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l-amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l-amino acids to the corresponding alpha-keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporum in the yeast Pichia pastoris with a 9His-tag and compare this with the recently characterized 6His-hcLAAO4 expressed in E. coli. The expression of the enzyme with an ER-signal sequence in P.pastoris resulted in a glycosylated, secreted protein. The enzymatic activity without activation was higher after expression in P.pastoris compared to E. coli. Due to treatment with acidic pH, a striking increase of activity could be detected for both expression systems resulting in similar specific activities after acid activation. Regarding the substrate spectrum, temperature stability, Km, and vmax values, hcLAAO4 showed very few differences when produced in these two expression systems. A higher yield of hcLAAO4 could be obtained by fermentation. © 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.