Molecularly Imprinted Biomimetic Carrier Regulation Promotes Biological Enzyme Catalysis

As a natural catalyst,enzyme has the characteristics of high efficiency,region and stereoselectivity,so it has attracted a lot of attention in chemical industry,food,medicine and other fields.The new challenge of enzyme catalyst is how to improve the activity and stability of enzyme in complex application environment.The immobilization of the enzyme can improve the stability of the enzyme,but the apparent activity of the enzyme is generally lost in the conventional immobilization process.Therefore,it is of great significance to design and develop new immobilized enzyme carriers and methods to improve the activity and stability of enzyme in complex application environment.In this paper,based on the biomimetic molecular design method,a series of flexible molecularly imprinted vectors containing"imprinted holes" of enzyme molecules were synthesized to regulate the microenvironment of the enzyme,and the activation effect of selective imprinting recognition sites was used to significantly improve the enzyme activity and broaden the application range of enzyme pH.The "molecular chaperone" function of the selective recognition site of molecular imprinting was further revealed.The main research achievements of this paper include:(1)Design and synthesis of molecularly imprinted biomimetic carriers.D-amino acid oxidase(DAAO,optimal catalysis pH 8.5)and cytochrome C(Cyt C,optimal catalysis pH 4.5)were selected as the research objects,and flexible hydrogel biomimetic carriers were prepared by precipitation polymerization of different charged(3-acrylamide propyl)trimethylammonium chloride(ATC),acrylic acid(AAC)functional monomer and hydrophobic functional monomer N-tert-butylacrylamide(TBAm).Scanning electron microscope showed that the biomimetic carrier was regular spherical,and the particle size of 100 nm,had positive and negative charge of 15.2 mV and-9.8 mV,respectively.After imprinting,the immobilization capacity of DAAO and Cyt C could reach 0.825 mg/mg and 0.205 mg/mg.(2)The performance optimization of molecularly imprinted biomimetic carrier.The catalytic activity of DAAO and Cyt C is affected by the hydrophobicity and electrostatic interaction of biomimetic carrers,and the charge and hydrophobicity of molecularly imprinted biomimetic carriers are optimized.Through scanning electron microscope and biofilm interference technique,it was found that when the ratio of functional monomers of AAC and ATC was 10 mol%,TBAm,35 mol%and 40 mol%respectively,the spherical biomimetic carrier with uniform morphology and distinct particles could be synthesized.The affinity KD for enzyme molecules reached 1.41×10-8 and 1.71 ×10-8,and the enzyme activity was increased by 1.77 times and 1.51 times,respectively.(3)The regulation of molecularly imprinted biomimetic carrier promotes enzyme catalysis and its molecular chaperone function.The molecularly imprinted biomimetic carrier was prepared by using DAAO and Cyt C as template molecules.The selective "imprinted hole" of the carrier can provide an alkaline/acidic microenvironment for DAAO/Cyt C.the enzyme activity is 2.59 times higher than that of free enzyme(DAAO)and 3.44 times higher than that of free enzyme.DAAO/Cyt-C double enzyme cascade catalytic system was constructed based on molecular imprinting biomimetic carrier,and its catalytic efficiency(Kcat/Km)was 7.24 times higher than that of free enzyme.And the stability is also significantly improved under extreme pH and high temperature.At the same time,the selective "imprinted hole" of the biomimetic carrier will induce the correct folding of the denatured enzyme and restore the enzyme activity,which has the function of molecular chaperone.This method has good universality and has significant effect on D-amino acid oxidase,Cytochrome C,Lipase and Horseradish peroxidase.