Advertisement

Applied Microbiology and Biotechnology

, Volume 102, Issue 16, pp 6987–6996 | Cite as

Biochemical characterization of an ulvan lyase from the marine flavobacterium Formosa agariphila KMM 3901T

  • Lukas Reisky
  • Christian Stanetty
  • Marko D. Mihovilovic
  • Thomas Schweder
  • Jan-Hendrik Hehemann
  • Uwe T. BornscheuerEmail author
Biotechnologically relevant enzymes and proteins
  • 601 Downloads

Abstract

Carbohydrates are the product of carbon dioxide fixation by algae in the ocean. Their polysaccharides are depolymerized by marine bacteria, with a vast array of carbohydrate-active enzymes. These enzymes are important tools to establish biotechnological processes based on algal biomass. Green tides, which cover coastal areas with huge amounts of algae from the genus Ulva, represent a globally rising problem, but also an opportunity because their biomass could be used in biorefinery processes. One major component of their cell walls is the anionic polysaccharide ulvan for which the enzymatic depolymerization remains largely unknown. Ulvan lyases catalyze the initial depolymerization step of this polysaccharide, but only a few of these enzymes have been described. Here, we report the cloning, overexpression, purification, and detailed biochemical characterization of the endolytic ulvan lyase from Formosa agariphila KMM 3901T which is a member of the polysaccharide lyase family PL28. The identified biochemical parameters of the ulvan lyase reflect adaptation to the temperate ocean where the bacterium was isolated from a macroalgal surface. The NaCl concentration has a high influence on the turnover number of the enzyme and the affinity to ulvan. Divalent cations were shown to be essential for enzyme activity with Ca2+ likely being the native cofactor of the ulvan lyase. This study contributes to the understanding of ulvan lyases, which will be useful for future biorefinery applications of the abundant marine polysaccharide ulvan.

Keywords

Ulvan Lyase Polysaccharides Green tide Formosa agariphila Enzyme characterization 

Notes

Acknowledgements

We thank Frank Unfried for providing biomass of F. agariphila.

Funding information

We kindly thank the German Research Foundation (DFG) for funding through the Research Unit FOR2406. J. H.H. acknowledges funding by the Emmy-Noether-Program of the DFG, grant number HE 7217/1-1.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9142_MOESM1_ESM.pdf (2.9 mb)
ESM 1 (PDF 2.88 mb)

