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菌株分泌的碱性弹性蛋白酶在高碱性范围内具有较高的活性。用携带碱性弹性蛋白酶结构基因的质粒单独转化的枯草芽孢杆菌不导致活性弹性蛋白酶的产生和/或分泌。B。然而，用携带克隆的弹性蛋白酶基因及其5 ′-侧翼区（约800 bp）的质粒转化的枯草杆菌可分泌活性弹性蛋白酶。我们测定了该基因5 '端侧翼区的核苷酸序列，发现一个开放阅读框，编码49个氨基酸。aleR基因可能是弹性蛋白酶基因在B细胞上表达所必需的。枯草杆菌。我们推测AleR蛋白是弹性蛋白酶原的激活酶，而碱性弹性蛋白酶则是一种潜在的肉嫩化剂。据了解，生肉的pH值为5-6。在pH6.0的条件下，测定了碱性弹性蛋白酶的嫩化活性。该酶对胶原蛋白和弹性蛋白仍有很大的活性，对肌原纤维的影响很小。最后，对弹性蛋白酶的结构与活性之间的关系进行了研究。碱性弹性蛋白酶与枯草杆菌蛋白酶BPN ′和枯草杆菌蛋白酶E具有55%的序列同源性。通过对枯草杆菌蛋白酶E一级氨基酸序列的比较，我们将4个氨基酸的缺失引入到枯草杆菌蛋白酶E中，观察到枯草杆菌蛋白酶E明显改变为弹性蛋白酶样酶。

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。”