Characterization and Pilot-Scale Application of the ZMS-2 Serine Protease with Novel Properties for the Eco-friendly...

Microbial alkaline proteases are dominating the global enzyme market with a share of over 65% due to their multifarious catalytic potentials. Hence, production of proteases with novel properties of commercial significance is highly desirable to meet the global enzyme demand. Here, we report the purification, characterization, and pilot-scale application of a serine protease from the desert soil bacterium Bacillus subtilis ZMS-2 with novel properties as dehairing agent in leather processing. The enzyme was purified 16.5-fold with a specific activity of 1543.5 U mg^-1 and recovery percentage of 33.6% using ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. The purified enzyme was characterized as a metal ion-, surfactant-, and denaturant-compatible alkaline serine protease having a molecular weight of 36.1 kDa with an optimum activity at pH 8.5 and 60 °C. The catalytic activity of the enzyme was enhanced by Zn⁺² (204%), Ag⁺ (110%), H₂O₂ (123%), Triton X-100 (110%), iso-octane (109%), chloroform (110%), ethanol (105%), ethyl acetate (110%), and acetonitrile (128%). During pilot-scale applications, the optimum condition was found to be a combination of enzyme (1.5%, 460 U mL^-1), sodium sulfide (2%), and calcium hydroxide (lime) (3%). Under this condition, the time required for complete dehairing was 90 min. Chemoenzymatically processed skins exhibited better physical properties than chemically processed skin, including tensile strength (16.35 ± 6.68 N/mm), ball burst (452.88 ± 6.06 N/mm), percent elongation (38.85 ± 1.06 N), tear strength (50.16 ± 4.42 N/mm), and softness (6.5 mm). Electron microscopy analysis of the treated skin showed complete removal of hairs with roots, confirming the keratin specificity of the enzyme. Moreover, the enzyme-assisted dehairing process reduced chemical oxygen demand (COD), biochemical oxygen demand (BOD), total dissolved solids (TDS), and total suspended solids (TSS) by 68, 77, 34, and 39%, respectively. Thus, the alkaline serine protease from B. subtilis ZMS-2 is a potential dehairing agent for the eco-friendly processing of animal skins on industrial scales.