Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Biochemical Characterization of a Novel GH86 β-Agarase Producing Neoagarohexaose from Gayadomonas joobiniege G7

  • Lee, Yeong Rim (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Jung, Subin (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Chi, Won-Jae (Biological and Genetic Resource Assessment Division, National Institute of Biological Resource) ;
  • Bae, Chang-Hwan (Biological and Genetic Resource Assessment Division, National Institute of Biological Resource) ;
  • Jeong, Byeong-Chul (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Hong, Soon-Kwang (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Lee, Chang-Ro (Department of Bioscience and Bioinformatics, Myongji University)
  • Received : 2017.10.16
  • Accepted : 2017.11.16
  • Published : 2018.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

A novel ${\beta}$-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be $30^{\circ}C$ and 4.5, respectively. AgaJ5 was an acidic ${\beta}$-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at $10^{\circ}C$. AgaJ5 required monovalent ions such as $Na^+$ and $K^+$ for its maximum activity, but its activity was severely inhibited by several metal ions. The $K_m$ and $V_{max}$ of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type ${\beta}$-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing ${\beta}$-agarase having specific biochemical features that may be useful for industrial applications.

Keywords

References

  1. Chi WJ, Chang YK, Hong SK. 2012. Agar degradation by microorganisms and agar-degrading enzymes. Appl. Microbiol. Biotechnol. 94: 917-930. https://doi.org/10.1007/s00253-012-4023-2
  2. Araki C. 1959. Seaweed polysaccharides, pp. 15-30. In Wolfrom ML (ed.). Carbohydrate Chemistry of Substances of Biological Interests. Pergamon Press, London.
  3. Jung S, Lee CR, Chi WJ, Bae CH, Hong SK. 2017. Biochemical characterization of a novel cold-adapted GH39 ${\beta}$-agarase, AgaJ9, from an agar-degrading marine bacterium Gayadomonas joobiniege G7. Appl. Microbiol. Biotechnol. 101: 1965-1974. https://doi.org/10.1007/s00253-016-7951-4
  4. Potin P, Richard C, Rochas C, Kloareg B. 1993. Purification and characterization of the ${\alpha}$-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. Eur. J. Biochem. 214: 599-607.
  5. Day DF, Yaphe W. 1975. Enzymatic hydrolysis of agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl ${\alpha}$-galactoside hydrolase. Can. J. Microbiol. 21: 1512-1518. https://doi.org/10.1139/m75-223
  6. Frey PA. 1996. The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose. FASEB J. 10: 461-470. https://doi.org/10.1096/fasebj.10.4.8647345
  7. Yun EJ, Lee S, Kim HT, Pelton JG, Kim S, Ko HJ, et al. 2015. The novel catabolic pathway of 3,6-anhydro-L-galactose, the main component of red macroalgae, in a marine bacterium. Environ. Microbiol. 17: 1677-1688. https://doi.org/10.1111/1462-2920.12607
  8. Fu XT, Kim SM. 2010. Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Mar. Drugs 8: 200-218. https://doi.org/10.3390/md8010200
  9. 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. https://doi.org/10.1271/bbb.61.162
  10. Hong SJ, Lee JH, Kim EJ, Yang HJ, Park JS, Hong SK. 2017. Anti-obesity and anti-diabetic effect of neoagarooligosaccharides on high-fat diet-induced obesity in mice. Mar. Drugs 15: 90. https://doi.org/10.3390/md15040090
  11. Chi WJ, Park DY, Seo YB, Chang YK, Lee SY, Hong SK. 2014. Cloning, expression, and biochemical characterization of a novel GH16 ${\beta}$-agarase AgaG1 from Alteromonas sp. GNUM-1. Appl. Microbiol. Biotechnol. 98: 4545-4555.
  12. Hsu PH, Wei CH, Lu WJ, Shen F, Pan CL, Lin HT. 2015. Extracellular production of a novel endo-${\beta}$-agarase AgaA from Pseudomonas vesicularis MA103 that cleaves agarose into neoagarotetraose and neoagarohexaose. Int. J. Mol. Sci. 16: 5590-5603.
  13. Liao L, Xu XW, Jiang XW, Cao Y, Yi N, Huo YY, et al. 2011. Cloning, expression, and characterization of a new ${\beta}$-agarase from Vibrio sp. strain CN41. Appl. Environ. Microbiol. 77: 7077-7079. https://doi.org/10.1128/AEM.05364-11
  14. Dong J, Tamaru Y, Araki T. 2007. A unique ${\beta}$-agarase, AgaA, from a marine bacterium, Vibrio sp. strain PO-303. Appl. Microbiol. Biotechnol. 74: 1248-1255. https://doi.org/10.1007/s00253-006-0781-z
  15. Park DY, Chi WJ, Park JS, Chang YK, Hong SK. 2015. Cloning, expression, and biochemical characterization of a GH16 ${\beta}$-agarase AgaH71 from Pseudoalteromonas hodoensis H7. Appl. Biochem. Biotechnol. 175: 733-747.
