Improvement of uricase production from Bacillus subtilis RNZ-79 by solid state fermentation of shrimp shell wastes

Among various strains of Bacillus subtilis, the strain RNZ-79 was selected for this study due to the highest uricase productivity in solid state cultures containing shrimp shell wastes, highest V-max (0.42 mu M mg(-1)min(-1)) and lowest value of Km (56 mu M). Maximum productivity was observed at pH 7.6 and 45 degrees C with an inducer concentration of 0.4% (w/v) uric acid and moisture content of 80% (v/w). An inoculum of 7% (v/v) and particle size of 354-500 mu m for substrate were optimal for productivity after 54 h of fermentation. None of the tested carbon and inorganic nitrogen sources had stimulatory effect on uricase productivity. KH2PO4 at 0.12% (w/v) was the best source of phosphorus. Final uricase productivity was 5.05-fold more than the initial productivity. Enzyme purification increased the specific activity to 25-fold with a recovery of 36%. Furthermore, the purified enzyme showed a molecular mass of 33.7 kDa. Purified uricase was optimally active at pH 8.0 and 40 degrees C and maximally stable at pH 7.0-10.0 and till 70 degrees C for 30 min. The half-life (t(1/2)) at 60, 70, 80, 90 and 100 degrees C were 87.0, 56.6, 30.0, 24.2 and 15.6 min, respectively, and the calculated midpoint temperature (T-m) was 66.85 degrees C. Interestingly, purified enzyme exhibited a good storage stability for 3 months and none of uric acid analogues were competitive inhibitor, indicating a high specificity of uricase.