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Pyruvate is a valuable chemical intermediate in the production of fine chemicals used by agrochemical, pharmaceutical, and food industries. Current technology for production of pyruvic acid is based on conversion from tartaric acid and results in environmentally incompatible byproducts. An enzymatic approach to making pyruvate was developed by cloning the glycolate oxidase (GO) gene from spinach into Pichia pastoris (Payne, et al., (1995). High-level production of spinach glycolate oxidase in the methylotrophic yeast Pichia pastoris: Engineering a biocatalyst. Gene, 167(1-2), 215-219). GO is a flavoprotein (FMN dependent) which catalyzes the conversion of lactate to pyruvate with the equimolar production of hydrogen peroxide. Hydrogen peroxide can lower GO activity and make non-catalytic byproducts, so catalase was also cloned into P. pastoris to create a double transformant.
Process development work was completed at the University of Iowa's Center for Biocatalysis and Bioprocessing. High-density P. pastoris fermentation (7.2 kg cells/L) was completed at the 100 L scale. Critical fermentation set-points were confirmed at 14 h glycerol feeding followed by methanol induction at 2 - 10 g/L for 30 h. After fermentation, these cells were permeabilized with benzalkonium chloride (BAC) to enable whole-cell biocatalysis and increase enzyme activity, yielding 100 U/g for GO. In 30 L enzyme reactions, permeabilized cells were recycled three times for over 92% conversion of 0.5 M lactate with an "enzyme to product" ratio of approximately 1:2 (Gough, et al., (2005). Production of pyruvate from lactate using recombinant Pichia pastoris cells as catalyst. Process Biochemistry, 40(8), 2597-2601). Though effective, the post-fermentation process for GO recovery involved several unit-operations, including multiple washing steps to remove residual BAC.
The present work has focused on minimizing unit-operations by spray-drying the fermentation product to create a powdered biocatalyst. Optimal spray-drying conditions for the Buchi B-190 instrument were 150°C drying air, 15 mL/min liquid feed rate, and 600 mg cells/mL liquid feed. These conditions resulted in P. pastoris biocatalyst with activities of 80 - 100 U/g for GO and 180,000 - 220,000 U/g for catalase. The spray-dried cells retained nearly 100% of the enzyme activity compared to BAC treated cells as reported by Gough et al. Additionally, the spray-dried biocatalyst was stable at room temperature for 30 days, and no measurable enzyme leaching was observed. Then, P. pastoris was spray-dried under optimal conditions and tested for conversion of lactate to pyruvate for an improved "enzyme to product" ratio.
Enzyme reaction optimization was done at the one-liter scale in DASGIP reactors. The DASGIP system contained four parallel reactors with control of temperature, pH, and dissolved oxygen. Other key variables included substrate loading, conducting the reaction in buffer or water, minimizing enzyme concentration, and maximizing the number of enzyme recycles. Optimal performance was achieved in water at pH 7.0 with an operating temperature of 25°C and 1.0 M substrate loading. Enzyme loading was at 12 g/L for the first two cycles, and subsequently, 2 - 3 g/L of fresh cells were added every alternate cycle to reach 15 cycles. Under these conditions, 75 - 95% conversion of lactate to pyruvate was accomplished for every 12 - 16 h reaction cycle. Based on these parameters, an "enzyme to product" ratio of 1:41 was achieved.