Comparative study of the structure/function relationship of wild-type and structurally modified maltopentaose-producing amylase.
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.
Candussio
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A
Lehrstuhl für Mikrobiologie der Universität München, Federal Republic of Germany.
Schmid
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G
Böck
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A
D000595:Amino Acid Sequence; D000681:Amylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D009844:Oligosaccharides; D011994:Recombinant Proteins; C019193:maltopentaose; D000681:Amylases; C070287:amylase A 180