Báo cáo khoa học: The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A ppt

The relationship between thermal stability and pH optimum
studied with wild-type and mutant
Trichoderma reesei
cellobiohydrolase Cel7A
Harry Boer and Anu Koivula
VTT Biotechnology, Espoo, Finland
The major cellulase secreted by the filamentous fungus
Trichoderma reesei is cellobiohydrolase Cel7A. Its three-
dimensional structure has been solved and various mutant
enzymes produced. In order to study the potential use of
T. reesei Cel7A in the alkaline pH range, the thermal sta-
bility of Cel7A was studied as a function of pH with the wild-
type and two mutant enzymes using different spectroscopic
methods. Tryptophan fluorescence and CD measurements
of the wild-type enzyme show an optimal thermostability
between pH 3.5–5.6 (T
m
,62±2°C), at which the highest
enzymatic activity is also observed, and a gradual decrease in
the stability at more alkaline pH values. A soluble substrate,
cellotetraose, was shown to stabilize the protein fold both at
optimal and alkaline pH. In addition, unfolding of the
Cel7A enzyme and the release of the substrate seem to
coincide at both acidic and alkaline pH, demonstrated by
a change in the fluorescence emission maximum. CD
measurements were used to show that the five point muta-
tions (E223S/A224H/L225V/T226A/D262G) that together
result in a more alkaline pH optimum [Becker, D., Braet, C.,
Brumer, H., III, Claeyssens, M., Divne, C., Fagerstro
¨
m,

R.B.,Harris,M.,Jones,T.A.,Kleywegt,G.J.,Koivula,A.,
et al. (2001) Biochem. J. 356, 19–30], destabilize the protein
fold both at acidic and alkaline pH when compared with the
wild-type enzyme. In addition, an interesting time-depend-
ent fluorescence change, which was not observed by CD, was
detected for the pH mutant. Our data show that in order to
engineer more alkaline pH cellulases, a combination of
mutations should be found, which both shift the pH opti-
mum and at the same time improve the thermal stability at
alkaline pH range.
Keywords: cellulase; circular dichroism; fluorescence; pH
optimum; thermostability.
In the filamentous fungus Trichoderma reesei the major
component of the secreted cellulase system and a key
enzyme for efficient crystalline cellulose degradation is the
family 7 cellobiohydrolase, Cel7A, formerly named CBHI
[1,2]. Similar to many other cellulases, T. reesei Cel7A is
composed of a large catalytic and a small cellulose-binding
domain (CBD; now also called a carbohydrate-binding
module, CBM), which are joined by an O-glycosylated
linker peptide. Three-dimensional structures of both the
isolated catalytic and cellulose-binding domain of wild-type
and various mutated forms of Cel7A cellobiohydrolase have
been solved [3,4–8]. The catalytic domain of Cel7A has a
large concave b-sandwich fold where individual b-strands
are joined by long surface loops enclosing the active site in a
tunnel, which spans through the whole catalytic domain.
The tunnel is 50 A
˚
long and contains at least 9 subsites

()7 fi +2) for the sugar units of a cellulose chain [5]. This
active site topology is assumed to account for the high
activity of Cel7A on crystalline substrates. Cel7A cleaves
cellobiose units by an acid hydrolysis mechanism mainly
from the reducing end of a cellulose chain. Cel7A is also a
processive enzyme, which makes multiple cuts before
dissociating from the cellulose chain [5]. The CBD is
essential for the high activity on insoluble substrates. Cel7A
CBD is a small, 36-amino acid peptide composed of three
short b-strands which are held together by two disulfide
bridges. The cellulose-binding surface of Cel7A CBD has
been shown to be formed by three tyrosine residues and
some polar residues [6,8,9].
T. reesei is an acidophilic fungus and most of its secreted
cellulases function optimally at around pH 5. Cellulases are
very potent industrial enzymes in various processes based
on renewable materials, such as bio-ethanol production
from lignocellulosic material. One of the goals in cellulase
engineering for these processes is to understand and alter the
operative pH of these enzymes. Recently a variant of
T. reesei Cel7A containing five point mutations (E223S/
A224H/L225V/T226A/D262G) and having a more alkaline
pH optimum was produced [3]. The mutations were
designed based on the sequence comparisons of family 7
cellulases having different pH behaviours. The comparison
revealed that a histidine residue near the acid/base catalyst
could account for the higher pH optimum of the Humicola
insolens Cel7B endoglucanase and therefore a mutation
A224H was designed. Modelling studies further suggested
that four additional amino acid changes (E223S/L225V/

