Production and characterization of the Talaromyces stipitatus feruloyl esterase FAEC in Pichia pastoris: identification of the nucleophilic serine.
Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. We report the over-expression and characterization of a novel feruloyl esterase exhibiting broad substrate specificity from Talaromyces stipitatus (FAEC) in Pichia pastoris. Using various gene constructions, we have investigated the use of alternative signal peptides to produce an authentic feruloyl esterase featuring the N-terminal sequence determined for the native enzyme. We demonstrate that additional amino acids at the N-terminus of the FAEC sequence do not influence the catalytic capacity of the enzyme, and that the nature of the signal sequence has a limited effect on the yield of the secreted enzyme, with the T. stipitatus FAEC signal sequence producing 297 mgL(-1), the Neurospora crassa Fae-1 260 mgL(-1), and the Saccharomyces cerevisiae alpha-factor secretion signal 214 mgL(-1). Mature FAEC contains two internal peptide sequences that correspond with the consensus motif G-X-S-X-G that contains the catalytic serine nucleophile, which is conserved in the esterase enzyme superfamily. The serine residues at the center of these peptide motifs have been independently mutated and the corresponding enzymes have been over-expressed in P. pastoris to identify the candidate nucleophilic residue responsible for catalyzing the enzymatic reaction. Purified recombinant FAEC containing S465A retained the esterase activity and appeared unaffected by the amino acid modification. In contrast, FAEC activity containing S166A was below the HPLC detection limit, suggesting that serine 166 constitutes the nucleophile.
Crepin
Valerie F
VF
University of Nottingham, School of Biosciences, Division of Food Sciences, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Faulds
Craig B
CB
Connerton
Ian F
IF
D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D032901:Talaromyces / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins; D012694:Serine; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase