Study On Flexible Immobilization Papain

At present traditional covalent immobilization is one of the hottestimmobilization ways because of firm combination, continuous using in long time. Butdisadvantage of this immobilization method is low recovery of immobilized enzyme,one of the reasons may be: in the process of covalent immobilization, the collisioncaused by impact between enzyme protein and support surface can make enzymeprotein natural conformation not maintain, therefore recovery of immobilized enzymecan be improved through meliorating characteristic of support surface from molecularlevel. Based on Lin Sicong's hypothesis about relationship between character andstructure of biomaterial surface and it's nonthrombogenicity, Bayer expatiatingflexible chain mechanism in the synthesis of peptides summarization, "Flexibleimmobilized enzyme"model is indicated in this paper, moreover feasibility of thismodel can proved by design, preparation and application of flexible support.In the first part, the development and application of immobilization technology isgeneralized; disadvantage and advantage, principle and method of traditional covalentimmobilization are summed up. It is found that covalent immobilization is one ofhottest ways, but this way can cause to less recovery of immobilized enzyme,therefore "flexible immobilized enzyme"model is indicated in this study on theformer groundwork of "rigid"immobilization and "arm spacer"immobilization. Themajor idea of this model is: one flexible and hydrophilic chain (flexible chain) islinked between the support and enzyme protein, then rigidity and hydrophobicproperty of the support can be meliorated, so that the surface of support become moreflexible and hydrophilic. In this model, the loss of enzyme activity caused by rigidimpact can be reduced and most of homogeneous catalytic activity of free enzyme isreserved. Accordingly, this model settles the problem about low recovery ofimmobilization by covalent immobilization.In the second part, because starch molecule is provided with flexibility,hydrophilic property and biocompatibility, dialdehyde starch (a kind of modifiedstarch including much aldehyde, simply called DAS) is chose to be flexible chain in"Flexible immobilized enzyme"model and it can be prepared by oxidation. In thepreparation of DAS, this paper adopted a new reaction system called "virtualhomogeneous phase" between homogeneous phase and heterogeneous phase andstudied the mechanism of this system. The experiment results show that in the "virtualhomogeneous phase"system, at a rather lower temperature(0℃), neutral pH(4.4), andwith fewer oxidant and time, oxidation reaction velocity can be improved, sidereaction can be avoided and the properties of products can be ameliorated.In the third part, DAS as flexible chain and glutaraldehyde as arm spacer arecoupled with the three supports (amino polystyrene micro-sphere, carboxylicpolystyrene micro-sphere and chitosan), then papain is immobilized on the flexiblesupport and arm spacer support, and immobilization conditions (means ofimmobilization, functional group on the support, amount of DAS and enzyme, and Ⅲ南京工业大学硕士学位论文immobilization time, temperature, pH etc.) are optimized. The experiment resultsshow that recovery of immobilized enzyme can reach 70% when chitosan as supportand recovery of immobilized enzyme is 40~50% when amino polystyrene orcarboxylic polystyrene as support, whereas only 20~30% with arm spacer support.Therefore, the "Flexible Immobilized Enzyme"model can overcome the deficiency ofarm spacer immobilization and recovery of immobilized enzyme can be enhanced 2~3times. There are three reasons bringing increasing recovery of immobilized enzyme atleast. First, flexible support can reduce the change of enzyme conformation. Second,flexible support can improve immobilization velocity. Third, flexibly immobilizedenzyme can reserve the homogeneous catalytic activity of free enzyme. At last,through the re...