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Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Lönn, Anna LU ; Gárdonyi, Márk LU ; van Zyl, Willem; Hahn-Hägerdal, Bärbel LU and Otero, Ricardo Cordero (2002) In European Journal of Biochemistry 269(1). p.157-163
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... (More)
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. (Less)
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organization
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Contribution to journal
publication status
published
subject
keywords
Hydrogen-Ion Concentration, Kinetics, Magnesium : pharmacology, Manganese : pharmacology, Mutagenesis, Polymerase Chain Reaction, Support Non-U.S. Gov't, Xylitol : pharmacology, Thermus thermophilus : enzymology, Enzyme Stability, Cold, Aldose-Ketose Isomerases : chemistry : genetics : metabolism
in
European Journal of Biochemistry
volume
269
issue
1
pages
157 - 163
publisher
Wiley-Blackwell
external identifiers
  • wos:000173398100018
  • scopus:0036150978
ISSN
0014-2956
DOI
10.1046/j.0014-2956.2002.02631.x
language
English
LU publication?
yes
id
cecb01a0-2423-4b2c-89e2-730d15d9dfb6 (old id 106747)
date added to LUP
2007-06-28 16:10:39
date last changed
2017-09-10 04:35:44
@article{cecb01a0-2423-4b2c-89e2-730d15d9dfb6,
  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.},
  author       = {Lönn, Anna and Gárdonyi, Márk and van Zyl, Willem and Hahn-Hägerdal, Bärbel and Otero, Ricardo Cordero},
  issn         = {0014-2956},
  keyword      = {Hydrogen-Ion Concentration,Kinetics,Magnesium : pharmacology,Manganese : pharmacology,Mutagenesis,Polymerase Chain Reaction,Support Non-U.S. Gov't,Xylitol : pharmacology,Thermus thermophilus : enzymology,Enzyme Stability,Cold,Aldose-Ketose Isomerases : chemistry : genetics : metabolism},
  language     = {eng},
  number       = {1},
  pages        = {157--163},
  publisher    = {Wiley-Blackwell},
  series       = {European Journal of Biochemistry},
  title        = {Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.},
  url          = {http://dx.doi.org/10.1046/j.0014-2956.2002.02631.x},
  volume       = {269},
  year         = {2002},
}