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http://dx.doi.org/10.4014/jmb.1810.09065

Isolation and Characterization of an Eosinophilic GH 16 β-Agarase (AgaDL6) from an Agar-Degrading Marine Bacterium Flammeovirga sp. HQM9  

Liu, Yan (College of Marine Science, Shandong University)
Tian, Xiaoxu (National Facility for Protein Science in Shanghai, Zhangjiang Lab)
Peng, Chao (National Facility for Protein Science in Shanghai, Zhangjiang Lab)
Du, Zongjun (College of Marine Science, Shandong University)
Publication Information
Journal of Microbiology and Biotechnology / v.29, no.2, 2019 , pp. 235-243 More about this Journal
Abstract
A special eosinophilic agarase exo-type ${\beta}$-agarase gene, AgaDL6, was cloned from a marine agar-degrading bacterium, Flammeovirga sp. HQM9. The gene comprised 1,383-bp nucleotides encoding a putative agarase AgaDL6 of 461 amino acids with a calculated molecular mass of 52.8 kDa. Sequence analysis revealed a ${\beta}$-agarase domain that belongs to the glycoside hydrolase family (GH) 16 and a carbohydrate-binding module (CBM_4_9) unique to agarases. AgaDL6 was heterologously expressed in Escherichia coli BL21 (DE3). Enzyme activity analysis of the purified protein showed that the optimal temperature and pH of AgaDL6 were $50^{\circ}C$ and 3.0, respectively. AgaDL6 showed thermal stability by retaining more than 98% of activity after incubation for 2 h at $50^{\circ}C$, a feature quite different from other agarases. AgaDL6 also exhibited outstanding acid stability, retaining 100% of activity after incubation for 24 h at pH 2.0 to 5.0, a property distinct from other agarases. This is the first agarase characterized to have such high acid stability. In addition, we observed no obvious stimulation or inhibition of AgaDL6 in the presence of various metal ions and denaturants. AgaDL6 is an exo-type ${\beta}$-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products. These characteristics make AgaDL6 a potentially valuable enzyme in the cosmetic, food, and pharmaceutical industries.
Keywords
Agarase; GH16; CBM_4_9; eosinophilic; thermostable enzyme;
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1 Ramos KRM, Valdehuesa KNG, Nisola GM, Lee WK, Chung WJ. 2018. Identification and characterization of a thermostable endolytic $\beta$-agarase Aga2 from a newly isolated marine agarolytic bacteria Cellulophaga omnivescoria W5C. New Biotechnol. 40: 261-267.   DOI
2 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.   DOI
3 Araki C. 1959. Seaweed polysaccharides. pp 15-30. In: Wolfrom ML (ed) Carbohydrate chemistry of substances of biological interests. Pergamon Press, London.
4 Lin B, Lu G, Zheng Y, Xie W, Li S, Hu Z. 2012. Gene cloning, expression and characterization of a neoagarotetraose-producing beta-agarase from the marine bacterium Agarivorans sp. HZ105. World J. Microbiol. Biotechnol. 28: 1691-1697.   DOI
5 van der Meulen HJ, Harder W. 1976. Characterization of the neoagarotetraase and neoagarobiase of Cytophaga flevensis. Antonie van Leeuwenhoek 42: 81-94.   DOI
6 Morrice LM, McLean MW, Long WF, Williamson FB. 1983. $\beta$-Agarases I and II from Pseudomonas atlantica. Substrate specificities. Eur. J. Biochem. 137: 149-154.   DOI
7 Fu W, Han B, Duan D, Liu W, Wang C. 2008. Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6. J. Ind. Microbiol. Biotechnol. 35: 915-922.   DOI
8 Vera J, Alvarez R, Murano E, Slebe JC, Leon O. 1998. Identification of a marine agarolytic Pseudoalteromonas isolate and characterization of its extracellular agarase. Appl. Environ. Microbiol. 64: 4378-4383.   DOI
9 Wang JX, Mou HJ, Jiang XL, Guan HS. 2006. Characterization of a novel $\beta$-agarase from marine Alteromonas sp. SY37-12 and its degrading products. Appl. Microbiol. Biotechnol. 71: 833-839.   DOI
10 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.   DOI
11 Kim JH, Yun EJ, Seo N, Yu S, Kim DH, Cho KM, et al. 2017. Enzymatic liquefaction of agarose above the sol-gel transition temperature using a thermostable endo-type $\beta$-agarase, Aga16B. Appl. Microbiol. Biotechnol. 101: 1111-1120.   DOI
12 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.   DOI
13 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 its purification and in vitro skin-whitening anti-inflammatory activities. Appl. Microbiol. Biotechnol. 97: 2961-2970.   DOI
14 Kim SW, Hong CH, Jeon SW, Shin HJ. 2015. High-yield production of reducing sugars from Gracilaria verrucosa by acid and enzymatic hydrolysis processes. Bioresour. Technol. 196: 634-641.   DOI
15 Wargacki AJ, Leonard E, Win MN, Regitsky DD, Santos CN, Kim PB, et al. 2012. An engineered microbial platform for direct biofuel production from brown macroalgae. Science 335: 308-313.   DOI
