Functional expression and characterization of a glucuronoyl esterase from the fungus Neurospora crassa: identification of novel consensus sequences containing the catalytic triad
Functional expression and characterization of a glucuronoyl esterase from the fungus Neurospora crassa: identification of novel consensus sequences containing the catalytic triad
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DOI：
10.2323/jgam.2016.03.004
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发表时间：
2016-01-01
影响因子：
1.2
通讯作者：
Arioka, Manabu
中科院分区：
文献类型：
--
作者：
Hung Hiep Huynh;Arioka, Manabu
木质纤维素的完全水解需要多种酶的作用，包括切割木质素和半纤维素之间的键的酶。葡萄糖醛酸酯酶（GE）构成了碳水化合物酯酶的新家族，CE 15，已经显示出切割木质素醇和半纤维素的木聚糖结合的4-O-甲基-D-葡萄糖醛酸之间的酯键的独特能力。我们在此报告的鉴定，表达和功能特性的一个新的GE，NcGE，从丝状真菌粗糙脉孢菌。C-末端c-myc和六组氨酸标记的NcGE在甲基营养型酵母巴斯德毕赤酵母中异源表达。通过Ni-NTA和阴离子交换层析从培养物上清液纯化的NcGE显示出水解底物3-（4-甲氧基苯基）丙基甲基4-O-甲基-a-D-吡喃葡萄糖醛酸的能力，其模拟木质素-碳水化合物复合物（LCC）中4-O-甲基-D-葡萄糖醛酸的酯键。该酯酶具有嗜中温酶的特性，最适温度为40-508 ℃，最适pH为7，并具有较宽的pH稳定性。基于NcGE与其他GE的比对，迄今为止的特点，我们提出了新的共识序列的GE含有催化三联体。
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-Omethyl-a-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 40-508 degrees 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.