The Preparation Of Activated Silica Particles And Its Properties For Papain And Cofactor NAD~+Immobilization

The aim of this study was to evaluate the immobilized efficacy ofcarboxyl-activated silica particles for papain EC:3.4.22.2and cofactorNAD+. Carboxyl-activated silica particles were prepared by two steps. First,γ-Aminpropyltriethoxysilane (APTES) was coupled to silica nanoparticlesand then treated with glutaric anhydride to produce acarboxylate-terminating immobilized spacer arm. Some parameters of thethe activated carrier were studied and compared. The results showed thatthe highest loading of amino-group was obtained using1.8ml APTES/gparticles; the highest loading of carboxyl groups was obtained using2.5g/g SiO2; and the optima reaction time was3h. Thermogravimetric analysis(TGA) and a fourier transform infrared spectrometer (FT-IR) was used forsample analysis. As indicated by the TGA and FT-IR, the modification forthe silica particle surface was successful.The procedure of immobilized papain was under mild conditions viaEDC. The results showed that the amount of immobilized papain on thesupport was measured to be4.96mg/g-particle; the highest activity recovery was obtained by using10mg papain/g-particle, the optimareaction pH was7.0and the optima reaction temperature was70.The properties of both immobilized papain and free papain wereinvestigated. The results showed that the thermal and pH stabilities of theimmobilized papain were higher than those of free enzyme, the resistanceto four inorganic salts and four organic solvent were improved considerably.In our research, the immobilized papain was used to hydrolyze proteinextracted from tea and release amino acides. The results showed that theconcentration of protein decreased from119.39to86.60g/ml, theconcentration of amino acides increased from116.58to123.45g/ml.We studied that cofactor NAD+covalently attached tocarboxyl-activated silica nanoparticles successfully by direct coupling ofNAD+to a carboxylate-terminating spacer arm using carbodiimidepromoted coupling. The carboxyl-activated carriers produced reactivecarboxyl groups which then react with the free amino groups of NAD+togive peptide bonds. Some parameters of the the immobilized NAD+werestudied and analysed. The results showed that the optima reactiontemperature was35; the optima reaction pH was5.0; the optima reactiontime was28h and the highest activity recovery was obtained by using500mg NAD+/g-particle. Compared with free NAD+, the immobilized NAD+had better stability. The immobilized NAD+retained certain activity after five reuses continuously.