催化学报

催化学报 ›› 2012, Vol. 33 ›› Issue (10): 1717-1723.DOI: 10.3724/SP.J.1088.2012.20606

• 研究论文 • 上一篇    下一篇

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

李贵祥, 徐铮, 李莎, 徐虹*   

  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
  • 收稿日期:2012-06-06 修回日期:2012-07-26 出版日期:2012-09-28 发布日期:2012-09-28

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

LI Guixiang, XU Zheng, LI Sha, XU Hong*   

  1. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Food Science and Light Industry, Nanjing University of Technology, Nanjing 210009, Jiangsu China
  • Received:2012-06-06 Revised:2012-07-26 Online:2012-09-28 Published:2012-09-28

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1052

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4

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.

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

Abstract: 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.

Key words: L-arabinose isomerase, D-tagatose, Lactobacillus fermentum, homology modeling, site-directed mutagenesis