Thermal Stability of Humicola insolens Cutinase in aqueous SDS

Cutinase from Humicola insolens (HiC) has previously been shown to bind anomalously low amounts of the anionic surfactant sodium dodecylsulfate (SDS)...
0 downloads 0 Views 121KB Size
Download PDF
J. Phys. Chem. B 2007, 111, 2941-2947

2941

Thermal Stability of Humicola insolens Cutinase in aqueous SDS Anders D. Nielsen,†,‡ Kim Borch,§ and Peter Westh*,† Department of Life Sciences and Chemistry, Roskilde UniVersity, 1 UniVersitetsVej, DK-4000 Roskilde, Denmark, and NoVozymes A/S, KoghshøjVej 36, DK-2880 BagsVaerd, Denmark ReceiVed: September 10, 2006; In Final Form: December 14, 2006

Cutinase from Humicola insolens (HiC) has previously been shown to bind anomalously low amounts of the anionic surfactant sodium dodecylsulfate (SDS). In the current work, we have applied scanning and titration calorimetry to investigate possible relationships between this weak interaction and the effect of SDS on the equilibrium and kinetic stability of HiC. The results are presented in a “state-diagram,” which specifies the stable form of the protein as a function of temperature and SDS concentration. In comparison with other proteins, the equilibrium stability HiC is strongly decreased by SDS. For low SDS concentrations (SDS:HiC molar ratio, MR < 8) this trait is also found for the kinetically controlled thermal aggregation of the protein. At higher MR, however, SDS stabilizes noticeably against irreversible aggregation. We suggest that this relies on electrostatic repulsion of the increasingly negatively charged HiC-SDS complexes. The combined interpretation of calorimetric and binding data allowed the calculation of the changes in enthalpy and heat capacity for the association of HiC and SDS near the saturation point. The latter function was about -410 J mol-1 K-1 or similar to the heat capacity change for micelle formation (-470 J mol-1 K-1). This suggests that SDS is hydrated to a similar extent in the micellar and protein associated forms. The results are discussed in terms of the Wyman theory for linked equilibria. Quantitative analysis along these lines suggests that the reversible thermal unfolding of the protein couples to the binding of 2-3 additional SDS molecules. This corresponds to a 15-20% increase in the binding number. Wyman theory also rationalizes relationships between low affinity and high susceptibility observed in this study.

Introduction The industrial use of enzymes is rapidly proliferating. The largest application is their use as detergent additives for the removal of specific stains.1 One inherent problem in this application, however, is the limited compatibility with surfactants, which are abundant in commercial detergents. It follows that there is a considerable interest in the development of enzyme variants with low susceptibility to surfactantss particularly ionic species which are in general strong denaturants. This design of surfactant resistant enzymes may exploit different avenues including so-called directed evolution, Nature’s diversity or rational design.1 As far as the latter approach is concerned, the primary requirement for progress is a detailed insight into the nature of protein-surfactant interaction, along with knowledge on how these interactions modulate protein properties. This area, however, remains only partially elucidated. Most work has used the sodium salt of the anionic surfactant dodecyl sulfate (SDS), and many reports have emphasized strong similarities of SDS’s interactions with a number of globular proteins. Thus, pronounced binding of SDS occurs even at submM concentrations, and as [SDS] is raised, the adsorption reaches a plateau at ∼0.4 g SDS/g protein.2-8 Further increase in the SDS concentration brings about a steep increase in the binding number which eventually saturates at a level of about 1.4 g/g for proteins with reduced cysteines.3,8-11 For proteins with intact S-S bonds the saturation level is somewhat lower * Corresponding author., Email: [email protected], Fax +45 4674 3011 † Roskilde University. ‡ Present address: Novo Nordisk A/S, Novo Alle ´ , DK-2880 Bagsvaerd, Denmark. § Novozymes A/S.

