Abstract
A high-alkaline, salt-activated alginate lyase is produced by Agarivorans sp. JAM-A1m from a deep-sea sediment off Cape Nomamisaki on Kyushu Island, Japan. Purified to homogeneity, as judged by SDS-PAGE, the enzyme (A1m) had a molecular mass of approximately 31 kDa. The optimal pH was around 10 in glycine–NaOH buffer, and the activity was increased to 1.8 times by adding 0.2 M NaCl. However, when the optimal pH in the presence of 0.2 M NaCl was shifted to pH 9.0, the activity was more than 10 times compared with that at pH 9 in the absence of NaCl. A1m showed the optimal temperature at around 30°C and was stable to incubation between pH 6 and 9. The enzyme degraded favorably mannuronate–guluronate and guluronate-rich fragments in alginate. Shotgun cloning and sequencing of the gene for A1m revealed a 930-bp open reading frame, which encoded a mature enzyme of 289 amino acids (32,295 Da) belonging to polysaccharide lyase family 7. The deduced amino acid sequence showed the highest similarity to that of a Klebsiella enzyme, with only 54% identity.
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References
Boyen C, Bertheau Y, Barbeyron T, Kloareg B (1990a) Preparation of guluronate lyase from Pseudomonas alginovora for protoplast isolation in Laminaria. Enzyme Microb Technol 12:885–890
Boyen C, Kloareg B, Polne-Fuller M, Gibor A (1990b) Preparation of alginate lyases from marine molluscs for protoplast isolation in brown algae. Phycologia 29:173–181
Brown BJ, Preston JF (1991) l-guluronan-specific alginate lyase from a marine bacterium associated with Sargassum. Carbohydr Res 211:91–102
Evans LR, Linker A (1973) Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacteriol 116:915–924
Gacesa P (1988) Alginates. Carbohydr Polym 8:161–182
Gorin PAJ, Spencer JFT (1966) Exocellular alginic acid from Azotobacter vinelandii. Can J Chem 44:993–998
Haug A, Larsen B, Smidsrød O (1966) A study of the constitution of alginic acid by partial acid hydrolysis. Acta Chem Scand 20:183–190
Haug A, Larsen B, Smidsrød O (1967) Studies on the sequence of uronic acid residues in alginic acid. Acta Chem Scand 21:691–704
Horikoshi K, Akiba T (1982) Alkalophilic microorganisms. Japan Scientific Societies Press, Tokyo, p 137
Iwamoto Y, Araki R, Iriyama K, Oda T, Fukuda H, Hayashida S, Muramatsu T (2001) Purification and characterization of bifunctional alginate lyase from Alteromonus sp. strain No. 272 and its action on saturated oligomeric substrates. Biosci Biotechnol Biochem 65:133–142
Kawamoto H, Horibe A, Miki Y, Kimura T, Tanaka K, Nakagawa T, Kawamukai M, Matsuda H (2006) Cloning and sequencing analysis of alginate lyase genes from the marine bacterium Vibrio sp. O2. Mar Biotechnol 8:481–490
Kitamikado M, Tseng CH, Yamaguchi K, Nakamura T (1992) Two types of alginate lyases. Appl Environ Microbiol 58:2474–2478
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lange B, Wingender J, Winkler UK (1989) Isolation and characterization of an alginate lyase from Klebsiella aerogenes. Arch Microbiol 152:302–308
Matsubara Y, Kawada R, Iwasaki K, Oda T, Muramatsu T (1998) Extracellular poly (α-l-guluronate) lyase from Corynebacterium sp.: purification, characterization, and conformational properties. J Protein Chem 17:29–36
Min KH, Sasaki SF, Kashiwabara Y, Nishizawa K (1977) Substrate specificity of endo-polyguluronide lyase of Pseudomonas sp. J Biochem 81:547–553
Muramatsu T, Hashimoto H, Takahashi T (1984) Physicochemical characteristics and conformational features of alginate lyase isozymes form Turbo cornutus. Biosci Biotechnol Biochem 48:79–85
Osawa T, Matsubara Y, Muramatsu T, Kimura M, Kakuta Y (2005) Crystal structure of the alginate (poly-α-l-guluronate) lyase from Corynebacterium sp. at 1.2 Å resolution. J Mol Biol 345:1111–1118
Pereira L, Sousa A, Coelho H, Amado AM, Ribeiro-Claro PJA (2003) Use of FTIR, FT-Raman and 13C-NMR spectroscopy for identification of some seaweed phycocolloids. Biomol Eng 20:223–228
Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629
Sartori C, Finch DS, Ralph B (1997) Determination of the cation content of alginate thin films by FT i.r. spectroscopy. Polymer 38:43–51
Shirai T, Kobayashi T, Ito S, Horikoshi K (2008) Chapter IV-Alkaline adaptation of proteins. In: Siddiqui KS, Thomas T (eds) Protein adaptation in extremophiles. Nova Science Publishers Inc, Hauppauge, NY, pp 105–141
Suda K, Tanji Y, Hori K, Unno H (1999) Evidence for a novel Chlorella virus-encoded alginate lyase. FEMS Microbiol Lett 180:45–53
Sugimura I, Sawabe T, Ezura Y (2000) Cloning and sequence analysis of Vibrio halioticoli genes encoding three types of polyguluronate lyase. Mar Biotechnol 2:65–73
Suzuki H, Suzuki K, Inoue A, Ojima T (2006) A novel oligoalginate lyase from abalone, Haliotis discus hannai, that releases disaccharide from alginate polymer in an exolytic manner. Carbohydr Res 341:1809–1819
Svanem BIG, Strand WI, Ertesvåg H, Skjåk-Bræk G, Hartmann M, Barbeyron T, Valla S (2001) The catalytic activities of the bifunctional Azotobacter vinelandii mannuronan C-5-epimerase and alginate lyase AlgE7 probably originate from the same active site in the enzyme. J Biol Chem 276:31542–31550
Weissbach A, Hurwitz J (1959) The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli B. J Biol Chem 234:705–709
Wong TY, Preston LA, Schiller NL (2000) Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Ann Rev Microbiol 54:289–340
Xiao L, Han F, Yang Z, Lu XZ, Yu WG (2006) A novel alginate lyase with high activity on acetylated alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03. World J Microbiol Biotechnol 22:81–88
Yamasaki M, Moriwaki S, Miyake O, Hashimoto W, Murata K, Mikami B (2004) Structure and function of a hypothetical Pseudomonas aeruginosa protein PA1167 classified into family PL-7. J Biol Chem 279:31863–31872
Yamasaki M, Ogura K, Hashimoto W, Mikami B, Murata K (2005) A structural basis for depolymerization of alginate by polysaccharide lyase family-7. J Mol Biol 352:11–21
Acknowledgments
We are grateful to the captain and crew of the R/V Natushima and the ROV Hyper Dolphin operation team, for their technical support in sampling. We also thank the chief scientist, Prof. K. Kubokawa of the Ocean Research Institute, University of Tokyo, as well as all scientists aboard the NT05-12 cruise. We thank Dr. S. Aihara and Dr. H. Minegishi of Bio-Nano Electronics Research Center, Toyo University, for the IR spectra.
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Communicated by L. Huang.
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Kobayashi, T., Uchimura, K., Miyazaki, M. et al. A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp.. Extremophiles 13, 121–129 (2009). https://doi.org/10.1007/s00792-008-0201-7
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DOI: https://doi.org/10.1007/s00792-008-0201-7