来自鱿鱼（美洲大赤鱿）肝胰腺的一种羧肽酶的纯化与特性分析

Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus).

作者信息

Raksakulthai R, Haard N F

机构信息

Department of Food Science and Technology, University of California at Davis, Davis, California 95616, USA.

出版信息

J Agric Food Chem. 2001 Oct;49(10):5019-30. doi: 10.1021/jf010320h.

DOI:10.1021/jf010320h

PMID:11600061

Abstract

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.

摘要

基于对N - CBZ - 二肽底物的水解作用，鱿鱼（Illex illecebrosus）提取物的肝胰腺含有多种羧肽酶（CP）活性。以N - CBZ - Phe - Leu为底物的SDS - PAGE酶谱显示出三个活性区（CP - I，23 kDa；CP - II，29 kDa；CP - III，42 kDa）。通过DEAE - 纤维素柱层析、凝胶过滤和色谱聚焦，基于N - CBZ - Ala - Phe活性，CP - I被纯化了225倍，回收率为86.20%。纯化后的酶对N - CBZ - 二肽具有广泛的特异性；然而，它更倾向于C末端带有疏水氨基酸的底物。CP - I对N - CBZ - Ala - Phe的活性最高（比活性 = 7104单位/毫克蛋白质，K(m) = 0.40 mM，生理效率 = 22863）。CP - I的pI为3.4，是一种金属蛋白酶，被Co(2+)激活，并被丝氨酸蛋白酶抑制剂Pefabloc部分抑制。对于N - CBZ - Ala - Phe和Gly - Phe，它在pH 8和70℃时具有最佳活性。鱿鱼CP - I的氨基酸组成与其他物种的CP A相似。