Pyrroloquinoline quinone (PQQ) is an
-quinone cofactor of several prokaryotic oxidases. Widely available in the diet and necessary for the correct growth of mice, PQQ has been suspected to be a ...vitamin for eukaryotes. However, no PQQ-dependent eukaryotic enzyme had been identified to use the PQQ until 2014, when a basidiomycete enzyme catalyzing saccharide dehydrogenation using PQQ as a cofactor was characterized and served to define auxiliary activity family 12 (AA12). Here we report the biochemical characterization of the AA12 enzyme encoded by the genome of the ascomycete
(
AA12). Surprisingly, only weak activity against uncommon carbohydrates like l-fucose or d-arabinose was measured. The three-dimensional structure of
AA12 reveals important similarities with bacterial soluble glucose dehydrogenases (sGDH). The enzymatic characterization and the structure solved in the presence of calcium confirm the importance of this ion in catalysis, as observed for sGDH. The structural characterization of
AA12 was completed by modeling PQQ and l-fucose in the enzyme active site. Based on these results, the AA12 family of enzymes is likely to have a catalytic mechanism close to that of bacterial sGDH.
Pyrroloquinoline quinone (PQQ) is an important cofactor synthesized by prokaryotes and involved in enzymatic alcohol and sugar oxidation. In eukaryotes, the benefit of PQQ as a vitamin has been suggested but never proved. Recently, the first eukaryotic enzyme using PQQ was characterized in the basidiomycete
, demonstrating that fungi are able to use PQQ as an enzyme cofactor. This discovery led to the classification of the fungal PQQ-dependent enzymes in auxiliary activity family 12 (AA12) of the Carbohydrate-Active Enzymes (CAZy) database (www.cazy.org) classification. In the present paper, we report on the characterization of the ascomycete AA12 enzyme from
(
AA12). Our enzymatic and phylogenetic results show divergence with the only other member of the family characterized, that from the basidiomycete
The crystallographic structure of
AA12 shows similarities to the global active-site architecture of bacterial glucose dehydrogenases, suggesting a common evolution between the two families.
Two laccase-encoding genes from the marine-derived fungus
sp. have been cloned in
for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical ...properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the presence of sea salt. The optimal pH and temperature of
Lac1 and
Lac2 differed in relation to the substrates tested, and both enzymes were shown to be extremely stable at temperatures up to 50 °C, retaining 100% activity after 3 h at 50 °C. Both enzymes were stable between pH 4-6. Different substrate specificities were exhibited, and the lowest
and highest catalytic efficiency values were obtained against syringaldazine and 2,6-dimethoxyphenol (DMP) for
Lac1 and
Lac2, respectively. The industrially important dyes-Acid Yellow, Bromo Cresol Purple, Nitrosulfonazo III, and Reactive Black 5-were more efficiently decolorized by
Lac1 in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). Activities were compared in saline conditions, and
Lac2 seemed more adapted to the presence of sea salt than
Lac1. The overall surface charges of the predicted
Lac three-dimensional models showed large negatively charged surfaces for
Lac2, as found in proteins for marine organisms, and more balanced solvent exposed charges for
Lac1, as seen in proteins from terrestrial organisms.
Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the ...use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization.
Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum, Stemphylium lucomagnoense and Aspergillus nidulans. Among these five isolates, one T. asperellum strain (T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator.
The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO
as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed ...to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H
O
-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS
fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.
Cellobiose dehydrogenases (CDHs) are extracellular glycosylated haemoflavoenzymes produced by many different wood-degrading and phytopathogenic fungi. Putative cellobiose dehydrogenase genes are ...recurrently discovered by genome sequencing projects in various phylogenetically distinct fungi. The genomes from the basidiomycete
Coprinopsis cinerea
and the ascomycete
Podospora anserina
were screened for candidate
cdh
genes, and one and three putative gene models were evidenced, respectively. Two putative
cdh
genes were selected and successfully expressed for the first time in
Aspergillus niger
. CDH activity was measured for both constructions (CDH
cc
and CDH
pa
), and both recombinant CDHs were purified to homogeneity and subsequently characterised. Kinetic constants were determined for several carbohydrates including β-1,4-linked di- and oligosaccharides. Optimal temperature and pH were 60 °C and 5 for CDH
cc
and 65–70 °C and 6 for CDH
pa
. Both CDHs showed a broad range of pH stability between 4 and 8. The effect of both CDHs on saccharification of micronized wheat straw by an industrial
Trichoderma reesei
secretome was determined. The addition of each CDH systematically decreased the release of total reducing sugars, but to different extents and according to the CDH concentration. Analytical methods were carried out to quantify the release of glucose, xylose and gluconic acid. An increase of glucose and xylose was measured at a low CDH
cc
concentration. At moderated and high CDH
cc
and CDH
pa
concentrations, glucose was severely reduced with a concomitant increase of gluconic acid. In conclusion, these results give new insights into the physical and chemical parameters and diversity of basidiomycetous and ascomycetous CDHs. These findings also demonstrated that CDH drastically influenced the saccharification on a natural substrate, and thus, CDH origin, concentration and potential enzymatic partners should be carefully considered in future artificial secretomes for biofuel applications.
