Cold adaptation of xylose isomerase from Thermus thermophilusthrough random PCR mutagenesis Gene cloning and protein characterization Anna LoÈnn1, MaÂrk GaÂrdonyi1, Willem van Zyl2, BaÈr
Trang 1Cold adaptation of xylose isomerase from Thermus thermophilus
through random PCR mutagenesis
Gene cloning and protein characterization
Anna LoÈnn1, MaÂrk GaÂrdonyi1, Willem van Zyl2, BaÈrbel Hahn-HaÈgerdal1and Ricardo Cordero Otero2,*
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 (kcat) forD-xylose compared with the wild-type
enzyme at 60 °C, but they did not show any increase in catalytic eciency (kcat/Km) For D-glucose, both the kcat
and the kcat/Km 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 (Ki) 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 the wild-type enzyme The results are discussed in terms of 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 kcatfor xylose than the wild-type enzyme at 60 °C are described
Correspondence to B Hahn-HaÈgerdal, 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)
Trang 2M A T E R I A L S A N D M E T H O D S
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 mM dATP, 0.2 mM
dGTP, 1 mMdCTP, 1 mMdTTP, 1.5 mMMgCl2, 0.5 mM
MnCl2, 0.15 lMof both primers, 0.02 nMtemplate DNA
and 5 U Taq DNA polymerase (BIOTAQä) in a total
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 °C and 45 s at 68 °C The PCR products were then
puri®ed using High 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 the coding and
the noncoding strands were sequenced to ensure the reliable
identi®cation of all mutations
Growth conditions and preparation of cell extract
fromE coli
E coli HB101 harbouring the plasmids pGEMÒ-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 mM triethanolamine,
pH 7.0, 65 kUámL)1 lysozyme, 0.25 mgámL)1 DNAse
and 1 mMphenylmethanesulfonyl¯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 g for 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 (SoÈdra 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 QUANTÒ 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 (Vmax) to calculate kcat Enzyme assays
A two-step XI standard assay (0.5 mL) was modi®ed from [26] A substrate concentration of 700 mM D-xylose was used at 60 °C in 200 mMtriethanolamine at pH 7.0 in the presence of 10 mMMnCl2and 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 2M Na2CO3 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 mMNADH 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)1ámg)1) was determined from the activity and the protein concentration of the crude enzyme preparations
Kinetic parameters The kinetic parameters, Vmax (lmolámin)1ámg)1) and Michaelis constant (Km, mM), 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 Kmwere used to determine the value of Km The
Trang 3concentration 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 (kcat)
from the relationship kcat Vmax/[E0], where [E0] total
enzyme concentration [28]
The Ki (mM) for xylitol was determined by incubating
crude enzyme preparations in different xylose
concentra-tions (20±600 mM) at different ®xed xylitol concentrations
By plotting the speci®c activities for each xylitol
concentra-tion against the xylose concentraconcentra-tions, Kiwas determined
using the equation Km¢ Kmá(1 + i/Ki) [29], where i is the
xylitol concentration (mM) and Km¢ the apparent Kmvalue
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 mM xylose, 10 mM MnCl2 and a buffer prepared by
mixing acetate, Pipes, Hepes and glycine, to a ®nal
concentration of 50 mM each [30] The pH was adjusted
at 60 °C with NaOH Above pH 7.0 corrections were made
for the chemical isomerization ofD-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 ofD-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
Mn2+, Mg2+ or Co2+ The effect of metal ions on XI
activity was determined by adding 10 mM®nal
concentra-tion of either CoCl2, MnCl2 or MgCl2 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 mM triethanolamine, pH 7.0 with
10 mMMnCl2in airtight tubes at 70 °C At different times,
100-lL samples were withdrawn and stored on ice until the
residual activity was determined
R E S U L T S
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 temperatempera-ture 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.
