A sensitive enzymic assay for benzylpenicillin

Abstract

A method has been developed for the determination of sodium benzylpenicillin concentrations in the range 3.3-33 micrograms/ml. 6-Aminopenicillanic acid is released from the benzylpenicillin by the action of the enzyme penicillin acylase and is estimated from its reaction with fluorescamine at pH 4. 7-Aminocephalosporanic acid shows a similar trend to 6-aminopenicillanic acid in its reaction at pH 4. The open beta-lactam ring form of each compound shows little fluorescence with fluorescamine at pH 4 but shows strong fluorescence in the pH range 7-9. 6-Aminopenicillanic acid and its open beta-lactam ring form give different fluorescent responses to increasing volumes of a solution of the fluorigenic agent at pH 7.8. This effect can be used to estimate concentrations in a mixture of the two components providing other amino material is absent.

Chemistry of some fluorescamine–amine derivatives with relevance to the biosynthesis of benzylpenicillin by fermentation

“…6‐APA and 7‐ACA were determined via the fluorescamine reaction at pH 4 12. Penicillin acylase was also measured via the fluorescamine reaction using a partly purified solution of the enzyme12, 13 prepared from Escherichia coli ATCC 9637. Some enzyme reactions were run from pH values as low as 4 to as high as pH 9.…”

“…During the preparation of the semi‐synthetic penicillins (or the cephalosporins), the side‐chain of benzylpenicillin is removed by penicillin acylase (or cephalosporin C hydrolase11) to yield either 6‐APA (or 7‐aminocephalosporanic acid (7‐ACA)). 6‐APA can be estimated, with great sensitivity and simplicity, by the fluorescamine reaction at pH 413 and the enzymic reaction may be used to estimate benzylpenicillin 13. 7‐ACA also reacts with fluorescamine at pH 412 and a technique was developed to estimate 7‐ACA hydrolase activity 14.…”

“…7‐ACA also reacts with fluorescamine at pH 412 and a technique was developed to estimate 7‐ACA hydrolase activity 14. The determination of benzylpenicillin by enzymic means during fermentations has not been seriously examined but it seemed that it, and some other analytical techniques used in this area,12–14 might enable more simplified separations and analyses for the pathway.…”

“…Before examining the metabolic controls involved in linking organic acid and phosphorylated intermediate metabolism to penicillin biosynthesis, it was desirable to know whether the fluorometric techniques12, 13 might be useful to investigate penicillin biosynthesis. Conditions were used where there were significant levels of amino compounds in the medium to represent potential for interference in the analytical reaction.…”

Fluorogenic Assay for Penicillin G Acylase Activity

“…Assay methods for the determination of penicillin acylase activity fall into two main groups. The first group of methods is based on the quantitative determination of products formed during enzymatic hydrolysis of penicillins and cephalosporins (3)(4)(5)(6)(7)(8)(9)(10). All these methods require the collection of aliquots and are multistage, laborious, and not very sensitive.…”