Abstract:Cold-adapted maltopentaose-forming amylases have relatively high catalytic activities at low temperatures and enable an efficient and specific conversion of starch to functional maltopentaose at room temperature, resulting in a broad applications in food, medical and pharmaceutical fields. In this study, SdG5A and SdG5A-CD from Saccharophagus degradans were expressed in Bacillus subtilis. Further, the cold adaptation of the recombinant enzymes was characterized and compared. The results showed that recombinant SdG5A retained 27.8% of its maximal activity at 0 ℃ and could produce 48.6% of maltopentaose at room temperature, indicating a strong cold adaptation of SdG5A. In contrast, SdG5A-CD, lacking the linker and starch-binding domain (SBD), did not show any cold adaptation. To investigate the mechanisms that might underlie the cold adaptation of SdG5A, the structure was predicted by RoseTTAFold and the structural flexibility was analyzed through molecular dynamics simulation. An extremely high flexibility in the C-terminal linker-SBD region was observed and its root-mean-square fluctuation at 0 ℃ was consistent with that at 45 ℃, indicating that the highly flexible linker-SBD region acted as a critical component for the cold adaptation of SdG5A.
