木素过氧化物酶的生产、固定化及其在染料降解中应用
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摘要
本文研究了利用鼓泡塔间歇培养固定化黄孢原毛平革菌合成木素过氧化物酶,通过初步分离纯化后,采用两种不同的材料进行酶的固定化,研究了固定化条件、固定化酶性质、固定化酶对染料的降解脱色。
研究发现,使用鼓泡式反应器进行固定化黄孢原毛平革菌培养合成木素过氧化物酶,更有利于产酶。考察了通气量对鼓泡式反应器合成木素过氧化物酶的影响,通气量在1.2vvm对产酶比较有利,酶活达到了234U/L,与在相同的培养基和相同的固定化载体用量下的摇瓶培养产酶相比,酶活提高了近50%。
发酵液经过冷冻除杂多糖、超滤、硫酸铵沉淀、透析等步骤进行初步分离纯化,比酶活提高1.85倍,酶活得率52.7%,酶活达到了1167U/L。
筛选出固定化效果较好的XAD7HP大孔树脂,研究了其固定化条件。结果表明,吸附4h,吸附温度25℃,酶液pH 4.5,戊二醛浓度0.2%、戊二醛处理时间120min、获得的效果最好,固定化酶活力可达16U/g(载体)。研究了木素过氧化物酶和葡萄糖过氧化物酶双酶的共固定化条件,最佳酶比为1:5。考察了固定化酶的最适pH值、最适温度和操作的稳定性。表明固定化酶具有更广的使用范围和稳定性。
利用聚氨酯泡沫共价交联法制备固定化酶,固定化木素过氧酶活力达到12.6U/g;共固定化双酶活为7.84U/g。探讨了固定化木素过氧酶和共固定化双酶最适反应温度、最适反应pH和操作的稳定性。多次的重复使用表明,木素过氧化物酶和共固定化酶都有良好的稳定性。
研究了固定化木素过氧化物酶对染料的降解条件。以酸性红37为研究对象,大孔树脂固定化酶降解染料pH值为4.0,聚氨酯固定化酶pH值为4.5,其他条件相同,H_2O_2的浓度为200μmol/L,最适温度为40℃,染料25mg/L,脱色率80%以上。共固定化木素过氧化物酶和葡萄糖氧化酶对染料的脱色,染料中葡萄糖浓度只要5g/L即可达到最佳降解效果,脱色率在80%左右。这两种载体固定化木素过氧化物酶经过分批重复降解实验,都表现出了良好的可持续降解能力。
In this thesis, Firstly, we studied lignin peroxidase(Lip) production in the air-bubble bioreactor by the immobilized white-rot fungus Phanerochaete chrysosporium on polyurethane foam. Secondly, Lip was simply purified, and then immobilized on two different carriers. Finally, we studied the conditions of Lip immobilization and properties of immobilized Lip, and its application in decoloring dyes .
We found it is more suitable for Lip production in the air-bubble bioreactor than in the shake-flask cultures. The influence of air-flux on Lip production was researched. In the optimal air flux of 1.2wm, the maximum enzyme activity reached 234U/L in the bioreactor which was increased approximately 50% than that in the shake-flask cultures in the same medium and the amount of porous polyurethane foams carriers.
Lip was simply purified by freeze, ultrafiltration, salting out, dialysis. The activity of Lip reached 1167U/L after purification, and was 1.85 times than the original one. Recovery of Lip was 52.7%.
Lip was immobilized on three kinds of microporous absorbent resins. Among them, XAD7HP resin showed the best result for Lip immobilization. The optimal conditions for Lip immobilization were as follow : the time and temperature for the absorption,4h and 25℃; pH4.5 of the enzyme solution; glutaraldehyde concentration, 0.2%; the cross-linked time with glutaraldehyde, 120min; Under the optimal conditions, the activity of immobilized enzyme was 16 U/g carrier. The conditions of Lip immobilization coupling with glucose oxidase was studied; the enzyme activity ratio was 1:5; we also investigated the properties of co-immobilized enzymes. It was showed that the immobilized enzymes were more stable.
We also immobilized enzymes on polyurethane foam. The activity of immobilized Lip was 12.6U/L, and the co-immobilized Lip coupling with glucose oxidase reached 7.84U/L. The properties of them were researched. After immobilization, Lip was more stable, and the range of pH and temperature were expanded.
Using immobilized Lip to degrade dyes was investigated. Acid 37 was chosen as the substrate. The optimal degrading conditions were follows: pH was 4.5 for the Lip immobilized on the resin, but pH was 4.5 for the Lip immobilized on polyurethane foam, the other conditions were the same: H_2O_2 200μmol/L, temperature 40℃, the dye concentration 25mg/L. The percentage of decoloriztion was more than 80%.When the dye was degraded by the co-immobilized enzyme, 5.0g/L glucose can be suitable concentration, and the percentage of dye decolorization was approximately 80%. The immobilized Lip on two differet kinds of carrier can degrade the dye efficiently in many batchs.
