Thermal Stabilizing Effect of Sugars on Proteins during Microwave Drying and Storage Periods

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15560653
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Properties in chemical engineering process/Transfer operation/Unit operation
• Research Institution: Kyoto University
• Principal Investigator: SUZUKI Tetsuo Kyoto University, Faculty of Engineering, Assistant Professor, 工学研究科, 助手 (50243043)
• Project Period (FY): 2003 – 2004
• Keywords: protein / sugar / amorphous / activity / thermal stabilization / microwave drying / molecular dynamics / hydrogen bond

Research Abstract

1.Retaining enzyme activity during drying and storage period
Sugars (glucose, sucrose, trehalose, maltose, etc.) were added to aqueous enzyme solutions and dried by commercial microwave oven. Concerning enzymes, alcohol dehydrogenase(ADH) and ascorbate oxidase(AOD) were treated. Dried samples were stored at 65℃ in vacuum, and residual enzyme activities were studied. All sugars showed thermal stabilizing effect on enzymes during drying, and all except glucose also did during storage period. Moreover, the ADH sample with maltose showed strange result that residual activity was increased during storage period. The mechanism was discussed from viewpoints of the crystallinity of sugars, glass transition temperature, and formation of hydrogen bonds. It was found that all sugars were amorphous in samples and that the degree of hydrogen bond formation was the lowest for the maltose-ADH sample. This suggests that the high order structure of ADH can be rather easily rebuilt through thermal vibration in the sample.
2.Molecular dynamics(MD) simulation concerning sugar enzyme solutions
MD simulation was carried out concerning sugar enzyme solutions. A protein surface model was constructed based on the structure of ADH. The surface model was placed in the middle of a unit cell and, 400 water molecules and 1 or 4 sugar molecules (glucose, sucrose, trehalose, etc.) were treated. NPT ensemble MD calculations were carried out under 3 periodic condition. Mean square displacement(MSD) was calculated for atoms which construct protein surface model. MSD was smaller in the case that sugars were in the system than that sugars were not. This suggests that protein is thermally stabilized by sugar addition. MSD seems useful as an index of thermal stability of proteins. Role of hydrogen bonds between water molecules and protein surface in thermal stabilization was also discussed.