Applied Microbiology and Biotechnology

, Volume 99, Issue 23, pp 10019–10029 | Cite as

Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH

  • Gen Li
  • Mingming Sun
  • Jun Wu
  • Mao Ye
  • Xincheng Ge
  • Wei Wei
  • Huixin Li
  • Feng Hu
Biotechnologically relevant enzymes and proteins


An agar-degrading bacterium, strain LGH, was isolated and identified as Cohnella sp. This strain had a capability of utilizing agar as a sole carbon source for growth and showed a strong agarolytic activity. A novel endo-type β-agarase gene agaW, encoding a primary translation product of 891 amino acids, including a 26 amino acid signal peptide, was cloned and identified from a genomic library of strain LGH. The AgaW belonged to the glycoside hydrolase (GH) GH50 family, with less than 39 % amino acid sequence similarity with any known protein, and hydrolyzed agarose into neoagarotetraose as the major end product and neoagarobiose as the minor end product through other neoagarooligosaccharide intermediates, such as neoagarohexaose.


Cohnella sp. LGH Endo-type β-agarase Neoagarotetraose Neoagarobiose 



This study was funded by the Fundamental Research Funds for the Central Universities of The People’s Republic of China (Project nos. KYZ201409 and KJQN201517) and the National Natural Science Foundation (Project nos. 31470551, 41401347, and 41401254).

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval

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

Supplementary material

253_2015_6869_MOESM1_ESM.pdf (218 kb)
ESM 1 (PDF 217 kb)


