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Abstract Pichia pastoris expression is a mature and efficient eukaryotic expression system. In this work, Aspergillus oryzae lipase (AOL, with the molecular mass of 28 kDa), which can perform highly stereoselective hydrolysis of (R, S)-methyl 2-(4-hydroxyphenoxy) propanoate, was expressed in P. pastoris X-33. The specific activity of AOL was 432 U/mg, which was obtained by fed-batch cultivation in a 5 L bioreactor using a methanol feeding strategy. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut-off membrane and purified with DEAE-Sepharose™ FF ion-exchange chromatography and phenyl Seflnose™ 6 FF hydrophobic interaction chromatography. The purified lipase activity reached 5509 U/mg. AOL showed high activity toward short-chain triacylglyceride (C 4), and the optimum temperature and pH were 40 °C and 8.0, respectively. The purified enzyme activity was inhibited by Zn2+ and Cu2+. Moreover, the K m and V max values were 1 mM and 32.89 mmol/min, respectively. Highlights • The recombinant strain P. pastoris X-33/pPICZɑA-aol was constructed. • The recombinant A. oryzae lipase was obtained in fed-batch cultivation. • The enzymatic properties of recombinant A. oryzae lipase were studied. [ABSTRACT FROM AUTHOR]