Bioconversion of colchicine into its pharmacologically active derivative 3-demethylated colchicine (3-DMC) mediated by P450BM3 enzyme is an economic and promising strategy for the production of this inexpensive and potent anticancer drug. Continuous stirred tank reactor (CSTR) and packed-bed reactor (PBR) of 3？L and 2？L total volumes were compared for the production of 3-demethylated colchicine (3-DMC) a colchicine derivative using Bacillus megaterium MTCC*420 under aerobic conditions. Statistical optimization technique was utilized with the most significant variables, that is, dissolved oxygen (DO), colchicine concentration, and process time for optimization. The validation of the model was performed by experiments on the predicted values in an individual run, and the optimum parameters were DO (~50%), colchicine concentration (7.5？g/L), and process time (39？h) resulted in a maximum bioconversion of 3-DMC 3.36？g/L. The PBR reactor achieved much higher productivity (6.58？g/L/h) as reported by earlier researchers. This is the first report on the use of PBR for bioconversion of colchicine. 1. Introduction Bioreactor design is of crucial importance in the development of bioprocesses. Once a microorganism is selected and the culture and/or production conditions are optimized at laboratory scale, the next issue is proceeding to larger scale so that bulk quantities of the desired product can be produced optimally in a cost-effective manner. The selection of a proper reactor type is critical to such processes and plays a major role in large-scale production. The bioreactors widely used in today’s fermentation industry are mainly those developed in the past decades purely for chemical reactions [1, 2]. Among others, Stirred Tank Reactors (STRs) and Packed-Bed Reactors (PBRs) find the widest application. The application of these reactors for biological processes has been intensively studied with aerobic cultures [3–5]. Colchicine is a well-documented pseudo alkaloid obtained from Colchicum autumnale L. and Gloriosa superba, widely used in therapy for the treatment of gout pain persisting for a very long time [6, 7]. It is too toxic to be of value as an antitumor drug, in its native form. Derivatives of colchicine, that is, 3-demethylcolchicine, colchicoside, and thiocolchicoside with improved therapeutic properties for anti-inflammatory and anti-tumor drugs, have good commercial demand as these compounds are known to have clinical significance for the treatment of certain forms of leukemia and solid tumors [8, 9]. Colchicine and a number of its prepared
References
[1]
M. A. Al-Juaied, D. Lafarga, and A. Varma, “Ethylene epoxidation in a catalytic packed-bed membrane reactor: experiments and model,” Chemical Engineering Science, vol. 56, no. 2, pp. 395–402, 2001.
[2]
C. F. Torres, F. Munir, L. P. Lessard, and C. G. Hill Jr., “Lipase-mediated acidolysis of tristearin with CLA in a packed-bed reactor: a kinetic study,” Journal of the American Oil Chemists' Society, vol. 79, no. 7, pp. 655–661, 2002.
[3]
D. Chen, Z. Lewandowski, F. Roe, and P. Surapaneni, “Diffusivity of Cu2+ in calcium alginate gel beads,” Biotechnology and Bioengineering, vol. 41, no. 7, pp. 755–760, 1993.
[4]
A. Sánchez, F. Valero, J. Lafuente, and C. Solà, “Highly enantioselective esterification of racemic ibuprofen in a packed bed reactor using immobilised Rhizomucor miehei lipase,” Enzyme and Microbial Technology, vol. 27, no. 1-2, pp. 157–166, 2000.
[5]
H. Tanaka, M. Matsumura, and I. A. Veliky, “Diffusion characteristics of substrates in Ca-ALGINATE gel beads,” Biotechnology and Bioengineering, vol. 26, no. 1, pp. 53–58, 1984.
[6]
T. Tateishi, P. Soucek, Y. Caraco, F. Peter Gnengerich, and A. J. J. Wood, “Colchicine biotransformation by human liver microsomes,” Biochemical Pharmacology, vol. 53, no. 1, pp. 111–116, 1997.
[7]
M. R？sner, H.-G. Capraro, A. E. Jacobson et al., “Biological effects of modified colchicines. Improved preparation of 2-demethylcolchicine, 3-demethylcolchicine, and (+)-colchicine and reassignment of the position of the double bond in dehydro-7-deacetamidocolchicines,” Journal of Medicinal Chemistry, vol. 24, no. 3, pp. 257–261, 1981.
[8]
A. Muzaffar, A. Brossi, C. M. Lin, and E. Hamel, “Antitubulin effects of derivatives of 3-demethylthiocolchicine, methylthio ethers of natural colchicinoids, and thioketones derived from thiocolchicine. Comparison with colchicinoids,” Journal of Medicinal Chemistry, vol. 33, no. 2, pp. 567–571, 1990.
[9]
P. Kerekes, P. N. Sharma, A. Brossi, C. F. Chignell, and F. R. Quinn, “Synthesis and biological effects of novel thiocolchicines. 3. Evaluation of N-acyldeacetylthiocolchicines, N-(alkoxycarbonyl) deacetylthiocolchicines, and O-ethyldemethylthiocolchicines. New synthesis of thiodemecolcine and antileukemic effects of 2-demethyl- and 3-demethylthiocolchicine,” Journal of Medicinal Chemistry, vol. 28, no. 9, pp. 1204–1208, 1985.
[10]
K. K. Dubey, A. Jawed, and S. Haque, “Enhanced bioconversion of colchicine to regiospecific 3-demethylated colchicine (3-DMC) by whole cell immobilization of recombinant E. coli harboring P450 BM-3 gene,” Process Biochemistry, vol. 48, pp. 1151–1158, 2013.
[11]
K. K. Dubey, A. Jawed, and S. Haque, “Enhanced extraction of 3-demethylated colchicine from fermentation broth of Bacillus megaterium: optimization of process parameters by statistical experimental design,” Engineering in Life Sciences, vol. 11, no. 6, pp. 598–606, 2011.
[12]
K. K. Dubey and B. K. Behera, “Statistical optimization of process variables for the production of an anticancer drug (colchicine derivatives) through fermentation: at scale-up level,” New Biotechnology, vol. 28, no. 1, pp. 79–85, 2011.
[13]
K. K. Dubey, A. R. Ray, and B. K. Behera, “Production of demethylated colchicine through microbial transformation and scale-up process development,” Process Biochemistry, vol. 43, no. 3, pp. 251–257, 2008.
[14]
K. K. Dubey, S. Haque, A. Jawed, B. P. Singh, and B. K. Behera, “Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70l bioreactor level,” Process Biochemistry, vol. 45, no. 7, pp. 1036–1042, 2010.
[15]
A. E. Klein and P. J. Davis, “Determination of colchicine and colchiceine in microbial cultures by high-performance liquid chromatography,” Analytical Chemistry, vol. 52, no. 14, pp. 2432–2435, 1980.
[16]
P. Ducommun, P.-A. Ruffieux, A. Kadouri, U. Von Stockar, and I. W. Marison, “Monitoring of temperature effects on animal cell metabolism in a packed bed process,” Biotechnology and Bioengineering, vol. 77, no. 7, pp. 838–842, 2002.
[17]
H. Treichel, D. de Oliveira, M. A. Mazutti, M. Di Luccio, and J. V. Oliveira, “A review on microbial lipases production,” Food and Bioprocess Technology, vol. 3, no. 2, pp. 182–196, 2010.
[18]
X. Xu, H. Mu, C. E. H？y, and J. A. Nissen, “Production of specifically structured lipids by enzymatic interesterification in a pilot enzyme bed reactor: process optimization by response surface methodology,” Lipid/Fett, vol. 101, pp. 207–214, 1999.
