Strains of Comamonas acidovoras have been reported to have vital role in degradation of natural as well as complex organic compounds. Comamonas acidovoras MTCC 3364 has been routinely reported for steroid bioconversion by activated beads in aqueous system. Previously studies observed that progesterone was converted into AD and ADD steroids. These compounds were higher valuable steroids which were mainly used in different types of drugs. Novel system was used for the bioconversion of progesterone in to AD and ADD. Aqueous two-phase system was initially optimized for the progesterone solubility, separation of two phases and extraction of product steroids from the system. Even 1% of PEG can easily dissolved progesterone but 5% of PEG needed for separating phases and for extraction purposes. Beads was easily tolerate up to 20% of PEG system even for 120 hours. Hexane used as better extraction solvent among different type of solvent system. Products were shown in PEG 6000 system after 96 hours in fewer amounts. PEG 8000 system showed products 48 hours in minor amount but increased in 96 hours. Advantage of aqueous twophase system in progesterone bioconversion was better stability of progesterone, extraction of steroid products and stability of the system.

1. International Journal of Biotechnology. Photon 111 (2013) 211-214
https://sites.google.com/site/photonfoundationorganization/home/international-journal-of-biotechnology
Original Research Article. ISJN: 3352-7304
International Journal of Biotechnology
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Bioconversion of Progesterone Using Aqueous Two Phase System by
Comamonas acidovorans MTCC 3364
Darshan M. Rudakiya, Kirti Pawar*
Ashok & Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences, New Vallabh
Vidya Nagar, Anand, Gujarat, 388121 India
Article history:
Received: 16 May, 2013
Accepted: 26 May, 2013
Available online: 09 June, 2013
Keywords:
Comamonas acidovorans MTCC 3364, Progesterone,
Aqueous Two Phase System, HP-TLC
Abbreviation:
MTCC: Microbial Type Culture Collection, PEG:
Polyethylene glycol, AD: androst-4-ene-3, 17-dione, ADD:
androsta-1, 4-diene-3, 17- dione
Corresponding Author:
Pawar K.*
Associate Professor
Email: drkirtipawar@rediffmail.com
Fax No: +912692229189
Rudakiya D.M.
Email: darshan1420@gmail.com
Phone No: +919714370084
Fax No: +912692229189
Abstract
Strains of Comamonas acidovoras have been
reported to have vital role in degradation of natural
as well as complex organic compounds.
Comamonas acidovoras MTCC 3364 has been
routinely reported for steroid bioconversion by
activated beads in aqueous system. Previously
studies observed that progesterone was converted
into AD and ADD steroids. These compounds were
higher valuable steroids which were mainly used in
different types of drugs. Novel system was used for
the bioconversion of progesterone in to AD and
ADD. Aqueous two-phase system was initially
optimized for the progesterone solubility, separation
of two phases and extraction of product steroids
from the system. Even 1% of PEG can easily
dissolved progesterone but 5% of PEG needed for
separating phases and for extraction purposes.
Beads was easily tolerate up to 20% of PEG system
even for 120 hours. Hexane used as better
extraction solvent among different type of solvent
system. Products were shown in PEG 6000 system
after 96 hours in fewer amounts. PEG 8000 system
showed products 48 hours in minor amount but
increased in 96 hours. Advantage of aqueous twophase system in progesterone bioconversion was
better stability of progesterone, extraction of steroid
products and stability of the system.
Citation:
Rudakiya D.M., Pawar K., 2013. Bioconversion of
Progesterone Using Aqueous Two Phase System by
Comamonas acidovorans MTCC 3364. International
Journal of Biotechnology. Photon 111, 211-214.
1. Introduction
Strains of Comamonas acidovoras have been
reported to have vital role in degradation of
natural as well as complex organic
compounds. Comamonas acidovoras MTCC
3364 has been routinely reported for steroid
bioconversion. Progesterone also known as
P4 (pregn-4-ene-3, 20-dione) is a C-21 steroid
hormone involved in the female menstrual
cycle, pregnancy and embryogenesis of
human and other
species. Chemical
modification of the steroid ring structure
causes many problems due to steric hindrance
and the complexity of the molecule.
Conversion of progesterone to AD and ADD
has higher value steroidal compounds. An
alternative approach for chemical modification
of pharmaceutical active steroids has been to
use microbial system (Ahmad et al, 1992).
