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Enzymatic characterization of a recombinant xyloglucan endotransglycosylase PttXET16-35 from Populus tremula x tremuloides
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH.
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0002-8576-4370
Show others and affiliations
(English)Manuscript (Other academic)
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-5705OAI: oai:DiVA.org:kth-5705DiVA: diva2:10156
Note
QC 20100624Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2010-09-03Bibliographically approved
In thesis
1. Heterologous expression, characterization and applications of carbohydrate active enzymes and binding modules
Open this publication in new window or tab >>Heterologous expression, characterization and applications of carbohydrate active enzymes and binding modules
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Wood and wood products are of great economical and environmental importance, both in Sweden and globally. Biotechnology can be used both for achieving raw material of improved quality and for industrial processes such as biobleaching. Despite the enormous amount of carbon that is fixed as wood, the knowledge about the enzymes involved in the biosynthesis, re-organization and degradation of plant cell walls is relatively limited. In order to exploit enzymes more efficiently or to develop new biotechnological processes, it is crucial to gain a better understanding of the function and mechanism of the enzymes. This work has aimed to increase the knowledge about some of the enzymes putatively involved in the wood forming processes in Populus. Xyloglucan endotransglycosylases and a putative xylanase represent transglycosylating and hydrolytic enzymes, respectively. Carbohydrate binding modules represent non-catalytic modules, which bind to the substrate.

Among 24 genes encoding for putative xyloglucan endotransglycosylases or xyloglucan endohydrolases that were identified in the Populus EST database, two were chosen for further studies (PttXTH16-34 and PttXTH16-35). The corresponding proteins, PttXET16-34 and PttXET16-35, were expressed in P. pastoris, purified and biochemically characterized. The importance of the N-glycans was investigated by comparing the recombinant wild-type proteins with their deglycosylated counterparts. In order to obtain the large amounts of PttXET16-34 that were needed for crystallization and development of biotechnological applications, the conditions for the large-scale production of PttXET16-34 in a fermenter were optimized.

In microorganisms, endo-(1,4)-β-xylanases are important members of the xylan degrading machinery. These enzymes are also present in plants where they might fulfill a similar, but probably more restrictive function. One putative endo-(1,4)-β-xylanase, denoted PttXYN10A, was identified in the hybrid aspen EST library. Sequence analysis shows that this protein contains three putative carbohydrate-binding modules (CBM) from family 22 in addition to the catalytic module from GH10. Heterologous expression and reverse genetics were applied in order to elucidate the function of the catalytic module as well as the binding modules of PttXYN10A.

Just as in microorganisms, some of the carbohydrate active enzymes from plants have one or more CBM attached to the catalytic module. So far, a very limited number of plant CBMs has been biochemically characterized. A detailed bio-informatic analysis of the CBM family 43 revealed interesting modularity patterns. In addition, one CBM43 (CBM43PttGH17_84) from a putative Populus b-(1,3)-glucanase was expressed in E. coli and shown to bind to laminarin (β-(1,3)-glucan), mixed-linked β-(1,3)(1,4)-glucans and crystalline cellulose. Due to their high specificity for different carbohydrates, CBMs can be used as probes for the analysis of plant materials. Generally, they are more specific than both staining techniques and carbohydrate-binding antibodies. We have used cellulose- and mannan binding modules from microorganisms as tools for the analysis of intact fibers as well as processed pulps.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 69 p.
Keyword
Populus, xyloglucan endotransglycosylase, carbohydrate binding modules, endo-(1, 4)-b-xylanase, Escherichia coli, Pichia pastoris, N-glycosylation, fiber analysis
National Category
Industrial Biotechnology Wood Science
Identifiers
urn:nbn:se:kth:diva-3950 (URN)91-7178-349-0 (ISBN)
Public defence
2006-05-24, FR4, Albanova University Center, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100903Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2011-11-23Bibliographically approved
2. Xyloglucan-active enzymes: properties, structures and applications
Open this publication in new window or tab >>Xyloglucan-active enzymes: properties, structures and applications
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [sv]

Cellulosabaserade material är världens rikligast förekommande förnyelsebara råvara. Växters cellväggar är naturliga kompositmaterial där den kristallina cellulosan är inbäddad i en väv av hemicellulosa, strukturproteiner och lignin. Xyloglukaner är en viktig hemicellulosagrupp som omger och korslänkar den kristallina cellulosan i cellväggarna. I denna avhandling undersöks undersöks sambanden mellan struktur och funktion hos olika xyloglukan-aktiva enzymer.

