Enzyme and Microbial Technology 37 (2005) 582–588 Activity and stability of Caldariomyces fumago chloroperoxidase modified by reductive alkylation, amidation and cross-linking Camilo E. La Rotta Hernandez a , Stephan Lütz b , Andreas Liese c , Elba P.S. Bon a,∗ a Chemistry Institute, Federal University of Rio de Janeiro, CT Bloco A, Ilaha do Fundao, CEP 21949-900 Rio de Janeiro, RJ, Brazil b Institute for Biotecnology II, Forschungzentrum Jülich GmbH, D-52425 Jülich, Germany c Institute of Biotechnology II, Technical University of Hamburg-Harburg, D-21071 Hamburg, Germany Received 4 August 2004; accepted 1 February 2005 Abstract Caldaryomyces fumago chloroperoxidase (CPO) was treated with sodium cyanoborohydride and 9-antraldehyde (9A) for reductive alkylation and with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or with hexamethylendiamine (HMDA) for amidation. Furthermore, native CPO and amidated derivatives were cross-linked with glutaraldehyde (GA). The CPO derivatives highest overall activity levels were obtained in reaction mixtures presenting a molar excess of 1:100 where activity retention was 80% for alkylation and amidation and 70% for cross-linking reactions. The 9A and GA treatments resulted in 4 and 8% decrease of free amino groups while the EDAC and HMDA treatments resulted in an increase of 9–11%. Further GA cross-linking treatments decrease the free amino groups around 20%. CPO derivatives pH and temperature profiles were similar to that of the native CPO, presenting maximum activity at pH 6.0 and 30 ◦ C. CPO–EDAC and CPO–HMDA and all GA cross-linked derivatives presented 40% residual activity after incubation for 120 min at 60 ◦ C in pH 6.0, and during 60 min at 30 ◦ C in pH 7.0, conditions that completely inactivated the native CPO. The CPO–9A derivative presented a four-fold hydrophobicity increase and the CPO–GA showed to be 30% more stable than the native enzyme in 60% tert-butanol. © 2005 Elsevier Inc. All rights reserved. Keywords: Caldariomyces fumago; Chloroperoxidase; Enzyme stabilisation; Chemical modification; Cross-linking; Reductive alkylation 1. Introduction Among the haloperoxidases the enzyme chloroperoxidase (CPO) produced by Caldariomyces fumago has been more thoroughly investigated. CPO is a glycoenzyme with 42 kDa molecular weight (312 amino acids residues, predominantly acidic) and pI in the range of 3.2–4.0. The enzyme, which is stable up to 40 ◦ C and in the pH range 3.0–5.5, exhibits an uncommon dual halogenase–peroxidase activity that depends on pH and the presence of halide ions [1–3]. Although CPO has been studied for a wide range of applications, from fine chemicals synthesis to environmental biocatalysis, its use has been hindered by instability under industrial conditions, as frequently observed for biocatalysts in general [2,4–8]. The active conformation of a biocatalyst can be stabilised by chemically strengthening its structure. As such chemical ∗ Corresponding author. Tel.: +55 21 25627358; fax: +55 21 25627266. E-mail address: elba1996@iq.ufrj.br (E.P.S. Bon). 0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.02.025 modification reactions including cross-linking, amidation, reductive alkylation and also immobilisation have been investigated. Cross-linking reactions of proteins can be achieved by the use of bifunctional compounds such as glutaraldehyde that react with the nucleophilic side chains of aminoacid residues such as the free amino group of lysine and that of the N-terminal amino acid and the sulphydryl group of cysteine [2,9]. Chemical modification can be carried out by carbodiimide and diamines which target carboxyl groups of aspartic and glutamic acid, the imidazolyl group of histidine, and the thioether group of methionine [10]. Bifunctional compounds can be classified into zero-length, homobifunctional and heterobifunctional reagents, varying in structure, reactivity and degree of specificity (Table 1). The zero-length cross-linkers, such as carbodiimide, isoazolium derivatives, chloroformates and carbonyldiimidazoles, that induce direct bonds of two chemical components, can be used individually or with another bifunctional reagent such as glutaraldehyde [9,10]. Reactivity of an amino acid side chain and the rela- 583 C.E. La Rotta Hernandez et al. / Enzyme and Microbial Technology 37 (2005) 582–588 Table 1 Examples for zero-length, homobifunctional and heterobifunctional cross-linking and reductive alkylation reagents [10,14,15] Type of reagent Examples Coupling group Zero-length Carbodiimides Isoxazolium derivatives Chloroformates Carbonyldiimidazole Cupric di(1,10-phenanthroline) 2,2′ -Dipyridylsulfide Carboxyl Carboxyl Amino Carboxyl Sulphidryl and thiol Sulphidryl and thiol Homobifunctional Glutaraldehyde (Low specificity) Hexamethylendiamine Hexandiamine Dissuccinimidyl suberate Dimethyl malonimidate 1,4-Dicyanatobenzene p-Phenylene-diisocyanate N,N′ -methylenebismaleimide ␣,␣′ -Diiodo-pxylene sulfonic acid Di(2-chloroethyl) sulfone Bis(3-nitro-4-fluophenyl) sulfone Amino Carboxyl Carboxyl Amino Amino Amino Amino Amino Amino, sulfide and thiol Amino, sulfide and thiol Amino, sulfide and thiol Heterofunctional N-succinimidyl 3-maleimidopropionate N-succinimidyl iodoacetate 4-Maleimidobenzoyl chloride Ethyl iodoacetimidate Amino Amino, sulfide and thiol Amino, sulfide and thiol Sulfide Reductive alkylation Reductive arylation Brig, Tween, PEG, PG aldehydes plus reducing agent (e.g. sodium cyanoborohydride) Nitrobenzaldehyde, 