荧光假单胞菌过氧化物酶 Dyp1B 的蛋白质工程化用于氧化酚类和聚合木质素底物。
Protein engineering of Pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates.
机构信息
Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK; Department of Bioengineering, University of Illinois at Urbana-Champaign, USA.
School of Life Sciences, Coventry University, Coventry, CV1 5FB, UK.
出版信息
Enzyme Microb Technol. 2019 Apr;123:21-29. doi: 10.1016/j.enzmictec.2019.01.002. Epub 2019 Jan 9.
Directed evolution was applied to dye-decolourizing peroxidase Dyp1B from Pseudomonas fluorescens Pf-5, in order to enhance the activity for oxidation of phenolic and lignin substrates. Saturation mutagenesis was used to generate focused libraries at 7 active site residues in the vicinity of the heme cofactor, and the libraries were screened for activity towards 2,6-dichlorophenol. Mutants N193 L and H169 L were found to show 7-8 fold enhanced k/K towards DCP, and replacements at Val205 and Ala209 also showed enhanced activity towards alkali Kraft lignin. Residues near the predicted Mn(II) binding site were also investigated by site-directed mutagenesis, and mutants S223 N and H127R showed 4-7-fold increased k/K for Mn(II) oxidation. Mutant F128R also showed enhanced thermostability, compared to wild-type Dyp1B. Testing of mutants for low molecular weight product release from Protobind alkali lignin revealed that mutant H169 L showed enhanced product release, compared with WT enzyme, and the formation of three low molecular weight metabolites by this mutant was detected by reverse phase HPLC analysis.
定向进化被应用于荧光假单胞菌 Pf-5 的染料脱色过氧化物酶 Dyp1B，以提高其氧化酚类和木质素底物的活性。在靠近血红素辅基的 7 个活性位点残基处使用饱和诱变生成聚焦文库，并针对 2,6-二氯苯酚筛选文库的活性。突变体 N193L 和 H169L 被发现对 DCP 的 k/K 提高了 7-8 倍，而 Val205 和 Ala209 的取代也显示出对碱性 kraft 木质素的增强活性。通过定点突变还研究了预测的 Mn(II)结合位点附近的残基，突变体 S223N 和 H127R 对 Mn(II)氧化的 k/K 提高了 4-7 倍。与野生型 Dyp1B 相比，突变体 F128R 还显示出增强的热稳定性。对突变体从 Protobind 碱性木质素中释放低分子量产物的测试表明，与 WT 酶相比，突变体 H169L 显示出增强的产物释放，并且通过反相 HPLC 分析检测到该突变体形成了三种低分子量代谢物。
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