Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Characterization of a Novel 1,3-α-3,6-Anhydro-L-Galactosidase, Ahg943, with Cold- and High-Salt-Tolerance from Gayadomonas joobiniege G7

  • Seo, Ju Won (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Tsevelkhorloo, Maral (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Lee, Chang-Ro (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Kim, Sang Hoon (Department of Animal Resources Science, Dankook University) ;
  • Kang, Dae-Kyung (Department of Animal Resources Science, Dankook University) ;
  • Asghar, Sajida (Department of Bioscience and Bioinformatics, Myongji University) ;
  • Hong, Soon-Kwang (Department of Bioscience and Bioinformatics, Myongji University)
  • Received : 2020.08.10
  • Accepted : 2020.08.27
  • Published : 2020.11.28
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Abstract

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-L-galactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6-anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20℃, respectively. In particular, rAhg943 could maintain enzyme activity at 10℃ (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca2+ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. Km and Vmax of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 μM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.

Keywords

References

  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-402. https://doi.org/10.1093/nar/25.17.3389
  2. Araki C. 1959. Seaweed polysaccharides, pp. 15-30. In: Wolfrom ML (ed.), Carbohydrate chemistry of substances of biological interests, Pergamon Press, London.
  3. Asghar S, Lee CR, Chi WJ, Kang DK, Hong SK. 2019. Molecular cloning and characterization of a novel cold-adapted alkaline 1,3-α3,6-anhydro-l-galactosidase, Ahg558, from Gayadomonas joobiniege G7. Appl. Biochem. Biotechnol. 188: 1077-1095. https://doi.org/10.1007/s12010-019-02963-w
  4. Asghar S, Lee CR, Park JS, Chi WJ, Kang DK, Hong SK. 2018. Identification and biochemical characterization of a novel coldadapted 1,3-a-3,6-anhydro-L-galactosidase, Ahg786, from Gayadomonas joobiniege G7. Appl. Microbiol. Biotechnol. 102: 8855-8866. https://doi.org/10.1007/s00253-018-9277-x
  5. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1006/abio.1976.9999
  6. 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
  7. 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. https://doi.org/10.4014/jmb.1308.08007
  8. Choi U, Jung S, Hong SK, Lee CR. 2019. Characterization of a novel neoagarobiose-producing GH42 β-agarase, AgaJ10, from Gayadomonas joobiniege G7. Appl. Biochem. Biotechnol. 189: 1-12. https://doi.org/10.1007/s12010-019-02992-5
  9. Elkahlout K, Alipour S, Eroglu I, Gunduz U, Yucel M. 2017. Long-term biological hydrogen production by agar immobilized Rhodobacter capsulatus in a sequential batch photobioreactor. Bioprocess Biosyst. Eng. 40: 589-599. https://doi.org/10.1007/s00449-016-1723-5
  10. Ficko-Blean E, Duffieux D, Rebuffet E, Larocque R, Groisillier A, Michel G, et al. 2015. Biochemical and structural investigation of two paralogous glycoside hydrolases from Zobellia galactanivorans: novel insights into the evolution, dimerization plasticity and catalytic mechanism of the GH117 family. Acta. Crystallogr. D Biol. Crystallogr. 71: 209-223. https://doi.org/10.1107/S1399004714025024
  11. Guerrero C, Vera C, Serna N, Illanes A. 2017. Immobilization of Aspergillus oryzae b-galactosidase in an agarose matrix functionalized by four different methods and application to the synthesis of lactulose. Bioresour. Technol. 232: 53-63. https://doi.org/10.1016/j.biortech.2017.02.003
  12. Ha SC, Lee S, Lee J, Kim HT, Ko HJ, Kim KH, et al. 2011. Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40. Biochem. Biophys. Res. Commun. 412: 238-244. https://doi.org/10.1016/j.bbrc.2011.07.073
  13. Han Z, Zhang Y, Yang J. 2019. Biochemical characterization of a new β-agarase from Cellulophaga algicola. Int. J. Mol. Sci. 20: 2143. doi:10.3390/ijms20092143.
  14. 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. https://doi.org/10.1038/nature08937
  15. Hehemann JH, Smyth L, Yadav A, Vocadlo DJ, Boraston AB. 2012. Analysis of keystone enzyme in Agar hydrolysis provides insight into the degradation (of a polysaccharide from) red seaweeds. J. Biol. Chem. 287: 13985-13995. https://doi.org/10.1074/jbc.M112.345645
  16. Jumaidin R, Sapuan SM, Jawaid M, Ishak MR, Sahari J. 2016. Characteristics of thermoplastic sugar palm Starch/Agar blend: Thermal, tensile, and physical properties. Int. J. Biol. Macromol. 89: 575-581. https://doi.org/10.1016/j.ijbiomac.2016.05.028
