title: Cloning, sequencing, and overexpression in Escherichia coli of the Enterobacter sp. Px6-4 gene for ferulic acid decarboxylase.
abstract: 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.
authors.affiliation: Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, People's Republic of China.
authors.forename: Wen
authors.initials: W
authors.lastname: Gu
authors.forename: Xuemei
authors.initials: X
authors.lastname: Li
authors.forename: Jingwen
authors.initials: J
authors.lastname: Huang
authors.forename: Yanqing
authors.initials: Y
authors.lastname: Duan
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Meng
authors.forename: Ke-Qin
authors.initials: KQ
authors.lastname: Zhang
authors.forename: Jinkui
authors.initials: J
authors.lastname: Yang
mesh_terms: D001711:Biotransformation; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004754:Enterobacter / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006139:Guaiacol / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008628:Mercury / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D003373:Coumaric Acids; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D006139:Guaiacol; C014245:4-vinylguaiacol; D003300:Copper; C004999:ferulic acid; D002262:Carboxy-Lyases; C087194:phenylacrylic acid decarboxylase; D008628:Mercury