l ‐Amino acid oxidases (LAAOs) catalyze the oxidative deamination of l ‐amino acids to α‐keto acids. Recombinant production of LAAOs with broad substrate spectrum remains a formidable challenge. We previously achieved this for the highly active and thermostable LAAO4 of Hebeloma cylindrosporum ( Hc LAAO4). Here, we crystallized a proteolytically truncated surface entropy reduction variant of Hc LAAO4 and solved its structure in substrate‐free form and in complex with diverse substrates. The ability to support the aliphatic portion of a substrate's side chain by an overall hydrophobic active site is responsible for the broad substrate spectrum of Hc LAAO4, including l ‐amino acids with big aromatic, acidic and basic side chains. Based on the structural findings, we generated an E288H variant with increased activity toward pharmaceutical building blocks of high interest.

An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid co-substrates of (S)-selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1 mol % of the co-substrate was required and l-amino acids instead of α-keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)-selective ATA from Vibrio fluvialis (ATA-Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co-substrate channeling between ATA-Vfl and hcLAAO4 due to their close proximity. Co-immobilization allowed further reduction of the co-substrate amount to 0.1 mol % most likely due to a more efficient H2 O2 -removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co-immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)-1-PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA-Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA-Vfl-8M was used in the co-immobilized enzyme cascade to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate, with a 1,000 fold lower input of the co-substrate.

Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co‐substrate. A dual‐enzyme recycling system overcomes this limitation: l‐amino acid oxidases (LAAO) recycle the accumulating co‐product of (S)‐selective transaminases in the kinetic resolution of racemic amines to produce pure (R)‐amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H2O2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using sub‐stoichiometric amounts of 1 mol% of the transaminase co‐substrate as well as the initial application of l‐amino acids instead of α‐keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90 mg) by the synthesis of highly enantiomerically pure (R)‐methylbenzylamine (>99 %ee) at complete conversion (50 %).

Abstract 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. cgLAAO1 from Colletotrichum gloeosporioides and ncLAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His‐hcLAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His‐frLAAO1 from Fibroporia radiculosa and 9His‐laLAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N‐glycosylated. We generated mutants of 9His‐hcLAAO4 lacking N‐glycosylation sites to further understand the effects of N‐glycosylation. All four predicted N‐glycosylation sites were glycosylated in 9His‐hcLAAO4 expressed in P. pastoris. Enzymatic activity was similar for fully glycosylated 9His‐hcLAAO4 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.