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

Abstract

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 Bacillus terquilensis 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 (Tm) 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.

Crystal structure and catalytic characterization of the dehydrogenase/reductase SDR family member 4 (DHRS4) from Caenorhabditis elegans

“…Indeed, disulfide bridges are known to increase the overall thermal stability of proteins . It has been reported that not only peptide bonds but also disulfide bridges do not resist temperatures of more than 80 °C.…”

Insights from molecular dynamics simulations for computational protein design

“…This study demonstrates the capacity of MD to aid in the rational selection of positions for the introduction of disulfide bonds into proteins. This approach improves on the prediction of amino acid pairs for the introduction of a disulfide bond by factoring in protein flexibility and conformational heterogeneity within the native state and has been used recently by others 72 .…”

Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability

“…Computer-assisted rational design is an attractive alternative to accelerate the enzyme engineering process. In the context of computational design to improve the thermostability of a biocatalyst, several approaches are widely used, such as the consensus sequence (comparing amino acid sequences with a higher thermostability), B-factor/RMSF analysis, molecular dynamics (MD) simulations, constraint network analysis (CNA), disulfide bonds (DSBs) design, and stabilizing salt-bridge design based on the sequence and structure of enzymes [18,19,20,21,22,23].…”

“…In recent years, a few studies have applied the DSB design approach to improve the thermostability of various enzymes, such as alkaline α-amylase [27], cellulases [28], and amadoriase [29]. Recently, the DSB engineering (N31C-T187C/P102C-N125C) of a mesophilic β-glucanase from Bacillus terquilensis showed an enhancement of the thermostability by 48.3% in increasing the half-life at 60 °C and a 4.1 °C rise in melting temperature compared to wild type [22]. However, DSB engineering to improve the thermostability remains to be unexplored for endoglucanase II (EGLII) from P. verruculosum.…”