Abstract
Two GH117 family α-neoagarobiose hydrolases (GH117A α-NABH and GH117B α-NABH) from the freshwater agar-degrading Cellvibrio sp. KY-GH-1 were expressed and purified as recombinant His-tagged proteins using an Escherichia coli expression system to compare activities. The amino acid sequence of GH117A α-NABH (364 amino acids, 40.9 kDa) showed 35% identity with that of GH117B α-NABH (392 amino acids, 44.2 kDa). GH117A α-NABH, but not GH117B α-NABH, could hydrolyze neoagarobiose (NA2) into monosaccharides 3,6-anhydro-L-galactose (L-AHG) and D-galactose. The presence of GH117A α-NABH homologues in all of the agar-degrading bacteria aligned suggests that GH117A α-NABH hydrolyzing NA2 into L-AHG and D-galactose is an essential component of the agar-degrading enzyme machinery. For GH117A α-NABH-catalyzed hydrolysis, NA2 was the sole substrate among various neoagaro-oligosaccharides (NA2~NA18). GH117A α-NABH appeared to exist as a dimer, and optimal enzymatic temperature and pH were 35 °C and 7.5, respectively. GH117A α-NABH was stable up to 35 °C and at pH 7.5 and unstable beyond 35 °C and outside pH 7.0~7.5. The kinetic parameters Km, Vmax, kcat, and kcat/Km for NA2 were 16.0 mM, 20.8 U/mg, 14.2 s-1, and 8.9 × 102 s-1 M-1, respectively. Combined addition of 5 mM MnSO4 and 10 mM tris(2-carboxyethyl)phosphine enhanced the enzyme activity by 2.4-fold. The enzyme-mediated hydrolysis of 5.0% NA2 into monosaccharide and purification of L-AHG from hydrolysis products by Sephadex G-10 column chromatography recovered ~ 192 mg L-AHG from 400 mg NA2 (~ 92% of the theoretical maximum yield). These results indicate that the recombinant GH117A α-NABH is NA2-specific and useful to produce L-AHG from NA2.
Key points
• Recombinant GH117A α-NABH (364 aa, 40.9 kDa) purified from E. coli forms a dimer.
• The enzyme hydrolyzes only NA2 among various neoagaro-oligosaccharides (NA2~NA18).
• The enzyme completely hydrolyzes up to 5% NA2 into monomers under optimal conditions.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All data generated or analyzed during this study are included in this published article.
References
Agbo JAC, Moss MO (1979) The Isolation and characterization of agarolytic bacteria from a lowland river. J Gen Microbiol 115(2):355–368. https://doi.org/10.1099/00221287-115-2-355
Alkotaini B, Han NS, Kim BS (2017) Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar. Appl Microbiol Biotechnol 101(4):1573–1580. https://doi.org/10.1007/s00253-016-8011-9
An K, Shi X, Cui F, Cheng J, Liu N, Zhao X, Zhang XH (2018) Characterization and overexpression of a glycosyl hydrolase family 16 β-agarase YM01-1 from marine bacterium Catenovulum agarivorans YM01T. Protein Expr Purif 143:1–8. https://doi.org/10.1016/j.pep.2017.10.002
Ariga O, Okamoto N, Harimoto N, Nakasaki K (2014) Purification and characterization of α-neoagarooligosaccharide hydrolase from Cellvibrio sp. OA-2007. J Microbiol Biotechnol 24(1):48–51. https://doi.org/10.4014/jmb.1307.07018
Asghar S, Lee CR, Park JS, Chi WJ, Kang DK, Hong SK (2018) Identification and biochemical characterization of a novel cold-adapted 1,3-α-3,6-anhydro-L-galactosidase, Ahg786, from Gayadomonas joobiniege G7. Appl Microbiol Biotechnol 102(20):8855–8866. https://doi.org/10.1007/s00253-018-9277-x
