Random mutagenesis and process optimization of bacterial co-culture for hyperproduction of 1, 4-α-D-glucan glucanohydrolase using submerged fermentation

Abstract

The exponential increase in the application of 1,4-α-D-glucan glucanohydrolase (GGH) in various fields has placed stress and demand in both qualitative improvement and quantitative enhancement through strain improvement. In the present work, Bacillus subtilis LCBT-15 and Bacillus amyloliquefaciens LCBT-20 were subjected to physical as well as chemical mutagenesis for improving the GGH production potential. Applications of the UV light and ethidium bromide did not cause a significant increase in the enzyme production. However, Ethyl methane sulphonate (EMS) treated co-culture 10 gave 1.3-fold increase in the GGH production, in contrast to the wild co-culture. Different physicochemical parameters including fermentation media, rate of fermentation, temperature, pH, nitrogen and carbon sources and surfactants were also investigated. The M7 medium composition was optimized for GGH production after 48h of incubation at 37 o C and pH 6. The optimum inoculum size was 3.5 ml (1´10 6 cells/ml) in 50 ml of medium. The best carbon and nitrogen sources were lactose (2.5 %); ammonium chloride (1.75 %) and beef extract (1 %), respectively. Optimal GGH production (287 U/ml) was obtained when the medium was supplemented with 0.05% Tween 80. The novelty of this work was exploration of the synergistic phenomena of mutant bacterial co-culture for the enhancement of GGH production.