Thermostability of Native and Pegylated Myceliophthora thermophila

Bioconjugate Chem. 2006, 17, 1093−1098

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TECHNICAL NOTES Thermostability of Native and Pegylated Myceliophthora thermophila Laccase in Aqueous and Mixed Solvents J. I. Lo´pez-Cruz,† G. Viniegra-Gonza´lez,*,† and A. Herna´ndez-Arana‡ Laboratorio de Biologı´a Molecular y Enzimologı´a de Hongos Filamentosos, Departamento de Biotecnologı´a, and Laboratorio de Biofisicoquı´mica, Departamento de Quı´mica, Universidad Auto´noma Metropolitana Iztapalapa, CP. 09340, Avenida San Rafael Atlixco, 186, Me´xico D.F. . Received December 5, 2005; Revised Manuscript Received April 17, 2006

A commercial preparation of laccase (EC 1.10.3.2), cloned from Myceliophthora thermophila and expressed in Aspergillus oryzae (MtL), was purified and modified by conjugation with poly(ethylene glycol) (Mr ) 5000) and is labeled PEG-MtL. Native enzyme was found to have a molecular mass of 80 kDa, as determined by gel filtration, and 110 kDa, by SDS-PAGE. The oxidative dimerization of 2,6-dimethoxyphenol (DMP) to produce the corresponding dibenzoquinone was catalyzed by MtL in a manner comparable to that for a diffusion-controlled reaction (kcat/KM = 108 M-1 s-1 and Ea = 18 kJ M-1). PEG-MtL was found, by TNBS titration, to have blocked 54% of lysine groups; its hydrodynamic and charge properties were different from those of MtL. Catalytic efficiency (kcat/KM) of PEG-MtL was similar to that of MtL with DMP as substrate; however, kcat/KM was 2-fold reduced for the reaction in which 2′,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is oxidized to form a radical cation. Ea values were similar in both enzyme preparations when assayed in buffered solutions. Far-UV CD spectra were similar for MtL and PEG-MtL and consistent with a protein rich in β-sheet structure with negligible content of R-helices. A blue shift of near-UV CD spectrum for PEG-MtL as compared to MtL was consistent with the decreased polarity of the tyrosyl side chains upon PEG conjugation. Also the blue band of the copper active site was shifted from λ ∼ 610 nm (MtL) to λ ∼ 575 nm (PEG-MtL). Scanning microcalorimetry showed small denaturation enthalpies (6.3 and 7.5 J g-1 for MtL and PEG-MtL, respectively), indicating the high stability of the β-sheet folding pattern of laccases. However, PEG-MtL proved to be more stable, its half-denaturation temperature being 2 °C higher than that of MtL. In 30% alcohol, pegylated laccase showed slower enzymeactivity decay rates than the unmodified enzyme; this behavior was caused by a decrease in the activation entropy of the denaturation reaction. Results can be explained by entropic stabilization by PEG conjugation because of the restricted motion of some surface amino acid side chains, which results in a more stable active site.

INTRODUCTION Laccase is an enzyme (p-diphenol: dioxygen oxido-reductase, EC 1.10.3.2) that catalyzes the oxidation of a large variety of phenolic and polyaromatic compounds (1). It has been proposed as a potential bleaching agent of cellulose pulp and organic dyes (2, 3) and also as a catalyst for the oxidation of polluting polycyclic aromatic hydrocarbons (4, 5). This enzyme has four copper atoms in its active site that reduce O2 to water using an appropriate electron donor as substrate (6, 7). One of those copper atoms (T1) absorbs light near 620 nm due to a highly covalent bond between cysteine and Cu (8, 9). Another copper (T2) does not show strong absorption of light, and two other copper atoms (T3) are coupled through a hydroxide and shuttle electrons from substrate to the trinuclear T2/T3 cluster (11, 12). Among the future applications of laccase, the oxidation of dibenzothiophene derivatives in diesel fuel has been mentioned (13, 14), but the rates of oxidation of this compound were found too slow, perhaps because the substrate is sparsely soluble in water and the enzyme is inactivated by the presence of organic * Corresponding author. Tel. +52-5804-4719. Fax: +52-5804-6407. E-mail: [email protected]. † Departamento de Biotecnologı´a. ‡ Departamento de Quı´mica.

solvents (13). Garcı´a-Arellano et al. (15) showed that the conjugation of cytochrome c with poly(ethylene glycol) (PEG) improved the thermal stability of the catalytic site. Furthermore, Vandertol-Varnier et al. (16) showed that laccase from Coriolopsis gallica conjugated with PEG had a higher catalytic activity on syringaldazine than the native enzyme. In this work the thermal stability of laccase from Myceliophthora thermophila (17) conjugated with PEG was studied in relation to the presence of alcohols of different polarity using spectropolarimetric, calorimetric, and enzymological techniques as compared with the native preparation. The hypothesis of this work was that the thermal stability of PEG-laccase would be better than that of native laccase.

