Hyper-production of Alkaline Protease by Mutagenic Treatment of Bacillus subtilis M-9 using Agroindustrial Wastes in Submerged Fermentation

Abstract

Green chemistry technologies are the powerful tool for the management of environmental wastes challenges. Agro industrial residues are composed of complex polysaccharides that support the microbial growth for the production of useful products (enzymes, organic acids, drugs, etc.). Disposal and environment friendly management of these wastes has become a global priority. The aim of present investigation was to improve the alkaline protease yield by treating the parent Bacillus subtilis M-9 strain with different mutagens UV-irradiations, N-methyl-N-nitro-N-nitrosoguinidine (NTG), Ethidium bromide (EB), using agroindustrial wastes (banana stalk and corn stover) in submerged fermentation. Fifteen positive mutants were selected on skim milk agar plates for shake flask experiments. BSU-5 mutant strain showed 81.21± 3.24 PU/mL alkaline protease activity higher than parent strain (23.57 ± 1.19 PU/mL) in optimized fermentation medium. The fermentation profile like pH (9), temperature (45°C), inoculum size (2 mL), incubation time (24 hrs, and kinetic parameters such as u (h -1

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

“…The optimum temperature of a mutant of Bacillus licheniformis N-2 was found to be 60ºC [23]. While the maximum protease activity of the parent strain Bacillus subtilis M-92 was as 70.18 ± 1.25 PU/mL at optimal pH 9.0 and 37ºC, the protease activity of the mutant strain was reported as 75.75 ± 2.84 PU/mL at optimal pH 9.0 and 45ºC [24].…”

“…It has been reported to be used in combination with UV, NTG and EBr to increase the productivity of the parent strain B. subtilis M-9. Among the mutants obtained, it was determined that the BSU-5 mutant produced approximately 5.67 times more alkaline protease (81.21 ± 1.85 PU/mL) at 24 hrs of incubation than the parental strain (48 h) [24]. Bacillus subtilis S1-4 mutant obtained by combining UV and MNNG treatment achieved 2.5 times more protease production than the parental strain at the end of 24-hour production [25].…”

“…In the production of protease from mutant bacteria, a medium containing (g/L) 1 glucose, 0.2 yeast extract, 10 peptone, 0.1 CaCl2, 0.1 MgSO4 and 0.5 K2HPO4 (pH 7.0) was used [17]. The overnight culture with an optical density of 0.3 (600nm) was inoculated at 1% into 500 mL erlenmeyer flasks containing 150 mL of enzyme production medium, and incubated at 18,24,28,30,32,40,44,48,64 and 72 h in a shaking incubator at 37°C and 150 rpm. At the end of each incubation period, the cultures were centrifuged (6000 rpm, 10 minutes).…”