Hyperproduction of nattokinase from Bacillus subtilis VIT MS2 using random mutagenesis and statistical optimization through central composite design

Abstract

Background: An increase in worldwide death rates attributed to ischemic stroke and myocardial infarction explains the demand to search for new thrombolytic drugs. The current study investigates the therapeutic effect of Nattokinase, a fibrinolytic protein from the mutant strain Bacillus subtilis VITMS 2 isolated from fermented milk of Vigna unguiculata. Results: The enzyme production was improved using random mutagenesis combined with statistical optimization through composite central design (CCD) with agro-residual substrates. Among all the different combinations employed, 10% (v/v) cane molasses, 12.5 g/L soybean waste, 12.5 g/L eggshell powder, and 27.5 g/L brewer’s spent grain demonstrated a significant influence on fibrinolytic enzyme yield was 4639.43 ± 10.65 FU/mL (Fibrinolytic units/milliliter). This represents a ~ 37.75-fold increase when compared to the unoptimized wild-type strain. The CCD model demonstrated high significance (p < 0.0001) with a strong correlation (R2 = 0.9963), indicating a reliable model fit. The identity and purity of the enzyme was confirmed via MALDI-TOF. Conclusion: The combination of strain improvement through mutagenesis and media optimization enhanced Nattokinase production, offering a promising approach to develop an enzyme therapy for ischemic stroke and myocardial infarction.