Abstract
β-Agarases are mostly categorized into three glycoside hydrolase (GH) families 16, 50, and 86. Recent genomic analysis of Saccharophagus degradans 2–40 revealed the presence of five agarase genes belonging to these GH families. Among the five agarases, Aga50D (a member of GH50) had neither been functionally characterized nor overexpressed. In this report, we present soluble overexpression and molecular characterization of Aga50D. Aga50D was expressed in an active form resulting in a single major product from agarose without intermediates. While known GH50 agarases have both endo-lytic and exo-lytic activities, which produce neoagarobiose as a final product through the intermediate, neoagaro-oligosaccharides, identification and analysis of the reaction product by mass spectrometry and 13C NMR showed that Aga50D had unique exo-lytic activity and was able to produce neoagarobiose directly from agarose. The optimum pH and temperature for the activity were 7.0 and 30°C, respectively. The K m and V max for agarose were 41.9 mg/ml (4.2 mM) and 17.9 U/mg, respectively.
Similar content being viewed by others
References
Allouch J, Jam M, Helbert W, Barbeyron T, Kloareg B, Henrissat B, Czjzek M (2003) The three-dimensional structures of two β-agarases. J Biol Chem 278:47171–47180
Araki C (1956) Structure of the agarose consitituent of agar-agar. Bull Chem Soc Jpn 29:543–544
Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238
de Hoffmann E, Stroobant V (2007) Mass spectrometry: principles and applications. Wiley, Chichester
Ekborg NA, Gonzalez JM, Howard MB, Taylor LE, Hutcheson SW, Weiner RM (2005) Saccharophagus degradans gen. nov., sp. nov., a versatile marine degrader of complex polysaccharides. Int J Syst Evol Microbiol 55:1545–1549
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 72:3396–3405
Ensor LA, Stosz SK, Weiner RM (1999) Expression of multiple complex polysaccharide-degrading enzyme systems by marine bacterium strain 2–40. J Ind Microbiol Biotechnol 23:123–126
Fernandez LE, Valiente OG, Mainardi V, Bello JL, Velez H, Rosado A (1989) Isolation and characterization of an anitumor active agar-type polysaccaride of Gracilaria dominguensis. Carbohydr Res 190:77–83
Flament D, Barbeyron T, Jam M, Potin P, Czjzek M, Kloareg B, Michel G (2007) Alpha-agarases define a new family of glycoside hydrolases, distinct from beta-agarase families. Appl Environ Microbiol 73:4691–4694
Fu XT, Lin H, Kim SM (2008) Purification and characterization of a novel beta-agarase, AgaA34, from Agarivorans albus YKW-34. Appl Microbiol Biotechnol 78:265–273
Fu XT, Pan C-H, Lin H, Kim SM (2009) Gene cloning, expression, and characterization of a β-agarase, AgaB34, from Agarivorans albus YKW-34. J Microbiol Biotechnol 19:257–264
Horn SJ (2009) Seaweed biofuels: production of biogas and bioethanol from brown macroalgae. VDM Verlag, Saarbrücken
Hu B, Gong Q, Wang Y, Ma Y, Li J, Yu W (2006) Prebiotic effects of neoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe 12:260–266
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 385:703–713
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
Lee J, Kim S-H (2009) High-throughput T7 LIC vector for introducing C-terminal poly-histidine tags with variable lengths without extra sequences. Protein Expr Purif 63:58–61
Lee D-G, Park G-T, Kim NY, Lee E-J, Jang MK, Shin YG, Park G-S, Kim T-M, Lee J-H, Lee J-H, Kim S-J, Lee S-H (2006) Cloning, expression, and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate. Biotechnol Lett 28:1925–1932
Michel G, Nyval-Collen P, Barbeyron T, Czjzek M, Helbert W (2006) Bioconversion of red seaweed galactans: a focus on bacterial agarases and carrageenases. Appl Microbiol Biotechnol 71:23–33
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Morrice LM, McLean MW, Long WF, Williamson FB (1983) β-agarase I and II from Pseudomonas atlantica—substrate specificities. Eur J Biochem 137:149–154
Ohta Y, Hatada Y, Nogi Y, Li Z, Ito S, Horikoshi K (2004a) Cloning, expression, and characterization of a glycoside hydrolase family 86 β-agarase from a deep-sea Microbulbifer-like isolate. Appl Microbiol Biotechnol 66:266–275
Ohta Y, Hatada Y, Nogi Y, Miyazaki M, Li Z, Akita M, Hidaka Y, Goda S, Ito S, Horikoshi K (2004b) Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from a novel species of deep-sea Microbulbifer. Appl Microbiol Biotechnol 64:505–514
Ohta Y, Hatada Y, Ito S, Horikoshi K (2005) High-level expression of a neoagarobiose-producing β-agarase gene from Agarivorans sp. JAMB-AII in Bacillus subtilis and enzymic properties of the recombinant enzyme. Biotechnol Appl Biochem 41:183–191
Renn D (1997) Biotechnology and the red seaweed polysaccharide industry: status, needs and prospects. Trends Biotechnol 15:9–14
Rochas C, Lahaye M, Yaphe W, Phan Viet MT (1986) 13C-NMR-spectrocopic investigation of agarose oligomers. Carbohydr Res 148:199–207
Rochas C, Potin P, Kloareg B (1994) NMR spectroscopic investigation of agarose oligomer produced by an α-agarase. Carbohydr Res 253:69–77
Sugano Y, Matsumoto T, Kodama H, Noma M (1993) Cloning and sequencing of agaA, a unique agarase 0107 gene from a marine bacterium, Vibrio sp. strain JT0107. Appl Environ Microbiol 59:3750–3756
Suzuki H, Sawai Y, Suzuki T, Kawai K (2002) Purification and characterization of an extracellular alpha-neoagarooligosaccharide hydrolase from Bacillus sp MK03. J Biosci Bioeng 93:456–463
Suzuki H, Sawai Y, Suzuki T, Kawai K (2003) Purification and characterization of an extracellular beta-agarase from Bacillus sp. MK03. J Biosci Bioeng 95:328–334
Wang J, Jiang X, Mou H, Guan H (2004) Anti-oxidation of agar oligosaccharides produced by agarase from a marine bacterium. J Appl Phycol 16:333–340
Weiner RM, Taylor LE, Henrissat B, Hauser L, Land M, Coutinho PM, Rancurel C, Saunders EH, Longmire AG, Zhang H, Bayer EA, Gilbert HJ, Larimer F, Zhulin IB, Ekborg NA, Lamed R, Richardson PM, Borovok I, Hutcheson S (2008) Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2–40T. PLoS Genet 4:13
Zhang WW, Sun L (2007) Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp. strain V134. Appl Environ Microbiol 73:2825–2831
Author information
Authors and Affiliations
Corresponding author
Additional information
Hee Taek Kim and Saeyoung Lee contributed equally to this study.
Rights and permissions
About this article
Cite this article
Kim, H.T., Lee, S., Lee, D. et al. Overexpression and molecular characterization of Aga50D from Saccharophagus degradans 2-40: an exo-type β-agarase producing neoagarobiose. Appl Microbiol Biotechnol 86, 227–234 (2010). https://doi.org/10.1007/s00253-009-2256-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-009-2256-5