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 α-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 FeSO4·7H2O were identified as significant medium components using PB design. Further CCRD optimized medium components as wheat bran; 4.62 μg, peptone; 315.42 μg, FeSO4·7H2O; 9.04 μg. RSM methodological optimization increased the enzyme production from 13.17 to 207.33 U/g showing 15.74-fold enhancement. The α-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 α-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 °C, whereas its activity remains above 50% up to 2 h at 75 °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 Kmvalues of enzyme for substrates p-nitrophenyl-α-D-galactopyranoside, melibiose and raffinose were 0.20, 1.36, and 3.66 mM, respectively. Low Kmand stability to various physiological conditions of enzyme represents its potential which can be exploited in various industrial applications.
Department of Microbiology, Shivaji University, Kolhapur, 416004, Maharashtra, India.
Sanjivani B
SB
Gajdhane
Department of Microbiology, Shivaji University, Kolhapur, 416004, Maharashtra, India.
Prashant K
PK
Bhagwat
Department of Biochemistry, Shivaji University, Kolhapur, 416004, Maharashtra, India. pbd_biochem@unishivaji.ac.in.
Padma B
PB
Dandge
Central composite rotatable design; Fusarium moniliforme; Plackett–Burman design; Response surface methodology; SDS-PAGE; α-Galactosidase