Noncovalent immobilization of chloroperoxidase onto talc: Catalytic properties of a new biocatalyst

Noncovalent immobilization of chloroperoxidase onto talc: Catalytic properties of a new biocatalyst Salim Aoun, Chahrazad Chebli, and Michel Baboule`ne Laboratoire des IMRCP, Universite´ Paul Sabatier, Toulouse Ce´dex, France The noncovalent adsorption of chloroperoxidase (CPO) from the mold Caldariomyces fumago onto a talc, a mineral support that is available in various forms in industrial quantities and low cost, was investigated at two different values of pH (3.0 and 6.0). pH was found to affect both adsorption and enzymatic activity owing to the isoelectric point of CPO (pI ' 4) and the chemical characteristics of the absorbing surface of the talc tested. The hydrophobic and chemically neutral talc, type 15M00, favored adsorption (up to 8 mg CPO-g21 talc); however, it led to strong inhibition of enzymatic activity on adsorption at pH 3, although activity was conserved ('61% for halogenation and up to 72% for oxidation) by performing the adsorption at pH 6. In contrast, the hydrophilic and slightly acidic, calcined talc, type CLST, was less favorable to adsorption (#2.5 mg CPO-g21 talc); however, the CLST-CPO combination had excellent enzymatic activity (80 –126%) irrespective of the pH of adsorption and without modification of the pH optimum of the enzyme. The results were accounted for in terms of the interactions between CPO and the talc as a function of the adsorption pH. Adsorption onto talc may thus lead to an improvement in reaction selectivity of CPO particularly in the competing halohydroxylation-oxidation reactions, thereby widening the potential industrial applications of this enzyme. © 1998 Elsevier Science Inc. Keywords: Chloroperoxidase; talcs; adsorption; enzymatic activity

Introduction Halometabolites have been identified in many living organisms, although to date only two types of enzyme with halogenation activity have been isolated: methyl halide transferases1 and haloperoxidases.2 Among the latter group, the chloroperoxidase (CPO: EC.1.11.1.10) from the mold Caldariomyces fumago has been characterized as a hemecontaining glycoprotein with haloperoxidase activity.3 CPO has not been used industrially due in part to its high cost, a highly acid pH optimum (pH 3), instability at high temperatures, and deactivation at high concentrations of oxidizing agents.4,5 Furthermore, although numerous natural chiral halogenated compounds exist,6 the stereoselectivity of halogenation catalyzed by CPO has not been established. Although the advantages conferred by immobilization of CPO onto an inert support have not been thoroughly

Address reprint requests to Dr. M. Baboulene, Universite Paul Sabatier, UMR (CNRS) 5623, Laboratoire des IMRCP, 118 route de Norbonne, 31062 Toulouse Cedex, France Received 16 March 1997; revised 24 April 1998; accepted 27 April 1998

Enzyme and Microbial Technology 23:380 –385, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

studied,7,8 the influence of the reaction medium has been explored. It is known, for example, that CPO is rapidly denatured by water-miscible organic solvents,9,10 while micellar conditions have been found to be favorable. In a microemulsion composed of aqueous buffer pH 3, cetyl trimethyl ammonium bromide (CTAB), pentanol, and octane, the CPO halogenates monochlorodimedon and 1,3dihydroxybenzene, but a high concentration of oxidizing agent (H2O2) was found to inactivate the enzyme due to the fact that the hydrogen peroxide is almost completely confined to the water pool in the immediate vicinity of CPO.11 We have recently shown that talc can serve as a useful enzyme support in various applications in biotechnology. A combination of lipase and talc was found to give an excellent esterification yield of milk fat products (butter substitute) whereas the lipase alone was almost completely inactive.12,13 Furthermore, the adsorption of lipase or hydrolases to the surface of talc produces agents with antipitch properties used as additives in the manufacture of paper pulp.14 The combination of talc and horseradish peroxidase has also been proposed as a system for depollution of wastewater containing phenolic compounds based on bioperoxidation and the ability of the talc to adsorb polyphe-

