Rational engineering of Cel5E from Clostridium thermocellum to improve its thermal stability and catalytic activity
| Autor: | Ibrahim Torktaz, Jafar Hemmat, Ali Asghar Karkhane |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine beta-Glucans Glycoside Hydrolases Stereochemistry Mutant Cellulase medicine.disease_cause 01 natural sciences Applied Microbiology and Biotechnology Catalysis Clostridium thermocellum 03 medical and health sciences Affinity chromatography Bacterial Proteins 010608 biotechnology Catalytic Domain Enzyme Stability medicine Escherichia coli chemistry.chemical_classification biology Active site General Medicine biology.organism_classification 030104 developmental biology Enzyme chemistry Mutation biology.protein Xylanase Mutagenesis Site-Directed Biotechnology |
| Zdroj: | Applied microbiology and biotechnology. 102(19) |
| ISSN: | 1432-0614 |
| Popis: | The celH gene from Clostridium thermocellum encodes a protein containing 900 residues and three components, including Cel5E, Lic26a, and carbohydrate-binding domains. Cel5E is a member of the glycoside hydrolase-5 family and is a bifunctional xylanase/cellulase enzyme. We targeted a semi-hydrophobic pocket near the Cel5E active site and theoretically screened mutated variants for enhanced levels of thermal stability. Cel5E mutations were inserted into celH by overlapping polymerase chain reaction, followed by expression of wild-type and mutant enzymes in Escherichia coli BL21 (DE3) and purification by affinity chromatography. Thermal-stabilizing mutations were subjected to molecular dynamics simulation, and measurement of the in vacuo potential energy, van der Waals forces, electrostatic interactions, and net nonbonded potential energies obtained an overall binding affinity of − 64.964 KJ/mol for wild-type Cel5E and − 176.148, − 200.921, and − 120.038 KJ/mol for the N94F, N94W, and E133F mutants, respectively. Additionally, the N94W, N94F, E133F, and N94A variants exhibited 1.92-, 1.29-, 1.1-, and 1.15-fold better carboxymethyl cellulase (CMCase) and 1.46-, 1.29-, 1.11-, and 1.12-fold better β-glucanase activity on barley β-glucan relative to the wild-type enzyme. Interestingly, the optimal temperature for CMCase activity by the N94W variant was shifted 2 °C higher than that for the wild-type enzyme. Mutated variants showed improved CMCase and β-glucanase activity and shifted toward higher temperature of maximum activity. |
| Databáze: | OpenAIRE |
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