Co-immobilization Of Multi-enzymes System:Encapsulation Within MOF And Affinity Adsorption

Multienzyme cascade reaction system completes the catalysis in an efficient way,and the intermediate products of the reaction are transported between the active sites of different enzymes.This reaction mechanism maintains the high local concentration of intermediates and reduces the diffusion loss during transportation,thus promoting the overall catalytic efficiency.Inspired by this,biomimetic construction of multienzyme cascade reaction has attracted lot of researcher attention.The cascade reaction systems of methanol dehydrogenase?FDH?-formaldehyde dehydrogenase?FalDH?-Alcohol dehydrogenase?ADH?-glutamate dehydrogenase?GDH?and alkaline phosphatase?AKP?-tyrosinase?TYR?are important cascade reactions and have important research value.FDH-FalDH-ADH-GDH cascade reaction can efficiently reduce carbon dioxide to methanol under mild conditions,which is of great significance for the fixation of carbon dioxide and the mitigation of greenhouse effect.Besides,o-phosphoric acid-L-tyrosine,the substrate of AKP-TYR cascade reaction,can inhibit the growth of human kidney and breast cancer cells,which has important medical value.It is of great scientific significance and application value to develop a new comobilization technology for multi-enzyme cascade reaction system,through enzyme immobilization combined with efficient synergistic catalysis mechanism in multienzyme cascade reaction system.There are four main methods of enzyme immobilization:adsorption,covalent bonding,encapsulation and crosslinking.Encapsulation does not require modification of the enzyme,so the original structure of the enzyme is retained to a large extent,resulting in less loss of enzyme activity.metal-organic framework material is a good carrier material for enzyme immobilization because of its super high specific surface area,good biocompatibility,adjustable pore size,functional structure and good thermal stability.On this basis,the immobilized enzymes were combined with the separation membrane to construct the enzyme membrane reactor,which could make the enzyme catalysis and product separation proceed simultaneously and further improve the reaction efficiency.In this paper,two cascade reaction systems of multi-enzymes were constructed:FDH-FalDH-ADH-GDH cascade reaction system and AKP-TYR cascade reaction system.FDH and GDH,FalDH and GDH,ADH and GDH were embedded in ZIF-8 in situ respectively.A cascade reaction system of FDH-FalDH-ADH-GDH was constructed to catalyze the reduction of CO₂ to methanol.In addition,reductive coenzyme I and dehydrogenase were embedded in ZIF-8 to construct a cascade reaction.Based on the above immobilization method,the immobilized enzymes were further combined with the separation membrane,and a cascade reaction system of FDH-FalDH-ADH-GDH was constructed in the membrane to synchronize catalysis and separation.And the competitive reversible inhibitor is an amphiphilic molecule with long fat chains.The AKP-TYR cascade reaction system was constructed by immobilizing tyrosinase and alkaline phosphatase on slide by affinity adsorption.From the above research work,the following conclusions are drawn:?1?Co-immobilization of coenzymes and enzymes into ZIF-8 could solve the problems of slow mass transfer and high loss of NADH,and the catalytic efficiency increased about 15%.?2?The catalytic efficiency of FDH-FalDH-ADH-GDH cascade reaction was further improved when the enzyme membrane reactor was constructed.?3?For the cascade reaction system of FDH-FalDH-ADH-GDH,the catalytic activity of the disordered distribution enzyme membrane reactor was inferior to that of the ordered distribution enzyme membrane reactor.?4?The enzyme activity of immobilized multi-enzyme system prepared by combining with inhibitors was higher than that of immobilized multi-enzyme system prepared by ordinary adsorption,which increased about 72%.This might be explained by that the enzyme reacts with the inhibitor to make the active site of the enzyme upward,which is conducive to the enzymatic reaction.