Cloning, purification and biochemical characterization of beta agarase from the marine bacterium Pseudoalteromonas sp. AG4

  • Chulhong Oh
  • Chamilani Nikapitiya
  • Youngdeuk Lee
  • Ilson Whang
  • Se-Jae Kim
  • Do-Hyung Kang
  • Jehee Lee
Original Paper


A gene (agrP) encoding a β-agarase from Pseudoalteromonas sp. AG4 was cloned and expressed in Escherichia coli. The agrP primary structure consists of an 870-bp open reading frame (ORF) encoding 290 amino acids (aa). The predicted molecular mass and isoelectric point were determined at 33 kDa and 5.9, respectively. The signal peptide was predicted to be 21 aa. The deduced aa sequence showed 98.6% identity to β-agarase from Pseudoalteromonas atlantica. The recombinant protein was purified as a fusion protein and biochemically characterized. The purified β-agarase (AgaP) had specific activity of 204.4 and 207.5 units/mg towards agar and agarose, respectively. The enzyme showed maximum activity at 55°C and pH 5.5. It was stable at pH 4.5 to 8.0 and below 55°C for 1 h. The enzyme produced neoagarohexaose and neoagarotetraose from agar and in addition to that neoagarobiose from the agarose. The neoagarooligosaccharides were biologically active. Hence, AgaP is a useful enzyme source for use by cosmetic and pharmaceutical industries.


Beta agarase GHF-16 Neoagarooligosaccharides Pseudoalteromonas spp. 



This research was financially supported by the Ministry of Education, Science Technology (MEST) and Korea Institute for Advancement of Technology (KIAT) through the Human Resource Training Project for Regional Innovation, and by a research grant (PP00740) from Korea Ocean Research & Development Institute.


