Positional Assembly Of ATP Synthase Based On Hollow Nanofibers And Microcapsules

ATP synthase is a key enzyme for energy in organism and can synthesize ATP by proton gradient between inner and outer membrane.As a membrane protein,it is an important content of founding nano-biomachine that how to assemble ATP synthase onto materials by biomimetic method.As the biomimetic materials,liposomes and layer-by-layer self-assembly microcapsules have been applied to the field of assembling ATP synthase.However,this methed appears some disadvantage.For instance,complex operation and poor stability of enzyme.In this article,the hollow microcapsules/nanofibers prepared by coaxial-electrospray/electrospinning serve as the scaffold of assembling ATP synthase firstly.Simultaneously,GOD(Glucose oxidase)is embedded in Chamber of hollow microcapsules/nanofibers in site and supply proten gradient for ATP synthase by catalytic oxidation of glucose.Compared to the existing assembly ATP synthase in form layer-by-layer microcapsules,novel hollow microcapsules and nanofibers supported assembly ATP synthase were expected to offer several advantages:electrospray and electrospinning procedure enable an simple operation,the low cost of equipment and the rate of immobilized enzyme in inner microcapsules and hollow nanofibers achieve about 100%as well as enhanced stability due to confining of enzyme inside th nano-scale compartments.The thesis includes of the following parts:(1)Polyurethane(PU)hollow microcapsules were produced by co-axial electrospray.The effects of structure of the concentration of internal and external phase of electrospray solution and their flow rate on structure of hollow microcapsules were studied.The results showed that uniform hollow PU microcapsules could be successfully fabricated by co-axial electrospray glucerol containing 20%(?)water and N,N-Dimethylacetamide solution of 10%(?)PU as core and shell solution under the following condition:core-shell flow rate were 0.05 and 1mL/h,voltage was 22kV,distance between spinneret and collector was 48 cm,temperature was 30~oC,humility was 10%.With Candida antarctica lipase B(CALB)as a model enzyme,relationahip between performance of microcapsules CALB and the immobilization protocols applied as well as the structure of microcapsule were studied.CALB was dissolved in the inner electrospray solution under the above conditions of electrospray to achieve immobilized enzyme.Its hydrolytic activity was76.19%of that of native one,remain 60%of that of initial activity after reusing cycle10 times and half life at 60~oC improve 8 times than room temperature.(2)The microcapsule,which embedded GOD in situ,was prepared by adding GOD into internal phase of electrospray solution during Co-axial electrospray and serve as the carrier material of assembly ATP synthase.The ATP synthase,which extracted in thylakoid membrane of spinach chloroplast,was recombined on phospholipid bilayers.Then a lipid-modified microcapsule with incorporation of ATP synthase was obtained by the mixture of ATP synthase proteoliposomes and microcapsule dispersion.The influence that microcapsule with different electric charge on the yield of ATP synthesized and the stability of ATP synthase were studied systematically.The yield of ATP synthesized by ATP synthase which on microcapsule surface with positive charge was 9.6 nmol/mg in 2 hours,it was much more than 8nmol/mg compared with which on microcapsules surface with negative charge.The activity recovery remained about 60%of that of initial activity on microcapsule surface with positive charge after reusing cycle 8 times.Compared with positive charge,the activity recovery was below 50%on negative charge surface.The activity recovery were 49.13%and 40.56%of that of initial activity on microcapsule surface with positive and negative charge stored 8 h at 60~oC.(3)Co-axial electrospinning hollow nanofibrous Glucose oxidase(GOD)as the scaffold of assembly ATP synthase.Then a lipid-modified hollow nanofibers with incorporation of ATP synthase was obtained by the mixture of ATP synthase proteoliposomes and nanofiber dispersion for the first time.Relationship between fibrous scaffold and catalytic efficiency and stability of ATP synthase were studied.The result showed that yield of ATP synthesized by ATP synthase which on hollow nanofibers was only 1.68 nmol/mg in 2 hours,the activity recovery remained about 45%of that of initial activity after reusing cycle 8 times and was 42.24%of that of initial activity stored 8 h at 60~oC.In summary,in this thesis a novel carrier assembled ATP synthase prepared by co-axial electrospray and co-axial electrospinning and the relationship between carrier and catalytic efficiency and stability of ATP synthase were studied.It was believed that results from this study would greatly promote the application and development of biocatalysis,biomachine and many other related research fields.