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Cytochrome P450-mediated N-demethylation of noscapine by whole-cell biotransformation: process limitations and strategies for optimisation.
Journal of Industrial Microbiology & Biotechnology ( IF 3.4 ) Pub Date : 2020-06-07 , DOI: 10.1007/s10295-020-02283-7
Luke Richards 1, 2 , Ailsa Jarrold 3 , Tim Bowser 4 , Geoffrey W Stevens 1 , Sally L Gras 1, 2
Affiliation  

Cytochrome P450 enzymes catalyse reactions of significant industrial interest but are underutilised in large-scale bioprocesses due to enzyme stability, cofactor requirements and the poor aqueous solubility and microbial toxicity of typical substrates and products. In this work, we investigate the potential for preparative-scale N-demethylation of the opium poppy alkaloid noscapine by a P450BM3 (CYP102A1) mutant enzyme in a whole-cell biotransformation system. We identify and address several common limitations of whole-cell P450 biotransformations using this model N-demethylation process. Mass transfer into Escherichia coli cells was found to be a major limitation of biotransformation rate and an alternative Gram-positive expression host Bacillus megaterium provided a 25-fold improvement in specific initial rate. Two methods were investigated to address poor substrate solubility. First, a biphasic biotransformation system was developed by systematic selection of potentially biocompatible solvents and in silico solubility modelling using Hansen solubility parameters. The best-performing biphasic system gave a 2.3-fold improvement in final product titre compared to a single-phase system but had slower initial rates of biotransformation due to low substrate concentration in the aqueous phase. The second strategy aimed to improve aqueous substrate solubility using cyclodextrin and hydrophilic polymers. This approach provided a fivefold improvement in initial biotransformation rate and allowed a sixfold increase in final product concentration. Enzyme stability and cell viability were identified as the next parameters requiring optimisation to improve productivity. The approaches used are also applicable to the development of other pharmaceutical P450-mediated biotransformations.



中文翻译:

细胞色素P450介导的全细胞生物转化Noscapine的N-去甲基化:工艺限制和优化策略。

细胞色素P450酶催化具有重大工业意义的反应,但由于酶的稳定性,辅因子要求以及典型底物和产品的不良水溶性和微生物毒性,在大规模生物过程中未得到充分利用。在这项工作中,我们调查了全细胞生物转化系统中P450 BM3(CYP102A1)突变酶对罂粟生物碱Noscapine的制备规模N-脱甲基化的潜力。我们使用此模型N-去甲基化过程识别并解决了全细胞P450生物转化的几个常见限制。大量转移到大肠杆菌中发现细胞是生物转化率的主要限制和另一种革兰氏阳性表达宿主巨大芽孢杆菌比初始速率提高了25倍。研究了两种方法来解决较差的底物溶解度。首先,通过系统地选择潜在的生物相容性溶剂和使用Hansen溶解度参数进行计算机溶解度建模,开发了一种双相生物转化系统。与单相系统相比,性能最佳的双相系统最终产物滴定度提高了2.3倍,但由于水相中底物浓度低,生物转化的初始转化速率较慢。第二种策略旨在使用环糊精和亲水性聚合物提高底物在水中的溶解度。该方法使初始生物转化率提高了五倍,并使最终产物浓度提高了六倍。酶稳定性和细胞活力被确定为需要优化以提高生产率的下一个参数。所使用的方法也可用于开发其他药物P450介导的生物转化。

更新日期:2020-06-07
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