1887

Abstract

Maltose phosphorylase (MP) from was purified and the corresponding gene was cloned and expressed in . The isoelectric point of the pure enzyme was determined to be 70. According to zymogram analysis and SDS-PAGE, the native MP was shown to be a monomeric enzyme with a molecular mass of 75 kDa. A polyclonal antiserum was produced to assess the regulation of the gene encoding MP in . According to immunoblot analysis, synthesis of the enzyme was markedly repressed by both glucose and lactose in the growth medium. When the lactococci were cultivated in the presence of other sugars, including maltose, trehalose or galactose, there was a pronounced expression of the MP gene. In addition, when the cells were grown in media without any added sugar, there was also pronounced expression of the enzyme, according to immunoblot analysis and specific activity data. These results indicated that no particular sugar specifically induces the gene encoding MP. However, an effect of glucose on MP expression was demonstrated by performing fermentations in the presence of both maltose and glucose. When glucose was added to maltose-grown lactococci in the mid-exponential growth phase, both the specific activity and amount of MP per millilitre of cell extract decreased rapidly. The genetic locus for the MP gene was found to be in the vicinity of the region encoding a possible regulator belonging to the LacI-GalR family of transcriptional regulators. Furthermore, this genetic location was separated from the previously characterized maltose-inducible and glucose-repressible β-phosphoglucomutase (β-PGM) gene. The different genetic loci for the genes encoding MP and β-PGM explains the different gene regulation behaviour.

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2001-06-01
2021-10-24
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References

