白腐菌Coriolus versicolor漆酶及其在制浆造纸废水深度处理中的应用
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摘要
漆酶(Laccase,EC1.10.3.1)是白腐菌降解木质素酶系中的重要酶之一,具有高氧化还原电位、不需要H2O2启动而以O2为电子受体,因而具有比其他几种木质素酶更实际的应用价值。杂色云芝菌是白腐菌中重要的一类,具有较高的漆酶分泌能力。
论文首先从液体和固体两种培养体系出发,探索不同培养条件对白腐菌分泌漆酶的影响。液体培养体系下,白腐菌的最佳生长和漆酶分泌条件是缓冲体系起始pH5.8、摇瓶转速为180rpm、装液量为100mL/500mL;在所有测试碳源中,葡萄糖为最佳碳源,在二糖中以蔗糖为碳源,漆酶酶活相对较高,而多糖如淀粉不利于漆酶的生产。在所有测试氮源中,硝酸氨和酒石酸氨组合为最佳氮源,氯化铵和尿素的效果次之,蛋白胨的效果最差。当C/N>10:1即高碳低氮培养条件下,有利于漆酶的形成。芳香化合物诱导剂ABTS浓度为0.5-1mM时,对漆酶的生产影响最明显,与限氮培养基中产酶相比可提高酶活5倍,约为230IU/ml;0.5mM愈创木酚、1mM阿魏酸、0.05mM二甲基苯胺的添加也明显提高酶活,约提高2-2.5倍;0.01mM黎芦醇的添加可提高漆酶约1.6倍。由于酪氨酸具有酚类化合物的特点,也可作为一种无毒的天然漆酶诱导剂。菌株漆酶活力固体培养体系下明显低于液体培养体系下。
以液体基质培养为研究对象,对白腐菌漆酶的分离纯化和主要酶学性质进行研究。纯化倍数为15倍,回收率为20.4%,该漆酶分子量为30.03kD;最适反应pH为4.5,pH6.0左右稳定性最高;最适温度为65℃,在50℃以下稳定性好,当温度超过70℃时,漆酶开始失活,在80℃处理20分钟,漆酶完全失活。
对杂色云芝漆酶处理造纸废水条件的研究结果表明:杂色云芝菌高产漆酶优化培养基与杂色云芝漆酶对造纸废水脱色的最佳培养基一致,说明在造纸废水的脱色方面漆酶起到了至关重要的作用;研究结果表明,造纸废水脱色与菌体生物量不呈正相关。在适宜的条件下,杂色云芝菌培养产生的漆酶粗酶液可有效的对碱法木浆造纸厂二沉池出水进行深度处理。漆酶粗酶液处理废水的效率跟处理时间和酶用量的大小有关,同时也受反应体系的温度和pH值等因素的影响。在一定的反应体系下,废水中木素、CODcr和色度的去除率随处理时间的延长和酶用量的增加而增加。最佳处理条件为:处理时间5小时,酶液用量5IU/ml,pH 7,温度55℃。在进水CODcr为268mg/l,木素含量为100mg/l,色度为186倍的条件下,处理后出水CODcr为48mg/l,木素含量16mg/l,色度为10倍。CODcr、木素和色度的降解率分别达82.1%,84%和94.6%。通过对比粗漆酶和纯漆酶对造纸废水中的木素的去除效果,发现在同样处理条件下粗漆酶的效果优于纯漆酶。
最后通过凝胶渗透色谱法测定了造纸厂二沉池出水中木素的分子量为3.12万,经纯漆酶液处理1小时后的木素的分子量为4.66万,经纯漆酶液处理2小时后的木素分子量为5.86万。实验结果表明:经过漆酶处理后,废水中木素的分子量明显变大,漆酶在反应中对木素起到了聚合作用,白腐菌Coriolus versicolor漆酶液在一定的条件下可以将造纸厂二沉池出水中的木素及其衍生物催化聚合从而使其沉淀去除,废水中的木素及其衍生物减少,从而使废水的色度和COD降低,进而为造纸废水中水回用提供可能。
Laccase is an important enzyme of lignin degradation by white rot fungi. It has high redox potential and takes O2 as electron receptor without the aid of H2O2. Therefore, it has more application potential than other ligninases.Coriolus versicolor is a very important species of white rot fungi with good ability of Laccase production.
Firstly, the influence on the production of laccase by Coriolus versicolor in different fermentation conditions and culture systems were investigated. In the liquid culture system, the optimal fermentation condition for best fungus growth and laccase producing were as follows: buffering system of pH 5.8, rotating speed 180rpm, 100mL culture medium per 500ml flask.The glucose was the best carbon fountain in all the tested carbon fountains. The laccase activity was comparative high when saccharose was used as the carbon fountain in all the disaccharide.However,starch appeared minus effect on laccase production.The combination of tartaric ammonium and ammonium nitrate was found to be the best nitrogen fountain in all the tested nitrogen fountains. The effect of ammonium chloride and carbamide took second place.The effect of peptone was the worst.It was suitable to the production of laccase when the ratio of carbon and nitrogen is more than 10. The laccase activity could be improved to 230 IU/ml when the revulsant ABTS concentration is 0.5-1mM.The addition of 0.5mM guaiacol、1mM ferulic acid、0.05mM dimethylaniline could also improve the laccase activity obviously. The addition of 0.01mM veraryl alcohol could also improve the laccase activity for 1.6 times. Tyrosine could be a native revulsant of laccase because it has the characteristic of hydroxybenzene.The laccase activity was less in solid conditions than in liquid system.
