Cloning and expression of a thermostable keratinase gene from Thermoactinomyces sp. YT06 in Escherichia coli and characterization of purified recombinant enzymes
| Autor: | Wei Qishun, Chengbao Guo, Hongying Huang, Ying Zhou, Ying Huang, Lin Wang |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Physiology medicine.medical_treatment Enzyme Activators Gene Expression medicine.disease_cause 01 natural sciences Applied Microbiology and Biotechnology law.invention 03 medical and health sciences chemistry.chemical_compound law 010608 biotechnology Enzyme Stability Escherichia coli medicine Extracellular Cloning Molecular Enzyme Inhibitors Serine protease chemistry.chemical_classification 0303 health sciences Protease biology 030306 microbiology Temperature General Medicine Hydrogen-Ion Concentration Recombinant Proteins Thermoactinomyces Molecular Weight Enzyme Keratinase chemistry Biochemistry biology.protein Recombinant DNA PMSF Peptide Hydrolases Biotechnology |
| Zdroj: | World Journal of Microbiology and Biotechnology. 35 |
| ISSN: | 1573-0972 0959-3993 |
| DOI: | 10.1007/s11274-019-2710-1 |
| Popis: | The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6–11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and β-mercaptoethanol (β-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes. |
| Databáze: | OpenAIRE |
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