  16. Hehemann JH, Correc G, Thomas F, Bernard T, Barbeyron T, Jam M, et al. 2012. Biochemical and structural characterization of the complex agarolytic enzyme system from the marine bacterium Zobellia galactanivorans. J. Biol. Chem. 287: 30571-30584. https://doi.org/10.1074/jbc.M112.377184
  17. Cui F, Dong S, Shi X, Zhao X, Zhang XH. 2014. Overexpression and characterization of a novel thermostable ${\beta}$-agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01(T). Mar. Drugs 12: 2731-2747. https://doi.org/10.3390/md12052731
  18. Lee DG, Jeon MJ, Lee SH. 2012. Cloning, expression, and characterization of a glycoside hydrolase family 118 ${\beta}$-agarase from Agarivorans sp. JA-1. J. Microbiol. Biotechnol. 22: 1692-1697. https://doi.org/10.4014/jmb.1209.09033
  19. Li G, Sun M, Wu J, Ye M, Ge X, Wei W, et al. 2015. Identification and biochemical characterization of a novel endo-type ${\beta}$-agarase AgaW from Cohnella sp. strain LGH. Appl. Microbiol. Biotechnol. 99: 10019-10029. https://doi.org/10.1007/s00253-015-6869-6
  20. Li J, Sha Y, Seswita-Zilda D, Hu Q, He P. 2014. Purification and characterization of thermostable agarase from Bacillus sp. BI-3, a thermophilic bacterium isolated from hot spring. J. Microbiol. Biotechnol. 24: 19-25. https://doi.org/10.4014/jmb.1308.08055
  21. Ohta Y, Hatada Y, Miyazaki M, Nogi Y, Ito S, Horikoshi K. 2005. Purification and characterization of a novel ${\alpha}$-agarase from a Thalassomonas sp. Curr. Microbiol. 50: 212-216.
  22. 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 ${\beta}$-agarase-producing neoagarobiose. J. Bacteriol. 194: 142-149. https://doi.org/10.1128/JB.05978-11
  23. 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 ${\beta}$-agarase producing neoagarotetraose and neoagarohexaose. Appl. Microbiol. Biotechnol. 92: 749-759. https://doi.org/10.1007/s00253-011-3347-7
  24. Zhu Y, Zhao R, Xiao A, Li L, Jiang Z, Chen F, et al. 2016. Characterization of an alkaline ${\beta}$-agarase from Stenotrophomonas sp. NTa and the enzymatic hydrolysates. Int. J. Biol. Macromol. 86: 525-534. https://doi.org/10.1016/j.ijbiomac.2016.01.106
  25. Tawara M, Sakatoku A, Tiodjio RE, Tanaka D, Nakamura S. 2015. Cloning and characterization of a novel agarase from a newly isolated bacterium Simiduia sp. strain TM-2 able to degrade various seaweeds. Appl. Biochem. Biotechnol. 177: 610-623. https://doi.org/10.1007/s12010-015-1765-1
  26. Han W, Cheng Y, Wang D, Wang S, Liu H, Gu J, et al. 2016. Biochemical characteristics and substrate degradation pattern of a novel exo-type ${\beta}$-agarase from the polysaccharidedegrading marine bacterium Flammeovirga sp. strain MY04. Appl. Environ. Microbiol. 82: 4944-4954.
  27. Jung S, Jeong BC, Hong SK, Lee CR. 2017. Cloning, expression, and biochemical characterization of a novel acidic GH16 ${\beta}$-agarase, AgaJ11, from Gayadomonas joobiniege G7. Appl. Biochem. Biotechnol. 181: 961-971.
  28. Ariga O, Inoue T, Kubo H, Minami K, Nakamura M, Iwai M, et al. 2012. Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp. J. Microbiol. Biotechnol. 22: 1237-1244. https://doi.org/10.4014/jmb.1202.02020
  29. Ohta Y, Hatada Y, Nogi Y, Li Z, Ito S, Horikoshi K. 2004. Cloning, expression, and characterization of a glycoside hydrolase family 86 ${\beta}$-agarase from a deep-sea Microbulbiferlike isolate. Appl. Microbiol. Biotechnol. 66: 266-275. https://doi.org/10.1007/s00253-004-1757-5
  30. 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.
  31. Yan S, Yu M, Wang Y, Shen C, Zhang XH. 2011. Catenovulum agarivorans gen. nov., sp. nov., a peritrichously flagellated, chain-forming, agar-hydrolysing gammaproteobacterium from seawater. Int. J. Syst. Evol. Microbiol. 61: 2866-2873. https://doi.org/10.1099/ijs.0.027565-0
  32. Zor T, Selinger Z. 1996. Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal. Biochem. 236: 302-308. https://doi.org/10.1006/abio.1996.0171
  33. Segel IH. 1976. Enzyme kinetics, pp. 214-229. In: Biochemical Calculations. How to Solve Mathematical Problems in General Biochemistry, 2nd Ed. Wiley, New York.
  34. 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: e00144-00114.
  35. Belas R. 1989. Sequence analysis of the agrA gene encoding ${\beta}$-agarase from Pseudomonas atlantica. J. Bacteriol. 171: 602-605.
  36. Belas R, Bartlett D, Silverman M. 1988. Cloning and gene replacement mutagenesis of a Pseudomonas atlantica agarase gene. Appl. Environ. Microbiol. 54: 30-37.