  1. 1.
    Craigie JS (1990) Cell walls. In: Cole KM, Sheath RG (eds) Biology of the red algae. Cambridge University Press, Cambridge, pp 221–257Google Scholar
  2. 2.
    Usov AI (1998) Structural analysis of red seaweed galactans of agar and carrageenan groups. Food Hydrocol 12:301–308CrossRefGoogle Scholar
  3. 3.
    Araki C, Arai K (1967) Studies on the chemical constitution of agar-agar XXIV. Isolation of a new disaccharide as a reversion product from acidic hydrolysate. Bull Chem Societ Japan 40:1452–14564CrossRefGoogle Scholar
  4. 4.
    Potin P, Richard C, Rochas C, Kloareg B (1993) Purification and characterization of the alpha-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. Eur J Biochem 214:599–607CrossRefPubMedGoogle Scholar
  5. 5.
    Araki C (1959) Seaweed polysaccharides. In: Wolfrom ML (ed) Carbohydrate chemistry of substances of biological interests. Pergamon Press, London, pp 15–306Google Scholar
  6. 6.
    Hassairi I, Ben AR, Nonus M, Gupta BB (2001) Production and separation of alpha-agarase from Alteromonas agarlyticus strain GJ1B. Biores Technol 79:47–517CrossRefGoogle Scholar
  7. 7.
    Kobayashi R, Takisada M, Suzuki T, Kirimura K, Usami S (1997) Neoagarobiose as a novel moisturizer with whitening effect. Biosci Biotechnol Biochem 61:162–1638CrossRefPubMedGoogle Scholar
  8. 8.
    Aoki T, Araki T, Kitamikado M (1990) Purification and characterization of a novel beta-agarase from Vibrio sp. AP-2. Eur J Biochem 187:461–465CrossRefPubMedGoogle Scholar
  9. 9.
    Sugano Y, Terada I, Arita M, Noma M, Matsumoto T (1993) Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107. Appl Environ Microbiol 59:1549–155410PubMedGoogle Scholar
  10. 10.
    Araki T, Lu Z, Morishita T (1998) Optimization of parameters for isolation of protoplasts from Gracilaria verrucosa (Rhodophyta). J Mar Biotechnol 6:193–197PubMedGoogle Scholar
  11. 11.
    Fu W, Han B, Duan D, Liu W, Wang C (2008) Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6. J Ind Microbiol Biotechnol 35:915–922CrossRefPubMedGoogle Scholar
  12. 12.
    Groleau D, Yaphe W (1977) Enzymatic hydrolysis of agar: purification and characterization of beta-neoagarotetraose hydrolase from Pseudomonas atlantica. Can J Microbiol 23:672–679PubMedCrossRefGoogle Scholar
  13. 13.
    Morrice LM, McLean MW, Williamson FB, Long WF (1983) Beta-agarases I and II from Pseudomonas atlantica. Purifications and some properties. Eur J Biochem 135:553–558Google Scholar
  14. 14.
    Ha JC, Kim JT, Kim SK, Oh TK, Yu JH, Kong IS (1997) Beta agarase from Pseudomonas sp. W7: purification of the recombinant enzyme from Escherichia coli and the effects of salt on its activity. Biotechnol Appl Biochem 26:1–6PubMedGoogle Scholar
  15. 15.
    Vera J, Alvarez R, Murano E, Slebe JC, Leon O (1998) Identification of a marine agarolytic Pseudoalteromonas isolate and characterization of its extra cellular agarase. Appl Environ Microbiol 64:4378–4383PubMedGoogle Scholar
  16. 16.
    Schroeder DC, Jaffer MA, Coyne VE (2003) Investigation of the role of a beta (1–4) agarase produced by Pseudoalteromonas gracilis B9 in eliciting disease symptoms in the red alga Gracilaria gracilis. Microbiol 149:2919–2929CrossRefGoogle Scholar
  17. 17.
    Leon O, Quintana L, Peruzzo G, Slebe JC (1992) Purification and properties of an extracellular agarase from Alteromonas sp. strain C-1. Appl Environ Microbiol 58:4060–4063PubMedGoogle Scholar
  18. 18.
    Kirimura K, Masuda N, Iwasaki Y, Nakagawa H, Kobayashi R, Usami S (1999) Purification and characterization of a novel beta agarase from an alkalophilic bacterium, Alteromonas sp. E-1. J Biosci Bioeng 87:436–441CrossRefPubMedGoogle Scholar
  19. 19.
    Wang J, Mou H, Jiang X, Guan H (2006) Characterization of a novel beta-agarase from marine Alteromonas sp. SY37–12 and its degrading products. Appl Microbiol Biotechnol 71:833–839CrossRefPubMedGoogle Scholar
  20. 20.
    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
  21. 21.
    Hu Z, Lin BK, Xu Y, Zhong MQ, Liu GM (2009) Production and purification of agarase from a marine agarolytic bacterium Agarivorans sp. HZ105. J Appl Microbiol 106:181–190CrossRefPubMedGoogle Scholar
  22. 22.
    Duckworth M, Turvey JR (1969) The action of a bacterial agarase on agarose, porphyran and alkali treated porphyran. Biochem J 113:687–692PubMedGoogle Scholar
  23. 23.
    Van der Meulen HJ, Harder W (1975) Production and characterization of the agarase of Cytoplaga flevensis. Antonie van Leeuwenhoek 41:431–447CrossRefPubMedGoogle Scholar
  24. 24.
    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 72:3396–3405CrossRefPubMedGoogle Scholar
  25. 25.
    Suzuki H, Sawai Y, Suzuki T, Kawai K (2003) Purification and characterization of an extracellular beta agarase from Bacillus sp. MK03. J Biosci Bioeng 93:456–463Google Scholar
  26. 26.
    Lee DG, Park GT, Kim NY, Lee EJ, Jang MK, Shin YG, Park GS, Kim TM, Lee JW, Lee JH, Kim SJ, Lee SH (2006) Cloning, expression, and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate. Biotechnol Lett 28:1925–1932CrossRefPubMedGoogle Scholar
