Applied Microbiology and Biotechnology

, Volume 63, Issue 3, pp 286–292 | Cite as

Synthesis of α-galactooligosaccharides with α-galactosidase from Lactobacillus reuteri of canine origin

  • G. Tzortzis
  • A. J. Jay
  • M. L. A. Baillon
  • G. R. Gibson
  • R. A. RastallEmail author
Original Paper


Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, α-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 °C (K M 0.55 mM, V max 0.80 μmol min−1 mg−1 of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6′ α-galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.


Lactobacillus Oligosaccharide Raffinose Melibiose Stachyose 
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.


  1. Benno Y, Endo K, Shiragami N, Sayama K, Mitsuoka T (1987) Effect of raffinose intake on human fecal microflora. Bifidobact Microflora 6:59–63Google Scholar
  2. Bulpin PV, Gidley MJ, Jeffcoat R, Underwood DJ (1990) Development of a biotechnological process for the modification of galactomannan polymers with plant α-galactosidase. Carbohydr Polym 12:155–168Google Scholar
  3. Casas IA, Dobrogosz WJ (2000) Validation of the probiotic concept: Lactobacillus reuteri confers broad-spectrum protection against disease in humans and animals. Microbial Ecol Health Dis 12:247–285CrossRefGoogle Scholar
  4. El-Ziney MG, Debevere JM (1998) The effect of reuterin on Listeria monocytogenes and E. coli 0157:H7 in milk and cottage cheese. J Food Protect 61:1275–1280Google Scholar
  5. Fridjonsson O, Watzlawick H, Mattes R (2000) The structure of the alpha-galactosidase gene loci in Thermus brockianus ITI360 and Thermus thermophilus TH125. Extremophiles 4:23–33CrossRefPubMedGoogle Scholar
  6. Fuller R, Gibson GR (1998) Probiotics and prebiotics: microflora management for improved gut health. Clin Microbiol Infect 4:477–480Google Scholar
  7. Ganzle MG, Holtzel A, Walter J, Jung G, Hammes WP (2000) Characterization of reutericyclin produced by Lactobacillus reuteri LTH2584. Appl Environ Microbiol 66:4325–4333Google Scholar
  8. Garregg PJ (1990) Phase-transfer for selective substitution in carbohydrates and inositols. Abstr PAP AM Chem 199:10–15Google Scholar
  9. Garro MS, de Giori GS, de Valdez GF, Oliver G (1993) Characterization of alpha-galactosidase from Lactobacillus fermentum. J Appl Bacteriol 75:485–488Google Scholar
  10. Geel-Schuten GH van, Faber EJ, Smit E, Bonting K, Dijkhuizen L (1999) Biochemical and structural characterization of the glucan and fructan exopolysaccharides synthesized by the Lactobacillus reuteri wild type strain and by mutant strains. App Environ Microbiol 65:3008–3014Google Scholar
  11. Gibson GR, Roberfroid MB (1995) Dietary modulation of the human colonic microbiota. Introducing the concept of prebiotics. J Nutr 125:1401–1412PubMedGoogle Scholar
  12. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  13. Laere KMJ van, Hartemink R, Beldman G, Pitson S, Dijkema C, Schols HA, Voragen AGJ (1999) Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 α-galactosidase. Appl Microbiol Biotechnol 52:681–688PubMedGoogle Scholar
  14. Li X, Yang L, Yan P, Zuo F, Jin F (1997) Factors regulating production of alpha-galactosidase from Bacillus sp. JF(2). Lett Appl Microbiol 25:1–4PubMedGoogle Scholar
  15. Mital BK, Schallenberger RS, Steinkraus KH (1973) α-Galactosidase activity of lactobacilli. Appl Microbiol 26:783–788PubMedGoogle Scholar
