title: Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus.
abstract: Six protease fractions, namely FI, FII, FIII-1, FIII-2, FIII-3 and FIV, were isolated from Perionyx excavatus earthworm biomass by acetone precipitation, followed by serial chromatography using anion exchange, hydrophobic interaction and size exclusion chromatography. All fractions exhibited strong hydrolytic activity towards casein. The activity of six fractions towards fibrin, determined by fibrin plate assay, ranged from 44 to 831 plasmin unit.mg-1 and ranked as FIII-3 > FIII-2 > FI > FIII-1 > FIV > FII. Casein degradation was optimal at pH 7 and 11, and at 45-60°C. All fractions were considerably stable at high temperature and wide pH range. They were completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The molecular weights (MW) and isoelectric points (pI) determined by 2D-electrophoresis were 27.5-34.5 kDa, and 4.3-5.2, respectively. Tandem mass spectrometry (MS) analysis was used to deduce the amino acid sequences of some peptides from FIII-1 and FIII-2. The sequences shared 16.9% and 13.2% similarity, respectively, with the fibrinolytic enzymes from two related earthworm species, Lumbricus rubellus and Eisenia fetida. The P. excavatus proteases were classified as serine proteases. They could perform rapid hydrolysis on both coagulated fibrous fibrin and soluble fibrinogen monomers without the presence of activators such as tPA or urokinase.
authors.affiliation: Faculty of Food Processing Technology, Can Tho University of Technology, Can Tho, Vietnam. tienta2512@dhtcct.edu.vn.
authors.forename: Tram Thi Bich
authors.initials: TT
authors.lastname: Phan
authors.forename: Tien Duy
authors.initials: TD
authors.lastname: Ta
authors.forename: Dung Thi Xuan
authors.initials: DT
authors.lastname: Nguyen
authors.forename: Lambertus Am
authors.initials: LA
authors.lastname: Van Den Broek
authors.forename: Giang Thi Huong
authors.initials: GT
authors.lastname: Duong