Coriolus versicolor产漆酶及其对染料脱色的研究
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摘要
本文对彩绒革盖菌(Coriolus versicolor)产漆酶的工艺条件、漆酶的酶学性质、以及漆酶在染料降解方面的应用等进行了研究，取得了如下研究成果：
1)在摇瓶条件下对彩绒革盖菌的产酶工艺参数进行了研究，优化后的培养条件为：葡萄糖1.1g／L、蔗糖1.1g／L、豆饼粉1.429g／L、NaH_2PO_4 0.2g／L、MgSO_4·7H_2O 0.5g／L、微量元素20mL／L、pH4.5、30℃、180rpm，漆酶活力达22.37U／mL，生产周期由原来的14天缩短到8天。
(2)利用自絮凝菌丝球在自制的流化床生物反应器中分批发酵，确立了适宜的工艺条件为：30℃，菌丝球装填比例1:7，通气量1.0vvm。在维持一定产酶量的前提下，菌丝球至少可重复使用5次，平均漆酶产率可达5.9612U·mL~(-1)·d~(-1)，比摇瓶发酵2.7962U·mL~(-1)·d~(-1)的漆酶产率提高了一倍以上。流化床生物反应器动力消耗少、反应器内流化状态稳定、菌丝球可重复使用、产品易于分离纯化、生产成本较低，显示了明显的优越性。
(3)彩绒革盖菌固体培养试验表明：培养基质以麦草为佳，木屑其次。麸皮含有大量微量元素和生长因子，对固体发酵具有明显的促进作用。在麦草与麸皮比例为50:43、培养基质含水量70％、30℃条件下，漆酶活力可达924.71U／g；彩绒革盖菌除主要产漆酶外，还可形成一定量的CMC酶(90.85U／g)和木聚糖酶(242.29U／g)。
(4)综合考虑漆酶作用的最适温度与热稳定性，确立50℃为实际应用的最佳温度。采用漆酶对染料酸性橙进行脱色降解试验，不加任何介质条件下反应6h脱色率为45.8％；加入1％(V:V)的HBT溶液后，反应15min脱色率达94％以上。漆酶在无介质参与条件下不能使靛蓝脱色降解，当加入2％(V:V)的HBT溶液后，反应40min脱色率可达82.7％。
当前，印染废水对环境的危害日趋严重，由于其量大、色泽深、有害物质多，对于这类废水的治理一直被认为是相当棘手的问题。本文所取得的研究成果为酶法处理印染废水提供了可靠的实验依据，在环境治理方面具有良好的应用前景。
Studiesjn this thesis were focused on the fermentation process of laccase by Coriolus versicolor, the enzymatic characteristic of laccase, and the application in dyes decolorization by laccase and laccase-mediator system. The major research achievements were as follows:
(1) The optimal fermentation parameters of Coriolus versicolor in shaking flask were as follows: sucrose 1.1 g/L, glucose 1.1 g/L, bean cake powder 1.429 g/L, NaH2PO4 0.2 g/L, MgSO4.7H20 0.5 g/L and trace element 20 mL/L, initial pH 4.5; rotating speed 180 rpm, culture temperature 30℃. The activity of laccase reached 22.37 U/mL, and the production period of laccase was reduced from 14 days to 8 days.
(2) The repeated batch process for laccase production was carried out in a fluidized-bed bioreactor using Coriolus versicolor pellets. The optimal conditions in the bioreactor were as follows: 30℃, pH 4.5, pellets packing ratio 1:7, aeration rate 1.0vvm. The pellets could be reused successfully at least 5 batch cycles with an average laccase yield of 5.9612 U.mL-1.d-1, which was 113.1% higher than the laccase yield obtained in shaking flask. The fluidized-bed bioreactor has advantages such as good ability of mass transport, lower energy requirement and easier purification of the product.
(3) The solid-state fermentation process was studied for laccase production by Coriolus versicolor. The results showed: wheat straw was the best lignocellulosic substrate; wheat bran had an obvious stimulative effect on laccase production since it contained lots of trace elements and growth factors. The fermentation parameters were optimized as: the ratio between wheat straw and wheat bran, 50:43; water content, 70 %, fermentation temperature, 30℃. A high laccase activity (924.71 U/g) has been obtained under the optimal SSF conditions. In addition to laccase, another two kinds of enzyme (CMCase 90.85U/g and Xylanase 242.29U/g) were also detected.
(4) For the oxidation-reduction reaction catalyzed by laccase, the suitable temperature was 50℃, considering the optimum temperature for laccase activity and thermal stability. The laccase-mediator system was used to decolorize two general
commercial dyes: acrid orange and indigo. While adding 1% HBT solution (V:V) in the reactive system, 94% of acrid orange was removed in 15 minutes, which was over 48.2% higher than that without any redox mediator. Laccase couldn't decolor indigo singly, while 82.7% decolorization could be obtained with the cooperation of 2% HBT solution (V: V).
Nowadays, more and more people were concerned about environmental problems, especially the wastewater containing dyestuffs. A practical way using laccase mediator system to decolorize dyestuffs was built successfully in this thesis. It had some obvious advantages such as higher efficiency and wider range of substrates. The experimental data and the technology provided in this thesis were meaningful to large-scale application of laccase in environmental protection.
