Applied Microbiology and Biotechnology

, Volume 41, Issue 3, pp 302–308 | Cite as

Identification and characterisation of the extracellular sucrases of Zymomonas mobilis UQM 2716 (ATCC 39676)

  • R. G. Crittenden
  • H. W. Doelle
Original Papers
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Abstract

An investigation was conducted to isolate, and characterise the extracellular sucrases of Zymomonas mobilis UQM 2716. Levansucrase (EC 2.4.1.10) was the only extracellular sucrase produced by this organism. This enzyme was responsible for sucrose hydrolysis, levan formation, and oligosaccharide production. It had a molecular mass of 98 kDa, a Michaelis constant (Km) of 64 mm, and a pH optimum of 5.5. It was inhibited by glucose, but not by fructose, ethanol, sorbitol, NaCl, TRIS or ethylenediaminetetraacetic acid (EDTA). The formation of levan was the principal reaction catalysed by this enzyme at low temperatures. However, levan formation was thermolabile, being irreversibly lost when levansucrase was heated to 35°C. S This did not effect sucrose hydrolysis or oligosaccharide formation, which were optimal at 45°C. Sucrose concentration greatly influenced the type of acceptor molecule used in the transfructosylation reactions catalysed by levansucrase. At low sucrose concentration, the predominant reaction catalysed was the hydrolysis of sucrose to free glucose and fructose. At high sucrose concentrations, oligosaccharide production was the major reaction catalysed.

Keywords

Fructose Oligosaccharide Sorbitol Sucrose Concentration Acceptor Molecule 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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References

