Preparation And Application Of Enzyme-Metal Phosphate/Metal Organic Framework Immobilized Enzyme Based On Biomimetic Mineralization

Bioenzyme catalysts have the characteristics of high specificity,high catalytic performance and green pollution-free,but there are many limitations in practical application,such as harsh reaction conditions and inability to operate continuously.Biomineralization is a process of organic-inorganic composite materials formed by the connection of organic matter,inorganic matter,soft matter and hard material in organisms,which can provide good mechanical protection.Based on this principle,the immobilized enzyme with biomimetic mineralization without self-mineralization ability can be used to endue it with special properties and improve the stability of the enzyme.In this paper,the enzyme-copper phosphate nanomaterials and glucose oxidase-metal-organic framework materials were synthesized by a biomimetic mineralization method in one-step.They have excellent catalytic effect and can be applied to the extraction of active substances in chrysanthemum samples and the visual detection of glucose.The specific content is as follows:（1）The recombinantβ-glucosidase with His-tag（BglucLH）was directly separated and immobilized from cell lysate without the need to prepare the carrier in advance by one-step biomimetic mineralization method.BglucLH-Cu₃（PO₄）₂nanoflowers（BglucLH-NF）with micron scale flower-like structure assembled by nanometer petals was prepared experimentally.The BglucLH-Cu₃（PO₄）₂nanoflowers has the advantages of high enzyme loading,good stability and recyclability.The His-tag could enhanced the specific binding between the enzyme and copper ions and the activity recovery and separation efficiency of BglucLH-NF were improved by 253.2%and 290.1%in comparison with Bgluc-Cu₃（PO₄）₂nanoflowers（Bgluc-NF）prepared by glucosidase without His-tag（Bgluc）,indicating that His-tag could improve the efficiency of separation and immobilization of the enzyme.（2）The copper phosphate hybrid flowers with mixed enzyme（cellulase andβ-glucosidase）were successfully prepared based on the biomimetic mineralization method.When the dosage of Cellulase to Gluosidase was 25:25,the good enzyme activity recovery（95.1±2.81%）,encapsulation efficiency（82.6±2.36%）and relative enzyme activity（115.1±2.82%）were obtained.In this study,Cellulase&Glucosidase-Cu₃（PO₄）₂ hybrid nanoflowers（Cellu&Glu-CuP）were used to extract the flavonoids from chrysanthemum in 40 min at 50℃by enzymatic hydrolysis coupled with methanol solvent extraction.Compared with mixed free enzymes,Cellu&Glu-CuP showed excellent high temperature resistance and resistance to organic solvents.The yields of total flavonoids,quercitroside and quercetin in chrysanthemum were up to7.51%,0.1483%and 0.6231%,respectively.Compared with the condition without the addition of enzyme,it increased by 43%,39%and 74%,respectively.The Cellu&Glu-CuP enzymatic hydrolysis coupled with methanol solvent extraction technology had good application potential.（3）In this study,a 3-MPBA/glucose oxidase@ZIF-8 immobilized enzyme（3-MPBA/GOx@ZIF-8）was prepared to enhance the enrichment of substrate.On the one hand,based on the affinity adsorption between the phenylboric acid group and glucose,the strong enrichment of glucose substrate was realized.On the other hand,3-MPBA/GOx@ZIF-8 was rapidly prepared by accelerating nucleation based on the coordination of sulfhydryl functional groups with Zn²⁺.The optimum loading capacity of GOx was 470 mg/g,and the Michaelis constant K_m and catalytic efficiency k_cat/K_mthe 3-MPBA/GOx@ZIF-8 were 0.03±0.02 M and 63.87±1.96 s^-1 M^-1,respectively,which were more than 10 times the corresponding values of free GOx.In addition,3-MPBA/GOx@ZIF-8 showed good bioactivity in both chemical reagents and high temperature conditions and had good reusability.The relative activity of 3-MPBA/GOx@ZIF-8 remained about 80.8%after 7 cycles.The study have shown that the 3-MPBA/GOx@ZIF-8 has high encapsulation efficiency,catalytic efficiency and enhanced stability.Based on these characteristics,a new type of visual colorimetric sensor for diabetes has been realized,which provides a new strategy for glucose analysis.