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

Abstract

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 Bacillus terquilensis 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 T50 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.

Rational engineering of p‐hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway

“…Conversely, the other approach is rational design or semi‐rational design based upon the knowledge about the protein structure, catalytic mechanism, and the sequence similarity among the related homologues. Site‐directed mutagenesis based on rational design is becoming an efficient and economical way for improving enzyme activity and thermostability of target enzymes (Boehlein et al, ; Chen et al, ; Diao et al, ; Niu et al, ; Shen et al, ; Zhang et al, ).…”

“…Conversely, the other approach is rational design or semi-rational design based upon the knowledge about the protein structure, catalytic mechanism, and the sequence similarity among the related homologues. Site-directed mutagenesis based on rational design is becoming an efficient and economical way for improving enzyme activity and thermostability of target enzymes (Boehlein et al, 2015;Chen et al, 2013;Diao et al, 2016;Niu et al, 2015;Shen et al, 2015;Zhang et al, 2015). In this work, a new PobA mutant, based on the rational analysis of molecular docking models including wild-type PobA with 4-HBA or 3,4-DHBA and mutant PobAs with 3,4-DHBA, was designed and generated through site-directed mutagenesis.…”

Molecular characterization, overexpression and comparison of esterases-encoding LipRT, Lip4 and Lip20 from moderately thermophilic and mesophilic bacteria

“…In this study, the helix content of thermostable LipRT was higher than those of Lip20 and Lip4, and was an important role for thermal resistance (Table 1) [3]. Even if the helix content of MtEst45 from Microbulbifer thermotolerans was not as high as other thermally tolerant esterases, increasing β-sheet contents may contribute to the enzymatic stabilization [13]. In addition, some high frequency residues for facilitating thermostability, such as Val, Phe and Arg, were recognized in LipRT.…”

Engineering the thermostability of β-glucuronidase from Penicillium purpurogenum Li-3 by loop transplant

“…5, the backbone atoms of M1, M3, and M8 showed similar root mean square deviation (RMSD) values which were significantly lower than that of wild-type, which is consistent with the greater thermostability of mutants, as demonstrated in the experimental data earlier. The total energy is closely correlated to the stability of a protein: a lower total energy value indicates a more stable protein structure (Niu et al 2015). As shown in Table 3, the most thermostable mutant M1 had the lowest total energy of −13.2 kcal mol −1 , which was 1.5 kcal mol −1 lower than that of wild-type.…”

“…When analyzing the mutants, we found some introduced loops were not compatible with the enzyme structure and they were either severely twisted or showed unnatural conformation, so these mutants were directly excluded. In addition, we also considered the hydrogen bonds number, since many reports have shown that the hydrogen bonds can increase the thermostability of proteins (Niu et al 2015), so the mutants with less hydrogen bonds number than wild-type was also excluded. Based on the above-mentioned analysis, we identified two stable loops which were ideal segments for transplant: DYSTQKR and DYNTKR, which were compatible with the local structure of weak loops to be replaced of PGUS-E and formed more hydrogen bonds (shown in Table S1, Electronic Supplementary Material).…”