A coupled enzyme assay for isopenicillin N synthetase |
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| Authors: | J E Baldwin S E Moroney H H Ting |
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| Affiliation: | 1. The Life Science College, Henan Agricultural University, Zhengzhou 450002, China;2. National Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou 450002, China;3. The Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Henan Agricultural University, Zhengzhou 450002, China |
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| Abstract: | The development of a coupled enzyme assay for the determination of isopenicillin N synthetase activity in purified extracts from Cephalosporium acremonium was described. Isopenicillin N formed from its precursor, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), by the synthetase was hydrolyzed by beta-lactamase I to the corresponding penicilloic acid. Automatic titration of the acid with standard sodium hydroxide delivered by a pH-stat gave a continuous plot of product formed vs time. This assay has been used in kinetic studies and to determine the effects of pH, ionic strength, and temperature on the enzyme's activity. |
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