LAUSR

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. 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. 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.