Reaktion #1759298

ord-0026d7eb382d451e8099973e525b7a70

Reaktionsgleichung

O=c1cc[nH]c(=O)[nH]1
uracil
O=C(O)c1[nH]c(=O)[nH]c(=O)c1F
5-fluoroorotic acid
[Cl-].[K+]
KCl
O=P([O-])(O)O.[K+]
KH2PO4
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
O=c1cc[nH]c(=O)[nH]1
uracil
O=c1cc[nH]c(=O)[nH]1
uracil
O=c1ccn([C@@H]2O[C@H](CO)[C@@H](O)[C@H]2O)c(=O)[nH]1
uridine
O=C(O)c1[nH]c(=O)[nH]c(=O)c1F
5-FOA
→
O=C(O)c1cc(=O)[nH]c(=O)n1[C@@H]1O[C@H](COP(=O)(O)O)[C@@H](O)[C@H]1O
Orotidine-5′-Phosphate

Edukte

O=c1cc[nH]c(=O)[nH]1
uracil
O=C(O)c1[nH]c(=O)[nH]c(=O)c1F
5-fluoroorotic acid
[Cl-].[K+]
KCl
O=P([O-])(O)O.[K+]
KH2PO4
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
O=c1cc[nH]c(=O)[nH]1
uracil
—
MgSO4.7H2O
—
FeSO4.7H2O
O=c1cc[nH]c(=O)[nH]1
uracil
O=c1ccn([C@@H]2O[C@H](CO)[C@@H](O)[C@H]2O)c(=O)[nH]1
uridine
—
NaNO3
O=C(O)c1[nH]c(=O)[nH]c(=O)c1F
5-FOA

Reagenzien

Keine

Lösungsmittel

Cl.NC(CO)(CO)CO
Tris-HCl

Reaktionsbedingungen

Detaillierte Bedingungen
See reaction.notes.procedure_details.

Aufarbeitung

  1. 1
    SonstigeAfter UV irradiation of the suspension of Penicillium multicolor IAM7153 conidia
  2. 2
    workup.WAITthe media were incubated at 27° C. for 3 to 5 days
  3. 3
    Sonstigeincubated at 27° C.
  4. 4
    Temperaturcooled on ice
  5. 5
    Sonstigeto give
  6. 6
    workup.ADDITIONa suspension of the hypha
  7. 7
    Sonstigeg for 10 minutes, for separation of the supernatant

Vorschrift

After UV irradiation of the suspension of Penicillium multicolor IAM7153 conidia, the spores were coated on a 5-fluoroorotic acid (5-FOA) medium (Boeke et al., Mol. Gen. Genet. 197: 345-346, 1984) and incubated at 27° C. for 6 to 8 days. The 5-FOA-resistant strain grown was picked up respectively onto a minimal medium (0.3% NaNO3: 0.05% KCl, 0.05% MgSO4.7H2O, 0.001% FeSO4.7H2O, 0.1% KH2PO4, 2% glucose, 1.5% agar, pH 6.0) and a minimal medium containing 5 mM uracil respectively, and the media were incubated at 27° C. for 3 to 5 days. The uracil-demanding strain growing only on the minimal medium containing 5 mM uracil was inoculated into a YPM medium containing 5 mM uridine (1% yeast extract, 2% peptone, 2% maltose, pH 6.0) and incubated at 27° C. and 140 rpm for 3 days by shaking culture. 1 g of the hypha and 1 g of sea sand were placed in a mortar cooled on ice and were pulverized sufficiently therein, and 10 ml of 50 mM Tris-HCl (pH 8.0) was added to the mortar, to give a suspension of the hypha. The suspension was centrifuged at 36,000×g for 10 minutes, for separation of the supernatant. The orotic acid phosphoribosyltransferase (OPRTase) activity of the supernatant was determined according to the method of Belser et al., (Meth. Enzymol. 51: 155-167, 1978), and the active strain was regarded as an orotidine-5′-phosphate decarboxylase-deficient strain (IAM7153ΔpyrG) (Horiuchi et al., Curr. Genet. 27: 472-478, 1995).

Quelle

DOI: 10.6084/m9.figshare.5104873.v1Patent: US07998721B2uspto-grants-2011_08