Caldicellulosiruptor bescii, a bacterium known for its efficient cellulose degradation, utilizes multimodular enzymes like CelA. This enzyme, featuring both GH9 and GH48 catalytic domains, exemplifies a potential evolutionary intermediary between free enzymes and the more complex cellulosome systems. Similarly, CbXyn10C/Cel48B (Athe_1857), sharing a similar domain structure with CelA, is abundantly secreted during C. bescii’s growth on cellulose, suggesting a significant role in biomass breakdown. This study delves into the synergistic interaction between the GH10 and GH48 domains of CbXyn10C/Cel48B using three specifically engineered truncation mutants.
Constructing and Characterizing Truncation Mutants of CbXyn10C/Cel48B
To investigate the synergistic potential of the GH10 and GH48 domains, three truncation mutants (TMs) were designed and expressed in E. coli. TM1 consists of the N-terminal GH10 domain with an appended CBM3b, TM2 comprises the C-terminal GH48 domain linked to a CBM3b, and TM3 represents a minimized version of the full-length enzyme, containing both GH10 and GH48 domains separated by a single CBM3b. While full-length CbXyn10C/Cel48B proved difficult to express in sufficient quantities, TM3 offered a viable alternative for studying domain interactions. Following purification, these three constructs were subjected to activity assays using cellulose and xylan substrates.
Synergistic Hydrolysis of Cellulose by GH10 and GH48 Domains
Initial activity assays using the DNS method, while not revealing significant synergy in glucose release, highlighted the heterogeneous nature of the hydrolysis products. Subsequent HPAEC-PAD analysis demonstrated clear synergistic effects on cellobiose and cellotriose liberation from filter paper. Specifically, the TM1 + TM2 mixture exhibited a degree of synergy (DoS) of 2.3 and 1.7 for cellobiose and cellotriose, respectively. TM3, representing the intramolecular interaction, showed a DoS of 2.6 and 1.8. Interestingly, the predominant product in both inter- and intramolecular synergistic hydrolysis was cellobiose, a preferred carbon source for C. bescii. This observation suggests a coordinated mechanism where longer cellooligosaccharides produced by GH10 are efficiently processed into cellobiose by GH48.
Investigating Synergy in Xylan Hydrolysis
Although GH48 is typically considered an exocellulase, previous studies have indicated its activity on xylan. Analysis of xylan hydrolysis by the TMs revealed an intramolecular synergy in the early stages of the reaction, specifically for TM3. This synergy manifested as a more rapid release of shorter xylooligosaccharides compared to TM1 or the TM1 + TM2 mixture. However, this synergistic effect diminished over time, potentially due to product inhibition of GH48 by accumulating xylose and xylooligosaccharides.
Synergy on Complex Lignocellulose: Corn Straw
To assess the activity of the TMs on a more complex substrate, steam explosion-treated corn straw was used. While no synergy was observed for xylan degradation, likely due to the substrate’s recalcitrance, the synergistic pattern for cellulose hydrolysis mirrored that observed with pure cellulose. Both TM1 + TM2 and TM3 exhibited enhanced cellobiose and cellotriose release.
Conclusion
This study highlights the complex interplay between GH10 and GH48 catalytic domains in CbXyn10C/Cel48B, showcasing synergistic effects on both cellulose and xylan hydrolysis. These findings provide valuable insights into the efficient biomass degradation strategies employed by C. bescii and inform the development of tailored enzyme cocktails for biofuel production. The observed substrate promiscuity and the importance of domain organization underscore the need for further investigation into the functional diversity of multimodular enzymes.
