Pilot-Scale Production of Peroxygenase from Agrocybe aegerita

Abstract

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L^-1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H₂O₂ feed rate, linear product formation was observed for 7 days, producing more than 5 g L^-1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

Scheme 1. Comparison of the Overall Reaction Schemes of P450 Monooxygenase- (a) and Peroxygenase-Catalyzed Oxyfunctionalization Reactions (b); (c) Structure of the Oxygen-Transferring Heme Species (Compound I, Cpd I)

Scheme 2. Process Scheme of the Pilot-Scale Production of rAaeUPO Reported in This Contribution

Figure 1

Time profile of the main fermentation. Black squares: biomass as determined via the optical density at 600 nm, green diamonds: rAaeUPO_ABTS activity determined via the ABTS assay, blue line: smoothed glycerol feeding profile, red line: smoothed MeOH feeding profile.

Scheme 3. rAaeUPO-Catalyzed Stereoselective Hydroxylation of Ethyl Benzene to (R)-1-Phenyl Ethanol Using a 2LPS Approach

Figure 2

Time course of the semipreparative-scale hydroxylation of ethyl benzene to (R)-1-phenyl ethanol. Reaction conditions: the initial reaction mixture consisted of 50 mL of ethyl benzene and 50 mL of phosphate buffer (100 mM, pH 7) containing 500 nM biocatalyst. The reaction was thermostatted at 25 °C and stirred continuously at 150 rpm. H₂O₂ added continuously at a rate of 49 mmol h^–1 was applied (0.6 mL of a 82 mM stock solution), triangles: total product formed (sum of product amounts in the aqueous and organic layer), filled diamonds: (R)-1-phenyl ethanol found in the organic layer, open diamonds: (R)-1-phenyl ethanol found in the aqueous layer, dotted line: amount of H₂O₂ added to the reactor over time.