0141-0229/98/$19.00 PII S0141-0229(98)00061-1

Noncovalent immobilization of chloroperoxidase: S. Aoun et al. Table 1 Adsorption capacity as function of the physicochemical properties of talcs Adsorption ratioc (mg CPO-g21 talc)

Physicochemical properties of talcsb Talc samples

B.E.T. (m2 g21)

d50 (mm)

Chlorite (%)

pH 3

pH 6

8.6 10 10 6 10

3.7 9 – 3.8 9

5 30 60 99.5 –

8 (7.6) 4 7 7 2.5 (2.33)

4 (3.86) 3 3 1.5 1 (0.91)

15 M00 Steatrol Steopac C300 CLSTa a

Obtained by calcination of steatrol at 1,100°C for 1 h Abbreviations: B.E.T. 5 specific surface area; d50 5 grain size Determined from heme absorbance; in parentheses, quantity of protein by Bradford method19

b c

nols.15,16 The oxidative capacity of lipoxygenases also appears to be enhanced by the presence of talc.17 Our results suggest that utilization of CPO adsorbed to talc in biohalogenation reactions could enlarge the industrial applications of haloperoxidases. We report here our investigations on the use of talc as a support for chloroperoxidase (CPO).

Materials and methods Materials Monochlorodimedon and chloroperoxidase (EC.1.11.1.10) from C. fumago, as a freeze-dried powder, were from Sigma (St. Louis, MO). One unit of chloroperoxidase catalyzed the conversion of 1.0 mmol min21 monochlorodimedon to dichlorodimedon at pH 3, and 25°C in the presence of KCl and H2O2.18 Indole, hydrogen peroxide, and tert-butyl peroxide were supplied by Aldrich (Milwaukee, WI). The various mineral supports were kindly donated by the group Talc Europe (Toulouse, France). The manufacturer’s references are used (Table 1).

Apparatus

subtracting the absorbance of the supernatant plus washings from the initial absorbance of the enzyme solution (Table 1). From protein by the Bradford method, the protein adsorbed onto support is calculated from the protein obtained in the supernatant plus washings using the dye-binding method of Bradford with bovine serum albumin as a standard protein.19 Enzyme activity assays. Each point is a mean result of duplicate experiments. Concerning halogenation, in 2 ml of appropriate 0.1 m phosphate buffer containing 150 ml of an ethyl alcoholic solution of monochlorodimedon (0.013 mg ml21), 120 ml of a 0.1 m solution of KCl (0.45 mg ml21) and 1 mg of the talcchloroperoxidase pellet was added 140 ml of tert-butyl peroxide (0.63 mg ml21). The enzyme activity was calculated from the rate of disappearance of monochlorodimedon at 25°C determined by measuring the absorbance at 278 nm (S 5 12.2 m21cm21).18 Concerning oxidation, in 1.5 ml of appropriate 0.1 m phosphate buffer containing 470 ml of an ethyl alcoholic solution of indole (0.137 mg ml21) and 1 mg of the talc-chloroperoxidase pellet was added 10 ml of hydrogen peroxide (0.017 mg ml21) at 25°C. The enzyme activity was calculated from the rate of disappearance of indole determined by measuring absorbance at 270 nm (S 5 7m21cm21).20 The results were confirmed by HPLC analysis.

U.V. spectra were recorded on a Hewlett-Packard HP 8450 A spectrophotometer. Temperature and stirring were controlled using the adapted HP 89100 A controller. The HPLC analyses were performed on a C18 mBondapack column (water/methanol: 50/50 v/v at 1.5 ml min21) in Waters 600E equipment.

