KARITA Shuichi Mie University, Faculty of Bioresources, Associate Professor, 生物資源学部, 助教授 (90233999)
SENOO Keishi University of Tokyo, Graduate School of Agriculture and Life Sciences, Professor, 大学院・農学生命科学研究科, 教授 (40206652)
|Budget Amount *help
¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 2004 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 2003 : ¥1,400,000 (Direct Cost : ¥1,400,000)
We have tried to develop a convenient, general, and precise method for determining the enzyme kinetic parameters such as Michaelis constant and the molecular activity using reaction heat as a probe. For this purpose, we utilized high-sensitivity isothermal titration calorimetry. Two methods, "titration method" and "single-shot method" were employed. For the former case, a substrate concentration increases stepwise by injecting the substrate solution several times with short intervals into the enzyme solution and reaction heat was observed each time. For the latter, an enzyme solution and its substrate solution were mixed only once and the following total reaction is observed.
As model cases, ascorbate oxidase, glucoamylase, and lipase-catalyzed reactions were examined.
Satisfactory results were obtained for the cases of ascorbate oxidase and glucoamylase by the titration method, consisting with the previously reported data, indicating that this method is a promising approach for determining the enzyme kinetic parameters without skilled job. On the other hand, the single-shot method tends to give somewhat smaller k_<cat> and larger K_m values with unknown reason.
For the case of lipase, huge amount of dilution heat was observed when a substrate solution and the enzyme solution were mixed, which masked the enzyme reaction-heat. What is worse, the lipase molecules were adsorbed on the inner surface of the reaction cell of the titration calorimeter tightly, and it was not easy to remove the enzyme from the cell. As a result, the catalytic reaction started simply by filling the calorimeter cell with a substrate solution. This indicates that property of an enzymes as a protein is an important factor for this method.
We are planning to operate the data base of the parameters thus obtained, including reaction heat.