論文 - 詳細
| RRC ID | 20944 |
|---|---|
| 著者 | Mizutani K, Toyoda M, Otake Y, Yoshioka S, Takahashi N, Mikami B. |
| タイトル | Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris. |
| ジャーナル | Biochim Biophys Acta |
| Abstract |
The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K(M) and V(max) values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)(8) barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein. |
| 巻・号 | 1824(8) |
| ページ | 954-62 |
| 公開日 | 2012-8-1 |
| DOI | 10.1016/j.bbapap.2012.05.005 |
| PII | S1570-9639(12)00094-5 |
| PMID | 22613096 |
| MeSH | Amino Acid Sequence Animals Chlorides Crystallography, X-Ray Fish Proteins / chemistry* Fish Proteins / genetics Fish Proteins / isolation & purification Genetic Vectors / genetics Homologous Recombination Hydrogen-Ion Concentration Kinetics Molecular Sequence Data Oryzias* Pichia / genetics Protein Denaturation Protein Structure, Tertiary Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / isolation & purification Temperature alpha-Amylases / chemistry* alpha-Amylases / genetics alpha-Amylases / isolation & purification |
| IF | 3.411 |
| 引用数 | 7 |
| WOS 分野 | BIOPHYSICS BIOCHEMISTRY & MOLECULAR BIOLOGY |
| リソース情報 | |
| 酵母 | BY2778 |
| メダカ | full length cDNA (olvl8b03) |