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Over the years several β-glucan transferases from yeast and fungi have been reported, but enzymes with such an activity from bacteria have not been characterized so far. In this work, we describe the cloning and expression of genes encoding β-glucosyltransferase domains of glycosyl hydrolase family GH17 from three species of proteobacteria: Pseudomonas aeruginosa PAO1, P. putida KT2440 and Azotobacter vinelandii ATCC BAA-1303. The encoded enzymes of these GH17 domains turned out to have a non-Leloir trans-β-glucosylation activity, as they do not use activated nucleotide sugar as donor, but transfer a glycosyl group from a β-glucan donor to a β-glucan acceptor. More particularly, the activity of the three recombinant enzymes on linear (β1 → 3)-linked gluco-oligosaccharides (Lam-Glc(4-9)) and their corresponding alditols (Lam-Glc(4-9)-ol) was studied. Detailed structural analysis, based on thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and 1D/2D (1)H and (13)C nuclear magnetic resonance data, revealed diverse product spectra. Depending on the enzyme used, besides (β1 → 3)-elongation activity, (β1 → 4)- or (β1 → 6)-elongation, or (β1 → 6)-branching activities were also detected.

Chondroitin sulfate (CS) saccharides from cartilage tissues have potential application in medicine or as dietary supplements due to their therapeutic bioactivities. Studies have shown that depolymerized CS saccharides may display enhanced bioactivity. The objective of this study was to isolate a CS-degrading enzyme for an efficient production of CS oligo- or disaccharides. CS-degrading bacteria from marine environments were enriched using in situ artificial support colonization containing CS from shark cartilage as substrate. Subsequently, an Arthrobacter species (strain MAT3885) efficiently degrading CS was isolated from a CS enrichment culture. The genomic DNA from strain MAT3885 was pyro-sequenced by using the 454 FLX sequencing technology. Following assembly and annotation, an orf, annotated as family 8 polysaccharide lyase genes, was identified, encoding an amino acid sequence with a similarity to CS lyases according to NCBI blastX. The gene, designated choA1, was cloned in Escherichia coli and expressed downstream of and in frame with the E. coli malE gene for obtaining a high yield of soluble recombinant protein. Applying a dual-tag system (MalE-Smt3-ChoA1), the MalE domain was separated from ChoA1 with proteolytic cleavage using Ulp1 protease. ChoA1 was defined as an AC-type enzyme as it degraded chondroitin sulfate A, C, and hyaluronic acid. The optimum activity of the enzyme was at pH 5.5-7.5 and 40 °C, running a 10-min reaction. The native enzyme was estimated to be a monomer. As the recombinant chondroitin sulfate lyase (designated as ChoA1R) degraded chondroitin sulfate efficiently compared to a benchmark enzyme, it may be used for the production of chondroitin sulfate disaccharides for the food industry or health-promoting products.

Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (β1→3) linkages preferably and to a lesser extent (β1→6) or (β1→4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (β1→3)(β1→6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic β-glucans in B. diazoefficiens.