Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province, China.
J Microbiol Biotechnol. 2013 Jun 28;23(6):864-71. doi: 10.4014/jmb.1212.12057.
The recombinant strain P. pastoris GS115-lccC was used to produce laccase with high activity. Factors influencing laccase expression, such as pH, methanol concentration, copper concentration, peptone concentration, shaker rotate speed, and medium volume were investigated. Under the optimal conditions, laccase activity reached 12,344 U/L on day 15. The recombinant enzyme was purified by precipitating and dialyzing to electrophoretic homogeneity, and was estimated to have a molecular mass of about 58 kDa. When guaiacol was the substrate, the laccase showed the highest activity at pH 5.0 and was stable when the pH was 4.5~6.0. The optimal temperature for the laccase to oxidize guaiacol was 60°C, but it was not stable at high temperature. The enzyme could remain stable at 30°C for 5 days. The recombinant laccase was used to degrade chlorpyrifos in several laccase/mediator systems. Among three synthetic mediators (ABTS, HBT, VA) and three natural mediators (vanillin, 2,6-DMP, and guaiacol), vanillin showed the most enhancement on degradation of chlorpyrifos. Both laccase and vanillin were responsible for the degradation of chlorpyrifos. A higher dosage of vanillin may promote a higher level of degradation of chlorpyrifos, and the 2-step addition of vanillin led to 98% chlorpyrifos degradation. The degradation of chlorpyrifos was faster in the L/V system (kobs = 0.151) than that in the buffer solution (kobs = 0.028).
重组毕赤酵母菌株 P. pastoris GS115-lccC 被用来生产高活性的漆酶。考察了影响漆酶表达的因素，如 pH 值、甲醇浓度、铜浓度、蛋白胨浓度、摇床转速和培养基体积。在最佳条件下，第 15 天漆酶活性达到 12344 U/L。重组酶通过沉淀和透析达到电泳均一性进行纯化，估计分子量约为 58 kDa。当愈创木酚为底物时，漆酶在 pH 5.0 时表现出最高活性，在 pH 4.5~6.0 时稳定。漆酶氧化愈创木酚的最适温度为 60°C，但在高温下不稳定。该酶在 30°C 下可稳定 5 天。重组漆酶用于在几种漆酶/介体系统中降解毒死蜱。在三种合成介体（ABTS、HBT、VA）和三种天然介体（香草醛、2,6-DMP 和愈创木酚）中，香草醛对毒死蜱的降解表现出最大的增强作用。香草醛和漆酶都参与了毒死蜱的降解。香草醛的用量越高，毒死蜱的降解水平可能越高，两步添加香草醛可导致 98%的毒死蜱降解。在 L/V 体系（kobs=0.151）中，毒死蜱的降解速度比在缓冲溶液中（kobs=0.028）快。
