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In Vitro Inhibition of Sugarcance Invertase
Thesis Abstract:
Acid and neutral invertases in sugarcane milled juice were purified six fold by precipitation with (NH4)2SO4 at 20=40% saturation and 73-fold by Sephadex G-200 chromatography.
Two major elution peaks on Sephadex, A1 and N1 corresponding to the isoenzymes were observed. Acid invertase A1 had a MW (molecular weight) of 380,000 and invertase activity and multiple MW values suggested aggregation of the neutral enzyme. Carbohydrate contents of the major acid and neutral isoenxymes were 23.5 and 22.0%, respectively.
The (NH4)2SO4 – purified invertases were comepletely inhibited by 4 mM (millimolat) laurel sulfate, 20mM metasillicate and 0.2% detergent ‘Tide.’ Both invertase in filtered juice were completely inhibited by 12 mM laurel sulfate and 42 mM metasilicate, and were 15% inhibited by 0.25% ‘Tide.’ The acid invertase, whose optimum pH was 5.3, was more strongly inhibited by laurel sulfate than the neutral invertase. Laurel sulfate inhibition of both invertases was uncompetitive, while metasilicate inhibition was noncompetitive.
Initial velocity and kinetic inhibition studies on the (NH4)2SO4 – purified enzymes at 37°C showed that for acid invertase, the average Michaelis constant Km was 2.8 mM and the maximal velocity Vmax was 2.7 µ moles sucrose hydrolyzed per hour per mg protein. For neutral invertase, Km and Vmax values were 0.32 mM and 2.9, respectively.