Amylase A-180, which is secreted by a new alkaliphilic organism, isolate 163-26, consists of a single type of polypeptide chain of 186.5 kDa and hydrolyses starch by exo-attack releasing malto-pentaose as preferential product. The structure/function relationship of this unusual starch-degrading enzyme was analysed by introducing 3' deletions into the structural gene. It was found that removal of up to a 110-kDa portion from the C-terminus leaving 563 N-terminal amino acids still led to the formation of a fully active enzyme. The part of the structural gene coding for these 563 N-terminal amino acids was fused with the signal peptide-encoding segment of the cyclodextrin glucanotransferase gene from Klebsiella oxytoca and was cloned into an expression vector. The resulting truncated A-180 derivative, A-180/21, was efficiently transported through the cytoplasmic membrane and released into the medium by an Escherichia coli strain which 'leaks' periplasmatic components. A-180/21 was purified and its catalytic properties, i.e. specific activity and product specificity, proved to be identical to those of the wild-type enzyme; however, in contrast to the wild-type enzyme, it was unable to bind to raw starch and it displayed an altered temperature and pH dependence of activity.
Download Full PDF Version (Non-Commercial Use)