at about 1 g/g.10 Particularly cogent demonstrations of this twophase binding behavior based on parallel investigations of several model proteins include the works by Reynolds and Tanford11 and Takagi et al.6 While the conspicuous similarities of different SDS-protein systems have been crucial for the current understanding, further progress probably requires focus on the specifics of certain proteins. This was already discussed 30 years ago by Takagi and co-workers,6,12 and recent work has indeed highlighted SDS effects, which are characteristic to a certain proteins.13,14 One of the most important properties, which may display specific dependence of the protein, is the structure of the saturated protein-SDS aggregate and several structural arrangements have been discussed.15-18 We have recently investigated interactions of SDS and the lipolytic enzyme cutinase from Humicola insolens (HiC).19,20 This is a small (22kDa) and experimentally convenient protein, which is of direct interest to the detergent industry. It appeared that HiC interacts quite differently with SDS than other investigated proteins. Thus, the radius of gyration, determined by small angle neutron scattering, only increased marginally (from 17 to 18 Å) upon denaturation by SDS, and at room temperature the binding at saturation (∼0.5 g/g) was 2-3 times lower than typical values. These observations concurrently suggested that HiC “interacted weakly” with SDS. This property (although vaguely defined) makes HiC-SDS a relevant system in attempts to single out characteristics of importance for protein-surfactant compatibility, including possible relationships between “weak interactions” and the effects of SDS on the conformational stability. In light of this, we have undertaken an investigation on the thermal stability of HiC in SDS solutions.

10.1021/jp065896u CCC: $37.00 © 2007 American Chemical Society Published on Web 02/24/2007

2942 J. Phys. Chem. B, Vol. 111, No. 11, 2007

Nielsen et al.

Figure 1. Scanning calorimetric data for 53µM HiC in buffers with different SDS concentrations (given as the molar ratio, MR ) [SDS]/[HiC]). The peak in the excess heat capacity function reflects the thermal denaturation of the protein and the exothermic step is assigned to aggregation and precipitation.

Methods and Materials Enzyme and Chemicals. Recombinant HiC was expressed in Aspergillus oryzae and purified to >95%, determined by SDS/ PAGE, at Novozymes A/S, Bagsvaerd, Denmark.21 The protein was extensively dialyzed, at 5 °C, against 50 mM TRIS, 2 mM EDTA, pH 7.0. All measurements reported here are made in this buffer. The following chemicals were used: TRIS, (>99%, Merck, Darmstadt, Germany), ethylenediaminetetraacetic acid, EDTA (>99%, Merck, Darmstadt, Germany) and sodium dodecyl sulfate, SDS (>99%, Fluka, Buchs, Switzerland). Isothermal Titration Calorimetric (ITC). The calorimetric measurements were conducted on a MCS-ITC (MicroCal Inc., Northampton, MA) isothermal titration calorimetry equipment.22 The reference cell was filled with water. In a typical experiment, the sample cell was loaded with a solution of 63-86 µM cutinase solution. The cell solution was titrated with 50-104 aliquots of 5 µL of SDS solution. In experiments exceeding 50 injections, it was necessary to make an initial titration trial of approximately 50 injections. Upon refilling the syringe another 50 injections was made into the resulting cell solution obtained in the first trial. The two data files were merged into a single file using the ITC-merge software written by Dr. Bent W. Sigurskjold, University of Copenhagen. The obtained heat signals from the ITC were integrated using the Origin software supplied by MircoCal Inc. More details pertaining to the ITC protocols have been given elsewhere.19 Differential Scanning Calorimetry (DSC). Scanning calorimetric experiments were performed on a MicroCal VP-DSC (Northampton, MA). All HiC solutions were dialyzed against the desired buffer, and the dialyzate was used as reference. Prior to scanning all solutions were degassed by stirring under vacuum. A pressure of 2 atm was applied over the cells to avoid the formation of bubbles during scanning. The concentration of HiC was 53 µM in all trials while the SDS concentration was varied from 0 to 2 mM. The data was analyzed using the Origin software from MicroCal, Inc. supplied with the instrument. Molar excess heat capacities (CEp) were obtained by subtracting a baseline (buffer) run from the raw out-put and normalizing with respect to the HiC concentration and the volume of the calorimetric cell. Each SDS concentration was

