Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
New Mass Spectrometric Methods for Structure Analysis of Carboxymethyl Cellulose
KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Carboxymethyl cellulose (CMC) is a cellulose derivative, made from cellulose by introduction of carboxymethyl groups along the molecules. The properties of CMC are affected by several parameters where the molar mass and molar mass distribution, degree of substitution (DS) and the distribution pattern along, and between, the cellulose molecules are the most prominent.

In the present thesis we have investigated the use of various mass spectrometric techniques, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization MS (ESI-MS), for characterization of the chemical structure of CMC. We here present a refined sample preparation procedure for MALDI that greatly facilitates the spectrum acquisition and improves the quality of the mass spectrum when analyzing partially hydrolyzed CMC. The refined method allows for estimation of the DS in CMC samples as a complement to more expensive and laborious methods. We also show that by combining MALDI-TOFMS with selective hydrolysis, by purified endoglucanase, and size-exclusion chromatography (SEC) it is possible to differentiate between CMCs of similar monomer composition but differences in the substitution pattern. Our findings show that the CMC containing longer non-substituted cellulose segments along the polymer backbone also displayed the highest intermolecular interactions in solution, as measured by rheometry.

In order to take full advantage of the selective hydrolysis, provided by purified endoglucanases, more information regarding their selectivity on CMC must be investigated. We therefore present methods where chemical sample preparation of the oligomeric enzyme products is performed in order to facilitate the chemical structure characterization by ESI-MS/MS and liquid chromatography MS (LC-MS). These methods provides valuable information regarding the substituent distribution along the oligomeric enzyme products, and thereby gives indications regarding the selectivity of the endoglucanases on CMC substrates. From our finding we are able to present interesting information regarding the selectivity of four endoglucanases (Cel7B, Cel5A, Cel45A and Cel74A) from the filamentous fungus Trichoderma reesei and provide plausible explanations to their apparent differences in selectivity on CMC.

Abstract [sv]

Karboximetylcellulosa (CMC) är ett kommersiellt viktigt cellulosaderivat som bl.a. används som förtjockningsmedel och bindemedel i livsmedel och läkemedelsformuleringar. CMC framställs från cellulosa genom kemisk modifiering där karboximetyl grupper introduceras längs cellulosamolekylerna. Modifieringen ger materialet nya egenskaper, bl. a.  vattenlöslighet, vilket öppnar för nya användningsområden.  Egenskaperna hos CMC påverkas bl. a. av parametrar som molekylvikt och molekylviktsfördelning, substitutionsgraden (DS) och fördelning av substituenter över, och mellan, cellulosakedjorna.

I den presenterade avhandlingen så undersöker vi möjligheterna av att använda olika mass spektrometriska metoder, så som matris-assisterad laser-desorption/joniserings löptids-masspektrometri (MALDI-TOFMS) och elektrospray-jonisering masspektrometri (ESI-MS) för karaktärisering av den kemiska strukturen i CMC. Vi presenterar en förbättrad provprepareringsmetod för MALDI som både förenklar insamlingen av, och förbättrar kvaliteten på, masspektra från delvis hydrolyserad CMC. Den förbättrade metoden möjliggör även en estimering av DS-värdet i de intakta CMC proverna, vilket innebär att denna metod är ett komplement till dyrare och mer arbetskrävande tekniker. Vi visar också att MALDI-TOFMS, i kombination med enzymatisk nedbrytning, och andra sofistikerade analysmetoder (t ex storleksuteslutningskromatografi (SEC)), kan användas för att särskilja CMC-prover med liknande monomersammansättning men med olika substitutionsmönster. Resultaten från studien visar att CMC-prov innehållande längre osubstituerade cellulosasegment längs molekylerna, påvisar högre grad av intermolekylära interaktioner i lösning, mätt med reometri. 

