Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules

Abstract

Background: Efficient degradation of lignocellulosic biomass has become a major bottleneck in industrial processes which attempt to use biomass as a carbon source for the production of biofuels and materials. To make the most effective use of the source material, both the hemicellulosic as well as cellulosic parts of the biomass should be targeted, and as such both hemicellulases and cellulases are important enzymes in biorefinery processes. Using thermostable versions of these enzymes can also prove beneficial in biomass degradation, as they can be expected to act faster than mesophilic enzymes and the process can also be improved by lower viscosities at higher temperatures, as well as prevent the introduction of microbial contamination.

Results: This study presents the investigation of the thermostable, dual-function xylanase-glucuronoyl esterase enzyme CkXyn10C-GE15A from the hyperthermophilic bacterium Caldicellulosiruptor kristjanssonii. Biochemical characterization of the enzyme was performed, including assays for establishing the melting points for the different protein domains, activity assays for the two catalytic domains, as well as binding assays for the multiple carbohydrate-binding domains present in CkXyn10C-GE15A. Although the enzyme domains are naturally linked together, when added separately to biomass, the expected boosting of the xylanase action was not seen. This lack of intramolecular synergy might suggest, together with previous data, that increased xylose release is not the main beneficial trait given by glucuronoyl esterases.

Conclusions: Due to its thermostability, CkXyn10C-GE15A is a promising candidate for industrial processes, with both catalytic domains exhibiting melting temperatures over 70 °C. Of particular interest is the glucuronoyl esterase domain, as it represents the first studied thermostable enzyme displaying this activity.

Keywords: Biomass; Caldicellulosiruptor kristjansonii; Carbohydrate-active enzyme; Carbohydrate-binding module; Glucuronoyl esterase; Lignin–carbohydrate complexes; Thermostability; Xylan; Xylanase.

Fig. 1

Domain architecture of CkXyn10C-GE15A outlining the seven domains investigated in this study as well as the three additional C-terminal domains that were not. The surface layer homology (SLH) domains are predicted to anchor the enzyme to the cell wall, and thereby enable both binding to substrates and catalysis in the proximity of the cell

Fig. 2

Relative activity of the GH10 domain (red square) and the GH10 domain with both N-terminal CBM22 domains (blue circle) over a range of different pHs. Sodium acetate buffer was used up to pH 5.5, sodium phosphate from pH 6 to 8, and Tris for pH 8.5 and 9. The results are presented as the average of triplicate experiments with standard errors

Fig. 3

Activity of CBM22-CBM22-CkXyn10C (500 nM; blue) and the single CkXyn10C domain (50 nM; red) as an effect of temperature on the beechwood xylan substrate. A clear increase in both activity and thermostability can be observed when the CBM22 domains are attached to the catalytic xylanase domain. The results are presented as the average of triplicate experiments with standard errors

Fig. 4

Binding of CBM22.1-CBM22.2 (a), CBM22.2 (b), CBM9.1 (c), CBM9.2 (d), and CBM 9.3 (e). Differences in binding no substrate (Co), birch xylan (BiX), beech xylan (BeX), cellulose (Cel), and mannan (Man) are seen by comparing the relative intensity of the bands, determined using the Image Lab software

Fig. 5

Assays on complex biomass using the C. kristjansonii catalytic domains and commercial xylanases in various combinations. Corn cob biomass at 30 °C (a) and 60 °C (b), and wheat straw at 60 °C (c). No activity could be detected on the wheat straw at 30 °C, and the CkGE15A enzyme on its own resulted in no release of detectable sugars in any of the conditions. The results are presented as the average of triplicate experiments with standard errors