233 research outputs found
Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger
Dolichol is not a necessary moiety for lipid-linked oligosaccharide substrates of the mannosyltransferases involved in in vitro N-linked-oligosaccharide assembly
Enzymatic synthesis of N-acetyllactosamine from lactose enabled by recombinant β1,4-galactosyltransferases
Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial β1,4-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-β-LacNAc from lactose using β1,4-galactosyltransferase NmLgtB-B as the only biocatalyst
A novel mono-branched lipid phosphate acts as a substrate for dolichyl phosphate mannose synthetase
Biocatalytic Transfer of Pseudaminic Acid (Pse5Ac7Ac) Using Promiscuous Sialyltransferases in a Chemoenzymatic Approach to Pse5Ac7Ac-Containing Glycosides
Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni, and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host–pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein, we describe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors, enabling the enzymatic synthesis of Pse5Ac7Ac-containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late-stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot two-enzyme process using biosynthetic enzymes. Finally, screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac-terminated glycosides.</p
Development of Continuous Flow Systems to Access Secondary Amines Through Previously Incompatible Biocatalytic Cascades**
A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof-of-principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed-bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross-reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O-methylnorbelladine
Lipase-catalysed acylation of starch and determination of the degree of substitution by methanolysis and GC
Background: Natural polysaccharides such as starch are becoming increasingly interesting as renewable starting materials for the synthesis of biodegradable polymers using chemical or enzymatic methods. Given the complexity of polysaccharides, the analysis of reaction products is challenging. Results: Esterification of starch with fatty acids has traditionally been monitored by saponification and back-titration, but in our experience this method is unreliable. Here we report a novel GC-based method for the fast and reliable quantitative determination of esterification. The method was used to monitor the enzymatic esterification of different starches with decanoic acid, using lipase from Thermomyces lanuginosus. The reaction showed a pronounced optimal water content of 1.25 mL per g starch, where a degree of substitution (DS) of 0.018 was obtained. Incomplete gelatinization probably accounts for lower conversion with less water. Conclusions: Lipase-catalysed esterification of starch is feasible in aqueous gel systems, but attention to analytical methods is important to obtain correct DS values
Non-secreting pituitary tumours characterised by enhanced expression of YAP/TAZ
Tumours of the anterior pituitary can manifest from all endocrine cell types but the mechanisms for determining their specification are not known. The Hippo kinase cascade is a crucial signalling pathway regulating growth and cell fate in numerous organs. There is mounting evidence implicating this in tumour formation, where it is emerging as an anti-cancer target. We previously demonstrated activity of the Hippo kinase cascade in the mouse pituitary and nuclear association of its effectors YAP/TAZ with SOX2-expressing pituitary stem cells. Here we sought to investigate whether these components are expressed in the human pituitary and if they are deregulated in human pituitary tumours. Analysis of pathway components by immunofluorescence reveals pathway activity during normal human pituitary development and in the adult gland. Poorly differentiated pituitary tumours (null cell adenomas, adamantinomatous craniopharyngiomas (ΑCPs) and papillary craniopharyngiomas (PCPs)), displayed enhanced expression of pathway effectors YAP/TAZ. In contrast, differentiated adenomas displayed lower or absent levels. Knock-down of the kinase-encoding Lats1 in GH3 rat mammosomatotropinoma cells suppressed Prl and Gh promoter activity following an increase in YAP/TAZ levels. In conclusion, we have demonstrated activity of the Hippo kinase cascade in the human pituitary and association of high YAP/TAZ with repression of the differentiated state both in vitro and in vivo. Characterisation of this pathway in pituitary tumours is of potential prognostic value, opening up putative avenues for treatments
Quasi-continuous parallel online scattered light, fluorescence and dissolved oxygen tension measurement combined with monitoring of the oxygen transfer rate in each well of a shaken microtiter plate
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