Purification and characterization of a cephalexin-synthesizing enzyme fromGluconobacter oxydans CCRC10383

A cephalexin-synthesizing enzyme which catalyzes the synthesis of cephalexi n from two substrates, 7-amino-3-deacetoxy cephalosporanic acid (7-ADCA) an d D-phenylglycine methyl ester (D-MEPG) was isolated, purified and characte rized from G. oxydans CCRC 10383 by ammonium sulfate precipitation, CM-Frac togel 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-PAG E, the enzyme is a tetramer and consists of two 53 kd subunits and two 73 k d subunits. The isoelectric point of the enzyme was estimated to be 7.5. Th e optimal pH and temperature for the synthetic reaction of cephalexin were 5-7 and 30-50 degrees C, respectively, with a maximum reaction rate at pH 6 or 50 degrees C. Metal ions are not essential for the enzymatic activity b ecause 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 x 10(4) s (-1), 30 unit/mg of protein and 6.2 x 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, res pectively. The conversion of cephalexin was found to be 60% when the synthe sis was carried out in the 0.1 M phosphate buffer solution (pH 6.2) contain ing 40 mM of 7-ADCA and 118 mM of MEPG at 37 degrees C.