Isolation of L-Asparginase from Marine Bacterium Bacillus subtilis and its Characterization

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 °C), 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 °C and its stability was observed upto 16 hrs at 40 °C 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.