  1. Aisaka K., Masuda T., Chikamune T., Kamitori K. 1998; Purification and characterization of trehalose phosphorylase from Catellatospora ferruginea. . Biosci Biotechnol Biochem 62:782–787 [CrossRef]
    [Google Scholar]
  2. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1996 Current Protocols in Molecular Biology , 2nd edn. New York: Wiley;
    [Google Scholar]
  3. Bolotin A., Mauger S., Malarme K., Ehrlich S. D., Sorokin A. 1999; Low-redundancy sequencing of the entire Lactococcus lactis IL1403 genome. In Proceedings of the Sixth Symposium on Lactic Acid Bacteria: Genetics, Metabolism and Applications pp 27–76 Edited by Konings W. N. Kuipers O. P., J J. H. Huis in’t Veld; Veldhoven:Kluwer:
    [Google Scholar]
  4. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254 [CrossRef]
    [Google Scholar]
  5. Bunte A. 1998 Genes and enzymes involved in maltose metabolism in Lactococcus lactis Licentiate thesis, Lund University; Sweden;
    [Google Scholar]
  6. Egeter O., Bruckner R. 1995; Characterization of a genetic locus essential for maltose-maltotriose utilization in Staphylococcus xylosus . J Bacteriol 177:2408–2415
    [Google Scholar]
  7. Ehrmann M., Vogel R. F. 1998; Maltose metabolism of Lactobacillus sanfranciscensis : cloning and heterologous expression of the key enzymes, maltose phosphorylase and phosphoglucomutase. FEMS Microbiol Lett 169:81–86 [CrossRef]
    [Google Scholar]
  8. Eis C., Niedetzky B. 1999; Characterization of trehalose phosphorylase from Schizophyllum commune . Biochem J 341:385–393 [CrossRef]
    [Google Scholar]
  9. Fitting C., Doudoroff M. 1952; Phosphorolysis of maltose by enzyme preparations from Neisseria meningitidis . J Biol Chem 199:153–163
    [Google Scholar]
  10. Gasson M. 1983; Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154:1–9
    [Google Scholar]
  11. Häggström M. H. 1981 Studies of parameters influencing cell and product yields in bacterial cultivations. PhD thesis, Lund University; Sweden:
    [Google Scholar]
  12. Hüwel S., Haalck L., Conrath N., Spener F. 1997; Maltose phosphorylase from Lactobacillus brevis : purification, characterization, and application in a biosensor for ortho -phosphate. Enz Microb Technol 21:413–420 [CrossRef]
    [Google Scholar]
  13. Inoue H., Nojima H., Okayama H. 1990; High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28 [CrossRef]
    [Google Scholar]
  14. Kamogawa A., Yokobayashi K., Fukui T. 1973; Purification and properties of maltose phosphorylase from Lactobacillus brevis . Agric Biol Chem 37:2813–2819 [CrossRef]
    [Google Scholar]
  15. Koga T., Nakamura K., Shirokane Y., Mizusawa K., Kitao S., Kikuchi M. 1991; Purification and some properties of sucrose phosphorylase from Leuconostoc mesenteroides . Agric Biol Chem 55:1805–1810 [CrossRef]
    [Google Scholar]
  16. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  17. Law J., Buist G., Haandrikman A., Kok J., Venema G., Leenhouts K. 1995; A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J Bacteriol 177:7011–7018
    [Google Scholar]
  18. Luesink E. J. 1998 Carbon catabolite repression and global control of the carbohydrate metabolism in Lactococcus lactis PhD thesis, Wageningen University; The Netherlands:
    [Google Scholar]
  19. Moustafa H. H., Collins E. B. 1968; Role of galactose or glucose 1-phosphate in preventing the lysis of Streptococcus diacetilactis . J Bacteriol 95:592–602
    [Google Scholar]
  20. Puyet A., Ibanez A. M., Espinoza M. 1993; Characterization of the Streptococcus pneumoniae maltosaccharide regulator MalR, a member of the LacI-GalR family of repressors displaying distinctive genetic features. J Biol Chem 268:25402–25408
    [Google Scholar]
  21. Qian N., Stanley G. A., Hahn-Hägerdal B., Rådström P. 1994; Purification and characterization of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells. J Bacteriol 176:5304–5311
    [Google Scholar]
  22. Qian N., Stanley G. A., Bunte A., Rådström P. 1997; Product formation and phosphoglucomutase activities in Lactococcus lactis : cloning and characterization of a novel phosphoglucomutase gene. Microbiology 143:855–865 [CrossRef]
    [Google Scholar]
  23. Reichenbecher M., Lottspeich F., Bronnenmeier K. 1997; Purification and properties of a cellobiose phosphorylase (CepA) and a cellodextrin phosphorylase (CepB) from the cellulolytic thermophile Clostridium stercorarium . Eur J Biochem 247:262–267 [CrossRef]
    [Google Scholar]
  24. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  25. Scott M., Russell J. B. 1987; Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis . Appl Environ Microbiol 53:2388–2393
    [Google Scholar]
  26. Silverstein R., Voet J., Reed D., Abeles R. H. 1967; Purification and mechanism of action of sucrose phosphorylase. J Biol Chem 242:1338–1346
    [Google Scholar]
  27. Sjöberg A., Hahn-Hägerdal B. 1989; β-Glucose 1-phosphate, a possible mediator for polysaccharide formation in maltose-assimilating Lactococcus lactis . Appl Environ Microbiol 55:1549–1554
    [Google Scholar]
  28. Sjöberg A., Persson I., Quednau M., Hahn-Hägerdal B. 1995; The influence of limiting and non-limiting growth conditions on glucose and maltose metabolism in Lactococcus lactis ssp. lactis strains. Appl Microbiol Biotechnol 42:931–938 [CrossRef]
    [Google Scholar]
  29. Tangney M., Buchanan C. J., Priest F. G., Mitchell W. J. 1992; Maltose uptake and its regulation in Bacillus subtilis . FEMS Microbiol Lett 97:191–196 [CrossRef]
    [Google Scholar]
  30. Thompson J., Chassy B. M. 1985; Intracellular phosphorylation of glucose analogs via the phosphoenolpyruvate: mannose-phosphotransferase system in Streptococcus lactis. . J Bacteriol 162:224–234
    [Google Scholar]
  31. Vieira J. D., Messing J. 1982; The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268 [CrossRef]
    [Google Scholar]
  32. de Vos W. M., Boerrigter I., Reiche B., Hengstenberg W., van Rooyen R. J. 1990; Characterization of the lactose-specific enzymes of the phosphotransferase system in Lactococcus lactis. . J Biol Chem 265:22554–22560
    [Google Scholar]
  33. Wannet W. J., Wisselink H. W., Vogels G. D., Op den Camp H. J., van der Drift C., van Griensven L. J. 1998; Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus. . Biochim Biophys Acta 1425177–188 [CrossRef]
    [Google Scholar]
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