Enhancement of the thermostability of Streptomyces kathirae SC-1 tyrosinase by rational design and empirical mutation

•PoPMuSiC algorithm was applied to predict more thermostable tyrosinase.•Site-directed mutagenesis was applied to obtain more thermostable tyrosinase.•Mutants showed 3-fold and 10 °C increase in half-life and optimal temperature.•The additional hydrogen bonds may be of value to improve the thermostability.•The improved thermostability may be due to the newly formed favorable interaction.

This study aimed to improve the thermostability of a newly cloned tyrosinase from Streptomyces kathirae SC-1. The POPMuSiC algorithm was applied to predict the folding free energy change (ΔDG) of amino acid substitution. Site-directed mutagenesis was used to construct mutants (Q7K, G234P, and Q7K/G234P), and the mutant, and wild-type enzymes were expressed in Escherichia coli (DE3). Compared to the wild-type tyrosinase, all three mutant enzymes showed improved thermal properties. The mutant with combined substitution (Q7K/G234P) showed the most pronounced shifts in temperature optima, about 10 °C upward, and the half-life for thermal inactivation at 60 °C, and melting temperatures were increased by 3 times and approximately 10 °C, respectively. Finally, the mechanisms responsible for the increased thermostability were analyzed through comparative analysis of structure models. The structure-based rational design strategies in this study may also provide further insight into the thermostability of other industrial enzymes and suggest further potential industrial applications.