Thermostabilization of a thermophilic 1,4-α-glucan branching enzyme through C-terminal truncation

Abstract Thermophilic proteins are useful for the detailed investigation of thermostability because they function efficiently at high temperatures. Comparison of the amino acid sequences and three-dimensional structures of mesophilic and thermophilic 1,4-α-glucan branching enzymes (GBEs) shows that the amino acid sequence of the last 26 residues at the C-terminal end of the GBE from Geobacillus thermoglucosidans STB02 (GBE Gt , GenBank accession no. KJ660983 ) are not conserved, and that their 3-dimensional structure is flexible. These residues appear to be modified based upon a balance between flexibility and rigidity that is related to thermostability. In this study, a truncated mutant of GBE Gt made by removing the last 26 residues from its C-terminal end was found to have increased thermostability and solubility, compared with the wild-type enzyme. Additionally, truncation of a portion of the C-terminus resulted in a decrease in aqueous stability. The circular dichroism spectra of GBE Gt and GBE Gt ΔC were also found to be different. These results suggest that deletion of flexible residues at the C-terminal end of GBE Gt , which are located on the surface of the enzyme, enhances the thermostability of the enzyme without significantly compromising its enzymatic activity.