investigated in two separate DSC trials which gave nearly identical results in all cases. Results The effects of SDS on the thermal stability of HiC are illustrated in Figure 1, which presents typical traces from the DSC trials. In general the scans showed two characteristics: first an endothermic peak, and second a strong exothermic deflection, which occurred either in immediate succession to the peak or following an intermediate baseline. An analogous behavior (in surfactant-free buffer) has recently been reported for cutinase from Fusarium solani pisi.23 In accordance with this latter work we assign the peak to the thermal denaturation of the native conformation and the exothermic “step” to the aggregation and precipitation of the denatured protein. The temperatures of these transitions are designated, respectively, Td and Ta. We define Td as the maximum of the excess heat capacity trace and Ta as the crossover of the baseline and an extrapolation of the almost vertical decrease in the trace at high-temperature (Figure 1). Inspection of the peak location shows that the stability of HiC is strongly affected by SDS and that a decrease in Td can be readily detected with as little as 0.5 mol SDS/mol HiC. For molar ratios (MR ) [SDS]/[HiC]) less than 6-8, the aggregation occurs immediately after the completion of the denaturation peak. Thus, both Td and Ta decrease with MR in a near linear fashion with a slope of 1.5 °C per MR unit (see Figure 5 discussed below). (MR is the total (free + bound) number of moles of SDS in the calorimetric cell per mole of protein. It should not be confused with the stoichiometry of proteinsurfactant adducts.) Interestingly, at still higher SDS concentrations, Td continues to decrease with approximately the same slope (Figure 5) while Ta passes through a minimum at MR∼8 and increases strongly to reach 85 °C at the highest SDS concentrations investigated here (MR ) 40). Analysis of the peak areas showed that the enthalpy change, ∆Hdenat, for the denaturation of HiC in (SDS-free) buffer was 360 kJ/mol. Figure 2 illustrates that this value decreases quickly with increasing SDS concentration, and that no endothermic peak could be detected in the DSC trials when MR exceeded ∼15.

Thermal Stability of Cutinase in SDS

Figure 2. Dependence of the measured enthalpy change, ∆Hdenat, for the denaturation of HiC on the concentration of SDS. The abscissa is in units of molar ratio (MR ) mol SDS/mol HiC).

Figure 3. Integrated data from the ITC measurementssso-called enthalpogramssfor the titration of 63 µM HiC with SDS at four temperatures. The abscissa is the molar ratio (see Figure 2). The ordinate is the enthalpy change, ∆H, produced per mol of SDS injected. The inset shows the value of ∆H for MR ) 60 plotted as a function of temperature.

Selected data from the titration calorimetry are presented in Figure 3. It appears that the enthalpy change, ∆H (in kJ per mole of SDS injected) shows a number of characteristic traits as [SDS] is gradually raised. For 22 °C, the molecular origin of these have recently been discussed in some detail.19,20 In short, the exothermic effect (∆H < 0) at very low SDS concentration (MR < ∼2) was ascribed to the specific binding of SDS, possibly to the active site of HiC. The strong endothermic peak which occur in the 2 < MR < 25 range (Figure 3) was assigned to two processes namely the formation of dimeric HiC (with a number of bound SDS molecules) followed by the denaturation of the protein molecule at the higher end of this interval. In the intermediate range of SDS concentrations (25 < MR < 75), it was found that SDS adsorbed to denatured HiC without introducing detectable structural changes in the protein molecule. Eventually the binding saturated at the sigmoidal part of the isotherms (MR ∼ 90 in Figure 3). At this point of the titration, the bulk (“free”) concentration of SDS reached the critical micelle concentration (CMC). These earlier conclusions for 22 °C are substantiated by the temperature dependence illustrated in Figure 3. The most conspicuous effect of increasing temperature is that the “dena-

J. Phys. Chem. B, Vol. 111, No. 11, 2007 2943

Figure 4. Results from ITC trials where 83 µM HiC was titrated with an 8 mM SDS solution. This protocol provides better resolution for the peak at low MR, which is also seen in Figure 3. The initial exothermic enthalpy change (MR ∼ 1-2) was assigned to specific binding of SDS to HiC while the minimum at MR ) 3-4 coincided with the formation of a dimeric protein adduct detected by SANS.20 The endothermic peak around MR ) 3-15 was interpreted as the denaturation of the protein conformation.

turation peak” at low SDS increases dramatically. This is in accord with the general trait that the unfolding of globular proteins is accompanied by a large increase in heat capacity, and thus that ∆Hdenat increases strongly with temperature. Hence, many small soluble proteins show a 2-4 fold increase in ∆Hdenat over the temperature interval (22-45 °C) investigated here.24 To test this further, we conducted a number of ITC experiments with lower SDS concentration in the syringe. This approach provides better resolution in the low MR range. The results in Figure 4 clearly show that the peak at low MR consist of two partially resolved contributions. On the basis of the data at 22 °C,19,20 the former is suggested to reflect both the specific binding of SDS molecule to HiC and the formation of a proteinSDS adduct with two HiC molecules. The second contribution, which increases strongly with temperature and starts at a lower MR the higher the temperature (Figure 4) reflects the denaturation of the protein. Integration of this major peak at 45 °C (Figure 4) from MR ) 3 to 14 and normalization with respect to the amount of HiC suggest an enthalpy change of about 140 kJ/mol HiC. This value compares favorably with the average of the ∆Hdenat function (measured by DSC) over the same interval (3 < MR < 14) in Figure 2. Thus, the current data support the interpretation that the second endothermic peak in Figure 4 reflects the SDS-induced breakdown of the native HiC structure. To discuss temperature effects in the intermediate MR range we now return to Figure 3. At 22 °C the signal here (25 > MR > 90) is negative (exothermic) but it changes to practically athermal (∆H ) 0) at 30 °C and endothermic values at still higher temperatures. As discussed above, the signal in this range is governed by the adsorption of surfactant to the denatured protein molecule. The enthalpy change at MR ) 60 was read off Figure 3 and plotted as a function of the temperature in the inset of the figure. It appears that ∆H (MR ) 60) increases linearly with temperature and regression suggested a slope ∆H/∆T of 235 ( 30 J mol-1 K-1. The inflection point around MR ) 90, which was assigned to the saturation of the protein-surfactant binding,19 depends only weakly on the temperature. Closer inspection of enlarged copies of Figure 3 suggested that the location of the inflection point increased from about MR ) 91 to 94 over the 22 to 45 °C interval. In other words, the ITC data suggests a very

2944 J. Phys. Chem. B, Vol. 111, No. 11, 2007

Nielsen et al. equilibrium constant K ) [D]/[N], linkage theory stipulates that addition of SDS perturbs the equilibrium as26

(

∂ ln K ∂ ln aSDS

Figure 5. State diagram for a HiC solution of about 60µM. The abscissa gives the total (bulk + protein bound) concentration of SDS in mM (upper) and MR (lower) units. Open circles represent the onset of the aggregation specified by the distinct downward bent in the DSC traces (Figure 1). The denaturation of HiC is indicated by filled circles (from DSC data) and squares (ITC data). Dimerization of HiC, originally detected at 22 °C by SANS and specified by the minima in the ITC data in Figure 4, is indicated by the asterisks. Finally, saturation of SDS binding to HiC, specified by the inflection point in the ITC traces (Figure 3) is given by triangles.

limited temperature dependence of the saturation binding number. Interestingly, CMC for SDS in the buffer used here increased by 30% in the same temperature range,20 and this challenges the strict coupling between saturation of HiC-surfactant binding on one hand and micelle formation in the bulk on the other, which was suggested earlier on the basis of data at room temperature.19 Discussion Interactions of proteins and surfactants are highly complex and involve a number of linked equilibria such as binding, denaturation, micellization, and oligomer formation. In addition, aggregation and precipitation of protein molecules may occur under kinetic control far from equilibrium. In an attempt to provide an overview of these processes for HiC-SDS, we have compiled the data in a “state diagram” in Figure 5. (Obviously Figure 5 is related to a phase diagram, but we refrain from this term since the aggregation process most likely occur far from equilibrium. Figure 5 thus specifies the dominant state of HiC on a typical experimental time scale.) This plot identifies the stable state of HiC as a function of temperature and SDS concentration. In the following we discuss different aspects of the interactions and transformations which underlie the boundaries in this figure. Thermal Stability. The thermal denaturation of a related cutinase has been studied in detail by Creveld et al.,23 and it was concluded that the protein (in surfactant-free buffer) went through one (or two) initial equilibrium step(s) followed by an irreversible aggregation. We interpret the DSC data on HiC analogously, and the boundary specified by the solid circles in Figure 5 thus represents the reversible transition. The clear negative slope of this boundary even at very low MR implies that HiC is strongly destabilized by SDS. This may be interpreted along the lines of the so-called linkage theory.25 If we consider the denaturation equilibrium, NTD with the

)

) νD - νN ) ∆ν

(1)

T,P,aHiC

where aSDS is the thermodynamic activity of SDS and T and P have their usual meaning. The function ν, is the preferential binding parameter, ν ) (∂mSDS/∂mHiC)T,P,aSDS, where mi denotes the molal concentration of component i (i ) HiC or SDS). To within a negligible approximation,26 ν is the binding number (mol SDS per mol protein) measured in a dialysis equilibrium experiment. On a qualitative level eq 1 reflects the principle of LeChateliersaddition of SDS will displace the equilibrium toward the state of protein (N or D) to which the surfactant binds the most. This is a purely thermodynamic approach which does not require any considerations of the complex’s structure or how the bound surfactants might disturb forces in the protein conformation. The negative slope in Figure 5 (filled circles) signifies that the average affinity of SDS is higher for the denatured than for the native state of HiC. As mentioned above, SDS has been suggested to bind specifically to the lipid binding site (in the native state) of both this and another cutinase.19,20,23,27 If this binding site was disrupted upon denaturation, the concomitant release of SDS would contribute negatively to ∆ν. It follows (eq 1) that SDS would decrease K and thus stabilize the native conformation at the lowest MR where nonspecific effects are weak. This type of behavior has indeed been observed for other proteins,28,29 particularly bovine serum albumin (BSA), which strongly binds about 10 SDS.30 As a result, ∆ν is negative and SDS induces a remarkable 20-25 °C increase in the thermal stability of BSA for MR ) 10-20.31-33 In contrast to these examples, the clear negative slope in Figure 5, even for MR < 1, suggests that a possible SDS-binding site in HiC remains intact in the D-state. This implies a high degree of residual structure in the D state in accord with earlier structural investigations.20,27 As mentioned in the introduction HiC interacts weakly with SDS in the senses that (i) the binding isotherm saturates at a low level (on the g/g scale) and (ii) that the overall dimensions of the protein molecule hardly change even in high SDS. It is of interest to investigate relationships of this and the protein’s compatibility with surfactant. If we use thermal stability as a measure of compatibility it is clear that the low affinity of HiC for SDS does not confer any particular resistance toward denaturation by the surfactant. In fact, comparisons with the thermal stability of other proteins in SDS solutions suggest that HiC is one of the more susceptible proteins.28,34-38 Earlier reports on the compatibility of other cutinases and anionic surfactants provide a picture of high to medium susceptibility. Egmont39 found that F. pisi cutinase was denatured by as little as 0.5 mM lithium dodecylsulfate. Thus, this system is even less compatible than HiC-SDS. Conversely, Creveld et al.23 found that F. pisi cutinase remained quite stable (Td)48 °C) in a 5 mM solution of sodium taurodeoxycholate. Also using cutinase from F. pisi, Goncalves et al.40 reported that AOT (sodium bis[2-ethylhexyl]ester sulfosuccinic acid) had no effect on the stability for MR < 5. Higher surfactant concentrations, however, strongly destabilized the protein, which was denatured for MR ∼ 30; i.e. similar to what was found for HiC in this work. Also in line with the current observations, Kolattukudy reported that F. pisi cutinase with a covalently linked fluorescent probe was denatured by about 1 mM SDS.27 We conclude that low affinity for SDS (exhibited by HiC) is not associated with high resistance toward surfactant-induced

Thermal Stability of Cutinase in SDS

J. Phys. Chem. B, Vol. 111, No. 11, 2007 2945

destabilization. This is readily rationalized through the linkage theory expressed in eq 1. Thus, designing a protein with a native state with very low affinity for SDS (i.e., νN ∼ 0) might be counterproductive since any binding to the D-state (νD > 0) would give a significant positive ∆ν and thus a high susceptibility toward SDS denaturation (eq 1). The key parameter, ∆ν, may be numerically estimated from the slope, dTd/dmSDS, of the denaturation boundary in Figure 541,42

∆ν ) -

∆Hdenat(dTd/dmSDS) RTm2(∂ ln aSDS/dmSDS)Tm

(2)

where ∆Hdenat is the enthalpy change of the NTD transition at the transition temperature, Td. Since the concentration of SDS does not exceed a few mM in the solutions investigated here, we may consider them ideal dilute mixtures (aSDS ∼ mSDS). Solving the differential in the denominator of eq 2 then yields

