|Title||Enhancing the thermostability of Rhizopus chinensis lipase by rational design and MD simulations.|
|Publication Type||Journal Article|
|Year of Publication||2020|
|Authors||Wang R, Wang S, Xu Y, Yu X|
|Journal||Int J Biol Macromol|
|Date Published||2020 Oct 01|
To improve the thermostability of r27RCL from Rhizopus chinensis and broaden its industrial applications, we used rational design (FoldX) according to ΔΔG calculation to predict mutations. Four thermostable variants S142A, D217V, Q239F, and S250Y were screened out and then combined together to generate a quadruple-mutation (S142A/D217V/Q239F/S250Y) variant, called m31. m31 exhibited enhanced thermostability with a 41.7-fold longer half-life at 60 °C, a 5 °C higher of t, and 15.8 °C higher of T compared to that of r27RCL expressed in Pichiapastoris. Molecular dynamics simulations were conducted to analyze the mechanism of the thermostable mutant. The results indicated that the rigidity of m31 was improved due to the decreased solvent accessible surface area, a newly formed salt bridge of Glu292:His171, and the increased ΔΔG of m31. According to the root-mean-square-fluctuation analysis, three positive mutations S142A, D217V, and Q239F located in the thermal weak regions and greatly decreased the distribution of thermal-fluctuated regions of m31, compared to that of r27RCL. These results suggested that to simultaneously implement MD simulations and ΔΔG-based rational approaches will be more accurate and efficient for the improvement of enzyme thermostability.
|Alternate Journal||Int J Biol Macromol|