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Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification, characterization, and insights towards thermal denaturation.

Ramadevi, M. and Deepesh, Panwar and Juby Elsa, J. and Gaurav Singh, Kaira and Mukesh, Kapoor (2018) Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification, characterization, and insights towards thermal denaturation. International Journal of Biological Macromolecules, 119. pp. 770-778. ISSN 0141-8130

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Abstract

A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal) from germinating black gram (Vigna mungo) seeds was purified to homogeneity (432 U/mg specific activity, 1542-fold purification) using ion-exchange (DEAE-cellulose, CMsepharose) and affinity (Con-A Sepharose 4B) chromatography. VM-αGal appeared as a single band ~ 45 kDa on SDS-PAGE and showed optimal activity at pH 5 and 550C. Hg2+ and SDS completely inhibited VM-αGal activity. The Km, Vmax and catalytic efficiency (kcat/Km) of VM-αGal for pNPG and raffinose was found to be 0.99, 17.23 mM, 0.413 s-1 mM-1 and 1.66, 0.146 mM ml-1 min-1, 0.0026 s-1 mM-1, respectively. VM-αGal was competitively inhibited by galactose (Ki 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-510C showed that the enzyme was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in β-sheet content at 45-650C, and above 550C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing the flatulence-causing raffinose family oligosaccharides [26.5% and 18.45% decrease in stachyose and raffinose, respectively).

Item Type: Article
Uncontrolled Keywords: Vigna mungo, α-galactosidase, Purification, Characterization, Thermal stability soymilk hydrolysis
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 28 Polysaccharide Chemistry
600 Technology > 08 Food technology > 22 Legumes-Pulses
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 10 Jan 2019 08:59
Last Modified: 10 Jan 2019 08:59
URI: http://ir.cftri.com/id/eprint/13900

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