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Purification, Properties and Immobilization of Peroxidase from Sweet Potato Leaf (Ipomoea Batatas Lam.)

Author: FuWeiLi
Tutor: TangYunMing
School: Southwestern University
Course: Biochemistry and Molecular Biology
Keywords: Peroxidase Purification Properties Function group Immobilization
CLC: S531
Type: Master's thesis
Year: 2010
Downloads: 118
Quote: 2
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Abstract


Peroxidase（POD,EC1.11.1.7）widely exsits in all kinds of organisms. Belonging to the oxidase containing heme, it can catalyze a variety of oxidation-reduction reaction which H2O2 takes part in. As a type of testing reagent, peroxidase can be used in food testing, medical testing and industry dection,and can also be used in treament of environmental pollution, etc.Electrophoresis pure peroxidase was obtained from sweet potato leaf by means of tissues mashing, buffer solution extraction, ammonium sulfate precipitation, ultrafiltration, ion exchange chromatography on DEAE-Sepharose Fast Flow column and gel filtration chromatography on Sephacryl S-200HR. Its specific activity was 91923.14 U/mg, the purification fold was 255.69, and the recovery rate is 1.59%. The molecular weight of this POD was worked out about 35kD by the means of SDS-PAGE and gel filtration chromatography on Sephacryl S-200HR.It was found that the optimal pH and temperature for the enzyme was pH5.6 and 60℃respectively when the substrates were H2O2 and 2-Methoxyphenol.The peroxidase appeared to be stable in 20～50℃and pH 4-8. The apparent Km values of the enzyme with different concentrations of H2O2 as the substrate was 0.291mol/L at 25℃and pH 7.2. Its activity was enhanced by urea, Li+、Na+、K+、Mg2+ and low concentration of Oxalic acid, yet inhibited by SDS、KSCN、AsA and Mn2+. The organic solvents such as methanol, ethanol, glycol and isopropanol can all inhibited POD activity and the order according to their inhibitory effects was isopropyl alcohol >ethanol>methanol> glycol.By modificating the function groups of the enzyme, it was discovered that Sulfydryl and disulfid bond might be essential function groups of the POD, but Arg, Met, His, Lys, Ser andTyr residues might not be.The enzyme was immobilized with polyvinyl alcohol and sodium alginate as carrier, saturated boric acid solution as crosslinking agent and CaCl2 as fixer. The effects of the sodium alginate concentration, enzyme quantity, CaCl2 concentration and immobilizing time on immobilizing were discussed through single factor experiment, and also the difference between them as well as the optimal level combination of them through orthogonal optimized experiments. The optimal levels of the four factors were 0.2%PVA-3%CA,the amount of carrier and enzyme were 3:1,saturated boric acid-4%CaCl2 and 15min immobilizing time. After being immobilized with the the optimal level combination, some physicochemical property, reuse stability and storage stability of the enzyme were tested. It was found that the optimal tempetature and storage stability increased, but the reuse stability was not fine.

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