1)在摇瓶条件下对彩绒革盖菌的产酶工艺参数进行了研究，优化后的培养条件为：葡萄糖1.1g／L、蔗糖1.1g／L、豆饼粉1.429g／L、NaH_2PO_4 0.2g／L、MgSO_4·7H_2O 0.5g／L、微量元素20mL／L、pH4.5、30℃、180rpm，漆酶活力达22.37U／mL，生产周期由原来的14天缩短到8天。
(2)利用自絮凝菌丝球在自制的流化床生物反应器中分批发酵，确立了适宜的工艺条件为：30℃，菌丝球装填比例1:7，通气量1.0vvm。在维持一定产酶量的前提下，菌丝球至少可重复使用5次，平均漆酶产率可达5.9612U·mL~(-1)·d~(-1)，比摇瓶发酵2.7962U·mL~(-1)·d~(-1)的漆酶产率提高了一倍以上。流化床生物反应器动力消耗少、反应器内流化状态稳定、菌丝球可重复使用、产品易于分离纯化、生产成本较低，显示了明显的优越性。
(3)彩绒革盖菌固体培养试验表明：培养基质以麦草为佳，木屑其次。麸皮含有大量微量元素和生长因子，对固体发酵具有明显的促进作用。在麦草与麸皮比例为50:43、培养基质含水量70％、30℃条件下，漆酶活力可达924.71U／g；彩绒革盖菌除主要产漆酶外，还可形成一定量的CMC酶(90.85U／g)和木聚糖酶(242.29U／g)。
(4)综合考虑漆酶作用的最适温度与热稳定性，确立50℃为实际应用的最佳温度。采用漆酶对染料酸性橙进行脱色降解试验，不加任何介质条件下反应6h脱色率为45.8％；加入1％(V:V)的HBT溶液后，反应15min脱色率达94％以上。漆酶在无介质参与条件下不能使靛蓝脱色降解，当加入2％(V:V)的HBT溶液后，反应40min脱色率可达82.7％。
当前，印染废水对环境的危害日趋严重，由于其量大、色泽深、有害物质多，对于这类废水的治理一直被认为是相当棘手的问题。本文所取得的研究成果为酶法处理印染废水提供了可靠的实验依据，在环境治理方面具有良好的应用前景。
Studiesjn this thesis were focused on the fermentation process of laccase by Coriolus versicolor, the enzymatic characteristic of laccase, and the application in dyes decolorization by laccase and laccase-mediator system. The major research achievements were as follows:
(1) The optimal fermentation parameters of Coriolus versicolor in shaking flask were as follows: sucrose 1.1 g/L, glucose 1.1 g/L, bean cake powder 1.429 g/L, NaH2PO4 0.2 g/L, MgSO4.7H20 0.5 g/L and trace element 20 mL/L, initial pH 4.5; rotating speed 180 rpm, culture temperature 30℃. The activity of laccase reached 22.37 U/mL, and the production period of laccase was reduced from 14 days to 8 days.
(2) The repeated batch process for laccase production was carried out in a fluidized-bed bioreactor using Coriolus versicolor pellets. The optimal conditions in the bioreactor were as follows: 30℃, pH 4.5, pellets packing ratio 1:7, aeration rate 1.0vvm. The pellets could be reused successfully at least 5 batch cycles with an average laccase yield of 5.9612 U.mL-1.d-1, which was 113.1% higher than the laccase yield obtained in shaking flask. The fluidized-bed bioreactor has advantages such as good ability of mass transport, lower energy requirement and easier purification of the product.
(3) The solid-state fermentation process was studied for laccase production by Coriolus versicolor. The results showed: wheat straw was the best lignocellulosic substrate; wheat bran had an obvious stimulative effect on laccase production since it contained lots of trace elements and growth factors. The fermentation parameters were optimized as: the ratio between wheat straw and wheat bran, 50:43; water content, 70 %, fermentation temperature, 30℃. A high laccase activity (924.71 U/g) has been obtained under the optimal SSF conditions. In addition to laccase, another two kinds of enzyme (CMCase 90.85U/g and Xylanase 242.29U/g) were also detected.
(4) For the oxidation-reduction reaction catalyzed by laccase, the suitable temperature was 50℃, considering the optimum temperature for laccase activity and thermal stability. The laccase-mediator system was used to decolorize two general
commercial dyes: acrid orange and indigo. While adding 1% HBT solution (V:V) in the reactive system, 94% of acrid orange was removed in 15 minutes, which was over 48.2% higher than that without any redox mediator. Laccase couldn't decolor indigo singly, while 82.7% decolorization could be obtained with the cooperation of 2% HBT solution (V: V).
Nowadays, more and more people were concerned about environmental problems, especially the wastewater containing dyestuffs. A practical way using laccase mediator system to decolorize dyestuffs was built successfully in this thesis. It had some obvious advantages such as higher efficiency and wider range of substrates. The experimental data and the technology provided in this thesis were meaningful to large-scale application of laccase in environmental protection.
引文
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