  16. Mitsutomi M, Ohtakara A (1988) Isolation and identification of oligosaccharides produced from raffinose by transgalactosylation reaction of thermostable α-galactosidase from Pycnoporus cinnabarinus. Agric Biol Chem 52:2305–2311Google Scholar
  17. Molin G, Jeppsson B, Johansson ML, Ahrne S, Nobaek S, Stahl M, Bengmark S (1993) Numerical taxonomy of Lactobacillus spp. associated with healthy and diseased mucosa of the human intestines. J Appl Bacteriol 74:314–323PubMedGoogle Scholar
  18. Ohshima T, Murray GJ, Swaim WD, Longenecker G, Quirk JM, Cardarelli CO, Sugimoto Y, Pastan I, Gottesman MM, Brady RO, Kulkarni AB (1997) α-Galactosidase A deficient mice: a model of Fabry disease. Proc Natl Acad Sci USA 94:2540–2544CrossRefPubMedGoogle Scholar
  19. Peterbauer T, Richter A (2001) Biochemistry and physiology of raffinose family oligosaccharides and galactosyl cyclitols in seeds. Seed Sci Res 11:185–197Google Scholar
  20. Rabiu BA, Jay AC, Gibson GR, Rastall RA (2001) Synthesis and fermentation properties of novel galactooligosaccharides by β-galactosidases from Bifidobacterium spp. Appl Environ Microbiol 67:2526–2530CrossRefPubMedGoogle Scholar
  21. Rastall RA, Bucke C (1992) Enzymatic synthesis of oligosaccharides. Biotechnol Gen Eng Rev 10:253–281Google Scholar
  22. Rolfe VE, Adams CA, Butterwick RF, Batt RM (2002) Relationships between fecal consistency and colonic microstructure and absorptive function in dogs with and without nonspecific dietary sensitivity. Am J Vet Res 63:617–622PubMedGoogle Scholar
  23. Rycroft CE, Jones MR, Gibson GR, Rastall RA (2001) A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. J Appl Microbiol 91:878–887CrossRefPubMedGoogle Scholar
  24. Sakai K, Tachiki T, Kumagai H, Tochikura T (1987) Hydrolysis of α-galactosyl oligosaccharides in soymilk by α-galactosidase of Bifidobacterium breve 203. Agric Biol Chem 51:315–321Google Scholar
  25. Sandine WE (1979) Roles of Lactobacillus in the intestinal tract. J Food Protect 42:259–262Google Scholar
  26. Savel'ev AN, Ibatyllin FM, Eneyskay EV, Kachurin AM, Neustroev KN (1996) Enzymatic properties of α-galactosidase from Trichoderma reesei. Carbohydr Res 296:261–273CrossRefGoogle Scholar
  27. Shibuya H, Nagasaki H, Kaneko S, Yoshida S, Park GG, Kusakabe I, Kobayashi H (1998) Cloning and high-level expression of alpha-galactosidase cDNA from Penicillium purpurogenum. Appl Env Microbiol 64:4489–4494Google Scholar
  28. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76–85PubMedGoogle Scholar
  29. Toone ESJ, Simon ES (1989) Enzyme-catalyzed synthesis of carbohydrates. Tetrahedron 45:5365–5422CrossRefGoogle Scholar
  30. Withers SG (2001) Mechanisms of glycosyl transferases and hydrolases. Carb Polym 44:325–337CrossRefGoogle Scholar
  31. Xiao M, Tanaka K, Qian XM, Yamamoto K, Kumagai H (2000) High yield production and characterization of α-galactosidase from Bifidobacterium breve grown on raffinose. Biotechnol Lett 22:747–751CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • G. Tzortzis
    • 1
  • A. J. Jay
    • 2
  • M. L. A. Baillon
    • 3
  • G. R. Gibson
    • 1
  • R. A. Rastall
    • 1
    Email author
  1. 1.School of Food BiosciencesThe University of ReadingReadingUK
  2. 2.Institute of Food ResearchNorwichUK
  3. 3.Waltham Centre for Pet NutritionMelton MowbrayUK

Personalised recommendations