Immobilization Of Enzyme Based On Organometallic Framework And Its Application Evaluation

Enzyme is a kind of important biocatalyst including of protein and RNA produced by organism,which show high efficiency and specific catalytic function.Enzyme catalysis shows high application value and potential in fine chemical industry and pharmaceutical industry.Enzyme catalysis shows many advantages,such as the high substrate specificity,the high reaction efficiency and the mild reaction conditions,which are in line with the development trend of upgrading chemical industry.However,due to enzyme's chemical nature,enzymes catalysis also shows obvious shortcomings.Enzymes are usually unstable under unnatural conditions,are difficult to separate and reuse,and some enzymes need expensive coenzyme factors.The immobilization technology of enzymes can overcome the above shortcomings.At present,many materials have been used fort the immobilization of enzymes,including inorganic carrier,polymer carrier and composite carrier.Metal organic framework?MOF?is a kind of typical composite material,which is an organic-inorganic hybrid material with intramolecular pores,which are formed by self-assembly of organic ligands and metal ions or clusters through coordination bonds.In recent years,,metal organic framework has been widely used in the immobilization of enzyme proteins as a new material.Many methods have been developed to prepare enzyme@MOFs composite materials,including the covalent bonding method,the surface adhesion method,the pore adsorption encapsulation method and the coprecipitation method.Nitrile hydratase?EC 4.2.1.84?is a key enzyme in the metabolic pathway of the nitriles degradation.It catalyzes the formation of amide?-CONH₂?from nitriles?-CN?.As an important industrial enzyme catalyst,it has been successfully applied to the large-scale production of acrylamide,nicotinamide,5-cyanopyramides and other bulk chemicals.Nicotinamide belongs to B group vitamin?VB?,and widely exists in dry yeast,grain germ,peanut and other species.Nicotinamide has a wide range of applications and market potential in many fields,particularly in the fields of cosmetics and feeds.In this paper,ZIF-67 was prepared by biomineralization in aqueous phase,and free nitrile hydratase was fixed in ZIF-67 for improving the thermal stability of nitrile hydratase.The main contents of this paper is as follows:Firstly,the stable MOF material ZIF-67 was synthesized in aqueous phase through optimizing the molar ratio and the concentrations of cobalt ion and2-methylimidazole,and the culture temperature.The synthesized materials were characterized by XRD,SEM,TEM,BET and IR.The results show that the synthesized material under the optimal conditions is agreement with the ZIF-67material reported in literature.In addition,we investigated the structural stability of ZIF-67 in the aqueous phase.The results show that the structure and morphology of ZIF-67 did not significantly change after two weeks of preservation in the phosphate buffer of p H 8.0.Secondly,a biomimetic mineralization strategy was developed to prepare the catalyst of nitrile hydratase@ZIF-67.Purified NHase1229 was added in the synthesis process of ZIF-67,and further immobilized in ZIF.The effects of cobalt ion and2-methylimidazole concentrations were investigated on the activity nitrile hydratase.The optimal immobilization conditions were as shown below:a 12 ml reaction system contained 0.044 g of cobalt nitrate,0.39 g of dimethylimidazole and 1.2 mg of nitrile hydratase.The nanocomposite catalyst NHase1229@ZIF-67 was incubated at 4?overnight.The results of XRD,SEM,TEM,BET and IR show that the crystal structure of NHase1229@ZIF-67 was similar to pure ZIF-67.The amount of immobilized NHase12299 was approximately 39.9mg protein/g ZIF-67.Thirdly,we investigated the enzymatic properties and catalytic function of NHase1229@ZIF-67.The optimal reaction temperature was 50-55°C,and it still maintained 70%and 40%of the maximum activity at 65°C and 70°C.Compared with the free NHase1229,the nano catalyst NHase1229@ZIF-67 showed better heat-resistance.The half-lives of NHase1229@ZIF-67 at 30?and40?are 102.0 h and26.5 h,respectively.Compared with the free enzyme,NHase1229@ZIF-67 shows a better thermal stability.The optimal p H value of NHase1229@ZIF-67 was 8.0,which was different from that of free NHase1229.This is due to the automatic degradation of ZIF-67 in weak acid or neutral aqueous solution.The activity of NHase1229@ZIF-67 was 38%of the maximum activity at p H 9.0.But free NHase1229 was about 20%of the maximum activity.The results show that NHase1229@ZIF-67 had a better alkali-resistance than free NHase1229.When3-cyanopyridine was used as substrate,the K_mvalue of NHase1229@ZIF-67 was 4.26mmol/L,and the maximum reaction rate was 33.95?mol/min/mg.Compared with free NHase1229,NHase1229@ZIF-67 showed lower K_mvalue,which suggests the increased affinity for substrate 3-cyanopyridine dut of the pore structure of ZIF-67.The cycle performance of NHase1229@ZIF-67 was further investigated.In the initial six cycles,0.9 mol/L 3-cyanopyridine was completely transformed to nicotinamide,and the catalytic efficiency did not significantly decrease.The results indicate that the nano catalyst NHase1229@ZIF-67 also possesses good recycle performance.The effect of the concentration of 3-cyanopyridine on the catalytic process was then investigated.The optimal substrate concentration was 0.9 mol/L.When the substrate concentration was?1.2 mol/L,the substrate inhibition was observed.Finally,a batch feeding strategy was used to produce nicotinamide for avoiding the substrate inhibition on the activity of nitrile hydratase.The yield of nicotinamide reached approximately 110 g/L/h.Therefore,The nano catalyst NHase1229@ZIF-67 shows a good catalytic performance and high application potential in the catalytic preparation of nicotinamide.