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Marine Biotechnology

, Volume 20, Issue 1, pp 75–86 | Cite as

Cloning, Expression, and Biochemical Characterization of Two New Oligoalginate Lyases with Synergistic Degradation Capability

  • Shangyong Li
  • Linna Wang
  • Xuehong Chen
  • Wenwen Zhao
  • Mi Sun
  • Yantao Han
Original Article
  • 264 Downloads

Abstract

Alginate, the most abundant carbohydrate presents in brown macroalgae, has recently gained increasing attention as an alternative biomass for the production of biofuel. Oligoalginate lyases catalyze the degradation of alginate oligomers into monomers, a prerequisite for bioethanol production. In this study, two new oligoalginate lyase genes, oalC6 and oalC17, were cloned from Cellulophaga sp. SY116, and expressed them in Escherichia coli. The deduced oligoalginate lyases, OalC6 and OalC17, belonged to the polysaccharide lyase (PL) family 6 and 17, respectively. Both showed less than 50% amino acid identity with all of the characterized oligoalginate lyases. Moreover, OalC6 and OalC17 could degrade both alginate polymers and oligomers into monomers in an exolytic mode. Substrate specificity studies demonstrated that OalC6 preferred α-L-guluronate (polyG) blocks, while OalC17 preferred poly β-D-mannuronate (polyM) blocks. The combination of OalC6 and OalC17 showed synergistic degradation ability toward both alginate polymers and oligomers. Finally, an efficient process for the production of alginate monomers was established by combining the new-isolated exotype alginate lyases (i.e., OalC6 and OalC17) and the endotype alginate lyase AlySY08. Overall, our work provides new insights for the development of novel biotechnologies for biofuel production from seaweed.

Keywords

Oligoalginate lyases Synergistic degradation Alginate monomer Cellulophaga sp. SY116 

Notes

Authors’ Contributions

SL and YH conceived the study; SL, LW, and WZ performed the experiments; SL, MS, XC, and YH analyzed the data; XC and SL wrote the paper. All the authors reviewed the manuscript.

Funding Information

This work was supported by National Natural Science Foundation of China (No. 81473384 and 81603337).

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no competing interest.

Supplementary material

10126_2017_9788_MOESM1_ESM.doc (2.4 mb)
ESM 1 (DOC 2483 kb)

