Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Culture Supernatant of Coprinus comatus

Abstract

A novel fibrinolytic enzyme was produced by the liquid fermentation of Coprinus comatus. The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn²⁺, K⁺, Ca²⁺, Mn²⁺, and Mg²⁺ while being inhibited by Fe²⁺, Fe³⁺, Al²⁺, and Ba²⁺. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases.

Keywords: Coprinus comatus; anticoagulant activity; fermentation; fibrinolytic enzyme.

Figure 1

Purification of the fibrinolytic enzyme from Coprinus comatus. The elution profile of (A) Octyl-Sepharose Fast Flow hydrophobic interaction chromatography, (B) SP-Sepharose High Performance ion exchange chromatography, and (C) Source 15 PHE hydrophobic interaction chromatography.

Figure 2

Molecular weight determination by SDS-PAGE under denaturing conditions. Lane 1: molecular weight standards; Lane 2: the purified enzyme.

Figure 3

Analysis of the purity of CFE. Lane 1: fibrin zymography; Lane 2: Native-PAGE; Lane 3: the imprint of CFE on a fibrin plate.

Figure 4

Effects of temperature and pH on fibrinolytic activity of CFE. (A) Optimal temperature. (B) Temperature stability. (C) Optimal pH. (D) pH stability.

Figure 5

Analysis of plasminogen activation by CFE on (A) plasminogen-positive fibrin plate and (B) plasminogen-negative fibrin plate. Circles 1–3 represent CFE; circles 4–5 represent urokinase.

Figure 6

Effect of simulated gastric and blood environments on the CFE. Sample 1, CFE in water (control); sample 2, CFE in artificial gastric juice; sample 3, CFE in gastric juices with a pH of 7.4; sample 4, CFE in artificial gastric juice and broth; sample 5, CFE in artificial gastric juice and saccharose; sample 6, CFE in artificial gastric juice, saccharose, and protein broth; sample 7, CFE in Locke solution. Different letters indicate significant differences (p < 0.05).

Figure 7

Analysis of thrombin-like activity of CFE. 1, control fibrin clot (human blood fibrinogen and human thrombin); 2, CFE, human blood fibrinogen, and human thrombin; 3, human blood fibrinogen and CFE.

Figure 8

Cleavage pattern of fibrin(ogen) by CFE; (A) SDS-PAGE analysis of human blood fibrinogen hydrolyzed by CFE. Lane C, control; Lanes 1–10, degradation pattern of fibrinogen at different time intervals of 1 min, 5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, and 5 h, respectively. (B) SDS-PAGE analysis of human fibrin hydrolyzed by CFE; Lane C, control; Lanes 1–9, degradation pattern of fibrin at different time intervals of 1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h, respectively.

Figure 9

Effects of CFE on some blood proteins. Lanes 1, 3, and 5 represent human thrombin, IgG, and human serum albumin (HSA), respectively. Lanes 2, 4, and 6 represent human thrombin, IgG, and HSA incubated with CFE, respectively.

Figure 10

Effects of CFE on coagulation index in vitro. The levels of (A) APTT, (B) PT, (C) PA, (D) TT, and (E) FIB in different experimental groups. Different letters indicate significant differences (p < 0.05).

Figure 11

Anticoagulant activity of CFE. (1) Plasma and heparin; (2) plasma and saline; (3) plasma and CFE.