Reaktion #717760

Reaktionsgleichung

ampicillin

TMS-diazomethane

IPTG

→

(R)-(−)-3-hydroxy-4-cyanobutyric acid

Lösungsmittel

—

Hexanes

EtOAc

Reaktionsbedingungen

Aufarbeitung

1. 1
   workup.ADDITIONThe overnight culture was diluted 2-fold
2. 2
   Sonstigeinduced at 37° C.
3. 3
   Waschenwashed in 100 mM pH 7 sodium phosphate buffer
4. 4
   workup.WAITto proceed for at least 36 h at 22° C. with gentle agitation
5. 5
   SonstigeReaction progress
6. 6
   SonstigeCells and other debris were removed by centrifugation
7. 7
   workup.ADDITIONthe treated with one volume methanol

Vorschrift

Putative nitrilase up-mutants were assayed in triplicate. Each transformant was grown in 5 mL LB (100 μg/mL ampicillin), at 37° C., 220 rpm for 18 h. The overnight culture was diluted 2-fold and nitrilase expression induced at 37° C., 220 rpm with 0.1 mM IPTG for 6 h. Cells were harvested by centrifugation, washed in 100 mM pH 7 sodium phosphate buffer and then re-suspended in 1 mL of 100 mM HGN in 100 mM pH 7 sodium phosphate buffer. Reactions were allowed to proceed for at least 36 h at 22° C. with gentle agitation. Reaction progress was monitored by TLC (1:1 EtOAc:Hexanes, Rf=0.5, nitrile; Rf=0.0, acid). Cells and other debris were removed by centrifugation and the treated with one volume methanol prior to lyophilization. The lyophilizate was re-suspended in methanol and treated with TMS-diazomethane (10 equivalents, 2 M solution in hexanes) until gas evolution ceased and yellow color persisted in order to prepare the methyl ester for GC analysis. Selected nitrilase variants producing (R)-(−)-3-hydroxy-4-cyanobutyric acid of 95% ee or greater were then evaluated for performance at 2.25 M HGN.

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