Functional expression and characterization of a glucuronoyl esterase from the fungus Neurospora crassa: identification of novel consensus sequences containing the catalytic triad.
The complete hydrolysis of lignocellulose requires the actions of a variety of enzymes, including those that cleave the linkage between lignin and hemicellulose. The enzyme glucuronoyl esterase (GE) that constitutes a novel family of carbohydrate esterases, CE15, has been shown to display a unique ability to cleave the ester linkage between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid of hemicellulose. We herein report identification, expression, and functional characterization of a new GE, NcGE, from the filamentous fungus Neurospora crassa. C-terminally c-myc and hexahistidine-tagged NcGE was heterologously expressed in the methylotrophic yeast Pichia pastoris. NcGE purified from the culture supernatant through Ni-NTA and anion exchange chromatographies showed the ability to hydrolyze the substrate 3-(4-methoxyphenyl) propyl methyl 4-O-methyl-α-D-glucopyranosiduronate, which mimics the ester linkage of 4-O-methyl-D-glucuronic acid in lignin-carbohydrate complexes (LCCs). This esterase showed the characteristic of a mesophilic enzyme with the temperature optimum at 40-50°C, and displayed the optimal activity at pH 7 and broad pH stability. Based on the alignment of NcGE with other GEs so far characterized, we propose novel consensus sequences for GEs containing the catalytic triad.
Department of Biotechnology, The University of Tokyo.
Hung Hiep
HH
Huynh
Manabu
M
Arioka
D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D016384:Consensus Sequence; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D020723:Glucuronic Acid / Q000378:metabolism*; D006868:Hydrolysis; D008031:Lignin / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D005656:Fungal Proteins; D009842:Oligopeptides; D011134:Polysaccharides; D011994:Recombinant Proteins; C036909:lignocellulose; C007916:hemicellulose; D020723:Glucuronic Acid; D008031:Lignin; D004950:Esterases