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Liquid Chromatography combined with Mass Spectrometry for the Investigation of Endoglucanase Selectivity on Carboxymethyl Cellulose
KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
VTT Technical Research Centre of Finland, Espoo.
KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.ORCID iD: 0000-0002-5394-7850
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.

Place, publisher, year, edition, pages
2009. Vol. 344, no 16, 2173-2181 p.
Keyword [en]
carboxymethyl cellulose, enzyme hydrolysis, cellulase, permethylation, liquid chromatography, mass spectrometry
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-9436DOI: 10.1016/j.carres.2009.08.009ISI: 000271254800009Scopus ID: 2-s2.0-70349776203OAI: oai:DiVA.org:kth-9436DiVA: diva2:113968
Note
QC 20100720. Uppdaterad från manuskript till artikel (20100720).Available from: 2008-11-03 Created: 2008-11-03 Last updated: 2017-12-14Bibliographically approved
In thesis
1. New Mass Spectrometric Methods for Structure Analysis of Carboxymethyl Cellulose
Open this publication in new window or tab >>New Mass Spectrometric Methods for Structure Analysis of Carboxymethyl Cellulose
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
carboxymethyl cellulose, substituent distribution, rheology, enzyme hydrolysis, cellulase, permethylation, liquid chromatography, MALDI, ESI, mass spectrometry
National Category
Polymer Chemistry
Identifiers
urn:nbn:se:kth:diva-9438 (URN)978-91-7415-162-6 (ISBN)
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

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