EXPERIMENTAL PROCEDURES Chemicals. The enzyme MtL was obtained from the company Novo Nordisk, in the form of a commercial preparation called Deni Lite IIs. Bovine serum albumin (BSA), cyanuric chlorideactivated monomethoxy poly(ethylene glycol) (Mr ) 5 kDa), 2′,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (DMP), and trinitrobenzenesulfonic acid (TNBS) were from Sigma (St. Louis, MO). Extraction and Purification of Laccase. Fifty grams of Deni Lite IIs was suspended in 200 mL of 50 mM potassium

10.1021/bc0503465 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/20/2006

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phosphate buffer, pH 6.0, and shaken for 24 h at 4 °C. Solids were removed by centrifugation at 10 000 rpm for 30 min. The supernatant was concentrated nearly 40-fold and dialyzed against the phosphate buffer using an ultrafiltration stirred system (Millipore) with a 10 kDa cutoff cellulose membrane pressurized at 5.95 bar with N2, on ice. The concentrate was applied to an anion exchange Q-Sepharose column (Hi-Prep 16/10 QFF, BioRad, 120 mg HSA/mL gel) preequilibrated with two column volumes of 50 mM Tris-HCl, pH 7.3, and eluted with a linear gradient from 0.0 to 0.5 M NaCl in the Tris-HCl buffer. Fractions with laccase activity were pooled and concentrated, dialyzed, and stored at -5 °C in phosphate buffer. This preparation was labeled as MtL (native enzyme). To determine the molecular weight of the enzyme a sample was dialyzed against 0.5 mM acetate buffer solution (pH 5.0) and applied to a Superdex 200 HR 10/30 column (Amersham Pharmacia); protein was eluted with buffer containing 0.5 M NaCl (0.5 mL min-1). Determination of Enzymatic Activity. Laccase activity was routinely assayed by measuring the rate of ABTS oxidation at room temperature. The reaction mixture contained 1.0 mL of 5 mM ABTS dissolved in 50 mM potassium phosphate buffer (pH 6.0) and 100 µL of appropriately diluted enzyme. ABTS oxidation was followed by an increase of absorbance at 420 nm (420 ) 36 000 M-1 cm-1). One unit of activity was defined as the amount of laccase that oxidized 1 µmol of substrate per minute. In assays carried out at other pH values, reaction solutions were buffered with citrate (pH 3.0 and 4.0), acetate (pH 5.0), and phosphate (pH 6.0, 7.0, and 8.0) solutions at 50 mM. For determination of catalytic constants, ABTS solutions were prepared in the phosphate buffer (pH 6.0) at concentrations varying from 0.01 to 5.0 mM. When DMP was the substrate, it was dissolved in a mixture of 10% propanol in the same buffer to a final concentration from 0.01 to 0.50 mM. Oxidation of DMP was followed by the absorption increment at 469 nm (469 ) 49 600 M-1 cm-1). Protein Concentration. The protein was assayed by the Bradford method (18) using the Bio-Rad protein reagent with bovine serum albumin as standard. Chemical Modification of Laccase. Chemical modification of the exposed lysine amino groups of laccase was carried out with 50-fold excess of monomethoxy poly(ethylene glycol) activated with cyanuric chloride (Sigma) in 40 mM borate buffer, pH 10.0. The reaction was allowed to proceed for 2.5 h at 25 °C (16). The resulting solution was dialyzed and concentrated 20-fold with 50 mM phosphate buffer, pH 6.0, by using the same procedure as for the native enzyme. This preparation was labeled as PEG-MtL. Determination of Modified Amino Groups. The degree of protein modification was estimated by titration of the residual free amino groups using the reagent TNBS in order to obtain the corresponding trinitrophenyl derivatives, according to the Habeeb method (19). The reaction was carried out at pH 8.5. Absorbance was measured at 335 nm against the appropriate blank. The molar extinction coefficient was taken to be 335 ) (1.09 ( 0.01) × 104 M-1 cm-1. Gel Electrophoresis. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) was performed in a MiniProtean II Cell (Bio-Rad), using a 10.0% gel containing 0.1% SDS according to the methodology of Laemmli (20). Protein bands were revealed with Coomassie Blue G-250 or the silver staining kit (Amersham Biosciences, Uppsala, Sweden). The samples were compared to high-range molecular weight markers of Sigma (St. Louis, MO). Other experiments were carried out using a 10.0% polyacrylamide gel, without SDS and mercaptoethanol.

