(2) Satapathy et al.. World Journal of Pharmaceutical Research. examine the transport system for L-methionine in the mutant Corynebacterium glutamicum X300. MATERIALS AND METHODS Selection of microorganism A regulatory mutant Corynebacterium glutamicum X1 (accumulated only 0.6 mg /ml Lmethionine) developed in our laboratory from its parent strain Corynebacterium glutamicum (basically a L-glutamic acid producing bacterium which does not accumulate L-methionine) which was isolated from North Bengal soil was subjected for mutational study. Optimum cultural conditions Volume of medium ,25 ml; initial pH ,7.0; shaker’s speed ,150 rpm; age of inoculum ,48 hours; optimum cell density ,4.0X108 cells/ml; temperature280C and period of incubation ,72 hours. Composition of basal salt medium for L-methionine fermentation L methionine production was carried out using the following basal salt medium (per litre): glucose, 60 g; (NH4)2SO4, 1.5 g ; K2HPO4, 1.4 g; MgSO4·7H2O, 0.9 g; FeSO4·7H2O, 0.01 g ;biotin, 60μg . Composition of synthetic medium(per Liter) Glucose, 100 gm; (NH4)2SO4, 8.0 gm (in terms of nitrogen) ; K2HPO4 , 2.2 gm; MgSO4.7 H2O, 1.5 gm ; FeSO4.7H2O, 0.03 gm; KH2PO4,2.0 gm ; ZnSO4.7H2O , 1.6 mg ; CaCO3, 1.5 gm;Na2MoO4.2H2O, 5.0 mg; MnSO4.4H2O , 2.5 mg; biotin ,80 mg and thiamine-HCl, 70 µg .. Analysis of L-methionine Descending paper chromatography was employed for detection of L-methionine in culture broth and was run for 18 hours on Whatman No.1 Chromatographic paper. Solvent system used includes n-butanol: acetic acid: water (2:1:1). The spot was visualized by spraying with a solution of 0.2 % ninhydrin in acetone and quantitative estimation of L-methionine in the suspension was done using colorimetric method . Preparation of cells for L-methionine transport assay Cells were grown in minimal salt medium at 280C to the mid logarithmic phase of bacterial growth. Then the cells were harvested by centrifugation at 10,000rpm for 10 minutes and www.wjpr.net. Vol 3, Issue 4, 2014.. 2029.
(3) Satapathy et al.. World Journal of Pharmaceutical Research. washed twice with double distilled deionized water containing 100µg/ml chloramphenicol. The cells were mixed with 200µM L-[14C] methionine at 280C. The cells were incubated for 5 minutes. After that, 0.2ml sample was collected and filtered through a 0.40 µm membrane filter (Millipore Corp., Bedford and Mass). The filter residues were washed twice by 5.0 ml deionized double distilled water. The cells were dried and placed in Scintillation counter containing 5.0 ml of cocktail (Venkerz, Muller Instrument, Telfon , India) for the determination of radioactivity. The initial vevelocity (vi) of L-methionine influx was estimated from the amount of radioactivity in 0.3 minutes .The exit of radio-labeled methionine was estimated by allowing the cells to accumulate methionine in a volume of 0.3 ml. The efflux of L-methionine was examined both by Chase and exit experiment in which an excess unlabelled L-methionine was added to the cells which accumulated [14C] methionine. 1.0 mM N-ethyl maleimide, an inhibitor of L-methionine influx was then added to the cells .. Estimation of intracellular amino acid pool The intracellular amino acid pool of the trichloroacetic acid soluble cellular extract was estimated. Strain was grown at 280C using a minimal salt medium. 50 ml broth was filtered (Containing 41 mg protein) on a 45 mm membrane filter covered by Whatman 1mm paper. Filter was washed twice using deionized double distilled water and then placed into 5 ml of cold 5% (40C) trichloroacetic acid containing 5.5 mM 2-mercaptoethanol. The supernatant was washed with diethyl ether and then dried. The amino acid analysis was done using an amino acid analyzer (Model no: ZERTKLCHD 601667) . Effect of metabolic inhibitors Cells were nutritionally starved by vigorously stirring in solutions of 10mM KCN, 2-heptyl4-hydroxy-quinoline-N-oxide, 2, 4 dinitrophenol, sodium arsenite, mercuric chloride, sodium arsenate, 2-thiouracil, sodium azide , malonic acid and sodium fluoride in separate sets for 3h .Cells were then washed twice with deionized double distilled water and incubated with synthetic medium and export of L-methionine were examined in different time intervals . Statistical analysis All the data were expressed as mean ± SEM. All the chemicals used in this study were analytical grade (AR) grade and obtained from E mark .Borosil glass goods and triple distilled water used throughout the study .. www.wjpr.net. Vol 3, Issue 4, 2014.. 2030.