T226A/D262G) would be required in order to fit the bulkier
Correspondence to H. Boer, VTT Biotechnology, PO Box 1500,
Espoo, Finland. Fax: +358 9 4552103, Tel.: +358 9 4565183,
E-mail: Harry.Boer@vtt.fi
Abbreviations: Cel7A, Trichoderma reesei cellobiohydrolase (former
CBHI); CBD, cellulose-binding domain; MULac, 4-Methylumbel-
liferyl-b-
D
-lactoside.
(Received 29 October 2002, accepted 16 December 2002)
Eur. J. Biochem. 270, 841–848 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03431.x
histidine side-chain. X-ray analysis showed that there were
no major differences between the mutant and wild-type
cellulase structures other than the designed point mutations.
Although the Cel7A pH mutant behaved in the desired way
with respect to the pH optimum, it had, however, lowered
overall activity on both soluble and insoluble substrates.
In order to investigate the thermal stability of T. reesei
wild-type Cel7A and the pH mutant E223S/A224H/L225V/
T226A/D262G in more detail as a function of pH, we report
here spectroscopic studies performed using both tryptophan
fluorescence and CD spectroscopy. These methods are
powerful tools to study the conformation of a protein as a
function of various environmental factors, such as pH
and temperature. We were particularly interested in study-
ing the alkaline stability of the wild-type Cel7A and mutant
protein.
Materials and methods
Protein production and purification
The purification of wild-type Cel7A enzyme and the E217Q

and E223S/A224H/L225V/T226A/D262G mutants from
T. reesei strain ALKO2877 has been published [3,7].
Concentrations of purified wild-type Cel7A and mutated
proteins were determined from UV absorbance at 280 nm
using a molar extinction coefficient, e ¼ 83000
M
)1
Æcm
)1
measured with total amino acid analysis of the wild-type
Cel7A enzyme (A. Koivula, unpublished data).
Enzyme activity measurements
The soluble substrate, 4-methylumbelliferyl-b-
D
-lactoside
(MULac) (Sigma), was used to determine the activity of
Cel7A essentially as described by van Tilbeurgh and
Claeyssens [10]. The assay conditions used in the experi-
ments are stated in the text or figure legends.
Fluorescence measurements
Unfolding studies based on monitoring the intrinsic tryp-
tophan fluorescence of wild-type Cel7A and the pH mutant
were performed on a Shimadzu RF-5000 spectrofluoro-
meter [11]. Emission and excitation spectra were recorded
with a bandwidth of 5 nm on both monochromators. A
thermostated cuvette holder connected to a water bath
controlled the temperature of the sample solution. Tem-
perature-induced unfolding was monitored by heating
samples gradually up to 80 °C at approximately 1 °CÆmin
)1

and measuring the fluorescence intensity [11]. The tempera-
ture of the sample solution was measured continuously
using a Fluke 52 electronic thermometer equipped with
K-type thermocouple that was immersed in the solution.
Fluorescence emission spectra from 300 to 500 nm were
collected at 2-°C intervals in the temperature range
25–85 °C. The excitation wavelength was 285 nm and the
spectra were corrected for the buffer spectrum. The change
in the fluorescence intensity of the sample at 340 nm was
plotted as a function of temperature and differentiated by
using Origin 6.0 graphics and data analyses software. The
culmination point of each curve was taken as the melting
temperature. All points were measured at least in duplicate.
The enzyme concentrations used in the measurements
varied from 0.5 to 1 l
M
. Buffers used were 50 m
M
sodium
acetate (pH 3.5–5.6), 50 m
M
potassium phosphate (pH 6–8)
and 50 m
M
Tris/HCl (pH 9.0).
CD spectroscopy
CD measurements were performed on a Jasco J-720 CD
spectrometer equipped with PTC-38WI Peltier type tem-
perature control system that controlled the temperature in
the cuvette. Spectra were recorded from 240 to 190 nm