∆ν ) -

mSDS∆Hdenat(dTd/dmSDS) RTd2

(3)

To put eq 3 to use, we determine the slope of the (near linear) denaturation boundary in Figure 5 (∆T/∆CSDS ∼ -2.5 × 104 K/M). Also, we read off values of Td (Figure 5) and ∆Hdenat (Figure 2) at 0.1 mM intervals of [SDS] in the range which could be investigated by DSC ( 15. Consequently, the SDS concentration required to denature HiC may become almost independent of temperature. The equilibrium binding of SDS to denatured HiC at intermediate MR shows a heat capacity change comparable to that of for micelle formation. This suggests a surprisingly effective molecular packing of small SDS aggregates on the protein molecule. Analysis of the kinetic stability of HiC shows that SDS provides significant stabilization against aggregation and precipitation for MR>10. We suggest this is due to electrostatic repulsion among negativily charged HiC-SDS complexes.

Nielsen et al. Acknowledgment. This work was supported by the Danish National Science Foundation through the establishment of the MEMPHYS centre of excellence. The financial support by the Carlsberg Foundation, Novozymes Ltd., and the Danish Research Agency (Grants 26-02-0160 and 21-04-0087) is gratefully acknowledged. References and Notes (1) Kirk, O.; Borchert, T. V.; Fuglsang, C. C. Cur. Opin. Biotech. 2002, 13, 345. (2) Bordbar, A. K.; Saboury, A. A.; Housaindokht, M. R.; MoosaviMovahedi, A. A. J. Colloid Interface Sci. 1997, 192, 415. (3) Jones, M. N. Chem. Soc. ReV. 1992, 21, 127. (4) Jones, M. N.; Manley, P. Int. J. Biol. Macromol. 1982, 4, 201. (5) Sen, M.; Mitra, S. P.; Chattoraj, D. K. Ind. J. Biochem. Biophys. 1980, 17, 370. (6) Takagi, T.; Tsujii, K.; Shirahama, K. J. Biochem. 1975, 77, 939. (7) Takeda, K.; Miura, M.; Takagi, T. J. Colloid Interface Sci. 1981, 82, 38. (8) Takeda, K.; Moriyama, Y.; Hachiya, K. Interaction of protein with ionic surfactants: Part 1. Binding of surfactant to protein and protein fragments, and conformational changes induced by binding. In Encyclopedia of Surface and Colloid Science; Somasundaran, P., Ed.; Marcel Dekker: New York, 2002; pp 2558-2574. (9) Fish, W. W.; Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 5166-5168. (10) Pittrivers. R. Impiombato. Fs, Biochem. J. 1968, 109, 825. (11) Reynolds, J. A.; Tanford, C. Proc. Nat. Acad. Sci. U.S.A. 1970, 66, 1002. (12) Shiraham. K; Tsujii, K.; Takagi, T. J. Biochem. 1974, 75, 309. (13) Gudiksen, K. L.; Gitlin, I.; Whitesides, G. M. Proc. Nat. Acad. Sci. U.S.A. 2006, 103, 7968. (14) Xu, Q.; Keiderling, T. A. Protein Sci. 2004, 13, 2949. (15) Gudiksen, K. L.; Gitlin, I.; Moustakas, D. T.; Whitesides, G. M. Biophys. J. 2006, 91, 298. (16) Guo, X. H.; Chen, S. H. Chem. Phys. 1990, 149, 129. (17) Ibel, K.; May, R. P.; Kirschner, K.; Szadkowski, H.; Mascher, E.; Lundahl, P. Eur. J. Biochem. 1990, 190, 311. (18) Turro, N. J.; Lei, X. G.; Ananthapadmanabhan, K. P.; Aronson, M. Langmuir 1995, 11, 2525. (19) Nielsen, A. D.; Arleth, L.; Westh, P. Biochim. Biophys. Acta 2005, 1752, 124. (20) Nielsen, A. D.; Arleth, L.; Westh, P. Langmuir 2005, 21, 4299. (21) Andersen, A.; Svendsen, A.; Vind, J.; Lassen, S. F.; Hjort, C.; Borch, K.; Patkar, S. A. Col. Surf. B 2002, 26, 47. (22) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem. 1989, 179, 131. (23) Creveld, L. D.; Meijberg, W.; Berendsen, H. J. C.; Pepermans, H. A. M. Biophys. Chem. 2001, 92, 65. (24) Makhatadze, G. I.; Privalov, P. L. AdV. Protein Chem. 1995, 47, 307. (25) Wyman, J.; Gill, S. J. Binding and Linkage: Functional Chemistry of Biological Macromolecules; University Science Books: Mill Valley, 1990. (26) Timasheff, S. N. AdV. Protein Chem. 1998, 51, 355. (27) Kolattukudy, P. E. Cutinases from fungi and pollen. In Lipases; Borgstrom, B., Brockman, H. L., Eds.; Elsevier: Amsterdam, 1984; pp 472504. (28) Boye, J. I.; Ma, C. Y.; Ismail, A., J. Dairy Res. 2004, 71, 207. (29) Dauria, S.; Rossi, M.; Nucci, R.; Irace, G.; Bismuto, E. Protein Struct. Func. Gen. 1997, 27, 71. (30) Nielsen, A. D.; Borch, K.; Westh, P. Biochim. Biophys. Acta 2000, 1479, 321. (31) Deep, S.; Ahluwalia, J. C. Phys. Chem. Chem. Phys. 2001, 3, 4583. (32) Giancola, C.; DeSena, C.; Fessas, D.; Graziano, G.; Barone, G. Int. J. Biol. Macromol. 1997, 20, 193. (33) Yamasaki, M.; Yano, H.; Aoki, K. Int. J. Biol. Macromol. 1992, 14, 305. (34) Jones, M. N.; Manley, P.; Midgley, P. J. W.; Wilkinson, A. E. Biopolymers 1982, 21, 1435. (35) Jones, M. N.; Manley, P.; Wilkinson, A. Biochem. J. 1982, 203, 285. (36) Miksovska, J.; Yom, J.; Diamond, B.; Larsen, R. W. Biomacromolecules 2006, 7, 476. (37) Prakash, V.; Nandi, P. K.; Jirgensons, B. Int. J. Pep. Protein Res. 1980, 15, 305. (38) Rao, K. S.; Prakash, V. J. Biol. Chem. 1993, 268, 14769. (39) Egmond, M. R. Action of Lipases. In Engeneering of/with Lipases; Malcata, F. X., Ed.; Klu¨ver Academic Press: Dordrecht, The Nederlands, 1996; pp 183-191.

