Glycolate Oxidase (GO), a Platform Technology

E. I. DuPont donated patents related to glycolate oxidase (GO) technology to The University of Iowa. CBB developed the process to produce high-cell-density fermentations of Pichia pastoris yielding large quantities of biocatalyst. (Recently, CBB added a manifold to supply oxygen from Dewars, enhancing capabilities for 100- and 1,000-liter fermentors to produce cells at high density.) During fermentation, glycerol and methanol are continuously monitored to maximize enzyme yield per cell and cell yield per unit volume. CBB has installed all the safety measures to handle solvent additions with oxygen sparging during fermentation.

A proprietary spray drying process has been developed to take the cells from the fermentor to biocatalysis reactors in 2-3 unit operations. The spray-dried cells are stable for >30 days and do not leach the enzymes involved in catalysis. Also, spray-drying renders the cells porous to the substrate and product. The reaction process for converting L-lactate to pyruvate (Figure 1) has been fully optimized. GO has been used to produce 1 Kg of pyruvate in water, at room temperature in neutral pH. The conversion of L-lactate is nearly 100% and the product does not require additional purification. Pyruvate is recovered as a pure white crystalline powder via either lyophilization or spray-drying of the reaction mixture. The cells can be recycled at least 15 times for maximum product yield per unit time and enzyme. Samples of pyruvate and complete certification of the product produced relative to commercial standard can be provided upon request. For further inquiries or for a sample of pyruvate produced with (GO) technology, see the contact information. GO can also be used to produce glyoxylic acid (vanillin) and to oxidize other several 2-hydroxy acids including long chain aliphatics and hetero-aromatics to their corresponding 2-keto acids (Figure 1).

Figure 1. GO catalyzed oxidation of 2-hydroxy acids to corresponding 2-keto acids
Figure 1. GO catalyzed oxidation of 2-hydroxy acids to corresponding 2-keto acids

Dynamic resolution of racemic 2-hydroxy acids to (R)-2-hydroxy acids and conversion of (S)-2-hydroxy acids to (R)-2-hydroxy acids by glycolate oxidase
Due to the absolute specificity of GO to (S)-enantiomers, it has been used for resolution of racemic 2-hydroxy acids to (R)-2-hydroxy acids by selective oxidation of (S)-enantiomers. However, such a resolution has a maximum theoretical yield of only 50% with respect to the (R)-acid. In order to obtain higher yield, GO-based resolution has been made dynamic as shown in Figure 2.

Figure 2. Dynamic resolution of racemic 2-hydroxy acids by double recombinant Pichia pastoris co-expressing glycolate oxidase and catalase.  Non-selective reduction of the 2-keto acids (2) creates a dynamic process for resolution of 2–hydroxy acids (1) with high yield of the (R)-enantiomers.
Figure 2. Dynamic resolution of racemic 2-hydroxy acids by double recombinant Pichia pastoris co-expressing glycolate oxidase and catalase. Non-selective reduction of the 2-keto acids (2) creates a dynamic process for resolution of 2–hydroxy acids (1) with high yield of the (R)-enantiomers.

In this Figure, 2-keto acids produced from (S)-2-hydroxy acids by GO, has been non-selectively reduced by sodium borohydride to racemic 2-hydroxy acids. This ensures continuous production and enrichment of (R)-2-hydroxy acids (Figure 2). Three substrates have been tested, including racemic lactic acid, 2-hydroxybutanoic acid, and 3-phenyllactic acid up to 500 mM concentration and 0.5 L scale. The reaction has been conducted in water at room temperature in neutral pH. As indicated in Figure 2, the reaction was conducted in one-pot with direct addition of stoichiometric addition of sodium borohydride into the reaction mixture. High yield of (R)-2-hydroxy acids was obtained by this dynamic process. Reduction of 2-keto acids by sodium borohydride is efficient and does not compromise the enzyme activity. The yields of the above (R)-2-hydroxyacids from their racemic mixture in the dynamic process range from 95% to 99%. GO has been recycled at least two times with more than 95% yield for the dynamic resolution of 2-hydroxybutanoic acids. The dynamic resolution of racemic 2-hydroxybutanoic acids to (R)-2-hydroxybutanoic acids with 99% yield is shown in the Figure 3.

Figure 3. One-pot chemo-enzymatic dynamic resolution of (RS)-2-hydroxybutanoic acids to (R)-2-hydroxybutanoic acid. D-HBA, L-HBA, KBA, and BH represent R-2-hydroxybutanoic acid, S-2-hydroxybutanoic acid, 2-ketobutanoic acid, and sodium borohydride, respectively.
Figure 3. One-pot chemo-enzymatic dynamic resolution of (RS)-2-hydroxybutanoic acids to (R)-2-hydroxybutanoic acid. D-HBA, L-HBA, KBA, and BH represent R-2-hydroxybutanoic acid, S-2-hydroxybutanoic acid, 2-ketobutanoic acid, and sodium borohydride, respectively.

Optically active R-2-hydroxy acids can be synthesized from cheaper S-2-hydroxy acids at close to 100% theoretical yield, via dynamic R-enantiomerization of S-2-hydroxy acids. An example of S-lactic acid to R-lactic acid in a chemo-enzymatic dynamic process is shown in Figure 4.

Figure 4. One-pot chemo-enzymatic dynamic R-enantiomerization of S-lactic acids to R-lactic acids.  (A) Freeze-dried white crystalline powder of R-lactate produced from 1.0 L of 250 mM S-lactate.  D-LA, L-LA, PA, and BH represent R-lactate, S-lactate, pyruvic acid, and sodium borohydride, respectively.Figure 4. One-pot chemo-enzymatic dynamic R-enantiomerization of S-lactic acids to R-lactic acids.  (A) Freeze-dried white crystalline powder of R-lactate produced from 1.0 L of 250 mM S-lactate.  D-LA, L-LA, PA, and BH represent R-lactate, S-lactate, pyruvic acid, and sodium borohydride, respectively.
Figure 4. One-pot chemo-enzymatic dynamic R-enantiomerization of S-lactic acids to R-lactic acids. (A) Freeze-dried white crystalline powder of R-lactate produced from 1.0 L of 250 mM S-lactate. D-LA, L-LA, PA, and BH represent R-lactate, S-lactate, pyruvic acid, and sodium borohydride, respectively.

The Technology for production of pyruvic acid from L-lactic acid using spray-dried P. pastoris coexpressing glycolate oxidase and catalase has been licensed to a company non-exclusively for a specific field. This technology is still available for production of pyruvic acid for pharmaceutical, agricultural, nutritional and industrial applications. CBB is ready for technology transfer to a company in terms of evaluating the economics of optically active (R)-2-hydroxy acid (lactic acid, 2-hydroxybutanoic acid, and 3-phenyllactic acid) production. For further inquiries or for a sample of above mentioned (R)-2-hydroxy acid and (R) Lactate produced with (GO) technology, see the contact information.

S. Gough, M. Deshpande, M. Scher and JPN Rosazza. 2001. Permeabilization of Pichia pastoris for glycolate oxidase activity. Biotechnol. Lett. 23:1535-1537.

V. Subramanian, J. H. Glenn IV and Shuvendu Das (2007) International Publication Number: WO 2009/064277 A1

Shuvendu Das, J. H. Glenn IV, Mani Subramanian (online published on Dec 15, 2009) Enantioselective oxidation of 2-hydroxy carboxylic acids by glycolate oxidase and catalase coexpressed in methylotrophic Pichia pastoris. Biotechnol. Prog. Pub# 10.1002/btpr.363 PDF

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