using a 1-mm cell and a bandwidth of 1 nm. The unfolding
curves were measured at 202 nm using the temperature scan
mode with a gradient of 1 °CÆmin
)1
until a temperature of
80 °C was reached. The measurements were performed at a
low salt concentration (a requirement for CD spectroscopy)
in 10 m
M
potassium phosphate buffer at pH 5.8 and 8.0.
Results and discussion
According to theoretical studies the curve representing the
melting temperature, i.e. the thermostability of an enzyme,
as a function of pH has a bell shape [12]. The activity profile
of an enzyme is also pH dependent, and, depending on the
enzyme, the pH stability and activity curves can in theory
overlap totally, partially or not at all. When thinking of
applying an enzyme at certain pH and temperature
conditions, one should consider both stability and activity
issues. We have studied earlier the temperature-induced
unfolding of T. reesei wild-type Cel7A enzyme at pH 5.0
and shown that the thermal inactivation at this (acidic) pH
appears to be related to the thermal unfolding of the protein
structure [11]. This unfolding study was based mainly on
monitoring tryptophan fluorescence. There are altogether
nine tryptophan residues in T. reesei Cel7A and all of them
are situated in the catalytic domain; five of them are buried
and four are situated in the active site tunnel, stacking
against the cellulose chain. The fluorescence emission
maximum and quantum yield are sensitive to the micro-

environment of tryptophan side-chains in a protein, and
both parameters can be used to monitor structural changes
and the unfolding of proteins.
In the series of experiments presented here, we study the
thermostability of T. reesei wild-type Cel7A and a mutant
with a more alkaline pH optimum, and focus especially on
the alkaline pH area. Fig. 1A shows the temperature-
induced unfolding of T. reesei wild-type Cel7A at both
pH 5.0 [11] and pH 8.0. Unfolding was monitored by
heating samples gradually up to 80 °C and measuring the
change in intrinsic protein fluorescence as described earlier
by Boer et al. [11]. The significant temperature dependency
of the baseline fluorescence, which leads to the steep slope of
the baseline, is an indication of many tryptophan residues in
Cel7A exposed to the solvent. The transition region for
unfolding is observed between 55 and 65 °CatpH5.0,
while at pH 8.0 it is shifted to lower temperature area
(Fig. 1A). This can be seen more clearly from the derivative
plot in Fig. 1B, from which the melting temperatures (T
m
)
were also calculated. When the unfolding of the intact
enzyme (containing the CBD) was compared to that of the
catalytic domain a very similar pH-dependent behaviour
was observed, indicating that the CBD does not have a large
842 H. Boer and A. Koivula (Eur. J. Biochem. 270) Ó FEBS 2003
effect on the overall stability of the catalytic domain of
Cel7A (data not shown).
The melting temperatures (T
m

) for wild-type Cel7A
enzyme were then measured over a pH range of 3.5–9.0 in a
manner demonstrated in Fig. 1. The results shown in Fig. 2
(j) seem to hold with the theoretical studies of the bell-
shaped curve, with Cel7A having an optimal thermostability
between pH 3.5 and 5.6 (T
m
¼ 62 ± 2 °C). This pH area
coincides with the highest enzymatic activity of Trichoderma
Cel7A measured on soluble methyl-umbelliferyllactoside
(Fig. 2, d). A pH optimum between pH 4 and 5 has also
been reported for wild-type Cel7A on other soluble
substrates at temperatures ranging from 25 to 37 °C
[3,11,13,14]. Thus it seems that T. reesei Cel7A has the
highest activity at acidic pH values, where the protein is also
most stable. At pH 8.0, which would be a desired pH
optimum in some cellulase applications, Cel7A has a clearly
reduced, or no activity at all depending on the substrate
used [3,11,13,14]. Although catalytically not active and
having a clearly reduced stability at pH 8.0, Cel7A seems to
be folded at this pH at 25 °C (Figs 1 and 2), confirmed also
with the CD measurements described below (Fig. 6).
The effect of a soluble substrate (cellotetraose) on the
stability of Cel7A was also studied at pH 5.0 and pH 8.0.
The binding at pH 5 was first checked with the wild-type
enzyme, but as it is fully active at pH 5, a slow decrease in
the fluorescence intensity and a red shift (due to degradation
of cellotetraose, see also below) was detected making
interpretation of the data difficult. However, with the
catalytically inactive acid/base mutant E217Q [5,7] binding

