title: Metagenomic mining of feruloyl esterases from termite enteric flora.
abstract: A metagenome expression library was created from Trinervitermes trinervoides termite hindgut symbionts and subsequently screened for feruloyl esterase (FAE) activities, resulting in seven recombinant fosmids conferring feruloyl esterase phenotypes. The amino acid sequence lengths of the seven FAE encoding open reading frames (ORFs) ranged from 260 to 274 aa and encoded polypeptides of between 28.9 and 31.4 kDa. The highest sequence identity scores for the seven ORFs against the GenBank database were between 45 and 59 % to a number of carboxyl ester hydrolyses. The seven FAE primary structures contained sequence motifs that correspond well with a classical pentapeptide (G-x-S-x-G) serine hydrolyse signature motif which harbours the catalytic serine residue in other FAE families. Six of the seven fae genes were successfully expressed heterologously in Escherichia coli, and the purified enzymes exhibited temperature optima range of 40-70 °C and the pH optima of between 6.5 and 8.0. The k(cat)/K(M) ratios for the six characterised FAEs showed the following order of substrate preference: methyl sinapate > methyl ferulate > ethyl ferulate. All six FAEs showed poor conversion rates against methyl p-coumarate and methyl caffeate, both of which lacked the methoxy (O-CH₃) group substituent on the aromatic ring of the ester substrates, emphasising the requirement for at least one methoxy group on the aromatic ring of the hydroxycinnamic acid ester substrate for optimal FAE activity.
authors: CSIR Biosciences, Building 18 Meiring Naudé Road, Brummeria, PO Box 395, Pretoria, 0001, South Africa, KRashamuse@csir.co.za.
authors: Konanani
authors: K
authors: Rashamuse
authors: Tina
authors: T
authors: Ronneburg
authors: Walter
authors: W
authors: Sanyika
authors: Kgama
authors: K
authors: Mathiba
authors: Edwin
authors: E
authors: Mmutlane
authors: Dean
authors: D
authors: Brady
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D041981:Gastrointestinal Tract / Q000382:microbiology; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D020049:Isoptera / Q000382:microbiology*; D054892:Metagenome; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase