Enhancing the Thermostability of Rhizomucor miehei Lipase with a Limited Screening Library by Rational-Design Point Mutations and Disulfide Bonds.


Abstract

Rhizomucor miehei lipase (RML), as a kind of eukaryotic protein catalyst, plays an important role in the food, organic chemical, and biofuel industries. However, RML retains its catalytic activity below 50°C, which limits its industrial applications at higher temperatures. Soluble expression of this eukaryotic protein in Escherichia coli not only helps to screen for thermostable mutants quickly but also provides the opportunity to develop rapid and effective ways to enhance the thermal stability of eukaryotic proteins. Therefore, in this study, RML was engineered using multiple computational design methods, followed by filtration via conservation analysis and functional region assessment. We successfully obtained a limited screening library (only 36 candidates) to validate thermostable single point mutants, among which 24 of the candidates showed higher thermostability and 13 point mutations resulted in an apparent melting temperature ([Formula: see text]) of at least 1°C higher. Furthermore, both of the two disulfide bonds predicted from four rational-design algorithms were further introduced and found to stabilize RML. The most stable mutant, with T18K/T22I/E230I/S56C-N63C/V189C-D238C mutations, exhibited a 14.3°C-higher [Formula: see text] and a 12.5-fold increase in half-life at 70°C. The catalytic efficiency of the engineered lipase was 39% higher than that of the wild type. The results demonstrate that rationally designed point mutations and disulfide bonds can effectively reduce the number of screened clones to enhance the thermostability of RML.

Submission Details

ID: bM53qrzH3

Submitter: Shu-Ching Ou

Submission Date: Nov. 12, 2018, 3:06 p.m.

Version: 1

Publication Details
Li G;Fang X;Su F;Chen Y;Xu L;Yan Y,Appl Environ Microbiol (2018) Enhancing the Thermostability of Rhizomucor miehei Lipase with a Limited Screening Library by Rational-Design Point Mutations and Disulfide Bonds. PMID:29101200
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
6QPR 2019-03-13 1.45 Rhizomucor miehei lipase propeptide complex, Ser95/Ile96 deletion mutant
6QPP 2019-03-13 1.49 Rhizomucor miehei lipase propeptide complex, native
3TGL 1993-07-15 1.9 STRUCTURE AND MOLECULAR MODEL REFINEMENT OF RHIZOMUCOR MIEHEI TRIACYLGLYCERIDE LIPASE: A CASE STUDY OF THE USE OF SIMULATED ANNEALING IN PARTIAL MODEL REFINEMENT
1TGL 1990-10-15 1.9 A SERINE PROTEASE TRIAD FORMS THE CATALYTIC CENTRE OF A TRIACYLGLYCEROL LIPASE
4TGL 1993-07-15 2.6 CATALYSIS AT THE INTERFACE: THE ANATOMY OF A CONFORMATIONAL CHANGE IN A TRIGLYCERIDE LIPASE
5TGL 1994-01-31 3.0 A MODEL FOR INTERFACIAL ACTIVATION IN LIPASES FROM THE STRUCTURE OF A FUNGAL LIPASE-INHIBITOR COMPLEX

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.3 Lipase P19515 LIP_RHIMI