Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents

Abstract

Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)₈-barrel structure, where each (βα) module is essential for the enzyme's stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A from Talaromyces emersonii (TeEgl5A), which has an optimal temperature of 90°C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 from Stegonsporium opalus (SoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20°C), the temperature at which the protein lost 50% of its activity (T₅₀) (15 and 19°C), and the melting temperature (T_m ) (16.5 and 22.9°C) and extended half-lives (t_1/2) (∼240- and 650-fold at 55°C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 from Aspergillus niger (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both the SoCel5 and TeEgl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between α₂ and α₃ is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parent SoCel5.IMPORTANCE Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure, SoCel5 and TeEgl5A, with different thermal properties, were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

Keywords: (βα)8-barrel structure; GH5 endoglucanase; hybrid enzymes; structure-based recombination; thermostability.

FIG 1

Sequence alignment of SoCel5, TeEgl5A, and their hybrid enzymes H8 and H9 and three crystallized GH5 cellulases, from Aspergillus niger (PDB 5I78) (11), Thermoascus aurantiacus (PDB 1H1N) (57), and Penicillium verruculosum (PDB 5L9C). Residue numbering below the alignment is according to the sequence from SoCel5, which is used consistently throughout the text when referring to residue numbers, including for TeEgl5A and the hybrids, to simplify comparison of interactions between enzymes. Module elements are labeled above the alignment. Residues highlighted in purple are identical across all sequences, and residues highlighted in blue are shared in at least 5 enzymes.

FIG 2

Enzyme properties of the purified recombinant SoCel5 and TeCel5 and their hybrid enzymes. The relative activities corresponding to 100% with CMC-Na as the substrate are 351 U/mg for SoCel5, 620 U/mg for TeEgl5A, 270 U/mg for H4, 175 U/mg for H5, 101 U/mg for H6, 721 U/mg for H8, and 917 U/mg for H9. (a) pH-activity profiles tested at the optimal temperature for each enzyme (60°C for SoCel5, 50°C for H4, H5, and H6, 80°C for H8, 70°C for H9, and 90°C for TeEgl5A). (b) pH-stability profiles. After incubation of the enzymes at 37°C for 1 h in buffers ranging from pH 3.0 to 12.0, the residual activities were determined in 100 mM citric acid–Na₂HPO₄ buffer at the optimal pH and optimal temperature of each enzyme. (c) Temperature-activity profiles tested at the optimal pH of each enzyme (pH 5.0 for SoCel5 and H4 and pH 4.0 for TeEgl5A, H5, H6, H8, and H9). (d) Temperature-stability profiles. Each enzyme was preincubated at 70°C and optimal pH in 100 mM citric acid–Na₂HPO₄ buffer for different periods of time and subjected to residual activity assay under the optimal conditions for each enzyme.

FIG 3

Comparative per-residue root mean square fluctuations (RMSFs) across all trajectories. The RMSF for each simulated GH5 is presented on three graphs (black, SoCel5; red, H8; blue, H9; gray, TeEgl5A), one for each temperature, as indicated. To highlight the disparity of RMSFs between structural elements and the intervening loops, the eight beta-strand regions are highlighted in pink and the eight alpha-helical regions are highlighted in green, as well as labeled in the 398 K graph. The elevated RMSF for SoCel5 at residue 52 is indicated by black arrows.

FIG 4

Simulation snapshots. In the barrel assemblies of SoCel5 (A) and TeEgl5A (B), we highlight specific interactions around the R52 residue that is present for each enzyme. For visual clarity, only heavy atoms and polar hydrogens for the selected residues that form an extended hydrogen bond network with R52 are shown. Along the interface between β₂α₂β₃α₃, sequence differences between SoCel5 (C) and TeEgl5A (D) lead to different hydrogen bonds and hydrophobic packing arrangements. The hydrogen bond interactions are shown as green dashed lines.

FIG 5

Atomic contact changes between different structural elements in going from 298 K to 398 K. Each of the four enzymes studied is labeled in the top right of each panel, with the color scale defined on the far right. Contacts were defined between individual atom pairs that were within 5 angstroms during simulation and weighted according to distance. The weighting function used was C(t) = ∑_pairs[1/1 + e^{5(d − 4 Å)}], where d is the distance between individual atoms in the pair.