För att kunna utnyttja den selektiva hydrolysen från enzymatisk nedbrytning på bästa sätt så bör selektiviteten på de nyttjade enzymerna undersökas. Vi presenterar därför metoder där de bildade oligomererna från den enzymatiska nedbrytningen genomgår en kemisk provpreparering, för att förenkla efterföljande analyser med ESI-MS/MS och vätskekromatografi MS (LC-MS). Med dessa metoder kan man undersöka substitutions positionerna på oligomererna och därigenom få indikationer på enzymets selektivitet mot CMC. Från våra resultat så kan vi presentera intressant information gällande fyra olika endoglukanaser (Cel7B, Cel5A, Cel45A and Cel74A)  från den fintrådiga svampen Trichoderma reesei och ge möjliga förklaringar till deras påvisade skillnader i selektivitet mot CMC.

Place, publisher, year, edition, pages
Stockholm: KTH , 2008. , 60 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2008:68
Keyword [en]
carboxymethyl cellulose, substituent distribution, rheology, enzyme hydrolysis, cellulase, permethylation, liquid chromatography, MALDI, ESI, mass spectrometry
National Category
Polymer Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-9438ISBN: 978-91-7415-162-6 (print)OAI: oai:DiVA.org:kth-9438DiVA: diva2:113970
Public defence
2008-11-28, F3, Lindstedtsvägen 26, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100720Available from: 2008-11-06 Created: 2008-11-03 Last updated: 2010-07-20Bibliographically approved
List of papers
1. Improved matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of carboxymethyl cellulose
Open this publication in new window or tab >>Improved matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of carboxymethyl cellulose
2006 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 20, no 24, 3693-3698 p.Article in journal (Refereed) Published
Abstract [en]

A refined sample preparation procedure for matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) was developed for the evaluation of the degree of substitution (DS) in partially depolymerised carboxymethyl cellulose (CMC). By adding ammonium sulphate to the sample mixture prior to the analysis, good quality mass spectra could be acquired. The usual time-consuming search for 'sweet-spots' at the crystalline rim of the MALDI target spot was also avoided. This quality improvement made it possible to investigate whether various positions on the target spot generated mass spectra in which the measured DS varied. The accuracy and reproducibility of the sample preparation procedure were tested by applying it on three commercial CMCs. The study shows that the DS values that were calculated from the spectra acquired from the centre region of the MALDI target spot were in better agreement with the DS provided by the supplier than were the values obtained from the large crystals at the target spot rim. This observation could be one reasonable explanation for the higher DS values reported in other publications. By applying our refined MALDI sample preparation procedure DS values that were in good agreement with the values provided by the manufacturer could be obtained. This indicates that MALDI-TOFMS of partially depolyrnerised CMCs can be used for an estimation of the DS as a complement to the more established methods, e.g. NMR, titrimetry, and chromatographic techniques.

Keyword
desorption, oligonucleotides, chromatography, substituents, ethers, acid
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-7104 (URN)10.1002/rcm.2786 (DOI)000242936400011 ()2-s2.0-33845565989 (Scopus ID)
Note
QC 20100720Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2017-12-14Bibliographically approved
2. A New Approach for Studying Correlations between the Chemical Structure and the Rheological Properties in Carboxymethyl Cellulose
Open this publication in new window or tab >>A New Approach for Studying Correlations between the Chemical Structure and the Rheological Properties in Carboxymethyl Cellulose
2007 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 8, no 10, 3253-3257 p.Article in journal (Refereed) Published
Abstract [en]

Two model sodium carboxymethyl celluloses (CMC) with similar monomer composition but with significant differences in the viscoelastic properties, that could not be assigned to variations in the average molar mass or molar mass distribution, were investigated with respect to the fraction of nonsubstituted cellulose segments in the polymers. The CMCs were hydrolyzed by a purified highly selective endoglucanase. The average molar mass and molar mass distribution of the enzyme products, as measured by size-exclusion chromatography with online multi-angle light scattering and refractive index detection (SEC/MALS/RI), revealed that the enzyme-catalyzed hydrolysis was more effective on one of the CMCs. To investigate whether this was due to a higher fraction of nonsubstituted cellulose segments in the polymer, the concentrations of nonsubstituted enzyme products, e.g., cellotetraose and cellopentaose, were measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was concluded that the two CMCs displayed significant differences in the fraction of nonsubstituted cellulose segments. Furthermore, the CMC with the strongest attractive intermolecular interactions, according to rheometry, also contained the highest fraction of nonsubstituted cellulose segments.

