漆酶在纳米金溶胶/多重壁碳纳米管复合载体上固定方法的比较及粒子尺寸效应

以纳米金溶胶（NGS）和多重壁碳纳米管（MWCNTs）的共混物(NGS/MWCNTs)作为固定漆酶的载体，研究了3种固定漆酶方法在酶固定量、比活力上的差异。 研究了不同的固定方法对固定酶热稳定性和重复使用性及纳米金溶胶颗粒粒径对酶固定量和固定酶动力学参数的影响。 实验结果表明，NGS/MWCNTs具有良好的固定漆酶能力和高固酶比活力，NGS/MWCNTs（NGS粒径37 nm）通过简单物理吸附法固定漆酶的量和固酶的比活力最高，分别可达33.80 mg/g和9.433 U/mg。 在NGS-MWCNTs上采用化学键合方法固定的漆酶在70 ℃放置2 h后仍然保持初始活力的75％，重复使用20次后仍保持初始活力的70％。 纳米金溶胶粒子越小（24 nm）,底物和固定漆酶间亲和力越好（KM=0.027 mmol/L），表观速率常数越大。

Immobilization Approaches of Laccase on Nanogold Sol/Multi-walled Carbon Nanotube Matrices and the Particle Size Effects

Mixtures of nanogold sol(NGS) and multi-walled carbon nanotubes(MWCNTs) were used as a carrier for laccase immobilization. Three immobilization approaches were evaluated via comparing the amount of immobilized laccase and their specific activity of immobilized enzyme. Thermal stability and reusability of immobilized laccase were investigated and the effects of particle size of nanogold sol particles on laccase immobilization and immobilized enzyme kinetics were examined. Results from experiments reveal that NGS/MWCNTs matrices have the most excellent capability of laccase immobilization and high specific activity of immobilized laccase. Amount of immobilized enzyme and specific activity reach 33.80 mg/g and 9.433 U/mg, respectively, through simple physical adsorption when NGS particle size is 37 nm. It is also shown that the strategy of physical adsorption is suitable for immobilization of laccase in practice according to comprehensive evaluation of the amount of immobilized laccase, thermal stability, specific activity and reusability. Immobilized laccase on NGS/MWCNTs through covalent coupling can retain about 75％of initial activity after 2 h at 70 ℃ and retain 70％of initial activity after 20 recycles of repeated use. Conclusion from experiments indicates smaller NGS particle(24 nm) makes the affinity between immobilized laccase and its substrate(KM=0.027 mmol/L) stronger and the apparent catalyzing rate constant(1088 min-1) greater.