RESUMO
ß-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 ß-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied ß-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc ß-1,4 and ß-1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-ß-Gal-1,4-ß-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: kcat/KM being 341 and 344 mM-1 s-1, respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide ß-Gal-1,4-ß-Glc-1,1-ß-GlcNAc, and in low amounts the ß-1,2- or ß-1,3-linked trisaccharide ß-Gal-1,4(ß-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 ß-N-acetylhexosaminidases.
Assuntos
Alteromonadaceae/enzimologia , Organismos Aquáticos/enzimologia , beta-N-Acetil-Hexosaminidases/biossíntese , Alteromonadaceae/genética , Organismos Aquáticos/genética , Biocatálise/efeitos dos fármacos , Estabilidade Enzimática , Genoma Bacteriano , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Octoxinol/farmacologia , Filogenia , Domínios Proteicos , Soroalbumina Bovina/farmacologia , Cloreto de Sódio/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Temperatura , Fatores de Tempo , beta-N-Acetil-Hexosaminidases/químicaRESUMO
Blooms of the microalga Prymnesium parvum cause devastating fish kills worldwide, which are suspected to be caused by the supersized ladder-frame polyether toxins prymnesin-1 and -2. These toxins have, however, only been detected from P. parvum in rare cases since they were originally described two decades ago. Here, we report the isolation and characterization of a novel B-type prymnesin, based on extensive analysis of 2D- and 3D-NMR data of natural as well as 90% (13)C enriched material. B-type prymnesins lack a complete 1,6-dioxadecalin core unit, which is replaced by a short acyclic C2 linkage compared to the structure of the original prymnesins. Comparison of the bioactivity of prymnesin-2 with prymnesin-B1 in an RTgill-W1 cell line assay identified both compounds as toxic in the low nanomolar range. Chemical investigations by liquid chromatography high-resolution mass spectrometry (LC-HRMS) of 10 strains of P. parvum collected worldwide showed that only one strain produced the original prymnesin-1 and -2, whereas four strains produced the novel B-type prymnesin. In total 13 further prymnesin analogues differing in their core backbone and chlorination and glycosylation patterns could be tentatively detected by LC-MS/HRMS, including a likely C-type prymnesin in five strains. Altogether, our work indicates that evolution of prymnesins has yielded a diverse family of fish-killing toxins that occurs around the globe and has significant ecological and economic impact.
Assuntos
Glicosídeos/química , Glicosídeos/isolamento & purificação , Haptófitas/química , Lipoproteínas/química , Lipoproteínas/isolamento & purificação , Polímeros/química , Polímeros/farmacologia , Piranos/química , Piranos/isolamento & purificação , Animais , Cromatografia Líquida , Relação Dose-Resposta a Droga , Lipoproteínas/metabolismo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , PolicetídeosRESUMO
Aggregation of reduced graphene oxide (RGO) due to π-π stacking is a recurrent problem in graphene-based electrochemistry, decreasing the effective working area and therefore the performance of the RGO electrodes. Dispersing RGO on three-dimensional (3D) carbon paper electrodes is one strategy towards overcoming this challenge, with partial relief aggregation. In this report, we describe the grafting of negatively charged 4-aminobenzoic acid (4-ABA) onto a graphene functionalized carbon paper electrode surface. 4-ABA functionalization induces separation of the RGO layers, at the same time leading to favorable orientation of the blue multi-copper enzyme Myrothecium verrucaria bilirubin oxidase (MvBOD) for direct electron transfer (DET) in the dioxygen reduction reaction (ORR) at neutral pH. Simultaneous electroreduction of graphene oxide to RGO and covalent attachment of 4-ABA are achieved by applying alternating cathodic and anodic electrochemical potential pulses, leading to a high catalytic current density (Δjcat:193 ± 4 µA cm-2) under static conditions. Electrochemically grafted 4-ABA not only leads to a favorable orientation of BOD as validated by fitting a kinetic model to the electrocatalytic data, but also acts to alleviate RGO aggregation as disclosed by scanning electron microscopy, most likely due to the electrostatic repulsion between 4-ABA-grafted graphene layers. With a half-lifetime of 55 h, the bioelectrode also shows the highest operational stability for DET-type MvBOD-based bioelectrodes reported to date. The bioelectrode was finally shown to work well as a biocathode of a membrane-less glucose/O2 enzymatic biofuel cell with a maximum power density of 22 µW cm-2 and an open circuit voltage of 0.51 V.
Assuntos
Técnicas Biossensoriais , Grafite , Eletrodos , Enzimas Imobilizadas , Hypocreales , Oxirredutases atuantes sobre Doadores de Grupo CH-CHRESUMO
The aim of this paper was to perform a comprehensive characterization of polysaccharides isolated from the interior (IOI) and exterior (IOE) parts of the fungus Inonotus obliquus. Pre-extraction with DCM and MeOH, followed by water and alkali extraction and ethanol precipitation gave two water extracts and two alkali extracts. Neutral and acidic polysaccharide fractions were obtained after anion-exchange chromatography of the water extracts. The neutral polysaccharides (60-73â¯kDa) were heterogeneous and branched and consisted of a (1â¯ââ¯3)-linked ß-Glc backbone with (1â¯ââ¯6)-linked kinks in the chain at approximately every fifth residue, with branches of (1â¯ââ¯6)-linked ß-Glc in addition to substantial amounts of (1â¯ââ¯6)-linked α-Gal with 3-O-methylation at about every third Gal residue. The acidic polysaccharide fractions (10-31â¯kDa) showed similar structural motifs as the neutral fractions differing mainly by the presence of (1â¯ââ¯4)-linked α-GalA and α-GlcA. ß-Xyl, α-Man and α-Rha were also present in varying amounts in all fractions. No major structural differences between the IOI and IOE fractions were observed. An alkaline polysaccharide fraction (>450â¯kDa) was obtained from the IOI alkali extract, and consisted mainly of (1â¯ââ¯3)- and (1â¯ââ¯6)-linked ß-Glc and (1â¯ââ¯4)-linked ß-Xyl. Several of the fractions showed in vitro immunomodulatory effect by increasing NO production in the murine macrophage and dendritic cell lines J774.A1 and D2SC/1. Most fractions managed to increase NO production only at the highest concentration tested (100⯵g/ml), while the neutral fraction IOE-WN activated potent NO production at 10⯵g/ml and was considered the most promising immunomodulating fraction in this study.