of the cellotetraose could be monitored at pH 5.0. At
pH 8.0 wild-type Cel7A enzyme could be used because of its
very low activity at this pH (Fig. 2). Fig. 3C shows that the
stability of the active site mutant E217Q was similar to that
of the wild-type Cel7A enzyme shown in Fig. 1 (d). In
Fig. 3B and D the fluorescence emission maximum wave-
length is plotted against temperature in the presence and
absence of substrate at pH 8 and pH 5, respectively.
Interestingly, upon binding of the substrate, an increase in
the fluorescence intensity (data not shown) and a blue shift
of the emission maximum was observed. This blue shift
(Fig. 3B and D) and change in fluorescence intensity can be
explained by a change in polarity of some, or all of the four
tryptophans in the active site tunnel of Cel7A, when
cellotetraose is bound and can thus be used to monitor the
binding of the substrate. Cellotetraose (125 l
M
)wasusedin
these experiments, and this is assumed to be a saturating
concentration based on the measured K
m
value of 7 l
M
for
wild-type Cel7A enzyme [15,16]. Fig. 3A and C demon-
strate that cellotetraose has a small stabilizing effect on
Cel7A at both pH 8.0 and 5.0, respectively. When the
enzyme–substrate mixture was heated, a red shift in the
emission maximum occurred at both pH values (Fig. 3B
and D), indicative of the release of the substrate. Compari-

son of the Cel7A unfolding with and without the substrate
either at pH 8.0 (Fig. 3A and B), or pH 5 (Fig. 3C and D)
shows interestingly that the unfolding of the protein and the
release of the substrate coincide at both pH 5.0 and pH 8.0.
This would indicate that while the enzyme is folded the
substrate is bound in the active site tunnel.
Fig. 1. Thermostability of T. reesei wild-type Cel7A at pH 5.0 and pH 8.0 measured by tryptophan fluorescence. Fluorescence intensity was measured
with excitation at 285 nm and the two buffers used were 50 m
M
sodium acetate, pH 5.0 (d)and50m
M
potassium phosphate, pH 8.0 (s). The
concentration of the enzyme in the experiments was 1 l
M
. Samples were heated gradually to 75 °Catapproximately1°CÆmin
)1
and emission
intensity at 340 nm was recorded every 2 °C. (A) The relative fluorescence intensity (Int; 1.0 at 25 °C) was plotted as a function of temperature.
(B) The melting temperatures at different pH values were determined by plotting the differential, d(Int)/dT, against temperature.
Ó FEBS 2003 Stability of cellobiohydrolase Cel7A (Eur. J. Biochem. 270) 843
One of the most interesting and challenging questions on
the enzymatic action of cellobiohydrolases is how the tunnel
shaped active site architecture allows the direct access of the
enzyme to the glucan chains of cellulose. There have been
suggestions that the surface loops that form the active site
tunnel can open during the catalytic cycle for the release and
binding of the polymeric substrate [17–19]. Spectroscopic
methods, in particular, could be suitable for observing this
phenomena. In our steady-state fluorescence experiment
described above and shown in Fig. 3, we are not able to