研究发现,使用鼓泡式反应器进行固定化黄孢原毛平革菌培养合成木素过氧化物酶,更有利于产酶。考察了通气量对鼓泡式反应器合成木素过氧化物酶的影响,通气量在1.2vvm对产酶比较有利,酶活达到了234U/L,与在相同的培养基和相同的固定化载体用量下的摇瓶培养产酶相比,酶活提高了近50%。
发酵液经过冷冻除杂多糖、超滤、硫酸铵沉淀、透析等步骤进行初步分离纯化,比酶活提高1.85倍,酶活得率52.7%,酶活达到了1167U/L。
筛选出固定化效果较好的XAD7HP大孔树脂,研究了其固定化条件。结果表明,吸附4h,吸附温度25℃,酶液pH 4.5,戊二醛浓度0.2%、戊二醛处理时间120min、获得的效果最好,固定化酶活力可达16U/g(载体)。研究了木素过氧化物酶和葡萄糖过氧化物酶双酶的共固定化条件,最佳酶比为1:5。考察了固定化酶的最适pH值、最适温度和操作的稳定性。表明固定化酶具有更广的使用范围和稳定性。
利用聚氨酯泡沫共价交联法制备固定化酶,固定化木素过氧酶活力达到12.6U/g;共固定化双酶活为7.84U/g。探讨了固定化木素过氧酶和共固定化双酶最适反应温度、最适反应pH和操作的稳定性。多次的重复使用表明,木素过氧化物酶和共固定化酶都有良好的稳定性。
研究了固定化木素过氧化物酶对染料的降解条件。以酸性红37为研究对象,大孔树脂固定化酶降解染料pH值为4.0,聚氨酯固定化酶pH值为4.5,其他条件相同,H_2O_2的浓度为200μmol/L,最适温度为40℃,染料25mg/L,脱色率80%以上。共固定化木素过氧化物酶和葡萄糖氧化酶对染料的脱色,染料中葡萄糖浓度只要5g/L即可达到最佳降解效果,脱色率在80%左右。这两种载体固定化木素过氧化物酶经过分批重复降解实验,都表现出了良好的可持续降解能力。
In this thesis, Firstly, we studied lignin peroxidase(Lip) production in the air-bubble bioreactor by the immobilized white-rot fungus Phanerochaete chrysosporium on polyurethane foam. Secondly, Lip was simply purified, and then immobilized on two different carriers. Finally, we studied the conditions of Lip immobilization and properties of immobilized Lip, and its application in decoloring dyes .
We found it is more suitable for Lip production in the air-bubble bioreactor than in the shake-flask cultures. The influence of air-flux on Lip production was researched. In the optimal air flux of 1.2wm, the maximum enzyme activity reached 234U/L in the bioreactor which was increased approximately 50% than that in the shake-flask cultures in the same medium and the amount of porous polyurethane foams carriers.
Lip was simply purified by freeze, ultrafiltration, salting out, dialysis. The activity of Lip reached 1167U/L after purification, and was 1.85 times than the original one. Recovery of Lip was 52.7%.
Lip was immobilized on three kinds of microporous absorbent resins. Among them, XAD7HP resin showed the best result for Lip immobilization. The optimal conditions for Lip immobilization were as follow : the time and temperature for the absorption,4h and 25℃; pH4.5 of the enzyme solution; glutaraldehyde concentration, 0.2%; the cross-linked time with glutaraldehyde, 120min; Under the optimal conditions, the activity of immobilized enzyme was 16 U/g carrier. The conditions of Lip immobilization coupling with glucose oxidase was studied; the enzyme activity ratio was 1:5; we also investigated the properties of co-immobilized enzymes. It was showed that the immobilized enzymes were more stable.
We also immobilized enzymes on polyurethane foam. The activity of immobilized Lip was 12.6U/L, and the co-immobilized Lip coupling with glucose oxidase reached 7.84U/L. The properties of them were researched. After immobilization, Lip was more stable, and the range of pH and temperature were expanded.
Using immobilized Lip to degrade dyes was investigated. Acid 37 was chosen as the substrate. The optimal degrading conditions were follows: pH was 4.5 for the Lip immobilized on the resin, but pH was 4.5 for the Lip immobilized on polyurethane foam, the other conditions were the same: H_2O_2 200μmol/L, temperature 40℃, the dye concentration 25mg/L. The percentage of decoloriztion was more than 80%.When the dye was degraded by the co-immobilized enzyme, 5.0g/L glucose can be suitable concentration, and the percentage of dye decolorization was approximately 80%. The immobilized Lip on two differet kinds of carrier can degrade the dye efficiently in many batchs.
引文
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