State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, P. R. China.
State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 360015, P. R. China.
Angew Chem Int Ed Engl. 2019 Jan 14;58(3):764-768. doi: 10.1002/anie.201812093. Epub 2018 Dec 4.
Hydroquinone (HQ) is produced commercially from benzene by multi-step Hock-type processes with equivalent amounts of acetone as side-product. We describe an efficient biocatalytic alternative using the cytochrome P450-BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi-rational protein engineering strategy was developed. Highly active mutants were obtained which transform benzene in a one-pot sequence first into phenol and then regioselectively into HQ without any overoxidation. A computational study shows that the chemoselective oxidation of phenol by the P450-BM3 variant A82F/A328F leads to the regioselective formation of an epoxide intermediate at the C3=C4 double bond, which departs from the binding pocket and then undergoes fragmentation in aqueous medium with exclusive formation of HQ. As a practical application, an E. coli designer cell system was constructed, which enables the cascade transformation of benzene into the natural product arbutin, which has anti-inflammatory and anti-bacterial activities.
对苯二酚（HQ）是由商业生产的苯通过多步 Hock 型工艺与丙酮作为副产物的当量。我们描述了使用细胞色素 P450-BM3 单加氧酶的有效生物催化替代方法。由于野生型酶不接受苯，因此开发了一种半理性的蛋白质工程策略。获得了高活性的突变体，它们可以在一锅序列中将苯首先转化为苯酚，然后区域选择性地转化为 HQ，而不会发生任何过度氧化。计算研究表明，P450-BM3 变体 A82F/A328F 对苯酚的选择性氧化导致 C3=C4 双键处形成环氧化物中间体，该中间体离开结合口袋，然后在水介质中进行片段化，仅形成 HQ。作为实际应用，构建了一种大肠杆菌设计细胞系统，该系统能够使苯进行级联转化为具有抗炎和抗菌活性的天然产物熊果苷。
