N-Linked Oligosaccharides of Aspergillus awamori Feruloyl Esterase Are Important for Thermostability and Catalysis
Access this Article
Search this Article
A unique <I>N</I>-linked glycosylation motif (Asn<SUP>79</SUP>-Tyr-Thr) was found in the sequence of type-A feruloyl esterases from <I>Aspergillus</I> spp. To clarify the function of the flap, the role of <I>N</I>-linked oligosaccharides located in the flap region on the biochemical properties of feruloyl esterase (AwFAEA) from <I>Aspergillus awamori</I> expressed in <I>Pichia pastoris</I> was analyzed by removing the <I>N</I>-linked glycosylation recognition site by site-directed mutagenesis. N79 was replaced with A or Q. <I>N</I>-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 <I>K</I><SUB>m</SUB> and decreased <I>k</I><SUB>cat</SUB> for N79A, and to a considerably decreased <I>k</I><SUB>cat</SUB> for N79Q. N79A and N79Q mutant enzymes also exhibited considerably reduced thermostability relative to the wild-type.
- Agricultural and Biological Chemistry
Agricultural and Biological Chemistry 70(10), 2476-2480, 2006-10-23
Japan Society for Bioscience, Biotechnology, and Agrochemistry