Cloning, purification and biochemical characterization of beta agarase from the marine bacterium Pseudoalteromonas sp. AG4
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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.
KeywordsBeta 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.Craigie JS (1990) Cell walls. In: Cole KM, Sheath RG (eds) Biology of the red algae. Cambridge University Press, Cambridge, pp 221–257Google Scholar
- 5.Araki C (1959) Seaweed polysaccharides. In: Wolfrom ML (ed) Carbohydrate chemistry of substances of biological interests. Pergamon Press, London, pp 15–306Google Scholar
- 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
- 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
- 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
- 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.Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
- 48.Chen HM, Zheng L, Yan XJ (2005) The preparation and bioactivity research of agaro-oligosaccharides. Food Technol Biotechnol 43(1):29–36Google Scholar