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Characterization of a F280N variant of l-arabinose isomerase from Geobacillus thermodenitrificans identified as a d-galactose isomerase

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Abstract

The double-site variant (C450S-N475K) l-arabinose isomerase (l-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of d-galactose to d-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to d-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for d-galactose isomerization. The k cat/K m of the triple-site variant enzyme for d-galactose was 2.1-fold higher than for l-arabinose, whereas the k cat/K m of the double-site variant enzyme for l-arabinose was 43.9-fold higher than for d-galactose. These results suggest that the triple-site variant enzyme is a d-galactose isomerase. The conversion rate of d-galactose to d-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of l-arabinose to l-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for d-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for d-galactose and l-arabinose, and the triple-site variant enzyme has the potential to produce d-tagatose on an industrial scale.

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Acknowledgments

This work was supported by the Basic Research Lab program (No. 2013-A423-0061) of the National Research Foundation, the Ministry of Education, Science and Technology, and by a grant (No. 311042-5) from the Korea Institute of Planning and Evaluation for Technology, the Ministry of Food, Agriculture, Forestry and Fisheries, South Korea.

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  1. Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, South Korea

    Baek-Joong Kim, Seung-Hye Hong, Kyung-Chul Shin, Ye-Seul Jo & Deok-Kun Oh

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  1. Baek-Joong Kim
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  2. Seung-Hye Hong
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Correspondence to Deok-Kun Oh.

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Kim, BJ., Hong, SH., Shin, KC. et al. Characterization of a F280N variant of l-arabinose isomerase from Geobacillus thermodenitrificans identified as a d-galactose isomerase. Appl Microbiol Biotechnol 98, 9271–9281 (2014). https://doi.org/10.1007/s00253-014-5827-z

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