Genome mining and expression of a β-agarase from marine bacterium <i>Microbulbifer</i> sp. ARAS-458.1 isolated in Vietnam

Bao Chau Nguyen; Nguyen Thanh Vu; Xuan Bach Cao; Thi Van Anh Trinh; Tuan Kiet Do; Tuan Minh Do; Anh Tuan Nguyen; Thanh Thuy Nguyen

doi:10.15625/vjbt-23338

Author affiliations

Authors

Bao Chau Nguyen \(^1\) Food Industries Research Institute, 301 Nguyen Trai, Khuong Dinh, Hanoi, Vietnam
Nguyen Thanh Vu \(^1\) Food Industries Research Institute, 301 Nguyen Trai, Khuong Dinh, Hanoi, Vietnam https://orcid.org/0000-0002-4911-6771
Xuan Bach Cao \(^1\) Food Industries Research Institute, 301 Nguyen Trai, Khuong Dinh, Hanoi, Vietnam
Thi Van Anh Trinh \(^1\) Food Industries Research Institute, 301 Nguyen Trai, Khuong Dinh, Hanoi, Vietnam https://orcid.org/0009-0000-8424-0359
Tuan Kiet Do \(^1\) Food Industries Research Institute, 301 Nguyen Trai, Khuong Dinh, Hanoi, Vietnam https://orcid.org/0009-0008-2643-222X
Tuan Minh Do \(^1\) Food Industries Research Institute, 301 Nguyen Trai, Khuong Dinh, Hanoi, Vietnam
Anh Tuan Nguyen \(^2\) Innovation Center, Amano Enzyme Inc., 1-6, Techno Plaza, Kakamigahara, Gifu 509-0109, Japan
Thanh Thuy Nguyen \(^1\) Food Industries Research Institute, 301 Nguyen Trai, Khuong Dinh, Hanoi, Vietnam

DOI:

https://doi.org/10.15625/vjbt-23338

Keywords:

β-agarase, Microbulbifer, marine, agar, oligosaccharides.

Abstract

Agarolytic microorganisms and their enzymes are of growing interest for producing bioactive agaro-oligosaccharides. From 169 environmental samples collected in Vietnam, 507 agarolytic bacterial strains were isolated. Among them, Microbulbifer sp. ARAS-458.1, obtained from red algae (Rhodophyta) in Quang Binh Province, exhibited remarkable agar-degrading activity. Genome sequencing revealed a 4.0 Mb draft genome containing 3,350 predicted protein-coding genes and a GC content of 57.1%. Comparative analysis showed 85.84% average nucleotide identity with Microbulbifer elongatus DSM 6810ᵀ, indicating that ARAS-458.1 represents a distinct species. Six putative agarase genes were identified, including MARAS458A, a β-agarase of the glycoside hydrolase family 16 (GH16). MARAS458A was cloned and expressed in Escherichia coli and Pichia pastoris. In E. coli, the enzyme formed insoluble inclusion bodies, whereas expression in P. pastoris yielded an active, extracellular enzyme with a specific activity of 3.25 U/mg. The enzyme hydrolyzed agar to produce neoagarotetraose (NA4) and neoagarohexaose (NA6) as final products, confirming its endo-agarase function. MARAS458A exhibited optimal activity at 45°C and pH 7–10, was stable below 40°C, and showed thermolability at 45 °C. The dual production of NA4 and NA6, together with its alkaline nature, distinguishes MARAS458A from previously reported Microbulbifer β-agarases, suggesting structural variations affecting substrate processing and product specificity. These findings highlight MARAS458A as a promising biocatalyst for sustainable production of functional agaro-oligosaccharides for food and health applications.

