Studies on protein engineering of a microbial elastase

微生物弹性蛋白酶的蛋白质工程研究

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03556011/
• 关键词: alkaline elastase; meat-tenderizer; subtilisin; protein engineering; 食肉軟化剤; サチライシンE; エラスタ-ゼ; 枯草菌; 大腸菌; 食肉軟化

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.

嗜碱芽孢杆菌Ya-B菌株分泌碱性弹性酶，在高碱性范围内具有较高的活性。单独携带碱性弹性酶结构基因的质粒转化枯草芽孢杆菌不会产生和/或分泌活性弹性酶。而枯草芽孢杆菌用携带克隆弹性酶基因及其5'侧区（约800 bp）的质粒转化后，可分泌活性弹性酶。我们测定了5'侧区整个核苷酸序列，发现一个编码49个氨基酸的开放阅读框。这个名为aleR的基因似乎是弹性蛋白酶基因在枯草芽孢杆菌上表达所必需的。我们假设AleR蛋白是前弹性酶的一种激活酶。另一方面，碱性弹性蛋白酶有望成为一种潜在的肉类嫩化剂。众所周知，生肉的pH值为5-6。在pH值为6.0的条件下，测定了碱性弹性蛋白酶的肉嫩化活性。该酶对胶原蛋白和弹性蛋白仍有较大的活性，对肌原纤维的作用较小。因此，这种酶被证明适合用于肉的嫩化。最后研究了弹性蛋白酶的结构与活性之间的关系。碱性弹性酶与枯草菌素BPN′和枯草菌素E的序列同源性为55%，通过一级氨基酸序列的比较，我们在枯草菌素E中引入了4个氨基酸缺失，观察到枯草菌素E明显改变为弹性酶样酶。

项目成果

H.Takagi,H.Matsuzawa,T.Ohta,M.Yamasaki,M.Inouye: "Studies on the structure and function of subtilisin E by protein engineering" Ann.N.Y.Acad.Sci.30. 52-59 (1992)

H.Takagi、H.Matsuzawa、T.Ohta、M.Yamasaki、M.Inouye：“通过蛋白质工程研究枯草杆菌蛋白酶 E 的结构和功能”Ann.N.Y.Acad.Sci.30。

H.Takagi,M.Kondou,T.Hisatsuka,S.Nakamori,M.Yamasaki: "Effects of an alkaline elastase from an alkalophilic Bacillus strain on the tenderization of beef meat" J.Agric.Food Chem.40. 2364-2368 (1992)

H.Takagi、M.Kondou、T.Hisatsuka、S.Nakamori、M.Yamasaki：“嗜碱性芽孢杆菌菌株的碱性弹性蛋白酶对牛肉嫩化的影响”J.Agric.Food Chem.40。

H.TAKAGI,H.MATSUZAWA,T.OHTA,M.YAMASAKI,M.INOUYE: "Studies on the structure and function of subtilisin E by protein engineering" Ann.N.Y.Acad.Sci.672. 52-59 (1992)

H.TAKAGI、H.MATSUZAWA、T.OHTA、M.YAMASAKI、M.INOUYE：“通过蛋白质工程研究枯草杆菌蛋白酶 E 的结构和功能”Ann.N.Y.Acad.Sci.672。

H.TAKAGI,M.KONDOU,T.HISATSUKA,S.NAKA-MORI,M.YAMASAKI: "Effects of an alkaline elastase from an alkalophilic Bacillus strain on the tenderization of beef meat" J.Agric. Food Chem.40. 2364-2368 (1992)

H.TAKAGI、M.KONDOU、T.HISATSUKA、S.NAKA-MORI、M.YAMASAKI：“嗜碱芽孢杆菌菌株的碱性弹性蛋白酶对牛肉嫩化的影响”J.Agric。

H.Takagi, S.Arafuka, M,Inouye, M.Yamasaki: "The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis." J. Biochem.111. 584-588 (1992)

H.Takagi、S.Arafuka、M、Inouye、M.Yamasaki：“基于与微生物碱性弹性蛋白酶的结构比较，枯草杆菌蛋白酶 E 中氨基酸缺失对其底物特异性和催化作用的影响。”