田菁根瘤菌嗜盐α-淀粉酶的基因克隆表达与分子改造

Rhizobium sesbania Halophilic Alpha-Amylase Gene Cloning and Enzymology Characteristics Research

【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 RhmoSiuni sesbadiu and surrounding soil. RSA-D205 site was found tohave 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,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 rela-tionship 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.