Development of a β-glucosidase hyperproducing mutant by combined chemical and UV mutagenesis

Abstract

The extracellular β-glucosidase from microorganisms is generally produced in low levels. Therefore, in this study, a β-glucosidase hyperproducing mutant was developed by multiple exposures of ethyl methyl sulfonate (EMS) and ultraviolet (UV) radiation (both individually and jointly) to Bacillus subtilis strain (PS). The developed mutants were screened, selected and characterized. The mutant, PS-UM1 developed after UV exposure alone, indicated a small increase in β-glucosidase production (718 U/l) in comparison to the wild-type strain, PS (675 U/l). The mutant, PS-CM5 developed after EMS exposure alone, displayed a slightly better production (762 U/l) than both the above strains. However, after exposure of the wild-type strain to both UV and EMS mutagens jointly, a better mutant (PS-CM5-UM3) was developed with 1.2-fold increase in production (806 U/l). Further, optimization of culture conditions by classical “one-variable-at-a-time” approach was done to determine the optimum, pH, temperature and nitrogen sources. The selected mutant (PS-CM5-UM3) produced up to 1,797 U/l enzyme and was found to be stable for ten generations. The β-glucosidase from the selected mutant (PS-CM5-UM3) was concentrated and purified using ammonium sulfate, dialysis and size-exclusion chromatography. The enzyme displayed maximal activity at 60 °C and it was found to be fairly stable at temperatures up to 70 °C for 30 min. Its molecular weight was determined to be around 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Production, purification, characterization and usage of a detergent additive of endoglucanase from isolated halotolerant Amycolatopsis cihanbeyliensis mutated strain Mut43

“…Similar observations have been reported by Rajoka et al (1998) and Kumar (2015). Although the mechanism through which random mutation increases enzyme production remains unclear, some researchers with similar results have suggested that the improvement in enzyme production might be due to the effect of mutagens on regulatory genes, modular proteins or mRNA stability (Agrawal et al 2012). …”

Rice straw as a feedstock for biofuels: Availability, recalcitrance, and chemical properties

“…Silica may have a negative effect on cellulase enzymes . A recent paper found nonproductive adsorption of cellulase (Novozymes CTec2) to pure amorphous silica with a maximum enzyme binding capacity of 244 mg per gram silica, and that adsorption increased with enzyme concentration and temperature.…”

“…111 Silica may have a negative eff ect on cellulase enzymes. [112][113][114][115] A recent paper found nonproductive adsorption of cellulase (Novozymes CTec2) to pure amorphous silica with a maximum enzyme binding capacity of 244 mg per gram silica, and that adsorption increased with enzyme concentration and temperature. In the enzymatic hydrolysis of Avicel mixed with amorphous silica, they found that addition of PEG1500 almost completely relieved inhibition caused by silica.…”

Immobilization of β-glucosidase onto silicon oxide nanoparticles and augment of phenolics in sugarcane juice

“…The b-glucosidase was previously purified from developed mutant of Bacillus subtilis strain (PS) in our laboratory (Agrawal et al 2012(Agrawal et al , 2013 b-Glucosidase assay b-Glucosidase activity was assayed by incubating the reaction mixture containing 10 mM p-nitrophenyl-b-Dglucopyranoside (pNPG) and purified b-glucosidase in 1:1 ratio for 1 h at 60°C. The reaction was stopped by adding 2 ml of 1 M Na 2 CO 3 .…”