Abstract

Biocatalysts are increasingly utilized in the synthesis of drugs and agrochemicals as an alternative to chemical catalysis. They are preferred in the synthesis of enantiopure products due to their high regioselectivity and enantioselectivity. Cytochrome P450 (P450) oxygenases are valuable biocatalysts, since they catalyze the oxidation of carbon–hydrogen bonds with high efficiency and selectivity. However, practical use of P450s is limited due to their need for expensive cofactors and electron transport partners. P450s can employ hydrogen peroxide (H₂O₂) as an oxygen and electron donor, but the reaction with H₂O₂ is inefficient. The development of P450s that can use H₂O₂ will expand their applications. Here, an assay that utilizes Amplex Red peroxidation, to rapidly screen H₂O₂-dependent activity of P450 mutants in cell lysate was developed. This assay was employed to identify mutants of CYP119, a thermophilic P450 from Sulfolobus acidocaldarius, with increased peroxidation activity. A mutant library of CYP119 containing substitutions in the heme active site was constructed via combinatorial active-site saturation test and screened for improved activity. Screening of 158 colonies led to five mutants with higher activity. Among improved variants, T213R/T214I was characterized. T213R/T214I exhibited fivefold higher k_cat for Amplex Red peroxidation and twofold higher k_cat for styrene epoxidation. T213R/T214I showed higher stability towards heme degradation by H₂O₂. While the K_m for H₂O₂ and styrene were not altered by the mutation, a fourfold decrease in the affinity for another substrate, lauric acid, was observed. In conclusion, Amplex Red peroxidation screening of CYP119 mutants yielded enzymes with increased peroxide-dependent activity.

Graphic abstract

中文翻译：

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改进的Amplex Red过氧化活性测定方法的开发，用于筛选细胞色素P450变体和鉴定嗜热CYP119的新型突变体。

摘要

生物催化剂在药物和农用化学品的合成中越来越多地被用作化学催化的替代方法。由于它们的高区域选择性和对映选择性，它们在对映纯产物的合成中是优选的。细胞色素P450（P450）加氧酶是有价值的生物催化剂，因为它们以高效率和选择性催化碳氢键的氧化。但是，由于P450需要昂贵的辅因子和电子传输伙伴，因此实际使用受到限制。P450可以使用过氧化氢（H ₂ O ₂）作为氧和电子供体，但与H ₂ O ₂的反应效率低下。可以使用H ₂ O ₂的P450的开发将扩展他们的应用程序。在这里，开发了一种利用Amplex Red过氧化作用来快速筛选细胞裂解物中P450突变体的H ₂ O ₂依赖性活性的测定方法。该测定法被用来鉴定CYP119突变体，该突变体是来自Sulfolobus acidocaldarius的嗜热P450 ，具有增加的过氧化活性。通过组合活性位点饱和度测试，构建了一个在血红素活性位点中包含取代基的CYP119突变体文库，并筛选了改善的活性。158个菌落的筛选导致5个具有较高活性的突变体。在改进的变体中，表征了T213R / T214I。T213R / T214I表现出五倍更高ķ_猫于Amplex Red，过氧化和高两倍ķ苯乙烯环氧化的_猫。T213R / T214I对H ₂ O _2引起的血红素降解显示出更高的稳定性。虽然H ₂ O ₂和苯乙烯的K _m不会因突变而改变，但与另一种底物月桂酸的亲和力却降低了四倍。总之，CYP119突变体的Amplex Red过氧化筛选产生具有增加的过氧化物依赖性活性的酶。

图形摘要