Báo cáo Y học: Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis Gene cloning and protein characterization doc

Cold adaptation of xylose isomerase from
Thermus thermophilus
through random PCR mutagenesis
Gene cloning and protein characterization
Anna LoÈnn
1
,Ma
Â
rk Ga
Â
rdonyi
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 x ylA 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 w ere cloned and expressed
in Escherichia coli HB101 using the v ector pGEM Ò-T

Easy, and their physicochemical and catalytic properties
were determined. T he optimum pH for xylose isomeriza-
tion activity for the mut ants was  7.0, which is similar to
the wild-type e nzy me. Compared w ith 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 s how any increase in
catalytic eciency ( k
cat
/K
m
). For
D
-glucose, both the k
cat
and the k
cat
/K
m
values for the mutants were increased
compared w ith the wild-type enzyme. Furth ermore, the
mutant enzymes exhibited up to 255 times higher inhibi-
tion constants (K
i
) for xylitol than the wild-type, indicat-

ing t hat they are less in hibited 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 g lobal 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 i n [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 x ylose, 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-
mentin g yeast Pichia stipiti s, have been introduced in to
S. cerevisae to allow xylose fermentation to ethanol [3±5].
Fermentations r esulted in l ow ethanol yields and consi d-
erable xylitol by-product f ormation. Xylose isomeras e ( XI)
(EC 5 .3.1.5) i s u sed 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 v ivo 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 succe ssfully 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 a ctivity 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 t he 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 c atalytic properties of
three c old-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
È
gerdal, Department of Applied
Microbiology, Lund University, PO Box 124, SE-221 00 Lund,
Sweden. Fax: + 46 46 2224203, Tel.: + 46 46 2228428,
E-mail:
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) Ó FEBS 2002
MATERIALS AND METHODS
Chemicals
All chemicals were obtained from commercial suppliers and
used as described by the manufacturer.
D
(+)-xylose was
obtained f rom S igma (Steinheim, Germany) and sorbitol
dehydrogenase from Boehringer Mannheim (Mannheim,
Germany).
Strains and plasmids
Escherichia coli HB101(F-hsdS20ara-1 recA13 proA12

lacY1 galK2 rspL20 mtl-1xyl-5) [20] was used for cloning
of the mutated XIs using pGEMÒ-T Easy vector (Promega,
Madison, WI, USA).
PCR mutagenesis
Random mutagenesis of the XI gene (xylA) was performed
under conditions described previously [21] using the PCR
primers 5¢-TGATC AATGTACGAGCCCAAACC-3¢ and
5¢-TGATCACCCCCGCACC-3¢, which directly ¯ank the
xylA gene. Both primers contained the restriction endonuc-
lease site for BclI (underlined). The PCR contained:
1 ´ PCR buffer (BIOTAQä), 0.2 m
M
dATP, 0.2 m
M
dGTP, 1 m
M
dCTP, 1 m
M
dTTP, 1.5 m
M
MgCl
2
,0.5m
M
MnCl
2
,0.15l
M
of both primers, 0.02 n
M

template DNA
and 5 U Taq DNA polymerase (BIOTAQä)inatotal
volume of 100 lL. PCR was performed in a Thermal Cycler
(PerkinElmer 2400) for nine cycles: 30 s at 94 °C, 30 s at
50 °Cand45sat68°C. The PCR products were then
puri®ed using H igh Pureä PCR Product (Boehringer
Mannheim).
DNA sequencing
Analysis of the mutated sequences was carried out using
ABI PRISMÒ Big Dyeä Terminator cycle sequencing
ready reaction kits with an ABI PRISMä 377 DNA
sequencer (PE/Applied Biosystems). Both t he coding and
the noncoding strands were sequenced to ensure the reliable
identi®cation of all mutations.
Growth conditions and preparation of cell extract
from
E. coli
E. coli HB101 h arbouring the plasmids pGEM
Ò
-T Easy
containing the wild-type and the mutated XI genes w ere
grown at 37 °C in 50 mL Luria±Bertani medium [22]
containing 100 lgámL
)1
ampicillin. The cells were har-
vested by centrifugation in the stationary phase of growth
and wash ed once w ith i ce-cold d istilled w ater. W ashed
cells were resuspended in 100 m
M
triethanolamine,

