l ‐amino acid oxidases (LAAOs) catalyze the oxidative deamination of l ‐amino acids to corresponding α‐keto acids. Here, we describe the heterologous expression of four fungal LAAOs in Pichia pastoris . cg LAAO1 from Colletotrichum gloeosporioides and nc LAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His‐ hc LAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His‐ fr LAAO1 from Fibroporia radiculosa and 9His‐ la LAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N‐glycosylated. We generated mutants of 9His‐ hc LAAO4 lacking N‐glycosylation sites to further understand the effects of N‐glycosylation. All four predicted N‐glycosylation sites were glycosylated in 9His‐ hc LAAO4 expressed in P . pastoris . Enzymatic activity was similar for fully glycosylated 9His‐ hc LAAO4 and variants without one or all N‐glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N‐glycans. This was caused by the absence of a hypermannosylated N‐glycan on asparagine residue N54. The lack of one or all of the other N‐glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N‐glycosylation during biosynthesis.
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