observe the opening of the loops and release of the substrate
when Cel7A is folded. As we used an inactive mutant in the
pH 5.0 experiments, we cannot, however, exclude the
possibility that turnover of the enzyme is required for loop
opening, or on the other hand, that loop opening occurs on
a fast time-scale not observable in our steady-state fluores-
cence experiments. The strong response of both the emission
maximum and fluorescence intensity to binding of the
substrate, might actually allow stopped-flow experiments to
study the loop opening hypothesis under conditions where
theenzymeisactive.
The Cel7A pH mutant enzyme (E223S/A224H/L225V/
T226A/D262G) in which four additional amino acids were
mutated to facilitate the introduction of a histidine residue
near the acid/base catalyst D217, has a pH optimum shifted
to a more alkaline pH almost by one unit [3]. This supported
the hypothesis that the histidine residue, which is observed
in cellulases with a higher pH optimum, such as Humicola
insolens Cel7B endoglucanase, has an influence on the
activity of the enzyme at more alkaline pH by interacting
with the catalytic acid/base D217. Introduction of these five
mutations near the active site of the Cel7A enzyme resulting
in a shift in the pH optimum, had little effect on the K
m
, but
the k
cat
value was lowered 10-fold when compared to the
wild-type Cel7A [3]. Only minor structural changes were
observed in the mutant, apart from the changed atoms of

the mutated side chains. In order to evaluate further the
design of the mutations, it was of interest how the stability
behaviour of the protein was changed by the mutations,
particularly those in neutral and alkaline pH regions, pH
values at which this enzyme could be applied. Unexpectedly,
when the fluorescence emission spectrum of the mutant
enzyme was measured at pH 8.0 and 25 °C, a time-
dependent decay of fluorescence intensity was noted.
Further studies on the fluorescence intensity as a function
of time showed that at pH 5.0 the fluorescence emission
spectraofthemutantandthewild-typeCel7A(Fig. 4A)are
constant over a period of hours. However, when diluted in
pH 8.0 buffer, the fluorescence intensity of the Cel7A
mutant spectrum changes on a minute time scale, while the
spectrum for the wild-type enzyme remained constant
(Fig. 4B). The fluorescence intensity also changed in a
time-dependent manner at pH 6 and 7, but at a lower rate
than in the experiment at pH 8. This phenomenon is
reversed within 5 min when the pH of the mutant protein
solution is changed again from pH 8 to 5. These observa-
tions suggested that at pH 8.0 the five point mutations cause
small changes in the structure or polarity of the micro-
environment of some tryptophan residues. This is observed
as a time-dependent change in fluorescence intensity of these
tryptophans in the Cel7A mutant catalytic domain.
Because of the rapid decrease in fluorescence intensity at
pH 8.0, this method could not be used to produce a plot of
T
m
as a function of pH for the Cel7A pH mutant. Instead,

the temperature-induced unfolding of the Cel7A pH mutant
(and wild-type enzyme) were measured by CD spectroscopy
at different pH values (Fig. 5). CD is a technique which can
be used to monitor the secondary structure content of a
protein as a function of different environmental parameters.
The enzyme samples were heated from 25 to 80 °Catarate
of 1 °CÆmin
)1
using a Peltier-type temperature control
system. The initial CD spectra at 25 °C of the wild-type and
the mutant measured from 240 to 190 nm at pH 5.8 and
pH 8.0 in 10 m
M
potassium phosphate buffer were very
similar, indicating no major conformational changes in the
mutant enzyme structure at those two pH values (Fig. 6). In
addition, no time-dependent change of the CD spectra
could be observed with either the wild-type Cel7A or the
mutant protein at either pH. Above the T
m
spectra with a
distinct minimum around 202 nm, which is typical for an
unfolded protein consisting mainly of random coil structure
[20], were found with both proteins. The CD unfolding
curves for both the Wild-type Cel7A and mutant protein
(Fig. 5A) at pH 5.8 resemble those observed for a single
transition two-state unfolding reaction, and the experimen-
tal data could be analysed using this model [21]. The results
Fig. 2. Melting temperatures (T
m

