The Study Of Extraction And Purification Of Protein With Modified Peanut Shells

Proteins are one of the most elementary materials of life phenomenon. Their purification hasbeen under extensive attention and related research has been done more and more by humanbeings. Reported purification methods, such as high performance liquid chromatography （HPLC）,affinity chromatography and capillary electrophoresis，are high cost and have trivial operationsteps，while the method of adsorption to separate and purify protein demonstrates muchadvantages of simple operation, short processing time and low cost. This thesis chooses to usenatural biomass-peanut shells as raw material，through the chemical reaction on their surfaces tograft with specific functional groups with adsorption effect or simple acidification and alkalizationprocessing, preparing peanut shells into different adsorbents. Results show that their adsorptionperformance are greatly improved and their application scope are widely broadened, successfullyachieving the reuse of the residue. The specific work are as follows:1. A new adsorbent was prepared by graft succinic anhydride to the glutaraldehyde crosslinkedpeanut shells through simple chemical reaction, then processed by saturated sodium bicarbonate.Weighing, infrared spectrum and XPS energy spectrum were used to characterize the products.The prepared absorbents were used in the adsorption experiments of alkaline protein cytochromeC. Factors affecting adsorption capacity, such as pH, temperature, contact time, initial cytochromec concentration and NaCl ionic strength were extensively investigated. Results showed that themaximum adsorption capacity of the succinic anhydride modified peanut shells was432. mg.g^-1atpH5.0,25℃for3h. Langmuir adsorption model can be well fitted the adsorption isotherm（R2>0.99） with theory adsorption capacity of454.5mg.g^-1, close to the experimental value.Themodified peanut shells were used to separate and purify the cytochrome c from pig myocardium.The desorption ratio reached89.9%when using1.5mol.L-1NaCNS as eluent. Purification in13.5-fold in a single step with a total enzyme activity recovery of74.0%was achieved.2.With1-Methylimidazole for modifier and glutaraldehyde as crosslinking agent, a noveladsorbent was prepared by using peanut shells. Through the weighing, infrared spectrum, and XPSenergy spectrum, the original, crosslinked, and modified peanut shells were characterizedrespectively. A series of adsorption experiments were carried out with lysozyme as adsorptiontargets. Adsorption capability of the original, crosslinked, and modified peanut shells werecompared. The experimental results showed that the modified peanut shells gave the largestadsorption capacity of194.6mg.g^-1, is1.9,1.3times higher than that of the original andcrosslinked one, respectively. And adsorption capability of the three adsorbent reached at pH10.7,indicating hydrophobic effect as the main adsorption mechanism. With Freundlich and Langmuiradsorption model to simulate the adsorption process, the results found to agree well withLangmuir model. With1-Methylimidazole modified peanut shells to purify the lysozyme in eggwhite, a high vitality yield and purification multiples were realized, and electrophoresis resultsshowed that high purity lysozyme was got after the adsorption process.3.Succinic anhydride modified peanut shells,1-Methylimidazole modified peanut shellswere used for catalase adsorption, and compared with the performance of glutaraldehydecrosslinking peanut shells. The effects of the solution acidity, initial concentration of catalase,absorbent dosage, NaCl concentration on adsorption were investigated. The results found that theinfluence of the acidity on the adsorption is larger. Only in close to catalase isoelectric point, thatis when at pH7.0, adsorption ratio is higher, indicating hydrophobic effect as the main adsorptionmechanism. Adsorption capacities of two modified peanuts shells is2times higher than that of crosslinked peanut shells, indicating modification was a effective way to enhance the adsorptionperformance. With Freundlich and Langmuir adsorption model to simulate the adsorption isothermof catalase, correlation coefficient showed that experiment agree well with Langmuir model. Alsoelution effect of different eluents on catalase were studied. Results showed that pH9.0phosphoricacid buffer solution containing0.5mol.L-1NaCNS as elution agents gaves the secondary elutionratio83.5%. Succinic anhydride modified peanut shells and1-Methylimidazole modified peanutshells were used to extract catalase from pig myocardium. Vitality yield were63.2%,71.6%andmultiple purification were17.7and11.3, respectively.4. Sodium hydroxide, citric acid and phosphoric acid were used to operate simple acid-basetreatment on the original peanut shells, Respectively. Then the alkali soluble and acid solublematerial were removed, making active groups exposed on the surface of peanut shells to increasethe effective adsorptive sites. FT-IR Spectrascopy, XPS energy spectrum were used to characterizethe prepared adsorbents. A series of adsorption experiments were carried out to explore theadsorption conditions with lysozyme as adsorption targets. The experimental results show that thehighest lysozyme adsorption capacity: phosphoric acid activated one> citric acid activated one>sodium hydroxide activated one>original peanut shells. With activated peanut shells to extractlysozyme in egg white, high vitality yield and purification multiples were realized.