References

  1. Badur AH, Jagtap SS, Yalamanchili G, Lee J-K, Zhao H, Rao CV (2015) Alginate lyases from alginate-degrading Vibrio splendidus 12B01 are endolytic. Appl Environ Microbiol 81:1865–1873.  https://doi.org/10.1128/AEM.03460-14 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Becker S, Scheffel A, Polz MF, Hehemann J-H (2017) Accurate quantification of laminarin in marine organic matter with enzymes from marine microbes. Appl Environ Microbiol 83:e03389–e03316.  https://doi.org/10.1128/AEM.03389-16 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bikker P, van Krimpen MM, van Wikselaar P, Houweling-Tan B, Scaccia N, van Hal JW, Huijgen WJJ, Cone JW, López-Contreras AM (2016) Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels. J Appl Phycol 28:3511–3525.  https://doi.org/10.1007/s10811-016-0842-3 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Cardoso S, Carvalho L, Silva P, Rodrigues M, Pereira O, Pereira L (2014) Bioproducts from seaweeds: a review with special focus on the Iberian Peninsula. Curr Org Chem 18:896–917.  https://doi.org/10.2174/138527281807140515154116 CrossRefGoogle Scholar
  5. Cesário MT, da Fonseca MMR, Marques MM, de Almeida MCMD (2018) Marine algal carbohydrates as carbon sources for the production of biochemicals and biomaterials. Biotechnol Adv 36:798–817.  https://doi.org/10.1016/j.biotechadv.2018.02.006 CrossRefPubMedGoogle Scholar
  6. Charlier RH, Morand P, Finkl CW (2008) How Brittany and Florida coasts cope with green tides. Int J Environ Stud 65:191–208.  https://doi.org/10.1080/00207230701791448 CrossRefGoogle Scholar
  7. Collén PN, Jeudy A, Sassi J-F, Groisillier A, Czjzek M, Coutinho PM, Helbert W (2014) A novel unsaturated β-glucuronyl hydrolase involved in ulvan degradation unveils the versatility of stereochemistry requirements in family GH105. J Biol Chem 289:6199–6211.  https://doi.org/10.1074/jbc.M113.537480 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Correc G, Hehemann J-H, Czjzek M, Helbert W (2011) Structural analysis of the degradation products of porphyran digested by Zobellia galactanivorans β-porphyranase A. Carbohydr Polym 83:277–283.  https://doi.org/10.1016/j.carbpol.2010.07.060 CrossRefGoogle Scholar
  9. Dickson AG (1993) The measurement of sea water pH. Mar Chem 44:131–142.  https://doi.org/10.1016/0304-4203(93)90198-W CrossRefGoogle Scholar
  10. Field CB (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281:237–240.  https://doi.org/10.1126/science.281.5374.237 CrossRefPubMedGoogle Scholar
  11. Foran E, Buravenkov V, Kopel M, Mizrahi N, Shoshani S, Helbert W, Banin E (2017) Functional characterization of a novel “ulvan utilization loci” found in Alteromonas sp. LOR genome. Algal Res 25:39–46.  https://doi.org/10.1016/j.algal.2017.04.036 CrossRefGoogle Scholar
  12. Glew MD, Veith PD, Peng B, Chen Y-Y, Gorasia DG, Yang Q, Slakeski N, Chen D, Moore C, Crawford S, Reynolds EC (2012) PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis. J Biol Chem 287:24605–24617.  https://doi.org/10.1074/jbc.M112.369223 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hehemann J-H, Correc G, Barbeyron T, Helbert W, Czjzek M, Michel G (2010) Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature 464:908–912.  https://doi.org/10.1038/nature08937 CrossRefPubMedGoogle Scholar
  14. Hehemann J-H, Boraston AB, Czjzek M (2014) A sweet new wave: structures and mechanisms of enzymes that digest polysaccharides from marine algae. Curr Opin Struct Biol 28:77–86.  https://doi.org/10.1016/j.sbi.2014.07.009 CrossRefPubMedGoogle Scholar
  15. Kim HT, Lee S, Kim KH, Choi I-G (2012) The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production. Bioresour Technol 107:301–306.  https://doi.org/10.1016/j.biortech.2011.11.120 CrossRefPubMedGoogle Scholar
  16. Kopel M, Helbert W, Belnik Y, Buravenkov V, Herman A, Banin E (2016) New family of ulvan lyases identified in three isolates from the Alteromonadales order. J Biol Chem 291:5871–5878.  https://doi.org/10.1074/jbc.M115.673947 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kraan S (2012) Algal polysaccharides, novel applications and outlook. In: Chang C-F (ed) Carbohydrates—comprehensive studies on glycobiology and glycotechnology. InTechGoogle Scholar
