Espírito Santo G M, Pedro A Q, Oppolzer D, Bonifácio M J, Queiroz J A, Silva F, Passarinha L A
CICS - UBI - Centro de Investigação em Ciências da Saúde, Faculdade de Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal.
Bial, Departamento de Investigação e Desenvolvimento, São Mamede do Coronado, Porto, Portugal.
Biotechnol Rep (Amst). 2014 May 27;3:34-41. doi: 10.1016/j.btre.2014.05.005. eCollection 2014 Sep.
Catechol--methyltransferase (COMT, EC 2.1.1.6) plays a crucial role in dopamine metabolism which has intimately linked this enzyme to some neurodegenerative diseases, such as Parkinson's disease. In recent years, in the attempt of developing new therapeutic strategies for Parkinson's disease, there has been a growing interest in the search for effective COMT inhibitors. In order to do so, large amounts of COMT in an active form are needed, and the best way to achieve this is by up-scaling its production through biotechnological processes. In this work, a fed-batch process for the biosynthesis of the soluble isoform of COMT in is proposed. This final process was selected through the evaluation of the effect of different dissolved oxygen concentrations, carbon and nitrogen source concentrations and feeding profiles on enzymatic production and cell viability, while controlling various parameters (pH, temperature, starting time of the feeding and induction phases and carbon source concentration) during the process. After several batch and fed-batch experiments, a final specific COMT activity of 442.34 nmol/h/mg with approximately 80% of viable cells at the end of the fermentation were achieved. Overall, the results described herein provide a great improvement on hSCOMT production in recombinant bacteria and provide a new and viable option for the use of a fed-batch fermentation with a constant feeding profile to the large scale production of this enzyme.
儿茶酚 - 甲基转移酶（COMT，EC 2.1.1.6）在多巴胺代谢中起关键作用，这使得该酶与某些神经退行性疾病（如帕金森病）密切相关。近年来，在开发帕金森病新治疗策略的尝试中，寻找有效的COMT抑制剂的兴趣日益浓厚。为了做到这一点，需要大量活性形式的COMT，而实现这一目标的最佳方法是通过生物技术过程扩大其生产规模。在这项工作中，提出了一种在 中生物合成COMT可溶性同工型的补料分批工艺。通过评估不同溶解氧浓度、碳源和氮源浓度以及补料曲线对酶产量和细胞活力的影响，同时在过程中控制各种参数（pH、温度、补料和诱导阶段的起始时间以及碳源浓度），选择了这个最终工艺。经过多次分批和补料分批实验，在发酵结束时获得了442.34 nmol/h/mg的最终比COMT活性，约80%的活细胞。总体而言，本文所述结果极大地改进了重组细菌中hSCOMT的生产，并为使用具有恒定补料曲线的补料分批发酵大规模生产该酶提供了一种新的可行选择。
