Purification and characterization of a dipeptidase from Pseudomonas fluorescens ATCC 948.
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 degrees C. Although activity decreased markedly at temperatures > 50 degrees C, about 50% of maximal activity was retained at 10 degrees 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.
Gobbetti
M
M
Department of Food Science and Nutrition, University of Perugia, Italy.
Fox
P F
PF
D002118:Calcium / Q000494:pharmacology; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003035:Cobalt / Q000494:pharmacology; D004150:Dipeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D015125:Oxyquinoline / Q000494:pharmacology; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D004791:Enzyme Inhibitors; D013439:Sulfhydryl Reagents; D003035:Cobalt; D015125:Oxyquinoline; D004492:Edetic Acid; D004150:Dipeptidases; C038937:dipeptidase; D002118:Calcium; D004229:Dithiothreitol