Gene identification and characterization of fucoidan deacetylase for potential application to fucoidan degradation and diversification

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Fucoidan is an α-l-fucopyranosyl polymer found in seaweeds with forms that have acetyl and sulfuric modifications and derivatives that are lower and/or diversified, with modifications that have attracted interest as potential bioactive substances. We identified the gene for a fucoidan deacetylase that cleaves acetyl moieties from fucoidan and thereby contributes to fucoidan utilization in the marine bacterium Luteolibacter algae H18. Fucoidan deacetylase was purified to homogeneity from a cell-free extract of L. algae H18, and used to determine the internal amino acid sequence and identify the gene, fud, in a draft genome sequence of the H18 strain. The gene product was heterologously produced in Escherichia coli and was demonstrated to catalyze fucoidan deacetylation, but not desulfation, and degradation into lower forms. In addition to fucoidan deacetylation, the enzyme catalyzed the hydrolysis of p-nitrophenyl esters with organic acids, and p-nitrophenyl acetate was the best substrate among those tested. The present study provides a new tool for fucoidan degradation, potentially expanding investigations on fucoidan derivatives.

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Purification of a deacetylase from L. algae H18

The high-molecular-weight fucoidan (SEA ALGA-F, Marine Products Kimuraya, Co., Ltd., Sakaiminato, Japan) used in this study was extracted and purified from C. okamuranus by the method of Kawamoto et al. (15). L. algae strain H18 was grown in medium F, a synthetic medium containing fucoidan as the sole carbon source at 30 °C for 48 h with reciprocal shaking at 100 strokes per min, and preparation of cell-free extracts was carried out as previously reported (13). The cell-free extracts prepared

Enzyme purification from the wild strain and N-terminal and internal amino acid sequences

The enzyme that catalyzed the deacetylation of fucoidan from C. okamuranus was purified by column chromatography of cell-free extracts of L. algae H18. The activity of the enzyme was estimated by measuring the amounts of acetate liberated from fucoidan with an Acetic Acid Test Kit during enzyme purification. SDS-PAGE showed that the enzyme was purified to almost homogeneity and that the molecular mass of the subunit was approximately 70 kDa (Fig. 1). Although the N-terminal amino acid sequence

Discussion

We demonstrated that L. algae H18 produced intracellular enzymes that are involved in the degradation of fucoidan from C. okamuranus. In addition, our findings showed that the deacetylation of fucoidan proceeded first, and without decreasing the molecular weight of fucoidan. Although some fucoidan-degrading enzymes have been reported in the literature, these studies performed fucoidan degradation in the absence of any other proteins or cofactors 5, 9, 12, 20. To the best of our knowledge, this

Acknowledgments

The analysis of the draft genome of strain H18 was performed by Professor Junichi Nakagawa at the Department of Food and Cosmetic Science, Tokyo University of Agriculture, Japan.

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