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. Study holds ProTherm entries: 16579, 16580, 16581, 16582, 16583, 16584 Extra Details: directed evolution; irreversible inactivation; lipase; protein aggregation; stability

Submission Details

ID: nh2RXzXM3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Zhang N;Suen WC;Windsor W;Xiao L;Madison V;Zaks A,Protein Eng. (2003) Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution. PMID:12968077
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5A71 2015-10-21 0.91 Open and closed conformations and protonation states of Candida antarctica Lipase B: atomic resolution native
4K5Q 2014-01-29 1.49 Crystal structure of CALB mutant DGLM from Candida antarctica
3ICV 2009-10-06 1.49 Structural Consequences of a Circular Permutation on Lipase B from Candida Antartica
4K6G 2014-01-29 1.5 Crystal structure of CALB from Candida antarctica
1TCA 1994-05-31 1.55 THE SEQUENCE, CRYSTAL STRUCTURE DETERMINATION AND REFINEMENT OF TWO CRYSTAL FORMS OF LIPASE B FROM CANDIDA ANTARCTICA
4K6K 2014-01-29 1.6 Crystal structure of CALB mutant D223G from Candida antarctica
4K6H 2014-01-29 1.6 Crystal structure of CALB mutant L278M from Candida antarctica
3ICW 2009-10-06 1.69 Structure of a Circular Permutation on Lipase B from Candida Antartica with Bound Suicide Inhibitor
6ISQ 2019-07-24 1.86 structure of Lipase mutant with oxided Cys-His-Asp catalytic triad
6ISP 2019-07-24 1.88 structure of Candida antarctica Lipase B mutant
4ZV7 2016-01-13 2.0 Crystal structure of hexagonal form of lipase B from Candida antarctica
1TCB 1994-05-31 2.1 THE SEQUENCE, CRYSTAL STRUCTURE DETERMINATION AND REFINEMENT OF TWO CRYSTAL FORMS OF LIPASE B FROM CANDIDA ANTARCTICA
5A6V 2015-10-21 2.28 Open and closed conformations and protonation states of Candida antarctica Lipase B: Xenon complex
1LBT 1995-10-15 2.5 LIPASE (E.C.3.1.1.3) (TRIACYLGLYCEROL HYDROLASE)
1TCC 1994-05-31 2.5 THE SEQUENCE, CRYSTAL STRUCTURE DETERMINATION AND REFINEMENT OF TWO CRYSTAL FORMS OF LIPASE B FROM CANDIDA ANTARCTICA
6ISR 2019-07-24 2.6 structure of lipase mutant with Cys-His-Asp catalytic triad
1LBS 1995-10-15 2.6 LIPASE (E.C.3.1.1.3) (TRIACYLGLYCEROL HYDROLASE)
3W9B 2014-04-02 2.9 Crystal structure of Candida antarctica lipase B with anion-tag

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Lipase B P41365 LIPB_PSEA2