title: Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100 strain.
abstract: The implications of Asn315 and Val450 in the atypical starch hydrolysis profile of Bacillus stearothermophilus Amy (a-amylase) US100 have been suggested previously [Ben Ali, Mhiri, Mezghani and Bejar (2001) Enzyme Microb. Tech. 28, 537-542]. In order to confirm this hypothesis, three mutants were generated. Of these two have a single mutation, N315D or V450G, whereas the third contains both mutations. Analysis of the starch breakdown-profile of these three mutants, as well as of the wild-type, allowed us to conclude that each single mutation induces a small variation in the hydrolysis product. However, the major end product produced by the double mutant shifts from maltopentaose/maltohexaose to maltose/maltotriose, confirming the involvement of these two residues in starch hydrolysis. The superimposition of AmyUS100 model with that of Bacillus licheniformis shows in AmyUS100 an additional loop containing residues Ile214 and Gly215. Remarkably, the deletion of these two residues increases the half-life at 100 degrees C from 15 min to approx. 70 min. Moreover, this engineered amylase requires less calcium, 25 p.p.m. instead of 100 p.p.m., to reach maximal thermostability.
authors.affiliation: Centre de Biotechnologie de Sfax, BP K 3038 Sfax, Tunisia.
authors.forename: Mamdouh
authors.initials: M
authors.lastname: Ben Ali
authors.forename: Bassem
authors.initials: B
authors.lastname: Khemakhem
authors.forename: Xavier
authors.initials: X
authors.lastname: Robert
authors.forename: Richard
authors.initials: R
authors.lastname: Haser
authors.forename: Samir
authors.initials: S
authors.lastname: Bejar
mesh_terms: D019943:Amino Acid Substitution; D000681:Amylases / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000145:classification* / Q000201:enzymology*; D006358:Hot Temperature; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013213:Starch / Q000378:metabolism*
chemical_list: D009844:Oligosaccharides; C016549:maltohexaose; D013213:Starch; D000681:Amylases; D002118:Calcium