Marine Biotechnology

, Volume 18, Issue 1, pp 133–143 | Cite as

Enzyme-Assisted Preparation of Furcellaran-Like κ-/β-Carrageenan

  • Aurélie Préchoux
  • Sabine Genicot
  • Hélène Rogniaux
  • William HelbertEmail author
Original Article


Carrageenans are sulfated galactans that are widely used in industrial applications for their thickening and gelling properties, which vary according to the amount and distribution of ester sulfate groups along the galactan backbone. To determine and direct the sulfation of κ-carrageenan moieties, we purified an endo-κ-carrageenan sulfatase (Q15XH1 accession in UniprotKB) from Pseudoalteromonas atlantica T6c extracts. Based on sequence analyses and exploration of the genomic environment of Q15XH1, we discovered and characterized a second endo-κ-carrageenan sulfatase (Q15XG7 accession in UniprotKB). Both enzymes convert κ-carrageenan into a hybrid, furcellaran-like κ-/β-carrageenan. We compared the protein sequences of these two new κ-carrageenan sulfatases and that of a previously reported ι-carrageenan sulfatase with other predicted sulfatases in the P. atlantica genome, revealing the existence of additional new carrageenan sulfatases.


Carrageenan Sulfatase Biotransformation Pseudoalteromonas atlantica 



The research leading to these results received funding from the European Community Seventh Framework Programme (FP7) under grant agreement no. 222628. Mass spectrometry analyses were conducted at the BIBS facility at the INRA Biopolymers Interaction Assemblies research unit ( We thank Mathilde Joint for her excellent technical assistance in LC-MS/MS. Special thanks to Nelly Kervarec from the “University of Bretagne Occidentale” for her expertise in the NMR analyses.

Supplementary material

10126_2015_9675_Fig9_ESM.jpg (576 kb)

(JPG 575 kb)

10126_2015_9675_Fig10_ESM.jpg (474 kb)

(JPG 474 kb)

10126_2015_9675_Fig11_ESM.jpg (594 kb)

(JPG 593 kb)

10126_2015_9675_Fig12_ESM.jpg (476 kb)

(JPG 476 kb)