Figure 1. Purification of commercial laccase by ion-exchange chromatography. Six milliliters of mediator-free Deni Lite IIs solution was applied to a Q-Sepharose column and eluted with a NaCl gradient (00.50 M). s, absorbance at 280 nm; - - -, laccase activity; - - - , concentration of NaCl in the eluent.

Stability Assays. The stabilities of MtL and PEG-MtL preparations were compared by measuring the decay of enzyme activities (assayed by ABTS oxidation at room temperature) when incubated at various temperatures (40-80 °C) in the presence of 0, 10.0, or 30.0% (v/v) of methanol, ethanol, propanol, or acetonitrile. Decay rate constants, ki, were estimated by fitting the first-order equation, A ) Ao exp(-kit), to the experimental data. Circular Dichroism (CD) Measurements. CD measurements were performed in a Jasco J-715 spectropolarimeter (Jasco Inc, Easton, MD) equipped with a PTC-348WI Peltier-type cell holder for temperature control. MtL or PEG-MtL samples (0.2 mg mL-1) were dissolved in 50 mM phosphate buffer (pH 6.0) and placed in a 0.10 cm cell. CD scans were run at 25 °C in the range from 190 to 250 nm. For the aromatic absorption region (above 250 nm) protein concentration was increased to 1.0 mg mL-1 and a 1.00 cm cell was used. Differential Scanning Calorimetry (DSC). Calorimetric endotherms were obtained with a Microcal MC2 differential scanning calorimeter (MicroCal Inc, Northampton, MA). Measurements were carried out with a degassed enzyme solution (2.0 mg mL-1, prepared in 5 mM citrate buffer, pH 5.0) in the sample cell. The reference cell was filled with the buffer alone. Both cells were pressurized to nearly 2.0 atm with N2 gas and heated from 25 to 109 °C at a rate of 1.0 °C min-1. The adiabatic jacket was kept at a partial vacuum of 0.0106 atm. Buffer (blank) tracings were recorded under the same conditions. The Origin software package (MicroCal) was used for data analysis including baseline subtraction and the calculation of specific enthalpies (∆Hcal).

RESULTS AND DISCUSSION Purification and Catalytic Properties of M. thermophyla Laccase (MtL). The reddish color of Deni Lite IIs, which is due to the presence of the redox mediator 10-phenotyazinpropionic acid (21), was completely eliminated by extensive dialysis through a 10 kDa cellulose membrane. This operation produced an apparent decrease of approximately 10.0% in the total laccase activity of the material. The elution profile from the ionexchange column (Figure 1) showed four peaks with absorbance at 280 nm, but all the laccase activity was contained in the peak that eluted last; fractions with enzyme activity were pooled and further submitted to PAGE, where a single major protein band was observed (Figure 2).

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Figure 2. Native PAGE of M. thermophila laccase (MtL) and its PEG derivative at pH 6.8. Lane 1, MtL; lane 2, PEG-MtL. The anode is on top. Table 1. Catalytic Constants for MtL and PEG-MtLa MtL substrate ABTS DMP

kcat (s-1)

KM (µM)

PEG5000-MtL kcat/KM (s-1 M-1)

kcat (s-1)

KM (µM)

kcat/KM (s-1 M-1)

6.01 × 102 49.5 1.21 × 107 3.76 × 102 71.2 5.29 × 106 3.48 × 102 5.02 6.93 × 107 8.73 × 102 9.06 9.63 × 107

a Michaelis-Menten constants (k , K ) were determined from activity cat M assays in 50 mM phosphate buffer (pH 6.0) at 25 °C.

