Studies on protein engineering of a microbial elastase

微生物弹性蛋白酶的蛋白质工程研究

基本信息

批准号：
03556011
负责人：
YAMASAKI Makari
金额：
$ 6.91万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

Alkalophilic Bacillus sp. Ya-B strain secretes alkaline elastase which has high activity in the high alkaline range. Bacillus subtilis transformed with a plasmid carrying the alkaline elastase structural gene alone does not result in the production and/or secretion of active elastase. B. subtilis transformed with a plasmid carrying the cloned elastase gene and its 5'-flanking region (about 800 bp), however, can secrete active elastase. We determined the whole nucleotide sequence of the 5'-flanking region and found an open reading frame encoding 49 amino acids. The gene named aleR seems to be necessary for the expression of elastase gene on B. subtilis. We postulate that AleR protein is an activating enzyme of pro-elastase.On the other hand, alkaline elastase is expected as a potential meat tenderizer. It is known that the pH of raw meat is 5-6. We tested the meat tenderizing activity of alkaline elastase at pH 6.0. The enzyme still shows substantial activities toward collagen and elastin with small effects to myofibril. The enzyme, therefore, is proven to be suitable for meat tenderizing use.Finally we studied the relationship between the structure and activity of elastase. Alkaline elastase has a sequence homology of 55% to subtilisin BPN' and subtilisin E. From the comparison of primary amino acid sequence, we introduced 4 acid amino deletion into subtilisin E, and observed a distinct alteration of subtilisin E to an elastase-like enzyme.

碱性芽孢杆菌YA-B菌株分泌碱性弹性酶，其在高碱性范围内具有较高的活性。枯草芽孢杆菌用仅带有碱性弹性蛋白酶结构基因的质粒转化并不能导致活性弹性酶的产生和/或分泌。枯草芽孢杆菌用带有克隆的弹性酶基因及其5'-垂直区域（约800 bp）的质粒转化，但是，可以分泌活性弹性酶。我们确定了5'Fancing区域的整个核苷酸序列，并找到了编码49个氨基酸的开放式阅读框架。名为Aler的基因似乎对于在枯草芽孢杆菌上表达弹性酶基因是必不可少的。我们假设Aler蛋白是一种丙酶的激活酶。另一方面，碱性弹性酶被预期为潜在的肉类嫩剂。众所周知，生肉的pH值为5-6。我们在pH 6.0时测试了碱性弹性蛋白酶的肉类嫩化活性。该酶仍然显示出对胶原蛋白和弹性蛋白的实质性活性，对肌原纤维产生小的作用。因此，该酶被证明适用于肉类嫩化的用途。在本文中，我们研究了弹性酶的结构和活性之间的关系。碱性弹性酶的序列同源性为55％，可对枯草脂蛋白BPN'和枯草脂蛋白E。根据原代氨基酸序列的比较，我们将4个酸氨基缺失引入了枯草脂中，并观察到枯草菌素E的明显变化对烯类酶样酶。