DETERMINATION OF NEUTRAL LIPIDS AND PHOSPHOLIPIDS IN THE CERCARIAE OF SCHISTOSOMA MANSONI BY HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

Schariter, Joseph; Pachuski, Janna; Fried, Bernard; Sherma, Joseph

doi:10.1081/jlc-120005708

Cited by 16 publications

(23 citation statements)

References 5 publications

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“…Furthermore, the influence of water was investigated in the case of ethanol by comparing absolute ethanol and 96% ethanol comprising 4% water. HPTLC has been widely used for lipid profile analyses (14). Compared with normal TLC, HPTLC gives much better and clearer separations.…”

Section: Resultsmentioning

confidence: 99%

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Enzymatic production of alkyl esters through alcoholysis: A critical evaluation of lipases and alcohols

Deng

Haraldsson

et al. 2005

J Americ Oil Chem Soc

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This paper focuses on a detailed evaluation of commercially available immobilized lipases and simple monohydric alcohols for the production of alkyl esters from sunflower oil by enzymatic alcoholysis. Six lipases were tested with seven alcohols, including straight and branched-chain primary and secondary alcohols. The reactions were conducted in a batch stirred reaction vessel using stoichiometric amounts of substrates under solvent-free conditions. Dramatic differences in alcoholysis performance were observed among the different lipases. For most of the alcohols, Novozym 435 produced the highest yield of FA alkyl esters, with yields well over 90% for methanol, absolute ethanol, and 1-propanol. Overall, 96% ethanol was the preferred alcohol for all lipases except Novozym 435, and ethanolysis reactions reached the maximal conversion efficiency. Increasing the water content in the system resulted in an increased degree of conversion for all lipases except Novozym 435. The secondary alcohol 2-propanol significantly reduced the alcoholysis reaction with all lipases; however, the branch-chain isobutanol was more advantageous than linear 1-butanol for Novozym 435, Lipozyme RM IM, and Lipase PS-C. Many commercial immobilized lipases are highly efficient and promising for the production of alkyl esters, offering high reaction yields and a simple operation process.Paper no. J11001 in JAOCS 82, 341-347 (May 2005). FIG. 3. Time courses of propyl ester formation catalyzed by different lipases in propanolysis. Reaction conditions: 40°C, 10 wt% lipase dosage, addition of propanol in four steps. (A) SFO and 1-propanol (1:3 mol/mol); (B) SFO and 2-propanol (1:3 mol/mol). For abbreviations see Figures 1 and 2. FIG. 4. Time courses of butyl ester formation catalyzed by different lipases in butanolysis. Reaction conditions: 40°C, 10 wt% lipase dosage, addition of butanol in four steps. (A) SFO and 1-butanol (1:3 mol/mol); (B) SFO and isobutanol (1:3 mol/mol). For abbreviations see Figures 1 and 2.

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Section: Resultsmentioning

confidence: 99%

“…The progress of alcoholysis was monitored by an HPTLC method with modifications for the developing solvent system (14). The HPTLC system (Desaga GmbH, Wiesloch, Germany) consisted of an AS 30 TLC applicator, a Densitometer CD 60 scanner, and ProQuant Control and Evaluation software.…”

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confidence: 99%

Enzymatic production of alkyl esters through alcoholysis: A critical evaluation of lipases and alcohols

Deng

Haraldsson

et al. 2005

J Americ Oil Chem Soc

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“…Although TLC-based assays have the benefit of directly showing changes in the substrate and product of interest, absolute quantification requires concurrently run standard curves (42,43). To determine specific activities for more quantitative comparisons of LpxH variants, UMP concentration was quantified with a UMP/CMP-Glo glycosyltransferase assay kit (Promega) as described by the manufacturer.…”

Section: Activity Assaysmentioning

confidence: 99%

The substrate-binding cap of the UDP-diacylglucosamine pyrophosphatase LpxH is highly flexible, enabling facile substrate binding and product release

Bohl

Ieong

Lee

et al. 2018

Journal of Biological Chemistry

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Gram-negative bacteria are surrounded by a secondary membrane of which the outer leaflet is composed of the glycolipid lipopolysaccharide (LPS), which guards against hydrophobic toxins, including many antibiotics. Therefore, LPS synthesis in bacteria is an attractive target for antibiotic development. LpxH is a pyrophosphatase involved in LPS synthesis, and previous structures revealed that LpxH has a helical cap that binds its lipid substrates. Here, crystallography and hydrogen-deuterium exchange MS provided evidence for a highly flexible substrate-binding cap in LpxH. Furthermore, molecular dynamics simulations disclosed how the helices of the cap may open to allow substrate entry. The predicted opening mechanism was supported by activity assays of LpxH variants. Finally, we confirmed biochemically that LpxH is inhibited by a previously identified antibacterial compound, determined the potency of this inhibitor, and modeled its binding mode in the LpxH active site. In summary, our work provides evidence that the substrate-binding cap of LpxH is highly dynamic, thus allowing for facile substrate binding and product release between the capping helices. Our results also pave the way for the rational design of more potent LpxH inhibitors.

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“…In addition, the amount of the lipids present on each plate could be compared by analyzing band intensity in ImageJ 64 . TLC plate charring is an established methodology for lipid quantification though absolute quantification requires standard curves of the lipids on interest to be run on the same plate as the samples 65 , 66 . The positive correlation between lipid amount and band intensity was confirmed for this study by running different amounts of UDP-DAG and lipid X and by reacting different amount of these reactants to completion with wild-type LpxB (Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of lipid A disaccharide synthase LpxB from Escherichia coli

Bohl

Shi²,

Lee

et al. 2018

Nat Commun

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Most Gram-negative bacteria are surrounded by a glycolipid called lipopolysaccharide (LPS), which forms a barrier to hydrophobic toxins and, in pathogenic bacteria, is a virulence factor. During LPS biosynthesis, a membrane-associated glycosyltransferase (LpxB) forms a tetra-acylated disaccharide that is further acylated to form the membrane anchor moiety of LPS. Here we solve the structure of a soluble and catalytically competent LpxB by X-ray crystallography. The structure reveals that LpxB has a glycosyltransferase-B family fold but with a highly intertwined, C-terminally swapped dimer comprising four domains. We identify key catalytic residues with a product, UDP, bound in the active site, as well as clusters of hydrophobic residues that likely mediate productive membrane association or capture of lipidic substrates. These studies provide the basis for rational design of antibiotics targeting a crucial step in LPS biosynthesis.

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DETERMINATION OF NEUTRAL LIPIDS AND PHOSPHOLIPIDS IN THE CERCARIAE OF SCHISTOSOMA MANSONI BY HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

Cited by 16 publications

References 5 publications

Enzymatic production of alkyl esters through alcoholysis: A critical evaluation of lipases and alcohols

Enzymatic production of alkyl esters through alcoholysis: A critical evaluation of lipases and alcohols

The substrate-binding cap of the UDP-diacylglucosamine pyrophosphatase LpxH is highly flexible, enabling facile substrate binding and product release

Crystal structure of lipid A disaccharide synthase LpxB from Escherichia coli

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