pH 7.0, 65 kUámL
)1
lysozyme, 0.25 mgámL
)1
DNAse
and 1 m
M
phenylmethanesulfonyl¯uoride in dimethylsulf-
oxide. The s olutions were kep t at room temperature for
1 h and then on ice for 2 h before storing in a freezer at
)20 °C. Cell extracts were thaw ed on ice, cell d ebris was
removed by centrif ugation (15 000 g for 1 5 min at 4 °C)
and the supernatant was used as the crude enzyme
preparation.
Protein determination
Protein concentration was determined using the Pierce
protein reagent with bovine serum albumin as standard [23].
Page
SDS/PAGE was performed as previously described [24].
Immunochemical determination of XI
Rabbit antiserum against XI from Streptomyces rubiginosus
was prepared by Antibody AB (So
È
dra Sandby, Sweden) and
immunoblotting was performed as described previously
[25]. Brie¯y, 2 lg of cell-free e xtract together with 2±50 ng
of puri®ed XI from S. rubiginosus were resolved by SDS/
PAGE and were then electrophoretically transferred onto a
poly(vinylidene di¯uoride) membrane (Bio-Rad, Hercules,
CA, USA). The blotted proteins were identi®ed immuno-

chemically by sequential addition of anti-XI serum followed
by goat anti-(rabbit IgG) Ig conjugated with alkaline
phosphatase (Bio-Rad, Hercules, CA, USA). The secondary
antibody was detected with a S torm 860Ò (Pharmacia
Amersham, Uppsala, Sweden) using a chemi¯uorescent
substrate ECF (Pharmacia Amersham). Data analysis was
performed using
IMAGE QUANT
Ò software (Pharmacia
Amershamm), giving a quantitative measurement of the
amount of XI in the cell-free extracts. These data were used
with the maximum velocity ( V
max
)tocalculatek
cat
.
Enzyme assays
A two-step XI standard assay (0.5 mL) was modi®ed from
[26]. A substrate concentration of 700 m
MD
-xylose was
used at 60 °C in 200 m
M
triethanolamine at pH 7.0 in the
presence of 10 m
M
MnCl
2
and crude enzyme preparations.
Glucose isomerase activity was assayed under the same

reaction conditions as those used in the XI assay, except that
glucose instead of xylose was used in the reaction mixture.
The reactions were stopped by adding 150 lL 50%
trichloroacetic acid, and then 2
M
Na
2
CO
3
was added to
neutralize the solutions. The isomerization products, xylu-
lose or fructose, were reduced at pH 7.0 (37 °C) with 0.04 U
sorbitol dehydrogenase (SDH) or 0.5 U SDH, respectively,
and 0.15 m
M
NADH using a COBAS MIRA plus (Roche,
Mannheim, Germany). The rate of disappearance of
NADH was followed at 340 nm and the amount of
D
-xylulose and
D
-fructose determined from calibration
curves. One unit o f isomerase activity was de®ned as the
amount of crude enzyme r equired to produce 1 lmol of
product per minute under the assay conditions employed.
The speci®c activity (Uámin
)1
ámg
)1
) was determined from

the a ctivity and the protein co ncentration of the crude
enzyme preparations.
Kinetic parameters
The kinetic parameters, V
max
(lmolámin
)1
ámg
)1
)and
Michaelis constant (K
m
,m
M
), were determined from
Michaelis±Menten plots of speci®c activities at various
substrate concentrations. Typically, duplicate measure-
ments at 6±10 concentrations of substrate s panning the
value of K
m
were used to determine the value of K
m
.The
158 A. Lo
È
nn et al. (Eur. J. Biochem. 269) Ó FEBS 2002
concentration of XI in the c ell-free extracts was determined
immunochemically using a molecular mass of 44 000 kDa
[27], to allow calculation of the catalytic rate constant (k
cat

)
from the relationship k
cat
 V
max
/[E
0
], where [E
0
]  tot al
enzyme concentration [28].
The K
i
(m
M
) for xylitol was d etermined by i ncubating
crude enzyme pre paration s in different xylose concentra-
tions (20±600 m
M
) at different ®xed xylitol concentrations.
By plotting the speci®c activities for each xylitol concentra-
tion against the xylose concentrations, K
i
was determined
using the equation K
m
¢  K
m
á(1 + i/K
i

)[29],wherei is the
xylitol concentration (m
M
)andK
m
¢ the a pparent K
m
value
at a certain concentration of xylitol.
PH pro®le
The effect of pH on the a ctivity of t he wild-typ e a nd
mutated enzymes was investigated in the pH range 5±10 in
700 m
M
xylose, 10 m
M
MnCl
2
and a buffer prepared b y
mixing acetate, Pipes, Hepes and glycine, to a ®nal
concentration o f 5 0 m
M
each [30]. The pH was adjusted
at 60 °C with NaOH. Above pH 7.0 corrections were made
for the chemical isome rization of
D
-xylose.
Temperature pro®le
The temperature pro®les for the wild-type XI and mutated
XIs were m easured at temperatures between 30 and 95 °C.

