Cell surface display of cold-active esterase EstPc with the use of a new autotransporter from Psychrobacter cryohalolentis K5T
| Autor: | Lada E. Petrovskaya, K. A. Novototskaya-Vlasova, D. A. Dolgikh, E. A. Kryukova, Mikhail P. Kirpichnikov, Elizaveta Rivkina |
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| Rok vydání: | 2014 |
| Předmět: |
Molecular Sequence Data
Permafrost Biology medicine.disease_cause Microbiology Esterase Escherichia coli medicine Amino Acid Sequence Cloning Molecular Ions chemistry.chemical_classification Sequence Homology Amino Acid Hydrolysis Cell Membrane Esterases Computational Biology Psychrobacter Biological Transport General Medicine Psychrobacter cryohalolentis biology.organism_classification Recombinant Proteins Cold Temperature Enzyme Biochemistry chemistry Solvents Autotransporter domain Molecular Medicine Cell fractionation Bacterial outer membrane Bacteria Peptide Hydrolases |
| Zdroj: | Extremophiles. 19:161-170 |
| ISSN: | 1433-4909 1431-0651 |
| Popis: | We have cloned the gene coding for AT877-a new predicted member of the autotransporter protein family with an esterase passenger domain from permafrost bacterium Psychrobacter cryohalolentis K5(T). Expression of AT877 gene in Escherichia coli resulted in accumulation of the recombinant autotransporter in the outer membrane fraction and at the surface of the induced cells. AT877 displayed maximum hydrolytic activity toward medium-chain p-nitrophenyl esters (C8-C10) at 50 °C and was resistant to the presence of several metal ions, organic solvents and detergents. Previously, we have described a cold-active esterase EstPc from the same bacterium which possesses high activity at low temperatures and relatively high thermal stability. To construct a cell surface display system for EstPc, the hybrid autotransporter gene coding for EstPc with the α-helical linker and the translocator domain from AT877 was constructed and expressed in E. coli. According to the results of the cell fractionation studies and esterase activity measurements, the EstPc passenger was successfully displayed at the surface of the induced cells. It demonstrated a temperature optimum at 15-25 °C and a substrate preference toward p-nitrophenyl butyrate (C4). Obtained results provide a new example of the biotechnologically relevant enzyme from the permafrost microbial community with potential applications for the conversion of short- and medium-chain ester substrates and a basis for the construction of a new cell surface display platform. |
| Databáze: | OpenAIRE |
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