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Purification and characterization of novel κ-carrageenase from marine Tamlana sp. HC4

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Abstract

We isolated a bacterial strain (HC4) that is able to degrade κ-carrageenan from a live specimen of the red alga Hyalosiphonia caespitosa. With 16S rRNA gene sequencing, we identified the strain as Tamlana sp., and then purified an extracellular κ-carrageenase from a culture of Tamlana sp. HC4 by ammonium sulfate precipitation, Sephadex G-200 gel filtration chromatography, and DE-cellulose 52 anion-exchange chromatography. The purified enzyme yields a single band on SDS-PAGE with a molecular mass of 66.4 kDa. The optimal pH and temperature for κ-carrageenase activity are at 8.0 and 30°C, respectively. The enzyme is stable over the range of pH 7.2–8.6 below 45°C. The enzyme activity is strongly inhibited by Zn2+ and Cu2+ at 1 mmol/L. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics with the Michaelis constant (K m ) at 7.63 mg/ml. Analysis of the degradation products of the κ-carrageenase by ESI-MS and 13C-NMR spectroscopy indicates that the enzyme degrades κ-carrageenan down to the level of κ-neocarrabiose sulfate.

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References

  • Antonopoulos A, Favetta P, Helbert W, Lafosse M. 2005. On-line liquid chromatography electrospray ionization mass spectrometry for the characterization of κ- and ι-carrageenans. Application to the hybrid ι-/ν-carrageenans. Anal. Chem., 77: 4 125–4 136.

    Article  Google Scholar 

  • Araki T, Higashitomo Y, Morishita T. 1999. Purification and characterization of κ-carrageenase from a marine bacterium Vibrio sp. CA-1004. Fisheries Sci., 65: 937–942.

    Google Scholar 

  • Barbeyron T, Michel G, Potin P, Henrissat B, Kloareg B. 2000. iota-Carrageenases constitute a novel family of glycoside hydrolases, unrelated to that of kappa-carrageenases. J. Biol. Chem., 275: 35 499–35 505.

    Article  Google Scholar 

  • Barbeyron T, L’Haridon S, Corre E, Kloareg B, Potin P. 2001. Zobellia galactanovorans gen. nov., sp. nov., a marine species of Flavobacteriaceae isolated from a red alga, and classification of [Cytophaga] uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Zobellia uliginosa gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol., 51: 985–997.

    Google Scholar 

  • Bradford M M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248–254.

    Article  Google Scholar 

  • Ekeberg D, Knutsen S H, Sletmoen M. 2001. Negative-ion electrospray ionization-mass spectrometry (ESI-MS) as a tool for analyzing structural heterogeneity in kappa-carrageenan oligosaccharides. Carbohydr. Res., 334: 49–59.

    Article  Google Scholar 

  • Gauthier G, Gauthier M, Christen R. 1995. Phylogenetic analysis of the genera Alteromonas, Shewanella, and Moritella using genes coding for small-subunit rRNA sequences and division of the genus Alteromonas into two genera, Alteromonas (emended) and Pseudoalteromonas gen. nov., and proposal of twelve new species combinations. Int. J. Syst. Bacteriol., 45: 755–761.

    Article  Google Scholar 

  • Guibet M, Colin S, Barbeyron T, Genicot S, Kloareg B, Michel G, Helbert W. 2007. Degradation of λ-carrageenan by Pseudoalteromonas carrageenovora λ-carrageenase: a new family of glycoside hydrolases unrelated to κ- and ι-carrageenases. Biochem. J., 404: 105–114.

    Article  Google Scholar 

  • Hu X K, Jiang X L, Aubree E, Boulenguer P, Critchley A. 2006. Preparation and in vivo anti-tumor activity of kappa-carrageenan oligosaccharides. Pharm. Biol., 44: 646–650.

    Article  Google Scholar 

  • Khambhaty Y, Mody K, Jha B. 2007. Purification and characterization of κ-carrageenase from a novel γ-Proteobacterium, Pseudomonas elongata (MTCC 5261) syn. Microbulbifer elongatus comb.nov. Biotechnol. Bioprocess Eng., 12: 668–675.

