Protocatechualdehyde and p-hydroxybenzaldehyde were oxidized in the same way as vanillin by both enzymes, and both strains

utilized these substrates as a carbon source for growth. Interestingly, the enzyme from strain TA1 exhibited much higher (about threefold) activity for isovanillin (3-hydroxy-4-methoxybenzaldehyde) than for vanillin, although strain TA1 did not grow on isovanillin as a carbon source. However, strain 5-Fluoracil TM1 grew weakly on isovanillin and the activity of its enzyme was only half of that with vanillin (data not shown). Syringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde), which has a 2-methoxyl group, was not oxidized by the enzyme from strain TA1, but was slightly oxidized by that obtained from strain TM1. Syringaldehyde was not utilized as a carbon source by both strains.

These results suggest that the position of the side chain within benzaldehyde derivatives affected the activity of these enzymes and the growth of the strains. The N-terminal amino acid and internal peptide sequences were determined by a protein sequencer as described previously (Mitsui et al., 2000). The N-terminal amino acid sequence of VDH from Micrococcus sp. TA1 was not obtained. The four internal peptide sequences obtained were FTAAAQSVK, FGDPAAEGLVGP, AEDEDHALQLANDXVCGLSS, and VNTDTNPFNDQVVARIRQA. The X in the above sequences indicates that the residue was not determined in the corresponding U0126 solubility dmso cycle. Some of these sequences showed similarities to aldehyde dehydrogenase or benzaldehyde dehydrogenase from Corynebacterium species. The N-terminal amino acid sequence of VDH from B. cepacia TM1 was obtained as MHEVSLLIDGVSRGASDXGTFDXIDPAT, and the six internal peptide sequences obtained were ARTLK, ASGYGRFGSK, QIESSGIEHINGPTVHDEAQMPFGGVK, VADAFVERLVAK, ASIAEFTDLRWITVQTT, and ASEGEPGVHRLIGSVLHDAGLGDGVVNVITHAPQDAPAIVERLIANPAVRRVNFTGSTS. These sequences showed a high similarity to aldehyde dehydrogenase from the strains classified in the B. cepacia complex. In our preliminary studies, we obtained partial gene sequences by amplifying the DNA fragment with degenerate primers derived from the above peptide sequences. The DNA fragment (about

500 bp) from strain Rutecarpine TA1 demonstrated the highest similarity (73%) to betaine-aldehyde dehydrogenase (accession number YP831378) from Arthrobacter sp. FB24 using the blastx program. On the other hand, the DNA fragment (about 600 bp) of strain TM1 appeared to be almost identical (99%) to the gene annotated as aldehyde dehydrogenase (accession number YP001583187) from Burkholderia multivorans ATCC 17616. These genes have a conserved domain of the NAD(P)-dependent aldehyde dehydrogenase superfamily (Perozich et al., 1999). In future experiments, we will try to obtain VDH-encoding genes using partial VDH genes from strain TA1 and TM1. Ferulic acid can be extracted from rice bran, which is an agricultural waste product, with hexane under alkaline conditions.