Réaction #1106662

ord-9e498f488fb2463a9a10c11eddcd2f9e

Équation de réaction

O=C1[C@@H](O)[C@H](O)C(O)[C@H](O)[C@H]1O
scyllo-inosose
CC#N
acetonitrile
O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
scyllo-inositol
O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
scyllo-inositol
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
→
OC1C(O)C(O)C(O)C(O)C1O
myo-inositol

Réactifs

O=C1[C@@H](O)[C@H](O)C(O)[C@H](O)[C@H]1O
scyllo-inosose
—
4E
CC#N
acetonitrile
O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
scyllo-inositol
O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
scyllo-inositol
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
—
product
—
strong base
—
strong acid

Réactifs

Aucun

Solvants

NC(CO)(CO)CO
Tris

Conditions de réaction

Conditions détaillées
See reaction.notes.procedure_details.

Traitement

  1. 1
    Températureby cooling
  2. 2
    workup.ADDITIONThen, the supernatant was diluted 2-fold
  3. 3
    Autreunder the condition of a column temperature of 40° C.

Mode opératoire

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.

Source

DOI: 10.6084/m9.figshare.5104873.v1Brevet: US08715974B2uspto-grants-2014_05