, Volume 21, Issue 6, pp 1027–1036 | Cite as

Expression and characterization of a new heat-stable endo-type alginate lyase from deep-sea bacterium Flammeovirga sp. NJ-04

  • Benwei Zhu
  • Fang Ni
  • Yun Sun
  • Zhong Yao
Original Paper


Alginate lyases play an essential role in the production of oligosaccharides by degrading alginate polysaccharide. Although many alginate lyases from various microorganisms have been characterized, reports on alginate lyases with special characteristics and commercial potential are still rather rare. In this study, a new alginate lyase, FsAlgA, was cloned from the deep-sea marine bacterium Flammeovirga sp. NJ-04. The recombinant enzyme was purified on Ni-NTA sepharose and then characterized in detail. It exhibited the highest activity (3343.7 U/mg) at pH 7.0 and 50 °C. Notably, the FsAlgA retained more than 80% of its maximum activity after incubation at 50 °C for 30 min, suggesting that FsAlgA was a heat-stable alginate lyase. Additionally, FsAlgA possessed broad substrate specificity, showing high activities toward both poly β-d-mannuronate (polyM) and poly α-l-guluronate (polyG). Furthermore, the K m values of FsAlgA toward sodium alginate (0.48 mM) and polyG (0.94 mM) were lower than that toward polyM (1.42 mM). The TLC and ESI–MS analyses indicated that FsAlgA endolytically degraded alginate polysaccharide and released oligosaccharides with degree of polymerization (DP) of 2–5. Therefore, it may be a potent tool to produce alginate oligosaccharides with low DPs.


Deep-sea bacterium Flammeovirga sp. NJ-04 Heat-stable Alginate lyase Oligosaccharides 



The work was supported by the National Natural Science Foundation of China (Grant No. 31601410), National Basic Research Program of China (973 Program) (2013CB733503), Key Research and Development Program of Jiangsu Province (BE2015305).


