Study On Synthesis Of Novel Flexible Carriers And Their Application In Immobilizing Papain

Immobilization of enzyme to maintain the high efficiency, mild and specialization catalytic characteristics with the properties of high stability, low environmental sensitivity, easy to be recovered and reused, easy to controlled, which has become one of the most active areas in biotechnology. The existing enzyme immobilization technologies have a certain deficiency. Therefore, developing immobilized enzyme carriers and methods are important and meaningful.In this paper, on the basis of the traditional enzyme immobilization methods and carrier material, it is found that covalent immobilization is one of hottest ways, but this way can cause to less recovery of immobilized enzyme, therefore flexible immobilized enzyme model is indicated in this study on the former groundwork of immobilization. In this model, the loss of enzyme activity caused by rigid impact can be reduced and most of homogeneous catalytic activity of free enzyme is reserved. Accordingly, this model settles the problem about low recovery of immobilization by covalent immobilization. In this paper, the macroporous type crosslinked polyacrylonitrile beads (PAN) with a larger specific surface area, high stability, high strength and an abundance of cyanogroups (C≡N) were grafted with Tetraethylenepentamine (TEPA) and Glucosamine (GA) by chemical modification method. The obtained PAN-TEPA and PAN-GA resin were used to study the flexible immobilization condition of papain. And in the present work, the modification conditions of resin, the factors of enzyme immobilization, free and immobilized enzyme characteristics were systematically investigated, which have achieved some good results. The main contents as follows:1. Tetraethylenepentamine (TEPA) and Glucosamine (GA) were selected as flexible functional groups to react with the macroporous type crosslinked polyacrylonitrile beads (PAN) by chemical modification method and two kinds of new functional flexible resin carriers (PAN-TEPA and PAN-GA) were synthesized. The optimum conditions such as reaction solvent, reaction time, reaction temperature and mass ratio of reagents have been investigated according to the change of resin weight gain rate. The synthesized resins structures were characterized by Elemental Analysis (EA), infrared spectra analysis (FT-IR) and thermogravimetry analysis (TGA). The paths of synthesis were confirmed to be nucleophilic addition reactions which amino of flexible functional groups with cyano of polyacrylonitrile beads. It was considered to be High Atom Economy, reflecting the characteristics of green chemistry well.2. To further study the immobilization enzyme process of two kinds of resin carriers (PAN-TEPA, PAN-GA), papain was chosen as a model enzyme. The factors affecting enzyme immobilization such as activation conditions of the carrier, pH, enzyme concentration, temperature and time was been investigated by the single factor experiment and Response surface methodology (RSM) to optimum the immobilized process. The optimum immobilized conditions obtained from RSM was as follows:the optimum conditions for PAN-TEPA was:immobilized pH6.7, the amount of enzyme1.0mg per10.0mg carrier, immobilization temperature31℃; the optimum conditions for PAN-GA was: immobilized pH7.3, the amount of enzyme1.47mg per10.0mg carrier, temperature31℃, immobilization time5h. The immobilized enzyme activity, amount of immobilized enzyme and recovery rate of enzyme activity obtained from the RSM optimum conditions for PAN-TEPA and PAN-GA was4.745U/mg,56.7mg/g,71.3%and5.855U/mg,72.9mg/g,80.6%, respectively, which reflect that the two modified resin have potential application value as enzyme immobilization carriers.3. The enzymatic activity of flexible immobilized papain was investigated by researching the enzymatic properties of free enzyme and immobilized enzyme, for which casein was chosen as substrate. The results showed that the optimum reaction temperature raised from50℃for free enzyme to60℃for immobilized papain, and the immobilized enzyme maintained a higher enzyme activity at50℃～80℃range. The optimum reaction pH for free and immobilized papain were6.5and7, and the pH ranges were widened from6～7(free enzyme) to5～9(immobilized enzyme), respectively. The decreased Km showed that the immobilized enzyme exhibited a higher affinity to the substrates. The half life time of papain t1/2was prolonged after the immobilization, prolonged from4.9d (free enzyme) to28.4d (PAN-TEPA) and26.7d (PAN-GA) at4℃. The thermal stability, storages stability and resistance to organic solvents of immobilized papain were higher than the free enzyme. The residual enzyme activity of immobilized enzyme was above60%after eight cycles, indicated that the immobilized enzyme with a better repeated operational stability.The novel flexible resin carrier and immobilization method obtained provides a friendly microenvironment for papain, and maintain the biological and catalytic activity of papain in a better level, indicating that the flexible resin carriers can be used as a good immobilizing carrier, which has great practical significance and applied value for immobilization enzyme.