Abstract
Biocatalytic transformations in organic synthesis often require the use of organic solvents to improve substrate solubility and promote the product formation. Halohydrin dehalogenases (HHDHs) are enzymes that catalyze the formation and conversion of epoxides, important synthetic class of compounds that are often sparingly soluble in water and prone to hydrolysis. In this study, the activity, stability, and enantioselectivity of HHDH from Agrobacterium radiobacter AD1 (HheC) in form of cell-free extract were evaluated in various aqueous-organic media. A correlation was discovered between the enzyme activity in the ring-closure reaction and logP of the solvent. Knowledge of such a relationship makes biocatalysis with organic solvents more predictable, which may reduce the need to experiment with a variety of solvents in the future. The results revealed a high enzyme compatibility with hydrophobic solvents (e.g., n-heptane) in terms of activity and stability. Regarding the HHDH applicability in an organic medium, inhibitions by a number of solvents (e.g., THF, toluene, chloroform) proved to be a more challenging problem than the protein stability, especially in the ring-opening reaction, thus suggesting which solvents should be avoided. In addition, solvent tolerance of the thermostable variant ISM-4 was also evaluated, revealing increased stability and to a lesser extent enantioselectivity compared to the wild-type. This is the first time such a systematic analysis has been reported, giving insight into the behavior of HHDHs in nonconventional media and opening new opportunities for the future biocatalytic applications.
Key points
• HheC performs better in the presence of hydrophobic than hydrophilic solvents.
• Enzyme activity in the PNSHH ring-closure reaction is a function of the logP.
• Thermostability of ISM-4 variant is accompanied by superior solvent tolerance.
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Data availability
The data that supports the findings of this study are available in the supplementary material of this article.
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Funding
This work was financially supported by the Croatian Science Foundation (HrZZ, IP-2018-01-4493). A part of the presented materials is the work supported by the Chinese-Croatian bilateral collaboration (2019-2021). N.M. is supported by a PhD scholarship from the Croatian Science Foundation through the Career Development Project for Young Researchers.
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NM, PŠ, and MME conducted experiments. NM, MS, and MME designed the research. MS, ZFB, and MME supervised the research. ZFB, LT, and MME acquired resources. MME acquired funding. All authors wrote, read, and approved the manuscript.
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Milčić, N., Švaco, P., Sudar, M. et al. Impact of organic solvents on the catalytic performance of halohydrin dehalogenase. Appl Microbiol Biotechnol 107, 2351–2361 (2023). https://doi.org/10.1007/s00253-023-12450-2
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DOI: https://doi.org/10.1007/s00253-023-12450-2