Additional salt bridges improve the thermostability of 1,4-α-glucan branching enzyme

The 1,4-alpha-glucan branching enzyme from Geobacillus thermoglucosidans STB02 (GtGBE, EC 2.4.1.18) does not possess the thermostability required by modified starch industry. To increase its thermostability, a rational design strategy was used to introduce additional salt bridges into GtGBE. The strategy involved in mutation of individual residues to form local two-residue salt bridges. Accordingly, five of local salt bridges (Q231R-D227, Q231K-D227, T339E-K335, T339D-K335, and 1571D-R569 mutants) were separately introduced into GtGBE. The half-times of these mutants at 60 degrees C were 17% to 51% longer than that of wild-type. Subsequently, these tworesidue salt bridges were extended to form salt bridge networks (Q231R/K-D227-D131H, T339D/E-K335-1291H, and 1571D-R569-R617H mutants). Among these mutants, except 1571D-R569-R617H, the half-times of Q231R/K-D227-D131H, T339D/E-K335-1291H mutants at 60 degrees C were 15%, 17%, 21% and 17% longer than those of the corresponding two-residue salt bridges, respectively. The results showed that design and introduction of salt bridges improves enzyme thermostability in GtGBE.