Isolation of L-asparginase from marine bacterium Bacillus subtilis and its characterization

Abstract

Microbial L-asparginases has wide range of applications as therapeutic agents and in industries. In the present study, 57 bacterial isolates from Konark beach, Bhubaneshwar were screened for L-asparginase production and KBI-13 isolate was found to be potential producer strain. KBI-13 was identified as Bacillus subtilis at molecular levels. During production optimization, pH (8.0), temperature (40 ºC), carbon and nitrogen sources (dextrose- 0.5 %; yeast extract 1 %), aeration conditions, metal salts (FeSO₄) and NaCl (4 %) were found to be optimum. The enzyme was produced under optimized conditions and was purified by sephadex G-50 column and the purification was obtained upto 61.54 fold. The activity of enzyme was increased upto pH 8.0 and temperature 40 ºC and its stability was observed upto 16 hrs at 40 ºC temperature and pH 8.0. Pretreatment of 0.5 mM CaCl₂ increased the enzyme activity upto 20 % while, 250 mM concentration of L-aspargine was suitable for optimum activity of enzyme which was further confirmed by values of Vmax (1.25 µM/min) and Km (0.05 mM). The reaction end products did not show any significant change in enzyme activity.