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References

Chumsook K., Praiboon J., and Fu X. (2023). Sulfated galactans from agarophytes: review of extraction methods, structural features, and biological activities. Biomolecules, 13(12). https://doi.org/10.3390/biom13121745

Cronin C. N., and Kirsch J. F. (1988). Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis. Biochemistry, 27(12), 4572-4579. https://doi.org/10.1021/bi00412a052

Eskandari A., Nezhad N. G., Leow T. C., Rahman M. B. A., and Oslan S. N. (2023). Current achievements, strategies, obstacles, and overcoming the challenges of the protein engineering in Pichia pastoris expression system. World Journal of Microbiology and Biotechnology, 40(1), 39. https://doi.org/10.1007/s11274-023-03851-6

Fu X. T., and Kim S. M. (2010). Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Marine Drugs, 8(1), 200-218. https://www.mdpi.com/1660-3397/8/1/200

Gran H. H. (1902). Studien über Meeresbakterien. II. Über die Hydrolyse des Agar-Agars durch ein neues Enzym, die Gelase. Bergens Museums Aarbog, 1902, 2.

Hehemann J.-H., Smyth L., Yadav A., Vocadlo D. J., and Boraston A. B. (2012). Analysis of keystone enzyme in agar hydrolysis provides insight into the degradation of a polysaccharide from red seaweeds. Journal of Biological Chemistry, 287(17), 13985-13995. https://doi.org/10.1074/jbc.M112.345645

Hong S. J., Lee J.-H., Kim E. J., Yang H. J., Chang Y.-K., Park J.-S., et al. (2017a). In vitro and in vivo investigation for biological activities of neoagarooligo- saccharides prepared by hydrolyzing agar with β-agarase. Biotechnology and Bioprocess Engineering, 22(4), 489-496. https://doi.org/10.1007/s12257-017-0049-8

Hong S. J., Lee J.-H., Kim E. J., Yang H. J., Park J.-S., and Hong S.-K. (2017b). Anti-obesity and anti-diabetic effect of neoagarooligosaccharides on high-fat diet-induced obesity in mice. Marine Drugs, 15(4), 90. https://www.mdpi.com/1660-3397/15/4/90

Jahromi S. T., and Barzkar N. (2018). Future direction in marine bacterial agarases for industrial applications. Applied Microbiology and Biotechnology, 102(16), 6847-6863. https://doi.org/10.1007/s00253-018-9156-5

James G. (2010). Universal bacterial identification by PCR and DNA sequencing of 16s rRNA gene. In M. Schuller T. P. Sloots, G. S. James, C. L. Halliday, and I. W. J. Carter (Eds.), PCR for Clinical Microbiology: An Australian and International Perspective (pp. 209-214). Springer Netherlands. https://doi.org/10.1007/978-90-481-9039-3_28

Jiang C., Liu Z., Cheng D., and Mao X. (2020). Agarose degradation for utilization: Enzymes, pathways, metabolic engineering methods and products. Biotechnology Advances, 45, 107641. https://doi.org/10.1016/j.biotechadv.2020.107641

Jiang F., Xu X. W., Chen F. Q., Weng H. F., Chen J., Ru Y., et al.(2023). Extraction, modification and biomedical application of agarose hydrogels: A review. Marine Drugs, 21(5). https://doi.org/10.3390/md21050299

Kumar S., Stecher G., Suleski M., Sanderford M., Sharma S., and Tamura K. (2024). MEGA12: Molecular evolutionary genetic analysis version 12 for adaptive and green computing. Molecular Biology and Evolution, 41(12). https://doi.org/10.1093/molbev/msae263

Li R.-K., Chen Z., Ying X.-J., Ng T. B., and Ye X.-Y. (2018). A novel GH16 beta-agarase isolated from a marine bacterium, Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris. International Journal of Biological Macromolecules, 119, 1164-1170. https://doi.org/10.1016/j.ijbiomac.2018.08.053

Long J., Ye Z., Li X., Tian Y., Bai Y., Chen L., et al. (2024). Enzymatic preparation and potential applications of agar oligosaccharides: a review. Critical Reviews in Food Science and Nutrition 64(17), 5818-5834. https://doi.org/10.1080/10408398.2022.2158452

Miller G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426-428. https://doi.org/10.1021/ac60147a030

