• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.329-332
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• 2001

It has been known that, in enzymatic synthesis of cephalexin, the conversion yield was reduced by high loading of ampicillin acylase. In order to elucidate this phenomena, pH-controlled synthesis of cephalexin was examined using a purified Acetobacter turbidans acylase. When the pH of the reaction mixture was maintained at $6.20{\pm}0.04$, the reduction of the maximal conversion rate was not observed even with high enzyme loading. The kinetic parameters also suggest that pH drop during the enzymatic synthesis of cephalexin was mainly attributed to the rapid hydrolysis of D-${\alpha}$-phenylglycine methyl ester to D-${\alpha}$-phenylglycine, rather than the disappearance of 7-amino-3-deacetoxycephalosporanic acid for cephalexin synthesis. At higher molar ratio of two substrates, [D-${\alpha}$-phenylglycine methyl ester]/[7-amino-3-deacetoxycephalosporanic acid], the conversion rate was also elevated under pH-controlled enzymatic synthesis, which implies that the main reason for the pH drop is due to the production of D-${\alpha}$-phenylglycine methyl easter, the effect of a water-methanol cosolvent system on the ester, the effect of a water-methanol cosolvent system on the conversion profile was also examined. Even the though the conversion rate was increased in 10% methanol solution, a higher than 16% methanol in the reaction mixture caused an inactivation of enzyme.

• Bulletin of the Korean Chemical Society
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• v.4 no.2
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• pp.72-76
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• 1983

Cephaloglycin was synthesized directly from D-${\alpha}$ -phenylglycine methyl ester and 7-aminocephalosporanic acid using whole cell enzyme of Xanthomonas citri (IFO 3835). Some optimal conditions for cephaloglycin synthesis were investigated, and yield improvements for its production by several methods were attempted. Using the whole cell enzyme system, the reaction kinetic model for cephaloglycin synthesis is proposed, and the kinetic constants for D-${\alpha}$ -phenylglycine methyl ester hydrolysis, cephaloglycin synthesis, and cephaloglycin hydrolysis were determined. The $K_m$ values of D-${\alpha}$-phenylglycine methyl ester, 7-aminocephalosporanic acid, and cephaloglycin were 11 mM, 24 mM, and 167 mM, and $K_i$ value of D-${\alpha}$-phenylglycine was 15 mM, respectively. The pattern of product inhibition was found to be competitive one.

• Archives of Pharmacal Research
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• v.3 no.1
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• pp.7-12
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• 1980

Ampliciline was synthesized from 6-amino-pencillanic acid (6-APA) and D-.alpha. phenylglycine methyl ester by using amplicilin synthesizing enzyme from Peudomonas melanogenum (IAM 1655). The whole cell enzyme was immobilized by entrapping it in the polyacrylamide gel lattices. The polymer used in the enzyme entrapment was made from 150 mg per ml of acrylamide monomer and 8 mg per ml of N, N'-methylenebisacrylamide. About 200 mg/whole cell enzyme was mixed in the polymer for entrapment. The maximal activity retention after immobilization was 56%. The optimal pH values for the whole cell enzyme and the immobilized whole cell enzyme were 6.0 and 5.9, respectively. The optimal temperature for the enzyme activity were the same for both type of preparations. The enzyme stabilities against pH and heat increased for immobilized whole cell enzyme. Immobilized cell was more stable especially in the acidic condition while both type were found to be very suceptible to thermal inactivation at a temperature above 4.deg.C. The kinetic constants obtained from Lineweaver-Burk plot based on two substate reaction mechanism showed somewhat higher value for immobilized whole cell enzyme as compared to the whole cell enzyme : the Km value for 6-APA were 7.0 mM and 12.5 mM while Km values for phenylglycine methyl ester were 4.5 mM and 8.2 mM, respectively. Using the immobilized whole cell enzyme packed in a column reactor, the productivity of ampiciline was studied by varying the flow rate of substrate solution. At the space velocity, SV, 0.14 hr$^{-1}$ the conversion was 45%. Operational stability found in terms of half life was 30 hr at SV = 0.2 hr.