SELECTION OF ORGANISMS

4. SELECTION OF ORGANISMS

The selection of strains either from its natural sources or from the various available cultures which are solely responsible for catalyzing the desired biotransformation reaction(s) is not only vital and critical but also of great importance. It has been observed that there are quite a few microorganisms that usually carry out the desired bioconversions with the help of a related chemical entity. In steroid one may encounter a rather difficult problem due to the lack of selective methods so as to identify the colonies precisely which usually perform the ear-marked specific activity.

Example : The ‘plate assay’ may be successfully employed to select such organisms which may aromatize several steroidal entities, for instance : 19-nor steroids; 19-substituted steroids; and sterols (e.g., β -sitosterol, ergosterol etc.) into equilin and related oestrogen.

p -N itro b e n z en e (A n o e s tro g e n )

d ia zo n iu m flu o b o ra te These ring A aromatic oestrogenic products I and II above usually react particularly with the

E q u ilin (I)

E q u ile n in (II)

(A re lated o e stro g e n )

reagent para-nitrobenzene diazonium fluoborate to give rise to the production of an intense red colouration. Therefore, the development of such colonies in a solid medium containing an appropriate

steroidal substrate, are duly replicated before the reagent is sprayed ; and thus, a red-ring gets developed all around the active colonies.

PHARMACEUTICAL BIOTECHNOLOGY

Modified Enrichment Method : The modified enrichment method is invariably used for the isolation of mutants blocked in the substrate dissimilation mechanism. In this specific instance, a steroid substrate is normally incorporated as the sole C-source exclusively in a ‘minimal medium’ seeded adequately with the soil dilutions. The cells that causes the degradation of the substrate will ultimately grow ; and are, therefore, subsequently transferred to the same medium but particularly enriched with another C-source, for instance : glucose. However, the mutants may be present which are strategically blocked at different stages in the process of degradation of the steroid substrate, but may consume glucose as the C-source.

Besides, the resulting intermediates may get accumulated, whereas the lesion-bearing mutants can be isolated conveniently. Furthermore, mutants may also be isolated which are incapable of accu- mulating an ‘undesirable compound’.

Example : An ‘undesirable reaction’ initiated by Aspergillus ochraceus which eventually blocked the ensuing transformation of 11 α αα αα -hydroxyprogesterone into 6 βββββ -11 αα αα α -dihydroxyprogesterone was

duly prepared that could only yield the derivative 11 α αα αα -hydroxy-progesterone.

∝ 11 -H y d ro x y p ro g es te ro n e β 6 , 11 -D ih y d ro x y p ro g es te rin e α It has been profusely established and reported that a fairly large number of microbial strains viz.,

eubacteria*, yeasts, molds, and streptomycetes may be stored and maintained strictly as per the recommended ‘standard methods’, such as : agar slant, soil culture, frozen culture, and lypholized culture preserved at temperatures ranging between – 20°C to – 170°C.

Filtration Enrichment Method : In this case, after mutagenesis the spores of filamentous

organisms e.g., actinomycetes, fungi, are made to develop in a liquid minimal medium. The ensuing microcolonies of prototrophs thus developed are meticulously separated by filtration, whereby the spores of auxotrophs** that were unable to grow left behind in the filtrate. The filtrate obtained in this manner in subsequently plated and the resulting colonies are adequately checked for auxotrophic char- acteristics .

Penicillin-Selection Procedure : In penicillin-selection procedure the prevailing growing cells are killed selectively by the ‘antibiotic’ treatment, thereby enriching the auxotrophs that are incapable of growing upon the ‘minimal medium’. Thus, exclusively based upon their mode of action a plethora of ‘inhibitors’ other than penicillin may also be employed effectively in this procedure, namely :

* Eubacterium : A genus of bacteria of the order Eubacteriales. ** An auxotrophic organism i.e., requiring a growth factor which is different from

MICROBIAL TRANSFORMATIONS

dihydrostreptomycin for Pseudomonas aeruginosa ; nystalin for Hansenula polymorpha, Penicillium chrysogenum, Aspergillus nidulans, and Saccharomyces cerevisiae ; nalidix acid for Salmonella

typhimurium ; colistin for the penicillin-resistant Hydrogenomonas strain H16. Sodium Pentachlorophenolate : The salt sodium pentachlorophenolate also affords enrich-

ment procedure by virtue of its greater toxicity particularly against the ‘germinating spores’ in com- parison to the ‘vegetative cells’.

Example : The above method has been successfully applied with several organisms, such as : Penicillium chrysogenum ; Streptomyces aureofaciens ; Streptomyces olivaceus ; and Bacillus subtilis.

It is, however, pertinent to state at this juncture that the applications of the aforesaid enrichment methods may cause an enhancement of ‘auxotrophs’ between 10 to 100 times, thus increasing consider- ably the probability of obtaining mutants. Importantly, one may observe that the variants of mutants present in the initial original population may get shifted to an appreciable extent, such as : an enhanced proportion of proline auxotrophs may be accomplished in E. coli after the due auxotroph enrichment.

Spraying with Reagents (or Incorporating Indicator Dyes) :

One may observe either the presence or absence of specific enzyme activities almost directly in the colonies that are allowed to grown on plates by employing either of the two available common procedures, namely : (a) spraying with appropriate reagents ; and (b) incorporating indicator-dyes right into the culture medium.

Inhibition of Assay Organisms : In this specific instance the antibiotically-active compounds may be detected quite easily and conveniently by measuring the inhibition of sensitive assay organ- isms. This procedure allows the precise determination (assay) of the ‘antibiotic content’ of an unknown solution using a reference standard simultaneously.

Agar Plug Method : The agar plug method is regarded to be one of the most reliable and precise techniques wherein the agar cylinders having ‘single-colonies’ are transferred to test plates after due incubation preferably in a moist chamber as depicted in Fig. 4.7 given below :

PHARMACEUTICAL BIOTECHNOLOGY

Spore suspension of kasugamycin-producing strain

Mutation

Plating (30-100 colonies/plate)

Incubation 29°C, 48 hr

Agar cylinder (6 mm diameter) is transfered to a sterile petridish

Incubation in a

moist chamber 29° C, 96-120 hr

Transfer of the cylinder to a test plate

Culture from cylinders with inhibition zones

Fig. 4.7. ‘Agar plug’ Method in Kasugamycin Strain Development (Ichikawa et al. 1971) [Adapted from : Crueger W and Crueger A, Biotechnology, 2004]

In fact, the actual observed diameter of the resulting ‘zones of inhibition’ invariably caters for a definite measure of the capability of ‘antibiotic production’ of each strain under investigation.

Suitability of Agar Plug Method : The method is fairly suitable for such processes where only

a differentiation between productivity and non-productivity is sufficient e.g., detecting the production of

specific constitutive enzymes.

MICROBIAL TRANSFORMATIONS

Drawbacks : This method has several drawbacks which may be summarized as given under : (1) There exists only a slight correlation between antibiotic formation in the ‘plate culture’ vis-

a-vis antibiotic production is submerged fermentation. (2) Strains that produce at high yields on being grown on plates may yield at only low yields or

even almost nil in the prevailing liquid culture. (3) When screening is adopted and initiated employing high-yielding strains, further enhance-

ments in yield invariably cannot be detected by the help of this procedure.