Improvement of Catalytic Activity and Thermostability of Alginate Lyase VxAly7B-CM via Rational Computational Design Strategies

Abstract

Alginate lyase degrades alginate through the β-elimination mechanism to produce alginate oligosaccharides (AOS) with notable biochemical properties and diverse biological activities. However, its poor thermostability limits large-scale industrial production. In this study, we employed a rational computational design strategy combining computer-aided evolutionary coupling analysis and ΔΔG_fold evaluation to enhance both the thermostability and catalytic activity of the alginate lyase VxAly7B-CM. Among ten single-point mutants, the E188N and S204G mutants exhibited increases in T_m from 47.0 °C to 48.9 °C and 50.2 °C, respectively, with specific activities of 3701.02 U/mg and 2812.01 U/mg at 45 °C. Notably, the combinatorial mutant E188N/S204G demonstrated a ΔT_m of 5 °C and an optimal reaction temperature up to 50 °C, where its specific activity reached 3823.80 U/mg-a 31% increase. Moreover, its half-life at 50 °C was 38.4 h, which is 7.0 times that of the wild-type enzyme. Protein structural analysis and molecular dynamics simulations suggested that the enhanced catalytic performance and thermostability of the E188N/S204G mutant may be attributed to optimized surface charge distribution, strengthened hydrophobic interactions, and increased tertiary structure stability. Overall, our findings provided valuable insights into enzyme stabilization strategies and supported the industrial production of functional AOS.

Keywords: alginate lyase; alginate oligosaccharides; molecular dynamics simulation; rational design; thermostability.

Figure 1

Prediction of mutation effects. (a) Epistatic effects of VxAly7B-CM mutants. The data were visualized using GraphPad Prism 8.0, with a color gradient representing values from −15 (blue) to 0 (white) to 5 (orange); (b) ΔΔG_fold of VxAly7B-CM mutants. The data were visualized using GraphPad Prism 8.0, with a color gradient representing values from −5 (red) to 0 (white) to 20 (purple).

Figure 2

Three-dimensional model and the evolutionary conservation of VxAly7B-CM. The amino acid residues were color-coded according to the conservation grades, with the mutated amino acid residues highlighted in stick models.

Figure 3

Optimal temperature and thermal stability of VxAly7B-CM and its mutants. (a) The optimal temperature of VxAly7B-CM and its mutants; (b) the thermal stability of VxAly7B-CM and its mutants at 45 °C; (c) the thermal stability of VxAly7B-CM and its mutants at 50 °C.

Figure 4

Optimal temperature and thermal stability of VxAly7B-CM and the mutant E188N/S204G. (a) The optimal temperature of VxAly7B-CM and the mutant E188N/S204G; (b) the thermal stability of VxAly7B-CM and the mutant E188N/S204G at 45 °C; (c) the thermal stability of VxAly7B-CM and the mutant E188N/S204G at 50 °C.

Figure 5

Analysis of degradation products. (a) TLC analysis of VxAly7B-CM degradation products (M: Marker, containing unsaturated alginate disaccharides and trisaccharides; lanes 1–7 corresponded to degradation products collected at 0, 1, 5, 15, 30, 60, and 120 min, respectively, ΔDP2, ΔDP3, ΔDP4, and ΔDP5 denoted unsaturated di-, tri-, tetra-, and penta-saccharides, respectively); (b) TLC analysis of the E188N/S204G mutant degradation products; (c) FPLC analysis of VxAly7B-CM end degradation products; (d) FPLC analysis of the E188N/S204G mutant end degradation products.

Figure 6

Surface charge and hydrophobicity distribution of proteins: (a) Surface charge distribution of VxAly7B-CM and the E188N/S204G mutant (left to right). Yellow sticks represent the tetrasaccharide substrate; (b) Surface hydrophobicity distribution of VxAly7B-CM and the E188N/S204G mutant (left to right).

Figure 7

Intramolecular interaction analysis. (a) Residue interactions at the 204 site in VxAly7B-CM (relevant amino acid residues are depicted as stick models; green dashed lines represent hydrogen bonds, yellow dashed lines represent polar bonds); (b) residue interactions at the 204 site in E188N/S204G; (c) sequence alignment of VxAly7B-CM with PL7 family alginate lyases and distribution of secondary structures (position 204 is marked in blue; pink triangles denote amino acids interacting with S204 in WT; purple triangles denote amino acids interacting with G204 in the E188N/S204G mutant).

Figure 8

Molecular dynamics simulation of VxAly7B-CM and its mutant E188N/S204G. (a) RMSD values of VxAly7B-CM and E188N/S204G during 100 ns MD simulations at 323 K; (b) RMSF values of VxAly7B-CM and E188N/S204G during 100 ns MD simulations at 323 K; (c) SASA values of VxAly7B-CM and E188N/S204G during 100 ns MD simulations at 323 K.