Direct evaluation of phenylacetyl-CoA : 6-Aminopenicillanic acid acyltransferase of Penicillium chrysogenum by bioassay.

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Abstract
The enzyme phenylacetyl-CoA: 6-Aminopenicillanic acid acyltransferase of Penicillium chrysogenum was evaluated by direct bioassay against Micrococcus luteus ATCC 9341. The enzyme required dithiothreitol, was inactivated by 0.2 mM Hg2+ (100%), Zn2+ (80%), Cu2+ (60%), 1 mM N-ethylmaleimide (80%), and showed maximal catalytic activity at pH 8.4 and 20.degree. C. The V50 values for phenylacetyl-CoA and 6-aminopenicillanic acid were 0.55 mM and 1 .mu.M, respectively. When octanoyl-CoA was employed as substrate similar results were obtained. In both cases the product generated showed strong antibacterial activity which was quickly lost when incubation was carried out with .beta.-lactamase. Reactions performed in the presence of Escherichia coli penicillin acylase did not generate active products when phenylacetyl-CoA was the substrate; they did with octanoyl-CoA. Time-course experiments revealed that the highest enzyme levels are found in 36 hours mycelium and remained almost constant from 48 to 96 hours, thereafter the level of the enzyme slowly decreased.