Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Enzyme Microb Technol. 2011 Jul 10;49(2):171-6. doi: 10.1016/j.enzmictec.2011.04.019. Epub 2011 Apr 30.
Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k(cat) values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190±10, 840±30, and 500±10 s(-1), respectively. The k(cat) values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240±60, 1450±30, and 2250±80 s(-1), respectively, at 1.0M. On the other hand, the k(cat) values at 0.05-1.0M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s(-1). These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K(m) values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K(m) value at 1.0M diethanolamine (0.83±0.15 mM) was 12-fold higher than that at 0.05M (0.07±0.01 mM), and that at 1.0M N-methylethanolamine (2.53±0.20 mM) was 14-fold higher than that at 0.2M (0.18±0.02 mM), while that at 1.0M triethanolamine (0.31±0.01 mM) was similar as that at 0.2M (0.25±0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.
牛肠碱性磷酸酶（BIALP）广泛用作敏感测定中的信号酶，例如酶免疫测定（EIA）。在这项研究中，我们评估了各种氨基醇和胺对 BIALP 在 pH9.8、20°C 下水解对硝基苯磷酸酯（pNPP）的活性的影响。在 0.05M 二乙醇胺、0.1M 三乙醇胺和 0.2M N-甲乙醇胺中的 k(cat)值分别为 190±10、840±30 和 500±10s(-1)。k(cat)值随二乙醇胺、三乙醇胺和 N-甲乙醇胺浓度的增加而增加，在 1.0M 时分别达到 1240±60、1450±30 和 2250±80s(-1)。另一方面，在 0.05-1.0M 乙醇胺、乙胺、甲胺和二甲胺中的 k(cat)值在 100-600s(-1)范围内。这些结果表明，二乙醇胺、三乙醇胺和 N-甲乙醇胺高度激活 BIALP，可能适合作为 EIA 中 BIALP 的稀释缓冲液。有趣的是，K(m)值随二乙醇胺和 N-甲乙醇胺浓度的增加而增加，但三乙醇胺则不然：在 1.0M 二乙醇胺（0.83±0.15mM）时，K(m)值比在 0.05M（0.07±0.01mM）时高 12 倍，在 1.0M N-甲乙醇胺（2.53±0.20mM）时高 14 倍，而在 0.2M（0.18±0.02mM）时相似，表明 BIALP 激活的机制在氨基醇之间不同。