Bannikova GE, Lopatin SA, Varlamov VP, Kuznetsov BB, Kozina IV, Miroshnichenko ML, Chernykh NA, Turova TP, Bonch-Osmolovskaia EA (2008) The thermophilic bacteria hydrolyzing agar: characterization of thermostable agarose. Appl Biochem Microbiol 45(4):366–371. https://doi.org/10.1134/S0003683808040054
Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active EnZyme database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37(Database issue):D233–D338. https://doi.org/10.1093/nar/gkn663
Chen HM, Yan XJ (2005) Antioxidant activities of agaro-oligosaccharides with different degrees of polymerization in cell-based system. Biochim Biophys Acta 1722(1):103–111. https://doi.org/10.1016/j.bbagen.2004.11.016
Chen ZW, Lin HJ, Huang WC, Hsuan SL, Lin JH, Wang JP (2018) Molecular cloning, expression, and functional characterization of the β-agarase AgaB-4 from Paenibacillus agarexedens. AMB Express 8(1):49. https://doi.org/10.1186/s13568-018-0581-8
Chi WJ, Chang YK, Hong SK (2012) Agar degradation by microorganisms and agar-degrading enzymes. Appl Microbiol Biotechnol 94(4):917–930. https://doi.org/10.1007/s00253-012-4023-2
Duckworth M, Yaphe W (1971) The structure of agar. Part I. Fractionation of a complex mixture of polysaccharides. Carbohydr Res 16(1):189–197. https://doi.org/10.1016/S0008-6215(00)86113-3
Enoki T, Tominaga T, Takashima F, Ohnogi H, Sagawa H, Kato I (2012) Anti-tumor-promoting activities of agaro-oligosaccharides on two-stage mouse skin carcinogenesis. Biol Pharm Bull 35(7):1145–1149. https://doi.org/10.1248/bpb.b12-00188
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4):783–791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
Flament D, Barbeyron T, Jam M, Potin P, Czjzek M, Kloareg B, Michel G (2007) α-Agarases define a new family of glycoside hydrolases, distinct from β-agarase families. Appl Environ Microbiol 73(14):4691–4694. https://doi.org/10.1128/AEM.00496-07
Fu XT, Kim SM (2010) Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Mar Drugs 8(1):200–218. https://doi.org/10.3390/md8010200
Ha SC, Lee S, Lee J, Kim HT, Ko HJ, Kim KH, Choi IG (2011) Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40. Biochem Biophys Res Commun 412(2):238–244. https://doi.org/10.1016/j.bbrc.2011.07.073
Han W, Cheng Y, Wang D, Wang S, Liu H, Gu J, Wu Z, Li F (2016) Biochemical characteristics and substrate degradation pattern of a novel exo-type β-agarase from the polysaccharide-degrading marine bacterium Flammeovirga sp. strain MY04. Appl Environ Microbiol 82(16):4944–4954. https://doi.org/10.1128/AEM.00393-16
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(4):90. https://doi.org/10.3390/md15040090
Hu B, Gong Q, Wang Y, Ma Y, Li J, Yu W (2006) Prebiotic effects of neoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe 12(5-6):260–266. https://doi.org/10.1007/s10068-015-0230-9
Jahromi ST, Barzkar N (2018) Future direction in marine bacterial agarases for industrial applications. Appl Microbiol Biotechnol 102(16):6847–6863. https://doi.org/10.1007/s00253-018-9156-5
Jiang C, Liu Z, Sun J, Mao X (2020) Characterization of a novel α-neoagarobiose hydrolase capable of preparation of medium- and long-chain agarooligosaccharides. Front Bioeng Biotechnol 7:470. https://doi.org/10.3389/fbioe.2019.00470
Jun DY, Kim MK, Kim YH (1996) Rabbit antibody against murine cyclin D3 protein overexpressed in bacterial system. J Micobiol Biotechnol 6(6):474–481.
