Abstract
The mature glucoamylase from Rhizopus oryzae consists of an N-terminal starch-binding domain (SBD, 106 aa), an o-glycosylated linker (L, 36 aa), and a C-terminal catalytic domain (CD, 437 aa). The SBD processes raw-starch binding ability and the CD can hydrolyze starch completely to glucose. As for the linker region, its function is not well characterized. We have used both E. coli and S. cerevisiae strains to express two recombinant clones, rSBD and rSBD-L. Each protein was directly purified by fast protein liquid chromatography. Saturation binding and amylolytic assays demonstrated that these recombinant proteins behave differently. In addition, Circular Dichroism analysis revealed that rSBD from E. coli and S. cerevisiae possesses a β-strand conformation and the existence of a linker region at the C-terminal end of SBD may influence the structural stability of rSBD. At high temperature, the rSBD-L was found to form amyloid-like structures as determined by Circular Dichroism, Congo Red assay and transmission electron microscopy. However, rSBD alone could only form insoluble aggregates under the same condition. We have demonstrated that the linker region strongly influenced the biochemical function and physical structure of the starch-binding domain. We have further expressed rSBD-s, rSBD-sk and rSBD-skptttta to investigate how many C-terminal residues are required to influence the conformation of starch-binding domain. The results showed that rSBD-s had similar structural properties as rSBD, whereas rSBD-sk and rSBD-skptttta formed β-amyloid-like conformation as rSBD-L. Therefore, we have confirmed that the linker region plays an important role in the structure and function of the starch-binding domain.