Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase.A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. ADI was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type.The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.%阿特拉津氯水解酶定向改造的关键是开发一种廉价的、表型改变明显的高通量筛选方法.利用高错误倾向PCR和DNA洗牌相结合的突变方法,对来源于假单胞菌ADP和节杆菌ADI的阿特拉津氯水解酶基因进行随机突变,以雨生红球藻为受体、以阿特拉津为选择压力对突变文库进行高通量筛选.筛选到的12个突变子序列分析显示,突变均为点替换,位点分散在全基因上,是在高错误倾向PCR及DNA洗牌过程中逐渐累积形成的.酶活力分析显示,突变子的酶活力均高于野生株,在添加1.0 mg/L阿特拉津培养液中的活力是野生株的1.9～3.6倍,在添加2.0 mg/L阿特拉津培养液中的活力是野生株的1.7～2.6倍,粗酶提取物的活力是野生株的1.7～2.7倍.上述结果表明,雨生红球藻表达系统是定向改造阿特拉津氟水解酶的高通量筛选平台.
展开▼
