| This paper studied the membrane immobilization of glucose oxidase (GOD) and L-glutamicoxidase (L-GOX), and the best immobilization conditions was achieved by optimizing theimmobilization carriers and some other immobilization conditions. The characteristic of twoimmobilized enzymes for biosensor was also investigated the present work.Glutaraldehyde and bovine serum albumin were used as cross-linking agent and asprotectant respectively. The effects of the carrier material, the carrier aperture, the GODconcentration, the BSA concentration and the glutaraldehyde concentration on theimmobilization of GOD were investigated. The best immobilization conditions were: a mixedcellulose membrane filter with a pore size of0.45μm as the carrier membrane,0.4%(v/v)glutaraldehyde as a cross-linking agent and3%(w/v) bovine serum albumin as a protectant, andthe concentration of glucose oxidase is3.75mg/mL. Under the above conditions the immobilizedenzyme has the highest activity. The suitable temperature and pH were about40℃and pH=7.0,respectively. And the Michaelis constant Km is20.317mmol/L, and the maximum velocity Vmaxis0.1176mmol/(L·min).The best immobilization conditions of the L-glutamic oxidase were: the mixed cellulosemembrane filter with a pore size of0.45μm,8mg/mL L-GOX, glutaraldehyde,0.3%(v/v) BSAconcentration. The suitable temperature was65～70℃, and the suitable pH was about pH=7.0,the Michaelis constant Km is1.063mmol/L, the maximum velocity Vmax is0.557mmol/(L·min).With the constructed glucose biosensor with the immobilized enzyme, the response timewas less than20s, and the linear response range is0～150mg/dL. And the biosensor showedextremely good selectivity and stability. With the constructed a L-glutamate biosensor with theimmobilized enzyme, the response time was less than20s, and a linear response arange isbetween0～160mg/dL (L-glutamate). Linear relative curve was Y=0.9715X+0.9966(R2=0.9994). And the biosensor showed extremely good selectivity and stability. |
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