Immobilization of Alkaline Protease From Bacillus brevis Using Ca-Alginate Entrapment Strategy for Improved Catalytic Stability, Silver Recovery, and Dehairing Potentialities

Abstract

Proteases are one of the most important biocatalysts with an industrial perspective due to their high production capability, cost-effective, and eco-friendly nature. In this study, an alkaline protease produced by Bacillus brevis (228.31 ± 6.2 U/mL) in liquid-state fermentation at pH 7.0 and 47 °C was further enhanced by optimizing various physical parameters. The maximum protease activity (631.09 ± 3.7 U/mL) was recorded at 45 °C, pH 8.0, and using 3 mL of inoculum size after 72 h. The optimally-produced alkaline protease was immobilized on Ca-alginate beads, and the process was optimized using a central composite design-based response surface methodology. The maximum immobilization yield (> 70%) was achieved using a 2–3.0% gelling agent (Na-alginate) and 2.5–3.0% binder (CaCl2), with 400–600 mg/L of protease concentration. Characterization revealed that immobilization improves the pH and thermal stability of protease, as the maximum activity was recorded at pH 10 and 65 °C. The kinetic studies of protease revealed higher Vmax (454.5 U/mL) and lower Km (0.09 μM) values after Ca-alginate immobilization as compared with the free enzyme (Vmax 333.3 U/mL and Km 0.16 μM). Ca-alginate immobilized protease also possessed good recyclability potential in several consecutive substrate hydrolysis experiments. By applying on goatskin and X-ray film, the immobilized biocatalyst showed improved activity than the free enzyme, revealing its potential biotechnological applications.