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通过定向进化提高环氧化物水解酶的水解活性和对映选择性。

Improving Hydrolytic Activity and Enantioselectivity of Epoxide Hydrolase from by Directed Evolution.

机构信息

College of Life Sciences, Sichuan Normal University, Chenglong Avenue, Chengdu 610101, China.

Sichuan Institute of Atomic Energy, Yidu West Road, Chengdu 610101, China.

出版信息

Molecules. 2024 Oct 14;29(20):4864. doi: 10.3390/molecules29204864.

DOI:10.3390/molecules29204864
PMID:39459231
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11509930/
Abstract

Epoxide hydrolases (EHs) catalyze the conversion of epoxides into vicinal diols. The epoxide hydrolase gene from was previously cloned and subjected to site-directed mutation to study its enzyme activity, but the results were unsatisfactory. This study used error prone PCR and DNA shuffling to construct a PchEHA mutation library. We performed mutation-site combinations on PchEHA based on enzyme activity measurement results combined with directed evolution technology. More than 15,000 mutants were randomly selected for the preliminary screening of PchEHA enzyme activity alongside 38 mutant strains with increased enzyme activity or enantioselectivity. Protein expression and purification were conducted to determine the hydrolytic activity of PchEHA, and three mutants increased their activity by more than 95% compared with that of the wt. After multiple rounds of screening and site-specific mutagenesis, we found that F3 offers the best enzyme activity and enantioselectivity; furthermore, the molecular docking results confirmed this result. Overall, this study uncovered novel mutants with potential value as industrial biocatalysts.

摘要

环氧化物水解酶（EHs）催化环氧化物转化为顺式二醇。先前已经克隆了 的环氧化物水解酶基因，并对其进行了定点突变以研究其酶活性，但结果并不理想。本研究使用易错 PCR 和 DNA 改组技术构建了 PchEHA 突变文库。我们根据酶活性测量结果以及定向进化技术对 PchEHA 进行了突变位点组合。随机选择了超过 15000 个突变体进行 PchEHA 酶活性的初步筛选，同时还筛选出了 38 株具有提高酶活性或对映选择性的突变株。进行了蛋白质表达和纯化以确定 PchEHA 的水解活性，与野生型相比，有三个突变体的活性提高了 95%以上。经过多轮筛选和定点突变，我们发现 F3 提供了最佳的酶活性和对映选择性；此外，分子对接结果证实了这一结果。总的来说，这项研究揭示了具有潜在工业生物催化剂价值的新型突变体。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/61f61b74c2a2/molecules-29-04864-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/ba192026ff59/molecules-29-04864-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/f5d7eb0522dc/molecules-29-04864-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/737706160fa0/molecules-29-04864-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/d987f16d3242/molecules-29-04864-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/34161e9b671a/molecules-29-04864-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/939d80bcc357/molecules-29-04864-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/61f61b74c2a2/molecules-29-04864-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/ba192026ff59/molecules-29-04864-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/f5d7eb0522dc/molecules-29-04864-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/737706160fa0/molecules-29-04864-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/d987f16d3242/molecules-29-04864-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/34161e9b671a/molecules-29-04864-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/939d80bcc357/molecules-29-04864-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca00/11509930/61f61b74c2a2/molecules-29-04864-g007.jpg

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Expert Opin Ther Pat. 2022 Jun;32(6):629-647. doi: 10.1080/13543776.2022.2054329. Epub 2022 Apr 12.
3
Significant improvement in catalytic activity and enantioselectivity of a Phaseolus vulgaris epoxide hydrolase, PvEH3, towards ortho-cresyl glycidyl ether based on the semi-rational design.
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Sci Rep. 2020 Feb 3;10(1):1680. doi: 10.1038/s41598-020-58693-1.
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Effect of Binding on Enantioselectivity of Epoxide Hydrolase.结合对环氧化物水解酶对映选择性的影响。
J Chem Inf Model. 2018 Mar 26;58(3):630-640. doi: 10.1021/acs.jcim.7b00353. Epub 2018 Feb 20.
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