Protein-engineered Bacillus alpha-amylases that have acquired both enhanced thermostability and chelator resistance

"Thermostability and chelator resistance of the liquefying alkaline alpha-amylase (AmyK) from alkaliphilic Bacillus sp. strain KSM-1378 were examined by deletion of either Argl81-Glyl82 or Thr183-Glyl84 on a loop in domain B. In the tertiary structure of Bacillus stearothermophius alpha-amylase (BSA), Ile181-Gly182 (Thr183-Gly184 in AmyK) pushes away a spatially contacting region containing Ca2+-coordinating Asp207 (Asp209 in AmyK). Therefore, the deletion of Ile181-Gly182 rather than Arg179-Gly180 was predicted to result in a higher thermostability of BSA. However, our results with AmyK were clearly contrary to this prediction. The resistance to EDTA of both mutant enzymes from AmyK was essentially equal, and the Arg181-Gly182-deleted mutant was more thermostable than the Thr183-Gly184-deleted one. It strongly implies that the microenvironmental topology around the loop containing these dipeptides in AmyK is different from that in BSA."