Engineering a Bacterial DyP-Type Peroxidase for Enhanced Oxidation of Lignin-Related Phenolics at Alkaline pH
Engineering a Bacterial DyP-Type Peroxidase for Enhanced Oxidation of Lignin-Related Phenolics at Alkaline pH
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DOI：
10.1021/acscatal.6b03331
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发表时间：
2017-05-01
期刊：
影响因子：
12.9
通讯作者：
Martins, Ligia O.
中科院分区：
文献类型：
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作者：
Brissos, Vania;Tavares, Diogo;Martins, Ligia O.
染料脱色过氧化物酶（DyPs）是一类含血红素的微生物过氧化物酶家族，由于其氧化木质素相关化合物的能力而显示出对木质纤维素生物精炼的重要性质。定向进化用于提高来自恶臭假单胞菌MET 94的细菌PpDyP对酚类化合物的效率。通过ppDyP基因的易错PCR进行三轮随机诱变，然后进行高通量筛选，从而鉴定出对2，6-二甲氧基苯酚（dichloromethyl phenol，dichloromethyl phenol，dichloromethyl phenol）显示出100倍增强的催化效率（k（ca）t/K-m）的6 E10变体，类似于真菌木质素过氧化物酶所表现出的催化效率（类似于10（5）M-1 s（-1））。进化的变体显示出对于许多异戊烯基型酚、愈创木酚、芳香胺、硫酸盐木质素和木质素酚模型二聚体愈创木基甘油-β-愈创木基醚的额外改进的效率。重要的是，变体6 E10在8.5显示最佳pH，与野生型相比上移4个单位，显示对过氧化氢失活的抗性，并且以2倍更高的产率产生。在进化过程中获得的突变影响了位于酶表面的三个氨基酸残基（E188 K，A142 V和H125 Y），位于血红素腔的第二层。从实验室进化的命中变体的生化分析，和使用定点诱变构建的单一变体，揭示了获得性突变的催化，稳定性和结构的观点的关键作用。我们发现A142 V和E188 K突变之间的上位性对于确定6 E10的底物特异性至关重要。有证据表明ABTS和ABTS氧化发生在血红素通道。通过瞬态动力学阐明了6 E10的催化循环的细节，为迄今为止研究的大多数血红素过氧化物酶中几乎未检测到的可逆酶过氧化氢复合物（化合物0）的形成提供了证据。
Dye-decolorizing peroxidases (DyPs) are a family of microbial heme-containing peroxidases that show important properties for lignocellulose biorefineries due to their ability to oxidize lignin-related compounds. Directed evolution was used to improve the efficiency of the bacterial PpDyP from Pseudomonas putida MET94 for phenolic compounds. Three rounds of random mutagenesis by error prone PCR of the ppDyP gene followed by high-throughput screening allow identification of the 6E10 variant showing a 100-fold enhanced catalytic efficiency (k(ca)t/K-m) for 2,6-dimethoxyphenol (DMP), similar to that exhibited by fungal lignin peroxidases (similar to 10(5) M-1 s(-1)). The evolved variant showed additional improved efficiency for a number of syringyl-type phenolics, guaiacol, aromatic amines, Kraft lignin, and the lignin phenolic model dimer guaiacylglycerol-beta-guaiacyl ether. Importantly, variant 6E10 displayed optimal pH at 8.5, an upshift of 4 units in comparison to the wild type, showed resistance to hydrogen peroxide inactivation, and was produced at 2-fold higher yields. The acquired mutations in the course of the evolution affected three amino acid residues (E188K, A142V, and H125Y) situated at the surface of the enzyme, in the second shell of the heme cavity. Biochemical analysis of hit variants from the laboratory evolution, and single variants constructed using site-directed mutagenesis, unveiled the critical role of acquired mutations from the catalytic, stability, and structural viewpoints. We show that epistasis between A142V and E188K mutations is crucial to determine the substrate specificity of 6E10. Evidence suggests that ABTS and DMP oxidation occurs at the heme access channel. Details of the catalytic cycle of 6E10 were elucidated through transient kinetics, providing evidence for the formation of a reversible enzyme hydrogen peroxide complex (Compound 0) barely detected in the majority of heme peroxidases studied to date.