Fluorogenic assay for penicillin G acylase activity.

Area of Science:

• Biochemistry
• Enzymology
• Microbiology

Background:

• Penicillin G acylase is a crucial enzyme in pharmaceutical production.
• Efficient screening methods are needed for identifying and characterizing penicillin G acylase variants.
• Existing assays may lack the sensitivity or throughput required for large-scale screening.

Purpose of the Study:

• To develop a simple, sensitive, and rapid assay for high-throughput screening of penicillin G acylase.
• To validate the assay for detecting penicillin G acylase-producing bacteria and enzyme activity.
• To demonstrate the utility of the assay for analyzing amidase activity in various contexts.

Main Methods:

• Development of a novel fluorescent substrate: phenylacetyl-4-methyl-coumaryl-7-amide.
• Assay principle based on the specific cleavage of the substrate by penicillin G acylase, releasing fluorescent 7-amino-4-methyl-coumarin.
• Application of the assay for screening bacterial cell extracts and nondenaturing polyacrylamide gels.

Main Results:

• The assay demonstrated high sensitivity and rapidity for detecting penicillin G acylase activity.
• Successfully screened penicillin G acylase from various bacterial sources, including Escherichia coli, Proteus rettgeri, and Kluyvera citrophila.
• Validated the assay for analyzing amidase activity directly in nondenaturing polyacrylamide gels, facilitating enzyme characterization.

Conclusions:

• The developed fluorescent assay provides an efficient tool for high-throughput screening of penicillin G acylase.
• This method is versatile, applicable to both pure enzymes and bacterial cell extracts.
• The assay's ability to analyze amidase activity in gels enhances its utility in enzyme research and development.