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Xylanases are hemicellulases that break down xylan to soluble pentoses. They are used for industrial purposes, such as paper whitening, beverage clarification, and biofuel production. The second-generation bioethanol production is hindered by the enzymatic hydrolysis step of the lignocellulosic biomass, due to the complex arrangement established among its constituents. Xylanases can potentially increase the production yield by improving the action of the cellulolytic enzyme complex. We prospected endo-ß-1,4-xylanases from meta-transcriptomes of the termite Heterotermes tenuis. In silico structural characterization and functional analysis of an endo-ß-1,4-xylanase from a symbiotic protist of H. tenuis indicate two active sites and a substrate-binding groove needed for the catalytic activity. No N-glycosylation sites were found. This endo-ß-1,4-xylanase was recombinantly expressed in Pichia pastoris and Escherichia coli cells, presenting a molecular mass of approximately 20 kDa. Enzymatic activity assay using recombinant endo-ß-1,4-xylanase was also performed on 1% xylan agar stained with Congo red at 30 °C and 40 °C. The enzyme expressed in both systems was able to hydrolyze the substrate xylan, becoming a promising candidate for further analysis aiming to determine its potential for application in industrial xylan degradation processes.

RESUMEN

Symbiotic protists in the hindgut of termites provide a novel enzymatic resource for efficient lignocellulytic degradation of plant biomass. In this study, two ß-mannanases, RsMan26A and RsMan26B, from a symbiotic protist community of the lower termite, Reticulitermes speratus, were successfully expressed in the methylotrophic yeast, Pichia pastoris. Biochemical characterization experiments demonstrated that both RsMan26A and RsMan26B are endo-acting enzymes and have a very similar substrate specificity, displaying a higher catalytic efficiency to galactomannan from locust bean gum (LBG) and glucomannan than to ß-1,4-mannan and highly substituted galactomannan from guar gum. Homology modeling of RsMan26A and RsMan26B revealed that each enzyme displays a long open cleft harboring a unique hydrophobic platform (Trp79) that stacks against the sugar ring at subsite - 5. The Km values of W79A mutants of RsMan26A and RsMan26B to LBG increased by 4.8-fold and 3.6-fold, respectively, compared with those for the native enzymes, while the kcat remained unchanged or about 40% of that of the native enzyme, resulting in the decrease in the catalytic efficiency by 4.8-fold and 9.1-fold, respectively. The kinetic values for glucomannan also showed a similar result. These results demonstrate that the Trp residue present near the subsite - 5 has an important role in the recognition of the sugar ring in the substrate.

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We investigated the effects of lignins as diet components on the physiological activities of a lower termite, Coptotermes formosanus Shiraki. Artificial diets composed of polysaccharides with and without purified lignins (milled-wood lignins) from Japanese cedar (softwood), Japanese beech (hardwood), and rice (grass), were fed to C. formosanus workers. The survival and body mass of the workers as well as the presence of three symbiotic protists in the hindguts of the workers were then periodically examined. The survival rates of workers fed on diets containing lignins were, regardless of the lignocellulose diet sources, significantly higher than those of workers fed on only polysaccharides. In addition, it was clearly observed that all the tested lignins have positive effects on the maintenance of two major protists in the hindguts of C. formosanus workers, i.e., Pseudotrichonympha grassii and Holomastigotoides hartmanni. Overall, our data suggest that the presence of lignin is crucial to maintaining the physiological activities of C. formosanus workers during their lignocellulose decomposition. Our data also suggested that some components, possibly minerals and/or non-structural carbohydrates, in grass lignocellulose negatively affect the survival of C. formosanus workers as well as the present rate of the symbiotic protists in their hindguts.

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Symbiotic protists in the gut of termites are prominent natural resources for enzymes involved in lignocellulose degradation. Here we report expression, purification, and biochemical characterization of a glycoside hydrolase family 26 mannanase RsMan26H from the symbiotic protist of the lower termite, Reticulitermes speratus. Biochemical analysis of RsMan26H demonstrates that this enzyme is an endo-processive mannobiohydrolase producing mannobiose from oligo- and polysaccharides, followed by a minor accumulation of oligosaccharides larger than mannobiose. To our knowledge, this is the first report describing the unique mannobiohydrolase enzyme from the eukaryotic origin.

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RESUMEN

Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of ß-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites -5 and -2, respectively, in addition to accommodation of both glucose and mannose at subsites -3 and -4. RsMan26C has a long open cleft with a hydrophobic platform of Trp(94) at subsite -5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met(85) specifically interacts with the equatorial O-2 of glucose at subsite -3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.

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