Is it possible to stabilize a thermophilic protein further using sequences and structures of mesophilic proteins: a theoretical case study concerning DgAS.

Incorporating structural elements of thermostable homologs can greatly improve the thermostability of a mesophilic protein. Despite the effectiveness of this method, applying it is often hampered. First, it requires alignment of the target mesophilic protein sequence with those of thermophilic homologs, but not every mesophilic protein has a thermophilic homolog. Second, not all favorable features of a thermophilic protein can be incorporated into the structure of a mesophilic protein. Furthermore, even the most stable native protein is not sufficiently stable for industrial applications. Therefore, creating an industrially applicable protein on the basis of the thermophilic protein could prove advantageous. Amylosucrase (AS) can catalyze the synthesis of an amylose-like polysaccharide composed of only α-1,4-linkages using sucrose as the lone energy source. However, industrial development of AS has been hampered owing to its low thermostability. To facilitate potential industrial applications, the aim of the current study was to improve the thermostability of Deinococcus geothermalis amylosucrase (DgAS) further; this is the most stable AS discovered to date. By integrating ideas from mesophilic AS with well-established protein design protocols, three useful design protocols are proposed, and several promising substitutions were identified using these protocols. The successful application of this hybrid design method indicates that it is possible to stabilize a thermostable protein further by incorporating structural elements of less-stable homologs.