Reaction #2161242

ord-b3540f376e2d4303b536f39d149380d8

Reaction equation

O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
Cc1ncc(C[n+]2csc(CCO)c2C)c(N)n1
thiamine
O=P([O-])(O)O.[K+]
KH2PO4
O=S(=O)([O-])[O-].[Mg+2]
MgSO4
CC[C@H](C)[C@H](N)C(=O)O
L-isoleucine
O=C([O-])[O-].[Ca+2]
CaCO3
O=C([O-])[O-].[Ca+2]
CaCO3
O=S(=O)([O-])[O-].[NH4+].[NH4+]
ammonium sulfate
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
Glucose
→
N[C@@H](CCC(=O)O)C(=O)O
L-glutamic acid

Reactants

O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
Cc1ncc(C[n+]2csc(CCO)c2C)c(N)n1
thiamine
O=P([O-])(O)O.[K+]
KH2PO4
O=S(=O)([O-])[O-].[Mg+2]
MgSO4
CC[C@H](C)[C@H](N)C(=O)O
L-isoleucine
O=C([O-])[O-].[Ca+2]
CaCO3
O=C([O-])[O-].[Ca+2]
CaCO3
O=S(=O)([O-])[O-].[NH4+].[NH4+]
ammonium sulfate
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
Glucose

Reagents

None

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    workup.ADDITIONcontaining 2 ml of fermentation medium
  2. 2
    workup.WAITCultivation can be carried out at 30° C. for 3 days

Procedure

Both strains, VL334thrC+ and VL334thrC+-ΔgeneX, can be grown for 18-24 hours at 37° C. on L-agar plates. Then, one loop of the cells can be transferred into test tubes containing 2 ml of fermentation medium. The fermentation medium contains glucose (60 g/l), ammonium sulfate (25 g/l), KH2PO4 (2 g/l), MgSO4 (1 g/l), thiamine (0.1 mg/ml), L-isoleucine (70 μg/ml), and CaCO3 (25 g/l). The pH is adjusted to 7.2. Glucose and CaCO3 are sterilized separately. Cultivation can be carried out at 30° C. for 3 days with shaking. After the cultivation, the amount of L-glutamic acid which is produced can be determined by paper chromatography (liquid phase composition of butanol-acetic acid-water=4:1:1) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0.5% CdCl2.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US07919283B2uspto-grants-2011_04