Co-immobilization of glucose oxidase and catalase in porous magnetic chitosan microspheres for production of sodium gluconate

In the process of immobilizing the enzyme, the overflow of enzyme and the decrease of enzyme activity are very serious. In order to improve the stable binding between enzyme and carrier, a kind of porous magnetic chitosan microsphere with appropriate pore size was successfully prepared by adjusting the size of pore-forming agent in this paper. The rough porous structure is favorable for the adsorption of enzyme and the catalytic action of enzyme on substrate. The results showed that when the pore size of the microspheres was at 790.15 +/- 250.91 nm, the protein loading and enzyme activity of GOD could be increased effectively, which could reach 58.28 +/- 2.64 mg/g and 16.93 +/- 0.14 U, respectively. The co-immobilization of CAT and GOD eliminated the harmful by-product H2O2 in time and effectively solved the problem of continuous deactivation of GOD in the reaction process. When the mass ratio of PMCSM/GOD/CAT was 100/6.02/10.96 (mg/mg/mg), the relative enzyme activity of GOD reached the highest (133.32 +/- 0.68%). The thermal stability and pH stability of the enzyme were greatly improved after co-immobilization. The relative enzyme activity of PMCSM@GOD@CAT was 57.27 +/- 3.04% at 60 degrees C, while that of free GOD was only 28.76 +/- 4.10%. The relative enzyme activity of PMCSM@GOD@CAT was above 63% at pH 5-10, while the relative enzyme activity of free GOD was only 4.98 +/- 0.72% at pH 10. The yield of sodium gluconate from 50 mL 250 mg/mL glucose catalyzed by PMCSM@GOD@CAT loading 60.2 mg GOD was 96.19 +/- 0.79% at pH 6.0 and 30 degrees C, and the reaction lasted 6 h. The relative enzyme activity of PMCSM@GOD@CAT remained 69.77 +/- 0.78% after repeated use for 10 times. After 30 days of storage, PMCSM@GOD@CAT maintained its initial activity of 76.52 +/- 1.41%. The immobilized process studied in this paper provides a theoretical basis for the production of sodium gluconate by double enzyme directly catalyzing and lays a certain foundation for the application of immobilized enzyme in the future chemical industry and food industry.