Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH3.5–5.6 Tm, 62 ± 2C, at which the highest enzymatic activity is also observed
Trang 1The relationship between thermal stability and pH optimum
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 reeseiCel7A in the alkaline pHrange, the thermal
sta-bility of Cel7A was studied as a function of pHwith 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 pH3.5–5.6 (Tm, 62 ± 2C), at which the highest
enzymatic activity is also observed, and a gradual decrease in
the stability at more alkaline pHvalues 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 pHoptimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerstro¨m, R.B., H arris, 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 pHwhen 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 pHmutant Our data show that in order to engineer more alkaline pHcellulases, a combination of mutations should be found, which both shift the pHopti-mum and at the same time improve the thermal stability at alkaline pHrange
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 reeseiis an acidophilic fungus and most of its secreted cellulases function optimally at around pH5 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 pHof these enzymes Recently a variant of
T reesei Cel7A containing five point mutations (E223S/ A224H/L225V/T226A/D262G) and having a more alkaline pHoptimum was produced [3] The mutations were designed based on the sequence comparisons of family 7 cellulases having different pHbehaviours The comparison revealed that a histidine residue near the acid/base catalyst could account for the higher pHoptimum of the Humicola insolens Cel7B endoglucanase and therefore a mutation A224Hwas 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)
Trang 2histidine 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 pHmutant behaved in the desired way
with respect to the pHoptimum, 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 pHmutant 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, e¼ 83000M )1Æcm)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 pHmutant
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 80C 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
25–85C 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 lM Buffers used were 50 mMsodium acetate (pH3.5–5.6), 50 mMpotassium phosphate (pH6–8) and 50 mMTris/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 temtem-perature 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 1CÆmin)1until a temperature of
80C was reached The measurements were performed at a low salt concentration (a requirement for CD spectroscopy)
in 10 mMpotassium phosphate buffer at pH5.8 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 pHhas a bell shape [12] The activity profile
of an enzyme is also pHdependent, and, depending on the enzyme, the pHstability and activity curves can in theory overlap totally, partially or not at all When thinking of applying an enzyme at certain pHand 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 pH5.0 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 pHoptimum, and focus especially on the alkaline pHarea Fig 1A shows the temperature-induced unfolding of T reesei wild-type Cel7A at both pH5.0 [11] and pH8.0 Unfolding was monitored by heating samples gradually up to 80C 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 65C at pH 5.0, while at pH8.0 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 (Tm) 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
Trang 3effect on the overall stability of the catalytic domain of
Cel7A (data not shown)
The melting temperatures (Tm) for wild-type Cel7A
enzyme were then measured over a pHrange 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 pH3.5 and 5.6 (Tm¼ 62 ± 2 C) This pHarea
coincides with the highest enzymatic activity of Trichoderma
Cel7A measured on soluble methyl-umbelliferyllactoside
(Fig 2, d) A pHoptimum between pH4 and 5 has also
been reported for wild-type Cel7A on other soluble
substrates at temperatures ranging from 25 to 37C
[3,11,13,14] Thus it seems that T reesei Cel7A has the
highest activity at acidic pHvalues, where the protein is also
most stable At pH8.0, 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 pH8.0, Cel7A seems to
be folded at this pHat 25C (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 pH5.0 and pH8.0
The binding at pH5 was first checked with the wild-type
enzyme, but as it is fully active at pH5, 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 pH5.0 At pH8.0 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 pH8 and pH5, 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 lM) was used in these experiments, and this is assumed to be a saturating concentration based on the measured Kmvalue of 7 lMfor wild-type Cel7A enzyme [15,16] Fig 3A and C demon-strate that cellotetraose has a small stabilizing effect on Cel7A at both pH8.0 and 5.0, respectively When the enzyme–substrate mixture was heated, a red shift in the emission maximum occurred at both pHvalues (Fig 3B and D), indicative of the release of the substrate Compari-son of the Cel7A unfolding with and without the substrate either at pH8.0 (Fig 3A and B), or pH5 (Fig 3C and D) shows interestingly that the unfolding of the protein and the release of the substrate coincide at both pH5.0 and pH8.0 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, pH5.0 (d) and 50 m M potassium phosphate, pH8.0 (s) The concentration of the enzyme in the experiments was 1 l M Samples were heated gradually to 75 C at approximately 1 CÆmin)1and 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 pHvalues were determined by plotting the differential, d(Int)/dT, against temperature.
