A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition
A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition
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DOI：
10.1042/bj20020917
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发表时间：
2003-03-01
影响因子：
4.1
通讯作者：
Connerton, IF
中科院分区：
文献类型：
--
作者：
Crepin, VF;Faulds, CB;Connerton, IF
阿魏酰酯酶是羧酸酯酶 (EC 3.1.1.1) 的一个亚类，能够水解植物细胞壁中存在的羟基肉桂酸和糖之间的酯键。根据其对芳香族部分的底物特异性，这些酶被分为 A 型或 B 型。我们表明，粗糙脉孢菌具有产生多种阿魏酸酯酶活性的能力，具体取决于用甜菜浆或麦麸底物发酵的时间长度。根据其在甜菜浆上的表达而鉴定的基因已在巴斯德毕赤酵母中克隆并过表达。该基因编码单域阿魏酸酯酶，这是非模块化 B 型酶的首次报道（fae-1 基因；GenBank 登录号：AJ293029）。纯化的重组蛋白已显示出浓度依赖性底物抑制作用（针对 3,4-二羟基肉桂酸甲酯，K-m 0.048 mM、K-i 2.5 mM 和 V-max 8.2 单位/毫克）。根据阿魏酸酯酶在植物细胞壁材料动员中的作用的多样性及其各自的作用模式，讨论了非模块化酶的动力学行为。
Feruloyl esterases, a subclass of the carboxylic acid esterases (EC 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell wall. The enzymes have been classified as type A or type B, based on their substrate specificity for aromatic moieties. We show that Neurospora crassa has the ability to produce multiple ferulic acid esterase activities depending upon the length of fermentation with either sugar beet pulp or wheat bran substrates. A gene identified on the basis of its expression on sugar beet pulp has been cloned and overexpressed in Pichia pastoris. The gene encodes a single-domain ferulic acid esterase, which represents the first report of a non-modular type B enzyme (fae-1 gene; GenBank accession no. AJ293029). The purified recombinant protein has been shown to exhibit concentration-dependent substrate inhibition (K-m 0.048 mM, K-i 2.5 mM and V-max 8.2 units/mg against methyl 3,4-dihydroxycinnamate). The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilization of plant cell wall materials and their respective modes of action.