Trang 4each mutant enzyme was measured as a function of pH
(Fig 3) The activity of each enzyme relative to the
maximum activity was plotted as a percentage against pH
The pH dependence of the enzyme activity was examined at
a substrate concentration well above Km, where the velocity
of the reaction is proportional to kcat The pH activity
pro®les of the mutants were broader, and extended into the
alkaline region, compared with the type XI The
wild-type showed no XI activity at pH 9 and 10 For M-1024 and
M-1026 the speci®c activity 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
Eect of metal ions XIs require 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], and XI
from T thermophilus requires either Mg2+ or Mn2+for
100% activity [30] Metal ions are not only essential for the catalytic mechanism, but they also contribute to the stabilization of the native structure, which is especially important for thermophilic enzymes
The effect of different bivalent metal ions (Mn2+, Mg2+
and Co2+) on the EDTA-treated enzymes was investigated (Table 1) The wild-type and mutated XIs were most effectively activated by Mn2+and, to a smaller degree, by
Mg2+and Co2+ The wild-type showed 88 and 74% of the maximum activity with Co2+and Mg2+, respectively The mutants, on the other hand, were less activated by Co2+
and Mg2+ Kinetic properties of D-xylose and D-glucose isomeriza-tion The kinetics ofD-xylose andD-glucose isomerization were determined from crude enzyme preparations at 60 °C,
pH 7.0, and at metal-ion saturation (Mn2+) (Table 2) The
Kmvalues forD-xylose were up to 26 times higher for the mutants, and the catalytic rate constants (kcat) were up to nine times higher than for the wild-type enzyme The catalytic ef®ciency (kcat/Km) 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 XI, the mutants had a lower Km and
higher kcat for D-xylose than for D-glucose The Km 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 Kmwas higher than that of the wild-type enzyme The kcatand the kcat/Kmvalues forD-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 acyclic forms of the substrates xylose and glucose have binding closely resem-bling that observed for the acyclic polyol inhibitor xylitol [34,35] Competitive inhibition is thus expected and has previously been reported [36,37] Kifor 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 the wild-type enzyme
Thermal stability To determine whether the mutations producing a change in the temperature dependence of XI activity also affected the thermal stability of the mutated enzymes, the residual activities were measured after heat treatment at 70 °C for various lengths of time (Fig 4) Investigations of the metal-free enzyme preparations in buffer at saturated metal concentration (Mn2+) showed that the wild-type XI and the mutated XIs retained almost
Fig 3 The relative activity at dierent 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 Eect of various bivalent cations (10 m M ) on the activity of EDTA-treated enzymes The % relative activity is shown compared to the speci®c activity with 10 m M MnCl2 at 60 °C which was set to 100% for each enzyme.
Temperature (oC)
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 dierent 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.
Trang 5full 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 The 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
D I S C U S S I O N
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 higher kcat values
than the wild-type XI forD-xylose at 60 °C
All mutations obtained were located on the enzyme
surface, and not close to the active site Amino-acid
substitution distant from the catalytic centre or in the
major substrate binding site of enzymes can 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 kcatand Kmin response to acute temperature
changes, these effects should play an important role [39] The effect of mutation in a single amino acid on the kinetic properties reported here has been seen before There are reports that almost all the psychrophilic 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±
Km 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 isomerase 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 The origin of the increased enzyme activity and reduced stability lies in a particular 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 balanced between the requirements for stability and dynamics We propose that the sequence changes underly-ing the adaptation of T thermophilus XI mutants to temperatures lower than their optimal temperature, allow
a higher degree of ¯exibility in areas that move during catalysis Higher ¯exibility in these areas should increase kcat
by reducing the energetic cost of a conformational change from the apoenzyme to the holoenzyme By increasing kcat
and Km, the catalytic ef®ciency of most cold-adapted enzymes increases, compared with the warm-adapted ones
kcatincreases because of the ability of cold-adapted enzymes
to reduce the free energy of activation compared with warm-adapted homologues The increased Kmis the result of a more ¯exible conformation [39] Kinetic analysis demon-strated that the increase in the relative activity in the mutated XIs for xylose at low temperatures was indeed caused by an increase in kcat andnot by a decrease in the Km
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 kcat/Kmvalues for xylose for the mutated XIs only improved for M-1026 This was due to the large increase in the Kmvalues for xylose The
Table 2 Kinetic properties of wild-type XI and mutated XIs.
Xylitol
Ki (m M )
Km (m M ) kcat(s )1 ) kcat/Km(s )1 ám M)1) Km(m M ) kcat(s )1 ) kcat/Km(s )1 ám M)1)
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 MnCl2, 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.
Trang 6speci®c activity, or turnover number, kcat, re¯ects the
catalytic potential at saturated substrate concentrations
The quantity, kcat/Km, is the catalytic ef®ciency that re¯ects
the overall conversion of substrate to product It has been
suggested that the catalytic ef®ciency, kcat/Km, provides a
better approximation of catalytic activity at physiological
substrate concentrations, which 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 other hand, remains unknown, and is
probably dependent on the xylose transporters In natural
xylose fermenting yeasts, the ®rst xylose converting enzyme
(XR) has a Kmfor xylose between 10 and 100 mM[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 the mutated XIs with Km for
xylose between 25 and 89 mM will be able to support a
functional xylose metabolic pathway
For glucose, all mutated XIs had both higher kcatand
kcat/Km values These results indicate that we obtained
improved kinetic constants at 60 °C forD-glucose
isomer-ization, but not to the same extent forD-xylose
isomeriza-tion
Clearly, the mutated XIs were also thermally sensitive at
70 °C, indicating that these mutations 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 amino acids are [49] It has also been
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 is 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 kcat at moderate temperatures is essential for
obtaining xylose fermentation rates compatible with
indus-trial processes [12]
All mutants showed a dramatic increase in Kifor 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 °C for the
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
A C K N O W L E D G E M E N T S
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 Energy 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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