Deletion of uncharacterized domain from α-1,3-glucanase of bacillus circulans KA-304 enhances heterologous enzyme production in escherichia coli

Abstract

α-1,3-Glucanase (Agl-KA) of Bacillus circulans KA-304 consists of an N-terminal discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), threonine and proline repeats (TP), a second discoidin domain (DS2), an uncharacterized conserved domain (UCD), and a C-terminal catalytic domain. Previously, we reported that DS1, CBM6, and DS2 have α-1,3-glucan-binding activity and contribute to α-1,3-glucan hydrolysis. In this study, UCD deletion mutant (Agl∆UCD) was constructed, and its properties were compared with those of Agl-KA. α-1,3-Glucan hydrolyzing, α-1,3-glucan binding, and protoplast-forming activities of Agl∆UCD were almost the same as those of Agl-KA. kcat/Km values of Agl∆UCD and Agl-KA were 11.4 and 11.1 s–1 mg–1 mL, respectively. Agl∆UCD and Agl-KA exhibited similar characteristics, such as optimal pH, pH stability, optimal temperature, and thermostability. These results suggest that UCD is not α-1,3-glucan-binding and flexible linker domain, and that deletion of UCD does not affect the affinity of N-terminal binding domains and the catalytic action of the C-terminal domain. Subsequently, heterologous enzyme productivity of Agl∆UCD in Escherichia coli was compared with that of Agl-KA. The productivity of Agl∆UCD was about 4-fold larger than that of Agl-KA after an 8-h induction at 30∞C. In the case of induction at 20∞C, the productivity of Agl∆UCD was also larger than that of Agl-KA. These findings indicate that deletion of only UCD enhances the enzyme productivity in E. coli.