title: Broad substrate affinity and catalytic diversity of fibrinolytic enzyme from Pheretima posthumous-Purification and molecular characterization study.
abstract: In this research, a serine protease was isolated and purified from Indian earthworm Pheretima posthumous by fractionation with ammonium sulfate followed by ion exchange and size exclusion chromatography. The molecular weight of purified protease was determined 29.5kDa by Maldi-TOF/MS. The enzyme exhibited a maximum proteolytic activity of 1.2U/ml with specific activity of 17.65U/mg at pH 8 and temperature 40°C. 2D electrophoresis study illustrated purity of enzyme, purified as a single peptide and isoelectric point (pI) 4.5. The enzyme has shown tremendous stability and proteolytic activity in the wide range of pH range (4-12) and temperatures (20-60°C). The kinetic constant Km and Vmax of purified protease were reported 0.09mg/ml and 23.25mg/ml/min. The enzyme also possesses excellent catalytic capacity with Kcat (341.9min-1) and catalytic efficiency (3798.88). The N-terminal sequence of purified protease Arg-Lys-Lys-Gly-Ala-Ser-Try-Phe-Try-Pro-Trp-Ser-Val-Lys-Lys-Arg, PMF and MS/MS studies had shown a partial homology with Lumbrokinase-P2 (2) from Lumbricus rubellus. The CD spectroscopy result provided an evidence for broad substrate affinity and stability of enzyme. The different forms of secondary structures determined in EFE result broad substrate affinity of enzyme.
authors.affiliation: Department of Biotechnology, Acharya Nagarjuna University, Guntur, 522 510, Andhra Pradesh, India.
authors.forename: Mahendra Kumar
authors.initials: MK
authors.lastname: Verma
authors.affiliation: Scientist, Department of Science and Technology, Ministry of Science and Technology, Govt. of India, New Delhi, 110 016, India. Electronic address: kkpulicherla@gmail.com.
authors.forename: K K
authors.initials: KK
authors.lastname: Pulicherla
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005591:Chemical Fractionation / Q000379:methods; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009835:Oligochaeta / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D005343:Fibrinolytic Agents; D010450:Endopeptidases; D057057:Serine Proteases; C083421:lumbrokinase
keywords: Earthworm fibrinolytic enzyme; Maldi-TOF; Maldi/MS; Serine protease; Thrombotic and vascular disorders