Effect of deglycosylation on the properties of thermophilic invertasepurified from the yeast Candida guilliermondii MpIIIa

Abstract

tInvertase from Candida guilliermondii MpIIIa was purified and biochemically characterized. The purifiedenzyme (INV3a-N) is a glycoprotein with a carbohydrate composition comprising nearly 74% of its totalmolecular weight (MW) and specific activity of 82,027 U/mg of protein. The enzyme displayed optimalactivity at pH 5.0 and 65◦C. The Km and Vmaxvalues for INV3a-N were 0.104 mM and 10.9 mol/min/mgof protein, respectively, using sucrose as the substrate. The enzyme retained 50% and 20% of its maximalactivity after 168 h and 30 days, respectively, at 50◦C. INV3a-N was fully active at sucrose concentrationsof 400 mM and the activity of the enzyme dropped slowly at higher substrate concentration. Interest-ingly, the deglycosylated form of INV3a-N (INV3a-D) displayed 76–92% lower thermostability than that ofINV3a-N at all temperatures assayed (50–70◦C), and was inhibited at sucrose concentrations of 200 mM.Findings here indicate glycosylation plays an important role, not only in the thermostability of INV3a-N,but also in the inhibition of the enzyme by sucrose. Since the enzyme is active at high sucrose concen-trations, INV3a-N may be considered a suitable candidate for numerous industrial applications involvingsubstrates with high sugar content or for improvement of ethanol production from cane molasses.