ThermoFAD, a Thermofluor®‐adapted flavin ad hoc detection system for protein folding and ligand binding

Cyclohexanone monooxygenase (CHMO) is ap romising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio-, chemo-, and enantioselectivity.However,the lowstability of many BaeyerVilliger monooxygenases is an obstacle for their exploitation in industry.C haracterization and crystal structure determination of ar obust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts avariety of aliphatic, aromatic,a nd cyclic ketones,a sw ell as prochiral sulfides.A compact substrate-binding cavity explains its preference for small rather than bulky substrates.S mall-scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO.T he exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology.

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“…[14] This confirmed that AcCHMO is only moderately stable since it exhibits a T m value of 37 8 8C (Table S4).…”

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confidence: 65%

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

et al. 2016

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“…The unfolding temperatures of purified HMFO, SUMO-HMFO, and the SUMO-HMFO H467A mutant enzyme were analyzed by the ThermoFAD method (17). By this method, the release of the flavin cofactor is monitored while heating the enzyme.…”

Section: Methodsmentioning

confidence: 99%

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

Dijkman

Fraaije

2014

Appl Environ Microbiol

129

200

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In the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein from Methylovorus sp. strain MP688 in Escherichia coli and subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.

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“…In order to check if the PAMO mutants lost significant thermostability due to the mutations, T m values were measured the ThermoFAD method (Forneris et al, 2009). …”

Section: Thermostability Of Wt Pamo and Evolved Mutantsmentioning

confidence: 99%

Directed evolution of phenylacetone monooxygenase as an active catalyst for the baeyer–villiger conversion of cyclohexanone to caprolactone

Parra

Acevedo

Reetz

2015

Biotech & Bioengineering

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Phenylacetone monooxygenase (PAMO) is an exceptionally robust Baeyer-Villiger monooxygenase, which makes it ideal for potential industrial applications. However, its substrate scope is limited, unreactive cyclohexanone being a prominent example. Such a limitation is unfortunate, because this particular transformation in an ecologically viable manner would be highly desirable, the lactone and the respective lactam being of considerable interest as monomers in polymer science. We have applied directed evolution in search of an active mutant for this valuable C-C activating reaction. Using iterative saturation mutagenesis (ISM), several active mutants were evolved, with only a minimal trade-off in terms of stability. The best mutants allow for quantitative conversion of 2 mM cyclohexanone within 1 h reaction time. In order to circumvent the NADP(+) regeneration problem, whole E. coli resting cells were successfully applied. Molecular dynamics simulations and induced fit docking throw light on the origin of enhanced PAMO activity. The PAMO mutants constitute ideal starting points for future directed evolution optimization necessary for an industrial process.

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ThermoFAD, a Thermofluor^®‐adapted flavin ad hoc detection system for protein folding and ligand binding

Cited by 177 publications

References 30 publications

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

Directed evolution of phenylacetone monooxygenase as an active catalyst for the baeyer–villiger conversion of cyclohexanone to caprolactone

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