Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase

Details Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase

2007-07-03

2007-07-03

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S069700

Reexamination Certificate

active

10900856

ABSTRACT:
The present invention relates to a novel cytochrome P450-like enzyme (Aspergillus ochraceus11 alpha hydroxylase) and an oxidoreductase (Aspergillus ochraceusoxidoreductase) isolated from cDNA library generated from the mRNA ofAspergillus ochraceusspores. When the cDNA encoding the 11 alpha hydroxylase was co-expressed inSpodoptera frugiperda(Sf-9) insect cells with the cDNA encoding human oxidoreductase as an electron donor, it successfully catalyzed the conversion of the steroid substrate 4-androstene-3,17-dione (AD) to 11 alpha-hydroxy-AD as determined by HPLC analysis. The invention also relates to nucleic acid molecules associated with or derived from these cDNAs including complements, homologues and fragments thereof, and methods of using these nucleic acid molecules, to generate, for example, polypeptides and fragments thereof. The invention also relates to the generation of antibodies that recognizes theA. ochraceus11 alpha hydroxylase and oxidoreductase and methods of using these antibodies to detect the presence of these native and recombinant polypeptides within unmodified and transformed host cells, respectively. The invention also provides methods of expressing theAspergillus11 alpha hydroxylase gene separately, or in combination with human orAspergillusoxidoreductase, in heterologous host cells, to facilitate the bioconversion of steroid substrates to their 11 alpha hydroxy-counterparts.

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