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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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author
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organization
publishing date
type
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
2016-04-01 16:37:51
date last changed
2022-01-28 21:01:22
@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}},
  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}},
  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}},
  doi          = {{10.1046/j.0014-2956.2002.02631.x}},
  volume       = {{269}},
  year         = {{2002}},
}