Chinese Journal of Oceanology and Limnology

, Volume 33, Issue 2, pp 319–327 | Cite as

Expression and enzymatic characterization of a cold-adapted β-agarase from Antarctic bacterium Pseudoalteromonas sp. NJ21

  • Jiang Li (李江)
  • Yujie Sha (沙玉杰)


An agar-degrading bacterium, designated as Pseudoalteromonas sp. NJ21, was isolated from an Antarctic sediment sample. The agarase gene aga1161 from Pseudoalteromonas sp. NJ21 consisting of a 2 382-bp coding region was cloned. The gene encodes a 793-amino acids protein and was found to possess characteristic features of the Glyco_hydro_42 family. The recombinant agarase (rAga1161) was overexpressed in Escherichia coli and purified as a fusion protein. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were 30–40°C and 8.0, respectively. rAga1161 was found to maintain as much as 80% of its maximum activity at 10°C, which is typical of a coldadapted enzyme. The pattern of agar hydrolysis demonstrated that the enzyme is an β-agarase, producing neoagarobiose (NA2) as the final main product. Furthermore, this work is the first proof of an agarolytic activity in Antarctic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.


Antarctic bacterium Pseudoalteromonas agarase expression enzymatic characterization 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Allouch J, Helbert W, Henrissat B, Czjzek M. 2004. Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose. Structure, 12: 623–632.CrossRefGoogle Scholar
  2. Aoki T, Araki T, Kitamikado M. 1990. Purification and characterization of a novel beta-agarase from Vibrio sp. AP-2. Eur. J. Biochem., 187: 461–465.CrossRefGoogle Scholar
  3. Araki T, Hayakawa M, Lu Z, Karita S, Morishita T. 1998. Purification and characterization of agarases from a marine bacterium, Vibrio sp. PO-303. J. Mar. Biotechnol., 6: 260–265.Google Scholar
  4. Araki T, Lu Z, Morishita T. 1998. Optimization of parameters for isolation of protoplasts from Gracilaria verrucosa (Rhodophyta). J. Mar. Biotechnol., 6: 193–197.Google Scholar
  5. Bokun L, Guoyong L, Yandan Z, Wei X, Shengkang L, Zhong H. 2012. Gene cloning, expression and characterization of a neoagarotetraose-producing b-agarase from the marine bacterium Agarivorans sp. HZ105. World J. Microbiol. Biotechnol., 28: 1 691–1 697.CrossRefGoogle Scholar
  6. Buttner M J, Fearnley I M, Bibb M J. 1987. The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis. Mol. Gen. Genet., 209: 101–109.CrossRefGoogle Scholar
  7. Chen H, Yan X, Zhu P, Lin J. 2006. Antioxidant activity and hepatoprotective potential of agaro-oligosaccharides in vitro and in vivo. Nutr. J., 5: 31.CrossRefGoogle Scholar
  8. Dong J, Tamaru Y, Araki T A. 2007. Unique beta-agarase, AgaA, from a marine bacterium, Vibrio sp. strain PO-303. Appl. Microbiol. Biotechnol., 74: 1 248–1 255.CrossRefGoogle Scholar
  9. Fu X T, Kim S M. 2010. Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Mar. Drugs, 8: 200–218.CrossRefGoogle Scholar
  10. Gerday C, Aittaleb M, Bentahir M, Chessa J-P, Claverie P, Collins T, D’Amico S, Dumont J, Garsoux G, Georlette D. 2000. Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol., 18: 103–107.CrossRefGoogle Scholar
  11. Hu Z, Lin B K, Xu Y, Zhong M Q, Liu G M. 2009. Production and purification of agarase from a marine agarolytic bacterium Agarivorans sp. HZ105. J. Appl. Microbiol., 106: 181–190.CrossRefGoogle Scholar
  12. Jang M K, Lee D G, Kim N Y, Yu K H, Jang H J, Lee S W, Jang H J, Lee Y J, Lee S H. 2009. Purification and characterization of neoagarotetraose from hydrolyzed agar. J. Microbiol. Biotechnol., 19: 1 197–1 200.Google Scholar
  13. Kim H T, Lee S, Kim K H, 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.CrossRefGoogle Scholar
  14. Kobayashi R, Takisada M, Suzuki T, Kirimura K, Usami S. 1997. Neoagarobiose as a novel moisturizer with whitening effect. Biosci. Biotechnol. Biochem., 61: 162–163.CrossRefGoogle Scholar
  15. Kong J Y, Hwang S H, Kim B J, Bae S K, Kim J D. 1997. Cloning and expression of agarase gene from a marine bacterium Pseudomonas sp. W7. Biotechnol. Lett., 19: 23–26.CrossRefGoogle Scholar
  16. Leon O, Quintana L, Peruzzo G, Slebe J C. 1992. Purification and properties of an extracellular agarase from Alteromonas sp. strain C-1. Appl. Environ. Microbiol., 58: 4 060–4 063.Google Scholar
  17. Lin N, Mao X Z, Yang M, Mu B Z, Wei D Z. 2014. Gene cloning, expression and characterisation of a new b-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801. World J. Microbiol. Biotechnol., 10.1007/s11274-013-1591-y.Google Scholar
  18. Long M, Yu Z, Xu X. 2010. A novel beta-agarase with high pH stability from marine Agarivorans sp. LQ48. Mar. Biotechnol., 12: 62–69.CrossRefGoogle Scholar
  19. 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: 3 747–3 754.CrossRefGoogle Scholar
  20. Margesin R, Schinner F. 1999. Cold-adapted organisms. In: Ecology, Physiology, Enzymology and Molecular Biology. Berlin: Springer.Google Scholar
  21. Marshall C J. 1997. Cold-adapted enzymes. TIBTECH, 15: 358–359.CrossRefGoogle Scholar
  22. Miller G L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 31: 426–428.CrossRefGoogle Scholar
  23. 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: 183–191.CrossRefGoogle Scholar
  24. 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. Microb. Biotechnol., 66: 266–275.CrossRefGoogle Scholar
  25. Russell N J. 2000. Towards a molecular understanding of cold activity of enzymes from psychrophiles. Extremophiles, 4: 83–90.CrossRefGoogle Scholar
  26. Sellek G A, Chaudhuri J B. 1999. Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb. Technol., 25: 471–482.CrossRefGoogle Scholar
  27. Soensen H P, Mortensen K K. 2005. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb. Cell Fact., 4: 1–8.CrossRefGoogle Scholar
  28. 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: 1 549–1 554.Google Scholar
  29. Wang J X, Mou H, Guan H. 2004. Anti-oxidation of agar oligosaccharides produced by agarase from a marine bacterium. J. Appl. Phycol., 16: 333–340.CrossRefGoogle Scholar
  30. Yoshizawa Y, Tsunehiro J, Nomura K, Itoh M, Fukui F, Ametani A, Kaminogawa S. 1996. In vivo macrophagestimulation activity of the enzyme-degraded water-soluble polysaccharide fraction from a marine alga (Gracilaria verrucosa). Biosci. Biotechnol. Biochem., 61: 1 667–1 671.CrossRefGoogle Scholar

Copyright information

© Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Key Lab of Marine Bioactive Substances, First Institute of OceanographyState Oceanic Administration (SOA)QingdaoChina
  2. 2.Institute of OceanologyChinese Academy of SciencesQingdaoChina

Personalised recommendations