Purification and characterization of a novel alkaline serine protease             secreted by Vibrio metschnikovii.
A novel extracellular alkaline serine protease secreted by Vibrio metschnikovii (V. metschnikovii) ATCC700040 cells was purified by three chromatographic steps and characterized in terms of enzymatic kinetics and substrate specificity. The purified enzyme (named AKP-Vm) was composed of a single polypeptide with an apparent molecular weight of 50 kDa on 12% SDS-polyacrylamide gel in the presence of CuCl₂. The optimal temperature and the pH for the enzyme were found to be 37˚C and 9.5, respectively. However, the enzyme activity was inhibited by inhibitors such as PMSF and aprotinin. AKP-Vm could hydrolyze a peptide bond at the carboxyl side of the arginine residue, as revealed by its amidolytic activity toward a chromogenic substrate, Boc-Val-Pro-Arg-pNA. The kinetic parameters of the enzyme were as follows: KM=0.91 mM, kcat=0.8 sec⁻¹ and kcat/KM=0.88 mM⁻¹sec⁻¹. AKP-Vm protease could cleave various blood coagulation-associated proteins, including fibrinogen, prothrombin and thrombin. In particular, the enzyme showed powerful fibrinogenolytic and fibrinolytic activities, as it could cleave all major chains of fibrinogen and also digest cross-linked fibrin. The results obtained suggest that AKP-Vm is a novel alkaline serine protease that can actively cleave fibrinogen and cross-linked fibrin.
Department of Biotechnology, College of Natural Sciences, Chosun                     University, Gwangju 501-759, Republic of Korea.
Jae Yeong
JY
Park
Jung Eun
JE
Park
Jong Woo
JW
Park
Seong Myeong
SM
Yoon
Jung Sup
JS
Lee
D002413:Cations, Divalent / Q000378:metabolism; D004789:Enzyme Activation; D005110:Extracellular Space / Q000201:enzymology; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D011489:Protein Denaturation; D059748:Proteolysis; D011516:Prothrombin / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology*
D002413:Cations, Divalent; D011516:Prothrombin; D005337:Fibrin; D005340:Fibrinogen; D012697:Serine Endopeptidases; D013917:Thrombin