基于同源建模和定点突变技术研究嗜热型L-阿拉伯糖异构酶与D-半乳糖的亲和作用  

Probing the Essential Catalytic Residues and Substrate Affinity in Thermophilic L-Arabinose Isomerase by Homology Modeling and Site-Directed Mutagenesis

作　　者：李贵祥[1] 徐铮[1] 李莎[1] 徐虹[1] 

机构地区：[1]南京工业大学食品与轻工学院材料化学工程国家重点实验室,江苏南京210009

出　　处：《催化学报》2012年第10期1717-1723,共7页

基　　金：国家高技术研究发展计划(863计划;2012AA021503);国家自然科学基金(20906050);高等学校博士学科点专项科研基金(20103221110006)~~

摘　　要：通过同源建模分析选取对Lactobacillus fermentum CGMCC2921来源的L-阿拉伯糖异构酶(简称L-AI酶)催化D-半乳糖生产D-塔格糖起重要作用的氨基酸位点进行突变,发现当Q16,M311,K423和Q438位点的氨基酸突变为丙氨酸时,突变酶Km值降低,其中突变酶M311A降至本体的51.6%,对D-半乳糖的转化率提高了18.7%.当K423位点的氨基酸残基分别突变为丙氨酸、天冬酰胺或精氨酸时,突变酶与底物的亲和力以及D-半乳糖的转化率随着423位点突变氨基酸侧链长度的增加而降低.运用计算机分子模拟技术分析表明,当M311位点氨基酸突变为丙氨酸以后,催化位点氨基酸残基与底物D-半乳糖之间的氢键作用增强,导致与底物亲和力增大,从而提高了酶活力.The L-arabinose isomerase from Lactobacillus fermentum CGMCC2921（named LFAI） was distinguished from other L-AIs by its outstanding thermostability,and was defined as a potential candidate for industrial D-tagatose production.By means of homologous modeling and structure analysis,some important amino acid residues influencing D-galactose isomerization of LFAI were selected and mutated.The results showed that when residues Q16,M311,K423,and Q438 mutated to alanine,the Km value of the mutant LFAI decreased.Among them,mutant enzyme M311A retained half of its original Km value,and the conversion rate for D-galactose raised approximately 20%.Furthermore,by comparing mutants K423R,K423N,K423A,and native LFAI,it was found that the side-chain length of residue K423 may determine the substrate affinity and D-galactose conversion rate of these mutated enzymes.Through computer molecular modeling,it was also found mutation M311A had an enhancement on hydrogen bonding with D-galactose,thus resulting in an enhancement on its substrate affinity and enzyme activity.

关 键 词：L-阿拉伯糖异构酶 D-塔格糖 发酵乳杆菌 同源建模 定点突变 

分 类 号：Q55[生物学—生物化学]