Cited by

  1. Identification and biochemical characterization of a novel cold-adapted 1,3-α-3,6-anhydro-l-galactosidase, Ahg786, from Gayadomonas joobiniege G7 vol.102, pp.20, 2018, https://doi.org/10.1007/s00253-018-9277-x
  2. Recombinant β-agarases: insights into molecular, biochemical, and physiochemical characteristics vol.8, pp.10, 2018, https://doi.org/10.1007/s13205-018-1470-1
  3. Biochemical Characterization of a New β-Agarase from Cellulophaga algicola vol.20, pp.9, 2018, https://doi.org/10.3390/ijms20092143
  4. Molecular Cloning and Characterization of a Novel Cold-Adapted Alkaline 1,3-α-3,6-Anhydro-l-galactosidase, Ahg558, from Gayadomonas joobiniege G7 vol.188, pp.4, 2019, https://doi.org/10.1007/s12010-019-02963-w
  5. Characterization of a Novel Neoagarobiose-Producing GH42 β-Agarase, AgaJ10, from Gayadomonas joobiniege G7 vol.189, pp.1, 2019, https://doi.org/10.1007/s12010-019-02992-5
  6. Biochemical characterization of a novel cold-adapted agarotetraose-producing α-agarase, AgaWS5, from Catenovulum sediminis WS1-A vol.103, pp.20, 2019, https://doi.org/10.1007/s00253-019-10056-1
  7. 한천분해효소의 재조합발현 : 기원, 활성조건, 분비신호와 게놈분석 등 vol.30, pp.3, 2018, https://doi.org/10.5352/jls.2020.30.3.304
  8. Implications of agar and agarase in industrial applications of sustainable marine biomass vol.104, pp.7, 2018, https://doi.org/10.1007/s00253-020-10412-6
  9. Expression and Characterization of a Methylated Galactose-Accommodating GH86 β-Agarase from a Marine Bacterium vol.68, pp.29, 2020, https://doi.org/10.1021/acs.jafc.0c02672
  10. Expression and Characterization of a Novel Cold-Adapted and Stable β-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9 vol.19, pp.8, 2018, https://doi.org/10.3390/md19080431