Fusion proteins of an enoate reductase and a Baeyer-Villiger monooxygenase facilitate the synthesis of chiral lactones
Fusion proteins of an enoate reductase and a Baeyer-Villiger monooxygenase facilitate the synthesis of chiral lactones
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DOI：
10.1515/hsz-2016-0150
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发表时间：
2017-01-01
影响因子：
3.7
通讯作者：
Bornscheuer, Uwe T.
中科院分区：
文献类型：
--
作者：
Peters, Christin;Rudroff, Florian;Bornscheuer, Uwe T.
大自然利用融合蛋白的优势进行多步反应，以促进细胞中的代谢，因为底物通过中间体转化为最终产物可以更快地发生，并且副产物形成较少。以类似的方式，对于酶级联反应，所涉及的生物催化剂的融合也是有利的。在本研究中，我们研究了醇脱氢酶（ADH），烯酸还原酶（ERED）和Baeyer-Villiger单加氧酶（BVMO）的融合，使（手性）内酯的合成从不饱和醇作为底物。研究了结构域顺序和各种接头，以找到表达水平和酶活性方面的最佳条件。对于来自恶臭假单胞菌的ERED异生物质还原酶B（XenB）和来自不动杆菌属的环己酮单加氧酶（CHMO），而所研究的ADH都不能成功地融合。该融合蛋白与单独提供的ADH一起在体内生物催化反应中产生相似的反应速率。1.5小时后，我们可以检测到40%以上的二氢香芹酮内酯在体内反应的融合蛋白和ADH，然后与单一的酶。
Nature uses the advantages of fusion proteins for multi-step reactions to facilitate the metabolism in cells as the conversion of substrates through intermediates to the final product can take place more rapidly and with less side-product formation. In a similar fashion, also for enzyme cascade reactions, the fusion of biocatalysts involved can be advantageous. In the present study, we investigated fusion of an alcohol dehydrogenase (ADH), an enoate reductase (ERED) and a Baeyer-Villiger monooxygenase (BVMO) to enable the synthesis of (chiral) lactones starting from unsaturated alcohols as substrates. The domain order and various linkers were studied to find optimal conditions with respect to expression levels and enzymatic activities. Best results were achieved for the ERED xenobiotic reductase B (XenB) from Pseudomonas putida and the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp., whereas none of the ADHs studied could be fused successfully. This fusion protein together with separately supplied ADH resulted in similar reaction rates in in vivo biocatalysis reactions. After 1.5 h we could detect 40% more dihydrocarvone lactone in in vivo reactions with the fusion protein and ADH then with the single enzymes.