易错PCR提高粘质沙雷氏菌磷脂酶A1活性  

Improving the Activity of Phospholipase A1 from Serratia Marcescs PL-06 by Error-prone PCR

作　　者：王玉满[1] 薛正莲[1] 王洲[1] 苏燕南[1] 朱昊[1] 

机构地区：[1]安徽工程大学生物与化学工程学院,安徽芜湖241000

出　　处：《安徽工程大学学报》2016年第4期6-11,共6页Journal of Anhui Polytechnic University

基　　金：国家自然科学基金资助项目(31471615)

摘　　要：利用易错PCR技术对来自粘质沙雷氏菌PL-06的磷脂酶A1基因plaB进行定向改造,通过引入不同浓度Mg^(2+)、Mn^(2+)得到的易错PCR产物与表达型质粒pET-28a(+)重组,构建磷脂酶A1突变文库,筛选出高酶活突变株EPB8.该突变酶相对野生酶酶活提高了22.5%,并对突变株EPB8产酶条件进行优化,优化后酶活较野生酶酶活提高41%.序列分析表明:plaB基因有11个碱基发生突变,导致7个氨基酸发生突变,其中磷脂酶A1蛋白有3个氨基酸突变,分别是Y^(97) C、P^(98)S、X^(228) L,辅助蛋白有4个氨基酸突变,分别是P^(46) L、Q^(58) L、N1^(36) D、H^(233) R.该实验结果为进一步在分子水平定向提高磷脂酶A1的活性奠定了基础,也为该酶在其他高表达系统中的表达提供了素材.In order to obtain phospholipase A1 with higher activity, error-prone PCR was used to evolve the phospholipase A1 from Serratia marcescens PL-06 by adding different concentration of Mg^2＋ and Mn^2＋.The gene was cloned into pET28a（＋）expression vector, and a mutant library was constructed. After the first and secondary screening, one mutant showed significant elevated activity, named EPB8. The enzyme activily of the mutants was 22.5N higher than that of the wild-type control.And increased by 41% after optimal Fermentation conditions. The sequence of the plaB gene of the mutants showed 7 mutated amino adds.It was found that phospholipase A1 protein revealed three amino acid substitutions： Y97 C, P98 S, X228 L, and auxiliary protein revealed four amino acid substitutions ： p46 L, Q58 L, N138 D, H233 R. These results provide a basis for further research of phospholipase A1.

关 键 词：粘质沙雷氏菌 磷脂酶A1 易错PCR 产酶条件优化 

分 类 号：Q786[生物学—分子生物学]