Study On Improving Enzyme Catalytic Efficiency By Immobilizing Dehaloperoxidase And Imine Reductase

Dehaloperoxidase(DHP),combines a heme cofactor in its active center,has dual catalytic functions of globin and peroxidase.When activated by H₂O₂,it can effectively oxidize halogenated phenols to obtain quinones,which has practical research significance and application prospects in the treatment of waste water and other directions.However,H₂O₂ will cause heme to fall off from the binding site,and change the secondary structure and conformation of DHP,thus resulting in enzyme inactivation.Biomineralization of DHP by forming metal-organic frameworks and metal-organic/inorganic hybrid nanoflowers can effectively improve the stability of the DHP reaction,and then enhance the catalytic activity of DHP.Imine reductase(IRED),which relies on the cofactor NADPH,can catalyze the asymmetric reduction of prochiral imines to prepare corresponding amines,has substantial application value in the preparation of drug intermediates.Combined with NADP⁺-dependent Glucose dehydrogenase(GDH)can realize the recycling of cofactors,effectively reducing the reaction cost and enhances its catalytic efficiency.The transfer efficiency of cofactor between the two enzyme active centers is one of the issues that affect the activity.Protein splicing technology can draw close the distance between the two enzyme active centers,reduce the mass transfer resistance of NADPH and NADP⁺between the active centers,and promote the recycling of cofactor.The immobilization of the two-enzyme cascade system by hybrid nanoflowers immobilization method is also beneficial to enhance the catalytic activity of the cascade system and promote the conversion.In this project,the free DHP was studied,the biomineralization method was used to immobilize DHP,the catalytic activity and the stability of immobilized enzymes were investigated.Furthermore,discussed the IRED and GDH two-enzyme systems,the two enzyme active centers were draw close by protein splicing technology,and the catalytic activity was improved by combining with biomineralization immobilization.The main research contents and results were summarized as follows:(1)Study of catalytic activity and stability of Dehaloperoxidase.The p ET28a-DHP recombinant plasmid was constructed,and the expression conditions were optimized to obtain high expression amount of DHP.The stability of free DHP was studied by UV-Vis spectrophotometer,fluorescence spectroscopy and circular dichroism spectroscopy.The results show that the free DHP conformation is in an open state and further becomes open under the influence of H₂O₂.H₂O₂ also has a great influence on the structure of free DHP,which easily leads to the shedding of heme in the enzyme activity center.In addition,the catalytic efficiency of DHP on substrates increased first and then decreased with the increase of H₂O₂ concentration in the system,and the catalytic results showed that the maximum substrate conversion of free DHP was 43.46%.(2)(DHP+2-methylimidazole)coordinates with zinc ions to form coordination polymers.The coordination polymer i-DHP@Zn-CP was successfully constructed by using DHP and 2-methylimidazole to coordinate with Zn²⁺,with optimizing the concentration of Zn²⁺and 2-methylimidazole.The surface morphology and DHP distribution of i-DHP@Zn-CP were studied by means of electron scanning electron microscopy,X-ray diffraction and Fourier infrared transform spectroscopy.By forming a coordination polymer,the problem of heme shedding from the active center is solved and the stability of DHP in the catalytic process is improved.The results showed that the catalytic activity of i-DHP@Zn-CP was 1.5 times that of free DHP.i-DHP@Zn-CP has good thermal stability and enhanced alkaline p H resistance,but is sensitive to acidic environment.Moreover,i-DHP@Zn-CP has good reusability,and the catalytic activity can be retained by 92.84%after five cycles of catalysis.(3)(DHP+PBS)coordinates with zinc ions to form nanoflower-like complex.The coral flower-like nano material DHP@Zn-NF with multi-layer structure and micron-scale pore channels was successfully constructed through coordination of DHP,PBS and Zn²⁺,by optimizing the concentration of Zn²⁺and DHP.DHP participated in the formation and assembly of hybrid nanoflower“petals”,and distributed uniformly with structurally stable in the mineralized products.FT-IR two-dimensional correlation analysis and Raman spectroscopy demonstrated that the preparation of hybrid nanoflowers had little effect on the structure of DHP and could maintain the catalytic activity of DHP well.By forming nanoflower complexes,heme shedding can be prevented and the catalytic effect of DHP is enhanced.The conversion catalyzed by DHP@Zn-NF was increased to 1.6 times higher than free DHP.DHP@Zn-NF has a good p H tolerance.The optimum p H shifted to acidity,and the higher catalytic activity is obtained at p H=5.6 with the conversion reached 83.46%.DHP@Zn-NF had a good tolerance to H₂O₂ and could catalyze 90.52%substrate conversion when the concentration of H₂O₂ reached30?M.Moreover,75-79%of the relative activity can be retained at 80?indicated that DHP@Zn-NF has excellent thermal stability,and more than90%relative activity can be retained after five cycles of catalysis with strong reusability.(4)(splicing product IRED&GDH+PBS)coordinates with zinc ions to form nanoflower-like complex.Imine reductase(IRED)and glucose dehydrogenase(GDH)were cascaded to achieve the circulation of the cofactor NADPH during catalysis.Protein splicing technology was used to draw close the active center distance between IRED and GDH,and splicing conditions were optimized to obtain the splicing product IRED&GDH,which makes IRED and GDH active center have a molecular level distance,improves the transmission efficiency of NADPH and NADP⁺,makes the cofactors recycle efficiently,and improved the catalytic activity of the two-enzyme system.The catalytic conversion of substrate 2-MPN by IRED&GDH was 1.5 times higher than that by(IRED+GDH).IRED&GDH@Zn-NF was obtained by Zn²⁺coordination immobilization of IRED&GDH,and the catalytic efficiency was1.51 times higher than that before immobilization and 2.14 times higher than that of(IRED+GDH).