Enhanced enzyme kinetic stability by increasing rigidity within the active site.

Area of Science:

• Biochemistry
• Protein Engineering
• Enzyme Stabilization

Background:

• Enzyme stability is crucial for protein engineers.
• Understanding active site rigidity's impact on kinetic stability offers insights into enzyme stabilization.

Purpose of the Study:

• To enhance the kinetic stability of Candida antarctica lipase B (CalB) by increasing the rigidity of its active site.
• To investigate the relationship between active site flexibility and enzyme stability.

Main Methods:

• Iterative saturation mutagenesis was performed on six flexible residues near the catalytic Ser(105) of CalB.
• Screened 2200 colonies to identify stabilizing mutations.
• Characterized the D223G/L278M mutant using thermal and chemical denaturation assays.
• Analyzed crystal structures and performed molecular dynamics simulations.

Main Results:

• The D223G/L278M mutant showed a 13-fold increase in half-life at 48°C and a 12°C higher T50(15).
• The mutant exhibited improved resistance to thermal and chemical denaturation.
• Crystal structure analysis revealed an additional hydrogen bond network in the mutant, enhancing rigidity.
• Molecular dynamics simulations confirmed reduced active site residue fluctuation at high temperatures.

Conclusions:

• Increasing the rigidity of flexible active site segments is an effective strategy for improving enzyme kinetic stability.
• The D223G/L278M mutation in CalB serves as a model for enzyme stabilization through rigidity enhancement.