Abstract
Agar is a galactan and a major component of the red algal cell wall. Agar is metabolized only by specific microorganisms. The final step of the β-agarolytic pathway is mediated by α-neoagarooligosaccharide hydrolase (α-NAOSH), which cleaves neoagarobiose to d-galactose and 3,6-anhydro-α-l-galactose. In the present study, two α-NAOSHs, SCO3481 and SCO3479, were identified in Streptomyces coelicolor A3(2). SCO3481 (370 amino acids, 41.12 kDa) and SCO3479 (995 amino acids, 108.8 kDa) catalyzed the hydrolysis of the α-(1,3) glycosidic bonds of neoagarobiose, neoagarotetraose, and neoagarohexaose at the nonreducing ends, releasing 3,6-anhydro-α-l-galactose. Both were intracellular proteins without any signal peptides for secretion. Similar to all α-NAOSHs reported to date, SCO3481 belonged to the glycosyl hydrolase (GH) 117 family and formed dimers. On the other hand, SCO3479 was a large monomeric α-NAOSH belonging to the GH2 family with a β-galactosidase domain. SCO3479 also clearly showed β-galactosidase activity toward lactose and artificial substrates, but SCO3481 did not. The optimum conditions for α-NAOSH were pH 6.0 and 25 °C for SCO3481, and pH 6.0 and 30 °C for SCO3479. Enzymatic activity was enhanced by Co2+ for SCO3481 and Mg2+ for SCO3479. The β-galactosidase activity of SCO3479 was maximum at pH 7.0 and 50 °C and was increased by Mg2+. Many differences were evident in the kinetic parameters of each enzyme. Although SCO3481 is typical of the GH117 family, SCO3479 is a novel α-NAOSH that was first reported in the GH2 family. SCO3479, a unique bifunctional enzyme with α-NAOSH and β-galactosidase activities, has many advantages for industrial applications.
Key points
• SCO3481 is a dimeric α-neoagarooligosaccharide hydrolase belonging to GH117.
• SCO3479 is a monomeric α-neoagarooligosaccharide hydrolase belonging to GH2.
• SCO3479 is a novel and unique bifunctional enzyme that also acts as a β-galactosidase.
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Data Availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.
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Funding
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through High Value-added Food Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number 322026–3).
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S.K. designed the research. M. performed most experiments. V. performed experiment on SCO3481. Y.S. constructed the recombined strain. C.R. performed mass spectrometry. S.K. wrote the manuscript. All authors reviewed the manuscript.
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Tsevelkhoroloo, M., Dhakshnamoorthy, V., Hong, YS. et al. Bifunctional and monofunctional α-neoagarooligosaccharide hydrolases from Streptomyces coelicolor A3(2). Appl Microbiol Biotechnol 107, 3997–4008 (2023). https://doi.org/10.1007/s00253-023-12552-x
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DOI: https://doi.org/10.1007/s00253-023-12552-x