Keyword
carboxymethyl cellulose, mass spectrometry, MALDI, substituent distribution, rheology, enzymatic hydrolysis
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-9433 (URN)10.1021/bm700547a (DOI)000250009900036 ()2-s2.0-35549002681 (Scopus ID)
Note
QC 20100720Available from: 2008-11-03 Created: 2008-11-03 Last updated: 2017-12-14Bibliographically approved
3. Investigation of endoglucanase selectivity on carboxymethyl cellulose by mass spectrometric techniques
Open this publication in new window or tab >>Investigation of endoglucanase selectivity on carboxymethyl cellulose by mass spectrometric techniques
2009 (English)In: Cellulose (London), ISSN 0969-0239, E-ISSN 1572-882X, Vol. 16, no 2, 271-280 p.Article in journal (Refereed) Published
Abstract [en]

The benefits of applying cellulose selective enzymes as analytical tools for chemical structure characterization of cellulose derivatives have been frequently addressed over the years. In a recent study the high selectivity of cellulase Cel45A from Trichoderma reesei (Tr Cel45A) was utilized for relating the chemical structure to the flow properties of carboxymethyl cellulose (CMC). However, in order to take full advantage of the enzymatic hydrolysis the enzyme selectivity on the cellulose substrate must be further investigated. Therefore, the selectivity of Tr Cel45A on CMC was studied by chemical sample preparation of the enzyme products followed by mass spectrometric chemical structure characterization. The results strongly suggest that, in accordance with recent studies, also this highly selective endoglucanase is able to catalyze hydrolysis of glucosidic bonds adjacent to mono-substituted anhydroglucose units (AGUs). Furthermore, the results also indicate that substituents on the nearby AGUs will affect the hydrolysis.

Keyword
carboxymethyl cellulose, selective hydrolysis, cellulase, permethylation, MALDI, ESI, mass spectrometry
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-9435 (URN)10.1007/s10570-008-9254-0 (DOI)000263352900012 ()2-s2.0-60549106198 (Scopus ID)
Note
QC 20100720. Uppdaterad från in press till published (20100720).Available from: 2008-11-03 Created: 2008-11-03 Last updated: 2017-12-14Bibliographically approved
4. Liquid Chromatography combined with Mass Spectrometry for the Investigation of Endoglucanase Selectivity on Carboxymethyl Cellulose
Open this publication in new window or tab >>Liquid Chromatography combined with Mass Spectrometry for the Investigation of Endoglucanase Selectivity on Carboxymethyl Cellulose
2009 (English)In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 344, no 16, 2173-2181 p.Article in journal (Refereed) Published
Abstract [en]

Endoglucanases are useful tools in the chemical structure analysis of cellulose derivatives. However, knowledge on the endoglucanase selectivity, which is of central importance for data interpretation, is still limited. In this study, new reverse-phase liquid chromatography mass spectrometry (LC-MS) methods were developed to investigate the selectivity of the endoglucanases Cel5A, cel7B, Cel45A, and Cel74A from the filamentous fungus Trichoderma reesei. The aim was to improve the identification of the regioisomers in the complex mixtures that are obtained after enzymatic hydrolysis. Reduction followed by per-O-methylation was performed in order to improve the separation in reverse-phase LC, increase MS sensitivity, and to facilitate structure analysis by MS/MS of O-carboxymethyl glucose and cellooligosaccharides. The cellulose selective enzymes that were investigated displayed interesting differences in enzyme selectivity on CMC substrates.

Keyword
carboxymethyl cellulose, enzyme hydrolysis, cellulase, permethylation, liquid chromatography, mass spectrometry
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-9436 (URN)10.1016/j.carres.2009.08.009 (DOI)000271254800009 ()2-s2.0-70349776203 (Scopus ID)
Note
QC 20100720. Uppdaterad från manuskript till artikel (20100720).Available from: 2008-11-03 Created: 2008-11-03 Last updated: 2017-12-14Bibliographically approved

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Enebro, Jonas
By organisation
Fibre and Polymer Technology
Polymer Chemistry

Search outside of DiVA

GoogleGoogle Scholar

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 5202 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf