Water → L-Phenylalanine

HRID 917283

反応式

HRID 917283 の反応式
O.CC#[N:4].N[C@H:6]([CH2:13][C:14]([OH:16])=[O:15])[C:7]1[CH:12]=[CH:11][CH:10]=[CH:9][CH:8]=1>C(O)C(N)(CO)CO.Cl>[NH2:4][C@H:13]([C:14]([OH:16])=[O:15])[CH2:6][C:7]1[CH:12]=[CH:11][CH:10]=[CH:9][CH:8]=1

参加物

5

反応物 3

HCID 962WaterHCID 6342AcetonitrileHCID 686703(3R)-3-amino-3-phenylpropanoic acid

生成物 1

HCID 6140L-Phenylalanine

試薬・触媒・溶媒 1

HCID 93573Tromethamine hydrochloride溶媒

実験手順

The standard PAM activity assay mixture contains 5 mM L-α-phenylalanine, 1 to 10 μg enzyme in 50 mM Tris-HCl buffer (pH 7.5) in a final volume of 0.5 ml. Enzyme reactions were carried out at 28° C., 150 rpm for 18 hours and terminated by addition of 50 μl ethanol to precipitate proteins. After centrifugation for 5 minutes, the supernatants were analyzed by a reverse phase HPLC method. The formation of β-phenylalanine was quantified based on peak area compared to the β-phenylalanine standard curve. Enzyme activity is expressed as μg β-phenylalanine formed per hour per mg protein. HPLC analysis used a YMC Ph column (4.6×150 mm; Waters, Milford, Mass.), and a flow rate of 1.2 ml/minutes. Absorbance was monitored at 210 nm. The method employed two solvents A, water; and B, CH3CN and a gradient consisting 0% to 12% B for 6 minutes, 12% to 90% in 1 minute and returning to 0% in 1 minute. Retention times of L-α-phenylalanine and β-phenylalanine are 4.3 minutes and 3.5 minutes, respectively.

後処理

  1. customwere carried out at 28° C.
  2. customterminated by addition of 50 μl ethanol
  3. customto precipitate proteins
  4. waitAfter centrifugation for 5 minutes
  5. customformed per hour per mg protein
  6. customfor 6 minutes
  7. wait12% to 90% in 1 minute
  8. waitreturning to 0% in 1 minute

出典と根拠

ORDレコード
ord-468c29ed91614976ba7609e0b3118347
出典データセット
uspto-grants-2007_09
特許番号
US07273755B2

関連ノート

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