Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    We will mail you new results for this query: keywords==Biocatalysis
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Editorial: Actinobacteria, a source of biocatalytic tools
Tischler, Dirk ; Berkel, Willem J.H. Van; Fraaije, Marco W. - \ 2019
Frontiers in Microbiology 10 (2019). - ISSN 1664-302X
Actinomycetes - Biocatalysis - Biotechnology - Extremophile actinobacteria - Germination - High GC genetics - Novel biocatalysts - Secondary metabolites
Discovering novel hydrolases from hot environments
Wohlgemuth, Roland ; Littlechild, Jennifer ; Monti, Daniela ; Schnorr, Kirk ; Rossum, Teunke van; Siebers, Bettina ; Menzel, Peter ; Kublanov, Ilya V. ; Rike, Anne Gunn ; Skretas, Georgios ; Szabo, Zalan ; Peng, Xu ; Young, Mark J. - \ 2018
Biotechnology Advances 36 (2018)8. - ISSN 0734-9750 - p. 2077 - 2100.
Biocatalysis - Enrichment - Enzyme characterization - Enzyme screening - Gene expression - Genomics - Hydrolases - Metagenomics - Sequencing - Thermophiles

Novel hydrolases from hot and other extreme environments showing appropriate performance and/or novel functionalities and new approaches for their systematic screening are of great interest for developing new processes, for improving safety, health and environment issues. Existing processes could benefit as well from their properties. The workflow, based on the HotZyme project, describes a multitude of technologies and their integration from discovery to application, providing new tools for discovering, identifying and characterizing more novel thermostable hydrolases with desired functions from hot terrestrial and marine environments. To this end, hot springs worldwide were mined, resulting in hundreds of environmental samples and thousands of enrichment cultures growing on polymeric substrates of industrial interest. Using high-throughput sequencing and bioinformatics, 15 hot spring metagenomes, as well as several sequenced isolate genomes and transcriptomes were obtained. To facilitate the discovery of novel hydrolases, the annotation platform Anastasia and a whole-cell bioreporter-based functional screening method were developed. Sequence-based screening and functional screening together resulted in about 100 potentially new hydrolases of which more than a dozen have been characterized comprehensively from a biochemical and structural perspective. The characterized hydrolases include thermostable carboxylesterases, enol lactonases, quorum sensing lactonases, gluconolactonases, epoxide hydrolases, and cellulases. Apart from these novel thermostable hydrolases, the project generated an enormous amount of samples and data, thereby allowing the future discovery of even more novel enzymes.

Two-component fad-dependent monooxygenases : Current knowledge and biotechnological opportunities
Heine, Thomas ; Berkel, Willem J.H. van; Gassner, George ; Pée, Karl Heinz van; Tischler, Dirk - \ 2018
Biology - open access biological sciences journal 7 (2018)3. - ISSN 2079-7737
Biocatalysis - Epoxidation - Flavin reductase - Flavoprotein monooxygenases - Halogenation - Heteroatom oxidation - Hydroxylation

Flavoprotein monooxygenases create valuable compounds that are of high interest for the chemical, pharmaceutical, and agrochemical industries, among others. Monooxygenases that use flavin as cofactor are either single-or two-component systems. Here we summarize the current knowledge about two-component flavin adenine dinucleotide (FAD)-dependent monooxygenases and describe their biotechnological relevance. Two-component FAD-dependent monooxygenases catalyze hydroxylation, epoxidation, and halogenation reactions and are physiologically involved in amino acid metabolism, mineralization of aromatic compounds, and biosynthesis of secondary metabolites. The monooxygenase component of these enzymes is strictly dependent on reduced FAD, which is supplied by the reductase component. More and more representatives of two-component FAD-dependent monooxygenases have been discovered and characterized in recent years, which has resulted in the identification of novel physiological roles, functional properties, and a variety of biocatalytic opportunities.

