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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

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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 two-phase 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).

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

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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. 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 SIGMA-Aldrich. 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

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. 2.5 Progesterone Bioconversion using Aqueous-Two Phase system Activated beads were weighed (3 gms) and again washed with sterile tris-buffer (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

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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 1: System was used for progesterone bioconversion

Figure 2: Proposed mechanism for Progesterone bioconversion

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

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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Figure 5: HP-TLC graph of progesterone bioconversion by PEG 8000

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 work. Authors also thank to MTCC for providing the cultures used in the study and SICART, Vallabh Vidhyanagar, India for extending analytical facilities for this study.

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