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A sensitive procedure for screening microorganisms for the presence of penicillin amidase
Abstract:
A procedure is described for screening bacteria for the presence of penicillin amidase. Cells, grown in the presence of phenylacetic acid, are incubated with phenoxymethylpenicillin (type I), benzylpenicillin (type II) or ampicillin and the 6-aminopenicillanic acid formed is detected and quantitatively estimated by its strong reaction with fluorescamine at pH 4. There is no requirement for separation of the penicillin substrate from the product but when alpha-aminobenzylpenicillin derivatives are used as enzyme substrates the amount of 6-aminopenicillanic acid formed must be determined by calculation. The procedure allowed positive and reliable identification of penicillin amidases in six organisms known to produce the enzyme and indicated that some of these enzymes had different properties in reactivity towards alpha-aminobenzylpenicillin derivatives.
Insights
This study presents a new method for detecting penicillin amidase in bacteria using fluorescamine. The assay reliably identifies the enzyme and its varying reactivity with different penicillin substrates.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- Penicillin amidase is a crucial enzyme in antibiotic production.
- Accurate screening methods are needed to identify enzyme-producing organisms.
- Existing methods may lack sensitivity or require complex sample preparation.
Purpose of the Study:
- To develop and validate a simple, reliable screening procedure for bacterial penicillin amidase.
- To assess the enzyme's reactivity with various penicillin substrates.
- To enable efficient identification of penicillin amidase-producing bacteria.
Main Methods:
- Bacteria were cultured with phenylacetic acid.
- Cell suspensions were incubated with phenoxymethylpenicillin, benzylpenicillin, or ampicillin.
- 6-aminopenicillanic acid production was detected and quantified using fluorescamine assay at pH 4.
- Calculations were used for alpha-aminobenzylpenicillin derivative substrates.
Main Results:
- The fluorescamine-based assay successfully identified penicillin amidase in six known producing organisms.
- The method demonstrated reliability without the need for substrate-product separation.
- Variations in enzyme reactivity towards different alpha-aminobenzylpenicillin derivatives were observed.
Conclusions:
- A robust and straightforward assay for screening bacterial penicillin amidase is established.
- The assay facilitates the study of enzyme properties and substrate specificity.
- This method aids in the discovery and characterization of novel penicillin amidases.