Response surface methodology-based optimization of production media and purification of α-galactosidase in solid-state fermentation by Fusarium moniliforme NCIM 1099

Response surface methodology was used to enhance the production of alpha-galactosidase from Fusarium moniliforme NCIM 1099 in solid-state fermentation. Plackett-Burman design was employed for selection of critical media constituents which were optimized by central composite rotatable design. Wheat bran, peptone and FeSO4.7H(2)O were identified as significant medium components using PB design. Further CCRD optimized medium components as wheat bran; 4.62 mu g, peptone; 315.42 mu g, FeSO4.7H(2)O; 9.04 mu g. RSM methodological optimization increased the enzyme production from 13.17 to 207.33 U/g showing 15.74-fold enhancement. The alpha-galactosidase was purified by 70% fractionation followed by DEAE anion exchange column chromatography which yields 23.33% with 28.68-fold purification. The molecular weight of alpha-galactosidase was 57 kDa which was determined by SDS-PAGE analysis. Purified enzyme has optimum pH of 4.0 and was found to be stable in wide pH range of 3.0-9.0. Its optimum temperature was 50 degrees C, whereas its activity remains above 50% up to 2 h at 75 degrees C. Hg2+ was found to be a potent inhibitor and Mg2+ acted as an activator of enzyme. No significant change was observed in enzyme activity for galactose concentration, ranging from 1 to 100 mM. The K-m values of enzyme for substrates p-nitrophenyl-alpha-D-galactopyranoside, melibiose and raffinose were 0.20, 1.36, and 3.66 mM, respectively. Low K-m and stability to various physiological conditions of enzyme represents its potential which can be exploited in various industrial applications.