Effect Of Enzyme Spacing On Cascade Activity In Magnetic DNA Co-immobilized Multienzyme System

Multi-enzyme cascade reaction plays an important role in catalytic process.It is widely used in many fields such as biochemistry,drug production and medical diagnosis.In order to obtain high enzyme activity,this study focused on the key factor of double enzyme spacing,using glucose oxidase(GOD)and horseradish peroxidase(HRP)as model enzymes,to prepare magnetic DNA scaffolds co-immobilized multi-enzyme system.First,GOD was immobilized by single strand DNA grafted onto the surface of magnetic microspheres.Then,the complementary base HRP-DNA complex was hybridized with the single strand DNA on the surface of the magnetic sphere and assembled to form a "Y" type DNA scaffold to achieve the directional co-immobilized of GOD and HRP.The "Y" type DNA scaffold enables the double enzyme to have better proximity and co-localization,which can produce substrate channel effect and improve the activity of the cascade enzyme.Moreover,DNA scaffolds are editable and can regulate enzyme spacing by changing DNA strand length.Compared with the DNA assembly(origami)co-immobilized multi-enzyme system reported in literature,the "Y" type DNA scaffold designed in this paper has the advantages of simple structure,easy preparation and low cost.This paper mainly carries out the following work:First,"Y" DNA scaffolds co-immobilized GOD and HRP.Two partially complementary DNA strands were designed to covalently connect GOD with DNA strand A to form GOD-A complex.HRP and complementary DNA strand B are connected by amide bond to form HRP-B complex.Two enzyme-DNA complexes GOD-A and HRP-B were assembled by base complementary pairing to form "Y" type DNA scaffold multi-enzyme complex.The "Y" type DNA scaffold,enzyme-DNA complex and multi-enzyme complex were successfully prepared by gel electrophoresis.The cascade activity of "Y" type DNA scaffold multi-enzyme complex was 448 U/mg,and the enzyme activity was basically unchanged after 6 days of storage at4 ℃,and its stability was better than free GOD and HRP.Second,the magnetic co-immobilization of GOD and HRP was mediated by "Y" type DNA scaffold.Using epoxy-modified polyvinyl acetate(PVAC)magnetic microspheres as the carrier,the "Y" type DNA scaffold multi-enzyme complex was covalently bound to the surface of the carrier,and the magnetic directional co-immobilization multi-enzyme system was constructed.Fluorescence characterization showed that GOD and HRP were successfully co-immobilization on the surface of microspheres.The Michaelis constant and enzyme activity of magnetic DNA scaffolds were 9.04 mg/m L and 967 U/mg,which were 3.5 times of GOD and HRP cascade enzyme activity.The spatial conformation of the enzyme molecules in the magnetic DNA scaffolds co-immobilized multi-enzyme system was stable and the enzymatic property was excellent.The optimal p H value of the catalytic reaction was4.0,which was lower than that of the free GOD and HRP cascade reaction of 7.0.The optimum reaction temperature was 45 ℃,which was higher than 35 ℃ for free double enzyme.After 7 h of high temperature treatment,the enzyme activity was no change.Compared with GOD and HRP,magnetic DNA co-immobilized multi-enzyme system had better applicability and thermal stability.Third,the effect of enzyme spacing on enzyme activity was investigated.The "Y" type DNA scaffolds with different lengths were designed to regulate the distance between GOD and HRP,and the magnetic DNA scaffolds with different enzyme spacing were constructed to co-immobilization multiple enzymes.The fluorescence resonance energy transfer(FRET)phenomenon was observed by laser confocal microscopy,and the fluorescence resonance energy transfer efficiency was measured.The actual enzyme spacing was calculated based on the transfer efficiency.The experimental results demonstrated that the multi-enzyme system with different enzyme spacing was successfully constructed.The results showed that when the enzyme spacing was 10.0 nm,the enzyme activity of GOD and HRP was 967 U/mg.The activity of the cascades at 7.5 nm and 16.0 nm was lower than that at 10.0 nm,indicating that the enzyme spacing was the key factor affecting the activity of the cascades.