Kobayashi R, Takisada M, Suzuki T, Kirimura K, Usami S (1997) Neoagarobiose as a novel moisturizer with whitening effect. Biosci Biotechnol Biochem 61(1):162–163. https://doi.org/10.1271/bbb.61.162
Koti BA, Shinde M, Lalitha J (2013) Repeated batch production of agar-oligosaccharides from agarose by an amberlite IRA-900 immobilized agarase system. Biotechnol Bioprocess Eng 18:333–341. https://doi.org/10.1007/s12257-012-0237-5
Kwon GH, Kwon MJ, Park JE, Kim YH (2019) Whole genome sequence of a freshwater agar-degrading bacterium Cellvibrio sp. KY-GH-1. Biotechnol Rep 23:e00346. https://doi.org/10.1016/j.btre.2019.e00346
Kwon MJ, Jang WY, Kim GM, Kim YH (2020) Characterization and application of a recombinant exolytic GH50A β-agarase from Cellvibrio sp. KY-GH-1 for enzymatic production of neoagarobiose from agarose. ACS Omega 5(45):29453–29464. https://doi.org/10.1021/acsomega.0c04390
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. https://doi.org/10.1038/227680a0
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. https://doi.org/10.1093/bioinformatics/btm404
Lee DG, Jang MK, Lee OH, Kim NY, Ju SA, Lee SH (2008) Over-production of a glycoside hydrolase family 50 β-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product. Biotechnol Lett 30(5):911–918. https://doi.org/10.1007/s10529-008-9634-4
Lee MH, Jang JH, Yoon GY, Lee SJ, Lee MG, Kang TH, Park YM (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(5):263–268. https://doi.org/10.5483/BMBRep.2017.50.5.014
Lee CH, Lee CR, Hong SK (2019) Biochemical characterization of a novel cold-adapted agarotetraose-producing α-agarase, AgaWS5, from Catenovulum sediminis WS1-A. Appl Microbiol Biotechnol 103(20):8403–8411. https://doi.org/10.1007/s00253-019-10056-1
Li G, Sun M, Wu J, Ye M, Ge X, Wei W, Li H, Hu F (2015) Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH. Appl Microbiol Biotechnol 99(23):10019–10029. https://doi.org/10.1007/s00253-015-6869-6
Liang Y, Ma X, Zhang L, Li F, Liu Z, Mao X (2017) Biochemical characterization and substrate degradation mode of a novel exotype β-agarase from Agarivorans gilvus WH0801. J Agric Food Chem 65(36):7982–7988. https://doi.org/10.1021/acs.jafc.7b01533
Liu N, Yang M, Mao X, Mu B, Wei D (2016) Molecular cloning and expression of a new α-neoagarobiose hydrolase from Agarivorans gilvus WH0801 and enzymatic production of 3,6-anhydro-L-galactose. Biotechnol Appl Biochem 63(2):230–237. https://doi.org/10.1002/bab.1363
Michel G, Nyval-Collen P, Barbeyron T, Czjzek M, Helbert W (2006) Bioconversion of red seaweed galactans. A focus on bacterial agarases and carrageenases. Appl Microbiol Biotechnol 71(1):23–33. https://doi.org/10.1007/s00253-006-0377-7
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428. https://doi.org/10.1021/ac60147a030
Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New York, pp 1–348
Rhee YJ, Han CR, Kim WC, Jun DY, Rhee IK, Kim YH (2010) Isolation of a novel freshwater agarolytic Cellvibrio sp. KY-YJ-3 and characterization of its extracellular β-agarase. J Microbiol Biotechnol 20(10):1378–1385. https://doi.org/10.4014/jmb.1007.07010
Sneath P, Sokal R (1973) Numerical taxonomy: the principles and practice of numerical classification. W.H. Freeman and Company, San Francisco, pp 1–573
Spriestersbach A, Kubicek J, Schafer F, Block H, Maertens B (2015) Purification of his-tagged proteins. Methods Enzymol 559:1–15. https://doi.org/10.1016/bs.mie.2014.11.003
