Production optimisation and characterisation of extracellular protease secreted by newly isolated Bacillus subtilis AU-2 strain obtained from Tribolium castaneum gut

• Published in: Biocatalysis and agricultural biotechnology 2019-05, Vol.19, p.101122, Article 101122
• Main Authors: Patel, Aniketa R., Mokashe, Narendra U., Chaudhari, Diptaraj S., Jadhav, Ajaykumar G., Patil, Ulhas K.
• Format: Article
• Language: English
• Subjects: Bacillus subtilis; Food-grade protease; Protease; Response surface methodology
• ISSN: 1878-8181; 1878-8181
• DOI: 10.1016/j.bcab.2019.101122

A novel protease secreting bacterial strain was isolated from the gut of Tribolium castaneum residing in stored Glycine max (soybean) seeds. The strain was identified as Bacillus subtilis AU-2. A response surface approach using Plakett-Burman Design (PBD) was used for the screening and a Central Composite Design (CCD) was used for the optimization of production parameters. An elevated protease production (580 U/mL) was achieved in optimised medium comprised of (g/L) soy bean meal, 10 g; glucose, 7.5 g; KH2PO4, 1.0 g; CaCl2, 1.0 g and pH, 7.0 by using 3.5% inoculum at 37°C temperature in agitated (60 rpm) batch culture after 48 h. The three step enzyme purification achieved 26.81 fold purified protease which yielded a specific activity of 22773 U/mg and 34% recovery. The purified extracellular protease of B. subtilis AU-2 has molecular mass 38 kDa. The protease was documented as metallo-protease having optimum pH 7.5 and temperature 40°C. The purified protease showed appreciable endurance in presence of sodium dodecyl sulphate, Triton X-100, hydrogen peroxide and sodium per-borate. The protease efficiently digests proteinaceous substrates like casein, soybean flour, bovine serum albumin, haemoglobin, and egg albumin. The protease of newly isolated B. subtilis is now available for various food applications like soybean hydrolysate preparation, meat tenderisation, casein lysate preparation, milk clotting, and food waste treatment. [Display omitted] •A new proteolytic Bacillus subtilis AU-2 strain isolated from Tribolium castaneum gut.•Protease production optimized using RSM approach.•Protease purified and characterized.•Protease suggested for food applications.