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1.
Enzyme Microb Technol ; 164: 110170, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36521309

RESUMO

ß-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members via conserved unique peptide patterns and phylogeny, revealed the occurrence of distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative ß-D-galactofuranosidase from A. niger which was recombinantly produced in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-nitrophenyl-ß-galactofuranoside (pNP-ß-Galf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 µM min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures.


Assuntos
Aspergillus niger , Glicosídeo Hidrolases , Glicosídeo Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Polissacarídeos , Especificidade por Substrato
2.
Food Chem ; 405(Pt B): 134939, 2023 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-36403470

RESUMO

Ferulic acid has antioxidant properties of interest to the food industry and can be released from natural plant fibres using feruloyl esterases. Esterases active at high temperatures are highly desirable but currently underrepresented. Here we report the biochemical characterization of the feruloyl esterase from Thermobacillus xylanilyticus. Specific activity of recombinant Tx-Est1 with ethyl ferulate was 29.2 ± 2.9 U mg-1, with a catalytic efficiency (Kcat/Km) of 393.7 ± 9.8 s-1mM-1. The temperature and pH optima were 60 °C and 7.5, whereby Tx-Est1 retains 70% activity after 25 h at 40 °C. MALDI-TOF MS revealed Tx-ESTI released ferulic acid from xylooligosaccharides with DP4-DP13, and from DP6-8 containing two ferulic acid groups. HPLC demonstrated ferulic acid release from destarched wheat bran was strongly potentiated by co-incubation with xylanase. These properties, especially the high activity at elevated temperatures, suggest Tx-Est1 can be employed for production of high-value compounds from agricultural waste or during plant polysaccharide saccharification.


Assuntos
Hidrolases de Éster Carboxílico , Temperatura
3.
Carbohydr Res ; 508: 108411, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34392134

RESUMO

Bottom-up synthesis of ß-glucans such as callose, fungal ß-(1,3)(1,6)-glucan and cellulose, can create the defined compounds that are needed to perform fundamental studies on glucan properties and develop applications. With the importance of ß-glucans and cellulose in high-profile fields such as nutrition, renewables-based biotechnology and materials science, the enzymatic synthesis of such relevant carbohydrates and their derivatives has attracted much attention. Here we review recent developments in enzymatic synthesis of ß-glucans and cellulose, with a focus on progress made over the last five years. We cover the different types of biocatalysts employed, their incorporation in cascades, the exploitation of enzyme promiscuity and their engineering, and reaction conditions affecting the production as well as in situ self-assembly of (non)functionalised glucans. The recent achievements in the application of glycosyl transferases and ß-1,4- and ß-1,3-glucan phosphorylases demonstrate the high potential and versatility of these biocatalysts in glucan synthesis in both industrial and academic contexts.


Assuntos
Celulose , beta-Glucanas , Fosforilases
4.
Org Biomol Chem ; 19(25): 5529-5533, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34105582

RESUMO

Promiscuous activity of a glycosyltransferase was exploited to polymerise glucose from UDP-glucose via the generation of ß-1,4-glycosidic linkages. The biocatalyst was incorporated into biocatalytic cascades and chemo-enzymatic strategies to synthesise cello-oligosaccharides with tailored functionalities on a scale suitable for employment in mass spectrometry-based assays. The resulting glycan structures enabled reporting of the activity and selectivity of celluloltic enzymes.


Assuntos
Glicosiltransferases
5.
Org Biomol Chem ; 17(24): 5920-5924, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31165848

RESUMO

Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial ß1,4-galactosyltransferases to catalyse the transgalactosylation from lactose to N-acetylglucosamine to form N-acetyllactosamine in the presence of UDP. The process is demonstrated by the preparative scale synthesis of pNP-ß-LacNAc from lactose using ß1,4-galactosyltransferase NmLgtB-B as the only biocatalyst.


Assuntos
Amino Açúcares/biossíntese , Galactosiltransferases/metabolismo , Lactose/metabolismo , Amino Açúcares/química , Biocatálise , Galactosiltransferases/química , Lactose/química , Estrutura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
6.
PLoS Pathog ; 15(5): e1007730, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31048927

RESUMO

Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires a complex network of transcriptional regulators and several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.


Assuntos
Antígenos de Superfície/imunologia , Enterococcus faecalis/patogenicidade , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/microbiologia , Muramidase/imunologia , Polissacarídeos/imunologia , Virulência , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/imunologia , Infecções por Bactérias Gram-Positivas/metabolismo , Muramidase/metabolismo , Mutagênese , Mutação , Polissacarídeos/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologia
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