Preparation Of Metal Organic Frameworks For Horseradish Peroxidase Immobilization And Its Application In Environment

Horseradish peroxidase（HRP）is a kind of green biocatalyst produced by biological organism.It has specificity and high efficiency in catalyzing hydrogen peroxide（H₂O₂）.In the presence of hydrogen peroxide,it can rapidly oxidize and degrade a variety of organic substrates.It has important application value in many fields,such as environmental analysis,wastewater treatment,food detection,chemical production and so on.However,due to its high cost,poor stability,easy denaturation and inactivation under adverse conditions,and soluble in water,it is difficult to recycle once put into use.These defects greatly restrict its large-scale application in industrial production.Immobilization of enzymes is considered to be the most effective method to improve the stability and reusability of enzymes.Therefore,the development of an efficient enzyme loading material and its application in environmental chemical analysis and environmental pollution control has become a research hotspot in the field of environmental engineering.In this paper,the synthesis of porous metal organic frameworks（MOFs）and the immobilization strategy of natural enzyme were explored and studied.A new type of functional iron-based metal organic frameworks immobilized enzyme composite was synthesized in normal temperature and atmospheric pressure water solvent.Through one pot immobilization of enzymes,macromolecular enzymes can be embedded in the porous structure of MOFs in the process of constructing the spatial topology of MOFs.MOFs still have a highly ordered crystal structure,and the three-dimensional structure of MOFs remains unchanged.Meanwhile,MOFs maintain a high biocatalytic activity.The main contents of this paper are as follows:（1）Mellitic acid as ligand had almost no degradation effect on phenol,and the degradation efficiency of the materials synthesized by the other two ligands H₂BDC and NH₂-H₂BDC could reach 90%and 92%respectively in 120 min;It was found that in sodium hydroxide solution,aminoterephthalic acid was easier to deprotonate than terephthalic acid;The amino terephthalic acid as ligand has two rich functional groups（amino group which can activate benzene ring and carboxyl group which is easy to form coordination bond with inorganic metal ions）.It has good biocompatibility and provides a good platform for enzyme immobilization.Therefore,the iron-based metal organic framework NH₂-MIL（Fe）synthesized with amino terephthalate as the ligand was selected as the carrier for immobilizing natural protein molecules,and horseradish peroxidase（HRP）was directly embedded in the MOF by one pot method HRP@NH₂-MIL（Fe）.The prepared NH₂-MIL（Fe）and NH₂-MIL（Fe）were characterized by SEM,TEM,XPS,XRD and FT-IR HRP@NH₂-MIL（Fe）characterization analysis was carried out.The scanning electron microscope showed that both NH₂-MIL（Fe）and HRP@NH₂-MIL（Fe）have abundant cellular structure;It can be clearly seen from the SEM that both the immobilized and non immobilized materials have uniform regular polyhedral structure,with good crystallinity,and the particle size is about 20 nm,especially for the immobilized materials HRP@NH₂-MIL（Fe）The regular configuration showed that the immobilization of enzyme did not significantly affect the highly ordered spatial structure of metal organic frameworks;In the XPS full frequency spectrum of NH₂-MIL（Fe）,the obvious signals of carbon,nitrogen,oxygen and iron can be detected;The characteristic peaks of HRP and MOF appeared simultaneously in the infrared spectrum of the composite.Combined with the immobilized enzyme,the degradation of phenol was highly efficient.These results proved that the biological enzyme was well immobilized in the metal skeleton material.（2）Accurate detection of hydrogen peroxide concentration is of great significance in the detection and application of medicine,food,environment and other fields.This section focuses on HRP@NH₂-MIL（Fe）,N,N-diethyl-p-phenylenediamine sulfate（DPD）was used as a chromogenic reagent.Under the optimum conditions:（a）the p H value was 6.0;（b）the reaction time was 20 min;（c）the loading concentration of HRP in immobilized enzyme was 80μg/L;（d）the concentration of DPD was 0.8 m M;（e）the reaction temperature was 25℃.A colorimetric method for the determination of hydrogen peroxide was established,within the range of 1×10^-7 M～1.2×10^-4 M,there was a good linear relationship between the absorbance（A）and the concentration of H₂O₂,A_550nm=14.29 c+0.101,the correlation coefficient was 0.9948,and the detection limit was 1×10^-7M.The method established in this paper has high specificity and sensitivity,and has good practical reference value in the detection of hydrogen peroxide.（3）Chlorophenol organic pollutants bring potential hazards,and it is difficult to remove them efficiently by traditional wastewater treatment technology.As an excellent environmental remediation agent,HRP can catalyze the rapid degradation of a variety of aromatic organic pollutants in the presence of hydrogen peroxide.In this section,three chlorophenols（2,4-dichlorophenol,2-chlorophenol and 4-chlorophenol）were used as substrates HRP@NH₂-MIL（Fe）.It was used to catalyze the oxidative degradation of phenolic pollutants by hydrogen peroxide.The content of residual phenols in the solution was determined by 4-aminoantipyrine method.The factors affecting the degradation efficiency of three chlorophenols were investigated in detail.The initial p H of the solution was 6.5,and the concentration of immobilized enzyme was 650μg/L The results showed that the removal rates of 2,4-dichlorophenol,4-chlorophenol and2-chlorophenol could reach 94%,79%and 54%under the conditions of 0.6 m M hydrogen peroxide concentration and 40 min degradation time.By comparing the degradation of 2,4-dichlorophenol by free enzyme and immobilized enzyme in different p H environment and different temperature,the immobilized enzyme showed good stability,which expanded the scope of application of the enzyme in a certain range.After centrifugation,theimmobilized enzyme was recovered and recycled for three times HRP@NH₂-MIL（Fe）,its activity can reach 57%of the original.These studies provide a reference for the application of immobilized enzyme in the treatment of environmental pollutants.