Thermostability modification of β-mannanase from Aspergillus niger via flexibility modification engineering

Abstract

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.

Computer-aided rational design strategy based on protein surface charge to improve the thermal stability of a novel esterase from Geobacillus jurassicus

“…In addition, an integrated rational design strategy to improve the enzymatic thermal stability would be bene cia (Tong et al 2021). Tan et al (Tan et al 2023) coupled B-factor and Gibbs unfolding free energy change (ΔΔG) for rational design of β-mannanase from Aspergillus niger to obtain mutants with increased thermal stability. The development of enzymes through rational engineering is capable of enhancing their e cacy, accuracy, e ciency, and productivity, thereby facilitating the evolution process at a faster rate.…”