Above 60 °C corrections were made for the chemical
isomerization of
D
-xylose.
Preparation of metal-free XI and metal ion effects
on enzyme activity
Metal-free enzymes were prepared as previously described
[26]. N o isomerase activity was observed in the absence of
Mn
2+
,Mg
2+
or Co
2+
. The effect of metal ions on XI
activity was determined by adding 10 m
M
®nal concentra-
tion of either CoCl
2
,MnCl
2
or MgCl
2
to the metal-free
enzyme preparations in the assay mixture.
Enzyme stability
The temperature stability of the wild-type XI and mutated
XIs was investigated by incubating metal-free crude enzyme
preparations in 200 m

M
triethanolamine, pH 7.0 with
10 m
M
MnCl
2
in airtight tubes at 70 °C. At different times,
100-lL samples were withdrawn and stored on ice until the
residual activity was determined.
RESULTS
Isolation of XI mutants with increased activity
at low temperatures
One-step mutagenesis was used to screen for mutant XIs
with improved activity at low temperatures. The mutated XI
fragments were cloned i nto the vector E. coli pGEMÒ-T
Easy and transformed into the E. coli HB101 (xyl-5)strain
to generate a mutant library. Transformants were replica
plated on McConkey agar plates, complemented with 1%
xylose and c ultivated at 3 7 °C overnight. A fter a further
2 days o f incubation at 30 °C, the p H indicator in t he
medium allowed detection and quanti®cation of red acid-
producing colonies. Three candidate mutants, termed
M-1021, M-1024 a nd M-1026 were identi®ed. Colonies of
these three were a d eeper red on the McConkey/xylose
medium than were wild-type xylA colonies (suggesting
higher XI activity). DNA s equencing revealed t hat the
mutants exhibited approximately 80% transitions (T to C)
and 20% transversions (A to C or T).
XI from T. thermophilus is a homotetrameric enzyme
with a 387-residue subunit. Each monomer c omprises two

domains: the larger N-terminal domain (domain I, residues
1±321), which f olds into a (b/a)
8
barrel, and t he smaller
C-terminal domain ( domain II, residues 322±387), which
consists of loops and helices (Fig. 1) [31]. Domain II extends
from domain I and makes extensive contacts with a
neighbouring subunit. M-1021 contained two mutations in
domain II; E372G and V 379A. M-1024 possessed two
mutations, one in domain I (F163L) and one in domain II
(E372G). M-1026 carries one mutation in domain II that is
shared by M-1024 and M-1021; E372G. The locations of the
amino-acid substitutions in the original tertiary structure of
XI are shown i n Fig. 1. Neither the substrate-binding sites
(H53, D56 and K 182) nor th e metal-binding sites (E180,
E216, H219, D244, D254, D256 and D286) were affected by
the mutations in the mutant enzymes.
Properties of the mutant enzymes
Temperature pro®les. XI from T. thermophilus has a
temperature optimum around 95 °C [30]. To investigate
whether the mutations caused any change in the tempera-
ture optimum the temperature pro®les were investigated
from 30 to 95 °C (Fig. 2). The temperature optimum for
M-1024 and M-1026 was around 5 °C higher t han t he
optimum for the wild-type (90 °C). For M-1021 the
temperature optimum was somewhat lower,  75 °C. At
30 °C the speci®c activity was higher for the mutants than
for the wild-type XI. Due to the overall low activity of the
enzymes at this temperature, the physicochemical and
kinetic characterization of the wild-type and mutant

