Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution.
To expand the functionality of lipase B from Candida antarctica (CALB) we have used directed evolution to create CALB mutants with improved resistance towards irreversible thermal inactivation. Two mutants, 23G5 and 195F1, were generated with over a 20-fold increase in half-life at 70 degrees C compared with the wild-type CALB (WT-CALB). The increase in half-life was attributed to a lower propensity of the mutants to aggregate in the unfolded state and to an improved refolding. The first generation mutant, 23G5, obtained by error-prone PCR, had two amino acid mutations, V210I and A281E. The second generation mutant, 195F1, derived from 23G5 by error-prone PCR, had one additional mutation, V221D. Amino acid substitutions at positions 221 and 281 were determined to be critical for lipase stability, while the residue at position 210 had only a marginal effect. The catalytic efficiency of the mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyl octanoate was also found to be superior to that of WT-CALB.
Zhang
Ningyan
N
Biotransformations Group and Structure Chemistry, Schering-Plough Research Institute, U-13-3000, 1011 Morris Avenue, Union, NJ 07083, USA. ningyan.zhang@spcorp.com
Suen
Wen-Chen
WC
Windsor
William
W
Xiao
Li
L
Madison
Vincent
V
Zaks
Aleksey
A
D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability / Q000235:genetics*; D005656:Fungal Proteins; D006207:Half-Life; D008049:Lipase / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D013696:Temperature
D005656:Fungal Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica