Cost-Effective Production and ProcessOptimization of Protease Enzymefrom Bacillus subtilis

Abstract

Background: The present study focuses on developing an economical and efficient process for producing
protease from Bacillus subtilis using low-cost agro-industrial residues. Rice bran, groundnut cake, and eggshell
powder were selected as nutrient sources and evaluated in submerged fermentation to assess their suitability
for enzyme production. Methodology: The fermentation conditions were systematically optimized by varying
inoculum size, substrate concentration, and nutrient supplementation. Results: Among the conditions tested,
a 3% inoculum and a 5% substrate concentration yielded the highest protease activity. Supplementation with
yeast extract produced only a marginal improvement, indicating that the agro-residues alone were sufficient to
support robust enzyme synthesis. The crude enzyme recovered from the broth was partially purified through
ammonium sulfate precipitation, dialysis, and cation exchange chromatography, resulting in a preparation with
enhanced specific activity of 310 IU/mL. Conclusion: The results demonstrate that inexpensive agricultural
by-products can effectively replace conventional media components, significantly reducing production costs
without compromising enzyme yield. This study supports the feasibility of adopting cost-efficient raw materials
and optimized fermentation strategies for sustainable, scalable protease production.