Methods Time-course adsorption. In 3 ml of appropriate 0.1 m phosphate buffer containing 0.14 mg of enzyme (0.047 mg ml21) were added 10 mg of mineral support. At regular intervals, the supernatant solution was analyzed by U.V. spectra recorded at 403 nm (S 5 75.2 m21cm21).3 The ratio of the adsorption was determined (Figure 1). Each point is a mean result of duplicate experiments. Adsorption isotherms. Each point is a mean result of duplicate experiments. Concerning heme adsorption, in 3 ml of appropriate 0.1 m phosphate buffer containing 0.27 mg CPO (0.09 mg ml21) was added increasing amounts of mineral support (5–25 mg). The mixture was stirred at 15°C for 90 min. It was then centrifuged and the U.V. spectrum of the supernatant recorded at 403 nm (S 5 75.2 m21cm21).3 The pellet was washed twice with water (2 ml) to remove any nonadsorbed enzyme. The amount of enzyme bound to the support was calculated from the U.V. absorbance by

Figure 1 Time-course adsorption. For experimental conditions, see Materials and methods. Adsorption at pH 3 with CLST (——‚——) and 15M00 (——V——). Adsorption at pH 6 with CLST (——f——) and 15M00 (——v——).

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Papers

Results

Adsorption at pH 6

Talc is a natural mineral composed of trioctahedral hydrated magnesium phyllosilicate.21,22 It shows considerable diversity in morphology, specific surface area (B.E.T.), and particle size (d50). Talcs of many different types are available in large quantities and at low cost. In the present study, we selected the supports on the basis of equivalent surface areas (Table 1); nevertheless, the specific areas were lower than those of other inorganic supports23 and grain size was variable, but these parameters are unimportant since our supports were nonporous. On the other hand, the different talcs differ in hydrophobicity or hydrophilicity of their surfaces. The hydrophobic-hydrophilic balance is affected by the presence in nature of differing amounts of chlorite [Mg6-x-y Fe3Alx (Si6-x Alx) O10 (OH)2]22 (Table 1). Furthermore, unlike organic supports, talc, which are insoluble in aqueous and organic media and are chemically inert, are also insensitive to bacterial contamination. This represents a distinct advantage for industrial applications.

Adsorption of CPO at pH 6 (Table 1) was three times less on the naturally hydrophilic talc C300 than on hydrophobic 15M00 talc. The lowest levels of adsorption were always observed with the CLST talc.

Adsorption of CPO onto different varieties of talc In general, adsorption (Table 1 and Figure 1) occurs mainly via electrostatic interactions between the external charges on the protein and on the support.24 It is obtained by simple addition of a solution of CPO to a suspension of talc. The time-course for CPO adsorption to various talcs was studied at pH 3 and pH 6 (Figure 1). Only results obtained with talcs 15M00 and CLST are presented. The responses of the other types of talc tested (steatrol, steopac, C300) produced similar-shaped plots within the boundaries of the graphs shown in Figure 1. In general, the time required for maximal adsorption (60 –90 min) was largely independent of the type of talc. No desorption was observed either with time or with dilution. On the other hand, the extent of adsorption was markedly affected by the nature of the support and the pH of the reaction medium which forms the subject of the present investigation.

Adsorption at pH 3 (optimum pH for halogenation) All the talcs tested (Table 1), had excellent adsorption properties. Determination of the extent of adsorption either from adsorption of CPO heme or from the amount of protein (Bradford’s method)19 in the supernatant and washings gave almost identical results. The insolubility of CPO-talc combinations and the high-adsorbing capacity of the talc rules out direct measurement of adsorption. Differences in the hydrophobic/hydrophilic balance of the mineral did not greatly affect the proportions of enzyme adsorbed. A strongly hydrophobic talc (15M00) adsorbed 8 mg CPO21 mineral whereas the hydrophilic type C300 adsorbed 7 mg CPO21. On the other hand, chemical modification of the surface of the support by calcination results in opening of certain Si-O-Si bonds,22,25 and adversely affected adsorption of CPO. This is the case for CLST which only bound 2.5 mg enzyme21. 382

