A Chimeric Styrene Monooxygenase with Increased Efficiency in Asymmetric Biocatalytic Epoxidation.
A Chimeric Styrene Monooxygenase with Increased Efficiency in Asymmetric Biocatalytic Epoxidation.
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DOI：
10.1002/cbic.201700653
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发表时间：
2018-04-04
期刊：
影响因子：
--
通讯作者：
Mutti FG
中科院分区：
文献类型：
--
作者：
Corrado ML;Knaus T;Mutti FG
来自假单胞菌的苯乙烯单加氧酶（SMO）系统由两种酶（StyA和StyB）组成。StyB以NADH为代价催化FAD的还原。在FADH 2从StyB转移到StyA之后，与O2反应生成FAD-OOH，其为环氧化剂。由于FADH 2的部分扩散转移导致的氧化还原当量的浪费、重组StyB的不溶性以及以1：1摩尔比表达StyA和StyB的不可能性降低了天然系统的催化效率。在此，我们提出了一种嵌合SMO（Fus-SMO），其通过柔性接头通过StyA和StyB的基因融合获得。由于以下因素的组合：1）还原酶和环氧酶单元的平衡和改善的表达水平，以及2）在某些情况下Fus-SMO的固有更高的比环氧化活性，表达Fus-SMO的大肠杆菌细胞具有比共表达StyA和StyB作为离散酶的大肠杆菌细胞高约50%的苯乙烯衍生物环氧化活性。 纯化的Fus-SMO的环氧化活性比双组分StyA/StyB（1：1，摩尔比）体系高多达3倍，比天然融合的SMO高多达110倍。耦合效率的测定和O2压力的影响进行了研究。最后，Fus-SMO和甲酸脱氢酶在大肠杆菌中共表达，并作为自给自足的生物催化系统用于大于500 mg规模的环氧化。
The styrene monooxygenase (SMO) system from Pseudomonas sp. consists of two enzymes (StyA and StyB). StyB catalyses the reduction of FAD at the expense of NADH. After the transfer of FADH2 from StyB to StyA, reaction with O2 generates FAD‐OOH, which is the epoxidising agent. The wastage of redox equivalents due to partial diffusive transfer of FADH2, the insolubility of recombinant StyB and the impossibility of expressing StyA and StyB in a 1:1 molar ratio reduce the catalytic efficiency of the natural system. Herein we present a chimeric SMO (Fus‐SMO) that was obtained by genetic fusion of StyA and StyB through a flexible linker. Thanks to a combination of: 1) balanced and improved expression levels of reductase and epoxidase units, and 2) intrinsically higher specific epoxidation activity of Fus‐SMO in some cases, Escherichia coli cells expressing Fus‐SMO possess about 50 % higher activity for the epoxidation of styrene derivatives than E. coli cells coexpressing StyA and StyB as discrete enzymes. The epoxidation activity of purified Fus‐SMO was up to three times higher than that of the two‐component StyA/StyB (1:1, molar ratio) system and up to 110 times higher than that of the natural fused SMO. Determination of coupling efficiency and study of the influence of O2 pressure were also performed. Finally, Fus‐SMO and formate dehydrogenase were coexpressed in E. coli and applied as a self‐sufficient biocatalytic system for epoxidation on greater than 500 mg scale.
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