N-Linked Oligosaccharides of Aspergillus awamori Feruloyl Esterase Are Important for Thermostability and Catalysis

KOSEKI, Takuya; Takahashi, Kenji; HANDA, Takashi; Yamane, Yūichi; FUSHINOBU, Shinya; HASHIZUME, Katsumi

N-Linked Oligosaccharides of Aspergillus awamori Feruloyl Esterase Are Important for Thermostability and Catalysis

2006

A unique N-linked glycosylation motif (Asn⁷⁹-Tyr-Thr) was found in the sequence of type-A feruloyl esterases from Aspergillus spp. To clarify the function of the flap, the role of N-linked oligosaccharides located in the flap region on the biochemical properties of feruloyl esterase (AwFAEA) from Aspergillus awamori expressed in Pichia pastoris was analyzed by removing the N-linked glycosylation recognition site by site-directed mutagenesis. N79 was replaced with A or Q. N-glycosylation-free N79A and N79Q mutant enzymes had lower activity than that of the glycosylated recombinant AwFAEA wild-type enzyme toward α-naphthylbutyrate (C4), α-naphthylcaprylate (C8), and phenolic acid methyl esters. Kinetic analysis of the mutant enzymes indicated that the lower catalytic efficiency was due to a combination of increased K ₘ and decreased k cₐₜ for N79A, and to a considerably decreased k cₐₜ for N79Q. N79A and N79Q mutant enzymes also exhibited considerably reduced thermostability relative to the wild-type.

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