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 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 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
MATERIALS AND METHODS
Chemicals
All chemicals were obtained from commercial suppliers and
used as described by the manufacturer.
D
(+)-xylose was
obtained from Sigma (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¢-TGATCAATGTACGAGCCCAAACC-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 High PureäPCR Product (Boehringer
Mannheim).
DNA sequencing
Analysis of the mutated sequences was carried out using
ABI PRISMÒBig DyeäTerminator cycle sequencing
ready reaction kits with an ABI PRISMä377 DNA
sequencer (PE/Applied Biosystems). Both the 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 harbouring the plasmids pGEM
Ò
-T Easy
containing the wild-type and the mutated XI genes were
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 washed once with ice-cold distilled water. Washed
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 solutions were kept at room temperature for
1 h and then on ice for 2 h before storing in a freezer at
)20 °C. Cell extracts were thawed on ice, cell debris was
removed by centrifugation (15 000 gfor 15 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
Èdra Sandby, Sweden) and
immunoblotting was performed as described previously
[25]. Brie¯y, 2 lg of cell-free extract 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 Storm 860Ò(Pharmacia
Amersham, Uppsala, Sweden) using a chemi¯uorescent
substrate ECF (Pharmacia Amersham). Data analysis was
performed using
IMAGE QUANT
Òsoftware (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 of isomerase activity was de®ned as the
amount of crude enzyme required 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 activity and the protein concentration 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 spanning the
value of K
m
were used to determine the value of K
m
.The
158 A. Lo
Ènn et al. (Eur. J. Biochem. 269)ÓFEBS 2002
concentration of XI in the cell-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
]total
enzyme concentration [28].
The K
i
(m
M
) for xylitol was determined by incubating
crude enzyme preparations 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],whereiis the
xylitol concentration (m
M
)andK
m
¢the apparent K
m
value
at a certain concentration of xylitol.
PH pro®le
The effect of pH on the activity of the wild-type and
mutated enzymes was investigated in the pH range 5±10 in
700 m
M
xylose, 10 m
M
MnCl
2
and a buffer prepared by
mixing acetate, Pipes, Hepes and glycine, to a ®nal
concentration of 50 m
M
each [30]. The pH was adjusted
at 60 °C with NaOH. Above pH 7.0 corrections were made
for the chemical isomerization of
D
-xylose.
Temperature pro®le
The temperature pro®les for the wild-type XI and mutated
XIs were measured 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]. No 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 into 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 cultivated at 37 °C overnight. After a further
2 days of incubation at 30 °C, the pH indicator in the
medium allowed detection and quanti®cation of red acid-
producing colonies. Three candidate mutants, termed
M-1021, M-1024 and M-1026 were identi®ed. Colonies of
these three were a deeper red on the McConkey/xylose
medium than were wild-type xylA colonies (suggesting
higher XI activity). DNA sequencing revealed that 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 comprises two
domains: the larger N-terminal domain (domain I, residues
1±321), which folds into a (b/a)
8
barrel, and the 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 V379A. 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 in Fig. 1. Neither the substrate-binding sites
(H53, D56 and K182) nor the 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 than the
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 whether 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.
ÓFEBS 2002 Mutant xylose isomerases (Eur. J. Biochem. 269) 159