(4) Satapathy et al.. World Journal of Pharmaceutical Research. RESULTS AND DISCUSSION Influx of L-methionine Incubation at 280C of chloramphenicol-treated cells Corynebacterium glutamicum X300 with L-methionine (52.1 mg/ml), resulted in a rapid accumulation of L-methionine into the mutant cells. The accumulation raised up to 8.0 minutes of incubation and then continues linearly (Fig.1).. Fig.1: Influx of L-methionine by chloramphenicol-treated cells Corynebacterium glutamicum X300 at different time intervals (Values were expressed as mean± SEM, where n=6). Kinetics of L-methionine influx Uptake constant were determined by Woolf plot for this mutant .The L-methionine transport system for this mutant has a low affinity for L-methionine ( Km =62µM ; Vmax = 15.1 nmols-1g-1). Regulation of L-methionine uptake Effect of L-methionine related compounds The effect of L-methionine related compounds on L-methionine fermentation were investigated as shown in table 1. Table 1 : Effect of L-methionine related compounds on L-methionine uptake by the mutant Corynebacterium glutamicum X300 L-methionine related compounds L-methionine uptake (%) ( 40 µmol) 1. Glycyl methionine 84.6±1.668 2. N-acetyl methionine 76.6±0,916 3. D-methionine 66.1±0.991 4. α-methyl D-methionine 71.3±0.861 5. S-methyl L-cysteine 92.1±1.313. www.wjpr.net. Vol 3, Issue 4, 2014.. 2031.
(5) Satapathy et al.. World Journal of Pharmaceutical Research. 6. L-methionine sulfoxide 68.1±0.981 7. L-methionine amide 81.9±0.965 8. DL-methionine S-methyl 74.6±1.683 sulfonium chloride 9. N-formyl L-methionine 100.4±0.911 10. L-methionine methyl ester 100.1±0.882 Values were expressed as mean± SEM (Values were expressed as mean± SEM, where n=6). . Effect of metabolic inhibitors The effect of different metabolic inhibitors on L-methionine fermentation was investigated as depicted in table 2. Table 2: Effect of metabolic inhibitors on L-methionine uptake by the mutant Corynebacterium glutamicum X300 Metabolic inhibitors (40 mM) L-methionine Uptake (%) 6-mercaptopurine 64.2±0.113 24-dinitrophenol 51.2±0.965 Sodium arsenite 89.2±1.681 Mercuric chloride 31.6±0.973 Sodium arsenate 66.1±0.961 2-thiouracil 81.2±1.882 Sodium azide 79.3±0.948 Malonic acid 85.2±1.882 Sodium fluoride 61.2±0.991 KCN 9.2±1.683 Values were expressed as mean± SEM(Values were expressed as mean± SEM, where n=6).. Thus, from this above table, it is clear that all the metabolic inhibitors examined showed drastic inhibitory effect on L-methionine uptake by the mutant. Effect of metal ions on L-mehionine uptake The effect of different metal ions on L-methionine uptake was investigated as shown in Table 3.Among the different metal ions investigated, K+ and Na+ showed potential acceleratory effect on L-methionine uptake , where as Ca2+ , Mg2+,Mn2+ , Rb2+, V5+, Zn2+ and Co2+ showed slight inhibitory effect on L-methionine uptake by this mutant. But Li2+, Cu2+, Cd2+ and Mo4+ did not show any significant impact on L-methionine fermentation.. www.wjpr.net. Vol 3, Issue 4, 2014.. 2032.
