Method for producing 9alpha-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion
US11001871B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Jan 24, 2019 |
| Grant date | May 11, 2021 |
| Priority date | — |
| Expiry date | Aug 4, 2039 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12Y114/13142
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
The present disclosure discloses a method for producing 9α-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion, and belongs to the fields of gene engineering and enzyme engineering. According to the present disclosure, oxidation subunit KshA, reduction subunit KshB and unknown active subunit KshC of 3-ketosteroid-9α-hydroxylase sourcing from Mycobacterium sp. Strain VKM Ac-1817D are successfully expressed in E. coli BL21, and KshC is identified as an oxidation subunit, the enzyme activity of which is far higher than that of KshA. BL21/pET-28a(+)-fdh constructed in the laboratory is used for expressing formate dehydrogenase (FDH), and by using crude enzyme liquid of KSH (KshB+KshC) and FDH engineering bacteria as a biocatalyst and a steroidal compound (AD) as a substrate, optimum reaction temperature is determined as 30° C. and optimum pH is determined as 7.0. In optimum conditions, AD is converted to produce a product 9-OH-AD, and within 20 hours, the output of 9-OH-AD is 4.7 g/L, and the molar conversion rate reaches 96.7%. According to the present disclosure, in production of 9-OH-AD, coupling of a 3-ketosteroid-9α-hydroxylase hydroxylation system and a coenzyme rec…
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.