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. 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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,. 8.0 and 25 °C. Spectra of the wild-type Cel7A at pH 5.8 (d) and pH 8.0 (s), and the pH mutant at pH 5.8 (j) and pH 8.0 (h) were recorded from 240 to 190 nm using a 1-mm path length cell and a bandwidth. 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