阿特拉津氯水解酶基因的定点诱变和酶活力检测

阿特拉津氯水解酶(AtzA)是一种对除草剂的生物降解和环境净化有重要意义的酶．采用定点诱变方法将假单胞菌ADP株的atzA基因第832位碱基(鸟嘌呤)诱变成腺嘌吟，然后将其插入表达载体pET21b( )，并在大肠杆菌中表达．表达的蛋白特性研究表明：AtzA或AtzA—NK融合蛋白系水溶性蛋白，很容易通过Ni—NTA Magnetic Agarose Beads分离纯化．采用阿特拉津脱氯反应产生HCl而引起pH指示剂颜色改变的测定方法能方便地对其酶活力进行定量．酶活力结果表明，突变酶的比活力与假单胞菌ADP菌株的AtzA相比没有明显改变，暗示突变位点(第278位缬氨酸突变成甲硫氨酸)不是酶的活性中心或底物结合部位。

SITE-DIRECTED MUTAGENSIS OF ATRAZINE CHLOROHYDRALASE GENE AND DETECTION OF ITS ACTIVITY

Atrazine chlorohydralase (AtzA) is an important enzyme, which biodegrade the herbicide and decontaminate the environment. The 832nd base (guanine) in the atzA gene from Pseudomonas sp. strain ADP was mutagenized into adenine by site - directed mutagenesis. Then it was inserted into an expression vector pET21b( + ) and expressed a water-soluble fusion protein AtzA or AtzA-NK in E. coli. Thus it was easy to isolate and purify the proteins by Ni-NTA Magnetic Agarose Beads. The activities were detected through the change of pH indicator color owing to the production of hydrochloric acid when atrazine was dechlorinated. The mutation AtzA-NK and AtzA from Pseudomonas sp. strain ADP have the same activity suggesting the mutation site, which is at 278th with valine mutagenized into methionine, is not an activity domain or a substrate-combing site.