[4] Separation of steroids by means of ion-exchange resins

Arihrobacter oxydans 317 which is able to bring about the complete degradation of 8-sitosterol was treated with acridine orange to obtain a plasmid cured strain Arthrobacter 31 7AL, incapable of producing steroid ring degradation. The strain was neither able to form 4-androstene-3,l 7-dione(AD) from 1-sitosterol nor to convert AD to 1,4-androstadiene-3,17-dione(ADD). It was, however, able to hydroxylate AD and ADD at the C-17 position to form testosterone and 1 ,Candrostadiene-l7~-0l-3-one respectively. The strain could grow on psitosterol presumably utilizing the degradation products of the sterol side chain. Partial degradation of the p-sitosterol side chain was demonstrated by the isolation of 27-norcholest-4-en-3,24-dione, a metabolic product of the side chain degradation. The existence of metabolic blocks in a further degradation of the side chain to AD, in 1,2-dehydrogenation and 9a-hydroxylation of the steroid ring structure was postulated.

“…Analytical methods: A quantitative estimation of 17-ketosteroids was made by a ZIMMERMAN reaction (SEKI 1969) and of B-sitosterol by LIBERMAN-BUCHARD reaction (KATAYAMA et a/. 1974).…”

Section: Methodsmentioning

confidence: 99%

Steroid transformations by a strain of Arthrobacter oxydans incapable of steroid ring degradation

Sarmah

Roy

Singh

1989

Metabolic blocks in the degradation of β‐sitosterol by a plasmid‐cured strain of Arthrobacter oxydans

1992

Plasmid-harbouring, sterol-decomposing organism Arthrobacter oxydans 317 was treated with sodium dodecylsulphate to obtain a plasmid-cured strain A. oxydans 317 A1 incapable of utilizing 4-androstene-3,17-dione (AD). The strain 317 A1 was unable to degrade beta-sitosterol side chain completely to form AD but could carry out partial degradation as shown by the accumulation of 3-oxochol-4-en-24-oic acid as a major metabolite and 27-norcholest-4-en-3,24-dione as a minor metabolite. The strain could form 1,4-androstadiene-3,17-dione (ADD) from 3-oxo-23,24-bisnorchol-1,4-dien-22- oic acid (BNC) to a limited extent. The existence of metabolic blocks in the conversion of 3-oxochol-4-en-24-oic acid to 3-oxo-23,24-bis-norchol-4-en-22-oic acid and further conversion to AD by the plasmid-cured strain 317 A1 was suggested. Neither the formation of ADD from AD nor the conversion of AD and ADD to 9 alpha-hydroxy derivatives leading to steroid ring opening could be done by the plasmid-cured strain but the 17 beta-reduction of AD and ADD and 1(2)-reduction of ADD were not affected by the absence of the plasmid. It was proposed that plasmid determines 1(2)-dehydrogenation and 9 alpha-hydroxylation of steroid ring structure in this organism.

Role of plasmid pJL1 of Arthrobacter oxydans 317 in the degradation of β‐sitosterol

Dutta

Roy

Singh

1992

Arthrobacter oxydans 3 17 is a plasmid (pJL1)-harbouring organism capable of complete degradation of p-sitosterol, 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). Plasmid-cured strain A . o.uvdaizs 317 A1 obtained by treatment of A. oxydans 317 with sodium dodecylsulphate (DUTTA et al. 1989) is capable of only partial degradation of 0-sitosterol side chain and incapable of utilizing AD and ADD. Conjugation experiments were carried out using A . oxyduns 317 Al as the recipient and A. o.xjukms 317 as the donor of pJLl plasmid. A transconjugant strain A . oxydans 317 TC containing pJLl plasmid was obtained. The transconjugant was able to form 9 a-hydroxy-4-androstene-3,17-dione (9a-hydroxy AD) from p-sitosterol and AD, and to utilize ADD for growth but it could not convert AD to ADD. It was proposed that pJLl is involved in the conversion of 3-oxochol-4-en-24-oic acid to AD and in the 9a-hydroxylation of the steroid nucleus but it is not connected with 1,2-dehydrogenation, a critical reaction step in steroid ring opening.Bacterial strains lacking either 9a-hydroxylase or 1,2-dehydrogenase or both are considered important in sterol biotransformation for production of AD and ADD provided that the strains are capable of complete elimination of C-17 alkyl sidechain of the sterol (MARTIN 1977). Though the enzymology of each step of sterol sidechain cleavage is not known, accumulation of intermediary sterol metabolites with intact steroid nucleus and partially cleaved sterol side chain have been reported (KNIGHT et al. 1980, HILL et al. 1982, JEKKEL et al. 1989. Plasmid mediated catabolic degradation of many organic compounds are known t'oday. However, there is an acute lack of information regarding the role of plasmids in sterol degradation, TENNESON et al. (1979) suggested the possible role of plasmids in complete degradation of the steroid ring structure.Arthrobucter oxyduns 3 17 is a plasmid harbouring organism (SARMAH e t ul. 1989) capable of complete degradation of P-sitosterol (GOSWAMI et al. 1982P-sitosterol (GOSWAMI et al. , 1983P-sitosterol (GOSWAMI et al. , 1984. Arthrobacter 317 AL, a plasmid-cured strain obtained from A . oxydans 317 by treatment with acridine orange was shown to be incapable of steroid ring degradation and complete cleavage of p-sitosterol side chain to form AD (SARMAH et al. 1989). In a previous communication (DUTTA et 01. 1992), the authors reported the work on the degradation of P-sitosterol by another plasmid-cured strain A . oxydans 317 Al and demonstrated the existence of metabolic blocks in the conversion of 3-oxochol-4-en-24-oic acid to AD and in 12-dehydrogenation and 9a-hydroxylation of the steroid ring structure. In this communication, the transfer of plasmid pJLl from A . oxyduns 3 17 to A . oxydans 3 17 Al through conjugation and the resulting changes in the metabolic pattern of p-sitosterol degradation in the transconjugant are reported providing clear evidence for the involvement of plasmid in sterol transformation. Materials and methodsOrganisms: 4. ...