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.
Submitter: Shu-Ching Ou
Submission Date: Nov. 29, 2018, 1:05 p.m.
|Number of data points||51|
|Assays/Quantities/Protocols||Experimental Assay: Thermal Inactivation: tm at 50 °C ; Experimental Assay: Thermal Inactivation: tm at 65 °C ; Experimental Assay: Thermal Inactivation: tm at 60 °C ; Experimental Assay: Kinetic Constant: kcat ; Experimental Assay: Kinetic Constant: Km ; Derived Quantity: Kinetic Constant: kcat/Km|
|Libraries||Variants for Enzyme-Substrate|
|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.|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|