High-level expression, purification, and some properties of a recombinant cephalosporin-C deacetylase
| Autor: | Shigeo Yagi, Kenji Mitsushima, Akio Takimoto |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
chemistry.chemical_classification
Expression vector Cephalosporin-C deacetylase Bioengineering Bacillus subtilis Biology medicine.disease_cause biology.organism_classification Applied Microbiology and Biotechnology law.invention Enzyme Plasmid Column chromatography Biochemistry chemistry law medicine Recombinant DNA Escherichia coli Biotechnology |
| Zdroj: | Journal of bioscience and bioengineering. 87(4) |
| ISSN: | 1389-1723 |
| Popis: | To maximize the expression of the cephalosporin-C deacetylase (CAH) gene isolated from Bacillus subtilis SHS 0133 in Escherichia coli, a series of expression plasmids was constructed with various spacings between the Shine-Dalgarno sequence and the ATG initiation codon. As the most efficient expression plasmid, we selected pCAH431, which has the trp promoter, a replication origin derived from pAT153, and a spacing of 13 nucleotides. E. coli JM103 with pCAH431 produced 4.9 g of CAH per liter on cultivation at 37 degrees C for 20 h in a 30-l jar fermentor. Since the amount of CAH reached about 70% of the total protein in the soluble fraction of the cells, and CAH was recovered from the cell extracts in an active form, the CAH was purified easily to homogeneity by only one column chromatography step. Twenty grams of 7-aminocephalosporanic acid was completely converted to deacetyl-7-aminocephalosporanic acid, a starting material for cefcapene pivoxil hydrochloride, by 12 mg of the purified enzyme without significant appearance of by-products. Thus, our expression and purification system has made the industrial production of CAH possible. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|