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

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.