Production, purification, and characterization of thermostable alkaline protease under solid state fermentation conditions for application in bio-detergent technology
Screening studies were carried out for one hundred, and fifty-three bacterial isolates with respect to their ability to produce protease(s), after growing on some industrial wastes at 55ºC, and pH 9.Two most potent thermophilic bacterial isolates concerning of alkaline thermostable protease(s) production were identified as Bacillus licheniformis B42 and Geobacillus stearothermophilus B78. Alkaline thermostable proteases productivity by the two most potent bacterial isolates was affected by substrate concentrations (solid substrate), carbon source, nitrogen source, mineral ions, vitamins, amino acid supplements, incubation temperature, incubation period, and inoculum size. Maximum both enzymes production by B. licheniformis B42 and G. stearothermophilus B78 were obtained on slaughter house wastes (SHW) concentrations,1.5,2.5 %;carbon sources; galactose, no tested sugar; nitrogen sources; diammonium hydrogen phosphate, ammonium molybdate; chelating agents; ethylene diamine tetra acetic acid (EDTA) (500 ppm), no tested ions; vitamins; thiamine pyridoxine; amino acids; arginine, DL-tyrosine at 55°C for 72 h. when inoculated by 0.5, and 2.5 ml respectively. The protease yield under all optimal conditions was increased many folds from 563.68 to 17825 U/ml (31 fold) by B. licheniformis and from 23.7 to 7096 U/ml (299 fold) by G. stearothermophilus. The purification fold of B. licheniformis B42 alkaline thermostable protease increased to 394.7 after applying Sephadex G200 and 411.9 times after Sephadex G100 column chromatography techniques. Fifteen amino acids were detected in the partially purified protease. Optimum conditions of the partially purified alkaline thermostable protease on Sephadex G200 followed by Sephadex G100 produced by B. licheniformis B42 were: incubation temperature, 50°C; thermostable between 50-70°C; pH,10; and incubation time,36 h exhibited maximal activity. The purified enzymes exhibited good stability towards chlorine. Compatibility of the purified enzyme with various commercial detergents reported that purified protease was unstable in presence of commercial detergents. Calcium chloride was able to restore and enhance the activity of the partially purified enzyme in the presence of commercial detergents. The alkaline serine proteases secreted by both strains are industrially important in respect of their abilities to act in alkaline pH and to show stability in broad pH ranges in addition to their stabilities toward sodium dodecyl sulphate(SDS), and hydrogen peroxide (H2O2), suggesting that it is a potentially candidate as an additive in the detergents formulations.
Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 111-129