title: Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution.
abstract: 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.
authors.affiliation: Biotransformations Group and Structure Chemistry, Schering-Plough Research Institute, U-13-3000, 1011 Morris Avenue, Union, NJ 07083, USA. ningyan.zhang@spcorp.com
authors.forename: Ningyan
authors.initials: N
authors.lastname: Zhang
authors.forename: Wen-Chen
authors.initials: WC
authors.lastname: Suen
authors.forename: William
authors.initials: W
authors.lastname: Windsor
authors.forename: Li
authors.initials: L
authors.lastname: Xiao
authors.forename: Vincent
authors.initials: V
authors.lastname: Madison
authors.forename: Aleksey
authors.initials: A
authors.lastname: Zaks
mesh_terms: 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
chemical_list: D005656:Fungal Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica