Fluorogenic assay for penicillin G acylase activity.
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.
Ninkovic
M
M
Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstrasse 8, Goettingen, 37077, Germany.
Riester
D
D
Wirsching
F
F
Dietrich
R
R
Schwienhorst
A
A
D003374:Coumarins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D007202:Indicators and Reagents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010399:Penicillin Amidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D003374:Coumarins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D010399:Penicillin Amidase; C028743:7-amino-4-methylcoumarin