Purification and Characterization of a Dipeptidase from Pseudomonas fluorescens ATCC 948

¹ Institute of Dairy Microbiology, Department of Food Science and Nutrition, University of Perugia, Italy
² Department of Food Chemistry, University College, Cork, Ireland

A dipeptidase from a cell extract of Pseudomonas fluorescens ATCC 948 was purified to homogeneity by ion-exchange chromatography, hydroxyapatite chromatography, gel filtration, and FPLC® Phenyl Superose chromatography. The enzyme was located in the cytoplasmic fraction. The dipeptidase appeared to be a monomer with a molecular mass of about 46 kDa, and activity was optimal at pH 8.0 and 50°C. Although activity decreased markedly at temperatures >50°C, about 50% of maximal activity was retained at 10°C. The activity was inhibited by EDTA, but could be reactivated with Co²⁺ and partially reactivated with Ca²⁺. Reducing agents, such as dithiothreitol and 8-hydroxyquinoline, also strongly inhibited the enzyme. The dipeptidase hydrolyzed only dipeptides and showed a particular aptitude for substrates containing a hydrophobic AA at the N-terminus. Dipeptides containing a proline residue were not cleaved. Kinetic studies indicated that the dipeptidase hydrolyzed Leu-Leu with a Michaelis-Menten constant of 0.43 mM and a turnover number of 1812/s.

Key Words: Pseudomonas fluorescens • dipeptidase • characterization • dairy products

Submitted on May 16, 1995
Accepted on October 23, 1995