广西科学2019，Vol.26Issue(4)：410-416,7.DOI:10.13656/j.cnki.gxkx.20190808.006
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 Cy-clodextrin Hydrolase cds1-3
摘要
Abstract
By analyzing the protein structure of the cyclodextrin hydrolase cdsl-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 cdsl-3 were located. Protein substrate binding analysis was performed using sybyl 1.2. The amino acids G1u66，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 PLR 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，cdsl-3 is a special cyclodextrin hydrolase capable of hydrolyzing macromolecular substrates，and amino acid G1u66 is one of the key amino acids of cdsl-3 hydrolyzed macromolecular amylopectin.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分类
生物科学引用本文复制引用
汤宏赤1,莫莉2,闭海2,林丽华1,郭媛1,庞浩1,2**..环糊精水解酶cds1-3底物通道氨基酸的定点突变研究[J].广西科学,2019,26(4):410-416,7.基金项目
广西科技基地和人才专项(桂科 AD16380016),广西重点研发计划(桂科 AB17190534)和广西自然科学基金项目(2018GXNSFAA294047)资助. （桂科 AD16380016）
