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KMID : 0545120140240020245
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Journal of Microbiology and Biotechnology
2014 Volume.24 No. 2 p.245 ~ p.253
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Purification and Characterization of a Novel Fibrinolytic Enzyme from Culture Supernatant of Pleurotus ostreatus
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Xiao-lan Liu
Xi-qun Zheng
Peng-zhi Qian
Narasimha-kumar Kopparapu
Yong-ping Deng
Masanori Nonaka
Naoki Harada
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Abstract
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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.
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KEYWORD
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Pleurotus ostreatus, Fibrinolytic enzyme, Fermentation, Purification, Characterization, Culture supernatant
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