Institute of Biochemistry, Saarland University, Campus B2.2, D-66123, Saarbruecken, Germany.
IFB Halle GmbH, Schiepziger Str. 35, 06120, Halle-Lettin, Germany.
J Biotechnol. 2021 Jan 10;325:355-359. doi: 10.1016/j.jbiotec.2020.09.027. Epub 2020 Oct 22.
Calcifediol (25(OH)VD3) is a physiologically very important vitamin D metabolite and of high pharmaceutical importance, due to its potential for treating not only vitamin D deficiencies but also coronary diseases and cancer. Previously, we established a whole-cell Bacillus megaterium-based system using the cytochrome P450 CYP109A2 for the biotransformation of vitamin D into its metabolite 25-hydroxyvitamin D In this study, we demonstrate the importance of the region between amino acids T103 and A106 for the catalytic activity of CYP109A2 towards vitamin D as a substrate. In order to increase the productivity of the system, reaction conditions (xylose, vitamin D, saponin, 2-hydroxypropyl-β-cyclodextrin) were optimized for the in vivo production of 25-hydroxyvitamin D. With cells producing the T103A mutant, a productivity of 282.7 mg/L/48 h was achieved under the optimized conditions. This value is two times higher than that obtained in the control reaction with the wild-type enzyme in this study and five times higher than that obtained in a previous study.
钙三醇（25（OH）VD3）是一种具有重要生理意义的维生素 D 代谢物，具有很高的药物学重要性，因为它不仅有治疗维生素 D 缺乏症的潜力，而且还有治疗冠心病和癌症的潜力。此前，我们利用枯草芽孢杆菌细胞内的细胞色素 P450 CYP109A2 建立了一个全细胞转化体系，将维生素 D 转化为其代谢产物 25-羟维生素 D。在本研究中，我们证明了氨基酸 T103 和 A106 之间的区域对于 CYP109A2 作为维生素 D 底物的催化活性的重要性。为了提高该系统的生产力，我们对反应条件（木糖、维生素 D、皂素、2-羟丙基-β-环糊精）进行了优化，以在体内生产 25-羟维生素 D。使用生产 T103A 突变体的细胞，在优化条件下，25-羟维生素 D 的产率达到了 282.7 mg/L/48 h。这一数值是本研究中野生型酶对照反应的两倍，是之前研究的五倍。
