TY - JOUR AU - Thuy Ngan, Nguyen Thi AU - Tuan, Le AU - Huong, Nguyen Lan PY - 2022/06/30 Y2 - 2024/03/29 TI - Purification and characterization of recombinant nattokinase from Bacillus subtilis R0H1 JF - Vietnam Journal of Biotechnology JA - Vietnam J. Biotechnol. VL - 20 IS - 2 SE - Articles DO - 10.15625/1811-4989/16027 UR - https://vjst.net/index.php/vjbt/article/view/16027 SP - 369-377 AB - <p>Nattokinase (NK) is a fibrinolytic enzyme with the potential for fighting cardiovascular diseases (CVD) thanks to its antithrombotic, antihypertensive, anticoagulant, anti-atherosclerotic, and neuroprotective effects. Nattokinase was first discovered and purified from soybean fermented food by <em>Bacillus subtilis </em>natto. To enhance NK’s activity and simplify downstream processes, production of recombinant NK using several microbial expression systems such as <em>Escherichia coli</em>, <em>B. subtilis</em>, and <em>Lactococcus lactic</em> has been studied. Among all of them, <em>B. subtilis</em> is a prominent host for overproduction of functional proteins which can be secreted directly into the culture medium. In this study, recombinant NK from <em>B. subtilis</em> R0H1 was purified using two-step membrane filtration. Results showed 3.2-fold increase in activity and a recovery rate of more than 80%. Molecular weight of NK was approximately 28 kDa and its fibrinolytic degradation capacity was proved according to SDS-PAGE. The optimal pH and temperature of this NK were 8.5 and 55°C, respectively. The enzyme activity was boosted by Mg<sup>2+</sup>, Ca<sup>2+</sup> and obviously inhibited by Co<sup>2+</sup>, Zn<sup>+2</sup>, Fe<sup>2+</sup>, and SDS. The apparent Km and Vmax with fibrin as the substrate were 3.08 mM and 6.7 nmol/min, respectively. The results suggested that membrane filtration is a useful method for purification of recombinant NK from <em>B. subtilis</em> R0H1. Therefore, application of membrane system is proposed to purify NK at the pilot scale. In addition, our findings indicated that recombinant NK produced in <em>B. subtilis</em> R0H1 showed high and stable proteolytic activity in slightly alkaline pH and at high temperature. It also exhibited strong fibrinolytic activity again both substrates: fibrinogen and fibrin.</p> ER -