Enhanced enzyme kinetic stability by increasing rigidity within the active site.
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.
From the State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China and.
Yuan
Y
Xie
Jiao
J
An
Guangyu
G
Yang
Geng
G
Wu
Yong
Y
Zhang
Li
L
Cui
Yan
Y
Feng
D002152:Calorimetry, Differential Scanning; D002175:Candida / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry*; D015723:Gene Library; D006860:Hydrogen Bonding; D007700:Kinetics; D008049:Lipase / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D011489:Protein Denaturation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
D005656:Fungal Proteins; D011994:Recombinant Proteins; D012694:Serine; D008049:Lipase; C431349:lipase B, Candida antarctica
Active Site; Crystal Structure; Lipase; Local Rigidity; Mutagenesis; Protein Design; Protein Stability