Rational design of disulfide bonds to improve the thermostability and γ-cyclodextrin production capacity of γ-cyclodextrin glycosyltransferase

The ability of γ-cyclodextrin(γ-CD)to form inclusion complexes with various guest compounds, together with its relatively high solubility, gives γ-CD advantages over both α-cyclodextrin(α-CD) and β-cyclodextrin(β-CD). The thermostability of γ-cyclodextrin glycosyltransferases (γ-CGTase) which can produce γ-CD discovered so far is poor, which is one of the problems limiting its industrial application. To enhance the thermostability of γ-CGTase from Bacillus clarkii 7364, potential disulfide bond mutants were predicted by Disulfide by Design. Five pairs of mutants were constructed by analyzing the predicted results. The t1/2 of S172C-R183C was 2.25 h, which was 3.17 times of WT. Molecular dynamics simulations confirmed that the increase of thermostability of S172C-R183C may be due to the formation of disulfide bond, which leads to the increase of structure rigidity of protein. Then the ability of S172C-R183C mutant to produce cyclodextrins(CDs) was tested. Compared with WT, the maximum CD yield, γ-CD yield and γ-CD specificity of S172C-R183C in the non-complexant catalysed reaction were increased by 6.9 %, 8.8 %, and 2.4 % respectively. In the complexant catalyzed reaction, the CDs yield of S172C-R183C increased by 17.0 %, in which the γ-CD yield increased by 21.7 %, and the γ-CD specificity increased by 3.9 %. Molecular docking results showed that S172C-R183C formed more hydrogen bonds with γ-CD, especially Y186, which was the key central site affecting product specificity. This work involving rational protein engineering provided a new method for adjusting γ-CGTase thermostability.