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A colorimetric assay for penicillin-V amidase
1Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Analytical Biochemistry
|March 1, 1993
Summary
A new colorimetric assay effectively quantifies penicillin-V amidase activity using a novel substrate. This method is crucial for determining levels of the enzyme, essential for producing semisynthetic penicillins.
Area of Science:
- Biochemistry
- Enzymology
- Industrial Microbiology
Background:
- Penicillin-V amidase enzyme is vital for producing 6-aminopenicillanic acid, a key intermediate for semisynthetic penicillins.
- Accurate and rapid assays for penicillin-V amidase are needed for industrial applications.
Purpose of the Study:
- To develop a rapid colorimetric assay for penicillin-V amidase.
- To characterize the kinetic properties of the enzyme using a novel substrate.
Main Methods:
- A new substrate, 2-nitro-5-(phenoxyacetamido)-benzoic acid, was synthesized for penicillin-V amidase.
- The released chromophore, 2-amino-5-nitrobenzoic acid, was detected spectrophotometrically at 405 nm.
- Enzyme kinetics (Km, Vmax) and inhibition (Ki) were determined using purified enzyme from Fusarium oxysporum.
Main Results:
- The assay demonstrated linearity with enzyme concentration (0.5–10 µg/ml).
- Kinetic parameters for penicillin-V amidase with the new substrate were determined (Km = 0.89 mM, Vmax = 2.6 µmol/min/mg).
- Penicillin-V competitively inhibited the hydrolysis with a Ki of 4 mM.
Conclusions:
- The developed colorimetric assay is a sensitive and reliable method for quantifying penicillin-V amidase activity.
- This assay is suitable for determining the presence and levels of penicillin-V amidase in industrial settings.
- The novel substrate and assay facilitate enzyme characterization and quality control.