Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase-NADPH Oxidase Fusions for Continuous Oxidations.
Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase-NADPH Oxidase Fusions for Continuous Oxidations.
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DOI：
10.1002/cbic.201800421
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发表时间：
2019-01-02
期刊：
影响因子：
--
通讯作者：
Fraaije MW
中科院分区：
文献类型：
--
作者：
Aalbers FS;Fraaije MW
为了扩大工业上适用的氧化酶的库，设计了乙醇脱氢酶与 NADPH 氧化酶的融合物。三种不同的乙醇脱氢酶（LbADH、TbADH、ADHA）与热稳定性 NADPH 氧化酶融合伴侣 (PAMO C65D) 一起表达并纯化。由此产生的双功能生物催化剂保留了各个酶的催化特性，其作用基本上类似于醇氧化酶：通过使用双氧作为温和氧化剂将醇转化为酮，同时仅需要催化量的 NADP+。在小规模反应中，纯化的融合酶表现出良好的性能，转化率为 69-99%，外消旋底物的 ee 为 99%，辅因子和酶总周转数较高。由于融合酶本质上充当氧化酶，我们发现可以使用常用的高通量氧化酶活性筛选方法。因此，如果需要，可以轻松地对融合酶进行改造以调整其特性。 自给自足：醇脱氢酶 (ADH) 可以催化选择性醇氧化，可用于动力学分解外消旋醇，或生产羰基结构单元。通过将 ADH 与 NADPH 氧化酶融合，可以产生自给自足的生物催化剂，其作用类似于酒精氧化酶。这些酶融合体可用于转化和活性筛选以辅助酶工程。
To expand the arsenal of industrially applicable oxidative enzymes, fusions of alcohol dehydrogenases with an NADPH‐oxidase were designed. Three different alcohol dehydrogenases (LbADH, TbADH, ADHA) were expressed with a thermostable NADPH‐oxidase fusion partner (PAMO C65D) and purified. The resulting bifunctional biocatalysts retained the catalytic properties of the individual enzymes, and acted essentially like alcohol oxidases: transforming alcohols to ketones by using dioxygen as mild oxidant, while merely requiring a catalytic amount of NADP+. In small‐scale reactions, the purified fusion enzymes show good performances, with 69–99 % conversion, 99 % ee with a racemic substrate, and high cofactor and enzyme total turnover numbers. As the fusion enzymes essentially act as oxidases, we found that commonly used high‐throughput oxidase‐activity screening methods can be used. Therefore, if needed, the fusion enzymes could be easily engineered to tune their properties. Going self‐sufficient: Alcohol dehydrogenases (ADHs) can catalyze selective alcohol oxidations and can be applied to kinetically resolve racemic alcohols, or to produce carbonyl building blocks. By fusing ADHs with an NADPH oxidase, self‐sufficient biocatalysts can be produced that act like alcohol oxidases. These enzyme fusions can be used for conversions and for activity screening to aid enzyme engineering.
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影响因子：
16.6
作者：
Pazmino, Daniel E. Torres;Snajdrova, Radka;Fraaije, Marco W.
通讯作者：
Fraaije, Marco W.
影响因子：
3.7
作者：
Peters, Christin;Rudroff, Florian;Bornscheuer, Uwe T.
通讯作者：
Bornscheuer, Uwe T.
影响因子：
5
作者：
Pickl M;Fuchs M;Glueck SM;Faber K
通讯作者：
Faber K
DOI：
10.1002/anie.201800343
发表时间：
2018-07-20
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Dong J;Fernández-Fueyo E;Hollmann F;Paul CE;Pesic M;Schmidt S;Wang Y;Younes S;Zhang W
通讯作者：
Zhang W
影响因子：
5
作者：
Fraaije, MW;Wu, J;Janssen, DB
通讯作者：
Janssen, DB