Structural and biochemical analyses of glycoside hydrolase family 26 ß-mannanase from a symbiotic protist of the termite Reticulitermes speratus.

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.

Promotion of efficient Saccharification of crystalline cellulose by Aspergillus fumigatus Swo1.

Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.

Purification and characterization of termite endogenous beta-1,4-endoglucanases produced in Aspergillus oryzae.

Although termites are known to have a highly efficient lignocellulose-digesting system, mass production of native endogenous cellulases of termites has failed in Escherchia coli, and in Saccharomyces cerevisiae, and it has not been accomplished. Here we report the successful production, purification, and characterization of two termite endogenous beta-1,4-endoglucanases, RsEG and NtEG, from the salivary gland of Reticulitermes speratus and the midgut of Nasutitermes takasagoensis respectively, using Aspergillus oryzae as host. Thin-layer chromatography analysis showed that both enzymes hydrolyzed the beta-1,4-cellulosic linkage of cellodextrin into cellobiose and glucose. Kinetic studies indicated that the specific activity and Vmax values of the two enzymes were significantly higher than those of previously reported fungal and bacterial endoglucanases.