enzymes was carried out at 60 °C.
PH pro®les. XI from T. thermophilus shows a pH optimum
around 7.0 [30]. To examine w hether the mutations altered
the pH dependence for xylose isomerization, the activity of
Fig. 1. Structure of one subunit of T. thermophilus XI. The amino acids
372, 379 and 163 are identi®ed to show the position of the mutations.
Ó FEBS 2002 Mutant xylose isomerases (Eur. J. Biochem. 269) 159
each m utant enzyme was measured as a function of pH
(Fig. 3). The activity of each enzyme relative t o t he
maximum activity was plotted as a percentage against pH.
The pH dependence of the enzyme activity was examined at
a substrate concentration well above K
m
, where the velocity
of the r eaction is p roportional to k
cat
. The pH activity
pro®les of the mutants were broader, and extended into the
alkaline region, compared with the wild-type XI. The wild-
type showed no XI activity at pH 9 and 10. For M-1024 and
M-1026 the speci®c activit y at pH 9 and 10, was 66 and
45%, and 62 and 31%, of the maximum, respectively. The
pH optima for the mutant XIs were not signi®cantly
different from that of the wild-type, i.e. around 7.0.
Eect of metal ions. XIs r equire two metal ions to be
bound to the active site of each monomer in order to exhibit
enzyme activity [32]. However, XIs from different organisms
require different metals for optimal activity [33], a nd XI
from T. thermophilus requires e ither M g
2+

or Mn
2+
for
100% activity [30]. Metal ions are not only essential for the
catalytic mechanism, but they also co ntribute to the
stabilization of the native structure, which is especially
important for thermophilic enzymes.
The effect of different bivalent metal ions (Mn
2+
,Mg
2+
and Co
2+
) on the EDTA-treated enzymes was investigated
(Table 1). The wild-type and mutated XIs were most
effectively activated by Mn
2+
and, to a smaller degree, by
Mg
2+
and Co
2+
. The wild-type showed 88 and 74% of the
maximum activity with Co
2+
and Mg
2+
, respectively. The
mutants, on the other hand, were less activated by Co
2+

and Mg
2+
.
Kinetic properties of
D
-xylose and
D
-glucose isomeriza-
tion. The kinetics of
D
-xylose and
D
-glucose isomerization
were determined from crude enzyme preparations at 60 °C,
pH 7.0, and at metal-ion satu ration (Mn
2+
) (Table 2 ). The
K
m
values for
D
-xylose were up to 26 times higher for the
mutants, and the catalytic rate constants (k
cat
)wereupto
nine times higher than for the wild-type enzyme. The
catalytic ef®ciency (k
cat
/K
m

)for
D
-xylose for M- 1026 was
6% higher than that of the wild-type, while for the other
mutants it was lower.
As for the wild-type X I, the mutants had a lower K
mand
higher k
cat
for
D
-xylose than for
D
-glucose. The K
m
for
glucose for M-1021 and M-1024 was lower, by as much as
three times, than for the wild-type enzyme. For M-1026, on
the other hand, the K
m
was higher than that of the wild-type
enzyme. T he k
cat
and the k
cat
/K
m
values for
D
-glucose were

up to ®ve and seven times higher, respectively, for all the
mutants, than for the wild-type XI.
Inhibition by xylitol. The extended a cyclic forms of the
substrates xylose and glucose have binding closely resem-
bling that observed for the acyclic polyol inhibitor xylitol
[34,35]. Competitive i nhibition is thus expected and has
previously been reported [36,37]. K
i
for xylitol for the three
mutant enzymes was between seven ( M-1021) and 255
(M-1024) times higher, than for the wild-type enzyme
(Table 2), indicating that the mutant enzymes are not
inhibited by xylitol to the same extent as t he wild-type
enzyme.
Thermal stability. To determine whether the m utations
producing a change in the temperature dependence of XI
activity also affected the thermal stability of the mutated
enzymes, the r esidual a ctivities w ere m easured afte r heat
treatment at 7 0 °C for various lengths of t ime (Fig. 4).
Investigations of the m etal-free enzyme preparations in
buffer at saturated metal concentration (Mn
2+
) showed
that the wild-type XI and the mutated XIs retained almost
Fig. 3. The relative activity at dierent values of pH for the mutated XIs
and the wild-type XI: (e) wild-type; (d) M-1021; (,) M-1024; and (j)
M-1026. The scale of relative activity (%) indicates the percentage of
experimental value at various pH relative to the maximum value of
each enzyme.
Table 1. Eect of various bivalent cations (10 m

M
) o n the activity of
EDTA-treated enzymes. The % relative activity is shown compared to
the speci®c activity with 10 m
M
MnCl
2
at 60 °C which was set to 100%
for each enzyme.
Enzyme Co
2+
Mg
2+
Wild type 87.9 73.8
M-1021 7.4 23.2
M-1024 33.6 22.7
M-1026 34.6 38.9
Temperature (
o
C)
20 30 40 50 60 70 80 90 100
Relative activity
(% of maximum)
0
20
40
60
80
100
120

