9α-Hydroxy-4-androstene-3,17-dione (9-OH-AD) is one of the significant intermediates for the preparation of β-methasone, dexamethasone, and other steroids. In general, the key enzyme that enables the biotransformation of 4-androstene-3,17-dione (AD) to 9-OH-AD is 3-phytosterone-9α-hydroxylase (KSH), which consists of two components: a terminal oxygenase (KshA) and ferredoxin reductase (KshB). The reaction is carried out with the concomitant oxidation of NADH to NAD+. In this study, the more efficient 3-phytosterone-9α-hydroxylase oxygenase (KshC) from the Mycobacterium sp. strain VKM Ac-1817D was confirmed and compared with reported KshA. To evaluate the function of KshC on the bioconversion of AD to 9-OH-AD, the characterization of KshC and the compounded system of KshB, KshC, and NADH was constructed. The optimum ratio of KSH oxygenase to reductase content was 1.5:1. An NADH regeneration system was designed by introducing a formate dehydrogenase, further confirming that a more economical process for biological transformation from AD to 9-OH-AD was established. A total of 7.78 g of 9-OH-AD per liter was achieved through a fed-batch process with a 92.11% conversion rate (mol/mol). This enzyme-mediated hydroxylation method provides an environmentally friendly and economical strategy for the production of 9-OH-AD.

中文翻译：

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一种新型 3-Phytosterone-9α-Hydroxylase 氧化成分及其在 4-Androstene-3,17-Dione 生物转化为 9α-Hydroxy-4-Androstene-3,17-Dione 与 NADH 再生甲酸脱氢酶偶联中的应用

9α-Hydroxy-4-androstene-3,17-dione (9-OH-AD) 是制备β-米松、地塞米松等类固醇的重要中间体之一。一般来说，使 4-androstene-3,17-dione (AD) 生物转化为 9-OH-AD 的关键酶是 3-phytosterone-9α-hydroxylase (KSH)，它由两个成分组成：末端加氧酶(KshA) 和铁氧还蛋白还原酶 (KshB)。该反应伴随着 NADH 氧化成 NAD+ 进行。在这项研究中，来自分枝杆菌的更有效的 3-植物甾酮-9α-羟化酶加氧酶 (KshC)。菌株 VKM Ac-1817D 得到确认并与报道的 KshA 进行比较。为了评估 KshC 对 AD 生物转化为 9-OH-AD 的作用，构建了 KshC 的表征以及 KshB、KshC 和 NADH 的复合体系。KSH加氧酶与还原酶含量的最佳比例为1.5:1。通过引入甲酸脱氢酶设计了 NADH 再生系统，进一步证实了从 AD 到 9-OH-AD 的生物转化更经济的过程。通过以92.11%的转化率(mol/mol)的补料分批工艺获得每升总共7.78g 9-OH-AD。这种酶介导的羟基化方法为 9-OH-AD 的生产提供了一种环保且经济的策略。