Over-expression, characterization, and modification of highly active alkaline phosphatase from a Shewanella genus bacterium
| Autor: | Yoshihiro Ojima, Yoshiaki Nishiya, Hiroshi Aiba, Masayuki Azuma |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Shewanella Gene Expression medicine.disease_cause Applied Microbiology and Biotechnology Biochemistry Substrate Specificity Analytical Chemistry law.invention 03 medical and health sciences law Escherichia coli medicine Molecular Biology chemistry.chemical_classification biology Organic Chemistry Active site General Medicine Alkaline Phosphatase biology.organism_classification Molecular biology Recombinant Proteins Amino acid Kinetics 030104 developmental biology Enzyme chemistry biology.protein Recombinant DNA Alkaline phosphatase Bacteria Biotechnology |
| Zdroj: | Bioscience, Biotechnology, and Biochemistry. 81:1994-2001 |
| ISSN: | 1347-6947 0916-8451 |
| DOI: | 10.1080/09168451.2017.1356217 |
| Popis: | We isolated a Shewanella sp. T3-3 bacterium that yielded highly active alkaline phosphatase (APase). We then cloned the APase gene from Shewanella sp. T3-3 (T3-3AP), and expressed and purified the enzyme from Escherichia coli. Recombinant T3-3AP showed high comparative reactivity on colorimetric (pNPP) and luminescent substrates (PPD and ASP-5). Subsequently, we improved the residual activity after maleimide activation by introducing amino acid substitutions of two Lys residues that were located near the active site. The double mutant enzyme (K161S + K184S) showed much higher residual specific activity after maleimide activation than the wild type enzyme, and had approximately twofold increased sensitivity on sandwich enzyme linked immunosorbent assays (ELISA) compared with calf intestinal APase (CIAP), which is routinely used as a labeling enzyme for ELISA. |
| Databáze: | OpenAIRE |
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