Trang 4One 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
pH5.0 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
the enzyme is active
The Cel7A pHmutant 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 pHoptimum shifted
to a more alkaline pHalmost by one unit [3] This supported the hypothesis that the histidine residue, which is observed
in cellulases with a higher pHoptimum, such as Humicola insolens Cel7B endoglucanase, has an influence on the activity of the enzyme at more alkaline pHby 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 pHoptimum, had little effect on the Km, but the kcatvalue 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 pHregions, pH values at which this enzyme could be applied Unexpectedly, when the fluorescence emission spectrum of the mutant enzyme was measured at pH8.0 and 25C, a time-dependent decay of fluorescence intensity was noted Further studies on the fluorescence intensity as a function
of time showed that at pH5.0 the fluorescence emission spectra of the mutant and the wild-type Cel7A (Fig 4A) are constant over a period of hours However, when diluted in pH8.0 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 pH6 and 7, but at a lower rate than in the experiment at pH8 This phenomenon is reversed within 5 min when the pHof the mutant protein solution is changed again from pH8 to 5 These observa-tions suggested that at pH8.0 the five point mutaobserva-tions 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 pH8.0, this method could not be used to produce a plot of
Tmas a function of pHfor the Cel7A pHmutant Instead, the temperature-induced unfolding of the Cel7A pHmutant (and wild-type enzyme) were measured by CD spectroscopy
at different pHvalues (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 80C at a rate
of 1CÆmin)1 using a Peltier-type temperature control system The initial CD spectra at 25C of the wild-type and the mutant measured from 240 to 190 nm at pH5.8 and pH8.0 in 10 mM potassium phosphate buffer were very similar, indicating no major conformational changes in the mutant enzyme structure at those two pHvalues (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 Tmspectra 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 pH5.8 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 pH3.5–5.6, 50 m M
potassium phosphate pH6.0–8.0, and 50 m M Tris/HCl pH 9.0 The
activity/pHprofile (d) of Cel7A (0.8 l M ) was determined at 30 C by
incubating enzyme samples first for 15 min at different pHvalues.
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 pHprofile under
satur-ating conditions over the whole pHrange At best the 500 l M MULac
corresponds with two times the K m value for this substrate at optimal
pH5 The measured pH profile corresponds well with the other
reported pHactivity profiles [3,11,13,14].
Trang 5from the analysis can be found in the legend of Fig 5
showing that the mutations have lowered the Tmof Cel7A
at pH5.8 by 6C to 57 C The CD measurements further
showed that the Cel7A E223S/A224H/L225V/T226A/
D262G mutant starts to unfold at a lower temperature also
at pH8.0 (Fig 5) as compared with the wild-type enzyme
A broad and much more complex transition is observed at
pH8.0 (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 37C and that of the mutant around 30C (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 reesei wild-type Cel7A in50 m M potassium phosphate pH 8.0 (A and B) and catalytically inactive E217Q mutant in 50 m M NaAc pH 5.0 (C an d D) (A) and (C) The relative intrinsic fluorescence (Int; 1.0 at 30 C) as a function of temperature in the presence (d) and absence (j) of 125 l M cellotetraose (B) and (D) Change in the fluorescence emission maximum
as a function of temperature in the presence (d) and absence (j) of 125 l M cellotetraose The excitation wavelength in these experiments was
285 nm.
Trang 6alkaline pHoptimum, they also seem to lead to lower
thermostability of the mutant protein at both pH5.8 and
pH8.0, as compared with the wild-type enzyme This
lowered overall stability might also explain the smaller kcat
values [3] The stability of the pHmutant was checked
previously at pH2–7 by residual activity measurements A
lower thermostability at pH3 was detected for the mutant,
whereas no changes were observed at neutral pHas
compared with the wild-type enzyme [3] These kinetic
stability studies were performed so that the enzyme was first
preincubated at 37 or 50C for 1 h, after which the activity
was measured at optimal pHand 25C 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 pHmutant 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 sense 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 pKavalue 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
a gradient of 1 CÆmin)1until 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 pHmutant enzyme (h)
at (A) pH5.8 and (B) pH8.0 Analysis of heat denaturation of the wild-type and mutant protein at pH5.8 (solid lines) gave the following parameters: wild-type, T m ¼ 63 C ± 0.03, DH m ¼ 579 ± 6 kJ mol)1, Mutant, T m ¼ 57 C ± 0.08, DH m ¼ 344 ± 6 kJ mol)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 pH5.8
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) pH5.0 (no
time-dependent intensity change), (b) pH8.0, t ¼ 0 min, (c) pH8.0,
t ¼ 120 min (B) The emission spectra of Cel7A E223S/A224H/
L225V/T226A/D262G mutant at (a) pH5.0 (no time-dependent
intensity change), (b) at pH8.0, t ¼ 0, (c) at pH 8.0, t ¼ 120 min The
excitation wavelength was 285 nm and the concentration of enzyme in
the experiments was 1 l M
Trang 7position 224 [3] The pHrange (pH6–8) where the change
in fluorescence intensity is observed, fits well also with the
pKavalue 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 pHmutant having a more alkaline pH
optimum, was shown to have lowered thermostability
both at acidic and alkaline pHdemonstrating that the
kinetic stability measurements performed earlier did not
give a full picture of the stability behaviour Furthermore,
the decreased stability of the pHmutant might be the
reason for the decreased activity (kcatvalues) In order to
engineer more alkaline pHcellulases (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 pHoptimum,
and at the same time improve the thermostability at the
new pHoptimum 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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Fig 6 The far-UV CD spectra of T reesei wild-type Cel7A and pH
mutant E223S/A224H/L225V/T226A/D262G at pH 5.8 and pH 8.0
and 25 °C Spectra of the wild-type Cel7A at pH5.8 (d) and pH8.0
(s), and the pHmutant at pH5.8 (j) and pH8.0 (h) were recorded
from 240 to 190 nm using a 1-mm path length cell and a bandwidth of
1 nm.
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