  1. Anastyuk SD, Barabanova AO, Correc G, Nazarenko EL, Davydova VN, Helbert W, Dmitrenok PS, Yermak IM (2011) Analysis of structural heterogeneity of κ/β-carrageenan oligosaccharides from Tichocarpus crinitus by negative-ion ESI and tandem mass spectrometry. Carbohydr Polym 86:546–554CrossRefGoogle Scholar
  2. Appel MJ, Bertozzi CR (2015) Formylglycine, a post-translationally generated residue with unique catalytic capabilities and biotechnology applications. ACS Chem Biol 10:72–84PubMedCrossRefGoogle Scholar
  3. Barabanova AO, Yermak I, Glazunov VP, Isakov VV, Titlyanov EA, Solov’eva TF (2005) Comparative study of carrageenan from reproductive and sterile forms of Tichocarpus crinitus (Gmel.) Rupr. (Rhodophyta, Tichocarpaceae). Biochem Mosc 70:430–437CrossRefGoogle Scholar
  4. Benjdia A, Dehò G, Rabot S, Berteau O (2007) First evidences for a third sulfatase maturation system in prokaryotes from E. coli aslB and ydeM deletion mutants. FEBS Lett 581:1009–1014PubMedCrossRefGoogle Scholar
  5. Berteau O, Guillot A, Benjdia A, Rabot S (2006) A new type of bacterial sulfatase reveals a novel maturation pathway in prokaryotes. J Biol Chem 281:22464–22470PubMedCrossRefGoogle Scholar
  6. Bjerre-Petersen E, Christensen J, Hemmingsen P (1973) Chapter VII: furcellaran. In: Whistler RL (ed) Industrial gums, 2nd edn. Academic, New York, pp 123–136CrossRefGoogle Scholar
  7. Boltes I, Czapinska H, Kahnert A, von Bülow R, Dierks T, Schmidt B, von Figura K, Kertesz MA, Usón I (2001) 1.3 A structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family. Structure 9:483–491PubMedCrossRefGoogle Scholar
  8. Bond CS, Clements PR, Ashby SJ, Collyer CA, Harrop SJ, Hopwood JJ, Guss JM (1997) Structure of a human lysosomal sulfatase. Structure 5:277–289PubMedCrossRefGoogle Scholar
  9. Carlson BL, Ballister ER, Skordalakes E, King DS, Breidenbach MA, Gilmore SA, Berger JM, Bertozzi CR (2008) Function and structure of a prokaryotic formylglycine-generating enzyme. J Biol Chem 283:20117–20125PubMedPubMedCentralCrossRefGoogle Scholar
  10. Correc G, Barabanova A, Tuvikene R, Truus K, Yermak I, Helbert W (2012) Comparison of the structures of hybrid κ-/β-carrageenans extracted from Furcellaria lumbricalis and Tichocarpus crinitus. Carbohydr Polym 88:31–36CrossRefGoogle Scholar
  11. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O (2008) robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36(Web Server issue):W465–W469Google Scholar
  12. Dierks T, Schmidt B, von Figura K (1997) Conversion of cysteine to formyl-glycine: a protein modification in the endoplasmic reticulum. Proc Natl Acad Sci U S A 94:11963–11968PubMedPubMedCentralCrossRefGoogle Scholar
  13. Dierks T, Miech C, Hummerjohann J, Schmidt B, Kertesz MA, von Figura K (1998) Posttranslational formation of formylglycine in prokaryotic sulfatases by modification of either cysteine or serine. J Biol Chem 273:25560–25564PubMedCrossRefGoogle Scholar
  14. Dierks T, Lecca MR, Schlotterhose P, Schmidt B, von Figura K (1999) Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases. EMBO J 18:2084–2091PubMedPubMedCentralCrossRefGoogle Scholar
  15. Genicot S, Groisillier A, Rogniaux H, Meslet-Cladière L, Barbeyron T, Helbert W (2014) Discovery of a novel iota-carrageenan sulfatase isolated from the marine bacterium Pseudoalteromonas carrageenovora. Front Chem 2:1–15CrossRefGoogle Scholar
  16. Genicot-Joncour S, Poinas A, Richard O, Potin P, Rudolph B, Kloareg B, Helbert W (2009) The cyclization of the 3,6-anhydro ring of iota-carrageenan is catalyzed by two d-galactose-2,6-sulfurylases in the red alga Chondrus crispus. Plant Physiol 151:1609–1616PubMedPubMedCentralCrossRefGoogle Scholar
  17. Groisillier A, Hervé C, Jeudy A, Rebuffet E, Pluchon PF, Chevolot Y, Flament D, Geslin C, Morgado IM, Power D, Branno M, Moreau H, Michel G, Boyen C, Czjzek M (2010) Marine-express: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms. Microb Cell Factories 9:45–56CrossRefGoogle Scholar
  18. Guibet M, Boulenguer P, Mazoyer J, Kervarec N, Antonopoulos A, Lafosse M, Helbert W (2008) Composition and distribution of carrabiose moieties in hybrid κ-/ι-carrageenans using carrageenases. Biomacromolecules 9:408–415PubMedCrossRefGoogle Scholar
  19. Hatada Y, Mizuno M, Li ZJ, Ohta Y (2011) Hyper-production and characterization of the iota-crrageenase useful for iota-carrageenan oligosaccharide production from a deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94(T), and insight into the unusual catalytic mechanism. Mar Biotechnol 13:411–422PubMedCrossRefGoogle Scholar
  20. Henrissat B, Davies GJ (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7:637–644PubMedCrossRefGoogle Scholar
  21. Knutsen SV, Grasdalen H (1992) Analysis of carrageenans by enzymatic degradation gel filtration and 1H NMR spectroscopy. Carbohydr Polym 19:199–210CrossRefGoogle Scholar
