田菁根瘤菌嗜盐α-淀粉酶的基因克隆表达与分子改造

摘要

[目的]克隆表达田菁根瘤菌嗜盐α-淀粉酶基因,并对其进行基因改造,为探索与嗜盐性相关的氨基酸位点提供参考.[方法]从田菁根瘤菌及周围土壤的宏基因组中克隆到一个命名为RSA的极端嗜盐淀粉酶.通过与同源性为75.33%的嗜盐α-淀粉酶K6的序列比对发现,RSA-D205位点与已报道的K6-N204(嗜盐相关的氨基酸位点)相似,从而对其进行定点饱和突变以研究相关酶学性质.[结果]共获得17个突变子,其最适NaCl浓度较RSA均有不同程度的降低.RSA-D205R、RSA-D205C酶反应温度高达80℃,拓宽了其应用范围.[结论]RSA-D205氨基酸残基位点与Na+结合位点有一定的联系;另外,精氨酸(R)、半胱氨酸(C)对淀粉酶的高温改造具有参考价值.%[Objective]The study was to provide the reference for exploring the amino acid sites related to halophilic characteristics by cloning,expressing and genetically modifying the gene of Rhizobium sesbania.[Methods]An extreme halophilic amylase named RSA was cloned from the metagenome of Rhizobium sesbania and surrounding soil.RSA-D205 site was found to have a similarity to the reported K6-N204 (amino acid site related to halophilic characteristics) at sequence homology level about 75.33% with halophilic amylase K6.In order to study the relevant enzymatic properties,a site directed saturation mutagenesis was performed.[Results]A total of 17 mutants were obtained,and the optimum NaCl concentration was lower than that of RSA in various degree.However,the reaction temperature of RSA-D205R and RSA-D205C were both up to 80℃,which broadened their application.[Conclusion]There is a certain relationship between the amino acid residues of RSA-D205 and Na+ binding sites,and the arginine (R) and cysteine (C) have the reference value for the modification of amylase.