Expression, isolation, and identification of an ethanol-resistant ethyl carbamate-degrading amidase from Agrobacterium tumefaciens d3
| Autor: | Mianbin Wu, Jianping Lin, Lirong Yang, Kang Tingting |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine chemistry.chemical_classification Urethanase activity Ethanol biology Urethanase Chemistry Bioengineering Agrobacterium tumefaciens biology.organism_classification 01 natural sciences Applied Microbiology and Biotechnology Amidase 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Enzyme Biochemistry 010608 biotechnology Ethyl carbamate Specific activity Biotechnology |
| Zdroj: | Journal of Bioscience and Bioengineering. 132:220-225 |
| ISSN: | 1389-1723 |
| Popis: | Ethyl carbamate (EC), widely found in alcoholic beverages, has been revealed to be a probable carcinogen in humans. Urethanase (EC 3.5.1.75) is an effective enzyme for the degradation of EC; however, the previously identified urethanases exhibited insufficient acid and alcohol resistance. In this study, an enantioselective amidase (AmdA) screened from Agrobacterium tumefaciens d3 exhibited urethanase activity with excellent alcohol resistance. AmdA was first overexpressed in Escherichia coli; however, the recombinant protein was primarily located in inclusion bodies, and thus, co-expression of molecular chaperones was used. The activity of AmdA increased 3.1 fold to 307 U/L, and the specific activity of urethanase with C-terminal His-tags reached 0.62 U/mg after purification through a Ni–NTA column. Subsequently, the enzymatic properties and kinetic constants of AmdA were investigated. The optimum temperature for AmdA was 55 °C, it showed the highest activity at pH 7.5, and the Km was 0.964 mM. Moreover, after 1 h of heat treatment at 37 °C in a 5–20% (v/v) ethanol solution, the residual urethanase activity was higher than 91%, considerably more than that reported thus far. |
| Databáze: | OpenAIRE |
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