Secondly, the laccase production of Coriolus versicolor, purification and properties of laccase isoforms have been studied in the liquid system.The molecular weight of the purified laccase is 30.03kDa. the optimal pH for reaction and stability was 4.5 and 6.0 respectively. The optimal temperature was 65℃.The laccase began losing activity when the temperature was more than 70℃. The laccase lost activity completely when it was treated in 80℃for 20 minute.
The optimal conditions for treatment of papermaking effluent by coriolus versicolor laccase were studied . The results showed that the substrate of high-production laccase was the same with the optimal substrate of papermaking wastewater decolorization.It showed that the laccase had important effect in the decolorization of papermaking wastewater.However,the decolorization rate was not related to the biomass of coriolus versicolor directly.
The crude laccase from Coriolus versicolor could achieve advanced treatment of papermaking wasterwater effectively.The treatment efficiency of laccase was relational to the treatment time, laccase dosage, pH value and temperature.The removal rates of ligin,CODcr and chroma were improved when the treatment time and laccase amount increased.The optimal condition was as follows:5 hours of treatment time;5IU/ml of laccase amount; pH 7; 55℃.When the pollution index of enterwater was 268mg/L of CODCr ,100mg/l of lignin content and 186 times of chroma.The pollution duty of outwater decreased to CODCr 48mg/L, 100mg/l of lignin content and 10 times of the chroma.The removal rates of CODCr, lignin content and colority were 82.1%,84% and 94.6% respectively.The removal effect of lignin in papermaking wastewater by crude laccase was better than that of purified laccase .The reason was that some low molecular substance mediators could be remoaved during purifying process.
The molecular weight of the ligin which is from the outwater of the effluent seffling chamber taken from a pulp mill is 31200.The molecular weight of the ligin treated for one hour by laccase increased to 46600 while the molecular weight of the ligin treated for two hours by laccase reached 58600.The results showed that the molecular weight of the ligin increased obviously after treated by laccase.In suitable condition, the laccase produced by Coriolus versicolor could polymerize the ligin and its ramification in the wastewater of alkaline pulp mill.Then the CODcr and chroma of the wastewater would decrease.So the use of laccase could provides feasibility to reclaimed water reuse of the papermaking wastewater.
论文首先从液体和固体两种培养体系出发,探索不同培养条件对白腐菌分泌漆酶的影响。液体培养体系下,白腐菌的最佳生长和漆酶分泌条件是缓冲体系起始pH5.8、摇瓶转速为180rpm、装液量为100mL/500mL;在所有测试碳源中,葡萄糖为最佳碳源,在二糖中以蔗糖为碳源,漆酶酶活相对较高,而多糖如淀粉不利于漆酶的生产。在所有测试氮源中,硝酸氨和酒石酸氨组合为最佳氮源,氯化铵和尿素的效果次之,蛋白胨的效果最差。当C/N>10:1即高碳低氮培养条件下,有利于漆酶的形成。芳香化合物诱导剂ABTS浓度为0.5-1mM时,对漆酶的生产影响最明显,与限氮培养基中产酶相比可提高酶活5倍,约为230IU/ml;0.5mM愈创木酚、1mM阿魏酸、0.05mM二甲基苯胺的添加也明显提高酶活,约提高2-2.5倍;0.01mM黎芦醇的添加可提高漆酶约1.6倍。由于酪氨酸具有酚类化合物的特点,也可作为一种无毒的天然漆酶诱导剂。菌株漆酶活力固体培养体系下明显低于液体培养体系下。
以液体基质培养为研究对象,对白腐菌漆酶的分离纯化和主要酶学性质进行研究。纯化倍数为15倍,回收率为20.4%,该漆酶分子量为30.03kD;最适反应pH为4.5,pH6.0左右稳定性最高;最适温度为65℃,在50℃以下稳定性好,当温度超过70℃时,漆酶开始失活,在80℃处理20分钟,漆酶完全失活。
对杂色云芝漆酶处理造纸废水条件的研究结果表明:杂色云芝菌高产漆酶优化培养基与杂色云芝漆酶对造纸废水脱色的最佳培养基一致,说明在造纸废水的脱色方面漆酶起到了至关重要的作用;研究结果表明,造纸废水脱色与菌体生物量不呈正相关。在适宜的条件下,杂色云芝菌培养产生的漆酶粗酶液可有效的对碱法木浆造纸厂二沉池出水进行深度处理。漆酶粗酶液处理废水的效率跟处理时间和酶用量的大小有关,同时也受反应体系的温度和pH值等因素的影响。在一定的反应体系下,废水中木素、CODcr和色度的去除率随处理时间的延长和酶用量的增加而增加。最佳处理条件为:处理时间5小时,酶液用量5IU/ml,pH 7,温度55℃。在进水CODcr为268mg/l,木素含量为100mg/l,色度为186倍的条件下,处理后出水CODcr为48mg/l,木素含量16mg/l,色度为10倍。CODcr、木素和色度的降解率分别达82.1%,84%和94.6%。通过对比粗漆酶和纯漆酶对造纸废水中的木素的去除效果,发现在同样处理条件下粗漆酶的效果优于纯漆酶。
最后通过凝胶渗透色谱法测定了造纸厂二沉池出水中木素的分子量为3.12万,经纯漆酶液处理1小时后的木素的分子量为4.66万,经纯漆酶液处理2小时后的木素分子量为5.86万。实验结果表明:经过漆酶处理后,废水中木素的分子量明显变大,漆酶在反应中对木素起到了聚合作用,白腐菌Coriolus versicolor漆酶液在一定的条件下可以将造纸厂二沉池出水中的木素及其衍生物催化聚合从而使其沉淀去除,废水中的木素及其衍生物减少,从而使废水的色度和COD降低,进而为造纸废水中水回用提供可能。
Laccase is an important enzyme of lignin degradation by white rot fungi. It has high redox potential and takes O2 as electron receptor without the aid of H2O2. Therefore, it has more application potential than other ligninases.Coriolus versicolor is a very important species of white rot fungi with good ability of Laccase production.