  18. Kwon HK, Kang H, Oh YH, Park SR, Kim G (2017) Green tide development associated with submarine groundwater discharge in a coastal harbor, Jeju, Korea. Sci Rep 7:6325.  https://doi.org/10.1038/s41598-017-06711-0 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Ladner CL, Yang J, Turner RJ, Edwards RA (2004) Visible fluorescent detection of proteins in polyacrylamide gels without staining. Anal Biochem 326:13–20.  https://doi.org/10.1016/j.ab.2003.10.047 CrossRefPubMedGoogle Scholar
  20. Lahaye M (1998) NMR spectroscopic characterisation of oligosaccharides from two Ulva rigida ulvan samples (Ulvales, Chlorophyta) degraded by a lyase. Carbohydr Res 314:1–12.  https://doi.org/10.1016/S0008-6215(98)00293-6 CrossRefPubMedGoogle Scholar
  21. Lahaye M, Kuhlenkamp R (1999) Chemical composition and 13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales, Chlorophyta). J Appl Phycol 11:1.  https://doi.org/10.1023/A:1008063600071
  22. Lahaye M, Robic A (2007) Structure and functional properties of ulvan, a polysaccharide from green seaweeds. Biomacromolecules 8:1765–1774.  https://doi.org/10.1021/bm061185q CrossRefPubMedGoogle Scholar
  23. Lahaye M, Brunel M, Bonnin E (1997) Fine chemical structure analysis of oligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp. (Ulvales, Chlorophyta). Carbohydr Res 304:325–333.  https://doi.org/10.1016/S0008-6215(97)00270-X CrossRefPubMedGoogle Scholar
  24. Li C, Wen A, Shen B, Lu J, Huang Y, Chang Y (2011) FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnol 11:92.  https://doi.org/10.1186/1472-6750-11-92 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495.  https://doi.org/10.1093/nar/gkt1178 CrossRefPubMedGoogle Scholar
  26. Mann AJ, Hahnke RL, Huang S, Werner J, Xing P, Barbeyron T, Huettel B, Stüber K, Reinhardt R, Harder J, Glöckner FO, Amann RI, Teeling H (2013) The genome of the alga-associated marine flavobacterium Formosa agariphila KMM 3901T reveals a broad potential for degradation of algal polysaccharides. Appl Environ Microbiol 79:6813–6822.  https://doi.org/10.1128/AEM.01937-13 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Mao W, Zang X, Li Y, Zhang H (2006) Sulfated polysaccharides from marine green algae Ulva conglobata and their anticoagulant activity. J Appl Phycol 18:9–14.  https://doi.org/10.1007/s10811-005-9008-4 CrossRefGoogle Scholar
  28. McBride MJ, Zhu Y (2013) Gliding motility and Por secretion system genes are widespread among members of the phylum Bacteroidetes. J Bacteriol 195:270–278.  https://doi.org/10.1128/JB.01962-12 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Nedashkovskaya OI (2006) Formosa agariphila sp. nov., a budding bacterium of the family Flavobacteriaceae isolated from marine environments, and emended description of the genus Formosa. Int J Syst Evol Microbiol 56:161–167.  https://doi.org/10.1099/ijs.0.63875-0 CrossRefPubMedGoogle Scholar
  30. Nyvall Collén P, Sassi J-F, Rogniaux H, Marfaing H, Helbert W (2011) Ulvan lyases isolated from the Flavobacteria Persicivirga ulvanivorans are the first members of a new polysaccharide lyase family. J Biol Chem 286:42063–42071.  https://doi.org/10.1074/jbc.M111.271825 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786.  https://doi.org/10.1038/nmeth.1701 CrossRefPubMedGoogle Scholar
  32. Qin H-M, Xu P, Guo Q, Cheng X, Gao D, Sun D, Zhu Z, Lu F (2018) Biochemical characterization of a novel ulvan lyase from Pseudoalteromonas sp. strain PLSV. RSC Adv 8:2610–2615.  https://doi.org/10.1039/C7RA12294B CrossRefGoogle Scholar
  33. Reisky L, Büchsenschütz HC, Engel J, Song T, Schweder T, Hehemann J-H, Bornscheuer UT (2018) Oxidative demethylation of algal carbohydrates by cytochrome P450 monooxygenases. Nat Chem Biol 14:342–344.  https://doi.org/10.1038/s41589-018-0005-8 CrossRefPubMedGoogle Scholar
  34. Robic A, Gaillard C, Sassi J-F, Lerat Y, Lahaye M (2009) Ultrastructure of ulvan: a polysaccharide from green seaweeds. Biopolymers 91:652–664.  https://doi.org/10.1002/bip.21195 CrossRefPubMedGoogle Scholar
