嗜热子囊菌Thermoascus crustaceus JCM12803来源的低温α-淀粉酶功能验证及其适冷机制分析

Characterization and cold-adaptation mechanism of a cold-active α-amylase from Thermoascus crustaceus JCM12803

摘要:

[目的]挖掘新颖的低温α-淀粉酶基因资源，并对其适冷机制进行分析，可以加深我们对低温酶的认识并为酶分子改良提供科学依据。[方法]根据嗜热子囊菌（Thermoascus crustaceus） JCM12803全基因组序列信息，利用PCR的方法获得一个α-淀粉酶基因Tcamy，将其插入至表达载体pPIC9后，在巴斯德毕赤酵母（Pichia pastoris） GS115中异源表达，测定其酶学性质。同时采用氨基酸序列分析和同源建模的方法获得其三维结构，分别从蛋白序列-结构-功能层面上研究其适冷机制。[结果]TcAmy是一个典型的低温α-淀粉酶，最适温度35℃，在0℃下保持有27%的活性。序列和结构分析表明该酶N-糖基化修饰程度低，Arg和Pro含量低而Gly含量高且二硫键和离子键较少。[结论]本研究获得了一个低温α-淀粉酶，低N-糖基化以及特殊的氨基酸组成和蛋白分子内作用力是其适应低温的根本原因。

Abstract:

[Objective] To mine the genetic resource of novel cold-active α-amylases and reveal their cold-adaptation mechanism are of importance to deepen our understanding of cold-active enzyme and provide key information for the molecular improvement of α-amylase.[Methods] Based on the genome sequence of Thermoascus crustaceus JCM12803, we cloned an α-amylase-encoding gene (Tcamy), and inserted it into the expression vector pPIC9. The gene product was heterologously expressed in Pichia pastoris GS115 and characterized. By using amino acid sequence analysis and homologous modeling, we studied the cold-adaptation mechanism of TcAmy in viewpoint of sequence-structure-function relationship.[Results] TcAmy is a typical cold-active α-amylase, showing optimal activity at 35℃ and remaining 27% maximal activity even at 0℃. Sequence and structure analysis indicated that in comparison to thermostable counterparts, TcAmy has low N-glycosylation degree, decreased Pro and Arg contents and increased Gly content, and less disulfide bridges and ionic bonds.[Conclusion] We obtained a novel cold-active α-amylase, with low N-glycosylation degree, specific amino acid composition and intermolecular interactions, all contributing to its cold-adapted property.