Firstly, the influence on the production of laccase by Coriolus versicolor in different fermentation conditions and culture systems were investigated. In the liquid culture system, the optimal fermentation condition for best fungus growth and laccase producing were as follows: buffering system of pH 5.8, rotating speed 180rpm, 100mL culture medium per 500ml flask.The glucose was the best carbon fountain in all the tested carbon fountains. The laccase activity was comparative high when saccharose was used as the carbon fountain in all the disaccharide.However,starch appeared minus effect on laccase production.The combination of tartaric ammonium and ammonium nitrate was found to be the best nitrogen fountain in all the tested nitrogen fountains. The effect of ammonium chloride and carbamide took second place.The effect of peptone was the worst.It was suitable to the production of laccase when the ratio of carbon and nitrogen is more than 10. The laccase activity could be improved to 230 IU/ml when the revulsant ABTS concentration is 0.5-1mM.The addition of 0.5mM guaiacol、1mM ferulic acid、0.05mM dimethylaniline could also improve the laccase activity obviously. The addition of 0.01mM veraryl alcohol could also improve the laccase activity for 1.6 times. Tyrosine could be a native revulsant of laccase because it has the characteristic of hydroxybenzene.The laccase activity was less in solid conditions than in liquid system.
Secondly, the laccase production of Coriolus versicolor, purification and properties of laccase isoforms have been studied in the liquid system.The molecular weight of the purified laccase is 30.03kDa. the optimal pH for reaction and stability was 4.5 and 6.0 respectively. The optimal temperature was 65℃.The laccase began losing activity when the temperature was more than 70℃. The laccase lost activity completely when it was treated in 80℃for 20 minute.
The optimal conditions for treatment of papermaking effluent by coriolus versicolor laccase were studied . The results showed that the substrate of high-production laccase was the same with the optimal substrate of papermaking wastewater decolorization.It showed that the laccase had important effect in the decolorization of papermaking wastewater.However,the decolorization rate was not related to the biomass of coriolus versicolor directly.
The crude laccase from Coriolus versicolor could achieve advanced treatment of papermaking wasterwater effectively.The treatment efficiency of laccase was relational to the treatment time, laccase dosage, pH value and temperature.The removal rates of ligin,CODcr and chroma were improved when the treatment time and laccase amount increased.The optimal condition was as follows:5 hours of treatment time;5IU/ml of laccase amount; pH 7; 55℃.When the pollution index of enterwater was 268mg/L of CODCr ,100mg/l of lignin content and 186 times of chroma.The pollution duty of outwater decreased to CODCr 48mg/L, 100mg/l of lignin content and 10 times of the chroma.The removal rates of CODCr, lignin content and colority were 82.1%,84% and 94.6% respectively.The removal effect of lignin in papermaking wastewater by crude laccase was better than that of purified laccase .The reason was that some low molecular substance mediators could be remoaved during purifying process.
The molecular weight of the ligin which is from the outwater of the effluent seffling chamber taken from a pulp mill is 31200.The molecular weight of the ligin treated for one hour by laccase increased to 46600 while the molecular weight of the ligin treated for two hours by laccase reached 58600.The results showed that the molecular weight of the ligin increased obviously after treated by laccase.In suitable condition, the laccase produced by Coriolus versicolor could polymerize the ligin and its ramification in the wastewater of alkaline pulp mill.Then the CODcr and chroma of the wastewater would decrease.So the use of laccase could provides feasibility to reclaimed water reuse of the papermaking wastewater.
引文
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