Obtaining efficient mutant from the wild type bacillus subtilis E6-5 by physical and chemical mutagenesis for high efficiency protease production, optimizing the mutant's culture medium

Abstract

In this study, to enhance protease production, the wild type of Bacillus subtilis E6-5 was mutagenized by random mutagenesis using ultraviolet radiation and ethidium bromide. After combined treatment, several mutants were obtained. Among these mutants, the mutant strain with the largest proteolytic zone diameter (25 mm) was selected and named Bacillus subtilis ATA38. The enzyme production capacity of the obtained mutant was tested and the mutant strain (404 IU/mL at 24 hours) produced 6.7 times more enzyme than the parental strain (60 IU/mL at 32 hours). The effects of some important parameters in the growth medium on enzyme production were examined. The best carbon, organic nitrogen and metal ion were obtained with wheat starch (525 IU/mL), meat extract (850 IU/mL) and KCl+CaCl2 (548 IU/mL), respectively. pH 6.0, 37 degrees C, 200 rpm, inoculum age 18 hours and inoculation amount 1% were obtained as the best physical factors.To further increase the yield, the best nutritional and physical parameters were combined to create a new modified medium. It was determined that the enzyme yield with mutant strain increased 2.7 times in the modified medium (1096 IU/mL) compared to the control (404 U/mL). The mutant strain (1096 IU/mL) showed an 18.2 -fold increase in production compared to the wild type (60 IU/mL) in the modified medium. Protease enzyme obtained from ATA38 mutant strain may have great potential in industry for different purposes.