反应详情

温度

5 °C

Into a 3 oz. Fischer-Porter glass aerosol reaction vessel was placed a magnetic stirring bar and 10 mL of an aqueous solution containing sodium L-lactate (0.500M), KH2PO4 (0.50M), and isobutyric acid (HPLC internal standard, 0.100M) at pH 9.0 (adjusted with 50% NaOH), and the solution cooled to 5° C. To the vessel was then added 0.75 g (wet weight) of Pichia pastoris transformant GS115-MSP10 (6.52 IU/mL glycolate oxidase and 10,100 IU/mL catalase) which had been permeabilized by treatment with 0.1% benzalkonium chloride ("BARQUAT" OJ-50), then the reaction vessel was sealed and the reaction mixture was cooled to 5° C. The vessel was flushed with oxygen by pressurizing to 70 psig and venting to atmospheric pressure five times with stirring, then the vessel was pressurized to 70 psig of oxygen and the mixture stirred at 5° C. Aliquots (0.10 mL) were removed by syringe through a sampling port (without loss of pressure in the vessel) at regular intervals for analysis by HPLC to monitor the progress of the reaction. After 5 hours, the HPLC yields of pyruvate and acetate were 97.6% and 2.5%, respectively, and 0.3% lactate remained. The remaining permeabilized cell activity of glycolate oxidase and catalase were 104% and 105%, respectively, of their initial values.

1. customFischer-Porter glass aerosol reaction vessel
2. customthe reaction vessel was sealed
3. temperaturethe reaction mixture was cooled to 5° C
4. customThe vessel was flushed with oxygen
5. stirringthe mixture stirred at 5° C
6. customAliquots (0.10 mL) were removed by syringe through a sampling port (without loss of pressure in the vessel) at regular intervals for analysis by HPLC
7. customthe progress of the reaction