  1. Balken JAM van, Dooren ThJGM van, Tweel WJJ van den, Kamphuis J, Meijer EM (1991) Production of 1-kestose with intact mycelium of Aspergillus phoenicis containing sucrose-1F-fructosyltransferase. Appl Microbiol Biotechnol 35:216–221Google Scholar
  2. Carlsson J (1970) A levansucrase from Streptococcus mutans. Cariesv Res 4:97–113Google Scholar
  3. Cote GL, Imam SH (1989) Purification and properties of an extracellular levansucrase from Erwinia herbicola NRRL B-1678. Carbohydr Res 190:299–307Google Scholar
  4. Crittenden RG, Doelle HW (1993) Structural identification of oligosaccharides produced by Zymomonas mobilis levansucrase. Biotechnol Lett 15:1055–1060Google Scholar
  5. Dawes EA, Ribbons DW, Rees DA, (1966) Sucrose utilization by Zymomonas mobilis: formation of a levan. Biochem J 98: 804–812Google Scholar
  6. Dedonder R (1966) Levansucrase from Bacillus subtilis. Methods Enzymol 8:500–506Google Scholar
  7. Doelle HW, Greenfield PF (1985a) The production of ethanol from sucrose using Zymomonas mobilis. Appl Microbiol Biotechnol 22:405–410Google Scholar
  8. Doelle HW, Greenfield PF (1985b) Fermentation pattern of Zymomonas mobilis at high sucrose concentrations. Appl Microbiol Biotechnol 22:411–415Google Scholar
  9. Doelle MB, Greenfield PF, Doelle HW (1990a) Effect of mineral ions on ethanol formation during sugar cane molasses fermentation using Zymomonas mobilis ATCC 39676. Process Biochem 25:151–156Google Scholar
  10. Doelle MB, Greenfield PF, Doelle HW (1990b) The relationship between sucrose hydrolysis, sorbitol formation and mineral ion concentration during bioethanol formation using Zymomonas mobilis 2716. Appl Microbiol Biotechnol 34:160–167Google Scholar
  11. Doelle HW, Kennedy LD, Doelle MB (1991) Scale-up of ethanol production from sugar cane using Zymomonas mobilis. Biotechnol Lett 13:131–136Google Scholar
  12. Doelle HW, Kirk L, Crittenden R, Toh H, Doelle MB (1993) Zymomonas mobilis — science and industrial application. CRC Crit Rev Biotechnol 13:57–98Google Scholar
  13. Dziezak JD (1986) Special report: sweeteners and product development. Food Technol 40:111–128Google Scholar
  14. Elisashvili VI (1980) Purification and properties of levansucrase of Gluconobacter oxydans. Biokhimiya 45:20–27Google Scholar
  15. Gunasekaran P, Karunakaran BC, Mukundan AG, Preziosi L, Baratti J (1990) Cloning and sequencing of the sac A gene: characterization of a sucrase from Zymomonas mobilis. J Bacteriol 172:6727–6735Google Scholar
  16. Hestrin S, Feingold DS, Avigad G (1956) The mechanism of polysaccharide production from sucrose. 3. Donor-acceptor specificity of levansucrase from Aerobacter levanicum. Biochem J 64:340–351Google Scholar
  17. Johns MR, Greenfield PF, Doelle HW (1991) Byproducts from Zymomonas mobilis. Adv Biochem Eng/Biotechnol 44:97–121Google Scholar
  18. Jones CW, Doelle HW (1991) Kinetic control of ethanol production by Zymomonas mobilis. Appl Microbiol Biotechnol 35:4–9Google Scholar
  19. Kannan TR, Mukundan AG, Gunasekaran P (1993) Fermentation characteristics of levansucrase mutants of Zymomonas mobilis. J Ferment Bioeng 75:265–270Google Scholar
  20. Kirk LA, Doelle HW (1992) The effects of potassium and chloride ions on the ethanolic fermentation of sucrose by Zymomonas mobilis. Appl Microbiol Biotechnol 37:88–93Google Scholar
  21. Lampen JO (1971) Yeast and Neurospora invertases. In: Boyer PD (ed) The enzymes, vol 4, 3rd edn. Academic Press, London, pp 291–303Google Scholar
  22. Lyness EW, Doelle HW (1980) Effect of temperature on sucrose to ethanol conversion by Zymomonas mobilis strains. Biotechnol Lett 2:549–554Google Scholar
  23. Lyness EW, Doelle HW (1983) Levansucrase from Zymomonas mobilis. Biotechnol Lett 5:345–350Google Scholar
  24. Mortatti MDPL, Park YK (1988) Comparative fementation of glucose and sucrose by Zymomonas mobilis. Rev Microbiol 19:253–257Google Scholar
  25. Myrbäck K (1960) Invertases. In: Boyer PD, Lardy H, Myrback K (eds) The enzymes, vol 4, 2nd edn. Academic press, London, pp 379–396Google Scholar
  26. O'Mullan P, Szakacs-Dobozi M, Eveleigh DE (1991) Identification of saccharolytic enzymes of Zymomonas mobilis CP4. Biotechnol Lett 13:137–142Google Scholar
  27. Pabst MJ (1977) Levan and levansucrase of Actinomyces viscosus. Infect Immun 15:518–526Google Scholar
  28. Preziosi L, Michel GPF, Barrati L (1990a) Sucrose metabolism in Zymomonas mobilis: production and localization of sucrase and levansucrase activities. Can J Microbiol 36:159–163vGoogle Scholar
  29. Preziosi L, Michel GPF, Barrati L (1990b) Characterization of sucrose-hydrolyzing enzymes of Zymomonas mobilis. Arch Microbiol 153:181–186Google Scholar
  30. Reece ET, Avigad G (1966) Purification of levansucrase by precipitation with levan. Biochem Biophys Acta 113:79–83Google Scholar
  31. Strzelecki AT, Goodman AE, Rogers PL, Watson JM (1993a) Cloning and expression of a Bacillus α-glucosidase gene in Escherichia coli, Zymomonas mobilis, and Pseudomonas putida. J Biotechnol 29: 189–203Google Scholar
  32. Strzelecki AT, Goodman AE, Watson JM, Rogers PL (1993b) Stability and expression of a cloned α-glucosidase gene in Zymomonas mobilis grown in batch and continuous culture. Biotechnol Lett 15:679–684Google Scholar
  33. Tanaka T, Oi S, Iizuka M, Yamamoto T (1978) Levansucrase of Bacillus subtilis. Agric Biol Chem 42:323–326Google Scholar
  34. Tanaka T, Oi S, Tamamoto T (1979) Synthesis of levan by levansucrase. J. Biochem 85:287–293Google Scholar
  35. Viikari L (1984) Formation of levan and sorbitol from sucrose by Zymomonas mobilis. Appl Microbiol Biotechnol 19:252–255Google Scholar
  36. Viikari L (1988) Carbohydrate metabolism in Zymomonas mobilis. CRC Crit Rev Biotechnol 7:237–261Google Scholar
  37. Viikari L, Linko M (1986) Rate and yield limiting factors in continuous fermentation of sucrose by Zymomonas mobilis. Biotechnol Lett 8:139–144Google Scholar
  38. Yanase H, Fukushi H, Ueda N, Maeda Y, Toyoda A, Tonomura K (1991) Cloning, sequencing, and characterization of the intracellular invertase gene from Zymomonas mobilis. Agric Biol Chem 55:1383–1390Google Scholar
  39. Yanase H, Iwata M, Nakahigashi R, Kita K, Kato N, Tonomura K (1992) Purification, crystallization, and properties of the extracellular levansucrase from Zymomonas mobilis.Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • R. G. Crittenden
    • 1
  • H. W. Doelle
    • 1
  1. 1.Department of MicrobiologyThe University of QueenslandBrisbaneAustralia

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