  27. 27.
    Ohta Y, Hatada Y, Ito S, Horikoshi K (2005) High-level expression of a neoagarobiose-producing beta-agarase gene from Agarivorans sp. JAMB-A11 in Bacillus subtilis and enzymic properties of the recombinant enzyme. Biotechnol Appl Biochem 41:183–191CrossRefPubMedGoogle Scholar
  28. 28.
    Ohta Y, Hatada Y, Nogi Y, Li Z, Ito S, Horikoshi K (2004) Cloning, expression, and characterization of a glycoside hydrolase family 86 beta agarase from a deep-sea Microbulbifer-like isolate. Appl Microbiol Biotechnol 66:266–275CrossRefPubMedGoogle Scholar
  29. 29.
    Ohta Y, Hatada Y, Nogi Y, Miyazaki M, Li Z, Akita M, Hidaka Y, Goda S, Ito S, Horikoshi K (2004) Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from a novel species of deep-sea Microbulbifer. Appl Microbiol Biotechnol 64:505–514CrossRefPubMedGoogle Scholar
  30. 30.
    Ohta Y, Hatada Y, Nogi Y, Miyazaki M, Li Z, Hatada Y, Ito S, Horikoshi K (2004) Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta agarase from the novel marine isolate, JAMB-A94. Biosci Biotechnol Biochem 68(5):1073–1081CrossRefPubMedGoogle Scholar
  31. 31.
    Ma C, Lu X, Shi C, Shi C, Li J, Gu Y, Ma Y, Chu Y, Han F, Gong Q (2007) Molecular cloning and characterization of a novel beta-agarase, AgaB, from marine Pseudoalteromonas sp. CY24. J Biol Chem 282:3747–3754CrossRefPubMedGoogle Scholar
  32. 32.
    Lee S, Park J, Yoon S, Kim J, Kong I (2000) Sequence analysis of a beta-agarase gene (pjaA) from Pseudomonas sp. isolated from marine environment. J Biosci Bioeng 89:485–488CrossRefPubMedGoogle Scholar
  33. 33.
    Ryu SK, Cho SJ, Park SR, Lim WJ, Kim MK, Hong SY, Baed W, Park YW, Kim BK, Kim H, Yun HD (2001) Cloning of the cel9A gene and characterization of its gene product from marine bacterium Pseudomonas sp. SK38. Appl Microbiol Biotechnol 57:138–145CrossRefPubMedGoogle Scholar
  34. 34.
    Zhang WW, Sun L (2007) Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp. strain V134. Appl Environ Microbiol 73:2825–2831CrossRefPubMedGoogle Scholar
  35. 35.
    Kendall K, Cullum J (1984) Cloning and expression of an extracellular-agarase from Streptomyces coelicolor A3(2) in Streptomyces lividans 66. Gene 29:315–321CrossRefPubMedGoogle Scholar
  36. 36.
    Jam M, Flament D, Allouch J, Potin P, Thion L, Kloareg B, Czjzek M, Helbert W, Michel G, Barbeyron T (2005) The endo-beta-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours. Biochem J 385:703–713CrossRefPubMedGoogle Scholar
  37. 37.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  38. 38.
    Pearson WR (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol 183:63–98CrossRefPubMedGoogle Scholar
  39. 39.
    Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefPubMedGoogle Scholar
  40. 40.
    Kumar S, Tamura K, Nei M (2004) MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163CrossRefPubMedGoogle Scholar
  41. 41.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  42. 42.
    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  43. 43.
    Allouch J, Jam M, Helbert W, Barbeyron T, Kloareg B, Henrissat B, Czjzek M (2003) The three-dimensional structures of two beta agarases. J. Biol Chem 278:47171–47180CrossRefPubMedGoogle Scholar
  44. 44.
    Michel G, Chantalat L, Duee E, Barbeyron T, Henrissat B, Kloareg B, Dideberg O (2001) The kappa-carrageenase of Pseudoalteromonas carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of clan-B glycoside hydrolases. Structure 9:513–525CrossRefPubMedGoogle Scholar
  45. 45.
    Allouch J, Helbert W, Henrissat B, Czjzek M (2004) Parallel substrate binding sites in a β-agarase suggest a novel mode of action on double-helical agarose. Structure 12:623–632CrossRefPubMedGoogle Scholar
  46. 46.
    Keitel T, Meldgaard M, Heineman U (1994) Cation binding to a Bacillus (1, 3–1, 4)-β-glucanase Geometry, affinity and effect on protein stability. Eur J Biochem 222:203–214CrossRefPubMedGoogle Scholar
  47. 47.
    Lee DG, Jang MK, Lee OH, Kim NY, Ju S, Lee SH (2008) Over-production of a glycoside hydrolase family 50 β-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product. Biotechnol Lett 30:911–918CrossRefPubMedGoogle Scholar
  48. 48.
    Chen HM, Zheng L, Yan XJ (2005) The preparation and bioactivity research of agaro-oligosaccharides. Food Technol Biotechnol 43(1):29–36Google Scholar

Copyright information

© Society for Industrial Microbiology 2010

Authors and Affiliations

  • Chulhong Oh
    • 1
    • 4
  • Chamilani Nikapitiya
    • 1
  • Youngdeuk Lee
    • 1
  • Ilson Whang
    • 2
  • Se-Jae Kim
    • 2
  • Do-Hyung Kang
    • 4
  • Jehee Lee
    • 1
    • 3
  1. 1.Department of Marine Life ScienceJeju National UniversityJejuRepublic of Korea
  2. 2.Department of Life ScienceJeju National UniversityJejuRepublic of Korea
  3. 3.Marine and Environmental InstituteJeju National UniversityJejuRepublic of Korea
  4. 4.Korea Ocean Research and Development InstituteAnsanRepublic of Korea

Personalised recommendations