Abstract
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 K m values of enzyme for substrates p-nitrophenyl-α-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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0575-7) contains supplementary material, which is available to authorized users.
Highlights
IntroductionIntroduction aGalactosidase (a-D-galactoside galactohydrolase EC. 3.2.1.22) is an exoglycosidase which catalyses hydrolysis of terminal a-1,6-linked galactosyl residue from a wide range of substrates, including oligosaccharides of raffinose family sugars, such as stachyose, melibiose, verbascose and polysaccharides of galactomannans, locust bean, and guar gum (Anisha and Prema 2006, Talbot and Sygusch 1990).a-Galactosidase has been reported to various biotechnological and clinical applications like processing of soymilk (Tzortzis et al 2003), conversion of blood type (Goldstein et al 1982), and in the treatment of Fabry’s disease (Fuller et al 2004)
Response surface methodology was used to enhance the production of a-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
Summary
Introduction aGalactosidase (a-D-galactoside galactohydrolase EC. 3.2.1.22) is an exoglycosidase which catalyses hydrolysis of terminal a-1,6-linked galactosyl residue from a wide range of substrates, including oligosaccharides of raffinose family sugars, such as stachyose, melibiose, verbascose and polysaccharides of galactomannans, locust bean, and guar gum (Anisha and Prema 2006, Talbot and Sygusch 1990).a-Galactosidase has been reported to various biotechnological and clinical applications like processing of soymilk (Tzortzis et al 2003), conversion of blood type (Goldstein et al 1982), and in the treatment of Fabry’s disease (Fuller et al 2004). Solid-state (SSF) and submerged fermentations (SmF) are two types of fermentation techniques in which SSF has gained importance for the production of microbial enzymes, due to economical advantages, such as use of cheap and abundant agro-industrial waste as a substrate. It has special interest in the processes where the crude fermented product is used directly as the enzyme source (Pandey et al 1999). SSF is superior to SmF due to various advantages like simple media composition (cost-effective constituents), aeration process, releases negligible quantity of effluent, reducing pollution concerns
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