Purification and Characterization of a Cephalexin‐Synthesizing Enzyme from Gluconobacter oxydans CCRC10383

A cephalexin‐synthesizing enzyme which catalyzes the synthesis of cephalexin from two substrates, 7‐amino‐3‐deacetoxy cephalosporanic acid (7‐ADCA) and D‐phenylglycine methyl ester (D‐MEPG) was isolated, purified and characterized from G. oxydans CCRC 10383 by ammonium sulfate precipitation, CM‐Fractogel and Sephadex G‐200 chromatography. The molecular weight of the enzyme was estimated to be 270 kd by gel filtration. From the analysis of SDS‐PAGE, the enzyme is a tetramer and consists of two 53 kd subunits and two 73 kd subunits. The isoelectric point of the enzyme was estimated to be 7.5. The optimal pH and temperature for the synthetic reaction of cephalexin were 5–7 and 30–50°C, respectively, with a maxium reaction rate at pH 6 or 50°C. Metal ions are not essential for the enzymatic activity because EDTA (ethylenediaminetetraacetic acid) exerts no influence upon the enzyme activity. The growth medium containing 0.25% DL‐phenylglycine (DL‐PG) or 0.25% D‐phenylglycine (D‐PG) as inducers could obtain 1.4 times higher enzyme activity than the growth medium without inducers. The values of K m , K cat , V max and bimolecular constant K cat /K m were 19 mM, 1.2 × 10 4 s −1 , 30 unit/mg of protein and 6.2 × 10 5 M −1 s −1 , respectively. The K m values for D‐MEPG and 7‐ADCA were determined as 13.9 mM and 3.08 mM, respectively. The conversion of cephalexin was found to be 60% when the synthesis was carried out in the 0.1 M phosphate buffer solution (pH 6.2) containing 40 mM of 7‐ADCA and 118 mM of MEPG at 37°C.