Thermal Stability of Cutinase in SDS (40) Goncalves, A. M. D.; Aires-Barros, M. R.; Cabral, J. M. S. Enz. Microb. Technol. 2003, 32, 868. (41) Delpino, I. M. P.; Sanchezruiz, J. M. Biochem. 1995, 34, 8621. (42) Kovrigin, E. L.; Potekhin, S. A. Biochem. 1997, 36, 9195. (43) Livingstone, J. R.; Spolar, R. S.; Record, M. T. Biochem. 1991, 30, 4237. (44) Spolar, R. S.; Ha, J. H.; Record, M. T. Proc. Nat. Acad. Sci. U.S.A. 1989, 86, 8382. (45) Amidon, G. L.; Yalkowsk, Sh.; Leung, S. J. Pharm. Sci. 1974, 63, 1858. (46) Hermann, R. B. J. Phys. Chem. 1972, 76, 2754.

J. Phys. Chem. B, Vol. 111, No. 11, 2007 2947 (47) Gelamo, E. L.; Itri, R.; Tabak, M. J. Biol. Chem. 2004, 279, 33298. (48) Santos, S. F.; Zanette, D.; Fischer, H.; Itri, R. J. Colloid Interface Sci. 2003, 262, 400. (49) Shweitzer, B.; Zanette, D.; Itri, R. J. Colloid Interface Sci. 2004, 277, 285. (50) Ibel, K.; May, R. P.; Sandberg, M.; Mascher, E.; Greijer, E.; Lundahl, P. Biophys. Chem. 1994, 53, 77. (51) Pocalyko, D. J.; Tallman, M. Enz. Microb. Technol. 1998, 22, 647. (52) Valstar, A.; Brown, W.; Almgren, M. Langmuir 1999, 15, 2366. (53) Vasilescu, M.; Angelescu, D.; Almgren, M.; Valstar, A. Langmuir 1999, 15, 2635.