环糊精水解酶cds1-3底物通道氨基酸的定点突变研究

Site-directed Mutagenesis of Amino Acid in the Substrate of Cyclodextrin Hydrolase cds1-3

关键词:  环糊精水解酶  同源建模  底物通道  定点突变  蛋白结构  氨基酸

Key words:  cyclodextrin hydrolase  homology modeling  substrate channel  site-directed mutation  protein structure  amino acid

摘要:

本研究对具有大分子水解能力的环糊精水解酶cds1-3的蛋白结构进行分析，选取底物通道相关氨基酸进行定点突变。通过比较突变酶和野生酶的功能差异，定位决定cds1-3特殊功能的氨基酸。采用sybyl 1.2进行蛋白质底物结合分析，选取和多聚体形成、底物结合以及底物通道相关的氨基酸Glu66、Pro48、Phe289为突变位点，反向PCR构建pSE380/E66G、pSE380/P48H、pSE380/F289A表达质粒并进行表达，获得酶活突变体并与原始酶进行底物特异性比较分析。其结果显示，突变酶E66G降解大分子底物木薯淀粉和支链淀粉的相对酶活力分别提高26.96%和23.15%，而对小分子底物普鲁兰糖的水解能力下降13.14%。因此，cds1-3是一个能水解大分子底物的特殊环糊精水解酶，氨基酸Glu66是cds1-3水解大分子支链淀粉的关键氨基酸之一。

Abstract:

By analyzing the protein structure of the cyclodextrin hydrolase cds1-3 with macromolecular hydrolysis ability,the substrate-related amino acids were selected for site-directed mutagenesis.By comparing the functional differences between the mutant enzyme and the wild enzyme,the amino acids that determine the specific function of cds1-3 were located.Protein substrate binding analysis was performed using sybyl 1.2.The amino acids Glu66,Pro48,and Phe289 related to multimer formation,substrate binding,and substrate channel were selected as mutation sites.The pSE380/E66G,pSE380/P48H,pSE380/F289A expression plasmids were constructed by reverse PCR and expressed.Enzyme-activated mutants were obtained and compared with the original enzyme for substrate specificity analysis.The relative enzyme activities of the mutant enzyme E66G degrading macromolecular substrates such as tapioca starch and amylopectin were increased by 26.96% and 23.15%,respectively,while the hydrolysis ability of the small molecule substrate pullulan was decreased by 13.14%.Therefore,cds1-3 is a special cyclodextrin hydrolase capable of hydrolyzing macromolecular substrates,and amino acid Glu66 is one of the key amino acids of cds1-3 hydrolyzed macromolecular amylopectin.