(6) Satapathy et al.. World Journal of Pharmaceutical Research. Table 4: Effect of metal ions on L-methionine uptake by the mutant Corynebacterium glutamicum X300 (values were expressed as mean ± SEM , where n=6 , *p<0.05 , **p<0.01 when compared to control) Metal ions. Ca2+. Mg2+. Mn2+. V5+. Zn2+. Co2+. Na+. K+. www.wjpr.net. Concentration(s) in %. L-methionine uptake (mM /µl of cells). 0.10 0.15(Control) 0.20 0.25 0.30 0.10 0.15(Control) 0.20 0.25 0.30 0.20 0.25(Control) 0.30 0.35 0.40 0.0(Control) 0.05 0.10 0.15 0.20 0.25 0.10 0.16(Control) 0.20 0.25 0.30 0.00(Control) 0.05 0.10 0.15 0.20 0.00(Control) 0.05 0.10 0.15 0.20 0.00(Control) 0.05 0.10 0.15 0.20 0.25. **864.2 ±1.987 876.4± 1.913 *874.2± 1.891 **866.3± 0.991 **854.3± 1.913 **872.2±0.871 876.4±0.993 **871.4±0.792 **866.3±1.892 **859.3±1.113 **854.7±0.911 876.4±1.871 *874.8±1.913 **871.6±0.911 **866.3±0.891 876.4±1.115 *875.1±1.821 **871.2±1.113 **869.6±1.640 **866.3±0.992 **863.2±1.116 *874.9±1.897 876.4±1.986 **874.0±0.991 **871.8±0.964 **868.3±1.116 876.4±1.117 **874.3±0.917 **871.7±0.992 **869.8±1.113 **866.5±0.561 876.4±1.913 **879.5±1.991 **883.6±0.993 **887.5±1.993 **887.5±0.991 876.4±0.897 **879.8±1.118 **882.6±1.119 **882.6±0.992 **882.6±0.871 **882.6±0.914. Vol 3, Issue 4, 2014.. 2033.
(7) Satapathy et al.. World Journal of Pharmaceutical Research. 0.00(Control) 876.4±1.669 0.05 *874.8±0.992 0.10 **871.3±0.971 Rb2+ 0.15 **869.8±1,781 0.20 **866.5±0.892 0.25 **860.7±0.993 Values were expressed as mean± SEM (Values were expressed as mean± SEM, where n=6). Efflux of L-methionine by the mutant Corynebacterium glutamicum X300: The efflux of L-methionine from the mutant cells were investigated by chase and exit experiment. In this experiment, an excess unlabeled L-methionine was added to the cells which accumulating L[14C]-methionine. The effect of 6X10-8M N-ethyl maleimide (NEM), an inhibitor of Lmethionine influx was investigated by incubating the cells with this compound for 10 minutes. 68% inhibition of influx was resulted with NEM as shown in Table 4. Efflux of Lmethionine was investigated with the cells exposed to 0.70µM L-[14C]-methionine which had to be diluted at least 600 fold with synthetic medium before the efflux experiment. Addition of unlabeled L-methionine caused a rapid loss of radioactivity in L-[14C]-methionine labeled cells which were NEM-treated. Table 4: Efflux of L-methionine by the mutant Corynebacterium glutamicum X300 Conditions of the cells NEM-treated NEM-untreated Time (min) Uptake Efflux Uptake Efflux (mM /µl of cells) (mM /µl of cells) (mM /µl of cells) (mM /µl of cells) 0.0 0.0±0.000 0.0±0.000 0.0±0.000 0.0±0.000 1.0 694.6±0.861 224.6±0.719 1021.6±0.991 321.2±0.773 2.0 659.7±0.691 321.4±0.881 970.2±0.891 418.3±0.668 3.0 419.3±0.961 416.2±0.667 722.4±0.691 516.6±0.719 4.0 349.7±0.883 577.8±0.836 514.2±0.711 621.3±0.913 5.0 284.2±0.997 621.3±0.991 417.8±0.655 797.2±0.771 6.0 218.9±0.668 724.1±0.761 321.6±0.491 816.3±0.856 7.0 147.2±0.961 896.6±0.583 216.4±0.862 921.4±0.661 8.0 77.7±0.883 896.6±0.873 114.2±0.991 1084.3±0.991 9.0 51.9±0.661 896.6±0.881 76.4±0.691 1084.3±0.871 10.0 36.9±0.719 896.6±0.668 54.2±0.668 1084.3±0.881 Values were expressed as mean± SEM (Values were expressed as mean± SEM, where n=6). From this table, it is seen that uptake of L-methionine by NEM-treated cells potential the efflux of this amino acid by this mutant. Ferchichi et al. (1987) reported similar single Lmethionine transport system in Brevibacterium linens CNRZ 918. .. The rate of L-. methionine uptake is independent of physiological state of the microorganism. www.wjpr.net. Vol 3, Issue 4, 2014.. .. Kadner. 2034.
