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 glycolic acid (0.750M), ethylenediamine (0.863M), isobutyric acid (0.100M, HPLC internal standard), and flavin mononucleotide (0.01 mM) at pH 9.0, and the solution cooled to 5° C. To the vessel was then added 0.75 g of Pichia pastoris transformant strain GS115-MSP10 (31 IU glycolate oxidase and 38,100 IU catalase) which had been permeabilized by treatment with 0.1% "TRITON" X-100/1 freeze-thaw, and the reaction vessel sealed and the reaction mixture was cooled to 5° C. The vessel was flushed with oxygen by pressuring 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 (0.20 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 6 h, the HPLC yields of glyoxylate, formate, and oxalate were 98.2%, 0%, and 0% respectively, and no glycolate remained. The remaining permeabilized-cell glycolate oxidase and catalase activity were 85% and 117% respectively, of their initial values.

1. customFischer-Porter glass aerosol reaction vessel
2. customthe reaction vessel 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 (0.20 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