Protective Effects Of Temperature-sensitive Polymers On Enzymes In Confined Space

Enzymes, as a biocatalyst with high performance, have been widely applied in the researches and productions. However, many enzymes are inactivated by aggregations at hydrophobic sites which are exposed on heat denaturation, which hinders the applications of enzymes. Isolating denatured enzyme via hydrophobic interaction with other material is a significant method to prevent enzyme from aggregation. But temperature-sensitive polymer poly(N-isopropylacrylamide)(PNIPAAm), supposed to spontaneously protect enzyme at high temperature, can not efficiently complex denatured carbonic anhydrase B (CAB, as a model enzyme) in bulk aqueous solution due to different phase transition speed. Here we devise a novel method for protecting enzyme against heat inactivation.Firstly, PNIPAAm and CAB are encapsulated in a confined space constructed by reverse microemulsion. At high temperature, PNIPAAm forms nanoscale aggregates possessing both large specific surface area and hydrophobic surface, and then adsorbs denatured CAB via hydrophobic interaction to avoid intermolecular aggregation of CAB. With cooling, CAB is released spontaneously and recovers its activity. The assays for enzymatic activity demonstrate that CAB is effectively protected against heat inactivation through this method (protection efficiency is up to83.2%).Lipase is active at the water-oil interface and thus very useful for many applications in non-aqueous media. However, the use of lipase is also limited due to the heat inactivation which is mainly caused by the irreversible aggregation among lipase molecules. Using two kinds of temperature-sensitive polymers with different LCST, PNIPAAm and poly(2-(2-methoxyethoxy)ethyl methacrylate)(PME02MA), the thermal stability of lipase in microemulsion is effectively improved, and so is the stability of lipase at ambient temperature. Apart from proving the effectiveness and generality of this method, the temperature-sensitive polymers/lipase microemulsion represents a simple and efficient system which could be used in practical applications, since lipase retains the interfacial activity in this system. Moreover, the influences of some factors on the improvement are discussed and the mechanism of this method is suggested after exploring the process by dynamic light scattering and fluorescence measurements.The inactivation of multimeric enzyme is more complicated, the first step of which often is the dissociation of subunits. Hence temperature-sensitive copolymer chitosan-graft-PNIPAAm, with a reversible sol-gel transition depending on temperature was synthesized. Coexisting with chitosan-g-PNIPAAm in the confined space, the quaternary structure of multimeric enzyme can be stabilized and the dissociation of subunits is prevented. Because chitosan-g-PNIPAAm forms gels with network structure at high temperature and coats on the multimeric enzymes, the subunits can not dissociate even through the weakening of subunits interactions on heating. During cooling, chitosan-g-PNIPAAm dissolves in the confined space and has no impacts on the activity of multimeric enzymes. The heat inactivations of both glucose oxidase and catalase can be prevented via this method. It is a general protection strategy for multimeric enzymes.