References

  1. Carvalho AK, Rivaldi JD, Barbosa JC, Castro HF (2015) Biosynthesis, characterization and enzymatic transesterification of single cell oil of mucorcircinelloides a sustainable pathway for biofuel production. Bioresour Technol 181:47–53Google Scholar
  2. Chen XL, Dong S, Xu F, Dong F, Li PY, Zhang XY, Zhou BC, Zhang YZ, Xie BB (2016) Characterization of a new cold-adapted and salt-activated polysaccharide lyase family 7 alginate lyase from Pseudoalteromonas sp. SM0524. Front Microbiol 7:1120PubMedPubMedCentralGoogle Scholar
  3. Doi H, Tokura Y, Mori Y, Mori K, Asakura Y, Usuda Y, Fukuda H, Chinen A (2017) Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus. Appl Microbiol Biotechnol 101(4):1581–1592Google Scholar
  4. Enquist-Newman M, Faust AM, Bravo DD, Santos CNS, Raisner RM, Hanel A, Sarvabhowman PL, Regitsky DD, Cooper SR, Peereboom L, Clark A, Martinez Y, Goldsmith J, Cho MY, Donohoue PD, Luo L, Lamberson B, Tamrakar P, Kim EJ, Villari JL, Gill A, Tripathi SA, Karamchedu P, Paredes CJ, Rajgarhia V, Kotlar HK, Bailey RB, Miller DJ, Ohler NL, Swimmer C, Yoshikuni Y (2014) Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature 505(7482):239–243Google Scholar
  5. Ertesvåg H (2015) Alginate-modifying enzymes: biological roles and biotechnological uses. Front Microbiol 6:523PubMedPubMedCentralGoogle Scholar
  6. Gao B, Jin M, Li L, Qu W, Zeng R (2017) Genome sequencing reveals the complex polysaccharide-degrading ability of novel deep-sea bacterium Flammeovirga pacifica WPAGA1. Front Microbiol 8:600PubMedPubMedCentralGoogle Scholar
  7. Garron M, Cygler M (2010) Structural and mechanistic classification of uronic acid containing polysaccharide lyases. Glycobiology 20(12):1547–1573Google Scholar
  8. Han W, Gu J, Cheng Y, Liu H, Li Y, Li F (2015) Novel alginate lyase (Aly5) from a polysaccharide-degrading marine bacterium, Flammeovirga sp. strain MY04: effects of module truncation on biochemical characteristics, alginate degradation patterns, and oligosaccharide-yielding properties. Appl Environ Microbiol 82(1):364–374Google Scholar
  9. Hirayama M, Hashimoto W, Murata K, Kawai S (2016) Comparative characterization of three bacterial exo-type alginate lyases. Int J Biol Macromol 86:519–524Google Scholar
  10. Hu T, Li CX, Zhao X, Li GS, GL Y, Guan HS (2013) Preparation and characterization of guluronic acid oligosaccharides degraded by a rapid microwave irradiation method. Carbohydr Res 373:53–58Google Scholar
  11. Imran M, Pant P, Shanbhag YP, Sawant SV, Ghadi SC (2017) Genome sequence of microbulbifer mangrovi DD-13T reveals its versatility to degrade multiple polysaccharides. Mar Biotechnol (NY) 19(1):116–124Google Scholar
  12. Jagtap SS, Hehemann JH, Polz MF, Lee JK, Zhao HM (2014) Comparative biochemical characterization of three exolytic oligoalginate lyases from vibrio splendidus revealed complementary substrate scope, temperature and pH adaptations. Appl Environ Microbiol 80(14):4207–4214Google Scholar
  13. Jeong GT, Kim SK, Park DH (2015) Application of solid-acid catalyst and marine macroalgae Gracilaria verrucosa to production of fermentable sugars. Bioresour Technol 181:16CrossRefGoogle Scholar
  14. Kim HS, Lee CG, Lee EY (2011) Alginate lyase: structure, property, and application. Biotechnol Bioprocess Eng 16(5):843–851Google Scholar
  15. Kim HT, Chung JH, Wang D, Lee J, Woo HC, Choi IG, Kim KH (2012) Depolymerization of alginate into a monomeric sugar acid using Alg17C, an exo-oligoalginate lyase cloned from Saccharophagus degradans 2-40. Appl Microbiol Biotechnol 93(5):2233–2239Google Scholar
  16. Kim HS, Chu YJ, Park CH, Lee EY, Kim HS (2015) Site-directed mutagenesis-based functional analysis and characterization of endolytic lyase activity of N- and C-terminal domains of a novel oligoalginate lyase from Sphingomonas sp. MJ-3 possessing exolytic lyase activity in the intact enzyme. Mar Biotechnol (NY) 17(6):782–792Google Scholar
  17. Li SY, Yang XM, Zhang L, WG Y, Han F (2015) Cloning, expression, and characterization of a cold-adapted and surfactant-stable alginate lyase from marine bacterium Agarivorans sp. L11. J Microbiol Biotechnol 25(5):681–686Google Scholar
  18. Li SY, Wang LN, Han F, Gong QH, WG Y (2016) Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp. Kz7. J Biochem 159(1):77–86Google Scholar
  19. Li SY, Wang LN, Hao JH, Xing MX, Sun JJ, Sun M (2017) Purification and characterization of a new alginate lyase from marine bacterium Vibrio sp. SY08. Mar drugs. 15(1):1Google Scholar
  20. Liu H, Cheng Y, Gu J, Wang Y, Li J, Li F, Han W (2017) Draft genome sequence of Paenibacillus sp. strain MY03, a terrestrial bacterium capable of degrading multiple marine-derived polysaccharides. Genome Announc 5:29Google Scholar
  21. Mille GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428Google Scholar
  22. Miyake O, Hashimoto W, Murata K (2003) An exotype alginate lyase in Sphingomonas sp. A1: overexpression in Escherichia coli, purification, and characterization of alginate lyase IV (A1-IV). Protein Expr Purif 29(1):33–41Google Scholar
  23. Na W, Josh Q, Yong S (2012) Marine macroalgae: an untapped resource for producing fuels and chemicals. Trends Biotechnol 31:70–77Google Scholar
  24. Ochiai A, Hashimoto W, Murata K (2006) A biosystem for alginate metabolism in Agrobacterium tumefaciens strain C58: molecular identification of Atu3025 as an exotype family PL-15 alginate lyase. Res Microbiol 157(7):642–649Google Scholar
  25. Park HH, Kam N, Lee EY, Kim HS (2012) Cloning and characterization of a novel oligoalginate lyase from a newly isolated bacterium Sphingomonas sp. MJ-3. Mar Biotechnol (NY) 14(2):189–202Google Scholar
  26. Park D, Jagtap S, Nair SK (2014) Structure of a PL17 family alginate lyase demonstrates functional similarities among exotype depolymerases. J Biol Chem 289(12):8645–8655Google Scholar
  27. Qin HM, Miyakawa T, Inoue A, Nishiyama R, Nakamura A, Asano A, Sawano Y, Ojima T, Tanokura M (2017) Structure and polymannuronate specificity of a eukaryotic member of polysaccharide lyase family 14. J Biol Chem 292(6):2182–2190Google Scholar
  28. Tan G, Gao Y, Shi M, Zhang X, He S, Chen Z, An C (2005) SiteFinding-PCR: a simple and efficient PCR method for chromosome walking. Nucleic Acids Res 33(13):e122Google Scholar
  29. Thomas F, Lundqvist L, Jam M, Jeudy A, Barbeyron T, Sandström C, Michel G, Czjzek M (2013) Comparative characterization of two marine alginate lyases from Zobellia galactanivorans reveals distinct modes of action and exquisite adaptation to their natural substrate. J Biol Chem 288(32):23021–23037Google Scholar
  30. Wang Y, Guo EW, WG Y, Han F (2013) Purification and characterization of a new alginate lyase from marine bacterium Vibrio sp. Biotechnol Lett 35(5):703–708Google Scholar
  31. Wang LN, Li SY, Han F, WG Y, Gong QH (2015) Cloning, overexpression and characterization of a new oligoalginate lyase from marine bacterium, Shewanella sp. Kz7. Biotechnol Lett 37(3):665–671Google Scholar
  32. Wargacki A, Leonard E, Win M, Regitsky DD, Santos CN, 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(6066):308–313Google Scholar
  33. Wong T, Preston L, Schiller N (2000) Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annu Rev Microbiol 54(1):289–340Google Scholar
  34. Xu F, Dong F, Wang P, Cao HY, Li CY, Li PY, Pang XH, Zhang YZ, Chen XL (2017) Novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase AlyGC from polysaccharide lyase family 6. J Biol Chem 292(11):4457–4468Google Scholar
  35. Yang X, Li S, Wu Y, Yu W, Han F (2016) Cloning and characterization of two thermo- and salt-tolerant oligoalginate lyases from marine bacterium Halomonas sp. FEMS Microbiol Lett 363(9):fnw079Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Pharmacology, College of basic MedicineQingdao UniversityQingdaoChina
  2. 2.Yellow Sea Fisheries Research Institute, Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of AgricultureChinese Academy of Fishery SciencesQingdaoChina

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