Celotno besedilo
Dostopno za:
CEKLJ, DOBA, EMUNI, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK
Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative ...enzyme identified from the transcriptome of marine-derived fungus
. The laccase-encoding gene
Lac2 from
was cloned for heterologous expression in
D15#26 for protein production in the extracellular medium of around 30 mg L
. The extracellular recombinant enzyme
Lac2 was successfully produced and purified in three steps protocol: ultrafiltration, anion-exchange chromatography, and size exclusion chromatography, with a final recovery yield of 24%.
Lac2 was characterised by physicochemical properties, kinetic parameters, and ability to oxidise diverse phenolic substrates. We also studied its activity in the presence and absence of sea salt. The molecular mass of
Lac2 was about 75 kDa, consistent with that of most ascomycete fungal laccases. With syringaldazine as substrate,
Lac2 showed an optimal activity at pH 6 and retained nearly 100% of its activity when incubated at 50°C for 180 min.
Lac2 exhibited more than 50% of its activity with 5% wt/vol of sea salt.
Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene–gene interactions. ...Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein–protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller “incisor field” forms in Pax9+/−;Msx1+/− mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia.
The wastewater from hospitals, pharmaceutical industries and more generally human and animal dejections leads to environmental releases of antibiotics that cause severe problems for all living ...organisms. The aim of this study was to investigate the capacity of three fungal strains to biotransform the fluoroquinolone levofloxacin. The degradation processes were analyzed in solid and liquid media. Among the three fungal strains tested, Coriolopsis gallica strain CLBE55 (BRFM 3473) showed the highest removal efficiency, with a 15% decrease in antibiogram zone of inhibition for Escherichia coli cultured in solid medium and 25% degradation of the antibiotic in liquid medium based on high-performance liquid chromatography (HPLC). Proteomic analysis suggested that laccases and dye-decolorizing peroxidases such as extracellular enzymes could be involved in levofloxacin degradation, with a putative major role for laccases. Degradation products were proposed based on mass spectrometry analysis, and annotation suggested that the main product of biotransformation of levofloxacin by Coriolopsis gallica is an N-oxidized derivative.
Many vertebrate organs form through the sequential and reciprocal exchange of signaling molecules between juxtaposed epithelial and mesenchymal tissues. We undertook a systems biology approach that ...combined the generation and analysis of large-scale spatiotemporal gene expression data with mouse genetic experiments to gain insight into the mechanisms that control epithelial-mesenchymal signaling interactions in the developing mouse molar tooth. We showed that the shift in instructive signaling potential from dental epithelium to dental mesenchyme was accompanied by temporally coordinated genome-wide changes in gene expression in both compartments. To identify the mechanism responsible, we developed a probabilistic technique that integrates regulatory evidence from gene expression data and from the literature to reconstruct a gene regulatory network for the epithelial and mesenchymal compartments in early tooth development. By integrating these epithelial and mesenchymal gene regulatory networks through the action of diffusible extracellular signaling molecules, we identified a key epithelial-mesenchymal intertissue Wnt-Bmp (bone morphogenetic protein) feedback circuit. We then validated this circuit in vivo with compound genetic mutations in mice that disrupted this circuit. Moreover, mathematical modeling demonstrated that the structure of the circuit accounted for the observed reciprocal signaling dynamics. Thus, we have identified a critical signaling circuit that controls the coordinated genome-wide expression changes and reciprocal signaling molecule dynamics that occur in interacting epithelial and mesenchymal compartments during organogenesis.
The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical ...characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments, collected beneath either
or
trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the
area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51-100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in
. The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical-chemical properties, its ability to oxidize diverse phenolic substrates, and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 °C, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi.
PDF