  22. Knutsen SV, Myslabodski D, Larsen B, Usov A (1994) A modified system of nomenclature for red algal galactans. Bot Mar 37:163–169CrossRefGoogle Scholar
  23. Kolender AA, Matulewicz MC (2004) Desulfation of sulfated galactans with chlorotrimethylsilane. Characterization of β-carrageenan by 1H-NMR spectroscopy. Carbohydr Res 339:1619–1629PubMedCrossRefGoogle Scholar
  24. Kumar S, Dudley J, Nei M, Tamura K (2008) MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9:299–306PubMedPubMedCentralCrossRefGoogle Scholar
  25. Laos K, Ring S (2005) Characterisation of furcellaran from Furcellaria lumbricalis (Rhodophyta). J Appl Phycol 17:461–464CrossRefGoogle Scholar
  26. Larre C, Penninck S, Bouchet B, Lollier V, Tranquet O, Denery-Papini S, Guillon F, Rogniaux H (2010) Brachypodium distachyon grain: identification and subcellular localization of storage proteins. J Exp Bot 61:1771–1783Google Scholar
  27. McLean MW, Williamson FB (1979) Glycosulfatase from Pseudomonas carrageenovora—purification and some properties. Eur J Biochem 101:497–505PubMedCrossRefGoogle Scholar
  28. McLean MW, Williamson FB (1981) Neocarratetraose 4-O-monosulfate β-hydrolase from Pseudomonas carrageenovora. Eur J Biochem 113:447–456PubMedCrossRefGoogle Scholar
  29. Myette JR, Soundararajan V, Shriver Z, Raman R, Sasisekharan R (2009) Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum. Integrated structural and biochemical investigation of enzyme active site and substrate specificity. J Biol Chem 284:35177–35188PubMedPubMedCentralCrossRefGoogle Scholar
  30. Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217PubMedCrossRefGoogle Scholar
  31. Préchoux A, Genicot S, Rogniaux H, Helbert W (2013) Controlling carrageenan structure using a formylglycine-dependent sulfatase, an endo-4S-iota-carrageenan sulfatase. Mar Biotechnol 15:265–274PubMedCrossRefGoogle Scholar
  32. Raman R, Myette JR, Shriver Z, Pojasek K, Venkataraman G, Sasisekharan R (2003) The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum—a structural and biochemical study of the enzyme active site and saccharide substrate specificity. J Biol Chem 278:12167–12174PubMedCrossRefGoogle Scholar
  33. Renn DW, Santos GA, Dumont LE, Parent CA, Stanley NF, Stancioff DJ, Guisely KB (1993) Beta-carrageenan—isolation and characterization. Carbohydr Polym 22:247–250CrossRefGoogle Scholar
  34. Rivera-Colón Y, Schutsky EK, Kita AZ, Garman SC (2012) The structure of human GALNS reveals the molecular basis for mucopolysaccharidosis IV A. J Mol Biol 423:736–751PubMedPubMedCentralCrossRefGoogle Scholar
  35. Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42(Web Server issue):W320–W324Google Scholar
  36. Sidhu NS, Schreiber K, Proepper K, Becker S, Uson I, Sheldrick GM, Gaertner J, Kraetzner R, Steinfeld R (2014) Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA. Acta Crystallogr D70:1321–1335Google Scholar
  37. Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234PubMedCrossRefGoogle Scholar
  38. Takano R (2002) Desulfation of sulfated carbohydrates. Trends Glycosci Glycotechnol 14:343–351CrossRefGoogle Scholar
  39. Usov AI (2011) Polysaccharides of the red algae. Adv Carbohydr Chem Biochem 65:115–217PubMedCrossRefGoogle Scholar
  40. von Bülow R, Schmidt B, Dierks T, von Figura K, Usón I (2001) Crystal structure of an enzyme-substrate complex provides insight into the interaction between human arylsulfatase A and its substrates during catalysis. J Mol Biol 305:269–277CrossRefGoogle Scholar
  41. Weigl J, Yaphe W (1966) Glycosulfatase of Pseudomonas carrageenovora: desulfation of disaccharide from κ-carrageenan. Can J Microbiol 12:874–876CrossRefGoogle Scholar
  42. Yermak IM, Kim YH, Titlynov EA, Isakov VV, Solov’eva TF (1999) Chemical structure and gel properties of carrageenans from algae belonging to the Gigartinaceae and Tichocarpaceae, collected from the Russian Pacific coast. J Appl Phycol 11:41–48CrossRefGoogle Scholar
  43. ZoBell CE (1941) Studies on marine bacteria. I. The cultural requirements of heterotrophic aerobes. J Mar Res 4:41–75Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Aurélie Préchoux
    • 1
    • 2
  • Sabine Genicot
    • 1
    • 2
  • Hélène Rogniaux
    • 3
  • William Helbert
    • 1
    • 2
    • 4
    • 5
    Email author
  1. 1.Sorbonne Universités, UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de RoscoffRoscoffFrance
  2. 2.CNRS, UMR 8227, Integrative Biology of Marine ModelsStation Biologique de RoscoffRoscoffFrance
  3. 3.INRA, Biopolymers Interactions AssembliesNantesFrance
  4. 4.Centre de Recherches sur les Macromolécules Végétales (CERMAV, UPR-CNRS 5301)Affiliated with the Université Joseph Fourier (UJF)Grenoble Cedex 9France
  5. 5.Institut de Chimie Moléculaire de Grenoble (ICMG, FR-CNRS 2607)Grenoble Cedex 9France

Personalised recommendations