Hara M, Miyake J, Asada Y, Ohkawa H
National Institute of Bioscience and Human-Technology, AIST, MITI, Ibaraki, Japan.
Biosci Biotechnol Biochem. 1999 Jan;63(1):21-8. doi: 10.1271/bbb.63.21.
A genetically engineered fusion enzyme between rat P4501A1 and yeast P450 reductase in the microsomal fraction of the recombinant yeast AH22/pAFCR1 was purified. The purified enzyme showed a typical CO-difference spectrum of P4501A1 and a single band with an apparent molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This agreed with the molecular weight of 131,202 calculated from the amino acid sequence. The purified enzyme showed both 7-ethoxycoumarin o-deethylase activity and horse heart cytochrome c reductase activity in the presence of NADPH. The 7-ethoxycoumarin o-deethylase activity depended on the species of lipid used for the reconstitution of the purified fusion enzyme although the purified enzyme showed the activity without reconstitution. The purified fusion enzyme had the Km value of 26 microM for 7-ethoxycoumarin and the maximal turnover rate of 29 mol product/min/mol enzyme at 30 degrees C.
在重组酵母AH22/pAFCR1的微粒体部分纯化了一种大鼠P4501A1和酵母P450还原酶之间的基因工程融合酶。纯化后的酶显示出P4501A1典型的一氧化碳差光谱，并且在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上呈现出一条表观分子量为125,000的单带。这与根据氨基酸序列计算出的131,202的分子量相符。纯化后的酶在存在NADPH的情况下同时显示出7-乙氧基香豆素O-脱乙基酶活性和马心脏细胞色素c还原酶活性。尽管纯化后的酶在未重组的情况下也具有活性，但7-乙氧基香豆素O-脱乙基酶活性取决于用于重组纯化融合酶的脂质种类。纯化后的融合酶对7-乙氧基香豆素的Km值为26 microM，在30℃时的最大周转速率为29 mol产物/分钟/摩尔酶。
