Manganese-based Materials Immobilized Enzyme To Fabricate Multi-enzyme Catalytic System

Enzyme immobilization technology can improve the stability and recyclability of free enzymes and has been widely used in the field of biocatalysis.Compared with single enzyme immobilization,multi-enzyme immobilization synergistic catalysis has attracted great interest in an efficient and economical way.Multi-enzyme continuous catalysis effectively avoids the separation and purification of intermediate products and improves the catalytic efficiency of the reaction system.In this paper,manganese dioxide(MnO2)nanorods,MnO2-based hollow microspheres and manganese-based coordination polymers(Mn-CP)were used as carriers to immobilize enzymes and mimic catalase.D-amino acid oxidase(DAAO)and(R)-?--transaminase(RTA)were immobilized to mimics three-enzyme catalysis.Galactose oxidase(GaO)was immobilized to mimics two-enzyme catalysis.The synergistic catalysis between enzymes and enzymes as well as between enzymes and carriers was studied.The main results are as follows:(1)Immobilization of DAAO/RTA on MnO2 nanorods for catalyzing the conversion of(R)-1-phenylethylamine.Firstly,polydopamine was coated on the synthesized MnO2 nanorods(MnO2@PDA).Then RTA and DAAO are immobilized to MnO2@PDA respectively.RT A/MnO2@PDA and DAAO/MnO2@PDA were combined to construct a simulated three-enzyme catalytic system.RTA/MnO2@PDA catalyzes(R)-1-phenylethylamine to produce corresponding ketones(pyruvate as an amino receptor).DAAO/MnO2@PDA catalyzes D-alanine back to pyruvic acid with O2,and realizes the internal circulation of pyruvic acid.On the other hand,the generated H2O2 is decomposed into O2 by MnO2 nanorods.Two cycles of pyruvic acid ? D-alanine? pyruvic acid and O2?H2O2?O2 were formed during the catalytic reaction.The rapid conversion of(R)-1-phenylethylamine was accelerated by these two cycles.As a result,The immobilized enzymes achieved the conversions over 98 ± 1.8%,in contrast to corresponding conversions 69.6 ± 1.2%by free enzymes.(2)Mn-based coordination polymers encapsulate GaO to catalyze oxidation of benzylic alchohol.In this part of the experiment,In the mixed solution of GaO and dimethylimidazole,Cull and Mn? were added in turn to construct GaO/Cu/Mn-CP complex by co-precipitation.In the catalytic process,GaO/Cu/Mn-CP mimics catalase combined with immobilized GaO to construct a two-enzyme synergistic catalytic system.GaO/Cu/Mn-CP catalyzes the oxidation of benzyl alcohol with O2,and produces by-product H2O2.H2O2 is decomposed and converted into O2 by GaO/Cu/Mn-CP to realize the internal circulation of O2.The formation of a cycle from O2?H2O2 ? O2 accelerates the rate of the whole catalytic reaction.GaO/Cu/Mn-CP achieved the conversions over 99.3%,in contrast to corresponding conversions 20.9%by free GaO.Finally,GaO/Cu/Mn-CP exhibits excellent temperature stability and reusability.(3)Multifunctional hollow-shell microspheres derived from cross-linking of MnO2 nanoneedles by zirconium-based coordination polymer:enzyme mimicking,micromotors,and protein immobilization.A Zr-based coordination polymer(Zr-CP)composed of Zr? and dimethylimidazole was coated on the surface of MnO2 microspheres to construct MnO2@Zr-CP.Zr-CP successfully coated nanoneedles on the surface of MnO2 microspheres,and the bases of nanoneedles were cross-linked with Zr-CP.Hollow-shell h-MnO2@Zr-CP produced by etching the core MnO2 in MnO2@Zr-CP microspheres.h-MnO2@Zr-CP possesses oxidase-and catalase-like activities.h-MnO2@Zr-CP can work as a micromotor with H2O2 as the fuel.By the coordination between hexahistidine-tags and Zr-CP,enzymes with hexahistidine-tags can be selectively and and strongly absorbed onto the hollow-shell microspheres with a large capacity.DAAO was immobilized on the hollow-shell microspheres.The conjugate DAAO/h-MnO2@Zr-CP mimics two-enzyme catalysis,achieving a catalysis efficiency 225%times that of free DAAO.Further,RTA and DAAO are co-immobilized on the hollow-shell microspheres.The conjugate RTA/DAAO/h-MnO2@Zr-CP mimics three-enzyme catalysis.For converting(R)-1-phenylethylamine and(R)-1-aminoindan to corresponding ketones,RTA/DAAO/h-MnO2@Zr-CP achieved the conversions 99 and 95%,respectively,in contrast to corresponding conversions 48.6 and 32.3%by the mixed free enzymes RTA+DAAO.From the perspective of application potential,the multi-functional DAAO/h-MnO2@Zr-CP can also be used to immobilize other kinds of enzymes to construct a variety of catalytic systems,which is a very valuable carrier for enzyme immobilization.