Lignocellulosic biomass is a major renewable resource with the potential to replace petrochemicals in many areas. β-Mannan is a significant component of this biomass in e.g. spruce, but it is often considered a byproduct in wood pulp processing, and is removed in side-streams or with the waste waters. However, there is a growing interest in utilizing softwood β-mannan in biorefinery applications such as the production of second-generation bioethanol and specialty chemicals, including surfactants. Alkyl glycosides, non-ionic surfactants made from saccharides and alcohols, are a renewable, green, biodegradable and mild alternative to petrochemical surfactants. We have previously described how mass spectrometry (MS) can be used to analyze donor substrate binding to β-mannanase active sites and product (alkyl mannoside) formation (Rosengren et al. (2012) Biocatal Biotransform. 30: 338-352, Rosengren et al. (2014) Appl Microbiol Biotechnol. 98: 10091-10104). We now demonstrate the production of alkyl mannosides by β-mannanases from Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C), using MS and high-performance liquid chromatography (HPLC). All three enzymes produce methyl mannosides, whereas TrMan5A is the best choice of enzyme for synthesizing butyl and hexyl mannosides. Focusing on the synthesis of hexyl mannosides, we have also optimized reaction conditions to maximize yield, with a lower enzyme concentration and a moderate substrate concentration leading to a higher ratio of alcoholysis to hydrolysis. We furthermore lowered water activity with organic co-solvents, and scaled up the synthesis reaction to purify and characterize the alkyl mannosides using reversed-phase chromatography and nuclear magnetic resonance (NMR). Our results give useful insight into the influence of enzyme structural differences on alkyl mannoside formation. Future production of these alkyl mannoside surfactants could significantly valorize residual mannan-rich side-streams and contribute to the replacement of petrochemicals with renewable alternatives.

Enzymatic synthesis of alkyl mannoside surfactants by β-mannanases

251st National Meeting (2016)

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