Domain engineering of β-amylase

β-淀粉酶的结构域工程

基本信息

批准号：
10660084
负责人：
MIKAMI Bunzo
金额：
$ 2.43万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660084/
关键词：
β-amylase protein engineering x-ray crystal analysis starch binding starch-binding domain

项目摘要

In order to design new functional enzyme, point mutations and domain substitution are important techniques of protein engineering. We have determined the structures of β-amylase from soybean, wheat and Bacillus cereus by X-ray crystal analysis. The bacterial β-amylase has C-terminal starch binding domain and is able to digest low starch granules. Plant and bacterial enzymes have different pH optima which is ascribed to their environment of the catalytic residues. The possibility to design new function of β-amylase was explored by protein engineering and X-ray crystal analysis.1. X-ray crystallography and design of strach binding domain of Bacillus cereus β-amylase.The C-terminal starch binding domain of Bacillus cereus β-amylase was expressed in Escherichia coli. and crystallized. The structure determined at 1.95 A resolution by X-ray crystallography revealed that one of the starch binding site out of two does not functional owing to the conformation change of a loop structure. In order to increase the starch binding ability mutations of the loop are now in progress.2. X-ray crystallography of the mutant soybean β-amylases which have increased pH optimum.The comparison of active site between plant and bacterial β-amylase suggested that hydrogen bond network exist around one of the catalytic residue (Glu380 in soubean β-amylase) only in the plant β-amylase. The three mutants of soybean enzyme (Met51T, Asn340T, Glu 178Y) expressed in E. coli. had increased pHoptimum about 0.5-1.0 pH unit toward the bacterial enzyme. X-ray crystallographic analysis at 2.0 A resolution demonstrated that all have broken hydrogen bonds with Glu380. We are now trying to make a mutant of B. cereus enxyme which have reduced pH optimum and are useful for industrial application.

为了设计新的功能酶，点突变和域取代是蛋白质工程的重要技术。我们通过X射线晶体分析确定了大豆，小麦和蜡状芽孢杆菌的β-淀粉酶的结构。细菌β-淀粉酶具有C末端淀粉结合结构域，并能够消化低淀粉颗粒。植物和细菌具有不同的pH Optia，分配给其催化残留物的环境。通过蛋白质工程和X射线晶体分析探索了设计β-淀粉酶新功能的可能性。1。 X射线晶体学和蜡状芽孢杆菌β-淀粉酶的Strach结合结构域的设计。在大肠杆菌中表达了蜡状杆菌β-淀粉酶的C末端淀粉结合结构域。并结晶。在1.95确定的X射线晶体学分辨率下确定的结构表明，由于循环结构的会议变化，两个淀粉结合位点之一不起作用。为了增加循环的淀粉结合能力突变，现在正在进行中2。突变大豆β-淀粉酶的X射线晶体学，增加了pH值的最佳pH值。植物与细菌β-淀粉酶之间的活性位点的比较表明，仅在催化居住区（SOUBEANβ-淀粉酶中的GLU380）中存在氢键网络，仅在植物β-淀粉酶中存在。大豆酶的三个突变体（Met51t，Asn340t，Glu 178y）在大肠杆菌中表达。对细菌酶的phopt pH值增加了约0.5-1.0 pH。 X射线晶体学分析在2.0 A分辨率表明所有氢键与GLU380的氢键断裂。现在，我们正在尝试制造一个蜡状芽孢杆菌的突变体，该突变体降低了pH值的最佳，并且对工业应用很有用。