S. Ganguly et. al. / International Journal on Pharmaceutical and Biomedical Research (IJPBR) Vol. 2(1), 2011, 21-25 EFFECT OF SOME AMINO ACIDS ON THE GROWTH AND L-GLUTAMIC ACID FERMENTATION BY AN AUXOTROPHIC MUTANT Micrococcus glutamicus AB 100. S. Ganguly and A. K. Banik Department of Chemical Engineering, Biochemical Engineering Division, Biotechnology laboratory, University of Calcutta, Kolkata 700 009 Abstract An investigation was carried out on the effect of some amino acids on the growth and production of L- glutamic acid by a mutant Micrococcus glutamicus AB 100 using selected suitable synthetic medium. It was observed that among different amino acids studied, l-alanine, l-arginine, l-threonine, l-tryptophan, l-proline and l-hitidine had positive effect; l-leucine, l-valine, l-phenylalanine, l-serine, l-methionine, l-lysine, l-glycine, l- cystine and l-asparagine had no effect, where as l-glutamic acid had negative effect on growth and l-glutamic acid production. maximum growth and l-glutamic acid was obtained with l-alanine, 0.3 mg/ml; l-arginine, 0.5 mg/ml; l-threonine, 0.3 mg/ml; l-tryptophan, 0.5 mg/ml; l-proline 0.75 mg/ml; and l-hitidine, 0.75 mg/ml. [Key words : L-glutamic acid, mutant, Micrococcus glutamicus, amino acids, synthetic medium] Introduction Microbial fermentation whereby -ketoglutarate is produced and subsequently converted to l-glutamic acid by different microorganisms has received the most attention. l-glutamic acid is formed from - ketoglutarate by transamination or reductive amination by glutamic acid dehydrogenase [1]. L-alanine can be used as the amino-donor following l-ketoglutaric acid production by bacterium ketoglutaricum, a 70%. yield of l-glutamic acid was accumulated [2]. katagiri et al (1960) used a number of amino acids as amino donors for this conversion [3]. l-glutamic acid fermentation by reductive amination of l-ketoglutaratee has been reported by otsuka et al (1957) [4]. Furthermore, Good (1960) employed Aeromonas sp and Good and Gunesaleus (1960) used yeast for the conversion of -ketoglutarate to l-glutamic acid [5,6]. Considering all these reviews, in the present study, an extensive investigation has been undertaken to examine the potency of the mutant Micrococcus glutamicus AB 100 to utilize some amino acids for its growth and l-glutamic acid accumulation. Materials and methods Microorganism : Micrococcus glutamicus AB 100, a biotin requiring auxotrophic mutant derived from a regulatory mutant Micrococcus glutamicus AB 1, derived in our laboratory by induced mutation, was used throughout this study [7]. Synthetic medium for L-glutamic acid production : The composite of the synthetic medium for l-glutamic acid production by this mutant include : glucose, 9.0%; diammonium hydrogen phosphate, 1.4% dipotassium hydrogen phosphate, 0.15%; magnesium sulfate, hepta hydrate, 0.03%; calcium carbonate, 0.04%; ferrous sulfate, hepta hydrate, 5.0 g/ml; zinc sulfate, hepta hydrate, 1.0 g/ml; manganese sulfate tetra hydrate, 1.0 g/ml and biotin 0.2 g/ml ph was adjusted to 6.5[8]. Addition of amino acids : The effects of l-alanine, l-arginine, l-threonine, l-tryptophan, l-tryptophan, l-proline, l-histidine, l-asperagine, l-leucine, l-valine, l-phenylalanine, l-serine, l-methione, l-lysine, l-glycine and l-cystine at varying concentrations (0.3-1.5 mg/ml) on l-glutamic acid accumulation by this mutant were studied. Fermentation was carried out using the shake flask method on a rotary shaker (150 rpm) in 100 ml Erlenmayer conical flask contain 20 ml synthetic medium for 24 h at 29 o C. The medium was inoculated with 4.0% (v/v) of a 48h old seed culture (6 x 10 7 cells) of Micrococcus glutamicus AB 100 [9]. ISSN : 0976-285X 21
