Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, People's Republic of China.
Appl Microbiol Biotechnol. 2011 Mar;89(6):1797-805. doi: 10.1007/s00253-010-2978-4. Epub 2010 Nov 18.
Ferulic acid decarboxylase (FADase) can catalyze the transformation of ferulic acid into 4-vinyl guaiacol via decarboxylation in microorganisms. In this study, a gene encoding FADase was first isolated from the bacterium Enterobacter sp. Px6-4 using degenerate primers and a genome walking technique. The putative encoding gene (fad) of FADase consists of 507-bp nucleotides, coding a polypeptide of 168 amino acid residues. In addition, a putative gene encoding the transcriptional regulator was identified from the upstream of the fad gene. The deduced peptide sequence of the FADase from Enterobacter sp. Px6-4 showed a 51.2-53.3% sequence identity to decarboxylases from other bacteria. The gene fad was successfully expressed in Escherichia coli BL21, and the recombinant FADase was purified as a protein of ca. 23 kDa with an optimal activity at pH 4.0 and 28 °C. The purified FADase could convert ferulic acid to 4-vinyl guaiacol effectively, and its hydrolytic activity could be inhibited by Cu(2+) (99%) and Hg(2+) (99.5%). A phylogenetic analysis of the FADase protein from bacteria revealed several different clades. Our result provided a basis for further studies of the ferulic acid transformation pathway and for enhanced production of vanillin in the future.
阿魏酸脱羧酶（FADase）可以在微生物中通过脱羧作用将阿魏酸转化为 4-乙烯基愈创木酚。本研究首次利用简并引物和基因组步移技术从 Enterobacter sp. Px6-4 细菌中分离出编码 FADase 的基因。FADase 的假定编码基因（fad）由 507 个核苷酸组成，编码 168 个氨基酸残基的多肽。此外，还在 fad 基因的上游鉴定出一个假定的编码转录调节剂的基因。来自 Enterobacter sp. Px6-4 的 FADase 推导出的肽序列与其他细菌的脱羧酶具有 51.2-53.3%的序列同一性。fad 基因在大肠杆菌 BL21 中成功表达，重组 FADase 被纯化为约 23 kDa 的蛋白质，在 pH 4.0 和 28°C 时具有最佳活性。纯化的 FADase 可以有效地将阿魏酸转化为 4-乙烯基愈创木酚，其水解活性可被 Cu(2+)（99%）和 Hg(2+)（99.5%）抑制。对细菌 FADase 蛋白的系统发育分析揭示了几个不同的分支。我们的结果为进一步研究阿魏酸转化途径和未来提高香草醛产量提供了依据。