En modell för effektiv enzymatisk omvandling av biomassa ges av cellulosomen hos den anaeroba prokaryota organismen Clostridium thermocellum. Cellulosomen är ett proteinkomplex med hög molmassa och flera olika enzymaktiviteter, bl.a. det inverterande xyloglukan-endohydrolaset CtXGH74A. Proteinstrukturen för CtXGH74A har lösts i komplex med xyloglukanoligosackarider, som stabliliserar vissa loopar/slingor som är oordnade i apostrukturen. Ytterligare detaljerade kinetiska och produktananalyser har genomförts för att entydigt visa att CtXGH74A är ett endoxyloglukanas vars slutliga nedbrytningsprodukt är Glc4-baserade xyloglukanoligosackarider.

Som jämförelse innehåller glykosidhydrolasfamilj 16 (GH16) såväl hydrolytiska endoxyloglukanaser som xyloglukantransglykosylaser (XETs) från växter. För att utreda vad som bestämmer förhållandet mellan transglykosylering och hydrolys i xyloglukanaktiva enzymer från familj GH 16 jämfördes struktur och kinetik hos ett strikt transglykosylas, PttXET16-34 från hybridasp, med ett nära besläktat hydrolytiskt enzym, NXG1 från krasse. I NXG1 identifierades en viktig förlängningsloop, som vid trunkering gav ett muterat enzym med högre transglykosyleringshastighet och minskad hydrolytisk aktivitet. Kinetikstudierna genomfördes med hjälp av nyutvecklade känsliga provmetoder med väldefinerade XGO:er och ett antal kromogena XGO-arylglykosider.

En detaljerad förståelse av enzymologin inom GH16 möjliggjorde utvecklingen av en ny kemoenzymatisk metod för biomimetisk fiberytmodifiering med hjälp av PttXET16-34s translgykosyleringsaktivitet. Aminoalditolderivat av xyloglukanoligosackarider användes som nyckelintermediärer för att introducera ny kemisk funktionalitet hos xyloglukan, såsom kromoforer, reaktiva grupper, proteinligander och initiatorer för polymeriseringsreaktioner. Tekniken innebär ett nytt och mångsidigt verktyg för fiberytmodifiering.

Abstract [de]

Zellulosehaltige Materialien sind die häufigsten erneuerbaren Rohmaterialien auf der Welt. Pflanzenzellwände sind natürliche Kompositmaterialien, sie enthalten kristalline Zellulose, die in einer Matrix aus Hemizellulosen, Proteinen und Lignin eingebettet sind. Xyloglukane sind eine wichtige Gruppe der Hemizellulosen, sie ummanteln und verbinden Zellulose in der pflanzlichen Zellwand. In dieser Abhandlung werden Strukturen von drei Xyloglukanaktiven Enzymen in Beziehung zu ihrer Funktion untersucht.

Ein Paradigma für effizienter Nutzung von Biomasse ist das Cellulosom des anaerob lebenden Bakteriums Clostridium thermocellum. Das Cellulosom ist ein hochmolekularer Komplex von Proteinen mit vielen verschiedenen Aktivitäten, darunter ist auch die invertierende Xyloglukan Endohydrolase CtXGH74A. Die Proteinstruktur von CtXGH74A wurde im Komplex mit Xyloglukanoligosacchariden (XGO) gelöst, welche ungeordnete Loops der apo-Struktur stabilisierten. Durch weitere detaillierte Analyse der Kinetik und Reaktionsprodukte konnte schlüssig gezeigt werden, daß CtXGH74A eine Endoglukanase ist, die Glc4-basierte XGO produziert.

Im Vergleich dazu enthält die retentierende Glykosidhydrolasefamilie 16 (GH16) sowohl hydrolytische Endoxyloglukanasen als auch Transglykosidasen von Pflanzen. Um zu erklären welche Faktoren das Verhältnis zwischen Transglykosidase und Hydrolase Aktivität bei GH16 Xyloglukanaktiven Enzymen bestimmen wurde eine reine Transglykosidase PttXET16-34 von Hybridaspen mit einem nah verwandten hydrolytischen Enzym NXG1 von Kapuzinerkresse strukturell und kinetisch verglichen. Als Schlüsselstelle wurde eine Verlängerung eines Loops in NXG1 identifiziert, Verkürzung des Loops führte zu einer Mutante mit erhöhter Transglykosylierungsrate bei verminderter hydrolytischer Aktivität. Kinetische Studien wurden erleichtert durch neu entwickelte hochempfindliche Methoden für Aktivitätsmessung, die auf XGO oder chromogene Aryl-XGO als definierte Substrate zurückgreifen.

