Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence
Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence
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DOI：
10.1016/j.bbagen.2004.12.016
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发表时间：
2005-03-11
影响因子：
3
通讯作者：
Matsuzawa, H
中科院分区：
文献类型：
--
作者：
Koseki, T;Takahashi, K;Matsuzawa, H
我们克隆了AwfaeA （Aspergillus awamori）的阿铁酰酯酶A基因，并对其进行工程修饰，研究了该酶的底物特异性和pH依赖性。基于黑曲霉两种a型阿铁酰酯酶（FAE-III和AnFAEA）的晶体结构，我们将AwFAEA瓣区（Asp71、Thr72、Asp77和Tyr80）的残基分别替换为在米黑根霉（RmLIP）和葎草糖（HILIP）脂肪酶盖上发现的相应氨基酸残基（Ile、Arg、Asn和Phe）。此外，在曲霉FAEs和脂肪酶中保守的AwFAEA的Asp77被一个疏水残基（Ile）取代。对突变体酶的动力学分析表明，D77I和Y80F突变体对α -萘基丁酸酯（C4）和α -萘基辛酸酯（C8）的催化效率较高是由于K-m值较低。D77N对C4底物的高催化效率是由于k -m的降低和k（cat）的显著增加共同作用的结果。D71I和Y80F突变体对长酰基链酯具有一定的活性。另一方面，D77I突变体对酚酸甲酯和阿铁酰化阿拉伯木聚糖没有检测到的活性。D77I、D77N和Y80F的最适pH值分别从5.0提高到7.0 ~ 8.0、7.0和6.0。(c) 2004 Elsevier B.V.版权所有
We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei (RmLIP) and Humicola lanuginose (HILIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward alpha-naphthylbutyrate (C4) and alpha-naphthylcaprylate (C8), respectively, was due to a lower K-m value. The higher catalytic efficiency of D77N toward C4 substrate was due to a combination of decreased K-m and considerably increased k(cat). The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0-8.0, 7.0, and 6.0, respectively. (c) 2004 Elsevier B.V. All rights reserved.