After ion-exchange chromatography the specific activity was 29 U mg-1, approximately twice the value shown by the original, mediator-free Deni Lite IIs; the yield was about 60%, based on the amount of protein recovered. All further studies were performed with the enzyme obtained from the Q-Sepharose column. The molecular mass of MtL, as determined by SDSPAGE (data not shown), was 110 kDa, whereas gel filtration chromatography gave a Mr of 80 kDa. Such a difference in Mr determinations probably arises from differences in the glycosylated nature of MtL (17), because it is well-known that the presence of covalently attached carbohydrates often results in incorrect Mr values when using SDS or gel filtration techniques (15). It should be mentioned that the protein moiety of this enzyme has a mass of 61 781 kDa, according to its amino acid sequence (22). The isoelectric point of the laccase was 3.8, in agreement with previous determinations (17). MtL displayed good activity toward ABTS in the 3.0-7.0 pH range; however, the enzyme was more stable between pH 6.0 and 8.0. Determination of kinetic constants was carried out at pH 6.0. With either ABTS or DMP as substrate, MtL showed typical Michaelis-Menten kinetics (results not shown). The values obtained for kcat and KM (Table 1) indicate that the catalytic efficiency (kcat/KM) of the enzyme is comparable to that of diffusion-controlled enzyme reactions (i.e., about 1 × 108 M-1 s-1; 23) when the substrate is the relatively small DMP molecule. With ABTS as substrate, the value found for kcat/KM is about 5-fold smaller, but still close to that for diffusioncontrolled reactions, and 2 orders of magnitude larger than the value reported by Li et al (3). The apparent activation energy, Ea, for the MtL-catalyzed oxidation of ABTS was determined from activity assays in the 25-70 °C temperature range; at temperatures higher than 70 °C, thermal denaturation of the enzyme led to notable loss of activity (Figure 3). From the Arrhenius plot on the lowtemperature arm of Figure 3, Ea was determined as 18.0 kJ mol-1, in agreement with the low value observed for diffusioncontrolled reactions, which have Eas between 10 and 30 kJ mol-1 (23).

Figure 3. Arrhenius plots for the ABTS-oxidation reaction (pH 6.0) with MtL (2) and PEG-MtL (O) as catalysts.

Catalytic Properties of Chemically Modified MtL. Under the conditions described in the Experimental Procedures, chemical modification of MtL resulted in a protein material with 54% of its amino groups linked to PEG chains. As a consequence of the reduction in the number of free amino groups, the enzyme derivative (PEG-MtL) showed a smaller electrophoretic mobility toward the cathode than the unmodified laccase (Figure 2). Similarly, attachment of PEG chains notably affected the hydrodynamic properties of MtL, leading to a large reduction of its elution volume in the gel filtration column; the apparent Mr of PEG-MtL determined by this method was larger than 300 kDa, although the expected increase in molecular mass, as computed from the number of modified amino groups, amounts to only 40 kDa. Determination of the catalytic constants for PEG-MtL gave the results listed in Table 1. It can be seen that the kinetic constants kcat and KM of the enzyme suffered only minor changes after chemical modification. This is an important result, because with other enzymes the use of cyanuryl chloride-PEG has led sometimes to substantial loss of enzymatic activity due to the low selectivity of this reagent as a protein modifier (24, 25). In the case of MtL, PEG modification slightly affected the affinity of the enzyme (i.e., KM increased less than 2-fold) for both of the substrates tested. In contrast, PEG-MtL showed a value of kcat that was twice that for the unmodified laccase when DMP was the substrate; with ABTS as substrate, however, kcat was about 2-fold reduced. As a result of these changes in the individual kinetic constants, the catalytic efficiency (kcat/KM) of the laccase, with the substrate DMP, was practically unaltered by chemical modification; with ABTS, on the contrary, kcat/KM was more than 2-fold diminished. These findings indicate that the presence of PEG chains on the macromolecular surface somewhat reduces the binding rate of the larger, more hydrophilic ABTS molecule but not that of the small hydrophobic DMP molecule. In other words, PEG modification apparently increased the relative selectivity of the enzyme toward two of its substrates. On the other hand, the apparent activation energy (Ea) for the oxidation of ABTS, whose value is also shown in Table 1, keeps the characteristic small magnitude of diffusioncontrolled enzymatic reactions (see Figure 3). Conformational Characteristics of the Laccase Forms. At 20 °C, the far-UV CD spectrum of MtL (Figure 4) shows a negative band around 216 nm and a positive one at 194-195 nm, both of which are typical of proteins rich in β-strands and with poor helix content (26). This observation was expected, given the high sequence identity between MtL and the laccase from Melanocarpus albomyces (27), whose secondary structure

