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Purification and Characterization of Glucosyltransferase and Fructosyltransferase in Leuconostoc mesenteroides NRRL B-1149  

Lee Jin Ha (The Engineering Research Institute, Chonnam National University)
Park Jun Seong (Department of Materials and Biochemical Engineering, Chonnam National University)
Lee Hee Sun (Department of Molecular Biotechnology, Chonnam National University)
Kim Do Man (School of Biological Sciences and Technology and Research Institue for Catalysis, Chonnam National University)
Publication Information
KSBB Journal / v.19, no.5, 2004 , pp. 368-373 More about this Journal
Abstract
The optimal condition for the production of a glucan and a fructan synthesizing enzymes from Leuconostoc mesenteroides NRRL B-1149 were studied based on the different medium compositions. Response surface methodology was applied to find the optimistic condition showing the relationship between the fermentation response (enzyme activities) and the fermentation variable concentrations of yeast extract, peptone concentration, K2HP04 concentration and sucrose. Optimum medium composition for both enzymes production was $0.75\%$ yeast extract, $0.72\%$ peptone, $1\%$ K2HP04 and $2.17\%$ sucrose. Using this medium, the activities produced in culture was 0.90 U/m~ for glucosyltransferase (GTase) and 0.96 U/ml for fructosyltransferase (FTase). After purification of 1149FTase by consecutive chromatographies using Sephadex G-150 and DEAE-Sepharose, a 1149FTase of 210 kDa on $7\%$ polyacrylamide gel was isolated and it synthesized soluble fructan. The 1149GTase showed a band of 180 kDa on $8\%$ polyacrylamide gel after purification using Bio-Gel P-100 gel chromatography and DEAE-Sepharose ion exchange chromatography and it synthesized insoluble glucan. The linkages of polymers were determined by methylation using Hakomori reagent and following NMR analysis. The glucan was composed of a(1~6) and a(1~3) linkages and the fructan was levan.
Keywords
Purification; glucosyltransferase; fructosyltransferase; response surface methodology;
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1 Perez, M. A., L. Guereca, and L. A. Mungua (1996), Properties of levansucrase from Bacillus circulans, Appl. Microbiol. Biotechnol. 45, 465-471
2 Monsan, P., F. Paul. R. Remaud, and A. Lopez-Munguia (1989), Novel enzymatic synthesis of oligosaccharides, Food Biotechnol. 3, 111-130
3 Kim, M. S., S. 0. Lee, H. J. Ryu, H. K. Kang, S. K. Yoo, S. S. Chang, D. W. Kim, D. Kim, and S. H. Kim (2001), Synthesis of highly branched isomaltodextrin by acceptor reaction using dextransucrases from L. mesenteroides B-742CB and B-512FMCM, Kor. J. Biotechnol. Bioeng. 16, 200-206
4 Allene, J., W. C. Haynes, C. A. Wilham, J. C. Rankin, E. H. Melvin, M. J. Austin, J. E. Cluskey, B. E. Fisher, H. M. Tsuchiya, and C. E. Rist (1954), Characterization and classification of dextrans from ninety-six strains of $bacteria^1b$, J. Am. Chem. Soc. 76, 5041-5052
5 Lee, J. H., D. Kim, J. S. Baek, K. H. Park, N. S. Han, and J. F.Robyt (1998), ModiScation of starch using dextransucrase andcharacterization of the modiBed starch, Kor. J. Appt. Microbiot.Technot. 26, 143-150
6 Shiroza, T. and H. K. Kuraimtsu (1988), Sequence analysis of the Streptococcus mutans fructosyltransferase gene and flanking regions, J. Bacteriol. 175, 810-816
7 Milward, C. P. and N. A. Jacques (1990), Secretion of fructosyl-transferase by Stretococcus salivarius involves the sucrose-dependentrelease of the cell-bound form, J. Gen. MicrobioI. 136, 165-169
8 Funane, K., M. Yamada, M. Shiraiwa, H. Takahaia, N. Yamamoto, E. Ichishima, and M. Kobayashi (1995), Aggregated form of dextransucrases from Leuconostoc mesenteroides NRRL B-512F and its constitutive mutant, Biosci. Biotechnol. Biochem. 59, 776-780
9 Tomasic, J., H. J. Jennings, and C. P. J. Glaudemans (1978), Evidence for a single type of linkage in a fructofutan from Lolium perenne, Carbohydr. Res. 62, 127-133
10 Bradford, M. M. (1976), A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye bindmg, Anat. Biochem. 72, 248-254
11 Lee, H. S., S. H. Park, H. H. Lee, and H. K. Lee (2001), Effect of aeration rates on production of extracellular polysaccharides, EPS-R by marme bactehum Hahetta chejuensis, Biotechonot. BioprocessEne. 6, 359-362
12 Laemmli, U. K. (1970), Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature. 227, 680-685
13 Tanirseven, A., and J. F. Robyt (1993), Interperation of dextransucrase inhibition at high sucrose concentration, Carbohydr. Res. 245, 97-104
14 Shimamura, A., H. Tsumori, and H. Mukasa (1982), Purification andproperties of Streptococcus mutans extracellular glucosyltransferase, Biochem. Biophys. Acta. 702, 72-80
15 Skov, L., O. Mirza, A. Henriks en, G. Potocki de Montalk, M. Remaud-Simeon, P. Sarcabal, R. Willemot, P. Monsan, and M. Gajhede (2000), Crystallization and preliminary X-ray studies of recombinant amylosucrase from Neisseria polysaccharea, Acta Crystallogr. 56, 203-205
16 Steinmetz, M., D. Le Coq, S. Aymerich, G. Gonzy-Treboul, and P. Gay (1985), The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites, Mol. Gen. Genet. 200, 220-228
17 Baek, J. S., D. Kim, J. H. Lee, P. S. Chang, N. S. Han, and J. F. Robyt (1998), Enzymatic synthesis of new oligosaccharides using glucansucrases, Kor. J. Appl. Microbiol. Biotechnol. 26, 179-186
18 Giffard, P. M., C. L. Simpson, C. P. Milward, and N. J. Jacques (1991), Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC 25975,J. Gen. Microbiol. 137, 2577-2593
19 Chan, L., B. Jinghua, C. Zhaoling, and O. Fan (2002), Optimization of a cultural medium for bacteriocin production by Lactococcus lactis using response surface methodology, J. Biotechnol. 93, 27-34
20 Funane, K., M. Shiraiwa, K. Hashimoto, E. Ichishima, and M. Kobayashi (1993), An active-site peptide containing the second essential carboxyl group of dextransucrase from Leuconostoc mesenteroides by chemical modifications, Biochemistry32, 13696-13702
21 Mukerjea, R., Kim, D and J. F. Robyt (1996), Simplified and improved methylation analysis of saccharides, using a modified procedure and thin-layer chromatography, Carbohydr. Res. 292, 11-20