漆酶在对苯二甲酰氯-己二胺缩聚物载体上的固定:物理吸附及共价偶联的比较

Immobilization of Laccase from Trametes versicolor on Polymer of Paraphthaloyl Chloride and Hexane Diamine:Physical Adsorption and Covalent Coupling

以对苯二甲酰氯(PPTC)和己二胺(HD)的缩聚物作为固定云芝漆酶的载体,研究了物理吸附和化学偶联两种方法对漆酶固定量、固酶比活力、固定漆酶的热稳定性、重复使用性的影响。此外还进一步研究了不同固定策略对固定漆酶动力学参数的影响。实验结果表明:无论采用物理吸附法或是化学偶联法都具有较高的固酶量和固酶比活力。两种方法的固酶量和固定漆酶的比活力分别为:物理吸附法-126 mg/g,1.65 U/mg;化学偶联法-92 mg/g,0.65 U/mg。通过比较两种不同的固酶策略对固酶热稳定性、重复使用性及酶动力学参数的影响后发现:化学偶联法固定漆酶的热稳定性及重复使用性能均优于物理吸附法,但此种方法固酶的表观催化氧化反应速率常数kcat低于物理吸附法固定漆酶的kcat。综合比较,以对苯二甲酰氯与己二胺缩聚物为固酶基元采用化学偶联法固定漆酶应为固定游离漆酶的一种较好的可行方法。

Carrier of laccase from Trametes versicolor immobilization was prepared through condensation of paraphthaloyl chloride（PPTC） and hexane diamine（HD）.The influence of immobilization strategy,physical adsorption and covalent coupling,on laccase-loading amount,specific activity of immobilized laccase,thermal stability and reusability of laccase-entrapped in condensation polymer was studied.Further investigation was on the influence of immobilization strategies on kinetics of immobilized laccase.The result indicated immobilized laccase showed high specific activity and laccase-loading amount was high with this polymer as immobilization matrix when both strategy of physical adsorption or covalent coupling were used.Laccase-loading amount of immobilization matrix and specific activity of immobilized laccase through approach of physical adsorption and covalent coupling were：126 mg/g,1.65 U/mg（（physical adsorption）;92 mg/g,0.65 U/mg（covalent coupling）.The thermal stability and reusability of immobilized laccase through covalent coupling was superior to that of immobilized laccase through physical adsorption.However,immobilized laccase through covalent coupling showed inferior catalytic function toward oxidation of substrate.The apparent catalytic oxidation reaction rate constant kcat through covalent coupling was 885 min-1,whereas kcat of physical adsorption was 964 min-1.It could be concluded that it is viable to immobilize laccase through covalent coupling with the condensation polymer of paraphthaloyl chloride and hexane diamine as matrix.