Medium and culture conditions of escherichia coli BL21 (DE3) pLysS cultivation for Thermostable Alkaline 50a Protease Biosynthesis and its characterization

摘要

Thermostable alkaline protease is one of the most important enzymes with wide industrial applications especially in detergent industry. Due to the availability of newly cloned E.coli BL21 (DE3) pLysS harboring 50a protease gene and commercial importance of protease; optimization, purification and characterization of this recombinant 50a strain was developed and identified throughout this study. The fermentation conditions focused on their medium and cultural conditions were developed for overproduction of thermostable alkaline 50a protease. The optimum medium composition was achieved through the application of one-factor-at-time technique, Plackett-Burman design and response surface methodology resulted atud188.74 U/ml of thermostable alkaline 50a protease activity. Sorbitol (5 g/l), tryptone (20 g/l) and CaCl2.2H2O (2 g/l) were identified as the most significant components in fermentation medium. The thermostable alkaline 50a protease production was further increased with the optimization of cultural conditions resulted about 327.46 U/ml of the protease activity. The optimum cultural conditions was also achieved through the application of RSM with the corresponding conditions of agitation speed, incubation temperature and initial pH of production medium at 240 rpm, 30ºC and pH 8.63. About 500% increment in the protease production using optimum condition was successfully achieved compared to the non-optimized conditions. The purity of purified thermostable alkaline 50a protease was proven by molecular weight of approximately 37 kDa on SDS-PAGE analysis with purification fold of 21.97. The characterization studies showed that the thermostable alkaline 50a protease was stable at 70ºC and pH range of 8 to 10 with the highest activity at pH 9 and 80ºC. The characteristic of serine protease was confirmed by the inhibition at more than 90% of protease activity through PMSF. The thermostable alkaline 50a protease also showed stability in the presence of non- ionic surfactant and oxidizing agents at incubation temperature of 40ºC. Besides, this protease also showed compatibility and stability in the presence of various commercial laundry detergents at 40ºC. The wash performance analysis using thermostable alkaline 50a protease as a detergent additive in commercial detergent showed better results than wash performance using detergent or enzyme alone. In conclusion, according to optimum condition of fermentation process, stability, compatibility, and visual observation of wash performance analysis, this thermostable alkaline 50a protease had a potential for commercialization in detergent industry as a new detergent additive.