16 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.   DOI
17 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.   DOI
18 Rios G, Ferrando A, Serrano R. 1997. Mechanisms of salt tolerance conferred by overexpression of the HAL1 Gene in Saccharomyces cerevisiae. Yeast 13: 515-528.   DOI
19 Singh A, Nigam PS, Murphy JD. 2011. Renewable fuels from algae: an answer to debatable land based fuels. Bioresour. Technol. 102: 10-16.   DOI
20 Yun EJ, Kim HT, Cho KM, Yu S, Kim S, Choi IG, et al. 2016. Pretreatment and saccharification of red macroalgae to produce fermentable sugars. Bioresour. Technol. 199: 311-318.   DOI
21 Song T, Xu H, Wei C, Jiang T, Qin S, Zhang W, Cao Y, et al. 2016. Horizontal transfer of a novel soil agarase gene from marine bacteria to soil bacteria via human microbiota. Sci. Rep. 6: 1-10.   DOI
22 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.   DOI
23 Dong Q, Ruan LW, Shi H. 2016. A $\beta$-agarase with high pH stability from Flammeovirga sp. SJP92. Carbohydr. Res. 432: 1-8.   DOI
24 Li J, Hu Q, Li Y, Xu Y. 2015. Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain. Braz . J. Microbiol. 46: 683-690.   DOI
25 Chi WJ, Chang YK, Hong SK. 2012. Agar degradation by microorganisms and agar-degrading enzymes. Appl. Microbiol. Biotechnol. 94: 917-930.   DOI
26 Zhang WB, Xu JN, Liu D Liu H, Lu XZ, Yu WG. 2018. Characterization of an $\alpha$-agarase from Thalassomonas sp. LD5 and its hydrolysate. Appl. Microbiol. Biotechnol. 102: 2203-2212.   DOI
27 Yang JI, Chen LC, Shih YY, Hsieh C, Chen CY, Chen WM, et al. 2011. Cloning and characterization of $\beta$-agarase AgaYT from Flammeovirga yaeyamensis strain YT. J. Biosci. Bioeng. 112: 225-232.   DOI
28 Long M, Yu Z, Xu X. 2010. A novel $\beta$-agarase with high pH stability from marine Agarivorans sp. LQ48. Mar. Biotechnol. 12: 62-69.   DOI
29 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.   DOI
30 Liu N, Mao X, Du Z, Mu B, Wei D. 2014. Cloning and characterization of a novel neoagarotetraose forming beta-agarase, AgWH50A from Agarivorans gilvus WH0801. Carbohydr. Res. 388: 147-151.   DOI
31 Ekborg N, Taylor L, Weiner R, Hutcheson S. 2006. Genomic and proteomic analysis of the agarolytic system of Saccharophagus degradans strain 2-40. Appl. Environ. Microbiol. 72: 3396-3405.   DOI
32 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.   DOI
33 Fu X, Kim S. 2010. Agarases: review of major sources, categories, purification method, enzyme characteristics and application. Mar. Drugs. 8: 200-218.   DOI
34 Boraston AB, Bolam DN, Gilbert HJ, Davies GJ. 2004. Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem. J. 382: 769-781.   DOI
35 Du ZJ, Zhang ZW, Miao TT. 2011. Draft Genome sequence of the novel agar-digesting marine bacterium HQM9. J. Bacteriol. 193: 4557-4558.   DOI
36 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.   DOI
37 Fu XT, Pan CH, Lin H, Kim SM. 2009. Gene cloning, expression, and characterization of a $\beta$-Agarase, AgaB34, from Agarivorans albus YKW-34. J. Microbiol. Biotechnol. 19: 257-264.   DOI
38 Mai ZM, Su HF, Zhang S. 2016. Isolation and Characterization of a glycosyl hydrolase family 16 $\beta$-Agarase from a mangrove soil metagenomic library. Int. J. Mol. Sci. 17: 1-12.   DOI
39 Xu H, Fu Y, Yang N, Ding Z, Lai Q, Zeng R. 2012. Flammeovirga pacifica sp. nov., isolated from deep-sea sediment. Int. J. Syst. Evol. Microbiol. 62: 937-941.   DOI
40 Zhao J, Shi B, Jiang QR, Ke CH. 2012. Changes in gut-associated flora and bacterial digestive enzymes during the development stages of abalone (Haliotis diversicolor). Aquaculture 343: 147-153.   DOI
41 Henshaw J, Horne-Bitschy A, van Bueren AL. 2006. Family 6 carbohydrate binding modules in beta-agarases display exquisite selectivity for the non-reducing termini of agarose chains. J. Biol. Chem. 281: 17099-17107.   DOI
42 Alkotaini B, Han NS, Kim BS. 2016. Enhanced catalytic efficiency of endo-$\beta$-agarase I by fusion of carbohydrate-binding modules for agar prehydrolysis. Enzyme Microbial. Technol. 9: 142-149.   DOI
43 Ohta Y, Hatada Y, Nogi Y. 2004. Enzymatic properties and nucleotide and amino acid sequences of a thermostable $\beta$-agarase from a novel species of deep-sea microbulbifer. Appl. Microbiol. Biotechnol. 64: 505-514.   DOI
44 Hu Z, Lin BK, Xu Y, Zhong MQ, Liu GM. 2008. Production and purification of agarase from a marine agarolytic bacterium Agarivorans sp. HZ105. J. Appl. Microbiol. 106: 181-190.   DOI
45 McCarter JD, Withers SG. 1994. Mechanisms of enzymatic glycoside hydrolysis. Curr. Opin. Struct. Biol. 4: 885-892.   DOI