Exploration of two pectate lyases from Caldicellulosiruptor bescii reveals a crucial role of CBM66 module in pectic biomass degradation.

Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that encode for pectinolytic enzymes. Pectate lyases that cleave α-1,4-galacturonosidic linkage of pectin are widely used in industries, such as paper making and fruit softening. However, reports on pectate lyases with good thermostability are few. Here, two pectate lyases (CbPL3 and CbPL9) from a hyperthermophilic bacterium Caldicellulosiruptor bescii, respectively, belonged to family 3 and family 9 polysaccharide lyases, were investigated. Biochemical properties on two CbPLs were shown similarly under optimized conditions of 80°C–85°C and pH 8–9. However, the degradation products from pectin and polygalacturonic acids (pGA) were different. A family-66 carbohydrate-binding module (CbCBM66), located in the N-terminus of two CbPLs, shares 100% amino acid identity. CbCBM66-truncated mutant of CbPL9 showed lower activities than the wild-type, whereas the CbPL3 with CbCBM66 knock-out portion was reported with enhanced activities, thereby revealing the different effect of CbCBM66. Prediction by I-TASSAR server revealed CbCBM66 is structurally close to BsCBM66 from Bacillus subtilis, however, COFACTOR and COACH programs speculated that the substrate-binding sites between CbCBM66 and BsCBM66 are different. Furthermore, substrate-binding assay indicated that the catalytic domains in two CbPLs had strong affinities on pectate-related substrates, but CbCBM66 showed weak interaction with a number of lignocellulosic carbohydrates. Finally, scanning electron microscope (SEM) analysis and total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for degradation of plant biomass. Importance Thermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium Caldicellulosiruptor bescii. The two enzymes share high optimum temperature, low optimum pH, and good thermostability at evaluated temperature. A family-66 carbohydrate binding module (CbCBM66) was identified in two CbPLs with sharing 100% amino acid identity. Deletion of CbCBM66 dramatically decreased the activity of CbPL9, but increase the activity and thermostability of CbPL3, suggesting the different roles of CbCBM66 in two enzymes. Moreover, the degradation products by two CbPLs were different. These results revealed these enzymes could represent a potential pectate lyase for applications in paper and textile industries.