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Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1325-1337 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) Sequential Optimization of Production of Cephamycin C Using Nocardia lactamdurans: Effect of Nutritional Supplements, Metabolic Precursors and Inducers Lalit D. Kagliwal*, Shrikant A. Survase and Rekha S. Singhal Food Engineering and Technology Department, Institute of Chemical Technology Matunga, Mumbai 400 019, India *For Correspondence - lalitkagliwal@gmail.com 1325 Abstract In this study, Nocardia lactamdurans NRRL 3802 was optimized for the first time for production of cephamycin C by using submerged fermentation. The effects of various carbon source, nitrogen source, inoculum size, initial pH of culture medium, minerals and amino acids were investigated for the maximum production of cephamycin C by N. lactamdurans NRRL 3802 in submerged fermentation. Subsequently, selected fermentation parameters were further optimized by Taguchi orthogonal array method and response surface methodology (RSM). Glycerol and yeast extract as carbon and nitrogen sources with initial pH of culture medium of 5.5 and inoculum size of 10 9 CFU/ml at 28 ± 2 °C after 3 days gave maximum production of 3042.19 ± 62.36 mg/l of cephamycin C as compared to 243.03 ± 9.43 mg/l before optimization. Incorporation of 1,3- diaminopropane at 1.2 % v/v in RSM optimized medium further increased the yield to 3973.20 ± 57.96 mg/l. Keywords: Cephamycin C, Nocardia lactamdurans, Inducer, Taguchi, RSM Introduction Since the discovery of penicillin, a number of microbial species producing a variety of related antibiotic products have been isolated. In all, over 1,000 naturally occurring or chemically modified antibiotic products have been reported, of which over 100 are commercially produced. One important class of microbially produced compounds, the cephamycins, was separately isolated in the early 1970s by Merck and Co., Inc., Germany and by Eli Lilly and Co., IN, USA. Cephamycin C is one of the extracellularly produced â-lactam antibiotics having hydrophilic properties. They are produced by actinomycetes such as Nocardia lactamdurans (1) and several species of Streptomyces viz. Streptomyces cattleya and Streptomyces clavuligerus (2). The increasing occurrence of bacterial resistance to â-lactam antibiotics is through synthesis of the enzyme â- lactamases that hydrolyze the â-lactam ring. Cephamycins are substituted with a methoxy group at the 7á-position of the cephalosporin nucleus, a modification that considerably reduces inactivation by â-lactamases. It is currently being used as an intermediate for semi-synthetic antibiotics such as cefoxitin, cefametazole and cefotetan having better pharmacokinetic and pharmacodynamic properties. Development of economical medium requires selection of carbon, nitrogen, inorganic salts, and trace element sources. The optimization of nutritional and environmental conditions plays an important role in developing bioprocesses and improving their performance. Nutritional Sequential optimization of cephamycin C
Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1325 -1337 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1326 requirement can be manipulated by the conventional or statistical methods (3). The conventional method involves changing one independent variable at a time, while keeping other variables at a fixed level. Statistical methods, on the other hand, (a) shortlist significant variables, (b) help in understanding the interactions among the variables at different levels, and (c) reduce the total number of experiments resulting in saving time and resources. Hence these methods are more rapid and reliable (4). There are very few reports on production of cephamycin C from Nocardia lactamdurans using submerged fermentation (SmF) (5,6). To the best of our knowledge, no report on optimization of the fermentation media composition and conditions for cephamycin C production are available, which prompted us to undertake this study. This paper reports a detailed study on the optimization of cephamycin C production from Nocardia lactamdurans NRRL 3802 using SmF, initially by the conventional one factor at-a-time method and subsequently by statistical methods such as Taguchi L 16 - orthogonal array design and central composite rotatable design. Furthermore, the effects of precursors and inducer on the production of cephamycin C were also undertaken. Materials and Methods Materials: Nutrient broth, yeast extract, malt extract, peptone, L-glutamic acid, sucrose, fructose, lactose, maltose, galactose, myoinositol and 1,3-diaminopropane were purchased from Himedia Limited, Mumbai, India. Glucose, glycerol, starch, ammonium chloride, calcium carbonate, potassium dihydrogen orthophosphate, dipotassium hydrogen orthophosphate, sodium chloride, magnesium sulphate, ferrous sulphate, manganese sulphate, zinc sulphate, sodium thiosulphate, orthophosphoric acid, methanol, L-cysteine, L- valine, DL-methionine, L-lysine hydrochloride, heptanesulphonic acid sodium salt for HPLC and HPLC grade acetonitrile were purchased from s.d. Fine-Chem Limited, Mumbai, India. Soybean oil, sesame oil, coconut oil, groundnut oil and mustard oil were procured from local market of Matunga, Mumbai, India. All solvents used were of AR grade unless mentioned. Standard cephamycin C (authentic sample) was a gift sample through kind courtesy of Merck Research Laboratories, Rahway, NJ, USA. A cephamycin C producing strain, Nocardia lactamdurans NRRL 3802, was a gift sample from Northern Regional Research Laboratory, Peoria, Illinois, USA. Preparation of seed inoculums: The strain was maintained as glycerol stock by adding 1 ml of culture solution, prepared by growing 1 ml (1 x 10 9 CFU/ml) of the organism in 50 ml NYG medium [(g/l) nutrient broth 8, yeast extract 2, glucose 10, MgCl 2 4, pH 6.8) for 48 h (7)], to preautoclaved 1.5 ml microcentrifuge tube containing 0.5 ml of 50% glycerol solution and stored at -20°C. For seed inoculum, the glycerol stock (1 x 10 9 CFU/ml) prepared earlier was inoculated into 50 ml of sterilized NYG media and kept on orbital shaker at 180 rpm for 48 h. Fermentative production of cephamycin C: The production medium for cephamycin C contained (g/l) glucose, 10.0; L-glutamic acid, 4.25; NH 4 Cl, 1.0; CaCO 3 , 0.25; K 2 HPO 4 , 2.0; myo-inositol, 0.2; MgSO 4 .7H 2 O, 0.5; NaCl, 0.5; FeSO 4 .7H 2 O, 0.025; MnSO 4 .H 2 O, 0.005; ZnSO 4 .7H 2 O, 0.01; para-amino benzoic acid (PABA), 0.0001 and Na 2 S 2 O 3 .5H 2 O, 0.36. The pH of the production medium was adjusted to 6.8 ± 0.05 (5). 50 ml of production medium was taken in 250 ml Erlenmeyer flask and sterilized by autoclaving at 121°C for 20 min. After cooling, the media was inoculated with 1 ml of 48 h old seed culture Lalit D. Kagliwal et al
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Page 175: (December 7-9, 2011) Venue: Karunya
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