A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis
A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis
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DOI：
10.1128/aem.02911-14
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发表时间：
2015-03-01
影响因子：
4.4
通讯作者：
Berger, Ralf G.
中科院分区：
文献类型：
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作者：
Nieter, Annabel;Haase-Aschoff, Paul;Berger, Ralf G.
采用Q Sepharose FF柱阴离子交换层析、制备等电聚焦（IEF）、聚酰胺亲和层析和疏水相互作用层析等3个分离步骤纯化了一种绿原酸酯酶（UmChlE）。SDS-PAGE分析显示该蛋白为71 kDa的单体蛋白。纯化后的酶在pH 7.5和37℃时表现出最大的活性，并且在较宽的pH范围（3.5 ~ 9.5）内都有活性。先前描述的绿原酸酯酶对绿原酸表现出类似的亲和力，但来自黑木耳的酶对典型的阿魏酰酯酶底物也有活性。绿原酸、对香豆酸甲酯、咖啡酸甲酯和阿威酸甲酯的动力学常数分别为：Km值分别为19.6、64.1、72.5和101.8亩M， k(cat)/ k - M值分别为25.83mM(-1) s（-1）、7.63mM(-1) s（-1）、3.83mM(-1) s（-1）和3.75mM(-1) s（-1）。UmChlE从脱淀粉麦麸（DSWB）和咖啡浆（CP）等天然底物中释放阿威酸、对香豆酸和咖啡酸，证实对复杂植物生物量有活性。编码UmChlE的全长基因全长1758 bp，对应585个氨基酸的蛋白，在毕赤酵母GS115中功能性产生。序列比对与注释绿原酸和阿魏酰酯酶强调了该酶的独特性。
An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of similar to 71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37 degrees C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 mu M, 64.1 mu M, 72.5 mu M, and 101.8 mu M, respectively, and k(cat)/K-m values of 25.83mM(-1) s(-1), 7.63mM(-1) s(-1), 3.83mM(-1) s(-1) and 3.75mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.