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Aqueous biphasic systems occur when certain
solutes cause an aqueous solution to separate
into two aqueous phases. Both phases contain
mainly water (typically 70-90% w/w water) and
are enriched in one of the polymers. PEG
6000 and 8000 PEG is soluble in water and
mostly used for steroid bioconversion. Selvaraj
Raja et al (2011) observed that aqueous two
phase system is a liquid – liquid extraction
method which employs two aqueous phases
having
applications
in
the
field
of
biotechnology for the separation and
purification of biological materials such as
proteins, enzymes, steroids and nucleic acid.
The emphasis is used in industry to PEG/salt
two-phase systems because of the low-cost of
the system. Gharaei Fathabadand and Chabra
Aroona (2011)
gave
first report
of
211

2. progesterone to AD and ADD by a natural
isolate from Penicillium aurantiogriseum. Lalita
Wadhwa, Kelvin E. Smith (2000) showed that
the side-chain of progesterone was cleaved by
Bacillus sphaericus to produce two C-19
ketoandrostene steroids. The structures of
these metabolites were androstenedione and
1-dehydroandrostenedione.
the phase. Activated beads were kept for 96
hours at 37° C in 120 rpm rotary shaker at
different concentration of PEG 6000, PEG
8000 and PEG 400 [0.5-30% (w/v)] to check
the activity of
beads under
higher
concentration of PEG.
Various organic
chemicals (ethyl acetate, hexane, benzene
and toluene) were checked for extraction
system and separation system.
2. Materials and Methods
2.1 Materials
The general chemicals, media components
required for the study were purchased from Hi
media, Merck, Glaxo and Qualigens. The
progesterone was purchased from SIGMAAldrich.
2.2 Microorganism and Culture Conditions
Strain of Comamonas acidovorans was
purchased from MTCC, Institute of Microbial
Technology, Chandigarh, India. The culture
was maintained on nutrient agar slants and
sub-cultured every month. For Immobilization:
Medium containing Nutrient broth (13 gml-1)
was autoclaved and inoculated with 100 µl of
culture of Comamonas acidovorans MTCC
3364. As an inducer, progesterone (prepared
in ethyl acetate) was added in the flask. Flask
was incubated at 37° C with 120 rpm shaking
overnight, After 24 hour growth; cells were
harvested by centrifugation at 10,000 rpm for 5
min at 4° C. Cell pellet was washed with sterile
tris-buffer (0.01 M) of pH 7.0 (±0.02).
2.3 Immobilization and activation of cells in
Alginate gel
Cells were mixed in slurry of 5.5% sodium
alginate in tris-buffer; the slurry was drop wise
added in a beaker containing ice-cold solution
of calcium chloride for gelling. The beads were
allowed to mature and stored in refrigerator.
Beads were activated in peptone broth (5%)
and incubated overnight on shaker (120 rpm).
Beads were washed with tris-buffer (pH 7.0,
0.01 M) and used for bioconversion.
2.4 Optimization of Aqueous two phase
system for progesterone
Polyethylene glycol (PEG) 6000, PEG 8000
and PEG 400 were used as a polymer system
and K2HPO4, KH2PO4 and Na2HPO4 were
used as a salt system. Polymer and salt
system were prepared at 15% and 30% (w/v)
and equal amount of polymer system and salt
system added to the sterile sugar tubes.
Progesterone was added 50 µl in each system
to check solubility of progesterone in aqueous
two-phase system. Whole system had to
vortex for a minute and allowed it to separate
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2.5
Progesterone
Bioconversion
using
Aqueous-Two
Phase system Activated beads were weighed
(3 gms) and again washed with sterile trisbuffer (pH 7.0, 0.01 M) in aseptic condition.
Activated beads were transferred in 20 ml of
sterile PEG (6000 and 8000) [5 % (w/v)] in
sterile flasks. 200 µl of progesterone was
added to the system for bioconversion. All
flasks were taken for 96 hours at 37° C at 120
rpm rotary shaker. Samples were taken at 0,
48 and 96 hours and extracted in hexane. It
was further analyzed by Gas chromatography
and
High
Performance
Thin
Layer
Chromatography (HP-TLC).
