Irreversible thermoinactivation of ribonuclease-A by soft-hydrothermal processing.
Ribonuclease (RNase), which often represents molecular biological contamination, is a thermostable enzyme. When RNase is heated at 121 degrees C by autoclave sterilization for 20 min, it does not lose its activity. However, the nature of the molecular events by which the irreversible denaturation occurs remains unknown. The purpose of this study was to elucidate the molecular mechanisms of irreversible thermal denaturation of RNase A and to develop an advanced sterilization method using soft-hydrothermal processing, which has the advantages of improved safety and cost-efficiency. The enzymatic activity of RNase was measured using polyacrylamide gel electrophoresis with torula yeast RNA. We evaluated the temperature and time course of irreversible thermoinactivation of RNase by normal autoclaving, hot-air sterilization, and soft-hydrothermal processing that had been controlled to the desired steam saturation ratio. The results indicated that RNase A was deactivated by autoclave sterilization (121 degrees C, 20 min) immediately after treatment, but was reactivated over time. Hot-air sterilization (180 degrees C, atmospheric pressure, 60 min) produced results similar to that of autoclave sterilization. In contrast, RNase A was irreversibly thermoinactivated by soft-hydrothermal processing (110 degrees C, 20 min) at 100% steam saturation ratio. We also determined that the mechanism of irreversible thermoinactivation of RNase A involved hydrolysis and deamidation under this condition at a steam saturation ratio of more than 100%.
Miyamoto
Toru
T
Institute for Animal Experimentation, Graduate School of Medicine, Tohoku University, Japan. miyamoto@mail.tains.tohoku.ac.jp
Okano
Shinya
S
Kasai
Noriyuki
N
D000704:Analysis of Variance; D000818:Animals; D002417:Cattle; D003641:Deamination; D004340:Drug Contamination; D004789:Enzyme Activation; D004793:Enzyme Reactivators; D004795:Enzyme Stability; D006868:Hydrolysis; D011312:Pressure; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013227:Steam; D013696:Temperature
D004793:Enzyme Reactivators; D013227:Steam; D012259:Ribonuclease, Pancreatic