Protein engineering of beta-amylase

β-淀粉酶的蛋白质工程

基本信息

批准号：
06660104
负责人：
MIKAMI Bunzo
金额：
$ 1.28万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660104/
关键词：
beta-amylase protein engineering x-ray crystal analysis

项目摘要

For the study of protein engineering of enzymes, X-ray crystallographic analysis and the over-production of the mutated enzymes are inevitable techniques. The author has determined the SH-modified soybean beta-amylases as a model of the mutated enzyme and also developed the over-production system of soybean beta-amylase in Escherichia coli.1. X-ray crystallography of the SH-modified soybean beta-amylaseTwo sulfhydryl groups (Cys95 and Cys343) in soybean beta-amylase exist near the active site of the enzyme. The role of SH (S-S) groups of b-amylase from soybean and Bacillus cereus have been investigated by chemical modification. The three dimensional structures of the complexes of Cys95 and Cys343 blocked soybean b-amylase with maltose or glucose have been determined at around 2.0 A resolution. Two maltose molecules could bind to subsite 1-4 as is the case of the native enzyme complex except for the position of the flexible loop (residues from 96 to 103). In the modified enzyme, the loop position could not be determined owing to high temperature factors, suggesting that the modified side chain of Cys95 inhibit the loop movement from open to closed conformation. The X-ray crystal analysis of Cys343-blocked enzyme/maltose complex showed the normal maltose binding except for the main chain positions of Thr342 and Cys343. It seems that the altered Thr342 residue affect the catalysis of the enzyme by the break of hydrogen bond with catalytic water.2. Over-production system of soybean beta-amylaseSoybean beta-amylase cDNA has been efficiently expressed in Escherichia coli.with pkk233-2 vector. The purified enzyme from the extract of Escherichia coli.showed the same enzymatic properties as the enzyme from soybean seeds except for the N-terminal blocked residue. The qurified enzyme has been crystallized in the same way as the seed enzyme. The X-ray crystallographic study of the expressed enzyme is now in progress.

为了研究酶的蛋白质工程，X射线晶体学分析和突变酶的过度生产是不可避免的技术。作者已经确定了SH修饰的大豆β-淀粉酶作为突变酶的模型，并在大肠杆菌中开发了大豆β-淀粉酶的过度生产系统。 SH修饰的大豆 - 氨基甲基乙酰胺基团的X射线晶体学（Cys95和Cys343）在大豆β-淀粉酶中存在于酶的活性位点附近。已经通过化学修饰研究了大豆和蜡状芽孢杆菌的B-淀粉酶Sh（S-S）组的作用。 Cys95和Cys343的络合物的三维结构用麦芽糖或葡萄糖阻断了大豆B-淀粉酶，在2.0 A左右的分辨率下确定。除了柔性环的位置（残基96至103）外，两个麦芽糖分子可以与本机酶复合物的情况结合，就像天然酶复合物一样。在改良的酶中，由于高温因子，无法确定环位位置，这表明Cys95的修饰侧链抑制了从开放到封闭构型的环运动。 Cys343阻滞酶/麦芽糖复合物的X射线晶体分析显示出正常的麦芽糖结合，除了THR342和CYS343的主链位置。似乎改变的Thr342残基通过与催化水的键断裂会影响酶的催化。2。大豆β-淀粉酶β-淀粉酶cDNA的过量生产系统已在大肠杆菌中有效表达。pkk233-2载体。大肠杆菌提取物的纯化酶表现出与来自大豆种子的酶相同的酶特性，除了N末端阻塞的残基。 Qurized酶的结晶方式与种子酶相同。现在正在进行的表达酶的X射线晶体学研究正在进行中。