Abstract
A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21( DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 51 bioreactor with 31 working volume. In 51 bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 701 bioreactor and resulted into similar to 80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l(-1) h(-1). Scale-up factors were measured as volumetric oxygen transfer coefficient (k(L)a) i.e., 56 h(-1) and impeller tip velocity (V-tip) i.e., 7.065 m s(-1). respectively. The kinetic parameters K-m, k(cat), and k(cat)/k(m) of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 +/- 30 mu M, 8533 +/- 25 min(-1), and 31.49 mu M min(-1), respectively, when IPTG induced recombinant E. coli culture was used. (C) 2010 Elsevier Ltd. All rights reserved.