Study On Chemical Modification And Immobilization Of Papain

As an effective technique to improve the physical and chemical characteristics,such as enzyme activity,enzyme chemical modification has received considerable attention.The methods includes enzyme crosslinking,site-directed mutagenesis,small molecular compounds, homo-functionality polymerization and so on.Immobilization of papain after chemical modification will further increase its stability.Papain(EC 3.4.22.2) is a powerful proteolytic enzyme,belonging to the cysteine protease family.It is a minor constituent among the papaya endopeptidases.The enzyme has found many application in cell isolation,cosmetic,detergents,food and pharmaceutical industries.In this paper,Under the substrate protecting and non-substrate protecting,papain was firstly modified by using three novel anhydrides.The average ratio of modified -NH₂ was tested by a trinitrobenzenesulfonic acid method.The native and modified papain samples were purified by dialysis and their structures were characterized by UV and IR.The factors related with the activity of the modified papain,such as temperature,pH value,kinetic constant,thermodynamics,metal ion and the concentration of SDS were studied and compare with those of the native papain.The results showed that the optimum reaction temperature and pH for modifying papain were 80℃and 9.0,respectively.When the concentration of SDS was 5 mg/mL,about 50%of the activity of modified papain were maintained.Among these modified enzymes,the enzyme modified with pyromellitic dianhydride was found to be best in catalytic efficiency.ΔH*,ΔG andΔS*were found as 131.8 kJ/mol,80.17 kJ/mol and 151.9 J/mol·K,respectively.Compared with the native papain, the thermal stability and the resistance of modified enzyme to alkali and washing detergent were improved considerably.Several mPEG derivatives with different molecular weight were synthesized in this dissertation,by which papain was modified,and relative characteristics of the enzyme modified was determined.It has been discovered in this thesis that the enzyme activity is retained to a great degree through modification,and the thermal stability is improved greatly,too.And it is proved that the thermal stability is related directly with the molecular weight of modifiers.As the molecular weight of modifiers increased,the enzyme's rate of SDS-PAGE slowed down.The modified papain was immobilized on the cotton fabric with the aid of glutaraldehyde. The properties of immobilized enzyme were studied,the enzyme stability,moisture regain and air permeability fabric were also measured.The inhibiton of tyrosine by papain was firstly investigate. The results showed that after treated with glutaraldehyde,papain was immobilized to the support. Compared with immobilized nature papain,the thermal stability and the resistance to alkali were improved obviously.Storaged,the free enzyme only kept half of the original activity but the immobilized enzyme still retained 70%of the activity.The regain of the cotton fabric with enzyme immobilized have been up to scratch and has good air permeability.Papain inhibited both monophenolase and diphenolase activities of mushroom tyrosinase when _L-tyrosine and _L-DOPA were assayed spectrophotometrically,respectively.