An Experimental Study on Production of Intracellular β-Galactosidase at Different Conditions by Batch Process Using Isolated Bacillus safensis (JUCHE 1) and Characterization of Synthesized β-Galactosidase

Abstract

Production of intracellular β-galactosidase has been performed by isolated facultative anaerobic consortium Bacillus safensis (JUCHE1) in lab-scale fermentor by batch mode. Two types of microbial growth media, namely, lactose broth and modified de-Mann Rogosa Sharpe (MMRS) media were tested for microbial growth and β-galactosidase production. It was observed that microbial growth was high in MMRS medium compared with lactose broth. Under the present investigation, growth kinetic parameters, namely maximum specific growth rate (μmax), Monod constant (Ks), yield coefficient (Yx/s) and maintenance coefficient ( $$ m_{s} $$ ) of this organism have been determined to be 0.75/h, 12 g/l, 0.1 g biomass/g. substrate and 0.688/h respectively considering lactose as the limiting carbohydrate source. Optimum concentrations of lactose and tryptone, a nitrogen source, in microbial growth medium have been determined to be 20 g/l and 10 g/l, respectively. Optimum temperature, pH and stirrer speed for microbial growth have been found to be 37 °C, 7.0 and 170 rpm, respectively. To isolate intracellular β-galactosidase, sonication was adopted and optimum sonication time was found 300 s with inter stage cooling for 10 s after an operating time of 30 s. Intracellular β-galactosidase was characterized by enzymatic reaction kinetics, optimum pH and temperature. Maximum reaction velocity (vmax), Michaelis–Menten constant (Km), optimum temperature and pH were found 5.1 μmol/min mg/protein, 20 mM, 45 °C and 7, respectively using ortho-nitrophenyl-β-galactoside (ONPG) as a substrate.