β-Mannanases catalyze the conversion and modification of β-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), β-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable β-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 β-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate β-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three β-mannanases using methanol or 1-hexanol as acceptor. Among the three β-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1-hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted β-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using β-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable β-mannans.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-8997-2) contains supplementary material, which is available to authorized users.

the genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: β-N-acetyl-glucosaminidases, β-1,4-glucosidases/β-xylosidases and macrolide β-glucosidases. the RmNag3 with additional β-lactamase domain clustered with the deepest rooted GH3-lineage of β-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed β-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide β-glucosidases from Actinomycetes. the β-xylosidases, RmXyl3A and RmXyl3B, and the β-glucosidases RmBgl3A and RmBgl3C clustered within the major β-glucosidases/β-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed β-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed β-1,4-glucosidase/β-xylosidase activity while RmBgl3C was active on pnp-β-Glc and β-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252 t (homolog strain). The analysis showed that in the homolog strain DSM 4252 t Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization. Marine extremophilic biotopes, such as hot springs and hydrothermal vents, harbour diverse microbes hitherto underexploited and unexplored. Recent genomic studies show that many of the species, especially those found in coastal geothermal areas surrounded by profusion of carbohydrate rich biomass (seaweeds as well as terrestrial species), contain a wide array of novel glycoside hydrolases (GHs) 1,2. Thermostable GHs have numerous applications in different fields, making marine thermophiles targets for prospecting of industrially interesting enzymes 3,4. Rhodothermus marinus are Gram-negative marine thermophilic bacteria, previously classified under the phylum Bacteroidetes, but recently assigned to the new phylum Rhodothermaeota 5. The type-species was isolated from a coastal hot spring on the NorthWest coast of Iceland and has an optimum temperature of 65 °C and is slightly halophilic 6. R. marinus can utilize a variety of sugars as carbon sources and produces a wide range of GHs 6-12. Sequence analysis shows that the R. marinus genome contains a large number of genes encoding GH enzymes, many of which are secreted extracellularly; yet, several of them appear to be attached to the cell surface 13. These putative enzymes include six members of GH family 3 (GH3) (RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3), which have not yet been studied.

The importance of the gut microbiota in human health has led to an increased interest to study probiotic bacteria. Fermented food is a source of already established probiotics, but it also offers an opportunity to discover new taxa. Four strains of Weissella sp. isolated from Indian fermented food have been genome sequenced and classified into the species W. cibaria based on wholegenome phylogeny. The genome of W. cibaria strain 92, known to utilise xylooligosaccharides and produce lactate and acetate, was analysed to identify genes for oligosaccharide utilisation. Clusters including genes involved in transportation, hydrolysis and metabolism of xylooligosaccharides, arabinooligosaccharides and β-glucosides were identified. Growth on arabinobiose and laminaribiose was detected. A 6-phospho-β-glucosidase clustered with a phosphotransferase system was found upregulated during growth on laminaribiose, indicating a mechanism for laminaribiose utilisation. the genome of W. cibaria strain 92 harbours genes for utilising the phosphoketolase pathway for the production of both acetate and lactate from pentose and hexose sugars but lacks two genes necessary for utilising the pentose phosphate pathway. The ability of W. cibaria strain 92 to utilise several types of oligosaccharides derived from dietary fibres, and produce lactate and acetate makes it interesting as a probiotic candidate for further evaluation. In recent years there has been an increased interest in the human gut microbiota and how it is associated with health. The gut microbiota has been shown to be important for maintaining a healthy gut, for synthesis of vitamins, for proper immune function and for the metabolism 1. With increasing understanding of the gut microbiota, the possibility to improve health by modifying its composition increases. Probiotics are live microorganisms that, when administrated in adequate amounts, confer a health benefit on the host 2. Mainly strains of Bifidobacterium and Lactobacillus are considered as probiotic and widely distributed for human administration, e.g. directly in capsules or incorporated in food items, such as yoghurt and juices. Other claimed probiotics include strains belonging to Saccharomyces, Lactococcus, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus and Escherichia 3. A few strains of commensal gut microbial bacteria associated with improved host functions have also been considered probiotic 2. Fermented food is an attractive source of new bacteria with potential probiotic properties 4. Expanding the research of probiotic bacteria into new strains, species and genera is important, both in order to disentangle the complex world of the gut microbiota and its effect on the host, but also to provide a proper cataloguing of probiotic taxa and perhaps, identify probiotic bacteria with novel beneficial properties.