Enzymatic conversion of β-mannans through transglycosylation and hydrolysis: Synthesis of building blocks for novel biomaterials from renewables

Research output: ThesisDoctoral Thesis (compilation)


The major softwood hemicellulose is galactoglucomannan (GGM), an abundant polysaccharide which constitutes up to 25% of the dry weight of wood, much of which is discarded in current refinery practices. Among the enzymes responsible for the degradation of this hemicellulose are the β-mannanases. Apart from their natural hydrolytic action, some β-mannanases have the ability to catalyse the synthesis of glycoconjugates through the formation of a new glycosidic linkage between a saccharide donor and a hydroxyl-containing acceptor, such as an alcohol. Uncertainties remain as to the molecular determinants and reaction parameters which dictate if a β-mannanase is efficient in performing these synthesis reactions. The work in this thesis aims to provide further insight into some of the determinants for efficient enzymatic conversion of β-mannans through β-mannanase catalysed transglycosylation and hydrolysis.
A method for sequential purification of GGM contained in an industrial side stream was described in Paper I. Higher purity of GGM (with less soluble lignin) was beneficial to enzymatic conversion rates and yields of hydrolysis products. In Paper II it was shown that GGM, could act as donor substrate in transglycosylation reactions catalysed by the β-mannanase TrMan5A, from Trichoderma reesei, resulting in a complex product profile. β-Mannan oligomers and polymeric galactomannan were used to synthesise reactive glycoconjugates with the bifunctional acceptors allyl alcohol and hydroxyethyl methacrylate (HEMA) using GH5_7 β-mannanases as catalysts (Papers II-IV). Modifications to aglycone subsites can affect acceptor substrate specificity. In Paper III, a novel double (+1/+2 subsite) mutant variant, R171K/E205D, of TrMan5A had a significantly improved transglycosylation capacity early in reactions, but secondary hydrolysis occurred, which reduced transglycosylation yields over prolonged incubations. It is suggested that the additional space generated through the E205D substitution in the +1 subsite improved accommodation of the acceptor molecule, improving transglycosylation, but also secondary hydrolysis (Paper III). In a novel concept, enzyme synergy, through simultaneous application of TrMan5A and guar α-galactosidase, resulted in significantly increased transglycosylation yields with a galactomannan donor (Paper III). The removal of galactosyl side-groups improved yields of allyl-mannosides, additionally allyl-galactosides were obtained through α-galactosidase catalysis. α-Galactosidases were further studied in Paper V, showing that the α-galactosidase from guar can use a variety of acceptor molecules in transglycosylation reactions, increasing a potential product portfolio. Through scale-up of transglycosylation reaction with TrMan5A, glycoconjugates synthesised from galactomannan and HEMA were successfully polymerised into novel glyco-polymers (Paper II & IV). The pendent mannosyl groups influenced the thermoresponsive aggregation behaviour of a novel co-polymer (Paper IV).
The results of this thesis contribute to further the understanding of molecular determinants and reaction parameters which influence the efficient conversion of β-mannans into novel glycoconjugates. Thus, demonstrating the feasibility in applying β-mannanases in enzymatic synthesis of novel bio-materials.
Original languageEnglish
  • Stålbrand, Henrik, Supervisor
Award date2021 May 7
Place of PublicationLund, Sweden
Print ISBNs978-91-7422-798-7
Electronic ISBNs978-91-7422-799-4
Publication statusPublished - 2021

Bibliographical note

Defence details

Date: 2021-05-07

Time: 13:15

Place: Kemicentrum, Hall A, Lund, Sweden. Join via zoom: https://lu-se.zoom.us/s/63118084684

External reviewer(s)

Name: Jönsson, Leif

Title: Professor

Affiliation: Umeå University, Sweden


Subject classification (UKÄ)

  • Biochemistry and Molecular Biology


  • β-Mannanase, Transglycosylation, Enzymatic synthesis, Glycoconjugates, Enzyme synergy, α- Galactosidase, Galactoglucomannan, Enzyme engineering, MALDI-ToF MS, HPLC


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