Computer-Aided Targeted Mutagenesis of Thermoclostridium caenicola d-Allulose 3-Epimerase for Improved Thermostability

Abstract

D-Allulose 3-epimerase (DAEase) is a key enzyme in D-allulose bioproduction. DAEase from Thermoclostridium caenicola suffers from poor thermostability, hampering its large-scale applications in industry. In this study, mutants A70P, G107P, F155Y, and D162T with increased melting point temperature (T m ) were obtained by targeted mutagenesis based on the calculation of the free energy of folding. The optimal single-point mutant G107P showed 11.08 h, 5, and 5.70 °C increases in the values of halflife (t 1/2 ) at 60 °C, the optimum temperature (T opt ), and T m , respectively. Beneficial mutations were combined by ordered recombination mutagenesis, and the combinational mutant Var3 (G107P/F155Y/D162T/A70P) was generated with ΔT opt of 10 °C and ΔT m of 12.25 °C. Its t 1/2 value at 65 °C was more than 140 times higher than that of the wild-type enzyme. Molecular dynamics simulations and homology modeling analysis indicated that the enhanced overall rigidity, increased hydrogen bonds between subunits, and redistributed surface electrostatic charges might be responsible for the improved thermostability of the mutant Var3.

Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

“…The high thermostability of proteins are associated with their specific structural features, including shorter loops and increased helical content, more hydrogen bonds and salt bridges, smaller and less numerous cavities, and greater hydrophobicity or hydrophobic cores . Hence, various strategies have been suggested to enhance enzyme thermostability based on homologous alignment, protein surface charge, disulfide bonds, proline effect, temperature factor (B-factor), and free energy for protein folding. − These methods have mainly attempted to increase structural rigidity of proteins by modifying the interaction of the residues with covalent or noncovalent bonds. Of note, the free energy of protein folding has been lowered to increase the thermostability of some proteins with the help of the PoPMuSiC algorithm. , PoPMuSiC displays good prediction performance, which can predict the stability changes resulting from all possible mutations in a medium size protein in less than a minute .…”

Improving the thermostability of alginate lyase FlAlyA with high expression by computer-aided rational design for industrial preparation of alginate oligosaccharides

“…The free energy of folding (ΔG) is an important indicator for judging the thermodynamic stability of a protein ( Cao et al, 2019 ). The difference in free energy of folding between WT and its mutants indicates the mutational effect ( Chen et al, 2022 ). In this study, the ΔΔG of the mutant H71K and H176D were calculated used the DeepDDG web server ( http://protein.org.cn/ddg.html ).…”

“…Chen et al constructed a combinational mutant Var3 (G107P/F155Y/D162T/A70P) of T. caenicola DAEase, which increased the T m value by 12.25°C. Among them, the positive effect of the thermal stability of the mutant D162T may be due to the redistribution of the surface electrostatic charge due to the removal of the carboxyl group ( Chen et al, 2022 ). Therefore, as shown in Figure 5D , the positive effect of mutant H71K on thermal stability may be attributed to the redistribution of surface electrostatic charges caused by the addition of NH 3 + .…”

Fine-Tuning of Carbon Flux and Artificial Promoters in Bacillus subtilis Enables High-Level Biosynthesis of d-Allulose

“…Many researchers have made great efforts to improve the catalytic efficiency and thermostability of DAEases . An in vitro enzymatic process using ApDAE with excellent thermostability and activity was conducted, in which the conversion rate of d -allulose reached 27% with 500 mg/L d -fructose as a substrate .…”

“…Many researchers have made great efforts to improve the catalytic efficiency and thermostability of DAEases. 14 An in vitro enzymatic process using ApDAE with excellent thermostability and activity was conducted, in which the conversion rate of D-allulose reached 27% with 500 mg/L D-fructose as a substrate. 15 However, enzymatic conversion processes cannot compete with microbial fermentation in long-term and largescale industrial production due to the high maintenance costs for the continuous production of expensive purified enzymes.…”