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Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry
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Natural deep eutectic solvent-A novel green solvent for protein stabilization.

Yu Tian1, Manman Zhu1, Tiantian Hu1

  • 1College of Marine Life Sciences, Ocean University of China, No. 5 Yushan Road, Qingdao 266003, PR China.

International Journal of Biological Macromolecules
|June 19, 2023
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Summary

Natural deep eutectic solvents (NADESs) offer a stable environment for proteins. NADES 1-7 demonstrated excellent biocompatibility and enhanced trypsin enzyme activity, showing promise for biotechnological applications.

Keywords:
BetaineEnzyme activityNatural deep eutectic solventsStructure stabilityTrehaloseTrypsin

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Area of Science:

  • Biochemistry
  • Materials Science
  • Enzymology

Background:

  • Natural deep eutectic solvents (NADESs) are investigated for protein stabilization.
  • Trehalose and betaine were used to create NADESs with varying molar ratios (1:3 to 1:9).
  • Hydrogen bonding interactions between trehalose and betaine were analyzed.

Purpose of the Study:

  • To evaluate NADESs as a medium for protein stabilization.
  • To determine the optimal NADES composition for enzyme activity.
  • To investigate the mechanism of protein stabilization by NADESs.

Main Methods:

  • Synthesis and characterization of NADESs (NADES 1-3 to NADES 1-9).
  • Assessment of NADES properties: thermal stability, viscosity, and pH.
  • Enzyme activity assays of trypsin in NADES 1-7 under various conditions.
  • Kinetic parameter analysis and spectral analysis (e.g., conformational folding).

Main Results:

  • NADES 1-7 exhibited favorable properties: good thermal stability (-60-100 °C), low viscosity, and neutral pH.
  • Trypsin showed the highest relative enzyme activity in 50% (v/v) NADES 1-7.
  • Kinetic analysis indicated enhanced enzyme-substrate interaction and reaction rates.
  • Spectral analysis confirmed NADES 1-7 promotes protein conformational folding and protects natural structure.
  • NADES 1-7 demonstrated good biocompatibility.

Conclusions:

  • NADES 1-7 provides a superior stabilizing environment for trypsin.
  • The stabilizing effect is intrinsic to the NADES 1-7 formulation.
  • NADES 1-7's biocompatibility broadens its potential applications in biotechnology and enzyme stabilization.