Softwood hemicellulose (galactoglucomannan, GGM) is a major renewable resource with a wide variety of potential applications. However, GGM is often removed and discarded in waste streams in wood pulp processing, and is currently greatly underutilized. There is, though, a significant interest in the valorization and functionalization of GGM through its use in biorefinery applications such as the production of second-generation biofuels and novel high-value compounds, e.g. surfactants. Some b-mannanases have the potential to create these compounds by use of their synthetic (transglycosylation) capacity. The transglycosylation capacity of β-mannanases depends greatly on the structural conformation of the aglycone regions of their active sites, where the acceptor molecule binds. By site-directed mutagenesis of β-mannanases from the blue mussel (MeMan5A) and the fungus Trichoderma reesei (TrMan5A), we have shown that altering the aglycone region can significantly change both transglycosylation capacity and acceptor specificity. Thus, enzyme engineering provides an excellent way of optimizing reactions to maximize yields of specific compounds. We have demonstrated the production of several novel conjugates through transglycosylation by b-mannanases. By transferring alcohols onto manno-saccharide donors produced by partial hydrolysis of mannan, using variants of TrMan5A and the Aspergillus nidulans b-mannanase AnMan5C, we have produced alkyl mannosides with potential applications as renewable, biodegradable surfactants. Reaction optimization and protein engineering approaches were used to increase yield and control product formation as analyzed by mass spectrometry (MS) and high-performance liquid chromatography (HPLC) before scale-up and further product characterization with focus on structure and surfactant properties. By taking advantage of the capability of enzymes to transfer acceptors onto mannan substrates in this way, a wide variety of novel high-performance and high-value compounds could potentially be synthesized.

Biocatalytic functionalization of softwood galactoglucomannan through transglycosylation and enzyme engineering

253rd National Meeting (2017)

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