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89. https://doi.org/10.1016/0076-6879(90)85008-C
Sugano Y, Kodama H, Terada I, Yamazaki Y, Noma M (1994) Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107. J Bacteriol 176(22):6812–6818. https://doi.org/10.1128/jb.176.22.6812-6818.1994
Syazni YM, Kasuu M, Nakasaki K, Ariga O (2015) Draft genome sequence of the nonmarine agarolytic bacterium Cellvibrio sp. OA-2007. Genome Announc 3(3):e00468–e00415. https://doi.org/10.1128/genomeA.00468-15
Syrovy I, Hodny Z (1991) Staining and quantification of proteins separated by polyacrylamide gel electrophoresis. J Chromatogr B 569(1-2):175–196. https://doi.org/10.1016/0378-4347(91)80229-6
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. https://doi.org/10.1038/srep44252
Wang Q, Sun J, Liu Z, Huang W, Xue C, Mao X (2018) Coimmobilization of β-agarase and α-neoagarobiose hydrolase for enhancing the production of 3,6-anhydro-l-galactose. J Agric Food Chem 66(27):7087–7095. https://doi.org/10.1021/acs.jafc.8b01974
Watanabe T, Kashimura K, Kirimura K (2017) Purification, characterization and gene identification of a α-neoagarooligosaccharide hydrolase from an alkaliphilic bacterium Cellvibrio sp. WU-0601. J Mol Catal B Enzym 133:S328–S336. https://doi.org/10.1016/j.molcatb.2017.02.003
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(11):4907–4915. https://doi.org/10.1007/s00253-012-4385-5
Xie Z, Lin W, Luo J (2017) Comparative phenotype and genome analysis of Cellvibrio sp. PR1, a xylanolytic and agarolytic bacterium from the Pearl River. Biomed Res Int 2017:6304248. https://doi.org/10.1155/2017/6304248
Yu S, Yun EJ, Kim DH, Park SY, Kim KH (2020) Dual agarolytic pathways in a marine bacterium, Vibrio sp. strain EJY3: molecular and enzymatic verification. Appl Environ Microbiol 86(6):e02724–e02719. https://doi.org/10.1128/AEM.02724-19
Yun EJ, Lee S, Kim JH, Kim BB, Kim HT, Lee SH, Pelton JG, Kang NJ, Choi IG, Kim KH (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(7):2961–2970. https://doi.org/10.1007/s00253-012-4184-z
Yun EJ, Lee AR, Kim JH, Cho KM, Kim KH (2017) 3,6-Anhydro-l-galactose, a rare sugar from agar, a new anticariogenic sugar to replace xylitol. Food Chem 221:976–983. https://doi.org/10.1016/j.foodchem.2016.11.066
Zeng C, Zhang L, Miao S, Zhang Y, Zeng S, Zheng B (2016) Preliminary characterization of a novel β-agarase from Thalassospira profundimonas. Springerplus 5(1):1086. https://doi.org/10.1186/s40064-016-2748-6
Funding
This work was supported by a grant from Kyungpook National University, Daegu 41566, Republic of Korea, 2020.
Author information
Authors and Affiliations
Contributions
YHK conceived and designed the research. WYJ and MJK conducted the experiments. WYJ and KYK contributed analytical tools. WYJ and KYK analyzed the data. YHK wrote the manuscript. All authors read and agreed to the published version of the manuscript.
Corresponding author
Ethics declarations
Ethics approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
ESM 1
(PDF 269 kb)
Rights and permissions
About this article
Cite this article
Jang, W.Y., Kwon, M.J., Kim, K.Y. et al. Enzymatic characterization of a novel recombinant 1,3-α-3,6-anhydro-L-galactosidase specific for neoagarobiose hydrolysis into monosaccharides. Appl Microbiol Biotechnol 105, 4621–4634 (2021). https://doi.org/10.1007/s00253-021-11341-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-021-11341-8