Abstract
This study presents a multi-factor rational design strategy combined with molecular dynamics simulation to improve the thermostability of Streptomyces cyaneofuscatus strain Ms1 tyrosinase. Candidate mutation sites were identified using Discovery Studio and FoldX software, and the double mutant G124W/G137W was obtained. The mutant was heterogeneously expressed in Escherichia coli strain Rosetta2 (DE3), and its thermostability was verified. Results indicate that the rational design method, combined with molecular dynamics simulation and protein energy calculation, improved the enzyme's thermostability more accurately and effectively. The double mutant G124W/G137W had an optimum temperature of 60°C, about 5.0°C higher than that of the wild-type TYRwt, and its activity was 171.06% higher than the wild-type TYRwt. Its thermostability was enhanced, 42.78% higher than the wild-type at 50°C. These findings suggest that the rational design strategy applied in this study can facilitate the application of industrial enzymes in the pharmaceutical industry.