Introductionβ-Mannanases can hydrolyze mannans, which are widely available in nature. However, the optimum temperature of most β-mannanases is too low to be directly utilized in industry.MethodsTo further improve the thermostability of Anman (mannanase from Aspergillus niger CBS513.88), B-factor and Gibbs unfolding free energy change were used to modify the flexible of Anman, and then combined with multiple sequence alignment and consensus mutation to generate an excellent mutant. At last, we analyzed the intermolecular forces between Anman and the mutant by molecular dynamics simulation.ResultsThe thermostability of combined mutant mut5 (E15C/S65P/A84P/A195P/T298P) was increased by 70% than the wild-type Amman at 70°C, and the melting temperature (Tm) and half-life (t1/2) values were increased by 2°C and 7.8-folds, respectively. Molecular dynamics simulation showed reduced flexibility and additional chemical bonds in the region near the mutation site.DiscussionThese results indicate that we obtained a Anman mutant that is more suitable for industrial application, and they also confirm that a combination of rational and semi-rational techniques is helpful for screening mutant sites.
The enzyme β-mannanase is widely used in industrial applications. In this study, we heterologously expressed a mannanase (Anman) strain from Aspergillus niger CBS513.88 in Pichia pastoris and found that it had the highest activity at pH 3.0 and 72.5°C with a molecular weight of approximately 47 kDa. The wild-type mannanase Anman remains stable below 65°C but inactivates rapidly at 70°C. To further improve the thermostability of Anman, we screened multiple mutation sites using B-factor and Gibbs unfolding free energy change, and then combined with multiple sequence alignment and consensus mutation to generate five excellent single-point mutants, the thermostability of combined mutant mut5 (E15C/S65P/A84P/A195P/T298P) was 70% increased than wild type Amman at 70℃, and the melting temperature ( T) and half-life ( t) values were increased by 2°C and 7.8 folds, respectively. Molecular dynamics simulation showed reduced flexibility and additional chemical bonds in the region near the mutation site. These results indicate that we obtained a mannanase mutant that is more suitable for industrial application, and also confirm that a combination of rational and semi-rational techniques is helpful for screening mutant sites.
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