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.

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

“…Thus, the significant inhibition zone demonstrating the enzymatic activity obtained by fibrin plate assay reveals RSA1 strain as a potent source for the production of fibrinolytic protease. Similar significant results were obtained for fibrinolysis using fibrin plate assay performed by Kang et al and Liu et al [9,14].…”

“…The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.Biomolecules 2020, 10, 3 2 of 23 (active protease), converting soluble fibrinogen (glycoprotein) into fibrin polymer (insoluble blood clot) [5]. Fibrin clots are hydrolyzed by plasmin [12], which is stimulated from plasminogen by plasminogen activators (PAs) [13,14]. This natural dynamic equilibrium is disturbed when the process of natural fibrin clot hydrolysis undergoes pathophysiological shambles, leading to formation of fibrin clots.…”

Proteases of Wood Rot Fungi with Emphasis on the GenusPleurotus

“…The fibrinolytic proteases of Pleurotus spp. have received attention in recent years because those searching for new proteases with fibrinolytic capacity are interested in nontoxic and edible fungi [ 77 ]. A monomeric protease with fibrinolytic activity was purified 29.3-fold from the basidiome of P. eryngii produced in corn cob.…”

“…In a study by Liu et al [ 77 ] the fibrinolytic and fibrinogenolytic enzyme from P. pulmonarius grown in submerged state were efficient in degrading the α (3 min) and β (45 min) chains of fibrinogen, followed by γ after 10 h incubation. The enzyme was purified 147-fold and presented good stability at human body temperature, which enables it to be used as an alternative in thrombolytic treatments, including oral applications, because it is an edible fungus.…”

Purification and characterization of a novel metalloprotease from fruiting bodies of Oudemansiella radicata

“…Mushrooms are abundant in bioactive compounds comprising proteases (Hu et al, 2012;Moon et al, 2014;Zheng et al, 2011), lectins (Wang et al, 2013;Xu et al, 2014;Zhang et al, 2014b), angiotensin I-converting enzyme inhibitory peptides (Geng et al, 2015;Kang et al, 2013;Lau et al, 2014;Mohamad Ansor et al, 2013), ribonucleases (Xu et al, 2013;Zhang et al, 2014a), acid phosphatases (Wannet et al, 2000;Zhang et al, 2013), laccases (Tian et al, 2012;Xu et al, 2015a;Xu et al, 2015b), antifungal proteins (Suzuki et al, 2011;Wong et al, 2010), and polysaccharides (Liu et al, 2014a;Ma et al, 2014;Mao et al, 2014;Zhu et al, 2014). Many of these mushroom proteins have potential applications or health-promoting activities in human (Liu et al, 2014b;Wu et al, 2014). There are also many papers documenting that extracts and purified compounds from various edible mushrooms have significant anti-cancer properties, for example, Antrodia cinnamomea sulfated polysaccharide exhibited activity in suppressing growth and migration of lung cancer cells (Lu et al, 2016), Pro4X, an extract of the edible Grifola frondosa mushroom, displayed anti-cancer activity and prevented oncogenesis in BABLc mice (Roldan-Deamicis et al, 2016).…”