Restaino Odile Francesca, Marseglia Mariacarmela, De Castro Cristina, Diana Paola, Forni Pasquale, Parrilli Michelangelo, De Rosa Mario, Schiraldi Chiara
Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Via De Crecchio 7, 80138, Naples, Italy,
Appl Microbiol Biotechnol. 2014 Feb;98(3):1291-9. doi: 10.1007/s00253-013-5384-x. Epub 2013 Dec 11.
Streptomyces roseochromogenes is able to hydroxylate steroid compounds in different positions of their cycloalkane rings thanks to a cytochrome P-450 multi-enzyme complex. In this paper, the hydroxylation of the hydrocortisone in the 16α position, performed by bacterial whole cells, was investigated in both shake flask and fermentation conditions; the best settings for both cellular growth and transformation reaction were studied by investigating the optimal medium composition, the kinetic of conversion, the most suitable substrate concentration and the preferred addition timing. Using newly formulated malt extract- and yeast extract-based media, a 16α-hydrohydrocortisone concentration of 0.2 ± 0.01 g L(-1) was reached in shake flasks. Batch experiments in a 2-L fermentor established the reproducibility and robustness of the biotransformation, while a pulsed batch fermentation strategy allowed the production to increase up to 0.508 ± 0.01 g L(-1). By-product formation was investigated, and two new derivates of the hydrocortisone obtained during the bacterial transformation reaction and unknown so far, a C-20 hydroxy derivate and a C-21 N-acetamide one, were determined by NMR analyses.
玫瑰产色链霉菌能够借助细胞色素P - 450多酶复合体，在甾体化合物环烷环的不同位置进行羟基化反应。本文研究了在摇瓶和发酵条件下，细菌全细胞对氢化可的松16α位的羟基化反应；通过研究最佳培养基组成、转化动力学、最适宜的底物浓度和优选的添加时间，探索了细胞生长和转化反应的最佳条件。使用新配制的基于麦芽提取物和酵母提取物的培养基，在摇瓶中16α - 氢化氢化可的松的浓度达到了0.2±0.01 g L⁻¹。在2 L发酵罐中进行的分批实验确定了生物转化的可重复性和稳健性，而脉冲分批发酵策略使产量提高到了0.508±0.01 g L⁻¹。研究了副产物的形成情况，通过核磁共振分析确定了在细菌转化反应过程中获得的两种氢化可的松新衍生物，目前尚不清楚其结构，一种是C - 20羟基衍生物，另一种是C - 21 N - 乙酰酰胺衍生物。
