Development and Application of Accessory Hemicellulases for the Production of Engineered Polysaccharides

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School of Chemical Technology | Doctoral thesis (article-based) | Defence date: 2017-05-19
Degree programme
94 + app. 78
Aalto University publication series DOCTORAL DISSERTATIONS, 82/2017
Hemicelluloses such as xylans and galactoglucomannans are thought to serve as crosslinking and bridging macromolecules in nature, and have been considered for applications ranging from prebiotics to films, coatings, and hydrogels. Accessory hemicellulases can be used to control and improve the functional performance this major biomass fraction through fine-tuning branching group chemistry. Accordingly, the overall objective of this thesis was to discover and engineer accessory hemicellulases that could be used to control the branching substituents present in different plant polysaccharides. Herein, the utility of characterized enzymes was demonstrated through chemo-enzymatic synthesis of xylan-based cellulose coatings. First, a new acetyl xylan esterase (AnAcXE) from carbohydrate esterase (CE) family CE1 was biochemically characterized and then fused to a family 3 carbohydrate binding domain (CtCBM3) in an effort to increase catalytic performance on high molecular weight and insoluble substrates, including cellulose acetate and acetylated xylan. Whereas increased binding and increased catalytic performance was observed using cellulose acetate, activity on oligomeric substrates was not affected. AnAcXE was then compared in its action on galactoglucomann and glucuronoxylan with enzymes from families CE6 and CE16. This direct comparative analysis uncovered differences in substrate preference and regio-selectivity of corresponding enzymes. Finally, a new α-arabinofuranosidase from GH62 (SthAbf62A) was comprehensively characterized. Its action on polymeric wheat and rye arabinoxylans containing high degree of substitution was significantly higher than on arabinan and 4-nitrophenyl α-L-arabinofuranose. These analyses indicated that SthAbf62A was a good candidate for selective removal of arabinofuranose linked through α-(1->2) and α-(1->3) to singly substituted xylopyranose in arabinoxylans. Accordingly, SthAbf62A along with the commercial AXHd3 (specific for (1->3) on di-substituted xylopyranose) were used to modify wheat arabinoxylan (WAX) prior to grafting with glycidyl methacrylate. Action of SthAbf62A lowered the water solubility of WAX, which was correlated to higher grafting efficiency. Although the binding efficiency of grafted WAX to regenerated cellulose did not change compared to ungrafted WAX, treatment with SthAbf62A or AXHd3 prior to grafting increased the viscoelastic properties of the stably adsorbed layer, indicating potential usefulness of these enzymes in production of new xylan-based coatings.
Supervising professor
Frey, Alexander D., Prof., Aalto University, Department of Bioproducts and Biosystems, Finland
Thesis advisor
Master, Emma R., Prof., University of Toronto, Canada
lignocellulose, acetyl esterases, α-arabinofuranosideses, CBM-fusion, regio-selectivity
Other note
  • [Publication 1]: Mai-Gisondi G., Turunen O., Pastinen O., Pahimanolis N., Master E. R. (2015)Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module. Enzyme Microb Technol 79-80, 27–33.
    DOI: 10.1016/j.enzmictec.2015.07.001 View at publisher
  • [Publication 2]: Mai-Gisondi G., Maaheimo H., Chong S-L., Hinz S., Tenkanen M., Master E. R. (2016) Functional comparison of versatile carbohydrate esterases from families CE1, CE6 and CE16 on 4-O-methylglucuronoxylan and acetyl-galactoglucomannan. (submitted in BBA General Subjects)
  • [Publication 3]: Wang W., Mai-Gisondi G., Stogios P. J., Kaur A., Xu X., Cui H., Turunen O., Savchenko A., Master E. R. (2014) Elucidation of the molecular basis for arabinoxylan-debranching activity of a thermostable family GH62 α-Larabinofuranosidase from Streptomyces thermoviolaceus. Appl Environ Microbiol 80 (17), 5317-5329.
    DOI: 10.1128/AEM.00685-14 View at publisher
  • [Publication 4]: Littunen K., Mai-Gisondi G., Seppälä J., Master E. R. (2016) Enzymatically debranched xylans in graft copolymerization. Biomacromolecules, 18 (5), pp 1634–1641.
    DOI: 10.1021/acs.biomac.7b00229 View at publisher