Vanillate is converted to protocatechuate by an
O-demethylase consisting of VanA and VanB in
Streptomyces sp. NL15-2K. In this study, vanillate demethylase from this strain was functionally expressed in
Escherichia coli, and its substrate range for vanillate analogs was determined by an
in vivo assay using recombinant whole cells. Among aromatic methyl ethers, vanillate, syringate,
m-anisate, and veratrate were good substrates, whereas ferulate, vanillin, and guaiacol were not recognized by
Streptomyces vanillate demethylase. After vanillate, 4-hydroxy-3-methylbenzoate was a better substrate than
m-anisate and veratrate, and the 3-methyl group was efficiently oxidized to a hydroxymethyl group. These observations suggest that the combination of a carboxyl group on the benzene ring and a hydroxyl group in the
para-position relative to the carboxyl group may be preferable for substrate recognition by the enzyme.
1H-NMR analysis showed that the demethylation product of veratrate was isovanillate rather than vanillate. Therefore, it was concluded that
O-demethylation of veratrate by
Streptomyces vanillate demethylase occurred only at the
meta-position relative to the carboxyl group.
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