  1. Araki C (1959) Seaweed polysaccharides. In: Wolfrom ML (ed) Carbohydrate chemistry of substances of biological interest. Pergamon Press, London, pp. 15–30Google Scholar
  2. Chi WJ, Chang YK, Hong SK (2012) Agar degradation by microorganisms and agar-degrading enzymes. Appl Microbiol Biotechnol 94:917–930CrossRefPubMedGoogle Scholar
  3. Chi WJ, Park da Y, Seo YB, Chang YK, Lee SY, Hong SK (2014) Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1. Appl Microbiol Biotechnol 98:4545–4555CrossRefPubMedGoogle Scholar
  4. Dong J, Hashikawa S, Konishi T, Tamaru Y, Araki T (2006) Cloning of the novel gene encoding beta-agarase C from a marine bacterium, Vibrio sp. strain PO-303, and characterization of the gene product. Appl Environ Microbiol 72:6399–6401PubMedCentralCrossRefPubMedGoogle Scholar
  5. Duckworth M, Turvey JR (1969) The action of a bacterial agarase on agarose, porphyran and alkali-treated porphyran. Biochem J 113:687–692PubMedCentralCrossRefPubMedGoogle Scholar
  6. Duckworth M, Yaphe W (1972) The relationship between structures and biological properties of agars. In: Nisizawa K (ed) Proceedings of the 7th international seaweed symposium. Halstead Press, New York, pp. 15–22Google Scholar
  7. Ekborg NA, Taylor LE, Longmire AG, Henrissat B, Weiner RM, Hutcheson SW (2006) Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2–40. Appl Environ Microbiol 72:3396–3405PubMedCentralCrossRefPubMedGoogle Scholar
  8. Flament D, Barbeyron T, Jam M, Potin P, Czjzek M, Kloareg B, Michel G (2007) Alpha-agarases define a new family of glycoside hydrolases, distinct from beta-agarase families. Appl Environ Microbiol 73:4691–4694PubMedCentralCrossRefPubMedGoogle Scholar
  9. Fu XT, Kim SM (2010) Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Mar Drugs 8:200–218PubMedCentralCrossRefPubMedGoogle Scholar
  10. Fu XT, Lin H, Kim SM (2008) Purification and characterization of a novel beta-agarase, AgaA34, from Agarivorans albus YKW-34. Appl Microbiol Biotechnol 78:265–273CrossRefPubMedGoogle Scholar
  11. Ha SC, Lee S, Lee J, Kim HT, Ko HJ, Kim KH, Choi IG (2012) Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2–40. Biochem Biophys Res Commun 412:238–244CrossRefGoogle Scholar
  12. Hatada Y, Ohta Y, Horikoshi K (2006) Hyperproduction and application of alpha-agarase to enzymatic enhancement of antioxidant activity of porphyran. J Agric Food Chem 54:9895–9900CrossRefPubMedGoogle Scholar
  13. Hehemann JH, Smyth L, Yadav A, Vocadlo DJ, Boraston AB (2012) Analysis of keystone enzyme in agar hydrolysis provides insight into the degradation of a polysaccharide from red seaweeds. J Biol Chem 287:13985–13995PubMedCentralCrossRefPubMedGoogle Scholar
  14. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316PubMedCentralCrossRefPubMedGoogle Scholar
  15. Hosoda A, Sakai M, Kanazawa S (2003) Isolation and characterization of agar-degrading Paenibacillus spp. associated with the rhizosphere of spinach. Biosci Biotechnol Biochem 67:1048–1055CrossRefPubMedGoogle Scholar
  16. Kim HT, Lee S, Lee D, Kim HS, Bang WG, Kim KH, Choi IG (2010) Overexpression and molecular characterization of Aga50D from Saccharophagus degradans 2–40: an exo-type beta-agarase producing neoagarobiose. Appl Microbiol Biotechnol 86:227–234CrossRefPubMedGoogle Scholar
  17. Knutsen SH, Myslabodski DE, Larsen B, Usov AI (1994) A modified system of nomenclature for red algal galactans. Bot Mar 37:163–169CrossRefGoogle Scholar
  18. Lahaye M, Yaphe W, Viet MTP, Rochas C (1989) 13C NMR spectroscopic investigation of methylated and charged agarose oligosaccharides and polysaccharides. Carbohydrate Res 190:249–265CrossRefGoogle Scholar
  19. Lee DG, Park GT, Kim NY, Lee EJ, Jang MK, Shin YG, Park GS, Kim TM, Lee JH, Lee JH, Kim SJ, Lee SH (2006) Cloning, expression, and characterization of a glycoside hydrolase family 50 β-agarase from a marine Agarivorans isolate. Biotechnol Lett 28:1925–1932CrossRefPubMedGoogle Scholar
  20. Liao L, Xu XW, Jiang XW, Cao Y, Yi N, Huo YY, Wu YH, Zhu XF, Zhang XQ, Wu M (2011) Cloning, expression, and characterization of a new beta-agarase from Vibrio sp. strain CN41. Appl Environ Microbiol 77:7077–7079PubMedCentralCrossRefPubMedGoogle Scholar
  21. Lin B, Lu G, Zheng Y, Xie W, Li S, Hu Z (2012) Gene cloning, expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium Agarivorans sp. HZ105. World J Microbiol Biotechnol 28:1691–1697CrossRefPubMedGoogle Scholar
  22. Ma C, Lu X, Shi C, Li J, Gu Y, Ma Y, Chu Y, Han F, Gong Q, Yu W (2007) Molecular cloning and characterization of a novel beta-agarase, AgaB, from marine Pseudoalteromonas sp. CY24. J Biol Chem 282:3747–3754CrossRefPubMedGoogle Scholar
