Aiba Hiroshi, Nishiya Yoshiaki, Ojima Yoshihiro, Azuma Masayuki
a Institute of Biotechnology , TOYOBO CO., Ltd. , Tsuruga , Japan.
b Department of Applied Chemistry and Bioengineering, Graduate School of Engineering , Osaka City University , Osaka , Japan.
Biosci Biotechnol Biochem. 2017 Oct;81(10):1994-2001. doi: 10.1080/09168451.2017.1356217. Epub 2017 Jul 31.
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.
