Glycolic Acid → Glyoxylic Acid

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A 300-mL EZE-Seal stirred autoclave reactor equipped with Dispersimax Impeller (Autoclave Engineers) was charged with 100 mL of a solution containing glycolic acid (0.750M), ethylenediamine (0.863M), isobutyric acid (0.100M, HPLC internal standard), and flavin mononucleotide (0.01 mM), at pH 9.25, and the solution cooled to 5° C. To the reactor was then added 5.0 g of Pichia pastoris transformant strain GS115-MSP10 (423 IU glycolate oxidase and 869,000 IU catalase) which had been permeabilized by treatment with 0.1% benzalkonium chloride (Sigma), and the reactor purged with oxygen. The mixture was then stirred at 1000 rpm, which bubbled oxygen through the mixture via the action of the turbine impeller, and at 5° C. under 120 psig of oxygen. The reaction was monitored by taking a 0.4 mL aliquot of the reaction mixture at regular intervals, filtering the aliquot using a Millipore Ultrafree-MC 10,000 NMWL Filter Unit, and analyzing the filtrate by HPLC. After 1.0 h, the yields of glyoxylic acid, oxalic acid, and formic acid were 98.7%, 1.3%, and 0%, respectively, with no remaining glycolic acid. The recovered activities of permeabilized-cell glycolate oxidase and catalase were 87% and 84% of their initial values, respectively.

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