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Figure 4. Circular dichroism (CD) spectra of MtL and PEG-MtL at low and high temperature (pH 6.0). Far- and near-UV regions are shown in the left and right panel, respectively. Spectra of MtL (triangles) and PEG-MtL (circles) were recorded at low temperature (25 °C; open symbols) and at high temperature (recorded at 88 °C, after thermal denaturation; closed symbols). Shown in the inset is the difference spectrum obtained by subtracting the low-temperature CD curve of MtL from that of the heat-denatured enzyme.

contains about 33% β-sheet and only 8% helices. As seen in Figure 4, the spectrum of PEG-MtL looks similar to that of its parent enzyme, indicating that chemical modification introduced no major structural changes in the secondary structure of the protein. On the other hand, in the near-UV spectra it is seen that the band envelope at 276-282 nm, a region where tyrosine residues display intense CD bands, is blue-shifted in the modified enzyme; this spectral shift might be caused by a less polar environment for the tyrosine side chains (28) in PEGMtL owing to the presence of the hydrocarbon chains of PEG. Similar polarity changes seem to take place around the active site, as inferred from absorption spectra (results not shown): the absorption maximum in the visible region, characteristic of type 1 copper atoms present in laccase enzymes (29, 30), was shifted from 610 to 575 nm as a result of chemical modification. Thermal Denaturation. When MtL was heated to 88 °C until the enzyme activity was reduced to only 5% of the original value measured at room temperature, its far-UV CD bands were shifted toward shorter wavelengths (Figure 4). The spectrum of denatured MtL, however, differs from those observed in other heat-denatured proteins such as lysozyme and trypsin (31). Thus, although some structural changes occurred at high temperature, the laccase molecule likely possesses residual native secondary structure (31). That some protein segments became unstructured at high temperature is clearly discerned from the difference spectrum of heat-denatured versus native MtL, which shows the negative peak around 198-201 nm (inset to Figure 4) characteristic of irregular polypeptide structures (28). Heating had more drastic effects on the local environment of aromatic residues, as judged from the abatement of the near-UV bands (Figure 4). Regarding PEG-MtL, its thermal denaturation was accompanied by conformational changes resembling those observed with the unmodified enzyme, as judged by the CD spectra at high temperature. Thermal scannings of MtL and its PEG derivative were carried out to determine the heat absorbed upon denaturation, as well as the changes in stability introduced by PEG modification. Both laccase preparations denatured irreversibly, with small denaturation enthalpies of only 6.3-7.5 g-1 (1.5-1.8 cal g-1), but the modified enzyme proved to be more stable, its temperature of half-denaturation being approximately 2 °C higher than that of MtL (Figure 5). For comparative purposes, the scanning thermogram of hen’s egg lysozyme, recorded in the same instrument, is also shown in Figure 5. The specific

Figure 5. DSC profiles for the denaturation of the laccase enzymes at pH 5.0. Dashed line, MtL; solid line, PEG-MtL. For comparison, the scanning profile of hen’s egg lysozyme, at pH 3.0, is also shown (-O). Curves represent the excess heat capacity function, CPex, obtained by subtracting buffer-buffer tracings and neglecting any change in heat capacity that takes place after denaturation. The area under a CPex curve is the denaturation enthalpy. The protein concentration was ca. 2.0 mg mL-1; the scanning rate was 1.0 °C min-1.

heat of denaturation of this protein, which is representative of values found with other small proteins, was determined as 10.8 cal g-1, in accord with previously reported determinations (32). It is clear then that thermal unfolding of the laccase enzymes is much less extensive than those of other typical proteins. This result is in keeping with the aforementioned characteristics of the CD spectra at high temperature and suggests that the β-structure scaffolding of the laccase family is notably resistant to high temperatures. Additional stability for the protein molecule may come from disulfide bonds; indeed, three of these bonds are known to be present in the structure of M. albomyces laccase (27), and the six Cys residues involved in those S-S bonds are all conserved in the sequence of MtL. Inactivation of the Laccases in Mixed Solvents. The kinetics of inactivation of MtL and its PEG derivative in mixed aqueous-organic solvents was studied at several temperatures. The loss of enzyme activity followed first-order kinetics, with an apparent rate constant, ki, that increased with the amount of organic cosolvent. We also made some measurements of the time course of ellipticity changes, at 202 nm, under the same

