title: Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus.
abstract: OBJECTIVE
  A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized.
  
  
  RESULTS
  A total of 30 clones exhibiting activity on α-naphthyl acetate (α-NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase-coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro-organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p-nitrophenyl fatty acid esters and hydroxylcinnamic acid esters.
  
  
  CONCLUSIONS
  Fae1A exhibited a lower K(m) and higher catalytic efficiency (k(cat) /K(m) ) on ferulic acid esters than on α-NA or p-nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p-coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine-specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity.
  
  
  CONCLUSIONS
  To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.
authors.affiliation: Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, AB, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Qi
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.forename: L B
authors.initials: LB
authors.lastname: Selinger
authors.forename: L J
authors.initials: LJ
authors.lastname: Yanke
authors.forename: R J
authors.initials: RJ
authors.lastname: Forster
authors.forename: T A
authors.initials: TA
authors.lastname: McAllister
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003373:Coumaric Acids / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D008969:Molecular Sequence Data; D009284:Naphthols / Q000378:metabolism; D020085:Neocallimastigales / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D011422:Propionates; D012417:Rumen / Q000382:microbiology; D016415:Sequence Alignment
chemical_list: D003373:Coumaric Acids; D005656:Fungal Proteins; D009284:Naphthols; D011422:Propionates; C027876:alpha-naphthyl acetate; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; C495469:p-coumaric acid