Determination of α-Amylase activity and soluble starch content in normal and cork spotted D̀'Anjou' pear fruit Public

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/dz010s57c

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  • There was a linear relationship of [alpha]-amylase activity from 2 to 12 hours incubation in both freeze-dried and fresh 'd'Anjou' pear fruit tissue. Activity was greater, however, in fresh as compared with freeze-dried tissues. [alpha]-Amylase activity from both types of samples was not detected until 60 minutes incubation. After 90 minutes, activity increased significantly in both freeze-dried and fresh tissue extracts. Three different buffers (acetate, Tris-HCl and imidazole-HCl) were used at varying pH levels (from 4.60 to 8.23) to ascertain the optimum assay system. Highest specific activity was recorded with an acetate buffer at pH 5.64. The Km value in this system was 1.43 mg.ml⁻¹. Specific activity of [alpha]-amylase increased as Ca concentration in the reaction mixture increased from 1 to 15 mM CaCl₂. No changes in specific activity were found as the Ca concentration increased from 15 to 25 mM CaCl₂. Alpha-amylase from 'd'Anjou' pear fruit was purified 5.68 fold using ammonium sulfate fractionation and a desalting column (Bio-Gel p-6). Activity of [alpha]-amylase and protein, calcium and starch concentration were measured in preharvest calcium-treated normal and corkspotted 'd'Anjou' pear fruit at commercial harvest maturity. Activity and specific activity of [alpha]-amylase extracted from corkspotted fruit were higher as compared to [alpha]-amylase extracted from normal tissue. Protein concentration was greater in corkspotted fruit. Starch levels were less in corkspotted than in normal fruit as evidenced either by an iodine stain visual technique or by quantitative analysis of hot-water soluble starch. No interaction were found with any measured parameter between calcium treatment and fruit condition. Activity of [alpha]-amylase extracted from calcium-treated pears was greater than that extracted from normal pears. Corkspotted fruit had less calcium as compared to normal fruit.
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