Abstract
Gayadomonas joobiniege G7 is an agar-degrading marine bacterium belonging to a novel genus. Genomic sequencing of G. joobiniege revealed that AgaJ9 (formerly YjdB) belonging to the glycoside hydrolase (GH) 39 family. It showed the highest similarity (47% identity) to a putative β-agarase from Catenovulum agarivorans DS-2, an agar-degrading marine bacterium sharing the highest similarity in the nucleotide sequence of 16s rRNA gene with G. joobiniege G7. The agaJ9 gene encodes a protein (134 kDa) of 1205 amino acids, including a 23-amino acid signal peptide. The agarase activity of purified AgaJ9 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ9 activity were determined as 5 and 25 °C, respectively. Notably, AgaJ9 is a cold-adapted β-agarase retaining more than 80% of its activity even at a temperature of 5 °C. In addition, gel filtration chromatography revealed that AgaJ9 exists as two forms, dimer and monomer. Although the two forms had similar enzymatic properties, their kinetic parameters were different. The K m and V max of dimeric AgaJ9 for agarose was 0.68 mg/ml (5.7 × 10−6 M) and 17.2 U/mg, respectively, whereas the monomeric form had a K m of 1.43 mg/ml (1.2 × 10−5 M) and V max of 10.7 U/mg. Thin-layer chromatography and agarose-liquefying analyses revealed that AgaJ9 is an endo-type β-agarase that hydrolyzes agarose into neoagarotetraose and neoagarobiose. This study is the first report of a GH39 β-agarase with a cold-adapted enzymatic feature, a unique attribute, which may be useful for industrial applications.
Similar content being viewed by others
References
Araki C (1959) Seaweed polysaccharides. In: Wolfrom ML (ed) Carbohydrate chemistry of substances of biological interests. Pergamon Press, London, pp. 15–30
Ariga O, Inoue T, Kubo H, Minami K, Nakamura M, Iwai M, Moriyama H, Yanagisawa M, Nakasaki K (2012) Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp. J Microbiol Biotechnol 22:1237–1244
Bayer EA, Ehrlich-Rogozinski S, Wilchek M (1996) Sodium dodecyl sulfate-polyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin. Electrophoresis 17:1319–1324
Bhalla A, Bischoff KM, Sani RK (2014) Highly thermostable GH39 beta-xylosidase from a Geobacillus sp. strain WSUCF1. BMC Biotechnol 14:963
Chi WJ, Chang YK, Hong SK (2012) Agar degradation by microorganisms and agar-degrading enzymes. Appl Microbiol Biotechnol 94:917–930
Chi WJ, Park da Y, Seo YB, Chang YK, Lee SY, Hong SK (2014) Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1. Appl Microbiol Biotechnol 98:4545–4555
Chi WJ, Park JS, Kwak MJ, Kim JF, Chang YK, Hong SK (2013) Isolation and characterization of a novel agar-degrading marine bacterium, Gayadomonas joobiniege gen, nov, sp. nov., from the Southern Sea, Korea. J Microbiol Biotechnol 23:1509–1518
Correa JM, Graciano L, Abrahao J, Loth EA, Gandra RF, Kadowaki MK, Henn C, Simao Rde C (2012) Expression and characterization of a GH39 β-xylosidase II from Caulobacter crescentus. Appl Biochem Biotechnol 168:2218–2229
Cui F, Dong S, Shi X, Zhao X, Zhang XH (2014) Overexpression and characterization of a novel thermostable β-agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01(T). Mar Drugs 12:2731–2747
Czjzek M, Ben David A, Bravman T, Shoham G, Henrissat B, Shoham Y (2005) Enzyme-substrate complex structures of a GH39 b-xylosidase from Geobacillus stearothermophilus. J Mol Biol 353:838–846
Duckworth M & Yaphe W (1972) The relationship between structures and biological properties of agars. In Nisizawa K (ed), Proceedings of the 7th International Seaweed Symposium Halstead Press, New York, NY p 15–22
Fu XT, Kim SM (2010) Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Mar Drugs 8:200–218
Hassairi I, Ben Amar R, Nonus M, Gupta BB (2001) Production and separation of α-agarase from Altermonas agarlyticus strain GJ1B. Bioresour Technol 79:47–51
Hehemann JH, Correc G, Barbeyron T, Helbert W, Czjzek M, Michel G (2010) Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature 464:908–912
Hosoda A, Sakai M (2006) Isolation of Asticcacaulis sp. SA7, a novel agar-degrading alphaproteobacterium. Biosci Biotechnol Biochem 70:722–725
