Calcifediol (25(OH)VD3) is a physiologically very important vitamin D₃ metabolite and of high pharmaceutical importance, due to its potential for treating not only vitamin D₃ 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 D₃ into its metabolite 25-hydroxyvitamin D_3. In this study, we demonstrate the importance of the region between amino acids T103 and A106 for the catalytic activity of CYP109A2 towards vitamin D₃ as a substrate. In order to increase the productivity of the system, reaction conditions (xylose, vitamin D₃, saponin, 2-hydroxypropyl-β-cyclodextrin) were optimized for the in vivo production of 25-hydroxyvitamin D₃.

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.

Calcifediol (25(OH)VD3) 是一种生理学上非常重要的维生素 D ₃代谢物，具有很高的药学重要性，因为它不仅可以治疗维生素 D ₃缺乏症，还可以治疗冠心病和癌症。

以前，我们建立了一个基于全细胞巨大芽孢杆菌的系统，使用细胞色素 P450 CYP109A2 将维生素 D ₃生物转化为其代谢物 25-羟基维生素 D _3。在这项研究中，我们证明了氨基酸 T103 和氨基酸之间区域的重要性A106 用于 CYP109A2 对作为底物的维生素 D ₃的催化活性。为了提高系统的生产率，对反应条件（木糖、维生素 D ₃、皂苷、2-羟丙基-β-环糊精）进行了优化，以在体内生产 25-羟基维生素 D ₃。

对于产生 T103A 突变体的细胞，在优化条件下实现了 282.7 mg/L/48 h 的生产力。该值比在本研究中与野生型酶的对照反应中获得的值高两倍，比先前研究中获得的值高 5 倍。