    Article  Google Scholar 

  • Knutsen S H, Myslabodski D E, Larsen B, Usov A I. 1994. A modified system of nomenclature for red algal galactans. Bot. Mar., 37: 163–169.

    Article  Google Scholar 

  • Lane D J. 1991. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M ed. Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, New York. p. 115–175.

    Google Scholar 

  • Le Gall Y, Braud J P, Kloareg B. 1990. Protoplast production in Chondrus crispus gametophytes (Gigartinales, Rhodophyta). Plant Cell Rep., 8: 582–585.

    Article  Google Scholar 

  • Lineweaver H, Burk D. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc., 56: 658–666.

    Article  Google Scholar 

  • McLean M W, Williamson F B. 1979. κ-Carrageenase from Pseudomonas carrageenovora. Eur. J. Biochem., 93: 553–558.

    Article  Google Scholar 

  • Miller G L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal. Chem., 31: 426–428.

    Article  Google Scholar 

  • Mou H J, Jiang X L, Guan H S. 2003. A κ-carrageenan derived oligosaccharide prepared by enzymatic degradation containing anti-tumor activity. J. Appl. Phycol., 15: 297–303.

    Article  Google Scholar 

  • Mou H J, Jiang X L, Liu Z H, Guan H S. 2004. Structural analysis of kappa-carrageenan oligosaccharides released by carrageenase from marine Cytophaga MCA-2. J. Food Biochem., 28: 245–260.

    Article  Google Scholar 

  • Ohta Y, Hatada Y. 2006. A novel enzyme, λ-carrageenase, isolated from a deep-sea bacterium. J. Biochem., 140: 475–481.

    Article  Google Scholar 

  • Potin P, Sanseau A, Le Gall Y, Rochas C, Kloareg B. 1991. Purification and characterization of a new kappa-carrageenase from a marine Cytophaga-like bacterium. Eur. J. Biochem., 201: 241–247.

    Article  Google Scholar 

  • Sarwar G, Matayoshi S, Oda H. 1987. Purification of a kappa-carrageenase from marine Cytophaga species. Microbiol. Immunol., 31: 869–877.

    Google Scholar 

  • Smith R G, Bidwell R G S. 1989. Inorganic carbon uptake by photosynthetically active protoplasts of the red macroalga Chondrus crispus. Mar. Biol., 102: 1–4.

    Article  Google Scholar 

  • Weigl J, Yaphe W. 1966. The enzymic hydrolysis of carrageenan by Pseudomonas carrageenovora: purification of a kappa-carrageenase. Can. J. Microbiol., 12: 939–947.

    Article  Google Scholar 

  • Yamada T, Ogamo A, Saito T, Wantanabe J, Uchiyama H, Nakagawa Y. 1997. Preparation and anti-HIV activity of low-molecular-weight carrageenans and their derivatives. Carbohydr. Polym., 32: 51–55.

    Article  Google Scholar 

  • Zhou M H, Ma J S, Li J, Ye H R, Huang K X, Zhao X W. 2008. A κ-carrageenase from a newly isolated Pseudoalteromonas-like Bacterrium, WZUC10. Biotechnol. Bioprocess Eng., 13: 545–551.

    Article  Google Scholar 

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Authors and Affiliations

  1. Key Laboratory of Mariculture, Ministry of Agriculture, Dalian Ocean University, Dalian, 116023, China

    Feixue Sun  (孙飞雪), Yuexin Ma  (马悦欣), Ying Wang  (王颖) & Qian Liu  (刘倩)

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  1. Feixue Sun  (孙飞雪)
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  2. Yuexin Ma  (马悦欣)
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  3. Ying Wang  (王颖)
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  4. Qian Liu  (刘倩)
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Correspondence to Yuexin Ma  (马悦欣).

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Supported by the Open Project of Key Laboratory of Mariculture and Biotechnology, Ministry of Agriculture, Dalian Ocean University (No. K2006-12)

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Sun, F., Ma, Y., Wang, Y. et al. Purification and characterization of novel κ-carrageenase from marine Tamlana sp. HC4. Chin. J. Ocean. Limnol. 28, 1139–1145 (2010). https://doi.org/10.1007/s00343-010-9012-7

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