Fig. 2. The relative activity at dierent temperatures for the mutated
XIs and the wild-type XI: (e) wild-type; (d) M-1021; (,) M-1024; and
(j) M-1026. The scale of relative activity (%) indicates the percentage
of experimental values at various temperatures relative to the maxi-
mum value of each enzyme.
160 A. Lo
È
nn et al. (Eur. J. Biochem. 269) Ó FEBS 2002
full activity after 8 h of incubation. The mutants showed a
drop in residual activity after 24 h, and after 56 h of
incubation between 54 and 74% of their maximum activity
remained. T he wild-type still had 95% residual activity
after 56 h of incubation. Clearly, the mutated XIs were
more sensitive to heat treatment at 70 °C than the
wild-type XI.
DISCUSSION
The goal of the present study was to generate ef®cient cold-
adapted XIs from T. thermophilus, with improved kinetic
properties at low temperatures. Random PCR mutagenesis
was performed in the gene encoding the enzyme (xylA)and
a mutant library was constructed. When the resulting
proteins were screened, we obtained three cold-adapted
mutants: E372G/V379A (M-1021), E372G/F163L
(M-1024) and E372G ( M-1026), with hig her k
cat
values
than the wild-t ype XI for
D
-xylose at 60 °C.
All mutations obtained were located on the enzyme

surface, and not close to t he active site. Amino-acid
substitution distant f rom the catalytic ce ntre or in the
major substrate b inding site of enzymes c an lead to cold
adaptation [38]. It has been proposed that variations in the
enthalpy and entropy of conformational changes of impor-
tance in binding and catalysis can be due to sequence
changes outside the active sites. In the evolutionary
adaptation of k
cat
and K
m
in response to acute temperature
changes, these e ffects should play an i mportant role [39].
The effect of mutation in a single amino acid on the kinetic
properties r eported h ere has been seen before. There are
reports that almost all the p sychrophilic character of some
cold-adapted enzymes is due to a single amino-acid
substitution. A single difference in the sequence at a subunit
contact site was the cause of differences in the temperature±
K
m
relationship or stability between closely related ®sh
LDH [40]. In addition, nearly all the improvement in the
catalytic ef®ciency of a mutated Vibrio marinus triosephos-
phate is omerase was due to replacement of a completely
conserved Ser in the phosphate binding helix by Ala in the
psychrophilic enzyme [41]. There are, however, no structural
features that can be correlated exclusively to cold adapta-
tion. Structural explanations for cold adaptation can not be
generalized. There is no single structural characteristic that

accounts for the simultaneously appearing low stability and
increased catalytic ef®ciency, proposed to be a consequence
of high molecular ¯exibility. T he origin o f the increased
enzyme activity and red uced stability lies i n a partic ular
region of the molecule rather than, for example, a general
reduction in intramolecular interactions. A clear correlation
seems to exist between cold adaptation and a reduction in
the number of interactions between structural domains or
subunits [42].
There is a close relationship between molecular ¯exibility
and function. Thermophilic enzymes are rigid and require
elevated temperatures in order to gain suf®cient molecular
¯exibility for activity. Their molecular structure must thus
be balanc ed between the requirements f or stability and
dynamics. We propose that the sequence changes underly-
ing t he adaptation of T. thermophilus XI mutants to
temperatures lower than their optimal temperature, allow
a higher degree of ¯exibility in a reas that move during
catalysis. Higher ¯exibility in these areas should increase k
cat
by reducing the energetic cost of a conformational change
from the apoenzyme to the holoenzyme. By increasing k
cat
and K
m
, the catalytic ef®ciency of most cold-adapted
enzymes increases, compared with t he warm-adapted ones.
k
cat
increases because of the ability of cold-adapted enzymes

to reduce the free energy of activation compared with warm-
adapted homologues. The increased K
m
istheresultofa
more ¯exible conformation [39]. Kinetic analysis d emon-
strated that the increase in the relative activity in the
mutated XIs for xylose at low temperatures was indeed
caused by an increase in k
cat and
not by a decrease in the K
m
value. This suggests that the mutant enzymes did not
acquire higher af®nity for the substrate than the wild-type
enzyme at lower temperatures. The k
cat
/K
m
values for xylose
for the mutated X Is only improved for M-1026. This was
due to the large increase in the K
m
values for xylose. The
Table 2. K inetic properties of wild-type XI and mutated XIs.
Xylose Glucose
Xylitol
K
i
(m
M
)

