Recombinant production of horseradish peroxidase in E. coli and yeast – a quantitative and qualitative comparison

Abstract

Horseradish peroxidase (HRP) is an enzyme originating from the horseradish root and belongs to the enzyme group of plant peroxidases. It is most prominently used for the oxidation of various organic compounds in biosensors, immunoassays as well as for wastewater-remediation and possesses a global market value of around $175 million. Currently, HRP is dominantly produced via E. coli BL21DE3. This process yields unfolded protein aggregates called inclusion bodies (IB), which need to undergo a precisely designed refolding process in order to attain an active and functional form. This refolding process is highly influenced by the conformation of the aggregated HRP. Therefore, the optimal upstream parameters still need to be looked into. Still, the high demands of time and energy of the refolding process as well as the lack of glycosylation motivate the lookout for a more suitable host. The production of recombinant HRP (rHRP) in a P. pastoris SuperMan5 strain acts as a promising alternative, as it delivers secreted, active rHRP with a glycosylation chain similar to the human pattern. The unique glycosylation pattern may not only positively affect the protein stability, but also potentially pave the way for new biopharmaceutical applications. The aim of this work was first to compare four different induced-fed-batch (IFB) conditions for rHRP production in E. coli for the highest IB titer. Then, a given refolding protocol was executed on the IBs of all four processes to determine the process yielding the most active rHRP. The results suggest running an IFB for 8 h at 30°C with an exponential feeding regime at a specific substrate uptake rate (qS) of 0.25 g/g/h. This process had an IB outcome of 5.43 g/L, a space-time-yield (STY) of 31.2 mg/L/h of pure rHRP with a specific enzyme activity (sAct) of 1163 U/mg. The outcome of this E. coli process was directly compared to a P. pastoris process for rHRP production exercising the necessary steps of the rHRP E. coli purification protocol. The P. pastoris process yielded very low amounts of 0.006 mg/mL, a STY 0.0355 mg/L/h of total rHRP with an sAct 3-times lower compared to the rHRP produced in E. coli. Therefore, our findings show that presently the P. pastoris process cannot act as an economically relevant alternative to the production process in E. coli without further optimization. For rHRP production in E. coli, a shorter IFB time as well as feeding at higher qS might lead to even higher titers and STY’s.