Abstract
A β-glucosidase with high specific activity towards isoflavone glycosidic conjugates was purified from seeds of Guar (Cyamopsis tetragonoloba) by ammonium sulphate precipitation followed by size exclusion and ion exchange chromatography. The pH and temperature optima of the purified Isoflavones conjugate hydrolyzing β-glucosidase (ICHG) were found to be pH 4.5 and 37 °C, respectively. The enzyme was relatively stable at higher temperatures. Effect of different divalent metal ions was studied and it was found that Cobalt and Mercury ions completely inhibited the enzyme activity. Km and Vmax of the purified isoflavones conjugates hydrolyzing β-glucosidases (ICHG) was 0.86 mM and 6.6 IU/mg respectively. The enzyme was most likely a trimer (approximate Mr 150 kDa) with potential subunits of 50 kDa. The purified enzyme showed activity against isoflavone conjugate glycosides viz daidzin and genistin but was inactive towards other flavonoid conjugates. The product conversion was confirmed by HPTLC and HRMS analysis. The MALDI-TOF analysis of the ICHG showed a score greater than 78 with 20 matches in MASCOT software. The five resultant peptides obtained had highest similarity in sequence with β-glucosidase from Cicer arietinum. The β-glucosidase from the C. arietinum has also been reported to exhibit the isoflavone conjugate hydrolyzing properties thus confirming the nature of the enzyme purified from the Guar seeds.
Highlights
Flavonoids and isoflavonoids represent one of the largest and most studied classes of plant based secondary metabolites with an estimate of more than 10,000 different compounds
It is important to understand that these compounds many a times exist as two different forms; the one glycosylated which is water soluble, relatively inactive and preferred by plants for the storage purpose, another one is less soluble in water and the active form
These different forms of the secondary metabolites play a crucial role in the biological activities they carry out eventually; either within the plants themselves or inside the human system once consumed via food
Summary
Flavonoids and isoflavonoids represent one of the largest and most studied classes of plant based secondary metabolites with an estimate of more than 10,000 different compounds. It is important to understand that these compounds many a times exist as two different forms; the one glycosylated which is water soluble, relatively inactive and preferred by plants for the storage purpose, another one is less soluble in water and the active form These different forms of the secondary metabolites play a crucial role in the biological activities they carry out eventually; either within the plants themselves or inside the human system once consumed via food. Apart from the industrial applications, guar has been reported to have anti-ulcer, hypolipidemic, cell-protective, hypoglycemic, and anti-secretory bioactivities These health beneficiary activities can be accredited to the presence of secondary metabolites of polyphenolic class such as daidzein, naringenin, caffeic acid, gallotannins, genistein, gallic acid, catechol, luteolin, chlorogenic acid, ellagic acid, myricetin-7-glucoside-3-glycoside, quercetin, catechin, 2,4,3,-trihydroxybenzoic acid, kaempferol, rutin, hydroxycinnamic acid, texasin-7-O-glucoside, and p-coumaroylquinic acid [8,9,10,11]. The present study was undertaken with the view of purifying the specific β-glucosidase from seeds of the guar involved in the bioconversion of isoflavonoids glycosides conjugates to their aglycone form
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