A novel spray-drying process to stabilize glycolate oxidase and catalase in Pichia pastoris and optimization of pyruvate production from lactate using the spray-dried biocatalyst

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), 215219). 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