, Volume 22, Issue 6, pp 889–894 | Cite as

Improvement of the transfucosylation activity of α-l-fucosidase from Thermotoga maritima for the synthesis of fucosylated oligosaccharides in the presence of calcium and sodium

  • Francisco Guzmán-Rodríguez
  • Sergio Alatorre-Santamaría
  • Lorena Gómez-Ruiz
  • Gabriela Rodríguez-Serrano
  • Mariano García-Garibay
  • Alma Cruz-GuerreroEmail author
Original Paper


The influence of CaCl2 and NaCl in the hydrolytic activity and the influence of CaCl2 in the synthesis of fucosylated oligosaccharides using α-l-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-l-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (aw) of 0.9672 and of 138% in the presence of 1.1 M CaCl2 (aw 0.9581). In addition, the hydrolytic activity was higher when using CaCl2 compared to NaCl at aw of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl2 in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl2 favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl2 did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl2, the decrement in aw and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides.


Fucosidase Fucosylated oligosaccharides Thermotoga maritima Transfucosylation Water activity 



Fucosyl oligosaccharides


Water activity






Hydrolytic specific activity


Acceptor/donor molar ratio


Transfucosylation rate


Hydrolysis rate


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aminoff D, Furukawa K (1970) Enzymes that destroy blood group specificity. I. Purification and properties of alpha-l-fucosidase from Clostridium perfringens. J Biol Chem 245:1659–1669PubMedGoogle Scholar
  2. Bridiau N, Issaoui N, Maugard T (2010) The effects of organic solvents on the efficiency and regioselectivity of N-acetyl-lactosamine synthesis, using the β-galactosidase from Bacillus circulans in hydro-organic media. Biotechnol Prog 26:1278–1289CrossRefPubMedGoogle Scholar
  3. Bronnenmeier K, Kern A, Liebl W, Staudenbauer WL (1995) Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials. Appl Environ Microbiol 61:1399–1407PubMedPubMedCentralGoogle Scholar
  4. Cruz-Guerrero AE, Gomez-Ruiz L, Viniegra-Gonzalez G, Barzana E, Garcia-Garibay M (2006) Influence of water activity in the synthesis of galactooligosaccharides produced by a hyperthermophilic beta-glycosidase in an organic medium. Biotechnol Bioeng 93:1123–1129CrossRefPubMedGoogle Scholar
  5. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  6. Lezyk M, Jers C, Kjaerulff L, Gotfredsen CH, Mikkelsen MD, Mikkelsen JD (2016) Novel α-l-fucosidases from a soil metagenome for production of fucosylated human milk oligosaccharides. PLoS One 11:e0147438CrossRefPubMedPubMedCentralGoogle Scholar
  7. Liu S, Kulinich A, Cai ZP, Ma HY, Du YM, Lv YM, Liu L, Voglmeir J (2016) The fucosidase-pool of Emticicia oligotrophica: biochemical characterization and transfucosylation potential. Glycobiology 26:871–879CrossRefPubMedGoogle Scholar
  8. Matsuki T, Yahagi K, Mori H et al (2016) A key genetic factor for fucosyllactose utilization affects infant gut microbiota development. Nat Commun 7:11939CrossRefPubMedPubMedCentralGoogle Scholar
  9. Möller J, Schroer MA, Erlkamp M, Grobelny S, Paulus M, Tiemeyer S, Wirkert FJ, Tolan M, Winter R (2012) The effect of ionic strength, temperature, and pressure on the interaction potential of dense protein solutions: from nonlinear pressure response to protein crystallization. Biophys J 102:2641–2648CrossRefPubMedPubMedCentralGoogle Scholar
  10. Nagae M, Tsuchiya A, Katayama T, Yamamoto K, Wakatsuki S, Kato R (2007) Structural basis of the catalytic reaction mechanism of novel 1,2-alpha-l-fucosidase from Bifidobacterium bifidum. J Biol Chem 282:18497–18509CrossRefPubMedGoogle Scholar
  11. Newburg DS (2009) Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans. J Anim Sci 87:26–34CrossRefPubMedGoogle Scholar
  12. Palcic MM (2011) Glycosyltransferases as biocatalysts. Curr Opin Chem Biol 15:226–233CrossRefPubMedGoogle Scholar
  13. Rodríguez-Díaz J, Carbajo R, Pineda-Lucena A, Monedero V, Yebra M (2013) Synthesis of fucosyl-N-acetylglucosamine disaccharides by transfucosylation using α-L-fucosidases from Lactobacillus casei. Appl Environ Microbiol 79:3847–3850CrossRefPubMedPubMedCentralGoogle Scholar
  14. Stokes RH, Robinson RA (1949) Standard solutions for humidity control at 25 °C. Ind Eng Chem 41:2013CrossRefGoogle Scholar
  15. Tarling CA, He S, Sulzenbacher G, Bignon C, Bourne Y, Henrissat B, Withers SG (2003) Identification of the catalytic nucleophile of the family 29 alpha-l-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis. J Biol Chem 278:47394–47399CrossRefPubMedGoogle Scholar
  16. Vera C, Guerrero C, Wilson L, Illanes A (2017) Optimization of reaction conditions and the donor substrate in the synthesis of hexyl-β-d-galactoside. Process Biochem 58:128–136CrossRefGoogle Scholar
  17. Vetere A, Galateo C, Paoletti S (1997) All-aqueous, regiospecific transglycosylation synthesis of 3-O-alpha-l-fucopyranosyl-2-acetamido-2-deoxy-d-glucopyranose, a building block for the synthesis of branched oligosaccharides. Biochem Biophys Res Commun 234:358–361CrossRefPubMedGoogle Scholar
  18. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43CrossRefPubMedPubMedCentralGoogle Scholar
  19. Warmerdam A, Wang J, Boom RM, Janssen AE (2013) Effects of carbohydrates on the oNPG converting activity of β-galactosidases. J Agric Food Chem 61:6458–6464CrossRefPubMedGoogle Scholar
  20. Winterhalter C, Liebl W (1995) Two extremely thermostable xylanases of the hyperthermophilic bacterium Thermotoga maritima MSB8. Appl Environ Microbiol 61:1810–1815PubMedPubMedCentralGoogle Scholar
  21. Zeuner B, Jers C, Mikkelsen JD, Meyer AS (2014) Methods for improving enzymatic trans-glycosylation for synthesis of human milk oligosaccharide biomimetics. J Agric Food Chem 62:9615–9631CrossRefPubMedGoogle Scholar
  22. Zeuner B, Muschiol J, Holck J, Lezyk M, Gedde MR, Jers C, Mikkelsen JD, Meyer AS (2018) Substrate specificity and transfucosylation activity of GH29 α-l-fucosidases for enzymatic production of human milk oligosaccharides. New Biotechnol 41:34–45CrossRefGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Departamento de BiotecnologíaUniversidad Autónoma Metropolitana-IztapalapaIztapalapa, Mexico CityMexico
  2. 2.Departamento de Ciencias de la Alimentación, División de Ciencias Biológicas y de la SaludUniversidad Autónoma Metropolitana, Unidad LermaLerma de VilladaMexico

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