  1. Aarstad OA, Tøndervik A, Sletta H, Skjåk-Bræk G (2012) Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes. Biomacromolecules 13:106–116CrossRefPubMedGoogle Scholar
  2. Ack B, Da SM, Kieckbusch TG (2012) Natamycin release from alginate/pectin films for food packaging applications. J Food Eng 110:18–25CrossRefGoogle Scholar
  3. Alipour M, Suntres ZE, Omri A (2009) Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa. J Antimicrob Chemother 64:317–325CrossRefPubMedGoogle Scholar
  4. Alkawash MA, Soothill JS, Schiller NL (2006) Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms. APMIS 114:131–138CrossRefPubMedGoogle Scholar
  5. Augst AD, Kong HJ, Mooney DJ (2006) Alginate hydrogels as biomaterials. Macromol Biosci 6:623–633CrossRefPubMedGoogle Scholar
  6. Dong S, Wei TD, Chen XL, Li CY, Wang P, Xie BB, Qin QL, Zhang XY, Pang XH, Zhou BC, Zhang YZ (2014) Molecular insight into the role of the N-terminal extension in the maturation, substrate recognition, and catalysis of a Bacterial Alginate Lyase from Polysaccharide Lyase Family 18. J Biol Chem 289:29558–29569CrossRefPubMedPubMedCentralGoogle Scholar
  7. Duan G, Han F, Yu W (2009) Cloning, sequence analysis, and expression of gene alyPI encoding an alginate lyase from marine bacterium Pseudoalteromonas sp. CY24. Can J Microbiol 55:1113–1118CrossRefPubMedGoogle Scholar
  8. Ertesvåg H, Erlien F, Skjåkbraek G, Rehm BH, Valla S (1998) Biochemical properties and substrate specificities of a recombinantly produced Azotobacter vinelandii alginate lyase. J Bacteriol 180:3779–3784PubMedPubMedCentralGoogle Scholar
  9. Evangelista G, Falasca P, Ruggiero I, Masullo M, Raimo G (2009) Molecular and functional characterization of polynucleotide phosphorylase from the antarctic eubacterium Pseudoalteromonas haloplanktis. Protein Peptide Lett 16:999–1005CrossRefGoogle Scholar
  10. Gacesa P (1992) Enzymic degradation of alginates. Int J Biochem 24:545–552CrossRefPubMedGoogle Scholar
  11. Han W, Gu J, Cheng Y, Liu H, Li Y, Li F (2016) 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 82:864–874Google Scholar
  12. Hata M, Kumagai Y, Rahman MM, Chiba S, Tanaka H, Inoue A, Ojima T (2009) Comparative study on general properties of alginate lyases from some marine gastropod mollusks. Fish Sci 75:755–763CrossRefGoogle Scholar
  13. Inoue A, Kagaya M, Ojima T (2008) Preparation of protoplasts from Laminaria japonica using native and recombinant abalone alginate lyases. J Appl Phycol 20:633–640CrossRefGoogle Scholar
  14. Inoue A, Takadono K, Nishiyama R, Tajima K, Kobayashi T, Ojima T (2014) Characterization of an alginate lyase, FlAlyA, from Flavobacterium sp. strain UMI-01 and its expression in Escherichia coli. Mar Drugs 12:4693–4712CrossRefPubMedPubMedCentralGoogle Scholar
  15. Inoue A, Mashino C, Uji T, Saga N, Mikami K, Ojima T (2015) Characterization of an eukaryotic PL-7 alginate lyase in the marine red alga Pyropia yezoensis. Curr Biotechnol 4(3):240–258CrossRefPubMedPubMedCentralGoogle Scholar
  16. Inoue A, Anraku M, Nakagawa S, Ojima T (2016a) Discovery of a novel alginate lyase from Nitratiruptor sp. SB155-2 thriving at deep-sea hydrothermal vents and identification of the residues responsible for its heat stability. J Biol Chem 291:15551–15563CrossRefPubMedPubMedCentralGoogle Scholar
  17. Inoue A, Nishiyama R, Ojima T (2016b) The alginate lyases FlAlyA, FlAlyB, FlAlyC, and FlAlex from Flavobacterium sp. UMI-01 have distinct roles in the complete degradation of alginate. Algal Res 19:355–362CrossRefGoogle Scholar
  18. Iwamoto M et al (2005) Structure–activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells. FEBS Lett 579:4423–4429CrossRefPubMedGoogle Scholar
  19. Kawada A, Hiura N, Tajima S, Takahara H (1999) Alginate oligosaccharides stimulate VEGF-mediated growth and migration of human endothelial cells. Arch Dermatol Res 291:542–547CrossRefPubMedGoogle Scholar
  20. Kim HT et al (2012) Characterization of a recombinant endo-type alginate lyase (Alg7D) from Saccharophagus degradans. Biotechnol Lett 34:1087–1092CrossRefPubMedGoogle Scholar
