Glycolic Acid → Formate

Participants

Reaction Conditions

Reaction Steps

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 (0.500M), aminomethylphosphonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard), and flavin mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH), and the solution cooled to 5° C. To the vessel was then added 0.47 g of Hansenula polymorpha transformant GO1 (10 IU glycolate oxidase and 22,100 IU catalase) which had been permeabilized by treatment with 0.1% "TRITON" X-100/1 freeze-thaw, and 1.4×106 units of Aspergillus niger soluble catalase (Sigma). The pH of the resulting mixture was re-adjusted to 8.3 with 50% NaOH, 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 16 hours, the HPLC yields of glyoxylate, formate, and oxalate were 90.1%, 1.3%, and 5.9%, respectively, and 3.0% glycolate remained. The remaining glycolate oxidase activity and total catalase activity were 86% and 136%, respectively, of their initial values.

Provenance & Evidence