Discussion Although the time to equilibrium was somewhat longer than that observed for adsorption of proteases and horseradish peroxidase onto talc,13 it was comparable to the time for equilibrium adsorption of lipoxygenase.17 A relationship of the kind described by Messing26 for the adsorption rate of protein to porous glasses as a function of molar weight and pI was not observed here. The differences in chemical structures of the surfaces of the talcs tested with respect to other common supports (silicas, clays, . . .) led to differences in the adsorption of proteins. Our results were attributed to differences in the basal surfaces of the talc supports. It should be noted that the lateral surfaces, less well studied, have a much lower area than the basal surfaces and their influence was thus neglected. We therefore considered that the presence of two basal hydrophobic, chemically neutral surfaces was characteristic of the 15M00 talc. The calcined talc CLST has basal surfaces bearing silanol groups (Si-OH) conferring hydrophilic and acidic properties. Association of a basal hydrophobic surface (talc type) with a hydrophilic surface (chlorite-type rich in Si-OH groups) produces a support with intermediate properties (steopac, steatrol, C300) whose hydrophobic/hydrophilic balance is governed by the ratio of the two types of surface (Table 1). Depending on the chemical structure of the available surfaces for adsorption and the pI of CPO (around pH 4),27 alteration in the pH of the adsorbing medium will have an influence on the support-protein interactions. This was borne out by our experimental observations. An acid pH enhanced enzyme binding by the talcs 15M00, steopac, steatrol, and C300 irrespective of chemical composition. At pH 3, the protein is likely to be weakly charged positively and little destructured electronically (active enzyme).27,28 Furthermore it is known that the environment of the heme group of CPO is polar and that the core and the iron heme are fairly accessible.29 Under these conditions, the hydrophobic and chemically inert talc 15M00 can only bind to CPO via hydrophobic interactions that are likely to be close to the active site. In contrast, the hydrophilic and Si-OHrich talc will bind essentially via electrostatic interactions between the protein and the Si-O2 groups on the basal surfaces. Intermediate talcs, on the other hand, will bind via both type of interaction with relative strengths depending on the particular hydrophobic/hydrophilic balance. These considerations can account for the much higher adsorbing capacity of the 15M00 and C300 talcs than the CLST type. On adsorption at pH 6, CPO has an overall negative charge close to its stability limit,27,28 and will thus undergo structural deformation giving rise to an unfolding of the protein and a modification in the hydrophobic zones.30 Talc 15MOO, which is insensitive to pH, adsorbs via mainly

Enzyme Microb. Technol., 1998, vol. 23, November 1

Noncovalent immobilization of chloroperoxidase: S. Aoun et al. Table 2 Specific activities of CPO-talcs

Table 3 Specific activities of CPO-talcs

Specific activitya (U nmol21 heme) Enzyme samples Native CPO CPO 1 15M00 CPO 1 Steatrol CPO 1 CLST

Enzyme samples at pH 3 1.5 (100%) Trace 0.48 (32%) 1.14 (80%)

Specific activitya (U nmol21 heme)

Relative specific activity (%)

1.50 0.83 1.35 1.18 1.29 1.56

100 61 90 78.6 86 104

at pH 6 0.15 (10%) 0.12 (8%) 0.28 (19%) 0.35 (23%)

a

Halogenation of the monochlorodimedon. For the experimental conditions, see MATERIALS AND METHODS. b In parentheses, relative specific activity calculated from specific activity of native CPO at pH 3 as reference

hydrophobic interactions. Only the strength of hydrogen bonds between the negative charges on CPO and the undissociated silanol groups (pH close to neutral) adsorb CPO onto CLST, similar to the process described for the immobilization of proteins onto silica supports.23 Two types of binding are possible with the intermediate talcs, and the particular hydrophobic/hydrophilic balance can be exploited for the given application. From these considerations, we concluded that the decrease in the amount of CPO adsorbed at pH 6 relative to that observed at pH 3 stemmed from unfolding of the protein, thereby giving rise to access to more hydrophobic sites. If adsorption is performed after incubation of CPO with its hydrophobic substrate such as monochlorodimedon or indole, there is an almost 20% decrease in adsorption onto talc 15M00. Adsorption under these conditions was, however, unchanged with CLST talc at all values of pH tested, which is consistent with the interactions described above.

