Ampicillin synthesis by microbial enzyme

Abstract

Ampicillin was synthesized from 6-aminopenicillanic acid(6-APA) and D- α-phenylglycine methyl ester(PGM) by $\mbox{\underline{Pseudomonas}}$ $\mbox{\underline{melannogenum}}$ IAM 1655 containing the acylating enzyme. The specific activity of the enzyme was 2.7 unit per mg after 24 hour to 30 hour cultivation. The whole cell enzyme was immobilized into polyacrylamide gel lattices. The enzyme activity was highest when 200 mg per ml of cell (wet weight) was entrapped into the polymer which was made from 150 mg per ml of acrylamide monomer and 8 mg per ml of N,N``-methylenebisacrylamide. The maximal activity retention after immobilization was 56%. The optimum pH values for intact cells and immobilized cells were 6.0 and 5.9, respectively, and they were practically the same. The optimum temperatures for the enzyme activity were the same for both type of preparations. The enzyme stabilities against pH and thermal changes increased for immobilized whole cell enzyme. Immobilized whole cells were especially more stable in the acidic condition while both found to be very susceptible to thermal deactivation at a temperature above 40 ℃. The activation energy was estimated as 6.15 kcal/mole for the immobilized whole cell enzyme and that for the intact whole cell enzyme was 8,43 kcal/mole. The kinetic constants obtained from Lineweaver-Burk polt based on two substrate mechanism showed somewhat higher values for immobilized whole cell enzyme : Km values for 6-APA were 7.0 mM and 12.5 mM while for PGM were 4.5 mM and 8.2 mM, respectively. Substrate inhibition by PGM was observed ; the inhibition constant, Kis, for intact whole cell was 98 mM and that for immobilized whole cell enzyme was estimated as about 100 mM. From the experimental result of the product inhibition study the reaction seemed to obey the ordered mechanism. Namely, the leading substrate and product were PGM and methanol, respectively. The use of some organic solvents and detergents, especially, benzene, chloro...