Biochemistry and Biotechnology
Wiley InterScience Backfile Collection 1832-2000
Process Engineering, Biotechnology, Nutrition Technology
An enzyme which catalyzes the synthesis of cephalexin fromD-α phenylglycinemethylester (PGM) and 7-amino-3-desacetoxy-cephalosporanic acid (7-ADCA) was prepared from Xanthomonas citri (IFO 3835) and partially purified 30-fold by ammonium sulfate fractionation, DEAE-cellulose, and Sepharose-4B column chromatography. The Km values for 7-ADCA, PGM, and cephalexin were determined as 11.1, 2.1, and 1.61 mM, respectively. The enzymatic cephalexin synthesis follows the reversible bi-uni reaction kinetics. The equilibrium constant is influenced by the initial mole ratios of 7-ADCA and PGM. The cephalexin hydrolysis is catalyzed by the same cephalexin synthesizing enzyme, but methanol does not participate in the hydrolytic reaction. The amount of enzyme in the reaction mixture affects the initial rate but does not influence the equilibrium product concentration. This cephalexin-synthesizing enzyme was immobilized onto several adsorbents. Among these, Kaolin and bentonite showed a higher retention of enzyme activity and stability for reuse. The immobilized-enzyme reaction kinetics were investigated and compared with those of the soluble enzyme. A rate expression for the enzymatic synthesis of cephalexin was derived. The results of computer simulation showed good agreement with the experimental results.
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