title: Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.
abstract: Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.
authors.affiliation: Department of Applied Microbiology, Lund University, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Lönn
authors.forename: Márk
authors.initials: M
authors.lastname: Gárdonyi
authors.forename: Willem
authors.initials: W
authors.lastname: van Zyl
authors.forename: Bärbel
authors.initials: B
authors.lastname: Hahn-Hägerdal
authors.forename: Ricardo Cordero
authors.initials: RC
authors.lastname: Otero
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003080:Cold Temperature; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D016963:Thermus thermophilus / Q000201:enzymology*; D014993:Xylitol / Q000494:pharmacology
chemical_list: D008345:Manganese; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D008274:Magnesium; D014993:Xylitol