Ohta Y., Hatada Y., Nogi Y., Li Z., Zhang H.-M., Ito S., et al.(2004a). Thermostable beta-agarase from a deep-sea Microbulbifer isolate. Journal of Applied Glycoscience, 51(3), 203-210. https://doi.org/10.5458/jag.51.203

Ohta Y., Hatada Y., Nogi Y., Miyazaki M., Li Z., Akita M., et al. (2004b). Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from a novel species of deep-sea Microbulbifer. Applied Microbiology and Biotechnology, 64(4), 505-514. https://doi.org/10.1007/s00253-004-1573-y

Palmer I., and Wingfield P. T. (2012). Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Current Protocols in Protein Science, 70(1), 6.3.1-6.3.20. https://doi.org/10.1002/0471140864.ps0603s70

Sambrook J., Fritsch E. F., and Maniatis T. (1989). Molecular cloning: a laboratory manual. https://openlibrary.org/books/OL2404785M/Molecularcloning

Strohmeier M., Hrmova M., Fischer M., Harvey A. J., Fincher G. B., and Pleiss J. (2004). Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae. Protein Science 13(12), 3200-3213. https://doi.org/10.1110/ps.04828404

Takagi E., Hatada Y., Akita M., Ohta Y., Yokoi G., Miyazaki T., et al. (2015). Crystal structure of the catalytic domain of a GH16 β-agarase from a deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94. Bioscience, Biotechnology, and Biochemistry, 79(4), 625-632. https://doi.org/10.1080/09168451.2014.988680

Wang, H., Zhang, W., Cui Z., Lu Z., and Lu X. (2020). Characterization of the hydrolysate and catalytic cavity of α-agarase AgaD. Biotechnology Letters, 42(10), 1919-1925. https://doi.org/10.1007/s10529-020-02901-5

Waterborg, J. H. (2009). The Lowry Method for Protein Quantitation. In J. M. Walker (Ed.), The Protein Protocols Handbook (pp. 7-10). Humana Press. https://doi.org/10.1007/978-1-59745-198-7_2

Won-Jae C., Yong-Keun C., and Soon-Kwang, H. (2012). Agar degradation by microorganisms and agar-degrading enzymes.

Applied Microbiology and Biotechnology, 94(4), 917-930. https://doi.org/10.1007/s00253-012-4023-2

Yu S., Yun E. J., Kim, D. H., Park S. Y., and Kim, K. H. (2019). Anticariogenic activity of agarobiose and agarooligosaccharides derived from red macroalgae. Journal of Agricultural and Food Chemistry, 67(26), 7297-7303. https://doi.org/10.1021/acs.jafc.9b01245

Yu S., Yun Eun J., Kim Dong, H., Park So, Y., and Kim Kyoung, H. (2020). Dual agarolytic pathways in a marine bacterium, Vibrio sp. strain EJY3: molecular and enzymatic verification. Applied and Environmental Microbiology, 86(6), e02724-02719. https://doi.org/10.1128/AEM.02724-19

Yun E. J., Lee S., Kim, J. H., Kim, B. B., Kim, H. T., Lee S. H., 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. Applied Microbiology and Biotechnology, 97(7), 2961-2970. https://doi.org/10.1007/s00253-012-4184-z

Zhai L., Wu L., Li F., Burnham, R. S., Pizarro, J. C., and Xu B. (2016). A rapid method for refolding cell surface receptors and ligands. Scientific Reports, 6(1), 26482. https://doi.org/10.1038/srep26482

Zheng, J., Ge Q., Yan Y., Zhang, X., Huang, L., and Yin Y. (2023). dbCAN3: automated carbohydrate-active enzyme and substrate annotation. Nucleic Acids Research, 51(W1), W115-W121. https://doi.org/10.1093/nar/gkad328

Zhu La A. L. T., Li D., Cheng, Z., Wen Q., Hu D., Jin X., et al.(2024). Enzymatically prepared neoagarooligosaccharides improve gut health and function through promoting the production of spermidine by Faecalibacterium in chickens. Science of The Total Environment, 912, 169057. https://doi.org/10.1016/j.scitotenv.2023.169057