Display Of Glucose Oxidase On The Surface Of Bacillus Subtilis Spores And Preparation Of Enzyme Electrode

Glucose oxidase(GOD)is an aerobic dehydrogenase with high efficiency and specific biological characteristics.It can catalyze the reaction of glucose and oxygen,and can be combined with electrode to prepare biological enzyme electrode.Glucose oxidase electrode as a representative of enzyme electrode has a wide range of application prospects in food industry,biomedicine,environmental monitoring and so on,and has been a hot spot in the field of biosensor research.At present,purified GOD is often used in the preparation of glucose oxidase electrode,and the process of enzyme separation and purification further increases the cost of the electrode.In addition,the immobilization of GOD will cause the loss of enzyme activity and affect the stability of the electrode.Therefore,in this study the fusion expression of GOD and spore coat protein can make GOD displayed on the surface of B.subtilis spores,thus avoiding the separation and purification of GOD and reducing the preparation cost of glucose oxidase electrode.And the excellent stress resistance of spores is conducive to the application and stable storage of surface GOD in complex environment and improve the stability of the enzyme electrode.The main research contents were as follows:(1)The spore coat proteins Cot C,Cot X and Cot Y were selected as anchor proteins,and glucose oxidase was displayed on the surface of B.subtilis WB800n spore,respectively.Through Western Blot analysis,immunofluorescence analysis and enzyme activity detection,it is proved that glucose oxidase can be displayed on the surface of spore and express the enzyme activity.The glucose oxidase enzyme activities displayed on the surface of the unit cell dry spores of the recombinant B.subtilis WB800n-Cot C/X/Y-GOD were 20.33,23.28,and 21.78 U/g,respectively.And the glucose oxidase activity displayed by Cot X as the anchor protein was the highest.(2)In order to increase the amount of glucose oxidase displayed on the surface of the spores,without affecting the normal growth of B.subtilis,amy E,lac A and pyr D genes were selected as the insertion sites.By constructing a vector with the upstream and downstream homology arms of the insertion sites,the foreign genes were integrated into the different insertion sites of B.subtilis WB800n through the double exchange of the homology arms.The result of Western Blot analysis indicated that glucose oxidase was successfully expressed on the surface of B.subtilis spores surface,and its expression level increased with the increase of the number of integration sites.And when the foreign genes were inserted into three different sites at the same time,the glucose oxidase activity on the spores surface was the highest,which was 50.09 U/g.(3)Fermentation by starvation method produces recombinant spores displaying glucose oxidase on the surface,which were combined with graphene oxide material and Prussian blue electronic mediator to prepare enzyme electrode.In the range of 0.1-9.0mmol/L glucose,the cyclic voltammetry curve on the enzyme electrode showed a linear relationship with the glucose concentration.The calibration curve equation was I=1.2846 C_glucose+5.7907(R²=0.9971),and the prepare enzyme electrode had a detection limit of 4.2?mol/L(S/N=3)and a sensitivity of 41.94?A·m M^-1·cm^-2.The modified electrode can be used for the analysis and determination of glucose.