  35. Rydahl MG, Kračun SK, Fangel JU, Michel G, Guillouzo A, Génicot S, Mravec J, Harholt J, Wilkens C, Motawia MS, Svensson B, Tranquet O, Ralet M-C, Jørgensen B, Domozych DS, Willats WGT (2017) Development of novel monoclonal antibodies against starch and ulvan - implications for antibody production against polysaccharides with limited immunogenicity. Sci Rep 7:9326.  https://doi.org/10.1038/s41598-017-04307-2 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Salinas A, French CE (2017) The enzymatic ulvan depolymerisation system from the alga-associated marine flavobacterium Formosa agariphila. Algal Res 27:335–344.  https://doi.org/10.1016/j.algal.2017.09.025 CrossRefGoogle Scholar
  37. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG (2014) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539–539.  https://doi.org/10.1038/msb.2011.75 CrossRefGoogle Scholar
  38. Smetacek V, Zingone A (2013) Green and golden seaweed tides on the rise. Nature 504:84–88.  https://doi.org/10.1038/nature12860 CrossRefPubMedGoogle Scholar
  39. Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 83:1–11.  https://doi.org/10.1016/S0960-8524(01)00212-7 CrossRefPubMedGoogle Scholar
  40. Teeling H, Fuchs BM, Becher D, Klockow C, Gardebrecht A, Bennke CM, Kassabgy M, Huang S, Mann AJ, Waldmann J, Weber M, Klindworth A, Otto A, Lange J, Bernhardt J, Reinsch C, Hecker M, Peplies J, Bockelmann FD, Callies U, Gerdts G, Wichels A, Wiltshire KH, Glockner FO, Schweder T, Amann R (2012) Substrate-controlled succession of marine bacterioplankton populations induced by a phytoplankton bloom. Science 336:608–611.  https://doi.org/10.1126/science.1218344 CrossRefPubMedGoogle Scholar
  41. Terrapon N, Lombard V, Gilbert HJ, Henrissat B (2015) Automatic prediction of polysaccharide utilization loci in Bacteroidetes species. Bioinformatics 31:647–655.  https://doi.org/10.1093/bioinformatics/btu716 CrossRefPubMedGoogle Scholar
  42. Ulaganathan T, Boniecki MT, Foran E, Buravenkov V, Mizrachi N, Banin E, Helbert W, Cygler M (2017) New ulvan-degrading polysaccharide lyase family: structure and catalytic mechanism suggests convergent evolution of active site architecture. ACS Chem Biol 12:1269–1280.  https://doi.org/10.1021/acschembio.7b00126 CrossRefPubMedGoogle Scholar
  43. Ulaganathan T, Helbert W, Kopel M, Banin E, Cygler M (2018) Structure–function analyses of a PL24 family ulvan lyase reveal key features and suggest its catalytic mechanism. J Biol Chem jbc.RA117.001642.  https://doi.org/10.1074/jbc.RA117.001642
  44. van der Wal H, Sperber BLHM, Houweling-Tan B, Bakker RRC, Brandenburg W, López-Contreras AM (2013) Production of acetone, butanol, and ethanol from biomass of the green seaweed Ulva lactuca. Bioresour Technol 128:431–437.  https://doi.org/10.1016/j.biortech.2012.10.094 CrossRefPubMedGoogle Scholar
  45. Veith PD, Nor Muhammad NA, Dashper SG, Likić VA, Gorasia DG, Chen D, Byrne SJ, Catmull DV, Reynolds EC (2013) Protein substrates of a novel secretion system are numerous in the Bacteroidetes phylum and have in common a cleavable C-terminal secretion signal, extensive post-translational modification, and cell-surface attachment. J Proteome Res 12:4449–4461.  https://doi.org/10.1021/pr400487b CrossRefPubMedGoogle Scholar
  46. Wargacki AJ, Leonard E, Win MN, Regitsky DD, Santos CNS, Kim PB, Cooper SR, Raisner RM, Herman A, Sivitz AB, Lakshmanaswamy A, Kashiyama Y, Baker D, Yoshikuni Y (2012) An engineered microbial platform for direct biofuel production from brown macroalgae. Science 335:308–313.  https://doi.org/10.1126/science.1214547 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Biochemistry, Department of Biotechnology and Enzyme CatalysisGreifswald UniversityGreifswaldGermany
  2. 2.Institute of Applied Synthetic ChemistryViennaAustria
  3. 3.Institute of Pharmacy, Pharmaceutical BiotechnologyGreifswald UniversityGreifswaldGermany
  4. 4.Max Planck-Institute for Marine MicrobiologyBremenGermany
  5. 5.Center for Marine Environmental Sciences (MARUM)University of BremenBremenGermany

Personalised recommendations