  17. Jung S, Jeong BC, Hong SK, Lee CR. 2017a. Cloning, Expression, and Biochemical Characterization of a Novel Acidic GH16 βAgarase, AgaJ11, from Gayadomonas joobiniege G7. Appl. Biochem. Biotechnol. 181: 961-971. https://doi.org/10.1007/s12010-016-2262-x
  18. Jung S, Lee CR, Chi WJ, Bae CH, Hong SK. 2017b. 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. https://doi.org/10.1007/s00253-016-7951-4
  19. Kazimierczak P, Palka K, Przekora A. 2019. Development and optimization of the novel fabrication method of highly macroporous chitosan/agarose/nanohydroxyapatite bone scaffold for potential regenerative medicine applications. Biomolecules 9: 434. https://doi.org/10.3390/biom9090434
  20. Kim HT, Lee S, Kim KH, Choi IG. 2012. The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production. Bioresour. Technol. 107: 301-306. https://doi.org/10.1016/j.biortech.2011.11.120
  21. Kim HT, Yun EJ, Wang D, Chung JH, Choi IG, Kim KH. 2013. High temperature and low acid pretreatment and agarase treatment of agarose for the production of sugar and ethanol from red seaweed biomass. Bioresour. Technol. 136: 582-587. https://doi.org/10.1016/j.biortech.2013.03.038
  22. Klausen MS, Jespersen MC, Nielsen H, Jensen KK, Jurtz VI, Sonderby CK, et al. 2019. NetSurfP-2.0: improved prediction of protein structural features by integrated deep learning. Proteins 87: 520-527. https://doi.org/10.1002/prot.25674
  23. Knutsen SH, Myslabodski DE, Larsen B, Usov AI. 1994. A modified system of nomenclature for red algal galactans. Botanica Marina 37: 163-169. https://doi.org/10.1515/botm.1994.37.2.163
  24. 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. strain G7. J. Bacteriol. 194: 6961-6962. https://doi.org/10.1128/JB.01931-12
  25. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  26. Lam PL, Gambari R, Kok SH, Lam KH, Tang JC, Bian ZX, et al. 2015. Non-toxic agarose/gelatin-based microencapsulation system containing gallic acid for antifungal application. Int. J. Mol. Med. 35: 503-510. https://doi.org/10.3892/ijmm.2014.2027
  27. Lee YR, Jung S, Chi WJ, Bae CH, Jeong BC, Hong SK, et al. 2018. Biochemical Characterization of a Novel GH86 β-Agarase Producing Neoagarohexaose from Gayadomonas joobiniege G7. J. Microbiol. Biotechnol. 28: 284-292. https://doi.org/10.4014/jmb.1710.10011
  28. Lineweaver H, Burk D. 1934. The determination of enzyme dissociation constants. J. Amer. Chem. Soc. 56: 658-666. https://doi.org/10.1021/ja01318a036
  29. Liu J, Xue Z, Zhang W, Yan M, Xia Y. 2018. Preparation and properties of wet-spun agar fibers. Carbohydr. Polym. 181: 760-767. https://doi.org/10.1016/j.carbpol.2017.11.081
  30. Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428. https://doi.org/10.1021/ac60147a030
  31. Nurizzo D, Turkenburg JP, Charnock SJ, Roberts SM, Dodson EJ, McKie VA, et al. 2002. Cellvibrio japonicus alpha-L-arabinanase 43A has a novel five-blade beta-propeller fold. Nat. Struct. Biol. 9: 665-668. https://doi.org/10.1038/nsb835
  32. Pandit P, Nadathur GT, Maiti S, Regubalan B. 2018. Functionality and properties of bio-based materials. In Ahmed S. (eds.), Bio-based Materials for Food Packaging, Springer, Singapore.
  33. Park SH, Lee CR, Hong SK. 2020. Implications of agar and agarase in industrial applications of sustainable marine biomass. Appl. Microbiol. Biotechnol. 104: 2815-2832. https://doi.org/10.1007/s00253-020-10412-6
  34. Pons T, Naumoff DG, Martinez-Fleites C, Hernandez L. 2004. Three acidic residues are at the active site of a beta-propeller architecture in glycoside hydrolase families 32, 43, 62, and 68. Proteins 54: 424-432. https://doi.org/10.1002/prot.10604
  35. Ranalli G, Zanardini E, Rampazzi L, Corti C, Andreotti A, Colombini MP, et al. 2019. Onsite advanced biocleaning system for historical wall paintings using new agar-gauze bacteria gel. J. Appl. Microbiol. 126: 1785-1796. https://doi.org/10.1111/jam.14275
  36. Rebuffet E, Groisillier A, Thompson A, Jeudy A, Barbeyron T, Czjzek M, et al. 2011. Discovery and structural characterization of a novel glycosidase family of marine origin. Environ. Microbiol. 13: 1253-1270. https://doi.org/10.1111/j.1462-2920.2011.02426.x
  37. Saitou N and Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
  38. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729. https://doi.org/10.1093/molbev/mst197
  39. Yun EJ, Yu S, Kim KH. 2017. Current knowledge on agarolytic enzymes and the industrial potential of agar-derived sugars. Appl. Microbiol. Biotechnol. 101: 5581-5589. https://doi.org/10.1007/s00253-017-8383-5
  40. Zuckerkandl E, Pauling L. 1965. Evolutionary divergence and convergence in proteins. pp. 97-166, In Bryson V, Vogel HJ (eds.), Evolving Genes and Proteins, Academic Press, New York