scholarly journals Tandem mass spectrometric analysis of Aspergillus niger pectin methylesterase: mode of action on fully methyl-esterified oligogalacturonates

2000 ◽  
Vol 346 (2) ◽  
pp. 469-474 ◽  
Author(s):  
Harry C. M. KESTER ◽  
Jacques A. E. BENEN ◽  
Jaap VISSER ◽  
Maria Esteban WARREN ◽  
Ron ORLANDO ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Mode Of Action ◽  
High Performance ◽  
Methyl Esters ◽  
Pectin Methylesterase ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Spectrometric Analysis ◽  
Reaction Products ◽  
Tandem Mass Spectrometric

The substrate specificity and the mode of action of Aspergillus niger pectin methylesterase (PME) was determined using both fully methyl-esterified oligogalacturonates with degrees of polymerization (DP) 2-6 and chemically synthesized monomethyl trigalacturonates. The enzymic activity on the different substrates and a preliminary characterization of the reaction products were performed by using high-performance anion-exchange chromatography at neutral pH. Electrospray ionization tandem MS (ESI-MS/MS) was used to localize the methyl esters on the 18O-labelled reaction products during the course of the enzymic reaction. A. niger PME is able to hydrolyse the methyl esters of fully methyl-esterified oligogalacturonates with DP 2, and preferentially hydrolyses the methyl esters located on the internal galacturonate residues, followed by hydrolysis of the methyl esters towards the reducing end. This PME is unable to hydrolyse the methyl ester of the galacturonate moiety at the non-reducing end.

scholarly journals Autocatalytic Fractionation of Wood Hemicelluloses: Modeling of Multistage Operation

Catalysts ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 337
Author(s):  
Mar López ◽  
Valentín Santos ◽  
Juan Carlos Parajó
Keyword(s):  
Liquid Phase ◽  
High Performance ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Optimal Recovery ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Reaction Products ◽  
Flight Mass Spectrometry ◽  
Kinetic Coefficients

Eucalyptus globulus wood samples were treated with hot, compressed water (autohydrolysis) in consecutive stages under non-isothermal conditions in order to convert the hemicellulose fraction into soluble compounds through reactions catalyzed by in situ generated acids. The first stage was a conventional autohydrolysis, and liquid phase obtained under conditions leading to an optimal recovery of soluble saccharides was employed in a new reaction (second crossflow stage) using a fresh wood lot, in order to increase the concentrations of soluble saccharides. In the third crossflow stage, the best liquid phase from the second stage was employed to solubilize the hemicelluloses from a fresh wood lot. The concentration profiles determined for the soluble saccharides, acids, and furans present in the liquid phases from the diverse crossflow stages were employed for kinetic modeling, based on pseudohomogeneous reactions and Arrhenius-type dependence of the kinetic coefficients on temperature. Additional characterization of the reaction products by High Pressure Size Exclusion Chromatography, High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection, and Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry provided further insight on the properties of the soluble saccharides present in the various reaction media.

scholarly journals Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli

2002 ◽  
Vol 184 (2) ◽  
pp. 363-369 ◽  
Author(s):  
Bernd Kneidinger ◽  
Cristina Marolda ◽  
Michael Graninger ◽  
Alla Zamyatina ◽  
Fiona McArthur ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Anion Exchange ◽  
High Performance ◽  
Inner Core ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Biosynthesis Pathway ◽  
Reaction Products ◽  
Novel Gene

ABSTRACT The steps involved in the biosynthesis of the ADP-l-glycero-β-d-manno-heptose (ADP-l-β-d-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-d-β-d-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-d-β-d-heptose. Our results demonstrate that the synthesis of ADP-d-β-d-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional d-β-d-heptose 7-phosphate kinase/d-β-d-heptose 1-phosphate adenylyltransferase), and GmhB (d,d-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-l-β-d-heptose precursor utilized in the assembly of inner core LPS.

scholarly journals Partially esterified oligogalacturonides are the preferred substrates for pectin methylesterase of Aspergillus niger

2003 ◽  
Vol 372 (1) ◽  
pp. 211-218 ◽  
Author(s):  
Gert-Jan W. M. van ALEBEEK ◽  
Katrien van SCHERPENZEEL ◽  
Gerrit BELDMAN ◽  
Henk A. SCHOLS ◽  
Alphons G. J. VORAGEN
Keyword(s):  
Methyl Ester ◽  
Aspergillus Niger ◽  
Methyl Esters ◽  
Pectin Methylesterase ◽  
Degree Of Polymerization ◽  
Specific Pattern ◽  
Specific Product ◽  
Ionization Time ◽  
Active Site Cleft

Investigations on the mode of action of Aspergillus niger pectin methylesterase (PME) towards differently C6- and C1-substituted oligogalacturonides (oligoGalpA) are described. De-esterification of methyl-esterified (un)saturated oligoGalpA proceeds via a specific pattern, depending on the degree of polymerization. Initially, a first methyl ester of the oligomer is hydrolysed, resulting in one free carboxyl group. Subsequently, this first product is preferred as a substrate and is de-esterified for a second time. This product is then accumulated and hereafter de-esterified further to the final product, i.e. oligoGalpA containing one methyl ester located at the non-reducing end residue for both saturated and unsaturated oligoGalpA, as found by post-source decay matrix-assisted laser-desorption/ionization–time-of-flight MS. The saturated hexamer is an exception to this: three methyl esters are removed very rapidly, instead of two methyl esters. When unsaturated oligoGalpA were used, the formation of the end product differed slightly, suggesting that the unsaturated bond at the non-reducing end influences the de-esterification process. In vivo, PME prefers methyl esters, but the enzyme appeared to be tolerant for other C6- and C1-substituents. Changing the type of ester (ethyl esterification) or addition of a methyl glycoside (C1) only reduced the activity or had no effect respectively. The specific product pattern was identical for all methyl- and ethyl-esterified oligoGalpA and methyl-glycosidated oligoGalpA, which strongly indicates that one or perhaps two non-esterified oligoGalpA are preferred in the active-site cleft.

scholarly journals Isolation of Reaction Products Resulting from Heat-Induced Degradation of Inulin

2009 ◽  
Vol 27 (Special Issue 1) ◽  
pp. S166-S168 ◽  
Author(s):  
K. Trabs ◽  
N. Kasprick ◽  
T. Henle
Keyword(s):  
High Performance ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Flash Chromatography ◽  
Bakery Products ◽  
Reaction Products ◽  
Pulsed Amperometric Detection ◽  
Dfa Iii ◽  
Final Purification

From inulin which had been heated for 30 min at 200°C, four di-D-fructose dianhydrides (DFDAs) were isolated using flash chromatography and final purification by semipreparative HPLC, followed by identification via NMR spectroscopy. The DFDAs &alpha;-D-Fru<I>f</I>-1,2':2,3'-β-D-Fruf (DFA III), &alpha;-D-Fru<I>f</I>-1,2':2,1'-β-D-Fru<I>f</I> (DFA I), &alpha;-D-Fru<I>f</I>-1,2':2,1'-&alpha;-D-Fru<I>f</I> (DFA VII) and β-D-Fru<I>f</I>-1,2´:2,1´-β-D-Fru<I>f</I> were identified. The yield of the isolated DFDAs varied depending on the DP of the used inulin. Using the isolated DFDAs as reference compounds, quantification of the disaccharides in commercial bakery products via high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was possible.

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