Studies on fermentation conditions for the production of penicillin G acylase by bacillis megaterium

Abstract

Penicillin G acylase (EC 3.5.1.11, penicillin amidohydrolase), which is responsible for the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid (6-APA) and phenylacetic and (PAA), was produced by $\mbox{\underline{Bacillus}}$ $\mbox{\underline{megaterium}}$ ATCC 14945. The optimal conditions of enzyme reaction were set as: at pH 8.5 to 9; at 40℃. The Km value for benzylpenicillin was 4.55 mM. Hydroxylamine assay for the determination of 6-APA was modified by using "HCl addition technique" instead of n-butylacetate extraction step. Using above microorganism, the effects of fermentation conditions on enzyme production were studied. When the following medium composition was used, the enzyme productivity obtained was about 14.1 units per ml broth. The medium composition ranges were: casitone, 2.5 to 5%; yeast extract, 0.5 to 1.0%; glucose, 1.0 to 2.0%; benzoic acid, 0.1 to 0.2% or phenylacetic acid 0.15 to 0.3%; and silicone antifoam, 0.01 to 0.02%. Proper physicochemical conditions of fermentation or enzyme production were; initial pH of medium, 7.0; temperature of cultivation 28 to 30℃ ; and fermentation period, 2 to 3 days under highly aerobic condition.