【摘要】 由血红密孔菌（Pycnoporus sanguineus）产生的漆酶耐热性能良好,具有重要工业应用价值,但该菌的产酶性能低,不适于规模化生产。研究将该漆酶基因进行密码子优化后,与里氏木霉（Trichoderma reesei）强启动子Pcbh 1、终止子Tcbh 1和潮霉素抗性基因连接得到重组质粒pCH-lac,再通过农杆菌介导技术,将此质粒转化到里氏木霉细胞中,通过潮霉素抗性筛选及复筛得到9个重组转化子。摇瓶产酶试验显示:发酵液中漆酶活力在补料培养至第7天可达20.3IU×mL^-1,表明外源漆酶基因已在里氏木霉宿主中成功实现了高效表达和胞外分泌。重组转化子所产漆酶对含活性艳蓝KN-R的印染废水具有明显的脱色作用:在漆酶用量为0.7 IU×mL^-1、60℃、pH 3.6,且不加任何介体条件下,反应5 h脱色率可达到91.0%。该研究成果对于促进漆酶的规模化生产及其在环境治理方面的应用具有重要意义。更多还原

【Abstract】 Thermotolerant laccase produced by Pycnoporus sanguineus has great potential in industrial applications. However, low production capacity limits its large-scale production. In this study, codon-optimized laccase gene was inserted between Trichoderma reesei strong promoter Pcbh 1 and terminator Tcbh 1, which was further linked to hygromycin B resistant gene to form a recombinant plasmid pCH-lac. The palsmid was transformed into the conidia of Trichoderma reesei by Agrobacterium-mediated technique. Nine transformants were obtained through hygromycin resistance screening and secondary screening. The results of enzyme production in shaking flasks show that the laccase activity is 20.3 IU × mL^-1 after 7 days of fed-batch fermentation, which indicates the heterologous laccase gene is highly expressed and its expression products can be secreted extracellularly. The transformants can efficiently decolorize wastewater containing Brilliant Blue KN-R（a reactive dye）. 91.0% dye was degraded when using 0.7 IU×mL^-1 laccase for 5 hours at pH 3.6, 60℃.The results are helpful in promoting large-scale laccase production and wastewater treatment.更多还原