Abstract
The fungal secretome comprises various oxidative enzymes participating in the degradation of lignocellulosic biomass as a central step in carbon recycling. Among the secreted enzymes, aryl-alcohol oxidases (AAOs) are of interest for biotechnological applications including production of bio-based precursors for plastics, bioactive compounds, and flavors and fragrances. Aryl-alcohol oxidase 2 (PeAAO2) from the fungus Pleurotus eryngii was heterologously expressed and secreted at one of the highest yields reported so far of 315 mg/l using the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). The glycosylated PeAAO2 exhibited a high stability in a broad pH range between pH 3.0 and 9.0 and high thermal stability up to 55 °C. Substrate screening with 41 compounds revealed that PeAAO2 oxidized typical AAO substrates like p-anisyl alcohol, veratryl alcohol, and trans,trans-2,4-hexadienol with up to 8-fold higher activity than benzyl alcohol. Several compounds not yet reported as substrates for AAOs were oxidized by PeAAO2 as well. Among them, cumic alcohol and piperonyl alcohol were oxidized to cuminaldehyde and piperonal with high catalytic efficiencies of 84.1 and 600.2 mM−1 s−1, respectively. While the fragrance and flavor compound piperonal also serves as starting material for agrochemical and pharmaceutical building blocks, various positive health effects have been attributed to cuminaldehyde including anticancer, antidiabetic, and neuroprotective effects. PeAAO2 is thus a promising biocatalyst for biotechnological applications.Key points• Aryl-alcohol oxidase PeAAO2 from P. eryngii was produced in P. pastoris at 315 mg/l.• Purified enzyme exhibited stability over a broad pH and temperature range.• Oxidation products cuminaldehyde and piperonal are of biotechnological interest.Graphical abstract
Highlights
The pursuit of a sustainable and bio-based society includes the search for and development of environmentally friendly production routes for fine chemicals
aryl-alcohol oxidases (AAOs) play an essential role in degradation of lignocellulosic biomass and in carbon recycling
Activity of PeAAO2 towards 41 compounds was tested in a coupled assay making use of the generated hydrogen peroxide as product of AAO activity
Summary
The pursuit of a sustainable and bio-based society includes the search for and development of environmentally friendly production routes for fine chemicals. Appl Microbiol Biotechnol (2020) 104:9205–9218 can be further oxidized to the aromatic acids depending on the degree of hydration via gem-diol formation of the aldehyde (Ferreira et al 2010). AAOs offer great potential for application in biocatalytic processes, as they only require molecular oxygen for substrate oxidation and generate hydrogen peroxide as byproduct, without the need of added cofactors. AAOs play an essential role in degradation of lignocellulosic biomass and in carbon recycling. Wooddecaying fungi secrete a whole bunch of oxidative enzymes like laccases, ligninolytic peroxidases, and aryl-alcohol oxidases in order to break down lignin, the most recalcitrant component of lignocellulose (Kirk and Farrell 1987)
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