Stabilization of bovine intestine alkaline phosphatase by sugars.
Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay. In this study, we evaluated the effects of sugars on the kinetic stability of BIALP in the hydrolysis of p-nitrophenylphosphate (pNPP). The temperatures reducing initial activity by 50% in a 30-min incubation, T(50), of BIALP with 1.0 M disaccharide (sucrose and trehalose) or 2.0 M monosaccharide (glucose and fructose) were 55.0-55.5 °C, 4.7-5.2 °C higher than without sugar (50.3±0.1 °C). The T(50) of BIALP increased to 58.4±0.3 °C when the trehalose concentration was from 1.0 to 1.5 M, but did not change when the glucose concentration was from 2.0 to 3.0 M. Thermodynamic analysis revealed that the stabilization of BIALP by sugars was driven by the increase in the enthalpy change of activation for thermal inactivation of BIALP. No sugars affected the k(cat) of BIALP in the hydrolysis of pNPP. These results suggest that not only trehalose, which is considered the most effective stabilizer of enzymes, but also sucrose, glucose, and fructose can be used as stabilizers of BIALP.
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.
Satoshi
S
Sekiguchi
Yasuhiko
Y
Hashida
Kiyoshi
K
Yasukawa
Kuniyo
K
Inouye
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D055550:Protein Stability / Q000187:drug effects; D013696:Temperature
D002241:Carbohydrates; D000469:Alkaline Phosphatase