Enzyme Immobilization On Three-dimensionally Ordered Macroporous Material And Their Catalytic Performance

Three-dimensionally ordered macroporous materials, which possess an open, interconnected macropore structure, have potential applications in enzyme immobilization. In this paper, three-dimensionally ordered macroporous silica was used as carrier, and covalent binding and entrapment methods were employed to fabricate immobilized enzymes. The details were summarized as follows:(1) Coimmobilization of bienzyme in three-dimensionally ordered macroporous silica through biomimetic adhesion and study of the catalytic performance. The immobilization process were as follows: firstly, three-dimensionally ordered macroporous silica was employed as carrier, and was modified by dopamine; secondly, GOD and HRP were coimmobilized on the dopamine-modified carrier through covalent binding with dopamine, and the coimmobilized bienzyme was named GOD/HRP@3DOM-Si O2. Activity ratio of GOD and HRP for enzyme immobilization was optimized to be 50 U: 37.5 U; Compared with native enzymes, GOD/HRP@3DOM-SiO2 exhibited better thermal stability, which could maintain 21.41% of initial activity after 40 min immersion in PBS buffer at 65 oC; GOD/HRP@3DOM-SiO2 possessed high sensitivity for the detection of glucose, the limit of detection was 0.99 μmol/L; GOD/HRP@3DOM-SiO2 exhibited better catalytic performance than enzymes that in other forms, and had high selectivity to glucose.(2) In situ construction of enzyme-containing silica inverse opals and study of the catalytic performance. The immobilizing processes were as follows: firstly, water-soluble polyacrylamide was used as template, and enzyme-containing sol were filled into the voids of the template; secondly, gel aged for one day and PAM template was removed by PBS buffer, and the enzyme-containing silica inverse opals were obtained and called 3DOM-GOD/SiO2. SEM, TEM and N2 adsorption-desorption experiments indicated that the 3DOM-GOD/SiO2 possessed ordered macroporous and mesoporous structure, the pore diameter were 500 and 6.56 nm, respectively; CLSM experiments indicated that the enzymes were successfully entrapped in the silica; GOD concentration for enzyme entrapment was optimized to be 12 mg/mL; Compared with native enzymes, the enzyme-containing silica exhibited enhanced thermal and pH stability, as well as improved denaturant tolerance; Kinetic experiments demonstrated that the hierarchical porous structure was helpful to mass transportation and beneficial to the decrease of mass transfer resistance; Glucose oxidase and organophosphorus hydrolase were used as model enzymes to fabricate 3DOM-GOD/Si O2 and 3DOM-OPH/SiO2, then used to detect glucose and methyl parathion in solutions, respectively. The results showed good response to glucose and methyl parathion of low concentration, the limit of detection were 0.91 μmol/L and 0.46 μmol/L, respectively.(3) In situ construction of bienzyme-containing silica inverse opals and study of the catalytic performance. Bienzyme-containing silica inverse opals were obatined and used to remove glucose in isomaltooligosaccharide. Morphology characterization indicated that hierarchical porous structure was obatined and the pore diameter of macroporous and mesoporous were 500 and 6.8 nm, respectively; macropore were connected through windows of 150 nm; Enzymes were entrapped in silica that derived from sol-gel process; Glucose removal efficiency reached up to 98.67% under the conditions of enzyme activity ratio of 1: 30, enzyme dosage of 68.8 mg, reaction time of 8 h, reaction temprature of 35 oC and pH of 7.0; 75.19% of removal efficiency could be retained even after 6 times of reuse of the bienzyme system.