Catalytic performance of a class III Old yellow enzyme and its cysteine variants
Scholtissek, Anika ; Gädke, Eric ; Paul, Caroline E. ; Westphal, Adrie H. ; Berkel, Willem J.H. van; Tischler, Dirk - \ 2018
Frontiers in Microbiology 9 (2018)OCT. - ISSN 1664-302X
Actinobacteria - Biocatalysis - Cysteine modification - Ene reductase - Flavoprotein - Inactivation - Protein engineering - Rhodococcus opacus 1CP

Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81-84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

Deazaflavins as photocatalysts for the direct reductive regeneration of flavoenzymes
Schie, M.M.C.H. van; Younes, S.H.H. ; Rauch, M.C.R. ; Pesic, M. ; Paul, C.E. ; Arends, I.W.C.E. ; Hollmann, F. - \ 2018
Molecular Catalysis 452 (2018). - ISSN 2468-8231 - p. 277 - 283.
Biocatalysis - Flavin - Old yellow enzyme - Oxygen dilemma - Photochemistry
Deazaflavins are potentially useful redox mediators for the direct, nicotinamide-independent regeneration of oxidoreductases. Especially the O2-stability of their reduced forms have attracted significant interest for the regeneration of monooxygenases. In this contribution we further investigate the photochemical properties of deazaflavins and investigate the scope and limitations of deazaflavin-based photoenzymatic reaction systems.
Critical biofilm growth throughout unmodified carbon felts allows continuous bioelectrochemical chain elongation from CO2 up to caproate at high current density
Jourdin, Ludovic ; Raes, Sanne M.T. ; Buisman, Cees J.N. ; Strik, David P.B.T.B. - \ 2018
Frontiers in Energy Research 6 (2018)MAR. - ISSN 2296-598X
Biocatalysis - Bioelectrochemical chain elongation - Biofilm - Caproate - Carbon dioxide utilization - Microbial electrosynthesis
Current challenges for microbial electrosynthesis include the production of higher value chemicals than acetate, at high rates, using cheap electrode materials. We demonstrate here the continuous, biofilm-driven production of acetate (C2), n-butyrate (nC4), and n-caproate (nC6) from sole CO2 on unmodified carbon felt electrodes. No other organics were detected. This is the first quantified continuous demonstration of n-caproate production from CO2 using an electrode as sole electron donor. During continuous nutrients supply mode, a thick biofilm was developed covering the whole thickness of the felt (1.2-cm deep), which coincided with high current densities and organics production rates. Current density reached up to -14 kA melectrode -3 (-175 A m-2). Maximum sustained production rates of 9.8 ± 0.65 g L-1 day-1 C2, 3.2 ± 0.1 g L-1 day-1 nC4, and 0.95 ± 0.05 g L-1 day-1 nC6 were achieved (averaged between duplicates), at electron recoveries of 60-100%. Scanning electron micrographs revealed a morphologically highly diverse biofilm with long filamentous microorganism assemblies (~400 μm). n-Caproate is a valuable chemical for various industrial application, e.g., it can be used as feed additives or serve as precursor for liquid biofuels production.
Chaotropic heat treatment resolves native-like aggregation of a heterologously produced hyperthermostable laminarinase
Westphal, Adrie H. ; Geerke-Volmer, Astrid A. ; Mierlo, Carlo P.M. van; Berkel, Willem J.H. van - \ 2017
Biotechnology Journal 12 (2017)6. - ISSN 1860-6768
Biocatalysis - Endoglucanase - Glycosyl hydrolase - Protein expression - Protein stability
Production of hyperthermostable enzymes in mesophilic hosts frequently causes undesired aggregation of these proteins. During production of Pyrococcus furiosus endo-β-1,3 glucanase (LamA) in Escherichia coli, soluble and insoluble species form. Here, the authors address the composition of this mixture, including the nature of LamA conformers, and establish a method to increase the yield of native monomer. With gel electrophoresis, size-exclusion chromatography, light scattering, circular dichroism and enzyme kinetics the authors show that approximately 50 % of heterologously produced LamA is soluble, and that 40 % of this fraction constitutes native-like oligomers and non-native monomers. Soluble oligomers display, like native LamA monomer, substrate inhibition, although with poor activity. Treatment of soluble oligomers with 3 M guanidinium hydrochloride at 80 °C yields up to 75 % properly active monomer. Non-native monomer shows low specific activity without substrate inhibition. Incubating non-native monomer with 3 M guanidinium hydrochloride at 80 °C causes formation of 25 % native LamA. Also, a large amount of insoluble LamA aggregates can be converted into soluble native monomer by application of this procedure. Thus, chaotropic heat treatment can improve the yield and quality of hyperthermostable proteins that form aberrant species during production in E. coli.
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