Functional expression and characterization of a glucuronoyl esterase from the fungus Neurospora crassa: identification of novel consensus sequences containing the catalytic triad

• Published in: Journal of general and applied microbiology Vol. 62; no. 5; pp. 217 - 224
• Main Authors: Huynh, Hung Hiep, Arioka, Manabu
• Format: Journal Article
• Language: English
• Published: Japan Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 01.01.2016
• Subjects: Amino Acid Sequence; carbohydrate esterase family 15; Catalysis; Catalytic Domain; Consensus Sequence; Esterases - chemistry; Esterases - genetics; Esterases - isolation & purification; Esterases - metabolism; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Glucuronic Acid - metabolism; glucuronoyl esterase; hemicellulose; Hydrolysis; Lignin - metabolism; lignin-carbohydrate complex; Neurospora crassa; Neurospora crassa - enzymology; Neurospora crassa - genetics; Neurospora crassa - metabolism; Oligopeptides - chemistry; Oligopeptides - metabolism; Pichia - genetics; Polysaccharides - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Substrate Specificity
• ISSN: 0022-1260; 1349-8037
• DOI: 10.2323/jgam.2016.03.004

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.