Abstract
Symbiotic protists in the hindgut of termites provide a novel enzymatic resource for efficient lignocellulytic degradation of plant biomass. In this study, two β-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 β-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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Funding
This work was supported by a Grant-in-Aid for Scientific Research (No. 16K14879) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) and a research grant from the Institute for Fermentation, Osaka. H. H. H. is a recipient of the MEXT Scholarship.
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Hsu, Y., Koizumi, H., Otagiri, M. et al. Trp residue at subsite − 5 plays a critical role in the substrate binding of two protistan GH26 β-mannanases from a termite hindgut. Appl Microbiol Biotechnol 102, 1737–1747 (2018). https://doi.org/10.1007/s00253-017-8726-2
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DOI: https://doi.org/10.1007/s00253-017-8726-2