Lysine-Based Site-Directed Mutagenesis Increased Rigid β‑Sheet Structure and Thermostability of Mesophilic 1,3–1,4-β-Glucanase

1,3–1,4-β-Glucanase is widely applied in the food industry, while its low thermostability often reduces its performance. In a previous study, chemical modification of surface lysine residues was proved to increase the thermostability of β-glucanase. To improve the thermostability, the mesophilic β-glucanase from <i>Bacillus terquilensis</i> was rationally engineered through site-directed mutagenesis of the 12 lysines into serines. The results showed that the K20S, K117S, and K165S mutants could both enhance the specific activities and thermostability of β-glucanase. The triple mutant (K20S/K117S/K165S) could increase the optimal temperature and <i>T</i>₅₀ value by 15 and 14 °C, respectively. Five percent more structured residues were observed in the mutant, which formed new β-sheet structures in the concave side. Molecular dynamics simulation analysis showed that the flexibility in the mutation regions was decreased, which resulted in the overall rigidity of the β-glucanase. Therefore, the lysine-based site-directed mutagenesis is a simple and effective method for improving the thermostability of β-glucanase

Comparison of CD spectra of wild-type BglTM and N31C-T187C/P102C-N125C mutant in 20 mM phosphate buffer (pH6.5).

<p>Comparison of CD spectra of wild-type BglTM and N31C-T187C/P102C-N125C mutant in 20 mM phosphate buffer (pH6.5).</p

Comparison of RMSF curves (a), overall RMSD curves (b) and local RMSD curves in mutant regions N31C-T187C (c) and P102C-N125C (d) of wild-type BglTM and N31C-T187C/P102C-N125C mutant by MD simulation at 500K for 20 ns.

<p>Comparison of RMSF curves (a), overall RMSD curves (b) and local RMSD curves in mutant regions N31C-T187C (c) and P102C-N125C (d) of wild-type BglTM and N31C-T187C/P102C-N125C mutant by MD simulation at 500K for 20 ns.</p

The optimal pH (a) and pH stability (b) of wild-type BglTM and the mutants.

<p>The optimal pH (a) and pH stability (b) of wild-type BglTM and the mutants.</p

Comparison of the 3D structures of wild-type BglTM and N31C-T187C/P102C-N125C mutant (the mutant residues are shown in sticks).

<p>(a) Comparison of the overall 3D structures of wild-type BglTM and N31C-T187C/P102C-N125C mutant; (b) Comparison of the hydrogen bond network within the 5 Å region around the mutant site of N31C-T187C between wild-type BglTM and N31C-T187C/P102C-N125C mutant; (c) Comparison of the hydrogen bond network within the 5 Å region around the mutant site of P102C-N125C between wild-type BglTM and N31C-T187C/P102C-N125C mutant.</p

Comparison of the average RMSD values in the mutant regions (within 5Å distance from the disulfide bonds) between wild-type BglTM and the mutants.

<p>Comparison of the average RMSD values in the mutant regions (within 5Å distance from the disulfide bonds) between wild-type BglTM and the mutants.</p

Comparison of the average overall RMSD values between wild-type BglTM and the mutants.

<p>Comparison of the average overall RMSD values between wild-type BglTM and the mutants.</p

Comparison of electrostatic surface potential of wild-type BglTM and N31C-T187C/P102C-N125C mutant.

<p>Comparison of electrostatic surface potential of wild-type BglTM and N31C-T187C/P102C-N125C mutant.</p

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from <i>Bacillus terquilensis</i>

<div><p>1,3–1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from <i>Bacillus terquilensis</i> was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C) were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (<i>T</i>_m) compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.</p></div

The optimal temperatures (a), enzyme inactivation curves at 60°C (b) and kinetic stability curves (c) of wild-type BglTM and the mutants.

<p>The optimal temperatures (a), enzyme inactivation curves at 60°C (b) and kinetic stability curves (c) of wild-type BglTM and the mutants.</p