) and enzymatic activity of wild-type
Cel7A as a function of pH. For the T
m
(j) all points were measured at
least in duplicate. Buffers: 50 m
M
sodium acetate pH 3.5–5.6, 50 m
M
potassium phosphate pH 6.0–8.0, and 50 m
M
Tris/HCl pH 9.0 The
activity/pH profile (d)ofCel7A(0.8l
M
) was determined at 30 °Cby
incubating enzyme samples first for 15 min at different pH values.
MULac (500 l
M
) was then added, and the rate was determined during
the first 30 min by taking samples, stopping the reaction with 0.5
M
Na
2
CO
3
and measuring the fluorescence (excitation wavelength
365 nm, emission wavelength 446 nm). Due to the poor solubility of
the substrate it is not possible to measure the pH profile under satur-
ating conditions over the whole pH range. At best the 500 l
M
MULac

corresponds with two times the K
m
value for this substrate at optimal
pH 5. The measured pH profile corresponds well with the other
reported pH activity profiles [3,11,13,14].
844 H. Boer and A. Koivula (Eur. J. Biochem. 270) Ó FEBS 2003
from the analysis can be found in the legend of Fig. 5
showing that the mutations have lowered the T
m
of Cel7A
at pH 5.8 by 6 °Cto57°C. The CD measurements further
showed that the Cel7A E223S/A224H/L225V/T226A/
D262G mutant starts to unfold at a lower temperature also
at pH 8.0 (Fig. 5) as compared with the wild-type enzyme.
A broad and much more complex transition is observed at
pH 8.0 (Fig. 5B) for both the wild-type and the mutant
protein, and a single transition unfolding model can no
longer be used for analysis. The structure of the wild-type
Cel7A starts to change around 37 °C and that of the mutant
around 30 °C (Fig. 5B). These experiments confirm the
results from the fluorescence measurements, i.e. decreased
stability of the wild-type Cel7A enzyme at more alkaline
pH. Furthermore, although the five point mutations
(E223S/A224H/L225V/T226A/D262G) cause a more
Fig. 3. Effect of cellotetraose on the temperature-dependent unfolding of T. reese i wild-type Cel7A in 50 m
M
potassium phosphate pH 8.0 (A and B)
and catalytically inactive E217Q mutant in 50 m
M
NaAcpH5.0(CandD).(A) and (C). The relative intrinsic fluorescence (Int; 1.0 at 30 °C) as a

function of temperature in the presence (d)andabsence(j)of125l
M
cellotetraose. (B) and (D). Change in the fluorescence emission maximum
as a function of temperature in the presence (d) and absence (j)of125l
M
cellotetraose. The excitation wavelength in these experiments was
285 nm.
Ó FEBS 2003 Stability of cellobiohydrolase Cel7A (Eur. J. Biochem. 270) 845
alkaline pH optimum, they also seem to lead to lower
thermostability of the mutant protein at both pH 5.8 and
pH 8.0, as compared with the wild-type enzyme. This
lowered overall stability might also explain the smaller k
cat
values [3]. The stability of the pH mutant was checked
previously at pH 2–7 by residual activity measurements. A
lower thermostability at pH 3 was detected for the mutant,
whereas no changes were observed at neutral pH as
compared with the wild-type enzyme [3]. These kinetic
stability studies were performed so that the enzyme was first
preincubated at 37 or 50 °C for 1 h, after which the activity
was measured at optimal pH and 25 °C. From our earlier
studies we know that T. reesei wild-type Cel7A can refold
under these conditions due to the 12 disulfide bridges
remaining (fully or partially) intact in the protein [12]. Our
thermostability data confirms now that kinetic stability
measurements do not give a complete picture of the
behaviour of the Cel7A proteins.
The Cel7A pH mutant was reported to have greater
sensitivity for papain degradation and this was suggested to
relate to a small local structural change near the introduced

mutations observed in the X-ray structure [3]. Our fluores-
cence measurements give further indications for small
structural changes in the Cel7A mutant. The reversible
time-dependent changes in tryptophan fluorescence at
pH ‡ 6 (see also above and Fig. 4B) are suggestive that
one (or several) tryptophans in the catalytic domain near the
point mutations ÔsenseÕ the small structural changes. These
changes are relatively small as they cannot be detected in far
UV CD spectra (Fig. 6). Two tryptophan residues, in
particular, might be affected by the mutations: W263
situated next to a stretch of the five mutated amino acids
(E223S/A224H/L225V/T226A/D262G), and/or W216 situ-
ated next to the catalytic amino acid E217. As discussed
earlier, E217 is the residue whose microscopic pK
a
value is
likely to be affected by introducing a histidine residue at
Fig. 5. Temperature-induced unfolding of wild-type Cel7A and pH
mutant E223S/A224H/L225V/T226A/D262G at pH 5.8 and 8.0
measured by CD spectroscopy. Spectra were recorded from 240 to
190 nm using a 1-mm cell and a bandwidth of 1 nm. The unfolding
curves were measured at 202 nm using the temperature scan mode with
agradientof1°CÆmin
)1
until a temperature of 80 °C was reached. The
measurements were performed in 10 m
M
potassium phosphate buffer.
Unfolding of the wild-type Cel7A (s) and the pH mutant enzyme (h)
at (A) pH 5.8 and (B) pH 8.0. Analysis of heat denaturation of the

