Production and purification of fibrinolytic enzyme nattokinase in Aspergillus oryzae

A significant portion of deaths worldwide are caused by thrombotic diseases. Although thrombotic diseases are known as blood disorders, they can be the underlying cause of many common diseases today, with cardiovascular diseases being one of the most severe and life-threatening conditions among them. For the treatment of these diseases, anticoagulant and antiplatelet drugs have been used for many years, but they are not sufficiently effective in resolving thrombosis. However, fibrinolytic enzymes have the ability to break down fibrin clots within blood vessels and move on the clot. Therefore, fibrinolytic enzymes play a crucial role in the treatment of many thrombotic diseases, especially cardiovascular diseases. One such fibrinolytic enzyme is Nattokinase, which is encoded by the aprN gene of Bacillus subtilis natto. Nattokinase is produced by Bacillus subtilis during the fermentation of the soybean product natto. Nattokinase is a promising enzyme used in the treatment of thrombotic and cardiovascular diseases, exhibiting antithrombotic, antihypertensive, anticoagulant, anti-atherosclerotic, and neuroprotective effects. Additionally, Nattokinase is a natural product considered a "nutraceutical" in many countries. It is suitable for oral administration, has a proven safety profile, is cost-effective, and provides several advantages compared to other pharmaceutical products. Therefore, our study aims to produce and purify the Nattokinase enzyme in Aspergillus oryzae. This study is the first of its kind in the literature. A. oryzae possesses easy culturing and optimization, rapid growth, and strong protein secretion abilities. Furthermore, A. oryzae has been chosen for this study due to its high efficiency, safety, and low cost. Recently, it has been utilized as a host cell for enzyme production. This is attributed to A. oryzae's ability to produce large quantities of hydrolytic enzymes such as amylolytic and proteolytic enzymes. This ability makes A. oryzae an important source for both homologous and heterologous enzyme production. In this study, the Nattokinase gene was synthesized by GenScript, with the C-terminal region tagged with 8xHis-tag and adapted to Aspergillus codon optimization. It was then transformed into competent bacteria (Escherichia coli One Shot) using the calcium chloride method. Colony selection was performed from ampicillin-resistant LB medium, followed by plasmid isolation. The plasmid was linearized using restriction enzymes, and agarose gel electrophoresis was conducted. Target DNA was identified and isolated from the gel for transformation into A. oryzae (Protoplast-Mediated Transformation). Samples were inoculated onto CD Agar plates and incubated at 30°C for 5-7 days. Colonies were checked using SDS-PAGE, and selected colonies were cultured in DPY medium and incubated at 30°C for 5-7 days to allow for protein expression. Subsequently, the expressed proteins were purified using the IMAC method. Expression control and protein analysis were performed using SDS-PAGE followed by Western Blot to detect the protein. The Western Blot results demonstrated the successful fusion production and purification of the Nattokinase enzyme. 106.66 mg/L of NTK enzyme was produced as a result of the study. Two activity tests were conducted. In the prothrombin time test, it was observed that NTK slowed down coagulation by approximately 8 seconds, significantly reduced the percentage of clot formation, and significantly slowed down coagulation according to the INR result. The other activity test involved transferring fibrin fragments obtained from blood into microcentrifuge tubes containing titanium and observing whether fragmentation occurred. The observation revealed that fibrin was completely broken down within 5-7 days. Based on the results of the activity tests, it has been proven that NTK is a biocatalytic enzyme. Keywords: Aspergillus oryzae, Fibrinolytic enzyme, Nattokinase, Protein purification, Protein production, Transformation