Improving the thermostability of alpha-amylase by combinatorial coevolving-site saturation mutagenesis

Background: The generation of focused mutant libraries at hotspot residues is an important strategy in directed protein evolution. Existing methods, such as combinatorial active site testing and residual coupling analysis, depend primarily on the evolutionary conserved information to find the hotspot residues. Hardly any attention has been paid to another important functional and structural determinants, the functionally correlated variation information--coevolution.Results: In this paper, we suggest a new method, named combinatorial coevolving-site saturation mutagenesis (CCSM), in which the functionally correlated variation sites of proteins are chosen as the hotspot sites to construct focused mutant libraries. The CCSM approach was used to improve the thermal stability of α-amylase from Bacillus subtilis CN7 (Amy7C). The results indicate that the CCSM can identify novel beneficial mutation sites, and enhance the thermal stability of wild-type Amy7C by 8°C ( T 50 30), which could not be achieved with the ordinarily rational introduction of single or a double point mutation.Conclusions: Our method is able to produce more thermostable mutant α-amylases with novel beneficial mutations at new sites. It is also verified that the coevolving sites can be used as the hotspots to construct focused mutant libraries in protein engineering. This study throws new light on the active researches of the molecular coevolution.