Abstract
Glycoside hydrolase family 5 subfamily 8 (GH5_8) mannanases belong to Firmicutes, Actinomycetia, and Proteobacteria. The presence or absence of carbohydrate-binding modules (CBMs) present a striking difference. While various GH5_8 mannanases need a CBM for binding galactomannans, removal of the CBM did not affect activity of some, whereas it in other cases reduced the catalytic efficiency due to increased KM. Here, monomodular GH5_8 mannanases from Eubacterium siraeum (EsGH5_8) and Xanthomonas citri pv. aurantifolii (XcGH5_8) were produced and characterized to clarify if GH5_8 mannanases from Firmicutes and Proteobacteria without CBM(s) possess distinct properties. EsGH5_8 showed a remarkably high temperature optimum of 55 °C, while XcGH5_8 had an optimum at 30 °C. Both enzymes were highly active on carob galactomannan and konjac glucomannan. Notably, EsGH5_8 was equally active on both substrates, whereas XcGH5_8 preferred galactomannan. The KM values were comparable with those of catalytic domains of truncated GH5_8s, while the turn-over numbers (kcat) were in the higher end. Notably, XcGH5_8 bound to but did not degrade insoluble ivory nut mannan. The findings support the hypothesis that GH5_8 mannanases with CBMs target insoluble mannans found in plant cell walls and seeds, while monomodular GH5_8 members have soluble mannans and mannooligosaccharides as primary substrates.