯二酚是一种具有极其重要生物学功能活性的物质。为进一步提高ARS的酶活性,研究在成功实现ARS在大肠杆菌中进行温控自裂解异源表达的基础上,通过易错PCR及定点随机突变构建酶基因突变文库,并结合高通量筛选,获得酶活力显著提高的突变子ARSSd-3(A207S/M266A/T256S)。酶学性质研究发现:ARSSd-3的底物选择性向长链脂肪酰SNAC略有偏移;当以棕榈酰-SNAC为底物时,ARSSd-3的酶活力是ARS的1.55倍,米氏常数(Km)降低至(6.8±1.3)μmol/L,最大反应速率(Vmax)增加至(234.1±9.8)pmol/(min·nmol)。研究结果对探究ARS的催化机制及其生产应用具有一定意义。Alkylresorcinol synthase(ARS)belongs to the typeⅢpolyketone synthase family,which is a model in study of relationship between protein structure and function.In order to improve activity of ARS,a mutant ARS Sd-3(A207S/M266A/T)with enhanced enzyme activity was obtained by error-prone PCR,site-random mutation and high throughput screening on the basis of successful heat-inducible autolytic heterologous expression of ARS in E.coli.The studies showed that substrate selectivity of the mutant shifted slightly to long chain fatty acyl-SNAC.The enzyme activity of ARS Sd-3was1.55times higher than that of wild-type ARS with palmitoyl-SNAC as substrate.Meanwhile,the Michaelis constant(K m)and the V max of ARS Sd-3were(6.8±1.3)μmol/L and(234.1±9.8)pmol/(min·nmol)respectively.Our findings are helpful to explore the catalytic mechanism of ARS and expand its application.

关 键 词：烷基间苯二酚合酶 分子改造 易错PCR 定点随机突变 酶学性质 

分 类 号：TS201.3[轻工技术与工程—食品科学]