  23. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215PubMedCentralCrossRefPubMedGoogle Scholar
  24. Ohta Y, Hatada Y, Ito S, Horikoshi K (2005) High-level expression of a neoagarobiose-producing β-agarase gene from Agarivorans sp. JAMB-AII in Bacillus subtilis and enzymic properties of the recombinant enzyme. Biotechnol Appl Biochem 41:183–191CrossRefPubMedGoogle Scholar
  25. Park da Y, Chi WJ, Park JS, Chang YK, Hong SK (2015) Cloning, expression, and biochemical characterization of a GH16 β-agarase AgaH71 from Pseudoalteromonas hodoensis H7. Appl Biochem Biotechnol 175:733–747CrossRefPubMedGoogle Scholar
  26. Pluvinage B, Hehemann JH, Boraston AB (2013) Substrate recognition and hydrolysis by a family 50 exo-β-agarase, Aga50D, from the marine bacterium Saccharophagus degradans. J Biol Chem 288:28078–28088PubMedCentralCrossRefPubMedGoogle Scholar
  27. Rochas C, Lahaye M, Yaphe W, Viet MTP (1986) 13C-N.M.R.-spectroscopic investigation of agarose oligomers. Carbohydr Res 148:199–207CrossRefGoogle Scholar
  28. Rochas C, Potin P, Kloareg B (1994) NMR spectroscopic investigation of agarose oligomers produced by an alpha-agarase. Carbohydr Res 253:69–77CrossRefPubMedGoogle Scholar
  29. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  30. Song T, Zhang W, Wei C, Jiang T, Xu H, Cao Y, Cao Y, Qiao D (2015) Isolation and characterization of agar-degrading endophytic bacteria from plants. Curr Microbiol 70:275–281CrossRefPubMedGoogle Scholar
  31. Sugano Y, Matsumoto T, Kodama H, Noma M (1993) Cloning and sequencing of agaA, a unique agarose 0107 gene from a marine bacterium, Vibrio sp. strain JT0107. Appl Environ Microbiol 59:3750–3756PubMedCentralPubMedGoogle Scholar
  32. Sugano Y, Kodama H, Terada I, Yamazaki Y, Noma M (1994a) Purification and characterization of a novel enzyme, α-neoagrooligosaccharide hydrolase (α-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107. J Bacteriol 176:6812–6818PubMedCentralPubMedGoogle Scholar
  33. Sugano Y, Matsumoto T, Noma M (1994b) Sequence analysis of the agaB gene encoding a new beta-agarase from Vibrio sp. strain JT0107. Biochim Biophys Acta 1218:105–108CrossRefPubMedGoogle Scholar
  34. Temuujin U, ChiWJ LSY, Chang YK, Hong SK (2011) Overexpression and biochemical characterization of DagA from Streptomyces coelicolorA3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose. Appl Microbiol Biotechnol 92:749–759CrossRefPubMedGoogle Scholar
  35. Temuujin U, Chi WJ, Chang YK, Hong SK (2012) Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo- and endo-type β-agarase-producing neoagarobiose. J Bacteriol 194:142–149PubMedCentralCrossRefPubMedGoogle Scholar
  36. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTALX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882CrossRefGoogle Scholar
  37. Usov AI (1998) Structural analysis of red seaweed galactans of agar and carrageenan groups. Food Hydrocoll 12:301–308CrossRefGoogle Scholar
  38. van de Velde F, Knutsen SH, Usov AI, Rollema HS, Cerezo AS (2002) 1H and 13C high resolution NMR spectroscopy of carrageenans: application in research and industry. Trends Food Sci Technol 13:73–92CrossRefGoogle Scholar
  39. Weiner RM, Taylor 2nd LE, Henrissat B, Hauser L, Land M, Coutinho PM, Rancurel C, Saunders EH, Longmire AG, Zhang H, Bayer EA, Gilbert HJ, Larimer F, Zhulin IB, Ekborg NA, Lamed R, Richardson PM, Borovok I, Hutcheson S (2008) Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2–40 T. PLoS Genet 4:e1000087PubMedCentralCrossRefPubMedGoogle Scholar
  40. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703PubMedCentralPubMedGoogle Scholar
  41. Xie W, Lin B, Zhou Z, Lu G, Lun J, Xia C, Li S, Hu Z (2013) Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3. Appl Microbiol Biotechnol 97:4907–4915CrossRefPubMedGoogle Scholar
  42. Yun EJ, Lee S, Kim HT, Pelton JG, Kim S, Ko HJ, Choi IG, Kim KH (2015) The novel catabolic pathway of 3,6-anhydro-L-galactose, the main component of red macroalgae, in a marine bacterium. Environ Microbiol 17:1677–1688CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Gen Li
    • 1
  • Mingming Sun
    • 1
  • Jun Wu
    • 1
  • Mao Ye
    • 2
  • Xincheng Ge
    • 1
  • Wei Wei
    • 1
  • Huixin Li
    • 1
  • Feng Hu
    • 1
  1. 1.Soil Ecology Lab, College of Resources and Environmental SciencesNanjing Agricultural UniversityNanjingPeople’s Republic of China
  2. 2.State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil ScienceChinese Academy of SciencesNanjingPeople’s Republic of China

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