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Research on Purification Technology and Characteristics of α-amylase Inhibitor from Maize

Author: LiuWenZhi
Tutor: GuoDeJun
School: Heilongjiang Bayi Agricultural University
Course: Of Food Science
Keywords: Corn α-AI Extract Separation and purification Nature Dynamics
CLC: TS201.25
Type: Master's thesis
Year: 2009
Downloads: 17
Quote: 0
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Abstract


α-amylase inhibitor (α-amylase inhibitor, α-AI), is a natural biologically active substance, because of the insect digestive tract, mammalian saliva and pancreatic amylase enzyme has a specific inhibitory activity, and are referred to as \off by \Therefore, α-AI is used for medical, agricultural, and enzyme engineering fields. The purpose of the test is the screening of corn varieties with higher levels of α-AI, determine key technical parameters extracted corn α-AI, establishing corn α-AI purification process and proceeds corn physicochemical properties and dynamics of the α-AI preliminary analysis. Corn α-AI extraction. Screened 20 corn varieties with higher levels of α-AI varieties as experimental materials. The single factor experiment to study the extract, extraction time, extraction temperature, salt concentration of the leaching solution, the pH of the leaching solution, leaching liquid ratio, centrifuge speed 8 corn α-AI extraction rate . Pearson correlation, variance analysis of several factors that determine a significant impact on corn α-AI extraction rate using quadratic orthogonal rotation combination testing to determine the best extraction parameters of maize α-AI. The results show that: to test C272 corn varieties α-AI highest content; optimum extraction process parameters: 0.02 mol · L -1 phosphate buffer solution, NaCl concentration of 0.15 mol · L -1 , extracts material than 4.3:1, extracts of pH 6.9, extraction time 3.2 h, the extraction rate of maize α-AI 22.162 AIU g -1 verification experiment reached 21.996 AIU · g -1 ; separation of ammonium sulfate saturation of 35% to 65% corn α-AI best. Maize α-AI separation and purification process and its technical parameters optimization. Corn α-AI is further isolated and purified, to obtain higher purity corn α-AI, and determine a simple and feasible purification technology. DEAE-Sephadex A-25 and D301, D315, DCF-5 and other ion-exchange medium is a static adsorption test to determine the optimum column material; eluant and elution, the packing height test, L9 (34) orthogonal test best test conditions to optimize the packing height, flow rate, feed concentration and injection volume four factors to determine the optimum separation conditions; using Sephadex G-75 gel filtration of the purified corn α-AI separation desalination, and finally using SDS-PAGE, HPLC purity of α-AI were identified. The results show the DEAE-Sephadex A-25 as the best filled the media adsorption corn α-AI effect, at a flow rate of 30 mL · h -1 , packing height of 28 cm, the injection concentration of 2.0 mg · mL -1 , the injection volume was 1.5 mL corn purification of α-AI results are satisfactory; SephadexG-75 gel filtration chromatography further desalination, can reach a purity of 388.2 AIU · mL -1 Corn α-AI products, the recovery rate was 33.2%. Corn α-AI concentration was determined by HPLC, 33.32 mg · mL -1 . Nature of corn α-AI research. By analyzing the nature of the mentioned corn α-AI, it is determined this corn α-AI is a noncompetitive inhibitor inhibition constant Ki of 8.0 × 10 -4 mol · L -1 ; its optimum pH is 6.8, and a pH of 6.0 to 7.0 is more stable; good thermal stability, there is still a high inhibitory activity at 100 ° C under the effect of 40 min; specificity detect that This inhibitor is not only the inhibition of porcine pancreatic amylase, and the inhibition of the corn borer amylase and human salivary amylase; scanning electron microscope test results show that the proposed corn the surface structure of the α-AI and corn powder structure is completely different; determine the corn α-AI PAGE and SDS-PAGE electrophoresis determination of one subunit relative molecular weight of 17.9 KDa; polyacrylamide isoelectric focusing (IEF) for the determination of the corn α-AI isoelectric point is 5.96.

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CLC: > Industrial Technology > Light industry,handicrafts > Food Industry > General issues > Basic science > Food Chemistry > Enzymes
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