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http://dx.doi.org/10.48022/mbl.2105.05009

Purification and Biochemical Characterization of β-agarase Produced by Marine Microorganism Cellulophga sp. J9-3  

Kim, Da Som (Microorganism Resources Division, National Institute of Biological Resource)
Kim, Jong-Hee (Department of Food and Nutrition, Seoil University)
Chi, Won-Jae (Microorganism Resources Division, National Institute of Biological Resource)
Publication Information
Microbiology and Biotechnology Letters / v.49, no.3, 2021 , pp. 329-336 More about this Journal
Abstract
Cellulophga sp. J9-3, is a gram-negative, aerobic marine bacterium belonging to the family Flavobacteriaceae. In addition to cellulose degradability, the J9-3 strain is also capable of hydrolyzing agar in the solid and liquid medium, and the production of agarase in the presence of agarose can be remarkably induced by the bacterium. From the cell culture broth of Cellulophga sp. J9-3, ammonium sulfate precipitation and three kinds of column chromatography were successively performed to purify a specific agarase protein, the AgaJ93. Purified AgaJ93 showed the strongest hydrolyzing activity towards agarose (approximately 22%), and even displayed activity towards starch. AgaJ93 hydrolyzed agarose into neoagarotetraose and neoagarohexaose via various oligosaccharide intermediates, indicating that AgaJ93 is an endo-type β-agarase. AgaJ93 showed maximum activity at a pH of 7.0 and temperature of 35 ℃. Its activity increased by more than six times in the presence of Co2+ ions. The N-terminal sequence of AgaJ93 showed 82% homology with the heat-resistant endo-type β-agarase Aga2 of Cellulophaga sp. W5C. However, the biochemical properties of the two enzymes were different. Therefore, AgaJ93 is expected to be a novel agarose, different from the previously reported β-agarases.
Keywords
Cellulophga sp. J9-3; neoagarotetraose; neoagarohexaose; agarose; agar;
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1 Hong SJ, Lee JH, Kim EJ, Yang HJ, Park JS, Hong SK. 2017. Antiobesity and anti-diabetic effect of neoagarooligosaccharides on high-fat diet-induced obesity in mice. Mar. Drugs 15: 90-102.   DOI
2 Yun EJ, Lee S, Kim JH, Kim BB, Kim HT, Lee SH, et al. 2013. Enzymatic production of 3,6-anhydro-L-galactose from agarose and its purification and in vitro skin whitening and anti-inflammatory activities. Appl. Microbiol. Biotechnol. 97: 2961-2970.   DOI
3 Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.   DOI
4 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.   DOI
5 Wang W, Liu P, Hao C, Wu L, Wan W, Mao X. 2017. Neoagaro-oligosaccharide monomers inhibit inflammation in LPS-stimulated macrophages through suppression of MAPK and NF-κB pathways. Sci. Rep. 7: 44252.   DOI
6 Lee MH, Jang JH, Yoon GY, Lee SJ, Lee MG, Kang TH, et al. 2017. Neoagarohexaose-mediated activation of dendritic cells via Toll-like receptor 4 leads to stimulation of natural killer cells and enhancement of antitumor immunity. BMB Rep. 50: 263-268.   DOI
7 Jung S, Jeong BC, Hong SK, Lee CR. 2017. Cloning, expression, and biochemical characterization of a novel acidic GH16 β-agarase, AgaJ11, from Gayadomonas joobiniege G7. Appl. Biochem. Biotechnol. 181: 961-971.   DOI
8 Chen X, Lin H, Jin M, Zeng R, Lin M. 2019. Characterization of a novel alkaline β-agarase and its hydrolysates of agar. Food Chem. 295: 311-319.   DOI
9 Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428.   DOI
10 Kang DR, Yoon GY, Cho J, Lee SJ, Lee SJ, Park HJ, et al. 2017. Neoagarooligosaccharides prevent septic shock by modulating A20-and cyclooxygenase-2-mediated interleukin-10 secretion in a septic-shock mouse model. Biochem. Biophys Res. Commun. 486: 998-1004.   DOI
11 Minegishi H, Shimane Y, Echigo A, Ohta Y, Hatada Y, Kamekura M, et al. 2013. Thermophilic and halophilic β-agarase from a halophilic archaeon Halococcus sp. 197A. Extremophiles 17: 931-939.   DOI
12 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.   DOI
13 Duckworth M, Yaphe W. 1971. Structure of agar: Part I. Fractionation of a complex mixture of polysaccharides. Carbohydr. Res. 16: 189-197.   DOI
14 Han Z, Zhang Y, Yang J. 2019. Biochemical characterization of a new β-agarase from Cellulophaga algicola. Int. J. Mol. Sci. 20: 2143-2157.   DOI
15 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang A, Miller W, et al. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.   DOI
16 Ramos KRM, Valdehuesa KNG, Nisola GM, Lee WK, Chung WJ. 2018. Identification and characterization of a thermostable endolytic β-agarase Aga2 from a newly isolated marine agarolytic bacteria Cellulophaga omnivescoria W5C. N. Biotechnol. 40: 261-267.   DOI
17 Jung S, Lee CR, Chi WJ, Bae CH, Hong SK. 2017. 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.   DOI
18 Ramos KRM, Valdehuesa KNG, Banares AB, Nisola GM, Lee WK, Chung WJ. 2020. Overexpression and characterization of a novel GH16 β-agarase (Aga1) from Cellulophaga omnivescoria W5C. Biotechnol. Lett. 42: 2231-2238.   DOI
19 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.   DOI
20 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.   DOI
21 Kim DS, Chi W-J, Hong S-K. 2019. Molecular characterization of an endo-β-1,4-glucanase, CelAJ93, from the recently isolated marine bacterium, Cellulophaga sp. J9-3. Appl. Sci. 9: 4061-4073.   DOI
22 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.   DOI
23 Chi WJ, Chang YK, Hong SK. 2012. Agar degradation by microorganisms and agar-degrading enzymes. Appl. Microbiol. Biotechnol. 94: 917-930.   DOI
24 Knutsen SH, Myslabodski DE, Larsen B, Usov AI. 1994. A modified system of nomenclature for red algal galactans. Botanica Marina 37: 163-170.   DOI