High overexpression of dye decolorizing peroxidase TfuDyP leads to the incorporation of heme precursor protoporphyrin IX

Colpa, Dana I.; Fraaije, Marco W.

doi:10.1016/j.molcatb.2016.08.017

Cited by 10 publications

(11 citation statements)

References 31 publications

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“…Furthermore, the spectral responses of both Cbo DyP variants to both redox agents confirmed that the heme had been loaded with iron. Previous work had shown that in the case of sequence-related DyPs, a high overexpression can lead to incorporation of iron deficient heme (protoporphyrin IX), resulting in inactive enzyme species [5].…”

Section: Resultsmentioning

confidence: 99%

Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

2019

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DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

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Section: Resultsmentioning

confidence: 99%

Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

2019

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“…After deglycosylation by EndoHF, this band shifted to around 45 kDa, which indicated that the protein was probably not only N‐, but also O‐glycosylated (Figure S1). In some cases, incorporation of the heme in the apoprotein or the incorporation of iron in the heme is not sufficient during overexpression of hemoproteins . Thus, we examined the integrity of the heme by measuring UV/Vis spectra of oxidized and reduced MrMnP1.…”

Section: Resultsmentioning

confidence: 99%

Redesign of a New Manganese Peroxidase Highly Expressed in Pichia pastoris towards a Lignin‐Degrading Versatile Peroxidase

2018

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Manganese peroxidases, lignin peroxidases, and versatile peroxidases secreted by white rot fungi are supposed to play an essential role in lignin degradation. Thus, these enzymes have attracted significant attention as potential biocatalysts. Versatile peroxidases are the most interesting ones, since they comprise activities of manganese and lignin peroxidases. Herein, we demonstrate how the properties of a new manganese peroxidase from Moniliophthora roreri, designated MrMnP1, were shifted towards those of a versatile peroxidase. MrMnP1 was cloned in Pichia pastoris X‐33 and highly expressed in a fed‐batch fermentation, yielding 132 mg L−1 of active enzyme. To extend the substrate spectrum of MrMnP1, a catalytically active tryptophan present in lignin and versatile peroxidases was first introduced. Additionally, the role of five amino acids at positions adjacent to the catalytic tryptophan was elucidated through their replacement by those found in a versatile peroxidase from Pleurotus eryngii. The resulting mutants demonstrated new activities towards high‐redox‐potential substrates, such as lignin dimers, veratryl alcohol, and the azo dye Reactive Black 5.

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“…HRP, however, is still extracted from horseradish because of difficulties in the heterologous expression of this plant peroxidase . Therefore, for this study, we selected a recently discovered bacterial peroxidase, Svi DyP, which was easily produced by Escherichia coli …”

Section: Figurementioning

confidence: 99%

“…Cloning : The genes of oxidases ChitO* (ChitO triple mutant, Q268R/G270E/S410R), EugO, HMFO, and HotAldO were amplified and cloned C terminally to the SviDyP gene in vector pBAD His‐ Svi DyP; for original and new plasmids, see Table S1 in the Supporting Information. pBAD His‐ Svi DyP contains C terminally to the SviDyP gene, a stop codon, a HindIII restriction site, and another stop codon.…”

Section: Figurementioning

confidence: 99%

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Creating Oxidase–Peroxidase Fusion Enzymes as a Toolbox for Cascade Reactions

et al. 2017

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A set of bifunctional oxidase–peroxidases has been prepared by fusing four distinct oxidases to a peroxidase. Although such fusion enzymes have not been observed in nature, they could be expressed and purified in good yields. Characterization revealed that the artificial enzymes retained the capability to bind the two required cofactors and were catalytically active as oxidase and peroxidase. Peroxidase fusions of alditol oxidase and chitooligosaccharide oxidase could be used for the selective detection of xylitol and cellobiose with a detection limit in the low‐micromolar range. The peroxidase fusions of eugenol oxidase and 5‐hydroxymethylfurfural oxidase could be used for dioxygen‐driven, one‐pot, two‐step cascade reactions to convert vanillyl alcohol into divanillin and eugenol into lignin oligomers. The designed oxidase–peroxidase fusions represent attractive biocatalysts that allow efficient biocatalytic cascade oxidations that only require molecular oxygen as an oxidant.

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High overexpression of dye decolorizing peroxidase TfuDyP leads to the incorporation of heme precursor protoporphyrin IX

Cited by 10 publications

References 31 publications

Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

Redesign of a New Manganese Peroxidase Highly Expressed in Pichia pastoris towards a Lignin‐Degrading Versatile Peroxidase

Creating Oxidase–Peroxidase Fusion Enzymes as a Toolbox for Cascade Reactions

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