Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.
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.
Lönn
Anna
A
Department of Applied Microbiology, Lund University, Sweden.
Gárdonyi
Márk
M
van Zyl
Willem
W
Hahn-Hägerdal
Bärbel
B
Otero
Ricardo Cordero
RC
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
D008345:Manganese; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D008274:Magnesium; D014993:Xylitol