Enzyme activity CPO catalyzes two types of reaction: i) halogenation by halide ions at low pH; and ii) oxidation of aromatic compounds (peroxidase-like activity) at a pH near 5. A “catalase” activity is always present and often limits the applications of CPO.31

Halagenation of monochlorodimedon using CPO-Talc combination CPO is known to halogenate monochlorodimedon to dichlorodimedon.3 The results of the various tests performed are listed in Table 2. At pH 3, CPO bound to 15M00 (hydrophobic talc) was totally inhibited; however, hydrophilicity did not appear to be more advantageous since only 11% of the activity was conserved after adsorption onto talc C300 (strongly hydrophilic). Conservation of catalytic activity therefore seems to depend on the exact hydrophobic-hydrophilic balance as confirmed by 32% enzyme activity obtained with steatrol (a talc with 30% chlorite content) (Table 2). Unfortunately, owing to the natural origin of talcs, no mineral samples are available that would enable the ideal rate to be determined. Enzyme inactivation has been observed when some proteins adsorb to highly hydrophobic surfaces;32,33 how-

Native CPO CPO 1 15M00 CPO 1 Steatrol CPO 1 Steopac CPO 1 C300 CPO 1 CLST a

Calculated from halogenation of monochlorodimedon at pH 3 from CPO-talc prepared at pH 6. For the experimental conditions, see MATERIALS AND METHODS.

ever, Grabski et al.34 showed that manganese peroxidase, which normally acts in a hydrophobic environment, remains active after immobilization onto a hydrophobic support. Modifying the structure of the talc surface by calcination (opening of the Si-O-Si bonds)25 enhances biohalogenation, and the CPO-CLST combination was found to retain almost 80% of its activity (Table 2). In light of these findings on manganese peroxidase,34 the hydrophilic and acidic nature of the CLST support may favor CPO activity. It should be borne in mind that this enzyme normally operates at acid pH and is structured by several water molecules.29 At pH 6, native CPO loses 90% of its activity. Under these conditions, binding to talcs confers a protective effect since CPO–talcs exhibit higher enzyme activity than that of the native enzyme (Table 2). Inspection of the results in Table 3 and comparison of the rates of activity at pH 3 and pH 6 indicated that adsorption at pH 6 was less destructuring than at pH 3. It can be seen from the results in Table 3 that the inhibition noted with CPO-15M00 (adsorption at pH 3:Table 2) could be circumvented when CPO was bound to talc at pH 6. Under these conditions, the CPO-15M00 combination recovered 61% of its halogenation activity. We checked that there was no desorption of the CPO-talc association at the active pH (pH 3). In addition, 90% of the enzyme activity was conserved after adsorption to steatrol, a support that has both hydrophobic and hydrophilic characteristics (Table 1). Under the same conditions, the CPOCLST association gave rise to enhanced enzyme activity (104%). These results can perhaps be accounted for in terms of the previously described noncovalent immobilization. At pH 6, in view of the unfolded state of CPO, adsorption by hydrophobic interactions liberates the environment near the active site, thereby, shifting the hydrophobic zones30 while conferring sufficient rigidity to prevent denaturation35 and maintain good catalytic activity. On the other hand, the increased catalytic activity observed with the CPO-CLST combination is less readily accounted for. Experiments in progress are pointing to the participation of acid/basesensitive amino acids in the active site and heme environment of CPO. It is known that weakly acidic ligands do bind to this enzyme.36 In this respect at pH 6, interactions by hydrogen bonds between CPO and CLST would be less disruptive for the environment of the active site than are the electrostatic interactions occurring at pH 3; however, the insolubility of the CPO-talc association henders experimen-

