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Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system
Biochemical Engineering Journal ( IF 3.7 ) Pub Date : 2020-04-01 , DOI: 10.1016/j.bej.2020.107555
Karla Ilić Đurđić , Selin Ece , Raluca Ostafe , Simon Vogel , Stefan Schillberg , Rainer Fischer , Radivoje Prodanović

Abstract Pleurotus eryngii wild-type versatile peroxidase (wtVP) oxidizes structurally diverse substrates in an H2O2-dependent manner, but its ability to oxidize many pollutants is limited by suicidal enzyme inactivation in the presence of excess H2O2. To address this drawback, we generated random mutagenesis libraries containing 3 × 106 mutated VP genes and screened for enzymes with higher oxidative stability expressed on the surface of yeast cells. This was achieved by flow cytometry using the substrate fluorescein tyramide. After two rounds of sorting, the percentage of cells expressing variants with improved oxidative stability had increased from 1 % to 56 %. The most stable variants featured 3–5 amino acid substitutions and retained up to 70 % of their initial activity after incubation for 1 h in 30 mM H2O2 (conditions that completely inactivate wtVP). Selected variants were extracted from yeast cell walls and purified for kinetic characterization. We also prepared yeast cell walls with wtVP and the three most stable VP variants for multiple cycles of azo dye (Reactive black 5) degradation. After 10 cycles of 12 h, two of the variants retained more than 97 % of their initial activity, whereas the activity of wtVP declined by ∼30 %. These results confirm that our high-throughput screening system can improve the oxidative stability of versatile peroxidase, providing a source of novel enzymes for remediation applications.

中文翻译:

通过基于流式细胞术的高通量筛选系统提高通用过氧化物酶的氧化稳定性

摘要杏鲍菇野生型多功能过氧化物酶 (wtVP) 以 H2O2 依赖性方式氧化结构多样的底物,但在过量 H2O2 存在下,其氧化许多污染物的能力受到自杀酶失活的限制。为了解决这个缺点,我们生成了包含 3 × 106 突变 VP 基因的随机诱变文库,并筛选了在酵母细胞表面表达的具有更高氧化稳定性的酶。这是通过使用底物荧光素酪酰胺的流式细胞术实现的。经过两轮分选后,表达具有改善的氧化稳定性的变体的细胞百分比从 1% 增加到 56%。最稳定的变体具有 3-5 个氨基酸取代,并在 30 mM H2O2 中孵育 1 小时后保留高达 70% 的初始活性(完全灭活 wtVP 的条件)。从酵母细胞壁中提取选定的变体并纯化以进行动力学表征。我们还用 wtVP 和三种最稳定的 VP 变体制备了酵母细胞壁,用于偶氮染料(活性黑 5)降解的多个循环。在 12 小时的 10 个循环后,其中两个变体保留了其初始活性的 97% 以上,而 wtVP 的活性下降了约 30%。这些结果证实,我们的高通量筛选系统可以提高多功能过氧化物酶的氧化稳定性,为修复应用提供新型酶的来源。从酵母细胞壁中提取选定的变体并纯化以进行动力学表征。我们还用 wtVP 和三种最稳定的 VP 变体制备了酵母细胞壁,用于偶氮染料(活性黑 5)降解的多个循环。在 12 小时的 10 个循环后,其中两个变体保留了其初始活性的 97% 以上,而 wtVP 的活性下降了约 30%。这些结果证实,我们的高通量筛选系统可以提高多功能过氧化物酶的氧化稳定性,为修复应用提供新型酶的来源。从酵母细胞壁中提取选定的变体并纯化以进行动力学表征。我们还用 wtVP 和三种最稳定的 VP 变体制备了酵母细胞壁,用于偶氮染料(活性黑 5)降解的多个循环。在 12 小时的 10 个循环后,其中两个变体保留了其初始活性的 97% 以上,而 wtVP 的活性下降了约 30%。这些结果证实,我们的高通量筛选系统可以提高多功能过氧化物酶的氧化稳定性,为修复应用提供新型酶的来源。而wtVP的活性下降了约30%。这些结果证实,我们的高通量筛选系统可以提高多功能过氧化物酶的氧化稳定性,为修复应用提供新型酶的来源。而wtVP的活性下降了约30%。这些结果证实,我们的高通量筛选系统可以提高多功能过氧化物酶的氧化稳定性,为修复应用提供新型酶的来源。
更新日期:2020-04-01
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