
Cold adaptation of xylose isomerase from
Thermus thermophilus
through random PCR mutagenesis
Gene cloning and protein characterization
Anna LoÈnn
1
,Ma
Ârk Ga
Ârdonyi
1
, Willem van Zyl
2
,BaÈ rbel Hahn-HaÈ gerdal
1
and Ricardo Cordero Otero
2,
*
1
Department of Applied Microbiology, Lund University, Sweden;
2
Department of Microbiology, University of Stellenbosch,
Matieland, South Africa
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 pGEMÒ-T
Easy, and their physicochemical and catalytic properties
were determined. The optimum pH for xylose isomeriza-
tion activity for the mutants was 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 °C, but they did not show any increase in
catalytic eciency (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 inhibi-
tion constants (K
i
) for xylitol than the wild-type, indicat-
ing that they are less inhibited by xylitol. The thermal
stability of the mutated enzymes was poorer than that of
thewild-typeenzyme.Theresultsarediscussedintermsof
increased molecular ¯exibility of the mutant enzymes at
low temperatures.
Keywords: xylose isomerase; cold adaptation; random
mutagenesis; Saccharomyces cerevisiae; xylose fermentation.
The use of ethanol from renewable raw materials is an
attractive alternative for meeting increasing global demand
for liquid fuels because its combustion does not contribute
to the greenhouse effect. For the industrial production of
ethanol from pretreated and hydrolysed lignocellulose, the
yeast Saccharomyces cerevisiae is the prime choice
(reviewed in [1]). Between 10 and 40% of lignocellulosic
raw materials consists of pentoses [2], where xylose is the
predominant portion. However, S. cerevisiae can not
metabolize xylose, only
D
-xylulose, an isomerization
product of
D
-xylose. Xylose reductase (EC 1.1.1.21) and
xylitol dehydrogenase (EC 1.1.1.9) from the xylose-fer-
menting yeast Pichia stipitis, have been introduced into
S. cerevisae to allow xylose fermentation to ethanol [3±5].
Fermentations resulted in low ethanol yields and consid-
erable xylitol by-product formation. Xylose isomerase (XI)
(EC 5.3.1.5) is used in the production of high-fructose corn
syrup, where it catalyses the conversion of
D
-glucose to
D
-fructose [6]. The physiological function of the enzyme
in vivo is, however, the isomerization of the pentose
D
-xylose to
D
-xylulose. XI genes (xylA) from several
bacteria have been introduced into S. cerevisiae, including
xylA from Escherichia coli [7,8], Actinoplanes missouriensis
[9], Bacillus subtilis [9], Lactobacillus pentosus [10] and
Clostridium thermosulfurogenes [11]. However, none of
these attempts generated an active XI.
The only xylA gene successfully expressed in S. cerevi-
siae was cloned from T. thermophilus [12]. This thermo-
philic XI, with a temperature optimum at 85 °C, has a
low activity at 30 °C [12] which is the optimal growth
temperature for S. cerevisiae. It would therefore be
desirable to generate mutants of XI with improved kinetic
properties at low temperatures. Random chemical muta-
genesis has been used recently to obtain variants of the
T. thermophilus 3-isopropylmalate-dehydrogenase [13],
Sulfolobus solfataricus indolglycerol phosphate synthase
[14] and the mesophilic protease subtilisin BPN¢[15±17],
with increased activity at low temperatures. Error-prone
PCR followed by DNA shuf¯ing resulted in the arti®cial
evolution of cold-adapted mutants of a b-glycosidase from
Pyrococcus furiosus [18] and a subtilisin-like protease from
Bacillus sphaericus [19].
Here, we report on random PCR mutagenesis to
create cold-adapted T. thermophilus XI. The character-
ization of the physicochemical and catalytic properties of
three cold-adapted XIs that exhibited up to 9 times
higher k
cat
for xylose than the wild-type enzyme at 60 °C
are described.
Correspondence to B. Hahn-Ha
Ègerdal, Department of Applied
Microbiology, Lund University, PO Box 124, SE-221 00 Lund,
Sweden. Fax: + 46 46 2224203, Tel.: + 46 46 2228428,
E-mail: Barbel.Hahn-Hagerdal@tmb.lth.se
Abbreviations: XI, xylose isomerase.
*Present address: Institute for Wine Biotechnology, University of
Stellenbosch, Private Bag XI, Matieland 7602, South Africa.
(Received 28 May 2001, revised 23 October 2001, accepted 25 October
2001)
Eur. J. Biochem. 269, 157±163 (2002) ÓFEBS 2002