(8) Satapathy et al.. World Journal of Pharmaceutical Research. (1977) claimed that permease activity was partly controlled by the internal L-methionine pool in Escherichia coli. .. Ferchichi et al. (1987) also claimed that L-methionine transport in. Brevibacterium linens CNRZ 918 was potentiated by Na+ and K+ but inhibited by electron transport chain inhibitors like KCN. .. Actually, Na+ apparently did not alter the affinity of. the transport system of the bacterial cell membrane, leading to establishment of cellular energy capacity by creating Na+ gradient across the membrane . From this present study, it can be concluded that the mutant Corynebacterium glutamicum X300 had an L-methionine transport system with low affinity (Km=15.1 nmolS-1g-1). Though it is a multiple-analogue resistant mutant, mild effect of L-methionine related compounds on its cellular uptake was observed. Several metabolic inhibitors significantly reduced its influx. Among different metal ions studied, Na+, and K+ accelerated L-methionine uptake , where as Ca2+, Mn2+, Rb2+, V5+, Zn2+, Co2+ and Mg2+ slightly inhibited the influx. Li2+, Cu2+, Cd2+ and Mo4+ did not show any significant effect on L-methionine uptake by the mutant. The uptake by NEM-treated cells accelerated the cellular efflux of this amino acid by this mutant. ACKNOWLEDGEMENT We express our sincere gratitude to Prof.(Dr) Ajit Kumar Banik, Professor , Department of Chemical Engineering , University of Calcutta for his outstanding contribution and cooperation without which we could not able to finish the work. REFERENCES 1. Ayling, P.D., 1972. Methionine transport in wild-type and transport defective mutants of Salmonella typhimurium. J.Gen. Microbiol., 73:127-141. 2. Kadner, R., 1974. Transport systems for L-methionine in Escherichia coli. J. Bacteriol., 117: 232-241. 3. Kadner, R., 1965. Regulation of methionine transport activity in Escherichia coli. J. Bacteriol., 122: 110-119. 4. Montie, D.B. and Montie, T.C., 1975. Methionine transport in Yersinia pestis. J. Bacteriol., 124: 296-306. 5. Drapeau, G.R. and Macleod, R.A., 1963. Na+ dependent active transport of αaminoisobutyric acid into cells of a marine Pseudomonad. Biochem.Biophys. Res.Commun., 12:111-115.. www.wjpr.net. Vol 3, Issue 4, 2014.. 2035.
