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Related Experiment Videos

Fluorogenic assay for penicillin G acylase activity.

M Ninkovic1, D Riester, F Wirsching

  • 1Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstrasse 8, Goettingen, 37077, Germany.

Analytical Biochemistry
|May 18, 2001
PubMed
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A new fluorescent assay enables rapid, high-throughput screening of penicillin G acylase. This method detects penicillin G acylase-producing bacteria and enzyme activity in cell extracts and gels.

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.

Related Experiment Videos

  • 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.