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Designing cryo-enzymatic reactions in subzero liquid water by lipidic mesophase nanoconfinement


Cryo-enzymology provides the possibility to develop unconventional biological reactions and detect intermediates in ultrafast enzymatic catalysis processes, but also illuminates the understanding of life principles in extremely cold environments. The scarcity of biological or biomimetic host systems that provide liquid water at subzero temperatures inhibits the prosperity of cryo-enzymology. Here we introduce cryo-enzymatic reactions in subzero water nanoconfined within lipid mesophases formed by conventional lipids. We show that the enzymatic reactions that ensue outperform the homologue catalytic processes run at standard temperatures. We use phytantriol-based lipidic mesophases (LMPs), within which water remains in the liquid state down to −120 °C, and combine crystallization and dynamic studies of the confined water to provide a fundamental understanding of the physical status of water at subzero temperatures, which sets the stage for cryo-enzymatic reactions in these environments. In the model horseradish peroxidase oxidization, the cation free-radical product is stabilized in LMPs at −20 °C, in contrast to the fast-consuming reactions at temperatures above 0 °C. Furthermore, the LMP system also supports the cascade reaction and lipase reaction at subzero temperatures, at which enzymatic reactions with both hydrophilic and hydrophobic substrates are successfully carried out. Our designed LMP system opens access to the nature of confined water in the biomimetic environment and provides a platform for low-temperature biomacromolecule reconstitution and the cryogenic control of enzymatic reactions in bionanotechnology.

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Fig. 1: Low temperature stable LMPs.
Fig. 2: Thermal behaviour of water in LMPs.
Fig. 3: Dynamics of water and phytantriol in LMPs.
Fig. 4: State of nanoconfined water.
Fig. 5: Cryo-enzymatic reactions confined in LMP.

Data availability

All data generated and analysed during this study are included in the article and its Supplementary Information.


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We acknowledge M. Strach at the Chalmers Materials Analysis Laboratory (CMAL) for the performance of the in situ low-temperature X-ray measurements. We thank V. Lutz Bueno for the coordination of the X-ray experiments. We thank H.-J. Butt, A. Best, C.-H. Tu and P. Räder from the Max-Planck Institute for Polymer Research for the excellent support in the DSC and BDS experiments. R.M. acknowledges support from SNF grants 200020_178997. T.Z. acknowledges the China Scholarship Council for financial support.

Author information




R.M., Y.Y. and T.Z. conceived the idea, planned the experiments, wrote the paper and coordinated the overall research project. Y.Y. and T.Z. discovered the LMP at subzero temperatures. Y.Y. developed methods to study the crystallization and dynamics of water, performed the DSC and BDS experiments, analysed and interpreted the DSC and BDS data and coordinated the WAXS experiments. T.Z. produced the LMPs, performed the SAXS experiments, designed and performed the cryo-enzymatic reactions and performed the ultraviolet experiments. G.F. contributed to the analysis and interpretation of the BDS data. R.F. and U.G. coordinated the BDS experiments. All the authors discussed the results, provided comments and revised the manuscript.

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Correspondence to Raffaele Mezzenga.

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The authors declare no competing interests.

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Peer review information Nature Nanotechnology thanks Renata Bilewicz, Yi-Tao Long and other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–13, and description on the fitting procedure of dielectric spectra.

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Yao, Y., Zhou, T., Färber, R. et al. Designing cryo-enzymatic reactions in subzero liquid water by lipidic mesophase nanoconfinement. Nat. Nanotechnol. (2021).

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