Directed evolution of the aryl-alcohol oxidase: Beyond the lab bench

Viña‐Gonzalez, Javier; Alcalde, Miguel

doi:10.1016/j.csbj.2020.06.037

Cited by 11 publications

(5 citation statements)

References 68 publications

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“…Another nice example exemplifying the power of protein engineering comes from the Alcalde lab [39]. Here, the aryl alcohol oxidase is from Pleurotus eryngii.…”

Section: Substrate Scopementioning

confidence: 99%

Biocatalytic Oxidation of Alcohols

et al. 2020

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Enzymatic methods for the oxidation of alcohols are critically reviewed. Dehydrogenases and oxidases are the most prominent biocatalysts, enabling the selective oxidation of primary alcohols into aldehydes or acids. In the case of secondary alcohols, region and/or enantioselective oxidation is possible. In this contribution, we outline the current state-of-the-art and discuss current limitations and promising solutions.

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“…Another nice example exemplifying the power of protein engineering comes from the Alcalde lab [39]. Here, the aryl alcohol oxidase is from Pleurotus eryngii.…”

Section: Substrate Scopementioning

confidence: 99%

Biocatalytic Oxidation of Alcohols

et al. 2020

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“…Broader exploitation and protein engineering of AAOs are however challenged by their difficult accessibility due to low concentrations in original fungi and insufficient expression in recombinant microorganisms. Whereas we will not specifically focus on the methods of enzyme engineering of AAOs described in a recent review (Vina-Gonzalez and Alcalde 2020 ), we will include a number of examples focusing on synthetic and biotechnological applications of wild-type AAOs and engineered variants thereof, and discuss various heterologous hosts and strategies aiming to enhance heterologous expression of these enzymes.…”

Section: Introductionmentioning

confidence: 99%

Pecularities and applications of aryl-alcohol oxidases from fungi

Urlacher

Koschorreck

2021

Appl Microbiol Biotechnol

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Aryl-alcohol oxidases (AAOs) are FAD-containing enzymes that oxidize a broad range of aromatic as well as aliphatic allylic alcohols to aldehydes. Their broad substrate spectrum accompanied by the only need for molecular oxygen as cosubstrate and production of hydrogen peroxide as sole by-product makes these enzymes very promising biocatalysts. AAOs were used in the synthesis of flavors, fragrances, and other high-value-added compounds and building blocks as well as in dye decolorization and pulp biobleaching. Furthermore, AAOs offer a huge potential as efficient suppliers of hydrogen peroxide for peroxidase- and peroxygenase-catalyzed reactions. A prerequisite for application as biocatalysts at larger scale is the production of AAOs in sufficient amounts. Heterologous expression of these predominantly fungal enzymes is, however, quite challenging. This review summarizes different approaches aiming at enhancing heterologous expression of AAOs and gives an update on substrates accepted by these promising enzymes as well as potential fields of their application. Key points • Aryl-alcohol oxidases (AAOs) supply ligninolytic peroxidases with H2O2. • AAOs accept a broad spectrum of aromatic and aliphatic allylic alcohols. • AAOs are potential biocatalysts for the production of high-value-added bio-based chemicals.

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“…Many industrially relevant enzymes use cofactors, such as NADPH or FAD, − and thus may form trimolecular complexes. We chose the NADPH-dependent redox enzyme Gre2p as a challenging, model system with multiple reaction components to experimentally investigate the performance of our proposed workflow (Figure ).…”

Section: Resultsmentioning

confidence: 99%

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

et al. 2021

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To apply enzymes in technical processes, a detailed understanding of the molecular mechanisms is required. Kinetic and thermodynamic parameters of enzyme catalysis are crucial to plan, model, and implement biocatalytic processes more efficiently. While the kinetic parameters, K m and k cat , are often accessible by optical methods, the determination of thermodynamic parameters requires more sophisticated methods. Isothermal titration calorimetry (ITC) allows the label-free and highly sensitive analysis of kinetic and thermodynamic parameters of individual steps in the catalytic cycle of an enzyme reaction. However, since ITC is susceptible to interferences due to denaturation or agglomeration of the enzymes, the homogeneity of the enzyme sample must always be considered, and this can be accomplished by means of dynamic light scattering (DLS) analysis. We here report on the use of an ITC-dependent work flow to determine both the kinetic and the thermodynamic data for a cofactor-dependent enzyme. Using a standardized approach with the implementation of sample quality control by DLS, we obtain high-quality data suitable for the advanced modeling of the enzyme reaction mechanism. Specifically, we investigated stereoselective reactions catalyzed by the NADPH-dependent ketoreductase Gre2p under different reaction conditions. The results revealed that this enzyme operates with an ordered sequential mechanism and is affected by substrate or product inhibition depending on the reaction buffer. Data reproducibility is ensured by specifying standard operating procedures, using programmed workflows for data analysis, and storing all data in a F.A.I.R. (findable, accessible, interoperable, and reusable) repository (https://doi.org/10.15490/fairdomhub.1.investigation.464.1). Our work highlights the utility for combined binding and kinetic studies for such complex multisubstrate reactions.

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Directed evolution of the aryl-alcohol oxidase: Beyond the lab bench

Abstract: Graphical abstract

Cited by 11 publications

References 68 publications

Biocatalytic Oxidation of Alcohols

Biocatalytic Oxidation of Alcohols

Pecularities and applications of aryl-alcohol oxidases from fungi

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

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