Production and Characterization of Asparaginase from Bacillus subtilis Strain BNT Isolated from Natto

Abstract

Bacillus subtilis strain BNT, isolated from Japanese fermented soybean (natto),was found to produce asparaginase. The supernatant of the BNT culture grown in nutrient broth at 37oC for 24 hours was purified and characterized for the general properties. It was found that BNT asparaginase worked best at 50oC and pH8.0. The enzyme activity remained unchanged after 2 hours of incubation at 60oC and pH7.0, respectively. From SDS-PAGE, it was 45.6 kDa in size. The enzyme could tolerate NaCl up to a concentration of 5% where its activity was reduced by approximately 11% after 2 hours exposure. A low cost medium was developed which was composed of 2% (v/v) molasses, 1% soybean meal, 0.2% (w/v) CaCl2, 0.05% (w/v) KCl, 0.05% (w/v) MgSO4.7H2O,0.5% (w/v) NH4NO3, 0.001% (w/v) FeSO4.7H2O and 0.001% (w/v) MnSO4.H2O in distilled water. The crude enzyme produced from the newly developed medium was found to retain its properties in terms of the optimum temperature and pH for its activity as well as the stability pattern towards various temperatures and pHs. BNT asparaginase was found to deaminate glutamine at more than 10 times lower than its activity towards asparagines substrate.