Characterization of new Baeyer-Villiger monooxygenases for lactonizations in redox-neutral cascades
Characterization of new Baeyer-Villiger monooxygenases for lactonizations in redox-neutral cascades
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DOI：
10.1016/j.mcat.2019.02.006
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发表时间：
2019-05-01
影响因子：
4.6
通讯作者：
Kara, Selin
中科院分区：
文献类型：
--
作者：
Engel, Jennifer;Mthethwa, Katlego S.;Kara, Selin
特别是来自不动杆菌NCIMB 9871的环己酮单加氧酶（CHMO）的技术应用受到有限的酶稳定性的阻碍。此外，底物和产物抑制是使用CHMO的众所周知的挑战。通过定点诱变，设计了两种新的组合CHMO变体CHMO M15 L323 C-A325 C（M15 DS）和CHMO M16 L323 C-A325 C（M16 DS），通过将报道的二硫键并入已经公布的亲本CHMO变体CHMO M15和CHMO M16中来稳定酶。此外，新描述的来自黄曲霉的BVMO AFL 706的特征在于ε-己内酯（ECL）合成，与野生型相比，该酶显示出显著更高的底物和产物耐受性。将野生型CHMO和四种变体以与来自嗜热厌氧杆菌乙醇的醇脱氢酶偶联的会聚级联应用于ECL合成。在操作条件下，M15 DS和M16 DS变体的性能优于野生型和亲本变体。随后，通过实验设计进一步优化级联，使产物产率加倍（21 mM，27%），辅因子量减少0.5 mM NADP（+）。
Technical application of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 in particular, is hindered by limited enzyme stability. In addition, substrate and product inhibition is a well-known challenge of using CHMO. By site-directed mutagenesis two new combinatorial CHMO variants, CHMO M15 L323C-A325C (M15 DS) and CHMO M16 L323C-A325C (M16 DS), were designed to stabilize the enzyme, by incorporating a reported disulfide bridge into the already published parental CHMO variants: CHMO M15 and CHMO M16. Additionally, the newly described BVMO AFL706 from Aspergillus flavus was characterized for epsilon-caprolactone (ECL) synthesis, for which the enzyme showed significantly higher substrate and product tolerance compared to the wild type. The wild type CHMO and the four variants were applied in a convergent cascade coupled with an alcohol dehydrogenase from Thermoanaerobacter ethanolicus for ECL synthesis. M15 DS and M16 DS variants performed better than the wild type and the parental variants under operational conditions. Subsequently, the cascade was further optimized by means of Design of Experiments, doubling the product yield (21 mM, 27%) with a reduced cofactor amount of 0.5 mM NADP(+).