| Autor: |
Hua Xiang Gu, Xun Li, Hao Shi, Fei Wang |
| Rok vydání: |
2014 |
| Předmět: |
|
| Zdroj: |
Advanced Materials Research. :841-848 |
| ISSN: |
1662-8985 |
| DOI: |
10.4028/www.scientific.net/amr.1004-1005.841 |
| Popis: |
The α-glucosidase geneaglfromThermus thermophilusHB8 was cloned into expression vector pBV220. The phylogenetic trees of α-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. Evolution analysis indicated the α-glucosidase fromT. thermophileHB8 was distant from the other glycoside hydrolases 4 and 31 α-glucosidases. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of theaglwas increased 30-fold. The recombinant AGL was purified by the heat treatment, and had a molecular mass of 61 kDa. The optimal activity was at pH 7.8 and 95°C over a 10 min assay. The purified enzyme was stable over a pH range of 5.4-8.6, and had a 1-h half life at 85°C. Kinetic experiments at 90°C withp-nitrophenyl-α-D-glucoside as substrate gave aKm, andVmaxof 0.072 mM and 400 U/mg. Thus, this report provides an industrial means to produce the recombinant α-glucosidase inE. coli. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
