Biotechnological approach to the synthesis of 9alpha-hydroxylated steroids.
The steroid 9alpha-hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9alpha-hydroxyprogesterone. In 7 days, 43.6 mg 9alpha-hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4-androstene-3,17-dione in vivo. An extensive analytical method development, including LLE, HPLC-DAD, MS, and NMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion rate were unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.
Arnell
Robert
R
Department of Physical and Analytical Chemistry, Uppsala University, Uppsala, Sweden. robert.arnell@gmail.com
Johannisson
Rickard
R
Lindholm
Johan
J
Fornstedt
Torgny
T
Ersson
Bo
B
Ballagi
Andras
A
Caldwell
Karin
K
D000595:Amino Acid Sequence; D000735:Androstenedione / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D019149:Bioreactors / Q000382:microbiology; D001709:Biotechnology; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006639:Histidine / Q000737:chemistry; D006900:Hydroxylation; D006908:Hydroxyprogesterones / Q000378:metabolism*; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics; D009284:Naphthols / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011374:Progesterone / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012015:Reference Standards; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014169:Transformation, Bacterial
D006908:Hydroxyprogesterones; D009284:Naphthols; D011994:Recombinant Proteins; D000735:Androstenedione; D011374:Progesterone; D006639:Histidine; D006899:Mixed Function Oxygenases