K
m
(m
M
)
k
cat
(s
)1
)
k
cat
/K
m
(s
)1
ám
M
)1
)
K
m
(m
M
)
k
cat
(s
)1
)

k
cat
/K
m
(s
)1
ám
M
)1
)
Wild-type 3.44  0.4 46.6 13.6 146.8  12.3 16.3 0.11 4.6
M-1021 25.1  4.0 257.5 10.3 52.0  4.9 39.9 0.77 33.2
M-1024 89.4  8.4 381.6 4.3 130.8  17.1 66.9 0.51 1174
M-1026 28.7  3.3 412.4 14.4 171.8  10.0 88.7 0.47 68.7
Fig. 4. Thermal stability of wild-type XI and mutated XIs. Metal-free
enzyme preparations were incubated at 70 °C in 200 m
M
triethanol-
amine, pH 7.0, 10 m
M
MnCl
2
, and residual activities of aliquots were
recorded as a function of time using xylose as a substrate: (e) wild-
type; (d) M-1021; (,) M-1024; (j) M-1026.
Ó FEBS 2002 Mutant xylose isomerases (Eur. J. Biochem. 269) 161
speci®c activity, or turnover number, k
cat
, re¯ects the
catalytic potential at saturated substrate concentrations.

The quantity, k
cat
/K
m
, is the catalytic ef®ciency that re¯ects
the overall conversion of substrate to product. It has been
suggested that the catalytic ef®ciency, k
cat
/K
m
,providesa
better approximation o f catalytic activity at physiological
substrate concentrations, w hich are usually below satura-
tion [43].
In lignocellulosic hydrolysate the concentration of xylose
can vary considerably. The concentration of xylose inside
the cell, on the o ther hand, remains unknown, and i s
probably dependent on the xylose transporters. In natural
xylose fermenting yeasts, the ®rst xylose converting enzyme
(XR) has a K
m
for xylose between 10 and 100 m
M
[44±46].
Recombinant S. cerevisiae expressing XR from P. stipitis
has been shown to ferment xylose [3±5]. Therefore it is
reasonable to assume that t he mu tated XIs with K
m
for
xylose between 25 and 89 m

M
will be able to support a
functional xylose metabolic pathway.
For glucose, all mutated XIs had both h igher k
cat
and
k
cat
/K
m
values. These results indicate that we obtained
improved kinetic constants at 60 °Cfor
D
-glucose isomer-
ization, but not to the same extent for
D
-xylose isomeriza-
tion.
Clearly, the mutated XIs were also thermally sensitive at
70 °C, indicating that these m utations might confer ther-
molabile characteristics on the enzyme. It has been reported
previously that the thermostability of proteins can be altered
by single amino-acid substitution [47,48], but it is not yet
clear which these a mino acids are [49]. I t has also be en
suggested that higher catalytic ef®ciency in naturally
occurring cold-adapted enzymes is associated with lower
thermal stability, due to the higher molecular ¯exibility at
lower temperatures [ 43,50,51]. The low stability at high
temperatures is therefore regarded as a necessary conse-
quence of cold adaptation. The reduced thermal stability of

the mutated XIs i s not a problem for xylose fermentation
because fermentation occurs at moderate (30±40 °C) tem-
peratures and the yeast is continuously producing the
enzyme during the fermentation process. However, the
higher k
cat
at moderate temperatures is essential for
obtaining xylose fermentation rates compatible with indus-
trial processes [12].
All mutants showed a dramatic increase in K
i
for xylitol,
which is an inhibitor of XI. This may be a very important
trait in the fermentation of xylose to ethanol, as S. cerevisiae
produces xylitol from xylose via unspeci®c aldose reductases
[52,53].
Together the improved kinetic properties at 60 °Cforthe
mutated XIs make them promising for xylose fermentation.
To evaluate the physiological consequence of the changed
kinetic properties of the wild-type and mutated xylA genes
must, however, be expressed in S. cerevisiae.
ACKNOWLEDGEMENTS
We would like to thank Jonas Fast for his technical assistance, and the
Department of Biochemistry, Lund University, Sweden, for the use of
the Storm 860Ò. This work was ®nancially supported by The Swedish
National E nergy Administration (Energimyndigheten), the Swedish
Foundation for International Cooperation in Research and Higher
Education (STINT) and the National Research Foundation, South
Africa (NRF).
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