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.
Submitter: Shu-Ching Ou
Submission Date: Jan. 30, 2019, 5:25 p.m.
Due to numbering (starts from -1 in PDB) issue, D223G/L278M/A281E are shown as D225G/L280M/A283E. Variant A281E(A283E) was deactivated too rapidly to measure at 48 °C. The half-life was less than 5 min at only 45 °C.
|Number of data points||127|
|Assays/Quantities/Protocols||Experimental Assay: kd: first-order rate constants of inactivation ; Experimental Assay: t1/2 at 48 °C ; Experimental Assay: T50_15 ; Experimental Assay: C50 ; Experimental Assay: Tm ; Experimental Assay: ΔHu ; Experimental Assay: ΔSu ; Experimental Assay: Cm ; Experimental Assay: Km ; Experimental Assay: kcat ; Experimental Assay: Ea: Activation Energy ; Derived Quantity: SD of ΔS‡: Entropy of activation ; Derived Quantity: ΔS‡: Entropy of activation ; Derived Quantity: ΔG‡: Free energy of activation ; Derived Quantity: ΔH‡: Enthalpy of activation ; Derived Quantity: SD of ΔH‡: Enthalpy of activation ; Derived Quantity: SD of ΔG‡: Free energy of activation ; Derived Quantity: SD of Ea: Activation Energy ; Derived Quantity: SD of kcat/Km ; Derived Quantity: kcat/Km ; Derived Quantity: SD of kcat ; Derived Quantity: SD of Km ; Derived Quantity: SD of Cm ; Derived Quantity: SD of ΔSu ; Derived Quantity: SD of ΔHu ; Derived Quantity: SD of Tm ; Derived Quantity: SD of C50 ; Derived Quantity: SD of T50_15 ; Derived Quantity: SD of ΔΔG at 48 °C ; Derived Quantity: ΔΔG at 48 °C ; Derived Quantity: SD of t1/2 at 48 °C ; Derived Quantity: SD of kd: first-order rate constants of inactivation|
|Libraries||Variants for CalB_pNP ; Variants for CalB|
|Structure ID||Release Date||Resolution||Structure Title|
|4K6K||2013-04-16T00:00:00+0000||1.6||Crystal structure of CALB mutant D223G from Candida antarctica|
|5A71||2015-07-02T00:00:00+0000||0.91||Open and closed conformations and protonation states of Candida antarctica Lipase B: atomic resolution native|
|6J1T||2018-12-29T00:00:00+0000||1.78||Crystal structure of Candida Antarctica Lipase B mutant SR with product analogue|
|6J1S||2018-12-29T00:00:00+0000||1.83||Crystal structure of Candida Antarctica Lipase B mutant - SS|
|6J1R||2018-12-29T00:00:00+0000||1.6||Crystal structure of Candida Antarctica Lipase B mutant - RR|
|6ISP||2018-11-18T00:00:00+0000||1.88||structure of Candida antarctica Lipase B mutant|
|6ISR||2018-11-18T00:00:00+0000||2.6||structure of lipase mutant with Cys-His-Asp catalytic triad|
|6ISQ||2018-11-18T00:00:00+0000||1.86||structure of Lipase mutant with oxided Cys-His-Asp catalytic triad|
|6TP8||2019-12-12T00:00:00+0000||1.55||Substrate protein interactions in the limbus region of the catalytic site of Candida antarctica Lipase B|
|6J1P||2018-12-29T00:00:00+0000||1.76||Crystal structure of Candida Antarctica Lipase B mutant - SR|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|