wild-type and mutant protein at pH 5.8 (solid lines) gave the following
parameters: wild-type, T
m
¼ 63 °C±0.03,DH
m
¼ 579±6kJmol
)1
,
Mutant, T
m
¼ 57 °C ± 0.08, DH
m
¼ 344±6kJmol
)1
.These
parameters are derived from a nonlinear least-squares fit of the data
using the following equation describing a reversible two state unfolding
reaction:Y ¼ (a
F
+b
F
*T)/(1 + exp((–DH
m
/T + DH
m
/T
m
)/R)) +
(a
U

+b
U
*T)*(exp((–DH
m
/T + DH
m
/T
m
)/(R)/(1 + exp((– DH
m
/
T + DH
m
/T
m
)/(R))) with a
F
,b
F
,a
U
and b
U
describing the pre- and
post transitional baselines, DH
m
and T
m
as fitting parameters
[21,22,23]. Thermal unfolding of T. reesei Cel7A is reversible at pH 5.8

as demonstrated earlier by the activity and CD measurements [11].
Fig. 4. Time dependency of the fluorescence emission spectra of wild-
type Cel7A and pH mutant measured at two different pH values at
25 °C. (A) The emission spectra of wild-type Cel7A at (a) pH 5.0 (no
time-dependent intensity change), (b) pH 8.0, t ¼ 0 min, (c) pH 8.0,
t ¼ 120 min. (B) The emission spectra of Cel7A E223S/A224H/
L225V/T226A/D262G mutant at (a) pH 5.0 (no time-dependent
intensity change), (b) at pH 8.0, t ¼ 0,(c)atpH 8.0,t ¼ 120 min. The
excitation wavelength was 285 nm and the concentration of enzyme in
the experiments was 1 l
M
.
846 H. Boer and A. Koivula (Eur. J. Biochem. 270) Ó FEBS 2003
position 224 [3]. The pH range (pH 6–8) where the change
in fluorescence intensity is observed, fits well also with the
pK
a
value of a histidine residue.
Concluding remarks
We have demonstrated that both the thermal stability and
enzymatic activity of T. reesei Cel7A follow a bell-shaped
curve as a function of pH, and that a soluble substrate
stabilizes the protein fold both at acidic and alkaline pH.
The Cel7A pH mutant having a more alkaline pH
optimum, was shown to have lowered thermostability
both at acidic and alkaline pH demonstrating that the
kinetic stability measurements performed earlier did not
give a full picture of the stability behaviour. Furthermore,
the decreased stability of the pH mutant might be the
reason for the decreased activity (k

cat
values). In order to
engineer more alkaline pH cellulases (or other enzymes)
for applications, both activity and thermostability issues
should thus be considered. A combination of mutations
should be found, which both change the pH optimum,
and at the same time improve the thermostability at the
new pH optimum. Whether these changes can be achieved
by a rational design or by directed evolution approaches is
an open question.
Acknowledgements
We thank S. Auer, R. Fagerstro
¨
m, T. Kinnari, T. Liljankoski, M. Siika-
aho and R. Suihkonen for construction of the Cel7A mutant, and for
the purification of the wild-type and the mutant protein. K. Rautajoki
is thanked for the help with the fluorescence measurements. The
research was supported by a Marie Curie Research Training Grant
(BIO4-CT97-5037) of the European Community and by EU Biotech-
nology Program grant BIO4-CT96-0580.
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