(48c) Characterizing the Biocatalytic Properties of Cytosolically Expressed Penicillin G Acylase from E. coli for the Synthesis of Cephalexin and Comparison with ?F24A Mutant
AIChE Annual Meeting
Monday, November 16, 2020 - 8:30am to 8:45am
We have improved the expression of wild -type EcPGA 20-fold to 45 mg/L culture. Additionally, we have characterized the synthetic properties of wild type EcPGA, Î²F24A, and AssemblaseÂ®, a commercial variant of EcPGA for cephalexin synthesis. We found that AssemblaseÂ® possessed marginally improved synthetic parameters over Î²F24A and wild-type enzymes. Higher product accumulation during long-term reaction experiments confirmed this observation. Next, wild type EcPGA was immobilized on various epoxy-functionalized supports to enable recycling of the biocatalyst between reactions. Approximately 30% of free enzymatic activity was retained after immobilization with decreased apparent activity attributed to diffusion limitations, as suggested by the decreasing apparent specific activity with increasing particle sizes. The synthesis to hydrolysis ratio decreased slightly, possibly due to internal product accumulation due to diffusion limitations. We are currently modeling the reaction and diffusion within the carrier pores using a reaction diffusion model to predict optimum enzyme loading to achieve high productivity while minimizing reduction of the synthesis to hydrolysis ratio.