3. Results and Discussion
3.1 Optimization of Aqueous two phase
system for progesterone
PEG (Polyethylene glycol) 400, 6000 and
8000 were made up of different concentration
and salt system also made up of 0.5-30%
(w/v). Equal volume of polymer and salt
system was taken for optimization of aqueous
two-phase system. PEG 6000 has longer
stability with beads where PEG 8000 had
better progesterone solubility and higher
separation phases. When concentration of
PEG and salt system increased, it made
higher distribution coefficient which lead to
higher separation phases. It was the major
significant role in steroid solubility and helped
in
steroid
extraction
process.
When
concentration of PEG was increased, it made
efficiently solubilized progesterone. Minimum
1% (w/v) of PEG could easily dissolve
progesterone which was the major aspect for
the steroid solubility in the aqueous system.
KH2PO4 was used as efficient salt system.
Activated beads compressed or shrink under
higher concentration of 30% (w/v) of PEG
within 24 hour. No effects were observed up to
20% (w/v) of PEG.
Solubility of progesterone also affects the
major role in the system. Ethyl acetate was
used for the solubilizing agent because it did
not readily evaporate like hexane and
benzene. For efficient extraction system
212

3. hexane was used instead of ethyl acetate.
Hexane showed better separation of steroid
from polyethylene glycol system. When ethyl
acetate was used, some amount of
polyethylene glycol also extracted in the ethyl
acetate system. Problems occurred due to
solubility of PEG in the extraction system and
also presence of spot in the thin layer
chromatography (TLC). In that case, TLC did
not quantify the spot of progesterone and its
products so it had to basic necessary to check
on other chromatography techniques (GC &
HPTLC) and to change the extraction system.
3.2
Progesterone
Bioconversion
using
Aqueous Two Phase system
For steroid bioconversion PEG 6000 and PEG
8000 were used and analyzed by Gas
chromatography and HP-TLC. Figure 1
showed PEG 6000 and 8000 system including
activated beads and figure 2 showed proposed
mechanism of progesterone bioconversion.
Figures 3 and 4 depicted the results. It is
evident from the results on samples withdrawn
after 0, 48 and 96 hour incubation that there
was minor conversion of the precursor into
product steroids in these systems as there
was no change in profiles of the peaks
observed
after
different
times
of
incubation.The results highlight that this
system is not successful for bioconversion,
though it highly improved the solubility of
progesterone in the aqueous medium.
Figure 3: High Performance Thin Layer
Chromatograph of PEG 8000 mediated
progesterone bioconversion
Comparative studies showed that PEG 6000
bind
progesterone
so
conversion
of
progesterone showed in minor amount.
Products were showed in minor amounts after
96 hours. It proved that PEG 6000 has good
stability of progesterone stability and solubility
but poor in bioconversion. No toxicity effects
observed on activated culture beads because
of its low molecular weight.
Figure 4: HP-TLC graph of progesterone
bioconversion by PEG 6000
Figure 1: System was used for progesterone
bioconversion
Figure
2:
Proposed
mechanism
Progesterone bioconversion
for
Comparative studies showed that PEG 8000
bind
progesterone
so
conversion
of
progesterone showed in minor amount after 48
hours but but increased in 96 hours. It proved
that PEG 8000 has better stability of
progesterone stability and solubility. It had
better bioconversion than PEG 6000 in
bioconversion. Toxicity effects observed on
activated culture beads after 120 hours
because of its high molecular weight.
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213

4. Figure 5: HP-TLC graph of progesterone
bioconversion by PEG 8000
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Conclusion
Conversion of progesterone to AD and ADD
was reported for repeatedly by Comamonas
acidovorans MTCC 3364. Novel system was
used for the bioconversion of progesterone in
to AD and ADD. Aqueous two phase system
was initially optimized for the progesterone
solubility, separation of two phases and
extraction of product steroids from the system.
Even 1% of PEG (6000 & 8000) can easily
dissolve but 5% of PEG needed for separating
phases and for extraction purposes. Beads
was easily tolerate up to 20% of PEG system
even for 120 hours. Hexane was used as
better extraction solvent among different type
of solvent system. Products were showed in
PEG 6000 system after 96 hours in minor
amounts. PEG 8000 system showed products
48 hours in minor amount but increasing by 96
hours. It possibly used as a novel system can
easily soluble and separate progesterone and
other steroid products.
Acknowledgement
The authors acknowledge Charutar Vidya
Mandal, Vallabh Vidyanagar, Anand for
providing the infrastructure, Laboratory,
chemicals, computational and other necessary
facilities for the successful completion of this
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