論文 - 詳細
| RRC ID | 66732 |
|---|---|
| 著者 | Martínez-Tobón DI, Waters B, Elias AL, Sauvageau D. |
| タイトル | Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases. |
| ジャーナル | Microbiologyopen |
| Abstract |
Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta-gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation. |
| 巻・号 | 9(4) |
| ページ | e1001 |
| 公開日 | 2020-4-1 |
| DOI | 10.1002/mbo3.1001 |
| PMID | 32087608 |
| PMC | PMC7142370 |
| MeSH | Bacteria / enzymology* Bacteria / genetics Bacteria / metabolism Bioreactors / microbiology Carboxylic Ester Hydrolases / genetics* Comamonas testosteroni / enzymology Comamonas testosteroni / genetics Comamonas testosteroni / metabolism Cupriavidus / enzymology Cupriavidus / genetics Cupriavidus / metabolism Escherichia coli / enzymology Escherichia coli / genetics Escherichia coli / metabolism Marinobacter / enzymology Marinobacter / genetics Marinobacter / metabolism Pseudomonas stutzeri / enzymology Pseudomonas stutzeri / genetics Pseudomonas stutzeri / metabolism Ralstonia / enzymology Ralstonia / genetics Ralstonia / metabolism Recombinant Proteins / genetics |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 2 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 一般微生物 | JCM10169 JCM10168 JCM10171 |