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Discovery and investigation of glycoside hydrolase family 5 enzymes with potential use in biomass conversion
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:5
Keyword [en]
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
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-160538ISBN: 978-91-7595-439-4 (print)OAI: oai:DiVA.org:kth-160538DiVA: diva2:790149
Public defence
2015-03-20, FD41, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Foundation for Strategic Research Swedish Research Council Formas
Note

QC 20150224

Available from: 2015-02-24 Created: 2015-02-23 Last updated: 2015-02-24Bibliographically approved
List of papers
1. Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)
Open this publication in new window or tab >>Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)
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2012 (English)In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 12, no 1, 186- p.Article in journal (Refereed) Published
Abstract [en]

Background: The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on beta-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. Results: About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Conclusion: Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html.

Keyword
Protein evolution, Enzyme evolution, Functional prediction, Glycogenomics, Glycoside hydrolase family 5, Phylogenetic analysis, Subfamily classification
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:kth:diva-109742 (URN)10.1186/1471-2148-12-186 (DOI)000312732100001 ()22992189 (PubMedID)2-s2.0-84866500048 (Scopus ID)
Funder
Swedish Research Council FormasSwedish Foundation for Strategic Research
Note

QC 20130201

Available from: 2013-01-08 Created: 2013-01-08 Last updated: 2017-12-06Bibliographically approved
2. Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana
Open this publication in new window or tab >>Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana
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.

Keyword
GH5_7, beta-Mannanase, Glycoside hydrolase, Mannan, Plant cell wall, Carbohydrates
National Category
Plant Biotechnology
Identifiers
urn:nbn:se:kth:diva-143702 (URN)10.1007/s00425-013-2005-y (DOI)000331648500010 ()2-s2.0-84916929268 (Scopus ID)
Funder
Swedish Foundation for Strategic Research FormasSwedish Research Council
Note

QC 20140331

Available from: 2014-03-31 Created: 2014-03-27 Last updated: 2017-12-05Bibliographically approved
3. Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thaliana
Open this publication in new window or tab >>Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thaliana
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(English)Manuscript (preprint) (Other academic)
National Category
Biocatalysis and Enzyme Technology Plant Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-160487 (URN)
Funder
Swedish Research Council Formas
Note

QS 2015

Available from: 2015-02-20 Created: 2015-02-20 Last updated: 2015-02-24Bibliographically approved
4. Investigating the function and biochemical properties of Arabidopsis mannanase 5-6
Open this publication in new window or tab >>Investigating the function and biochemical properties of Arabidopsis mannanase 5-6
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(English)Manuscript (preprint) (Other academic)
National Category
Biocatalysis and Enzyme Technology Plant Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-160489 (URN)
Funder
Swedish Research Council Formas
Note

QS 2015

Available from: 2015-02-20 Created: 2015-02-20 Last updated: 2015-02-24Bibliographically approved

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