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Figure 6. Arrhenius plots for denaturation of MtL (a) and PEG-MtL (b) in mixed solvents. Values of the rate constant (ki) were determined by measuring the loss of laccase activity upon incubation in aqueous-organic solutions containing 30% v/v of the following organic solvents: methanol (0), ethanol (O), propanol (4), acetonitrile (]). ki is in min-1. Table 2. Activation Energies (Eai) and Preexponential Factors (A) for the Inactivation of MtL and PEG-MtL in Mixed Solventsa MtL

PEG-MtL

solvent

Eai (kJ/mol)

ln A

Eai (kJ/mol)

ln A

methanol ethanol propanol acetonitrile

33.0 ( 4.9 27.2 ( 1.4 14.6 ( 4.6 20.2 ( 1.8

51.0 42.7 23.6 32.4

38.8 ( 5.6 23.6 ( 2.6 11.7 ( 2.7 14.0 ( 1.5

59.0 36.2 18.6 22.2

a Values were determined from the linear fits shown in Figure 6. Solvents contained 30% (v/v) of the organic component in 50 mM phosphate buffer. b E i and A represent the activation energy and the preexponential factor, a respectively, in the Arrhenius equation (see the text).

solvent composition and temperature as those used to follow enzyme inactivation. Rate constants determined by both methods were nearly identical within experimental error, indicating that the loss of activity is accompanied by the appearance of disordered regions in the enzyme molecules. In solutions containing 30% of the organic component, the effect of temperature on ki showed a clearly distinct trend depending on the nature of the cosolvent being present. Results obtained are plotted in Figure 6 according to the Arrhenius equation: ln ki ) ln A - (Eai/R)(1/T). Values of the preexponential term (A) and the activation energy (Eai) for the inactivation reaction are given in Table 2. It is seen in Figure 6 that at temperatures below 70 °C the denaturing efficiency of linear alcohols increases with the length of the alkyl chain. The denaturing effect of n-alcohols is well-documented in the literature (33), where it has been attributed mainly to a decrease in the strength of “hydrophobic interactions” in water-alcohol solutions. The contribution of alcohols to protein denaturation has both enthalpic and entropic terms that compensate with each other in a rather complex way (34, 35), depending on the concentration and type of the organic cosolvent. With 30% alcoholwater mixtures, Velicelebi and Sturtevant (35) found that the greater denaturing action of 1-propanol, with respect to ethanol and methanol, on hen lysozyme is due to a smaller enthalpy of denaturation. A similar situation is observed for the inactivation of both laccase forms: the activation energy suffers a large drop in n-propanol (Table 2) that results in the largest kinetic destabilization of the enzymes. When acetonitrile was the cosolvent, the temperature dependence of ki was similar to that observed in the presence of n-propanol. In this case, however, it is difficult to rationalize the effect of the cosolvent on the basis of the hydrophobic effect alone. Other mechanisms, such as the influence of acetonitrile on hydrogen bonding and electrostatic interactions, may also play relevant roles.

It should be noted that, despite the similar inactivation behavior of both laccase samples in different solvents, the value of ki for the modified enzyme was 2-3-fold smaller than that for the unmodified enzyme. An inspection of data in Table 2 shows that the extra stability of PEG-MtL can be attributed to its smaller activation entropy, because the A term is proportional to the entropy difference between the transition and native states of a reaction (23). This entropic stabilization may have its origin in the restricted motion of some amino acid groups on the protein surface due to the shell-like structure formed by coiled PEG chains (36).

CONCLUSIONS Chemical modification of M. thermophila laccase (MtL) with PEG5000 yielded an enzyme derivative showing only minor changes in both secondary structure and enzyme activity, in comparison with the parent enzyme. Besides, pegylation led to a modest increase in the enzyme thermostability and to a decrease in the rate of inactivation in aqueous-organic solvents. To enhance these effects, an issue that would be of importance for the industrial applications of MtL, it would be interesting to carry out studies with bigger PEG molecules. Furthermore, it should be noticed that only eight out of the 16 lysine residues of MtL seemed to have been linked to PEG. A more extensive modification could be attempted by increasing the number of solvent-exposed lysines using directed mutagenesis of cloned and expressed versions of the corresponding gene.

ACKNOWLEDGMENT This work was supported in part by CONACyT (convenio SEP-2003-CO2-44681). We thank the Novo Co., for providing the sample of Deni Lite IIs. Also Dr. Rafael Vazquez Duhalt (Instituto de Biotecnologı´a, UNAM) helped us to do the pegylation reaction. L.-C.J.I. had a doctoral fellowship from Consejo Nacional de Ciencia y Tecnologı´a (CONACYT).

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