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Homology modeling of tyrosinase was performed using the SWISS-MODEL based on the known crystal structure of tyrosinase from S. castaneoglobisporus（PDB No. 2AHL）. The POPMuSiC algorithm was applied to predict the folding free energy change （ΔΔG） of amino acid substitution. Site-directed mutagenesis was used to construct mutants at Arg95Tyr and Gly123Trp. The mutant and wild-type enzymes were expressed in Escherichia coli（DE3）. As compared to the wild-type tyrosinase，all the mutant enzymes showed improved thermal properties. The mutant with combined substitution（Arg95Tyr/Gly123Trp） showed the most pronounced shifts in temperature optima，which was about 5 ℃ upward，and the half-life for thermal inactivation at 60 ℃ was increased by 1.5 times.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.
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