Detailliertes Verständnis von GH16 Enzymologie hat den Weg für die Entwicklung für eine neuartige Methode für biomimetische Oberflächenmodifikation von Zellulosefibern geebnet, dafür wurde die transglykosylierende Aktivität von PttXET16-34 angewendet. Aminoalditol-derivate von XGO wurden als wichtigste Zwischenprodukte angewendet, um neue chemische Funktionalitäten in Xyloglukan einzuführen, darunter waren Chromophore, reaktive Gruppen, Proteinliganden und Initiatoren für Polymerisationsreaktionen. Die modifizierten Xyloglukane wurden an eine Reihe von verschiedenen Zellulosematerialien gebunden und veränderten die Oberflächeneigenschaften dramatisch. Diese Methode ist ein neues wertvolles Werkzeug für Oberflächenmodifikation von Zellulosen.

Abstract [en]

Cellulosic materials are the most abundant renewable resource in the world; plant cell walls are natural composite materials containing crystalline cellulose embedded in a matrix of hemicelluloses, structural proteins, and lignin. Xyloglucans are an important group of hemicelluloses, which coat and cross-link crystalline cellulose in the plant cell wall. In this thesis, structure-function relationships of a range of xyloglucan-active enzymes were examined.

A paradigm for efficient enzymatic biomass utilization is the cellulosome of the anaerobic bacterium Clostridium thermocellum. The cellulosome is a high molecular weight complex of proteins with diverse enzyme activities, including the inverting xyloglucan endo-hydrolase CtXGH74A. The protein structure of CtXGH74A was solved in complex with xyloglucan oligosaccharides (XGOs) which stabilized disordered loops of the apo-structure. Further detailed kinetic and product analyses were used to conclusively demonstrate that CtXGH74A is an endo-xyloglucase that produces Glc4-based XGOs as limit digestion products.

In comparison, the retaining glycoside hydrolase family 16 (GH16) contains hydrolytic endo-xyloglucanases as well as xyloglucan transglycosylases (XETs) from plants. To elucidate the determinants of the transglycosylase/hydrolysis ratio in GH16 xyloglucan-active enzymes, a strict transglycosylase, PttXET16-34 from hybrid aspen, was compared structurally and kinetically with the closely related hydrolytic enzyme NXG1 from nasturtium. A key loop extension was identified in NXG1, truncation of which yielded a mutant enzyme that exhibited an increased transglycosylase rate and reduced hydrolytic activity. Kinetic studies were facilitated by the development of new, sensitive assays using well-defined XGOs and a series of chromogenic XGO aryl-glycosides.

A detailed understanding of GH16 xyloglucan enzymology has paved the way for the development of a novel chemo-enzymatic approach for biomimetic fiber surface modification, in which the transglycosylating activity of PttXET16-34 was employed. Aminoalditol derivates of XGOs were used as key intermediates to incorporate novel chemical functionality into xyloglucan, including chromophores, reactive groups, protein ligands, and initiators for polymerization reactions. The resulting modified xyloglucans were subsequently bound to a range of cellulose materials to radically alter surface properties. As such, the technology provides a novel, versatile toolkit for fiber surface modification.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. viii, 59 p.
Keyword
xyloglucan, xyloglucan endo-transglycosylase/hydrolase, xyloglucan endo-hydrolase, xyloglucan endo-transglycosylase, XET, xyloglucan oligosaccharides, synthesis, carbohydrate, cellulose, crystal structure, fiber, surface modification, Populus tremula x Populus tremuloides, Hybrid aspen, nasturtium, NXG
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-4314 (URN)978-91-7178-596-1 (ISBN)
Public defence
2007-04-13, SAL FD5, AlbaNova, Roslagstullbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20100624Available from: 2007-03-22 Created: 2007-03-22 Last updated: 2012-02-23Bibliographically approved

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