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Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
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2005 (English)In: Biochemical Journal, ISSN 0264-6021, Vol. 390, 105-113 p.Article in journal (Refereed) Published
Abstract [en]

The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula x tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the a-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn(93) to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn(93) -> Ser) mutant.

Place, publisher, year, edition, pages
2005. Vol. 390, 105-113 p.
Keyword [en]
glycoside hydrolase family 16 (GH16), hybrid aspen (Populus tremula x tremuloides), Pichia pastoris, protein glycosylation, protein MS, xyloglucan endotransglycosylase (XET)
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-5704DOI: 10.1042/BJ20041749ISI: 000231492800012Scopus ID: 2-s2.0-23944474570OAI: oai:DiVA.org:kth-5704DiVA: diva2:10155
Note
QC 20100903Available 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
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Supervisors
Note
QC 20100903Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2011-11-23Bibliographically approved

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Kallas, Åsa M.Piens, KathleenDenman, Stuart E.Henriksson, HongbinFäldt, JennyBrumer, HarryTeeri, Tuula T.
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