title: Purification and characterization of a novel fibrinolytic enzyme from culture supernatant of Pleurotus ostreatus.
abstract: A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDSPAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the α and β chains of fibrinogen followed by the γ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at 45°C and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.
authors.affiliation: Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China.
authors.forename: Xiao-Lan
authors.initials: XL
authors.lastname: Liu
authors.forename: Xi-Qun
authors.initials: XQ
authors.lastname: Zheng
authors.forename: Peng-Zhi
authors.initials: PZ
authors.lastname: Qian
authors.forename: Narasimha-Kumar
authors.initials: NK
authors.lastname: Kopparapu
authors.forename: Yong-Ping
authors.initials: YP
authors.lastname: Deng
authors.forename: Masanori
authors.initials: M
authors.lastname: Nonaka
authors.forename: Naoki
authors.initials: N
authors.lastname: Harada
mesh_terms: D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism*; D005341:Fibrinolysin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism; D020076:Pleurotus / Q000201:enzymology*; D020539:Sequence Analysis, Protein; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D005340:Fibrinogen; D010958:Plasminogen; D005341:Fibrinolysin