Liu, Xiao-Lan
(Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University)
,
Zheng, Xi-Qun
(Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University)
,
Qian, Peng-Zhi
(Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University)
,
Kopparapu, Narasimha-Kumar
(Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University)
,
Deng, Yong-Ping
(Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University)
,
Nonaka, Masanori
(Faculty of Agriculture, Niigata University)
,
Harada, Naoki
(Faculty of Agriculture, Niigata University)
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 c...
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 SDS-PAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the ${\alpha}$ and ${\beta}$ chains of fibrinogen followed by the ${\gamma}$ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at $45^{\circ}C$ 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.
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 SDS-PAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the ${\alpha}$ and ${\beta}$ chains of fibrinogen followed by the ${\gamma}$ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at $45^{\circ}C$ 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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대상 데이터
Slants of Pleurotus ostreatus, No. 4241 of China General Microbiological Culture Collection Center (CGMCC), stored at -80°C in 20% glycerol, were used to inoculate potato dextrose agar (PDA) plates.
이론/모형
The molecular mass of the purified enzyme under reducing denaturing conditions was determined by SDS-PAGE as described by Laemmli [12], using a 12% polyacrylamide gel. After electrophoresis, the protein bands were visualized by staining with Coomassie brilliant blue R-250.
2A and 2B). The molecular mass was calculated according to the method of Andrews [1]. The elution curve and co-relation between lgMr and K av of the markers on a prepacked Superdex 75 (16 × 60 cm) column are shown in Fig.
The protein concentration of the samples at every step of chromatography was determined spectrophotometrically at 280 nm. The protein concentration of pooled enzyme solution from each purification step was measured according to the Lowry method [18], using bovine serum albumin (BSA) as standard.
성능/효과
The molecular mass of fibrinolyic enzymes from mushrooms are reported in the range of 17-100 kDa [19]. The MW of the purified enzyme was smaller than that of fibrinolytic enzymes from Cordyceps militaris (52 kDa), Pleurotus ostreatus (32 kDa), and Flammulina velutipes (37 kDa) [10, 24, 28] but somewhat similar to fibrinolytic enzymes from Armillaria mellea (18.5kDa), Tricholoma saponaceum (18.1 kDa), and Schizophyllum commune (17 kDa) [8, 9, 23]. Because of its small size, the fibrinolytic enzyme from Pleurotus ostreatus culture fluid may result in less antigenicity.
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