clavuligerus in a culture medium containing about 100 mmol l-1 of lysine [14, 20, 21]. In spite of lysine degradation via 1-piperideine-6-carboxylate pathway producing the precursor alpha-aminoadipic acid [25,
26], complete lysine catabolism occurs via cadaverine [24, 29, 30]. Cadaverine and other diamines, such as diaminopropane and putrescine, promote beta-lactam antibiotic production in Nocardia lactamdurans or S. clavuligerus [31–34]. Nevertheless, it is difficult to determine the extent to which these compounds influence antibiotic biosynthesis, since diamines act as modulators of several cell functions [32, 33, 35]. Thus, there is scarce quantitative research on the use of lysine combined with other diamines or other compounds that can potentially enhance beta-lactam antibiotic production in S. clavuligerus [16, 23, 33]. This was explored selleck chemical in this study, Adriamycin in vitro which investigates increases in cephamycin C production by adding cadaverine, putrescine, 1,3-diaminopropane or alpha-aminoadipic acid in culture media containing lysine as compared to those obtained in culture media containing lysine
alone. Cultivations were performed in accordance with a central composite-based, face-centered experimental design (CCF) whereas concentrations of lysine combined with every compound were optimized using Response Surface Methodology. Best conditions were validated by means of batch cultivations in a stirred and aerated bench-scale bioreactor. Methods Microorganisms Streptomyces clavuligerus ATCC 27064 Glycogen branching enzyme was stored in the form of spore suspension (approximately 108 spores ml-1) at -80°C in 2 ml cryotube vials (glycerol at 20% w v-1). Escherichia coli ESS 2235 supersensitive to beta-lactam antibiotics was employed as test organism. The strain was cultivated in nutrient agar medium (Difco™ Nutrient Agar) at 37°C for 24 hours. The cells were stored at -80°C in 2 ml cryotube
vials. Culture media The seed medium contained (g l-1) tryptone (5.0), yeast extract (3.0), malt extract (10), and buffering agent 3-(N-morpholine) propanesulfonic acid (MOPS) (21). The inoculum medium consisted (g l-1) of soluble starch (10), cotton seed extract (PROFLO® – Traders Protein, USA) (8.5), yeast extract (1.0), K2HPO4 (0.80), MgSO4.7H2O (0.75), MOPS (21), and 10 ml of salt solution per l of medium. The salt solution contained (g l-1) MnCl2.4H2O (1.0), FeSO4.7H2O (1.0), and ZnSO4.7H2O (1.0). The basal production medium contained (g l-1) soluble starch (10), PROFLO® (8.5) boiled down and filtered (using a vacuum pump), yeast extract (0.50), K2HPO4 (1.75), MgSO4.7H2O (0.75), CaCl (0.20), NaCl (2.0), MOPS (21), the aforementioned salt solution (5.0 ml l-1), and sodium thiosulfate (1.0) added at 30 h after inoculation according to Inamine and Birnbaum . The initial pH of culture media was fitted to 6.8 ± 0.1. The proportion of filtered PROFLO® nitrogen corresponded to 40% of gross PROFLO®.