Abstract:Unspecific peroxygenase（UPO）was capable of catalyzing various oxidation reactions using hydrogen peroxide（H2O2）and had great potential for industrial applications.In order to address the low expression level of AaeUPO in Pichia pastoris and the unclear fermentation process，the fermentation conditions were optimized through shake flask fermentation and orthogonal tests，and further scaled up using a 5 L fermenter.The results showed that the basic fermentation conditions for high-efficiency expression of AaeUPO in P.pastoris shake flask fermentation were as follows：methanol concentration of 1.00%，initial pH value of 5.5，peptone concentration of 1.5%，and yeast powder concentration of 1.5%.Under the optimal fermentation conditions，the crude enzyme activity reached 18.2 U/mL.Based on the shake flask fermentation experiment，a 5 L fermenter was used for scale up，and the final process conditions were optimized with an inoculation volume of 10% ，induction temperature of 30 ℃，and methanol fed-batch addition.The resulting cell wet weight was 244 g/L，and the highest enzyme activity reached 25.1 U/mL，which was a 91.6% improvement compared with the non-optimized initial stage.AaeUPO was used for H2O2 detection and demonstrated good detection performance with a detection limit of 5 μmol/L and strong anti-interference capability.