Study On The Immobilization Of Papain With Layer Material-Layered Double Hydroxides

The immobilizations of papain using covalent method on the anion layer material－ layered double hydroxides (LDHs) have been studied. LDHs are activated by glutaraldehyed before bing used as the carrier of the immobilized enzyme.Firstly, The effects of the electrical density of the layered sheet on immobilized enzymes activities are discussed, and the factors related with the activity of the immobilized papain on LDHs, such as time, temperature and pH of the immobilized reaction, concentration of glutaraldehyde and amount of papain were studied. The optimum conditions for immobilization were as follows: the ratio of enzyme and carrier was 160mg/g, activating with 0.5% glutaraldehyde, immobilizing for 12～24h at 15℃ and pH7.0. The ratio of activity recovery was 55% respectively.Secondly, The characters of these immobilized papain were also studied and compared with native papain. The results showed that the optimum pH value is 8.0～9.0, the optimum temperature is 65℃; the stabilities of the immobilized enzyme of the pH value, the thermostability and the stored stability are obviously higher than those of native papain, and the operational stability of the immobilized papain is also better than that of native papain, and after 10 cycles batch reactions, the activity of the immobilized papain does not descent.Thirdly, LDHs are intercalated by amino acid ,and the immobilized enzymes on the intercalated LDHs are obtained. The characters of these immobilized enzymes have also been researched, such as ,the stabilities of the immobilized enzyme of the pH value ,of urea ,of organic solvent and the thermostability, the stored stability.fourthly, use XRD to analyse the above kinds of LDHs and immobilized enzymes. The Michaelis-Menton constant of the immobilized enzymes and native enzyme are measured. The values are: Km＝97.78mmol/L(native enzyme),Km(app)=18.01mmol/L(CO32-LDHs-IE),Km(app)=5.36mmol/L(GLU-LDHs-IE)。Finally, use the immobilized papain to treat beer. As result, it is definitely effective to prevent beer emerging precipitation, and the original flavour of the beer and other physical and chemical indices remained unchanged after being treated. After at storage 4℃ for 120 days, no obvious turbidness was observed in the beer treated with the immobilized papain.