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Archives of Microbiology

, Volume 143, Issue 3, pp 209–215 | Cite as

Purification and properties of trehalase in Frankia ArI3

  • Mary F. Lopez
  • John G. Torrey
Original Papers

Abstract

Trehalase was purified from cultures of Frankia strain ArI3 grown on media with or without NH4Cl. The purified enzyme was specific for trehalose, exhibited a broad pH optimum of pH 4.5 to 5.3 and had a Km for trehalose of 4.2 mM. The trehalase was inhibited in vitro completely by sucrose, glucose and mannose and partially by mannitol and sorbitol. In addition to the specific trehalase, a mixture of non-specific α- and β-glucosidases which exhibited some activity with α,α-trehalose as a substrate were also partially purified in Frankia extracts made from nitrogen-fixing cells. These enzymes were not detected in the purifications of crude extracts made from non-nitrogen-fixing cells (grown on media supplemented with NH4Cl). Trehalase activity in crude extracts increased over time when cells were induced to fix nitrogen, and the maximum specific activity of trehalase from nitrogen-fixing cultures was 4 times the maximum activity from non-fixing cultures. Trehalase activity was also examined in crude extracts made from Frankia vesicle clusters isolated from Alnus rubra nitrogen-fixing nodules infected with ArI3. The maximum activity of trehalase in these clusters was 6–7 times greater than in the nitrogenfixing pure cultures of ArI3 and 26–33 times greater than the non-fixing pure cultures.

