Controllable Preparation Of Microgel Immobilized Enzyme Reactor And Its Multiple Response Enzyme Activity Regulation

Enzymes are biocatalysts with high catalytic activity and substrate specificity,which are widely used in the fields of environment,industry,medicine and biosensing.However,the activity of enzymes is easily affected by the external environment(such as extreme p H,temperature and organic solvents).Therefore,the full potential of enzyme catalysis has not been fully exploited,and it is a major problem to find effective strategies which overcome these shortcomings in the field of enzyme catalysis.At present,enzyme immobilization(EI)is the most prominent and successful R&D strategy to improve enzyme performance.There are many methods for enzyme immobilization,such as covalent bonding,cross-linking,embedding,etc.Compared with several methods,the embedding method not only has a lower probability of destroying the enzyme structure but also effectively improves the enzyme activity.Stimulation-responsive polymers are stimulated by the external environment(temperature,light,electricity,p H,salt concentration,magnetic field,etc.),so,they have corresponding changes in physical and chemical properties or structure.They have high application value in sensors,military,construction,electronic devices,medical treatment and other fields.Enzyme catalytic reactors were prepared by combining stimulus-responsive polymer materials with biological enzymes in a chemical or physical way,so that the enzyme activity could be regulated reversible.These enzyme catalytic reactors have unique stimulus-responsive and efficient catalytic activity at the same time,which provides a basis for the development and application of intelligent catalytic materials.Poly(N-isopropylacrylamide)(P(NIPAAm))material is the most widely used in the thermal responsive polymer material.When the external temperature is low critical phase transition temperature(LCST),the material will show obvious phase transition.However,P(NIPAAm)only has a thermal response and a single stimulus response,which limits its use scope.Therefore,when used as immobilized enzyme carrier materials,multiple responsive materials have more practical prospects.Thus,in this paper,It was synthesized for temperature/p H responsive P(NIPAAm-co-Ac Ty-co-AAc)copolymer,and it was used as a horseradish peroxidase(HRP)immobilized enzyme carrier materials.It was prepared for two kinds of temperature/p H responsive micro gel immobilized enzyme reactor under the HRP/H₂O₂ catalyzed cross-linking,and respectively to study the catalytic behavior and its enzyme activity of multiple response control,the specific research work are as follows:(1)Temperature/pH responsive P(NIPAAm-co-Ac Ty-co-AAc)copolymers were synthesized by free radical polymerization of N-isopropylacrylamide(NIPAAm),acrylic acid(AAc),and tyrosyl acrylic acid monomers(Ac Ty),and its and sodium alginate(SA-Tyr)which was modified with tyramine(Tyr)was used to form a double cross-linked microgel immobilized enzyme reactor under Ca²⁺ion cross-linking and HRP/H₂O₂ enzymatic cross-linking.Scanning electron microscopy analysis revealed that the microgel-immobilized enzyme reactor has a double cross-linked network structure,which expands the attachment point for the immobilization of the enzyme.Using orange II and pyrogallol as catalytic substrates,they were analyzed for the catalytic parameters of the microgel-immobilized enzyme reactor in the polar solvent aqueous phase and the non-polar organic phase(toluene),and It was found that the microgel immobilized enzyme reactor provided a water environment for horseradish peroxidase(HRP),which not only had high activity in the water phase,but also solved the problem that water-soluble enzymes cannot be used in organic solvent reactions,and has a high catalytic rate in the organic phase.It could be used repeatedly in both aqueous phase and organic phase and has good thermal stability.And because the microgel reactor contains temperature-sensitive NIPAAm chain segments and p H-responsive AAc chain segments,the enzyme activity in the reactor could be regulated by temperature and p H.Kinetic analysis of free enzyme and immobilized enzyme showed that the immobilized enzyme reactor of microgel had a high affinity to substrate.(2)P(NIPAAm-co-Ac Ty-co-AAc)self-assembled in water to form micelles,and HRP/H₂O₂ enzymes were used to catalyze cross-linked tyrosol groups to prepare cross-linked micelles,and HRP was simultaneously immobilized.It was measured using pyrene as a fluorescent probe for the critical micelle concentration(CMC)of the micelles formed by P(NIPAAm-co-Ac Ty-co-AAc)polymers of different structures.The results show that the more hydrophilic monomer(AAc)in the polymer,the less likely it is to form micelles,and the lower the p H,the easier it is to form micelles.Compared with uncrosslinked micelles,the structure of micelles crosslinked by HRP is more stable.The effects of the amount of immobilized HRP,the concentration of H₂O₂,the initial concentration of orange II and the reaction time on the catalytic performance of immobilized enzyme were investigated by using orange II as the catalytic substrate model.The optimal catalytic conditions were as follows:10mg immobilized enzyme,0.4m M H₂O₂,the initial concentration of orange II was 0.05m M,and the reaction time was 200min.At the same time,we explored the influence of temperature and p H on the catalytic activity of micellar immobilized enzymes,indicating that the activity of micellar immobilized enzymes is better.The kinetic analysis of free HRP and micellar immobilized enzymes showed that the micro-reaction environment constructed by the enzyme cross-linked micellar vector has good compatibility with enzyme molecules,which is conducive to the expression of horseradish peroxidase activity.So micellar immobilized enzyme has a high reaction rate.