Directed evolution of beta -glucosidase A from Paenibacillus polymyxa to thermal resistance.


Abstract

The beta-glucosidase encoded by the bglA gene from Paenibacillus polymyxa has a half-life time of 15 min at 35 degrees C and no detectable activity at 55 degrees C. We have isolated random mutations that enhance the thermoresistance of the enzyme. Following a directed evolution strategy, we have combined some of the isolated mutations to obtain a beta-glucosidase with a half-life of 12 min at 65 degrees C, in the range of resistance of thermophilic enzymes. No significant alteration of the kinetic parameters of the enzyme was observed. One of the mutants isolated in the screening for thermoresistant beta-glucosidase had the same resistance to denaturation as the wild type. This mutation caused the accumulation of enzyme in E. coli, probably due to its lower turnover. The structural changes responsible for the properties of the mutant enzymes have been analyzed. The putative causes increasing thermoresistance are as follows: the formation of an extra salt bridge, the replacement of an Asn residue exposed to the solvent, stabilization of the hydrophobic core, and stabilization of the quaternary structure of the protein.

Submission Details

ID: dxm3HW4W4

Submitter: Shu-Ching Ou

Submission Date: Nov. 29, 2018, 1:05 p.m.

Version: 1

Publication Details
Gonzalez-Blasco G;Sanz-Aparicio J;Gonzalez B;Hermoso JA;Polaina J,J Biol Chem (2000) Directed evolution of beta -glucosidase A from Paenibacillus polymyxa to thermal resistance. PMID:10788490
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
1E4I 2001-07-05 2.0 2-deoxy-2-fluoro-beta-D-glucosyl/enzyme intermediate complex of the beta-glucosidase from Bacillus polymyxa
6R4K 2019-07-03 2.13 Structure of beta-glucosidase A from Paenibacillus polymyxa complexed with a monovalent inhibitor
1TR1 1999-04-20 2.2 CRYSTAL STRUCTURE OF E96K MUTATED BETA-GLUCOSIDASE A FROM BACILLUS POLYMYXA, AN ENZYME WITH INCREASED THERMORESISTANCE
1UYQ 2005-03-03 2.2 mutated b-glucosidase A from Paenibacillus polymyxa showing increased stability
1BGG 1998-05-27 2.3 GLUCOSIDASE A FROM BACILLUS POLYMYXA COMPLEXED WITH GLUCONATE
1BGA 1998-04-15 2.4 BETA-GLUCOSIDASE A FROM BACILLUS POLYMYXA
6QWI 2019-07-03 2.85 Structure of beta-glucosidase A from Paenibacillus polymyxa complexed with multivalent inhibitors.

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
400.0 A,B,C,D Beta-glucosidase A P22073 BGLA_PAEPO