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383

Papers Table 4 Influence of the support and the pH of CPO adsorption on the oxidation of indole by CPO Specific activitya (U nmol21 heme) from pH of COP adsorption pH 3 pH 4.2 pH 6

Native CPO

CPO 1 15M00

COP 1 Steatrol

CPO 1 Steopac

CPO 1 C300

CPO 1 CLST

– 1.2 (100%) –

0.13 (10%) None 0.87 (72%)

0.18 (15%) Trace 1.34 (111%)

0.18 (15%) None 0.94 (78%)

0.14 (11%) Trace 0.54 (45%)

0.48 (40%) Low 1.52 (126%)

a Oxidation of indole at pH 4.2. For experimental conditions, see MATERIALS calculated from specific activity of native CPO at pH 4.2 as reference

tal elucidation of the mechanisms involved. We checked that talk alone had no effect. Likewise, horseradish peroxidase adsorbed onto talc CLST exhibited a significant increase in its peroxidase activity.13,21 On the other hand, we could not rule out the involvement of substrate-talc interactions which are inherent in heterogeneous enzymatic catalysis. Although their influences are difficult to quantify, they need to be evaluated in order to throw more light on the processes involved.

Oxidation of indole by CPO-talc association In the absence of halides, CPO can oxidize indole into oxyindole.20 In the same way as for the monochlorodimedon halogenation study, various comparative tests were performed. The results are listed in Table 4. Here again, the importance of the pH of adsorption on the talc was apparent. Immobilization at pH 4.2 (optimal pH for the oxidation reaction with free CPO)28, irrespective of the type of talc almost completely inhibited catalytic activity. This drastic effect of adsorption at pH 4.2 is not readily accounted for especially since halogenation of monochlorodimedon by CPO immobilized at pH 4.2 on CLST occurred at 66% of the efficiency of the same enzymatic system at pH 3;17 nevertheless, all CPO-talcs prepared at pH 3, exhibited weak oxidation activity. The conformational constraints engendered by immobilization onto the support as described above may account for these findings. In view of the pI of CPO and the activity of silanol groups on the CLST talc at pH 4.2, a combination of ionic interactions and hydrogen bonds may force the protein into a shape that hinders approach of bulky organic substrates. The small C12 ion would thus be more accessible to the heme site than the more bulky indole substrate. This would tend to favor halogenation reactions. Of the talcs tested, CLST conferred the highest peroxidase activity to the CPO. Analysis of the reaction mixture by HPLC confirmed that oxyindole was the sole product. These observations indicate the importance of the conformation adopted by the CPO on binding to talc which will depend on the exact chemical composition of the support.

References 1. 2. 3. 4.

5. 6. 7. 8.

Conclusions

384

In parentheses, relative specific activity

phobic (e.g., CPO-15M00) or hydrophilic characteristics (e.g., CPO-CLST) while conserving catalytic activity at the normal pH optimum of the enzyme. The importance of the value of pH of adsorption was demonstrated. With a hydrophobic and chemically neutral talc (e.g., 15M00), hydrophobic interactions account for the inhibition of enzymatic activity at pH 3 and conservation of activity at pH 6. On the other hand, the hydrophilic and acidic nature of the calcined talc CLST is consistent with the formation of CPO-CLST combinations stabilized by electrostatic interactions at pH 3 and hydrogen bonds at pH 6, conferring excellent enzymatic activity without altering the optimum pH of the enzyme. The results obtained with intermediate talcs are in line with these findings. Restricted accessibility to the active site as a function of the interactions and the nature of the support may favor reaction selectivity. This extends the range of applications of this enzyme for synthesis of halometabolites and biooxidations. These applications are being actively pursued in our laboratory.

9.

The results reported here help define the conditions of formation (by noncovalent immobilization) and the applications of combinations of CPO with talcs of either hydro-

AND METHODS.

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