Production, purification and characterization of a novel antithrombotic and anticoagulant serine protease from food grade microorganism Neurospora crassa

Abstract

A novel thrombolytic enzyme was produced by food grade microorganism Neurospora crassa using agro-industrial by-products as substrates. Process parameters were optimized using Plackett–Berman and Box–Benhken design. Under the optimized fermentation conditions, high fibrinolytic activity of 403.59 U/mL was obtained. It was purified with a specific activity of 3572.4 U/mg by ammonium sulfate precipitation and SP Sepharose chromatography. The molecular weight of the enzyme was approximately 32 kDa. It exhibited maximum activity at 40 °C and pH 7.4. Its activity was enhanced by Cu²⁺, Na⁺, Zn²⁺, and completely inhibited by phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, which indicates it could be a serine protease. The enzyme could degrade fibrin clot directly without the need of plasminogen activator, and effectively cleaved Aα, Bβ, γ chains of fibrinogen. It could inhibit the formation of blood clots in vitro and acts as an anticoagulant. Compared to heparin the purified enzyme showed extended anticoagulant activity. Blood clots were dissolved effectively and dissolution rate was increased with time. Based on these results, this novel enzyme has the potential to be developed as a thrombolytic agent.

Keywords:

• Cardiovascular diseases
• fermentation
• fibrinolytic enzyme
• Neurospora crassa
• purification
• thrombolytic agent

Disclosure statement

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Additional information

Funding

This research work was supported by College of Food Science, Southwest University, Chongqing, China.

This research work was supported by College of Food Science, Southwest University, Chongqing, China.