【摘要】 【目的】红色亚栖热菌(Meiothermus ruber)海藻糖合酶(Trehalose synthase,M-TreS)将麦芽糖转化生成海藻糖只需一步反应,且具有很好的热稳定性及pH耐受性,是潜在的工业生产海藻糖的酶源。为了提高该酶的性能,有必要对其进行定向进化。【方法】M-TreS基因(M-treS)大小为2 889 bp。该蛋白质分子本身具有很大的进化空间,但是却不宜进行全长基因Shuffling。分段DNA shuffling是为大分子蛋白质(基因≥2 000 bp)的进化而设计的一种方法。该方法分为三步:(1)用两对引物分别扩增目的基因的上游片段和下游片段;(2)上下游片段各自进行Shuffling;(3)利用重叠延伸PCR连接上下游突变群,建立完整基因的突变文库。【结果】结合易错PCR,通过该方法经一轮进化获得一株酶活力是野生型1.6倍、催化效率是野生型2倍的突变株。序列分析表明,该突变株共有6个位点发生了氨基酸的替代,其中一个来自易错突变,2个来自同源重组,3个为随机突变。【结论】分段DNA shuffling是进化大分子蛋白质的有效方法。更多还原

【Abstract】 [Objective]The gene M-treS from Meiothermus ruber CBS-01 encodes a trehalose synthase of 962 amino acids,named M-TreS.To improve its catalytic activity,we constructed a method of molecular directed evolution,the sectionalized DNA shuffling.[Methods]Through two PCR steps with two pairs of partially complementary primers,the M-treS gene was parted into two sections.After the two sections shuffled respectively,a whole gene was obtained through the complementarity of the primers.This method was more feasible,with higher mutability than normal DNA shuffling.[Results]Mutants were obtained after one round of the sectionalized DNA shuffling,in combination with error-prone PCR.The best mutant enzyme contained 6 amino acid substitutions,whose catalytic activity and efficiency were 1.6-fold and 2-fold of that of the wild type,respectively.In the 6 amino acid substitutions,5 were caused by homologous recombination,and one by error-prone PCR.[Conclusion]This study indicates that the sectionalized DNA shuffling is an effective tool for molecular directed evolution of macromolecular proteins.更多还原