(A) study on penicillin acylase from bacillus megaterium

Abstract

In order to develop a program to obtain the high activity and high purity of penicillin acylase, mutation and affinity chromatographic technique were studied using $\mbox{\underline{Bacillus}}$ $\mbox{\underline{megaterium}}$ ATCC 14945. An extracellular penicillin acylase (EC 3.5.1.11) from the medium of $\mbox{\underline{B}}$. $\mbox{\underline{megaterium}}$ culture was partially purified overall 446 folds by means of adsorption on celite, followed by an affinity chromatography using a Sepharose-1,6-diaminohexyl-penicillin V column. The partially purified enzyme of a specific activity of 1521 U/mg protein on benzylpenicillin showed 3 bands by 7\% polyacrylamide gel electrophoresis. The enzyme was inhibited by both reaction products, phenylacetic acid and 6-aminopenicillanic acid. Phenylacetic acid inhibited the enzyme competitively and 6-aminopenicillanic acid noncompetitively and inhibition constants were 45 mM and 26 mM, respectively Michaelis constant of the enzyme on benzylpenicillin was 4.5mM. The enzyme showed the maximum activity at 45$^\circ$C and pH8.7. The activation energy calculated by Arrhenius`` method was 4.57 cal/mole. The enzyme productivity was improved by control of fermentation conditions. When the cells were fermented with 20\% inoculum in reciprocal shaker at 250 rpm the enzyme productivity was 36.5 U/ml which was higher productivity compared with that of routine fermentation conditions, 5\% inoculum in rotary shaker at 250 rpm, of 15 U/ml. The strain of $\mbox{\underline{B}}$. $\mbox{\underline{magaterium}}$ ATCC 14945 was improved by mutation with UV irradiation followed by methylmethanesulfonate treatment. Two strains of mutant showed higher productivity of 44 U/ml and 56.6 u/ml, respectively, when they were fermented with 20\% inoculum in reciprocal shaker at 250 rpm.