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重组大肠杆菌发酵产漆酶及其催化应用
NADIA ABDRABOU ABDELKHALIK SAMAK
Thesis Advisor刘春朝
2017-01
Degree Grantor中国科学院研究生院
Degree Name博士
Degree Discipline生物化工
KeywordCota漆酶 , 微需氧培养,氧化石墨烯,固定化
Abstract

漆酶是多铜氧化酶家族的重要成员。作为环保型生物催化剂,漆酶能够降解多种工业有毒污染物。近年来,细菌漆酶已经引起研究者的广泛关注,因为细菌漆酶在高pH和高温条件下具有良好的催化特性。但是,细菌漆酶(尤其是CotA漆酶)异源表达所面临的主要问题是产率低、成本高。因此,开发成本低廉、能够提高酶产量的高效技术是工业应用的必要条件。含有或不含TTA密码子的CotA漆酶基因连接到pET30a和PT7-FLAG-MAT-TAG-1的表达载体上,重组载体在大肠杆菌中进行表达。在所有转化子中,不含TTA密码子的pT7-FLAG-MAT-TAG-1-CotA载体表达的漆酶活性最高,发酵水平达到1474 U/L,并通过SDS-PAGE确定了漆酶基因的表达。为了提高CotA漆酶表达水平,提出了一种微需氧培养策略。在微需氧培养条件下,含有pT7-FLAG-MAT-TAG-1-CotA表达载体的大肠杆菌BL21(DE3)可以高产CotA漆酶,发酵水平产量达到13903 U/L。微需氧培养策略可以显著提高胞内活性氧(ROS)水平,从而导致CotA漆酶基因过表达。在pH 6和9条件下,生产的CotA漆酶对孔雀石绿、结晶紫、刚果红和溴酚蓝等染料表现出高效的脱色性能。合成了一种带有金属亲和配体的磁性石墨烯氧化物(MGO)载体。在MGO表面修饰Nα,Nα- 双(羧甲基)-赖氨酸水合物(NTA-NH2)并螯合Cu2+,通过金属亲和选择性吸附CotA漆酶。Cu2+螯合的MGO(MGO-NTA-Cu2+)具有较高的CotA漆酶吸附容量,达到177 mg/g-suppor。CotA漆酶的最大酶活回收率为114%。与游离漆酶比较,MGO-NTA-Cu-CotA漆酶的催化性能得到了明显改善。在60°C、pH 为8、反应时间为5 h的条件下,MGO-NTA-Cu-CotA漆酶对刚果红的脱色效率为100%。此外,MGO-NTA-Cu-CotA漆酶重复使用10次后仍保留了89.4%的活性。结果表明MGO-NTA-Cu2+复合纳米材料是高效固定化漆酶的理想材料。总之,本研究通过使用强启动子、高效表达载体和微需氧培养策略为CotA漆酶的生产提供了一个新策略。同时,纳米技术可以显著提高CotA漆酶的活性并扩大其应用。;Laccases are important members of the multi-copper oxidases family. As environmentally friendly biocatalysts, laccases can degrade a broad range of industrial toxic contaminants. Recently, bacterial laccases have drawn researchers’ interest due to their catalytic characteristics under high pH and temperature conditions. The major challenges for heterologous expression of bacterial laccases, especially CotA laccase, are the high cost and low production yield. Therefore, development of efficient technologies which are cost effective is necessary for enhancing CotA laccase production for the industrial applications. CotA laccase gene with/without TTA codon was ligated in pET30a and PT7-FLAG-MAT-TAG-1 expression vectors. The recombinant vectors were transformed into Escherichia coli. Among all transformed cells, the recombinant E. coli cells carrying pT7-FLAG-MAT-TAG-1-CotA without TTA codon insertion showed the highest volumetric activity of 1474 U/L. SDS-PAGE results confirmed efficient CotA laccase expression in the recombinant cells.An efficient micro-aerobic cultivation strategy for enhancing CotA laccase expression was developed. Micro-aerobic cultivation of E. coli BL21 (DE3) with pT7-FLAG-MAT-TAG-1-CotA significantly enhanced CotA laccase production up to 13903 U/L. The micro-aerobic cultivation strategy enhanced the intracellular reactive oxygen species (ROS) level that consequently led to the over-expression of CotA laccase gene. The resulted CotA laccase showed efficient decolorization performance on malachite green, crystal violet, congo red and bromophenol blue at pH 6 and 9. Functionalized magnetic graphene oxide (MGO) support attached with Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA-NH2) and chelated with Cu2+ was synthesized. The Cu2+-chelated MGO (MGO-NTA-Cu2+) exhibited the highest adsorption capacity of 177 mg/g-support among all synthesized nano-composites. The maximum activity recovery of laccase using MGO-NTA-Cu2+ was 114%. The catalytic properties of MGO-NTA-Cu-CotA laccase were significantly improved in comparison with those of free laccase. MGO-NTA-Cu-CotA laccase showed efficient decolorization rate for Congo red (CR) reached 100 % after 5 h reaction at 60 °C and pH 8. MGO-NTA-Cu-CotA laccase retained 89.4 % of its initial activity after 10 consecutive cycles. These results prove that MGO-NTA-Cu2+ nano-composite is a promising support for efficient CotA laccase immobilization. In conclusion, the above results provide efficient strategies to enhance CotA laccase expression using strong promoter, efficient expression vector, and novel micro-aerobic cultivation method. Furthermore, nanotechnology can significantly enhance CotA laccase activity for expanding its application in practice. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/26828
Collection中国科学院过程工程研究所
研究所(批量导入)
Recommended Citation
GB/T 7714
NADIA ABDRABOU ABDELKHALIK SAMAK. 重组大肠杆菌发酵产漆酶及其催化应用[D]. 中国科学院研究生院,2017.
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