A dual-function chymotrypsin-like serine protease with plasminogen activation and fibrinolytic activities from the GRAS fungus, Neurospora sitophila

https://doi.org/10.1016/j.ijbiomac.2017.11.142Get rights and content

Highlights

Abstract

In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6 U/mg protein. The native molecular mass of the enzyme was 49 kDa, while the denatured molecular mass was 30 kDa and 17.5 kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50 °C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent Km and Vmax values were 0.24 mM and 4.17 × 10−5 mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by β chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.

Introduction

In the blood coagulation cascade, insoluble fibrin clot is formed from fibrinogen by the proteolytic action of thrombin [1]. During fibrinolysis, fibrin is hydrolyzed into fibrin degradation products by the action of plasmin (EC3.4.21.7), which is generated from plasminogen by plasminogen activator. In an unhealthy state, over a period of time, fibrin can accumulate in the blood vessels, resulting in thrombosis. Thrombosis is a major health concern since it can lead to a number of serious diseases such as massive pulmonary embolism, stroke, and myocardial infarction, which are among the leading cause of deaths around the world. Thrombosis can be treated by thrombolytic drugs which dissolve the blood clot by activating plasminogen [1].

A variety of thrombolytic agents such as recombinant tissue-type plasminogen activators (alteplase, reteplase, tenecteplase), bacterial plasminogen activator (streptokinase) and urokinase-type plasminogen activator have been extensively applied for the treatment of thrombosis [2], [3]. Although they have been widely used, all these thrombolytic agents have been known to cause several problems such as allergic reactions, gastrointestinal bleeding, low specificity for fibrin and are rather expensive [4], [5]. It is therefore essential to search for safer, more efficient and cost-effective thrombolytic agents.

In the recent years, several fibrinolytic enzymes have been found from microorganisms isolated from food sources including fermented food, such as natto [6], [7], starter of fermented rice-wine [8], Asian traditional fermented shrimp paste [9], commercial koji [10], fish sauce [11], fermented red bean paste [12], douchi [13] and medical mushrooms [14], [15], [16], [17], [18], [19]. However, relatively few enzymes have been reported from GRAS fungi. Novel enzymes which can efficiently dissolve fibrin clots should be investigated from GRAS micro-organisms since they have been proved to be safe in humans [7]. Neurospora, a genus of ascomycete fungi, has been safely used in food processing since many years and species of the genus Neurospora are recognized under Food and Drug Administration (FDA) regulations as a GRAS fungus. No dangerous secondary metabolites are known to be produced by strains of any Neurospora species [20]. Neurospora sitophila is also used in preparing fermented soybean-based food as a local industry in the south of China and applied for conversion of solid cellulosic substrates to protein-rich materials for food and fodder [21]. Neurospora sitophila was isolated from the starter used for fermenting soybean paste in the north of China and cultured in our laboratory. In an earlier study, we have reported purification and characterization of a fibrinolytic enzyme from Neurospora sitophila [22]. In this study, we report fermentation, purification and characterization of a novel chymotrypsin-like serine protease having fibrinolytic activity from Neurospora sitophila.

Section snippets

Chemicals and reagents

Superdex 75 (16/60) and HR10/30 pre-packed columns, Octyl-Sepharose Fast Flow media, SP-Sepharose High Performance media, gel filtration low molecular weight (LMW) calibration kit, and an isoelectric focusing (IEF) calibration kit were purchased from GE life sciences (USA). Sodium dodecyl sulfate, agarose, bovine fibrinogen, N-α-tosyl-l-phenylalanine (TPCK), and synthetic chromogenic substrates (N-Succinyl-Ala-Ala-Pro-Phe-pNA, d-Ile-Phe-Lys-pNA, N-Benzoyl-DL-Arg-pNA, d-Ile-Pro-Arg-pNA, N

Purification of fibrinolytic enzyme from Neurospora sitophila culture

This study describes the purification and characterization of a novel fibrinolytic enzyme from the GRAS fungus, Neurospora sitophila, isolated from the starter of soybean fermentation paste. The fibrinolytic enzyme produced by solid state fermentation using soybean residue as the major material, was purified to electrophoretic homogeneity by ammonium sulfate precipitation, followed by a series of chromatographic columns which included Octyl-Sepharose column (Fig. 1A), SP-Sepharose column (Fig. 1

Conclusions

A novel 30 kDa fibrinolytic enzyme was purified from Neurospora sitophila, a GRAS fungus and characterized. The chymotrypsin-like serine protease exhibited good pH and thermal stabilities. The fibrinolytic enzyme, in this study, is a dual-function enzyme which degraded fibrin clot by direct fibrinolysis as well as by plasminogen activation. Hence, in view of these findings, the fibrinolytic enzyme from Neurospora sitophila may have potential application as a safe, natural fibrinolytic agent for

Acknowledgements

This work was supported by National Natural Science Foundation of China (NSFC) (no. 31301414), Natural Science Foundation of Heilongjiang province (no. ZD201305), PR China. The authors declare no commercial or financial conflict of interest.

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