Purification and Characterization of a Prolidase from Aureobacterium esteraromaticum

Abstract

An EDTA-insensitive prolidase (proline dipeptidase, EC 3.4.13.9) was isolated from a cell-free extract of Aureobacterium esteraromaticum IFO 3752. The enzyme was purified almost to homogeneity using acetone precipitation, hydrophobic chromatography, ion-exchange chromatography, and gel-permeation chromatography. The enzyme has a molecular weight of about 440, 000 by gel permeation chromatography, and about 40, 000 by SDS polyacrylamide gel electrophoresis. The isoelectric point was 4.6. The enzyme hydrolyzed aminoacylprolines such as Ser-Pro, Thr-Pro, Gly-Pro, Ala-Pro, Ile-Pro, Leu-Pro, and Pro-Pro. It also hydrolyzed Gly-Hyp and Pro-Hyp. The rate of hydrolysis for Pro-Hyp was the highest among the substrates tested. Optimum pH for hydrolyzing Pro-Hyp was 9.0 and the enzyme was stable in the pH range from 5 to 10. The optimum temperature was estimated to be 45°C using 10 min of reaction. At least 90% of the initial activity remained after 30 min of incubation at 60°C. p-Chloromercuribenzoic acid and o-phenanthrolin inhibited the enzyme's activity while EDTA did not. Addition of Mn²⁺ ion did not stimulate activity. These results suggest either that the metal ion in the enzyme may be tightly bound to the polypeptide chain, or that the enzyme is not a metallo-enzyme but a thiol-enzyme.