Key words

Frankia Trehalose Trehalase Nitrogen-fixation Carbon metabolism 

Abbreviations

pcv

packed cell volume

DTE

dithioerythritol

PMSF

phenylmethylsulphonylfluoride

EDTA

sodium ethylenediaminetetraacetate

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References

  1. Akkermans ADL, Huss-Danell K, Roelofsen W (1981) Enzymes of the tricarboxylic acid cycle and the malate-aspartate shuttle in the N2-fixing endophyte of Alnus glutinosa. Physiol Plant 53:289–294Google Scholar
  2. Avigad G, Ziv G, Newfeld EF (1965) Intracellular trehalase of a hybrid yeast. Biochem 97:715–722Google Scholar
  3. Benson DR (1982) Isolations of Frankia strains from alder actinorhiza root nodules. Appl Env Microbiol 44:461–465Google Scholar
  4. Benson DR, Eveleigh DE (1979) Ultrastructure of the nitrogenfixing symbiont of Myrica pensylvanica L. (Bayberry) root nodules. Bot Gaz 140:(Suppl) S15-S21Google Scholar
  5. Berry A, Torrey JG (1979) Isolation and characterization in vivo and in vitro of an actinomycetous endophyte from Alnus rubra Bong. In: Gordon JC, Wheeler CT, Perry DA (eds) Symbiotic nitrogen fixation in the management of temperate forests. Forest Research Lab., Oregon State University, Corvallis, ORGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein binding. Anal Biochem 72:248–254Google Scholar
  7. Crabbe LH, Gussin AES, Tyrell EA (1969) Trehalase induction in Bacillus cereus. Experientia 25:793Google Scholar
  8. Elbein A (1974) The metabolism of α,α-trehalose. Adv Carbo Chem Biochem 30:227–256Google Scholar
  9. Elbein AD, Mitchell M (1973) Levels of glycogen and trehalase in Mycobacterium smegmatis and the purification and properties of the glycogen synthetase. J Bacteriol 113:863–873Google Scholar
  10. Fontaine MF, Lancelle SA, Torrey JG (1984) Inftiation and ontogeny of vesicles in cultured Frankia sp. strain HFPArI3. J Bacteriol 160:921–927Google Scholar
  11. Gilby AR, Wyatt SS, Wyatt GR (1967) Trehalases from the cockroach, Blaberus discoidales: activation, solubilization and properties of the muscle enzyme and some properties of the intestinal enzyme. Acta Biochim Pol 14:83–100Google Scholar
  12. Guilloux E, Arcila MA, Courtois JE, Mourmrikoff V (1971) La trehalase de Pseudomonas fluorescens. Biochimie 53:853–857Google Scholar
  13. Gussin AES, Wyatt GR (1965) Membrane bound trehalase from Cecropia silk moth muscle. Arch Biochem Biophys 112:626–634Google Scholar
  14. Hey A, Elbein AD (1968) Partial purification and properties of a trehalase from Streptomyces hygroscopicus. J Bacteriol 96: 105–110Google Scholar
  15. Hey-Ferguson A, Mitchell M, Elbein AD (1973) Trehalose metabolism in germinating spores of Streptomyces hygroscopicus. J Bacteriol 116:1084–1085Google Scholar
  16. Hill EP, Sussmann AS (1963) Purification and properties of trehalase(s) from Neurospora. Arch Biochem Biophys 102:389–396Google Scholar
  17. Hirokazu I, Shimoda C (1981) Changes in trehalose content and trehalase activity during spore germination in fission yeast, Schizosaccharomyces pombe. Arch Microbiol 129:19–20Google Scholar
  18. Horikoshi K, Ikeda Y (1966) Trehalase in conidia of Aspergillus oryzae. J Bacteriol 91:1883–1887Google Scholar
  19. Lefebvre YA, Huber RE (1970) Solubilization, purification and some properties of trehalase from honeybee (Apis mellifera). Arch Biochem Biophys 140:514–520Google Scholar
  20. Lopez MF, Whaling CS, Torrey JG (1983) The polar lipids and free sugars of Frankia in culture. Can J Bot 61:2834–2842Google Scholar
  21. Lopez MF, Fontaine MS, Torrey JG (1984) Levels of trehalose and glycogen in Frankia sp. HFPArI3 (Actinomycetales) Can J Microbiol 30:746–752Google Scholar
  22. Marechal LR, Belocopitow E (1972) Metabolism of trehalose in Euglena gracilis. I. Partial purification and some properties of trehalose phosphorylase. J Biol Chem 247:3223–3228Google Scholar
  23. Murry MA, Fontaine MS, Torrey JG (1984) Growth kinetics and nitrogenase induction in Frankia sp. HFPArI3 grown in batch culture. Plant Soil 78:61–78Google Scholar
  24. Panek A (1963) Function of trehalose in backer's yeast (Saccharomyces cerevisiae). Arch Biochem Biophys 100:422–425Google Scholar
  25. Paulsen R (1971) Characterization of trehalase from labial glands of ants. Arch Biochem Biophys 142:170–176Google Scholar
  26. Rao PS, Niederpruem DJ (1969) Carbohydrate metabolism during morphogenesis of Corprinus lagopus (sensu Buller). J Bacteriol 100:1222–1228Google Scholar
  27. Streeter JG (1982) Enzymes of sucrose, maltose, and α,α-trehalose catabolism in soybean root nodules. Planta 155:112–115Google Scholar
  28. Thevelein JM (1984) Regulation of trehalose mobilization in fungi. Microbiol Rev 48:42–59Google Scholar
  29. Tjepkema JD, Ormerod W, Torrey JG (1980) Vesicle formation and acetylene reduction activity in Frankia sp. CpI1 cultured in defined nutrient media. Nature (Lond) 287:633–635Google Scholar
  30. VanLaere AJ, Hendrix P (1983) Cyclic AMP-dependent in vitro activation of trehalase from dormant Phycomyces blakesleeanus spores. J Gen Microbiol 129:3287–3290Google Scholar
  31. Zobel RW, DelTredici P, Torrey JG (1976) Method for growing plants aeroponically. Plant Physiol 57:344–346Google Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • Mary F. Lopez
    • 1
  • John G. Torrey
    • 2
  1. 1.Department of BotanyUniversity of MassachusettsAmherstUSA
  2. 2.Cabot FoundationHarvard UniversityPetershamUSA

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