Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production.
L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.
Rhimi
Moez
M
Laboratoire d'Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax, Sfax-Tunisie, Tunisia.
Aghajari
Nushin
N
Juy
Michel
M
Chouayekh
Hichem
H
Maguin
Emmanuelle
E
Haser
Richard
R
Bejar
Samir
S
D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006601:Hexoses / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D013696:Temperature
D006601:Hexoses; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C030192:tagatose