青霉素G酰化酶活性的荧光测定法。

Fluorogenic assay for penicillin G acylase activity.

作者信息

Ninkovic M, Riester D, Wirsching F, Dietrich R, Schwienhorst A

机构信息

Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstrasse 8, Goettingen, 37077, Germany.

出版信息

Anal Biochem. 2001 May 15;292(2):228-33. doi: 10.1006/abio.2001.5078.

DOI:10.1006/abio.2001.5078

PMID:11355855

Abstract

A simple, highly sensitive, and rapid assay for high-throughput screening of penicillin G acylase-producing bacteria is presented. The method is based on the specific release of fluorescent 7-amino-4-methyl-coumarin through cleavage of phenylacetyl-4-methyl-coumaryl-7-amide by penicillin G acylase. The present method is suitable for screening pure enzymes as well as various penicillin G acylases like those from Escherichia coli, Proteus rettgeri, and Kluyvera citrophila in cell extracts. In addition, the new substrate was used for rapid assay of amidase activity in nondenaturing polyacrylamide gels.

摘要

本文介绍了一种用于高通量筛选产青霉素G酰化酶细菌的简单、高灵敏度且快速的检测方法。该方法基于青霉素G酰化酶裂解苯乙酰-4-甲基-香豆素-7-酰胺，特异性释放荧光7-氨基-4-甲基香豆素。本方法适用于筛选纯酶以及细胞提取物中各种青霉素G酰化酶，如来自大肠杆菌、雷氏变形杆菌和嗜柠檬酸克吕沃氏菌的酶。此外，新底物还用于在非变性聚丙烯酰胺凝胶中快速检测酰胺酶活性。