Partial Purification and Characterization of Invertase - Philippine ...

Weerasooriya & Yatawaraof Woodfordia (Weerasooriya and Yatawara 2002),Allocasia leaves (Nakasone & Yusai 1982), sugarcaneleaf (Minouru 1942) and sugar beet (Bohuslavska etal. 1984 ) .Thermal inactivationBeyond 45ºC, invertase activity of Madhucaslowly declined and became almost nil by 80ºC (Fig.4)similar to that reported for invertases of Woodfordia(Weerasooriya and Yatawara 2002), Allocasia leaves(Chen & Black 1992) and potato tubers (Isla et al. 1998). However, invertase of Madhuca seems to be moreheat sensitive than invertases of muscat bailey A grapes(Porntaveewat et al. 1994), semillon grapes (Nakanishiet al. 1991) and yeast particularly, Saccharomycesecerevisae (Raheja et al. 1998) and Saccharomycesefragilis (Kulikora 1986) which were reported to be stableup to 80ºC.Kinetic constants (km)The Km values of purified Madhuca invertase forsucrose was found to be ~ 125 mM which was very closeto the values that reported for invertases of Woodfordia,(Weerasooriya and Yatawara 2002). invertases fromSolanum tuberosum tubers (Isla et al. 1999) and higherthan Km values that reported for invertases of ricegrain (Km ~ 70mM) (Chang et al. 1994), and potatotubers (Km ~ 100mM) (Isla et al. 1998).In a summary, Invertase from Madhuca longifoliafits many characteristics described for invertaseisolated from Woodfordia fruticosa (another invertasesource which used for the preparation of Arishta)such as optimum pH, optimum temeperature, thermalstability, molecular mass and Km value. Km ofWoodfordia invertase for sucrose is reported to be~160 mM whereas that of Madhuca invertase foundto be ~125mM. This indicates possibly substratesucrose shows higher affinity towards Madhucainvertase rather than that of Woodfordia. All arishtapreparations in Sri Lanka are carried out at roomtemperature which is around 35-37ºC . This is inagreement with the temperature 37ºC which showsthe optimum activity of the enzyme. pH of the solutionis also very importance during the preparation ofArishta. As described in Kroes 1990, all arishtapreparations favour the weak acidic condition.Alkaline conditions are unfavourable for the reaction.This is in agreement with optimum pH of the enzyme.As reported by Kroes 1992, high content of gallicacid, (a weak acid present in preparation medium)presumably maintain the pH of the solution around 4-60Invertase activity (nmole/min)5040302010050 60 70 80 90 100Temperature 0 CFigure 4. Thermal stability of invertase of Madhuca longifolia (Incubation of 1nkat of enzyme in 0.1M citrate buffer pH 5 for30 min at different temperature 40-100°C and then measured for activity under standard conditions; 0.1M citrate pH 5, 37°C,3min, Mean and standard deviation were determined from six replicates).134