Improving the low-temperature activity of Bacillus subtilis keratinase by site-directed mutagenesis

Abstract:

[Background] The keratinase KerZ1 could efficiently degrade keratin at the optimum temperature of 60℃. However, the poor activity below the optimum temperature makes the enzyme difficult to be applied in industrial production. [Objective] To improve the low-temperature activity of KerZ1. [Methods] Based on homologous alignment and analysis of folding free energy, site-directed mutagenesis was employed for KerZ1, and then the enzymatic properties of the mutants were characterized. [Results] The introduction of T210S, N211S, or T212G into the flexible loop region (loop 13) increased the low-temperature activity of KerZ1. Subsequently, the compound mutant T210S/N211S/T212G was constructed, which showed the activity 45.68% and 85.74% higher than that of KerZ1 at moderate (40℃) and low (20℃) temperatures, respectively. Meanwhile, the half-life (t_1/2) at 60℃ decreased by only 11.52%, indicating that the low-temperature activity of T210S/N211S/T212G was improved without serious loss of thermostability. [Conclusion] The reduction of hydrogen bonds and surface hydrophobicity may be the main reasons for the increased flexibility of the keratinase molecule, which enhances the low-temperature activity of T210S/N211S/T212G. In summary, this study modifies the keratinase to improve its low-temperature activity and lays a foundation for the practical application of this enzyme.

Key words:keratinase;low-temperature activity;molecular modification;homologous alignment;molecular dynamics simulation;molecular flexibility;hydrogen bond