A solvent-free delipidation method for functional validation of lipases.

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• Protein extraction requires lipid removal for functional characterization.
• Existing delipidation protocols can be time-consuming or use harsh solvents.
• Efficient lipid removal is essential for downstream applications like enzyme assays and inhibitor screening.

Purpose of the Study:

• To develop a rapid, solvent-free delipidation method using activated silica.
• To validate the functional integrity of enzymes after delipidation.
• To demonstrate the utility of the method for protein enrichment and inhibitor screening.

Main Methods:

• Developed a novel solvent-free delipidation protocol utilizing activated silica.
• Assessed sample optical clarity and endogenous lipid reduction.
• Validated enzyme functional integrity using in vitro enzyme assays with physiological substrates.
• Employed activity-based protein profiling (ABPP) to assess enzyme active site accessibility and detect serine hydrolases.

Main Results:

• The activated silica method provided rapid and effective lipid removal, improving sample optical clarity.
• Functional integrity of lipases was maintained, as confirmed by in vitro enzyme assays.
• Activity-based protein profiling showed enhanced detection of serine hydrolases and improved protein enrichment.
• Reduced polyphenol content facilitated small-molecule inhibitor screening via ABPP.

Conclusions:

• The developed solvent-free delipidation approach is efficient and rapid.
• This method is highly compatible with the functional characterization of enzymes, especially lipid hydrolases.
• The technique offers advantages for protein enrichment and inhibitor screening applications.