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Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana
KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Industrial Biotechnology.
KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.ORCID iD: 0000-0003-3572-7798
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0002-8576-4370
2014 (English)In: Planta, ISSN 0032-0935, E-ISSN 1432-2048, Vol. 239, no 3, 653-665 p.Article in journal (Refereed) Published
Abstract [en]

Each plant genome contains a repertoire of beta-mannanase genes belonging to glycoside hydrolase family 5 subfamily 7 (GH5_7), putatively involved in the degradation and modification of various plant mannan polysaccharides, but very few have been characterized at the gene product level. The current study presents recombinant production and in vitro characterization of AtMan5-1 as a first step towards the exploration of the catalytic capacity of Arabidopsis thaliana beta-mannanase. The target enzyme was expressed in both E. coli (AtMan5-1e) and P. pastoris (AtMan5-1p). The main difference between the two forms was a higher observed thermal stability for AtMan5-1p, presumably due to glycosylation of that particular variant. AtMan5-1 displayed optimal activity at pH 5 and 35 A degrees C and hydrolyzed polymeric carob galactomannan, konjac glucomannan, and spruce galactoglucomannan as well as oligomeric mannopentaose and mannohexaose. However, the galactose-rich and highly branched guar gum was not as efficiently degraded. AtMan5-1 activity was enhanced by Co2+ and inhibited by Mn2+. The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min(-1) mg(-1) mL for AtMan5-1e and AtMan5-1p, respectively. Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase.

Place, publisher, year, edition, pages
2014. Vol. 239, no 3, 653-665 p.
Keyword [en]
GH5_7, beta-Mannanase, Glycoside hydrolase, Mannan, Plant cell wall, Carbohydrates
National Category
Plant Biotechnology
URN: urn:nbn:se:kth:diva-143702DOI: 10.1007/s00425-013-2005-yISI: 000331648500010ScopusID: 2-s2.0-84916929268OAI: diva2:708946
Swedish Foundation for Strategic Research FormasSwedish Research Council

QC 20140331

Available from: 2014-03-31 Created: 2014-03-27 Last updated: 2015-02-24Bibliographically approved
In thesis
1. Discovery and investigation of glycoside hydrolase family 5 enzymes with potential use in biomass conversion
Open this publication in new window or tab >>Discovery and investigation of glycoside hydrolase family 5 enzymes with potential use in biomass conversion
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glycoside hydrolases (GHs) cleave glycosidic bonds in glycoconjugates, oligosaccharides and polysaccharides such as cellulose and various hemicelluloses. Mannan is a major group of hemicelluloses. In higher plants, they usually serve as storage carbohydrates in seeds and tubers or as structural polysaccharides cross-linking with cellulose/lignin in cell walls. In industrial fields, this renewable biomass component can be used in various areas such as production of biofuels and health-benefit manno-oligosaccharides; and mannan degrading enzymes, especially mannanases, are important molecular tools for controlling mannan polysaccharides properties in biomass conversion. In this thesis, the evolution, substrate specificity and subfamily classification of the most important GH family, i.e., glycoside hydrolase family 5 (GH5), are presented providing a powerful tool for exploring GH5 enzymes in search for enzymes with interesting properties for sustainable biomass conversion. Additionally, three GH5_7 mannanases from Arabidopsis thaliana (AtMan5-1, AtMan5-2 and AtMan5-6) were investigated in the present study. Bioinformatics tools, heterologous expression, and enzymology were applied in order to reveal the catalytic properties of the target enzymes, increase understanding of plant mannanase evolution, and evaluate their potential use in biomass conversion. This approach revealed: (1) AtMan5-1 exhibits mannan hydrolase/transglycosylase activity (MHT), (2) AtMan5-2 preferably degrades mannans with a glucomannan backbone, and (3) AtMan5-6 is a relatively thermotolerant enzyme showing high catalytic efficiency for conversion of glucomannan and galactomannan making this plant mannanase an interesting candidate for biotechnological applications of digesting various mannans. Moreover, these studies suggest an evolutionary diversification of plant mannanase enzymatic function.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. 51 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:5
plant cell wall, biomass, hemicellulose, mannans, glycoside hydrolase, subfamily classification, Arabidopsis, mannanase, transglycosylase, enzymatic characterization.
National Category
Biocatalysis and Enzyme Technology Plant Biotechnology Biochemistry and Molecular Biology
Research subject
urn:nbn:se:kth:diva-160538 (URN)978-91-7595-439-4 (ISBN)
Public defence
2015-03-20, FD41, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Swedish Foundation for Strategic Research Swedish Research Council Formas

QC 20150224

Available from: 2015-02-24 Created: 2015-02-23 Last updated: 2015-02-24Bibliographically approved

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