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  11. Improved Activity And Thermostability Of Glycosylating Biocatalysts Acting In Neat Natural Deep Eutectic Solvents: Pushing Boundaries Of Enzymes

Improved activity and thermostability of glycosylating biocatalysts acting in neat natural deep eutectic solvents: Pushing boundaries of enzymes

K Bautista-Rangel1, L F Losoya-Uribe2, M Rodríguez-Gonzalez2

  • 1Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Chamilpa, 62210 Cuernavaca, Morelos, Mexico.

International Journal of Biological Macromolecules
|June 12, 2025

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View abstract on PubMed

Summary
This summary is machine-generated.

Natural Deep Eutectic Solvents (NaDES) significantly enhance enzyme activity and thermostability. Enzymes like Levansucrase and β-fructofuranosidase showed remarkable improvements in non-aqueous NaDES, becoming thermophilic.

Area of Science:

  • Biocatalysis and Enzyme Engineering
  • Green Chemistry and Sustainable Solvents
  • Protein Science and Structural Biology

Background:

  • Non-aqueous solvents are crucial for modifying enzyme properties.
  • Natural Deep Eutectic Solvents (NaDES) offer a green alternative, but their impact on enzyme structure-function requires further investigation.
  • Understanding these changes is key to unlocking new biocatalytic applications.

Purpose of the Study:

  • To investigate the behavior of Levansucrase (SacB) and β-fructofructofuranosidase (Invertase) in near-anhydrous NaDES.
  • To compare enzyme activity, optimal temperature (Topt), and thermostability in NaDES versus traditional acetate buffer.
  • To elucidate the structural basis for observed enzyme modifications in NaDES.

Main Methods:

  • Enzyme activity, Topt, Tm, and t1/2 measurements in various NaDES and acetate buffer.
Keywords:
Enhanced activity and higher thermal stability in NaDESEnzymes-NaDES interactionsNeat NaDES design with COSMO-RS

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  • NaDES selection and design utilizing the COSMO-RS model.
  • Differential Scanning Calorimetry (DSC) and Circular Dichroism (CD) spectroscopy for structural analysis.

    • Main Results:

    • Both SacB and Invertase exhibited activity in NaDES at 50°C, with peak performance in sugar-polyol NaDES.
    • In Fructose-Sorbitol-Water (F-S-W) NaDES, enzyme activities increased up to 742% (SacB) and 580% (Invertase).
    • Enzymes achieved thermophilic classification with Topt of 70°C (SacB) and 80°C (Invertase), and activity above 100°C.
    • Thermostability dramatically increased, with t1/2 values rising 23,100-fold (SacB) and 2500-fold (Invertase).
    • DSC indicated increased Tm (31.2°C for SacB, 3.2°C for Invertase).
    • CD spectroscopy revealed a significant increase in β-strand structures, correlating with enhanced thermostability.

    Conclusions:

    • NaDES, particularly sugar-polyol formulations like F-S-W, can radically transform enzyme function, enhancing activity and thermostability.
    • The observed improvements are linked to increased β-strand content, suggesting a stabilization mechanism.
    • NaDES represent a promising green alternative for enzyme stabilization and preservation in biocatalysis.