Abstract
Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.
Highlights
Improving the kinetic stability of enzymes is a key issue for protein engineers
Construction of Iterative Saturation Mutagenesis Libraries— Candida antarctica lipase B (CalB) is a globular ␣/-hydrolase that is ϳ30 ϫ 40 ϫ 50 Å in size with a catalytic triad composed of Ser105, Asp187, and His224
To identify residues that play a role in enhancing the local stability of the CalB active site, we mutated amino acids based on two criteria: 1) location within a 10-Å radius around the Ser105 residue and 2) a relatively high B factor
Summary
Improving the kinetic stability of enzymes is a key issue for protein engineers. Results: Mutagenesis of residues with a high B factor located within 10 Å of the catalytic Ser105 residue enhances kinetic stability dramatically. Conclusion: Increasing the rigidity of the flexible segment within the active site improves enzymatic kinetic stability. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability
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