Hosoda A, Sakai M, Kanazawa S (2003) Isolation and characterization of agar-degrading Paenibacillus spp. associated with the rhizosphere of spinach. Biosci Biotechnol Biochem 67:1048–1055
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
Kwak MJ, Song JY, Kim BK, Chi WJ, Kwon SK, Choi S, Chang YK, Hong SK, Kim JF (2012) Genome sequence of the agar-degrading marine bacterium Alteromonadaceae sp. G7. J Bacteriol 194:6961–6962
Lee CR, Park YH, Kim M, Kim YR, Park S, Peterkofsky A, Seok YJ (2013) Reciprocal regulation of the autophosphorylation of enzyme INtr by glutamine and α-ketoglutarate in Escherichia coli. Mol Microbiol 88:473–485
Li G, Sun M, Wu J, Ye M, Ge X, Wei W, Li H, Hu F (2015a) Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH. Appl Microbiol Biotechnol 99:10019–10029
Li J, Hu Q, Li Y, Xu Y (2015b) Purification and characterization of cold-adapted β-agarase from an Antarctic psychrophilic strain. Braz J Microbiol 46:683–690
Liao L, Xu XW, Jiang XW, Cao Y, Yi N, Huo YY, Wu YH, Zhu XF, Zhang XQ, Wu M (2011) Cloning, expression, and characterization of a new β-agarase from Vibrio sp. strain CN41. Appl Environ Microbiol 77:7077–7079
Liu N, Mao X, Du Z, Mu B, Wei D (2014) Cloning and characterisation of a novel neoagarotetraose-forming-β-agarase, AgWH50A from Agarivorans gilvus WH0801. Carbohydr Res 388:147–151
Morrice LM, McLean MW, Long WF, Williamson FB (1983) Porphyran primary structure. An investigation using β-agarase I from Pseudomonas atlantica and 13C-NMR spectroscopy. Eur J Biochem 133:673–684
Nieman CE, Wong AW, He S, Clarke L, Hopwood JJ, Withers SG (2003) Family 39 α-L-iduronidases and β-D-xylosidases react through similar glycosyl-enzyme intermediates: identification of the human iduronidase nucleophile. Biochemistry 42:8054–8065
Ohta Y, Hatada Y, Miyazaki M, Nogi Y, Ito S, Horikoshi K (2005) Purification and characterization of a novel α-agarase from a Thalassomonas sp. Curr Microbiol 50:212–216
Park Y, Chi WJ, Park JS, Chang YK, Hong SK (2015) Cloning, expression, and biochemical characterization of a GH16 β-agarase AgaH71 from Pseudoalteromonas hodoensis H7. Appl Biochem Biotechnol 175:733–747
Potin P, Richard C, Rochas C, Kloareg B (1993) Purification and characterization of the α-agarase from Alteromonas agarlyticus (Cataldi) comb. Nov., strain GJ1B. Eur J Biochem 214:599–607
Segel IH (1976) Enzyme kinetics. In: Biochemical calculations how to solve mathmatical problems in general biochemistry, 2nd edn. Wiley, New York, pp. 214–229
Shan D, Li X, Gu Z, Wei G, Gao Z & Shao Z (2014) Draft genome sequence of the agar-degrading bacterium Catenovulum sp. strain DS-2, isolated from intestines of Haliotis diversicolor. Genome Announc 2(2). doi:10.1128/genomeA.00144-14
Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433
Temuujin U, Chi WJ, Chang YK, Hong SK (2012) Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo- and endo-type β-agarase-producing neoagarobiose. J Bacteriol 194:142–149
Temuujin U, Chi WJ, Lee SY, Chang YK, Hong SK (2011) Overexpression and biochemical characterization of DagA from Streptomyces coelicolor A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose. Appl Microbiol Biotechnol 92:749–759
Xie W, Lin B, Zhou Z, Lu G, Lun J, Xia C, Li S, Hu Z (2013) Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3. Appl Microbiol Biotechnol 97:4907–4915
Zhu Y, Zhao R, Xiao A, Li L, Jiang Z, Chen F, Ni H (2016) Characterization of an alkaline β-agarase from Stenotrophomonas sp. NTa and the enzymatic hydrolysates. Int J Biol Macromol 86:525–534
Zor T, Selinger Z (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem 236:302–308
Acknowledgements
This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR201530201).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Ethics approval
This article does not contain any studies with human participants performed by any of the authors.
Rights and permissions
About this article
Cite this article
Jung, S., Lee, CR., Chi, WJ. et al. Biochemical characterization of a novel cold-adapted GH39 β-agarase, AgaJ9, from an agar-degrading marine bacterium Gayadomonas joobiniege G7. Appl Microbiol Biotechnol 101, 1965–1974 (2017). https://doi.org/10.1007/s00253-016-7951-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-016-7951-4