Estimation of Dry cell weight (DCW) : After centrifugation, a few ml of 1.0(M) HCl was poured into the precipitate of the bacterial cells and calcium carbonate to dissolve calcium carbonate. The remaining bacterial cells were washed with water and dried at 100 o C until cell weight remain constant [10]. Analysis of Amino acid : Descending paper chromatography was employed for detecting L-glutamic acid in culture medium and was run for 18h on a watman No. 1 chromatographic paper. Solvent system used include, n- butanol : acetic acid : water (2 : 1 : 1). The spots were visualized by spraying with a solution of 0.2% ninhydrin in acetone by spraying with a solution of 0.2% ninhydrin in acetone and quantitative estimation of L-glutamic acid in the suspension was done using colorimetric estimation method [11,12]. Statistical analysis : All data were expressed as mean SEM, where n = 6. The data were analyzed by one way ANOVA followed by Dunett s post-hoc multiple comparison test using prism 4.0 software (Graph pad Ind., USA). A p value less than 0.05 was considered significant and less than 0.01 as a highly significant. Result and Discussion The effects of different amino acids on the growth and l-glutamic acid fermentation by this mutant have been depicted in fig. 1 16 including statistical analysis of the data among the different amino acids tested, l-alanine, l-arginine, l-threonine, l-tryptophan, l-proline and l-hitidine had positive effect; l-leucine, l-valine, l- phenylalanine, l-serine, l-rnethionine, l-lysine, l-glycine, l-cystine and l-asparagine had no effect, where as l- glutamic acid had negative effect on growth and l-glutamic acid production. maximum growth and l-glutamic acid was obtained with l-alanine, 0.3 mg/ml; l-arginine, 0.5 mg/ml; l-threonine, 0.3 mg/ml; l-tryptophan, 0.5 mg/ml; l-proline 0.75 mg/ml; and l-hitidine, 0.75 mg/ml. Under various conditions, Micrococcus glutamicus AB 100 is able to excrete l-glutamic acid. Some other amino acids were added to the resting cell system to determine whether or not they contribute to the growth and production of l-glutamic acid. At present we have no information regarding the mechanism of inhibition of l- glutamic acid fermentation by extrageneously added l-glutamic acid. However, accumulation of l-glutamic acid in the fermentation broth controls its own biosynthesis in E. coli, B. subtilis and B. licheniformis respectively by repression of one or more enzymes of TCA cycle or NADP specific glutamic acid dehydrogenase [13-15]. Thus, addition of l-glutamic acid to the resting cell of this mutant present in growth medium had negative effect on growth and l-glutamic acid accumulation in the fermentation broth, suggesting thereby the amino acids could regulate the TCA cycle enzymes or glutamate dehydrogenase in the same manner as it was found in E. coli, B. subtilis or B. licheniformis. Other amino acids named l-alanine, l-arginine, l-threonine, l-tryptophane, l-proline and l-histidine might serve as amino donor for the conversion for l-ketoglutaric acid to l-glutamic acid by this mutant. Conclusion From this present study it is tentatively concluded that, the production of l-glutamic acid by a mutant Micrococcus glutamicus can be improved by addition of l-alanine, 0.3 mg/ml; l-arginine, 0.5 mg/ml; l-threonine, 0.3 mg/ml; l-tryptophan, 0.5 mg/ml; l-proline 0.75 mg/ml; and l-hitidine, 0.75 mg/ml in the fermentation broth. Acknowledgement We express our sincere gratitude to the librarian of Bose Institute, Kolkata for his continuous cooperation in finding necessary information regarding this study without which we could not able to finish the work. References [1] Smythe, C. and Huang, H. T. (to Rohm and Han Co.)., U.S. Patent 2, 749, 279 (1956). [2] Masuo, E. and Wakisaka, K., Rept. To Ann. Meeting Agr. Chem Soc, Jpn (1955). [3] Katagiri, H. and Tochikara, T (to Ajinomoto Co., Inc)., U. S. Patent 2,953,499 (1960). [4] Otsuka, S., Yazaki, H., Nagase, H. and Sakaguchi, K., J. Gen. Appl. Microbiol 1957,3,35. [5] Good, R. C. (to International Minerals and Chemical Corp.)., U.S. Patent 2,933,434 (1960), British patent 8,426,7 (1960. [6] Good, R. C. and Gunsalus, I. C. (to International Minerals and Chemical Corp.)., U.S. Patent 2.927,059 (1960). [7] Ganguly, S. and Banik, A. K., J. Indian chem. Soc. 2010, 87(6), 711-721. [8] Banik, A. K. and Majumdar, S. K., Indian J. Exp. Biol 1975, 13, 510-512. [9] Ganguly, S. and Banik, A. K., International Journal of Pharma and Bio Sciences 2011, 2(2), 1-5. [10] Shah, A. H., Hameed, A., Ahmad, S and Khan, G. M., on line J. Biol. Sci 2002, 2(3), 151-156. [11] Chinard, F. P., J. Biol. Chem 1952, 199, 91. [12] Sbies, J. R., Methods in Enzymol 1957, 3, 468-471. [13] Flechtner, V. R. and Hanson R. S., Biochim. Biophys. Acta 1969, 184, 252-262. [14] Hanson, R. S. and Cox, D. P., J. Bacteriol 1967, 93, 1771-1787. [15] Varrichario, F., Biochim. Biophys. Acta 1969, 177, 560-564. ISSN : 0976-285X 22
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