Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence.
We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei (RmLIP) and Humicola lanuginose (HlLIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward alpha-naphthylbutyrate (C4) and alpha-naphthylcaprylate (C8), respectively, was due to a lower K(m) value. The higher catalytic efficiency of D77N toward C4 substrate was due to a combination of decreased K(m) and considerably increased k(cat). The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0-8.0, 7.0, and 6.0, respectively.
Koseki
Takuya
T
Materials Divison, National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-hiroshima 739-0046, Japan. tkoseki@nrib.go.jp
Takahashi
Kenji
K
Fushinobu
Shinya
S
Iefuji
Haruyuki
H
Iwano
Kimio
K
Hashizume
Katsumi
K
Matsuzawa
Hiroshi
H
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase