Stimulation Responsive Hydrogel Immobilized Multi-Enzyme Cascade Reactor

Multi-enzyme cascade reaction plays an important role in the catalytic reaction process and has been widely used in chemical,medical and biological fields.However,the multi-enzyme cascade reaction system in the free solution state displays some disadvantages such as unrepeatable,difficult separation,and poor stability.So it is necessary to create an immobilized multi-enzyme cascade reactor to immobilize a variety of enzymes in the carrier by chemical or physical means.The immobilized multi-enzymes in the cascade reactor work together to catalyze the reaction efficiently.However,the traditional chemical crosslinking methods for the immobilization show some disadvantages,for example,damage to the activity of the enzyme,poor reusability,poor to regulate the activity of the immobilized enzyme.Therefore,we designed and synthesized a temperature and p H dual-responsive polymer containing phenolic hydroxyl groups,and a stimuli-responsive dual-crosslinked hydrogel was synthesized using Ca²⁺crosslinking combined with the HRP-catalyzed crosslinking.The horseradish peroxidase（HRP）and glucose oxidase（GOX）was in situ immobilized without damage in the hydrogel.A multi-enzyme cascade reactor with high catalytic activity and reusable was prepared using the stimuli-responsive hydrogel,and the activity of the cascade reactor could be regulated by changing the temperature and p H to achieve the"on-off"of the cascade reaction.The main work of this paper is as follows:（1）The thermosensitive N-isopropyl acrylamide（PNIPAm）,p H-responsive acrylic acid（AAC）and tyrosol acrylate（Ac Ty）containing enzymatic crosslinked phenolic hydroxyl group were polymerized to obtain the temperature and p H responsive P（NIPAm-co-AAC-co-Ac Ty）copolymer.The low critical solution temperature of P（NIPAm-co-AAC-co-Ac Ty）copolymer is 33℃.Then tyramine modified alginate sodium（SA-Tyr）and P（NIPAm-co-AAC-co-Ac Ty）solution are used to prepare the hydrogel.The quick gelation between sodium alginate and calcium ions combined with the HRP-catalyzed crossling between the phenol hydroxyl groups in P（NIPAm-co-AAC-co-Ac Ty）formed a double cross-linking network hydrogel,and HRP and GOX were immobilized in hydrogel microspheres without damage to obtain a stimuli-responsive hydrogel immobilized multi-enzyme cascade reactor.The gelation time,swelling rate,and the suitable proportion of double enzyme were determined by orthogonal experiment,and the optimal formulation of immobilized multi-enzyme hydrogel was explored,that is,3 wt%P（NIPAm-co-AAC-co-Ac Ty）,200 U/m L HRP,4 m M H₂O₂,2 wt%SA-Tyr.The GOX/HRP ratio is 1:2.The immobilization rate of HRP was 70.61%,the immobilization rate of GOX was 71.23%,the loading capacity of 0.2g gel microspheres（10 spheres）was 0.2541 mg,and the enzyme activity was62.37 Units.The effects of time,temperature and p H on the activity of the multi-enzyme cascade reactor and free double enzyme were investigated.The optimal temperature and p H of the enzyme-immobilized multienzyme cascade reactor were 45℃and 4 respectively.Under this temperature and p H,the network of the hydrogel microspheres collapsed due to the thermosensitive and the protonation of the carboxyl group.The shranked volume of the hydrogel resulted in the shortened diffusion path of the substrate and the increase of the local concentration of the intermediate products around the enzyme.Therefore the cascade activity was increased.The switchability of immobilized multi-enzyme cascade reactor was controlled by the"acid-base"and"low-high temperature"cycle.The cascade activity was regulated by repeatedly changing the p H and temperature of the solution,and the switchable activity of the immobilized multi-enzyme cascade reactor was realized by the p H and temperature dual-responsiveness.（2）The immobilized multi-enzyme cascade reactor was applied to the degradation of three typical dyes.The optimum conditions for degradation of direct black 38 dye were as follows:concentration 60 mg/L,degradation time 90 min,glucose concentration 2 m M,reaction temperature 40℃,p H=6,the degradation rate of 92.9%.The optimum conditions for degradation of indigo carmine dye were as follows:concentration of 20 mg/L,degradation time of 240 min,the glucose concentration of 2m M,reaction temperature of 50℃,p H=3,the degradation rate of 94.9%.The optimum conditions for degradation of reactive brilliant blue dye were as follows:concentration of 150 mg/L,degradation time of 105 min,the glucose concentration of 2 m M,reaction temperature of 40℃,p H=5,the degradation rate of 94.9%.