NADPH → An inositol

Participants

Reaction Steps

Meanwhile, the enzyme reaction product was measured as follows: 10 mg of scyllo-inosose, 40 mg of NADPH, and 10 U of the enzyme were allowed to react in 1.0 ml of 100 mM Tris buffer (pH 8.0) at 36° C. for 4 hours, and a heat treatment was performed at 80° C. for 10 min, followed by cooling. Then, 100 μl of a strong base cation exchange resin, 100 μl of a strong acid anion exchange resin, and 10 mg of activated carbon were added, and the mixture was stirred and centrifuged. Then, the supernatant was diluted 2-fold, and measurement was performed by HPLC (Shodex Asahipak NH2P-50 4E Φ 4.6×250 mm: Shodex) using an RI detector under the condition of a column temperature of 40° C. and a mobile phase flow rate of 1.5 ml (80% acetonitrile). As a result, the ydgJ gene product was found to have a high scyllo-inositol dehydrogenase activity, and 100% of the product was scyllo-inositol obtained by reduction, while myo-inositol that is an isomer thereof was not detected. As a result, the solution of a recombinant enzyme derived from the ydgJ gene was found to have a high scyllo-inositol dehydrogenase activity, and the gene product was scyllo-inositol dehydrogenase. Meanwhile, in the product obtained by the reduction reaction of scyllo-inosose, only scyllo-inositol was detected, and the enzyme was found to stereospecifically reduce scyllo-inosose into scyllo-inositol. Meanwhile, the sequence of the ydgJ gene is shown in SEQ ID NO: 1, and the amino acid sequence corresponding thereto is shown in SEQ ID NO: 2.

Provenance & Evidence