(9) Satapathy et al.. World Journal of Pharmaceutical Research. 6. Thompson,J. and Macleod, R.A.,1971. Functions of Na+ and K+ in the active transport of O-aminobutyric acid in a marine Pseudomonad. J. Biol.Chem., 246: 4066-4074. 7. Lanyi, J.K., Yearwood-drayton, V. and MacDonald, R.E., 1976. Light- induced glutamate transport in Halobacterium halobium envelope vesicles. I. Kinetics of the light dependent and the sodium-gradient dependent uptake. Biochemistry, 15:1595-1602. 8. Schellenberg, G.D. and Furlog, C.E., 1977. Resolution of the multicity of the glutamate and aspartate transport systems of Escherichia coli. J.Biol.Chem., 252: 9055-9064. 9. Tsuchiya, T., Hasan, S.M. and Raven, J., 1977. Glutamate transport driven by electrochemical gradient of sodium ions in Escherichia coli. J.Biol. Chem., 246:40664074. 10. Niiya, S., Moriyama, Y., Futai, M. and Tsuchiya, T., 1980. Cation coupling to melibiose transport in Salmonella typhimurium. J. Bacteriol., 144: 192-199. 11. Kitada,M. and Horikoshi,K., 1977. Sodium ion stimulated α-(1-C) aminoisobutyric acid uptake in alkalophilic Bacillus species. J. Bacteriol., 131: 784-788. 12. Ferchichi, M., Hemme, D. and Nardi, M., 1987. Na+- stimulated transport of Lmethionine in Brevibacterium linens CNRZ918. Appl. Environ. Microbiol., 53: 21592164. 13. Boyaval, P., Moreira, E. and Demazeaud , M.J., 1983. Transport of aromatic amino acid by Brevibacterium linens.J.Bacteriol. 155: 1123-1129. 14. Boyaval, P., Moreira, E. and Demazeaud , M.J.,1984. Electrochemical proton gradient of Brevibacterium linens and its relationship to phenylalanine transport. Ann.Microbiol ., 135B:91-99. 15. Hamouy,D., Boyaval, E. and Desmazeand, M.J., 1985. Active transport of L-serine in Brevibacterium linens ATCC9175. Milichwissenschaft, 40: 133-136. 16. Subhadeep Ganguly, Kunja Bihari Satapathy and Ajit Kumar Ganguly, 2014. Induced mutation, Development of multiple analogue resistant strain and protoplast fusion for Lmethionine fermentation by Corynebacterium glutamicum. Research Journal of Pharmaceutical Dose forms and technology, 6:303-310. 17. Subhadeep Ganguly and Kunja Bihari Satapathy , 2013. Optimization of physical parameters for L-methionine fermentation by a multiple analogue resistant mutant Corynebacterium. glutamicum. X300.The. Journal. of. Bioprocess. Technology,. Photon98:303-307. 18. Kase, H. and Nakayama, K.,1975. L-methionine production by methionine analogue resistant mutants of Corynebacterium glutamicum. Agric. Biol. Chem., 39:153-160. www.wjpr.net. Vol 3, Issue 4, 2014.. 2036.
(10) Satapathy et al.. World Journal of Pharmaceutical Research. 19. Subhadeep Ganguly and Kunja Bihari Satapathy, 2013.Selection of suitable synthetic medium for L-methionine fermentation by a multiple analogue resistant mutant Corynebacterium. glutamicumX300.. International. Journal. of. Multi. disciplinary. Educational Research,2: 252-261. 20. Kadner, R.J., 1974. Transport systems for L-methionine in Eschrichia coli. J.Bacteriol.,117: 232-241. 21. Moor, S.,1963. On the determination of cystine as cysteic acid. J.Biol.Chem.,232: 235237. 22. Ganguly, S. and Banik, A.K., 2011. Effect of surface active agents on the conversion of glucose to L-glutamic acid with the resting cells of the mutant Micrococcus glutamicus AB100. International Journal of Universal Pharmacy and Life Sciences., 1:94-97. 23. Ferchichi, M., Hemme,D. and Nardi,M.,1987. Na+stimulated transport of L-methionine in Brevibacterium linens CNRZ918. Appl.Environ.Microbiol.,53:2159-2164. 24. Ferchichi, M., Hemme,D. and Nardi,M. and Pamboukdjan N., 1985. Production of Methanol. from. methionine. by. Brevibacterium. linens. CNRZ918.. J.Gen.. Microbiol.,131:715-723. 25. Montie, D.B. and Montie, T.C. 1975. Methionine transport in Yersinia pestis .J.Bacteriol.,124:296-306.. .. www.wjpr.net. Vol 3, Issue 4, 2014.. 2037.