Reaction #1379909

Reaction equation

NaOH

magnesium sulfate

TES

calcium carbonate

monopotassium phosphate

L-proline

2-ketoglutaric acid

L-ascorbic acid

→

Trans-4-hydroxy-L-proline

Solvents

xylene

Conditions

Workup

1. 1
   OtherOne ml of the above-mentioned seed culture was inoculated in the medium in each test tube under germ-free condition
2. 2
   workup.WAITcultivated at 28° C. for 2 days
3. 3
   workup.WAITg for 10 minutes at 4° C
4. 4
   OtherThe cells thus separated
5. 5
   Washwere washed with 80 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] buffer (pH 7.5)
6. 6
   Otherhad been prepared
7. 7
   workup.ADDITIONby adding
8. 8
   workup.WAITto stand at 30° C. for 2 hours
9. 9
   Otherthe enzymatic reaction

Procedure

Separately, Df1 medium comprising 5% soluble starch, 1.5% soybean meal, 0.05% monopotassium phosphate, 0.05% magnesium sulfate 7 hydrate and 0.5% calcium carbonate, and adjusted to pH 7.0 with 6N NaOH, was put in test tubes (diameter 25 mm×length 200 mm) in an amount of 10 ml each and sterilized at 120° C. for 20 minutes. One ml of the above-mentioned seed culture was inoculated in the medium in each test tube under germ-free condition and cultivated at 28° C. for 2 days with shaking. The thus-obtained culture was centrifuged at 7000×g for 10 minutes at 4° C. The cells thus separated were washed with 80 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] buffer (pH 7.5) and then recentrifuged. 150 mg of the thus-obtained wet cells was suspended in 1.5 ml of a reaction mixture which had been prepared by adding 1.4% (v/v) of NYMEEN solution [prepared by adding 4 g of NYMEEN S-215 (produced by Nippon Oils & Fats Co.) to 10 ml of xylene] to 80 mM TES buffer (pH 7.5) containing 4 mM L-proline, 8 mM 2-ketoglutaric acid, 4 mM L-ascorbic acid and 2 mM ferrous sulfate] and the mixture was allowed to stand at 30° C. for 2 hours to carry out the enzymatic reaction.

Source