Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions.
Based on sequence alignments and homology modeling, Gly 312 and Lys 436 of the maltogenic amylase from Bacillus sp. US149 (MAUS149) were selected as targets for site-directed mutagenesis to improve the thermostability of the enzyme. Variants of MAUS149 with amino acid substitutions G312A, K436R and G312A-K436R had substrate specificities, kinetic parameters and pH optima similar to those of the wild-type enzyme; however, the enzymes with substitutions K436R and G312A-K436R, had an optimal temperature of 45 °C instead of the 40 °C for the wild-type enzyme. The half-life time at 55 °C increased from 15 to 25 min for the double mutant. Molecular modeling suggests that the increase in thermostability was due to new hydrophobic interactions and the formation of a salt bridge and hydrogen bond in the G312A and K436R variants, respectively. The double mutant could be a potential candidate for application in the bread industry.
Ben Mabrouk
Sameh
S
Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, BP 1177, Sfax 3018, Tunisia.
Aghajari
Nushin
N
Ben Ali
Mamdouh
M
Ben Messaoud
Ezzedine
E
Juy
Michel
M
Haser
Richard
R
Bejar
Samir
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000596:Amino Acids / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
D000596:Amino Acids; D050505:Mutant Proteins; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase