Abstract
A carboxypeptidase (CPase) of black matpe seedlings which hydrolyzes carbobenzoxy-phenylalanylalanine (Z-Phe-Ala) was purified by means of ammonium sulfate fractionation (30-70%), Sephadex G-150 gel filtration, DEAE-Sepharose chromatography, 2nd Sephadex G-150 gel filtration, 2nd DEAESepharose chromatography and TOYOPEARL HW-50F gel filtration. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated to be about 70, 000 by gel filtration.
The optima conditions for pH and temperature of the CPase were obtained at 6.0 and 50°C when measured with Z-Phe-Ala. The CPase activity was stable at pH 5-8 for 2hr standing at room temperature. The enzyme was inhibited by heavy metal ions (Hg++, Ag+, Cu++), DFP and PCMB, but not affected by EDTA, ICH2COOH and 2-mercaptoethanol. As substrate specificity, Z-Phe-Ala was the most suitable substrate of those tested. The Km value for Z-Phe-Ala was 1.0mM at pH 6.0.
