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Poster Presentation 6-15
Partial Purification and Kinetic Characterization of Acid Phosphatase from Garlic Seedlings Begum Yenigun, Yuksel GuvenilirIstanbul Technical UniversityDepartment of Chemical Engineering, 80626 Maslak, Istanbul, Turkey
Telephone: (90) 212-285-35-39; Fax: (90) 212-285-29-25; E-mail: yenigun@itu.edu.tr
The objective of this study was to obtain purer acid phosphatases than those previously produced, by operating under conditions that improve the final product. The study featured the use of a mild non-ionic detergent, 40-80 % saturation with (NH4)2SO4, maintaining a low temperature to remove impurity, and the use of chromatographic columns to concentrate the acid phosphatase and remove non-acid phosphatase proteins with lower or higher molecular weights. Acid phosphatase was isolated and purified from garlic seedlings by a streamline method without the use of proteolitic and lipolytic enzymes, butanol, or other organic solvents. Grown garlic seedlings of 10-15 cm height were homogenized with 0.1 M acetate buffer containing 0.1 M NaCl and 0.1%Triton X-100. After homogenization, supernatant was filtered with paper filters. Filtrated supernatant was cooled to 4oC, followed by a 3-step fractionation of the proteins with ammonium sulfate. The crude enzyme was isolated as green precipitate which was dissolved in small amount of 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. Garlic seedling acid phosphatase was purified with ion exchange chromatography (DEAE-cellulose). The column was equilibrated with 0.1 M acetate buffer. Acid phosphatase was purified 40 fold from the starting material. The specific activity of the pure enzyme was 168 Units/mg. A variety of stability and activity profiles were determined for the purified garlic seedling acid phosphatase. These included optimum pH, optimum temperature, pH stability, temperature stability, thermal inactivation, substrate specificity, effect of enzyme concentration, effect of substrate concentration, activation energy, effect of substrate concentration to reaction time, and effect of inhibitor and activator. The molecular mass of acid phosphatase was estimated to be 58 kDa. The pH optimum was 5.7 and the optimum temperature was 50oC. The enzyme is stable at pH 4-10 and 40-60o C. Activation energy was between 10-20 kcal and for Michealis Menten coefficients, Vm for pNPP was 100mM/sn and Km is 21.27 mM, while Vm for pNP was 20 mM/s and Km was 8.33 mM. Studies of the effect of metal ions on enzyme activity showed both activating and deactivating effects. While Cu, Mo and Mn showed strong inhibitory effects, Na, Ca and K were the significant activators of acid phosphatase.
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