漆酶在纳米多孔金上的固定化及其酶学性质研究

利用纳米材料为载体对酶等生物大分子进行固定化近年来引起人们的浓厚兴趣. 以Au/Ag合金为原料, 通过控制浓硝酸的腐蚀时间再辅以退火处理得到了不同孔径的纳米多孔金(NPG), 利用扫描电镜(SEM)和N2气体吸附仪对孔性质进行了表征. 以NPG为载体, 用α-硫辛酸和N-乙基-N’-(3-二甲基氨基丙基)碳酰二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)对金表面进行活化, 通过化学共价偶联的方法对产自Trametes versicolor的漆酶进行了固定化. 比较了孔径大小对酶固定化量及比活力的影响. 发现小孔径更有利于对该漆酶的固定化. 与游离酶相比, 固定化酶的最适pH没有改变, 但最适温度却从原来的40 ℃升到了60 ℃. 固定化后, 漆酶的pH和热稳定性都明显提高了. 重复使用8次仍能保持初始活力的65%, 且在4 ℃下保存1个月几乎观察不到酶活力的下降. 此外, 失活的固定化酶经浓硝酸处理后, NPG载体可重复利用. 本结果初步显示出了NPG在生物技术领域中的应用潜力.

Immobilization of Laccase on Nanoporous Gold and Its Enzymatic Properties

• Immobilization of biomolecules such as enzyme on nanomaterials has aroused wide interest in recent years. Nanoporous gold (NPG) with different pore sizes was prepared by controlling the etching time of Au/Ag alloy with concentrated nitric acid and annealing. Scanning electron microscopy and a nitrogen adsorption technique were used to characterize the NPG. The surface of NPG was activated with α-lipoic acid and N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), and laccase from Trametes versicolor was then immobilized on it through chemical coupling. The effects of pore size on the specific activity and on the amount of immobilized laccase were studied. The results showed that the NPG with a smaller pore size was a better carrier for the laccase immobilization. Compared with free enzyme, the optimum pH of immobilized laccase did not change, however, the optimum temperature rose from 40 ℃ to 60 ℃. Both pH and thermal stabilities were improved markedly via the immobilization. After 8 times usage, 65% of its initial activity could still remain. In addition, no obvious activity loss was observed after 1 month storage at 4 ℃. For the inactivated immobilized laccase, its carrier NPG could be recycled by simply immersing it into concentrated nitric acid. All these results demonstrate that NPG as a carrier for enzyme has great potential applications to biotechnology area.