新規な熱安定性ヒスタミンオキシダーゼの検索, 特性評価, 遺伝子クローニング及びその応用に関する研究(博士論文要録)

A thermostable histamine oxidase was found in cell extracts of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The purified enzyme was homogeneous on Native-PAGE and SDS-PAGE. As a result of SDS-PAGE, the enzyme was shown to be a single protein with a molecular mass of about 81 kDa without subunit structure. The enzyme showed powerful activity toward histamine (100%) and had little activity toward tyramine (36%). The enzyme was show keep its activity of about 70% after heating at 70℃ for 60 min. On the other hand, the enzyme from A. globiformis IFO12137 was fully lost its activity upon heating at 70℃ for 30 min. The histamine oxidase from A. crystallopoietes KAIT-B-007 was very thermostable. The enzyme is copper/2, 4, 5-trihydroxyphenylalanyl quinone-containing protein having one atom of copper and one molecule of TPQ per one molecule of the enzyme protein. The enzyme gene was cloned, and its complete DNA was sequenced. The histamine oxidase gene consisted of an open reading frame of 2, 172-bp encoding a protein of 725 amino acids. The deduced amino acid sequence of this enzyme was 84%, similar to that of histamine oxidase from A. globiformis IFO12137. A chemiluminometric flow-through sensor for the determination of histamine was prepared on the basis of co-immobilized histamine oxidase and peroxidase. The calibration curve for histamine was linear from 0.1 to 50μM. The present method gave good agreement with values obtained by HPLC, and the calculated coefficient of correlation was 0.996. This sensor system was applicable to the determination of histamine in fish meat extracts.