Isolation and characterisation of dipeptidyl peptidase IV from Prevotella loescheii ATCC 15930

Abstract 

A proline-specific dipeptidyl aminopeptidase, dipeptidyl peptidase IV (EC 3.4.14.5), was purified from a cell sonicate soluble fraction of Prevotella loescheii ATCC 15930 by sequential column chromatography. The molecular mass of the native enzyme was estimated as 160 kDa by high-pressure liquid gel filtration column chromatography and unheated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The subunit molecular mass was 80 kDa when the enzyme was heated to 100°C in the presence of 2-mercaptoethanol before SDS-PAGE, suggesting that the native enzyme consists of two identical subunits and is folded in 2% SDS. The optimum pH, with glycyl-prolyl-4-methyl-coumaryl-7-amide as the substrate, was 8.0; the isoelectric point was 5.2. Purified enzyme showed a strong preference for dipeptide substrates containing proline and, less efficiently, alanine in the P1 position. The enzyme was markedly inhibited by Cd²⁺, Zn²⁺, Hg²⁺, Co²⁺, and serine proteinase inhibitor di-isopropylfluorophosphate.

Keywords:  Prevotella loescheii, Dipeptidyl peptidase IV, Serine proteinase, Periodontal disease

Abbreviations:  AMC, 7-amino-4-methyl-coumarin, DPP, dipeptidyl peptidase, MCA, 4-methyl-coumaryl-7-amide, SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis