Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG, Groningen, The Netherlands.
Chembiochem. 2019 Jan 2;20(1):51-56. doi: 10.1002/cbic.201800421. Epub 2018 Oct 4.
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.
为了扩充工业应用氧化酶的储备，设计了将醇脱氢酶与 NADPH 氧化酶融合。三种不同的醇脱氢酶（LbADH、TbADH、ADHA）与一种耐热的 NADPH 氧化酶融合伴侣（PAMO C65D）表达并纯化。所得的双功能生物催化剂保留了单个酶的催化特性，并且基本上表现为醇氧化酶：将醇转化为酮，同时使用氧气作为温和氧化剂，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%，外消旋底物的对映体过量值为 99%，辅酶和酶总周转率很高。由于融合酶基本上作为氧化酶起作用，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，融合酶可以很容易地进行工程改造以调整其性质。
