Steroid Bioconversion via Fermentative Procedures

5.2. Steroid Bioconversion via Fermentative Procedures

Steroids i.e., the highly biologically active compounds are usually produced by a host of vital organs in the human body, namely : testies, ovaries, placenta and the adrenal cortex. The nature of the steroids are exclusively dependent upon the various functional moieties attached on the steroid nucleus

MICROBIAL TRANSFORMATIONS

cal activities e.g., antiinflammatory agents, antifertility agents, contraceptives, oestrogenic agents, ste- rility profile and the like.

Microorganisms invariably do play a vital and pivotal role in the steroid bioconversions as enumerated below in a sequential order :

(a) Corynebacterium simplex aids in the dehydrogenation of cortisone to yield prednisone (b) Rhizopus arrhizus (a fungal sp.) helps in the hydroxylation of progesterone therapy giving

rise to the corresponding steroid 11 α αα αα -hydroxyprogesterone by introducing an O-atom at C-11 position.

(c) Rhizopus nigricans (a fungal sp.) is also able to hydroxylate progesterone to give rise to the

formation of 11 αα αα α -hydroxyprogesterone.

(d) Corynebacterium simplex may cause the effective dehydrogenation of either cortisol or

hydrocortisone to yield prednisolone.

(e) Nocardia restrictus affords the biotransformation of δδδδδ 4 –cholestene-19-hydroxy-3-one into the desired oestrone.

(f) Cunninghamella blakesleeana (a fungus sp.) aids the hydroxylation of cortexolone to form 11-hydroxy cortisone by the introduction of an O-atom at C-11 position strategically.

(g) Yeast strains also cause conversion of androstendione into the corresponding male-sex- hormone testosterone.

Interestingly, in the typical steroid bioconversion via fermentative procedures the desired micro- organism, Rhizopus nigricans, is allowed to grow in a fermentation tank charged with an appropriate growth culture medium along with optimized incubation parameters to accomplish biomass upto the maximum possible extent. It is, however, pertinent to mention here that the ensuing fermentation proce- dures essentially require both aeration and agitation to accomplish rapid growth. Hence, it is always preferred to incorporate steroid (e.g., progesterone) right into the bioreactor loaded with Rhizopus nigricans. The end-product is subsequently recovered with methylene chloride, adequately purified by chromatography, and ultimately obtained in its purest form by recrystallization.

Generally, the bioconversion of steroids is invariably accomplished by employing exclusively the submerged aeration technique carried out in SS-tanks (fermentors) charged with minimal nutri- tional quantities so as to permit the maximum ease and conversion with regard to the extraction and purification of the ultimate desired transformation substance.

In usual practice, the fermentative procedure is adopted efficiently by either of the two following phases, namely :

Phase-I : It represents predominantly the ‘growth phase’ whereby the maximum extent of the culture-growth is accomplished. And for the culture-growth one may require both

optimized temperature and aeration parameters. However, the duration of incubation solely depends upon the type of culture being employed, such as : bacteria : 12-24 hrs ; fungi : 24-72 hours. Besides, one must adhere to strict environmental conditions as per the laid-out norms.

Phase-II : When the ‘growth-phase’ almost comes to an end, the careful addition of the respectively steroid commences. In certain instances, one may carry out the addition of steroid very much alongwith the inoculation procedure. Importantly, the quantum of steroid need to be added solely depends upon three vital components, namely :

PHARMACEUTICAL BIOTECHNOLOGY

(a) transforming capacity of culture ; (b) potential toxicity of substrate ; and (c) nature of product. It has been duly observed that deoxycorticosterone possesses an appreciable antifungal activity. It is absolutely essential to contain the ‘problem of toxicity’ in the culture medium by subjecting to either continuous or periodic incorporation of substrate into the fermentation vessel. The organic solvents that are utilized profusely may include : acetone, ethanol, and methanol, which essentially contribute two main

advantageous factors, such as : (a) afford relatively low toxicity for the conversion enzymes ; and (b) cause appreciable solubility of steroids. Importantly, dimethylformamide (DMF) does possess an exceptional solubility characteristic feature for steroids, whereas its non-toxic level stands at below 2% concentration in the culture medium.

It is, however, pertinent to state here that the ‘bioconversion capacity’ actually based upon the load of the prevailing substrate, and the ability of the microorganism.