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- Title
Over-expression, characterization, and modification of highly active alkaline phosphatase from a Shewanella genus bacterium.
- Authors
Aiba, Hiroshi; Nishiya, Yoshiaki; Ojima, Yoshihiro; Azuma, Masayuki
- Abstract
We isolated aShewanellasp. T3-3 bacterium that yielded highly active alkaline phosphatase (APase). We then cloned the APase gene fromShewanellasp. T3-3 (T3-3AP), and expressed and purified the enzyme fromEscherichia 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. Finding of new APase that is superior to a commercial enzyme used routinely, CIAP, and enzyme improvement to increase sensitivity on sandwich ELISA.
- Subjects
SHEWANELLA; ALKALINE phosphatase; ENZYME analysis
- Publication
Bioscience, Biotechnology & Biochemistry, 2017, Vol 81, Issue 10, p1994
- ISSN
0916-8451
- Publication type
Article
- DOI
10.1080/09168451.2017.1356217