超好熱菌酵素を素子とするバイオセンサーの開発 : ポリアミン関連酵素の機能解析とD-プロリン脱水素酵素機能電極センサーの開発

An amperometric enzyme sensor give us higher sensitivity and specificity for the substrate
determination. In spite of advantages of enzyme sensor, many enzymes so far found have been too
labile to use as biosensor elements in artificial circumstances for a longer period.
Hyperthermophiles, which can grow above 90℃, have been known to produce much more stable
enzymes under various artificial conditions. In this work, we carried out screening, biochemical
characterization and improvement of production for hyperthermostable enzymes which are more useful
as the elements in the biosensors. We focused on the polyamines as one of the substrates of
biosensors. Polyamines have been known to play many important roles in cell proliferation,
differentiation and transformation. The concentration of the polyamines together with their acetyl
conjugates remarkably increases in the biological fluids and the affected tissues of cancer patients.
Therefore, their polyamines are listed as tumor markers. Gas and ion chromatographies have been
so far used for polyamine determination, but have some problems from the aspects of high sensitivity
and easy operation. Thus, we here developed biosensors using hyperthermostable enzymes for
polyamine determination. Such enzyme sensors are more useful for the simple and rapid
determination of polyamines and application for clinical analysis and food analysis In addition, we
tried the construction of biosensor using the hyperthermophilic enzyme, D-Proline dehydrogenase.
As the results, we found the thermostable agmatinase and spermidine dehydrogenase in
hyperthermophiles, Pyrococcus horikoshii and Sulfolobus tokodaii, respectively. We succeeded the
construction of novel amperometric sensor for D-proline determination using D-Proline dehydrogenase
derived from Pyrobaculum islandicum.