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Cloning, expression, and characterization of a glycoside hydrolase family 86 β-agarase from a deep-sea Microbulbifer-like isolate

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Abstract

The gene for a novel β-agarase from a deep-sea Microbulbifer-like isolate was cloned and sequenced. It encoded a mature protein of 126,921 Da (1,146 amino acids), which was a modular protein including two tandem carbohydrate-binding module (CBM)-like sequences and a catalytic module. The catalytic module resembled a glycoside hydrolase family 86 β-agarase, AgrA, from Pseudoalteromonas atlantica T6c with 31% amino acid identity. Its recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host and purified to homogeneity. The activity and stability were strongly enhanced by CaCl2. The maximal enzyme activity was observed at 45°C and pH 7.5 in the presence of 10 mM CaCl2. The enzyme was an endo-type β-agarase and degraded agarose and agarose oligosaccharides more polymerized than hexamers to yield neoagarohexaose as the main product. This is the first glycoside hydrolase family 86 enzyme to be homogeneously purified and characterized.

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Acknowledgements

We are grateful to Dr. William D. Grant of the University of Leicester and Dr. Y. Sakano of the Tokyo University of Agriculture and Technology for stimulating discussions.

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Authors and Affiliations

  1. Japan Agency for Marine–Earth Science and Technology (JAMSTEC, formerly Japan Marine Science and Technology Center), 2-15 Natsushima, Yokosuka, 237-0061, Japan

    Yukari Ohta, Yuji Hatada, Yuichi Nogi, Zhijun Li, Susumu Ito & Koki Horikoshi

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  1. Yukari Ohta
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  2. Yuji Hatada
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  3. Yuichi Nogi
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  4. Zhijun Li
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  5. Susumu Ito
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  6. Koki Horikoshi
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Correspondence to Yuji Hatada.

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Ohta, Y., Hatada, Y., Nogi, Y. et al. Cloning, expression, and characterization of a glycoside hydrolase family 86 β-agarase from a deep-sea Microbulbifer-like isolate. Appl Microbiol Biotechnol 66, 266–275 (2004). https://doi.org/10.1007/s00253-004-1757-5

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  • DOI: https://doi.org/10.1007/s00253-004-1757-5

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