【作者】 张树江；

【导师】 夏黎明；

【作者基本信息】 浙江大学 ， 生物化工， 2006， 硕士

【摘要】 研究了杂色云芝菌Coriolus versicolor固态发酵生产漆酶的工艺条件,发现玉米皮为固态发酵产漆酶的适宜原料,玉米皮和麸皮的最佳比例为6:4。培养基的最适含水量为65%,最适接种量和培养温度分别为10%和30℃。在优化后的固态发酵条件下,漆酶活力在第十天可达到75932.9 U/g。 对C.versicolor漆酶的酶学性质进行了研究:C.versicolor漆酶在55℃和pH4.5的条件下催化活性最高,在40℃以下和pH5-6范围内较为稳定;以ABTS作为漆酶反应底物,通过Linewear-Burk曲线得到漆酶的动力学Km值为0.013 mmol/L;1 mmol/L Cu²⁺对漆酶活力有明显的促进作用;漆酶不太稳定,在常温和4℃条件下,酶液的半衰期分别为30天和66天。将酶液制成酶粉,稳定性有了较大提高,而在酶液中添加4%-8%的Na₂SO₄,则可以提高酶液的稳定性。 采用活性碳吸附与海藻酸钙凝胶包埋相结合的方法使漆酶固定化。利用固定化漆酶对2,4-二氯酚进行脱氯反应,其最适pH值为4.5、最适温度为40℃。与游离酶相比,固定化酶反应的pH值和温度范围更宽,其稳定性得到了明显改善。使用柱式固定化酶反应器处理2,4-二氯酚,在批式反应工艺条件下,当底物浓度为1 mmol/L、反应3-5h,2,4-二氯酚的去除率可达99.5%以上（脱除的氯离子浓度达0.5 mmol/L）。连续8批反应的结果表明:固定化漆酶性能稳定、催化效率高,在环境污染废水治理方面具有良好的应用前景。 工业上使用酸性纤维素酶水洗牛仔布时存在返沾色问题,而在此过程中添加适量漆酶,基本上解决了上述问题。在小试研究中,漆酶用量为0.5%（即5.0g漆酶/kg牛仔布）,协同水洗时间60min,牛仔布返染程度减少了83%。放大试验显示:0.5%漆酶与2.0%酸性纤维素酶协同作用水洗牛仔布,不仅能防止返沾色、提高牛仔布的质量,也能使废水中的靛蓝染料含量减少80%,从而大大降低环境污染。从放大试验可以看出,漆酶和酸性纤维素酶协同水洗是牛仔服生物整理中具有实用价值的一种新方法。更多还原

【Abstract】 The laccase production by Coriolus versicolor was studied under solid-state fermentation. Cornhusk was found to be a good carbon source for laccase production and the optimal ratio of cornhusk and wheat bran was 6:4. The optimum moisture content of substrate, inoculation dosage, and culture temperature were 65%, 10% and 30°C, respectively. High laccase activity （75432.5U/g） was obtained by solid-state fermentation after 10 days.The characterization of laccase from Coriolus versicolor was studied. The laccase had the highest activity at 55℃ and pH4.5. It was stable under 40℃ and at pH5-6. The Km value was 0.013 mmol/L with ABTS as a substrate. lmmol/L Cu²⁺ could stimulate the enzyme. The half-lives of liquid laccase at constant temperature and 4℃ were 30d and 66d, respectively. Addition of 4%-8% Na₂SO₄ could improve the storage stability of liquid laccase. Laccase powder was more stable than liquid enzyme.Laccase from Coriolus versicolor was immobilized by the method of being adsorbed first on activated carbon, and then entrapped into calcium alginate gels. The dechlorination of 2, 4-dichlorophenol in wastewater was performed by the immobilized laccase in a columnar reactor. The optimal temperature and pH value for the reaction were 40℃ and 4.5, respectively. Compared with free laccase, the immobilized laccase had a better thermostability, and also had a broader range of pH value and temperature in which the enzymatic catalysis displayed. The removal efficiencies of 2, 4-dichlorophenol （1 mmol/L） were all above 99.5% with the concentration of 0.5mmol/L chloride ions released during repeated 8 batch reactions. The research results had shown a good prospect of application in the biochemical treatment of polluted wastewater.The backstaining caused by indigo blue was always a big problem in the process of biofinishing blue jeans using cellulase from Trichoderma reesei. Laccase could hydrolyze indigo blue and reduce the backstaining. With the laccase dosage of 0.50% （o.w.f）, the backstaining extent on white cotton fabric decreased 83% by 60min. The results of scale-up experiments showed that synergistic effect of laccase and cellulase not only reduced 82.2% of indigo blue in biofinishing effluent, but also weakened 83% of backstaining on blue jeans.更多还原