Functional and structural characterisation of a multimodular xylanase from the hindgut metagenome of the snouted harvester termite

Abstract

The degradation of hemicellulose is a vital step in the efficient utilisation of plant biomass, an abundant source of carbohydrates in nature. Xylan, the most prevalent hemicellulose, is digested by xylanases and much work has gone into improving the efficiency and stability of these enzymes. Interest has recently shifted to non-catalytic, accessory domains found in many xylanases, including carbohydrate binding modules (CBMs). CBMs may enhance enzyme properties, such as thermostability and efficiency. This effect has, however, not yet been fully explained. In this study, a novel multimodular xylanase isolated from the hindgut metagenome of the snouted harvester termite (Trinervitermes trinervoides) was characterised and the properties of its two identical CBMs analysed. Xylanase variants were produced in E. coli by deleting none, one or both CBMs. The variants were functionally characterised using a modified DNS assay in a thermocycler and structurally modelled using computational techniques. Deleting one CBM shifted the pH profile, slightly increasing activity at lower pH whereas removing both CBMs decreased activity at 60°C from 90% (SD = 1.3) to 56% (SD = 2.9). Removal of the CBMs did not statistically significantly affect the Michaelis constants (KM), but did reduce the turnover number (kcat). Structure modelling failed to accurately predict the structure of the complete enzyme and in particular any interactions between domains. The origin of the stabilising effect of the second CBM and hence the evolutionary benefit of two CBMs remains unresolved.