  21. Kobayashi T, Uchimura K, Miyazaki M, Nogi Y, Horikoshi K (2009) A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp. Extremophiles 13:121–129CrossRefPubMedGoogle Scholar
  22. Lamppa JW, Griswold KE (2013) Alginate lyase exhibits catalysis-independent biofilm dispersion and antibiotic synergy. Antimicrob Agents Chemother 57:137–145CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lee KY, Mooney DJ (2012) Alginate: properties and biomedical applications. Prog Polym Sci 37:106–126CrossRefPubMedPubMedCentralGoogle Scholar
  24. Lombard V, Bernard T, Rancurel C, Brumer H, Coutinho PM, Henrissat B (2010) A hierarchical classification of polysaccharide lyases for glycogenomics. Biochem J 432:437–444CrossRefPubMedGoogle Scholar
  25. Miyake O, Ochiai A, Hashimoto W, Murata K (2004) Origin and diversity of alginate lyases of families PL-5 and -7 in Sphingomonas sp. strain A1. J Bacteriol 186:2891–2896CrossRefPubMedPubMedCentralGoogle Scholar
  26. Shin JW, Choi SH, Dong EK, Kim HS, Lee JH, Lee IS, Lee EY (2011) Heterologous expression of an alginate lyase from Streptomyces sp. ALG-5 in Escherichia coli and its use for preparation of the magnetic nanoparticle-immobilized enzymes. Bioprocess Biosyst Eng 34:113–119CrossRefPubMedGoogle Scholar
  27. Smidsrød O, SkjåkBræk G (1990) Alginate as immobilization matrix for cells. Trends Biotechnol 8:71–78CrossRefPubMedGoogle Scholar
  28. Srimathi S, Jayaraman G, Feller G, Danielsson B, Narayanan PR (2007) Intrinsic halotolerance of the psychrophilic α-amylase from Pseudoalteromonas haloplanktis. Extremophiles 11:505–515CrossRefPubMedGoogle Scholar
  29. Swift SM, Hudgens JW, Heselpoth RD, Bales PM, Nelson DC (2014) Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A. PLoS One 9:e112939–e112939CrossRefPubMedPubMedCentralGoogle Scholar
  30. Takeuchi T, Nibu Y, Murata K, Yoshida S, Kusakabe I (1997) Characterization of a novel alginate lyase from Flavobacterium multivolum K-11. Food Sci Technol Int Tokyo 3:388–392CrossRefGoogle Scholar
  31. Thomas F et al (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 288:23021–23037CrossRefPubMedPubMedCentralGoogle Scholar
  32. Trivedi VD, Bharadwaj A, Varunjikar MS, Singha AK, Upadhyay P, Gautam K, Phale PS (2017) Insights into metabolism and sodium chloride adaptability of carbaryl degrading halotolerant Pseudomonas sp. strain C7. Arch Microbiol 907:907–916CrossRefGoogle Scholar
  33. Tusi SK, Khalaj L, Ashabi G, Kiaei M, Khodagholi F (2011) Alginate oligosaccharide protects against endoplasmic reticulum- and mitochondrial-mediated apoptotic cell death and oxidative stress. Biomaterials 32:5438–5458CrossRefPubMedGoogle Scholar
  34. Wong TY, Preston LA, Schiller NL (2000) ALGINATE LYASE: review of major sources and enzyme characteristics, structure–function analysis, biological roles, and applications. Microbiology 54:289–340CrossRefGoogle Scholar
  35. Yagi H, Fujise A, Itabashi N, Ohshiro T (2016) Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae. Biosci Biotechnol Biochem 80:1–9CrossRefGoogle Scholar
  36. Zhang H, Wang ZY, Yang L, Yang X, Wang X, Zhang Z (2011) In vitro antioxidant activities of sulfated derivatives of polysaccharides extracted from Auricularia auricular. Int J Mol Sci 12:3288–3302CrossRefPubMedPubMedCentralGoogle Scholar
  37. Zhang Y et al (2017) Identification and characterization of a novel salt-tolerant esterase from the deep-sea sediment of the South China sea. Front Microbiol 8:441PubMedPubMedCentralGoogle Scholar
  38. Zhu BW, Yin H (2015) Alginate lyase: review of major sources and classification, properties, structure–function analysis and applications. Bioengineered 6:125–131CrossRefPubMedPubMedCentralGoogle Scholar
  39. Zhu B, Tan H, Qin Y, Xu Q, Du Y, Yin H (2015) Characterization of a new endo-type alginate lyase from Vibrio sp. W13. Int J Biol Macromol 75:1385–1391CrossRefGoogle Scholar

Copyright information

© Springer Japan KK 2017

Authors and Affiliations

  1. 1.College of Food Sciences and Light IndustryNanjing Tech UniversityNanjingChina

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