Noda Development Group, Planning & Administration Department, Kikkoman Biochemifa Company, 376-2, Kamihanawa, Nodashi, Chiba, 278-0033, Japan.
Biotechnol Lett. 2012 Feb;34(2):321-8. doi: 10.1007/s10529-011-0772-8. Epub 2011 Oct 19.
Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55-60 kDa, as determined by SDS-PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.
碱性磷酸酶能催化磷酸单酯的水解，在分子生物学技术和临床诊断中被广泛应用。我们在大肠杆菌 BL21 (DE3) 中表达了海洋细菌 Cobetia marina 的重组碱性磷酸酶。该重组蛋白的比活为 12700 U/mg 蛋白，是迄今为止研究过的任何细菌碱性磷酸酶中活性最高的。SDS-PAGE 测定重组蛋白的相对分子质量为 55-60 kDa，凝胶过滤分析表明其为二聚体。该酶在 45°C 时活性最佳，能有效水解发光和荧光底物中的磷酸酯。因此，这种酶可以被认为是一种非常有用的抗体标记物。