5-hydroxy-2-nitrobenzaldehyde, 2-naftaldehyde or 9-antraldehyde plus reducing agent (e.g. sodium cyanoborohydride) Amino Amino tive reactivity of the nucleophile vary according to the electronic structure, the pK of the relevant functional group and the microenvironment. Therefore, several amino acids side chains may react with the same bifunctional reagent. Although the extension of the modification may improve the enzyme stability, it often lessens the activity of the biocatalyst. Although CPO has been modified with several crosslinkers, only glutaraldehyde was able to produce catalytically active soluble CPO and insoluble crystals [9,11]. However, the high reactivity of this reagent precludes the use of an adequate molar excess to avoid over cross-linking within the protein molecule or the occurrence of cross-linking among individual proteins that could impair enzyme activity. A possible strategy to overcome this would involve a pre-treatment with carbodiimides or diamines to augment the enzyme free amino groups prior to the subsequent GA cross-linking step [9,12,13]. CPO variants with higher stability could also be obtained with a single carbodiimide cross-linking step. Indeed, as chloroperoxidase contains a large number of superficial aspartic and glutamic amino acids residues, it would be feasible to design bifunctional amines for intermolecular bonds between carboxylic groups [9]. The three-dimensional arrangement of the target protein molecule must be known to carry out such procedure. Based on the information given by X-ray diffraction, it is also possible to find an appropriate diamine to cover the distance between two carboxylic groups in adjacent protein molecules [3,10]. Changes in the superficial characteristics of CPO have also been performed through the removal of its carbo- hydrate moieties wherein deglycosylation was performed by the enzyme N-glycosidase-F (peptide N4-[N-acetyl]-pglucosaminyl) previously used for other peroxidases [14]. Deglycosylation increases hydrophobicity, a feature that also results from reductive alkylation reaction as it eliminates free amino groups by coupling them with a hydrophobic group (aryl or alkyl aldehydes) followed by treatment with a reducing agent such as cyanoborohydride [14,15]. In this study we compared the chemical modification of CPO by cross-linking with glutaraldehyde, amidation with 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) and hexamethylendiamin (HMDA) and amidation followed by glutaraldehyde (GA) cross-linking. Reductive alkylation using sodium cyanoborohydride and 9-anthraldehyde (9A) was also performed. The molar excess of the reagents was evaluated to identify reaction conditions that would conciliate the improvement of the biocatalyst stability towards pH, temperature and the presence of organic solvents to activity retention. 2. Materials and methods 2.1. Biocatalysts Two preparations of C. fumago chloroperoxidase (EC. 1.11.1.10) were used. The commercial preparation (activity of 7.5 kU/mL in respect to 2,4 DCP) and Rz(A403 /A280 ) = 1.5) was purchased form Fluka Chemie GmbH (Buchs, Switzerland). A second preparation (activity of 3.2 kU/mL in respect 584 C.E. La Rotta Hernandez et al. / Enzyme and Microbial Technology 37 (2005) 582–588 to 2,4 DCP) and Rz(A403 /A280 ) = 1.4), was produced by C. fumago CMI 89362 in our laboratories according to previous reports [2]. However, the growth medium composition was optimised (fructose 40.0 g/L, urea 10.0 g/L, NaNO3 2.0 g/L, KCl 2.0 g/L, KH2 PO4 2.0 g/L, MgSO4 ·7H2 O 1.0 g/L and FeSO4 ·7H2 O 0.02 g/L) to increase enzyme production. Supernatants were collected at the peak enzyme concentration and submitted to freeze and thaw cycles for pigment separation. The enzyme preparation was subsequently centrifuged, diafiltrated and concentrated using an AMICON ultrafiltration system with membranes of 10 and 30 kDa (Micropore® ). This crude preparation was treated with cold ethanol up to 65% saturation for a further pigment removal and up to 85% saturation for enzyme precipitation. The precipitate was subsequently removed, and the solution was diafiltrated, concentrated and stored at −4 ◦ C. tions were performed in zinc acetate buffer 100 mM, pH 6.5, and amidation reactions, using EDAC or HDMA, occurred in the presence of potassium phosphate buffer 10 mM, pH 5.5. Reductive alkylations, performed with 9-anthraldehyde and sodium cyanoborohydride, were carried out in potassium phosphate buffer 10 mM, pH 6.0 and 50% (v/v) ethanol. The cross-linking and amidation reactions occurred in the presence of 5% (w/v) polyethylene glycol 1500. All reactions were stirred a 4 ◦ C for one hour (2 h for the amidation reactions). After completion of the reactions, the derivatives were diafiltrated, using a 10–30 kDa membrane, against a mixture of potassium phosphate buffer 10 mM, pH 6.5 and ethanol, 95:5 (v/v). Amidated derivatives were also submitted to a further cross-linking treatment according to the above-described methodology. 2.6. Free amino groups determination 2.2. CPO characterization The average value for the enzyme concentration given as mmol L−1 was obtained utilizing the UV-absorption of the enzyme solution (potassium phosphate buffer 10 mM, pH 6.0) at 403, 515, 542 and 650 nm. For the transformation of the absorptions to the respective concentrations the corresponding extinction coefficients were considered (75.3, 11.5, 10.8 and 4.2 mM−1 cm−1 ). The Rz value (that expresses the purity degree of the CPO preparations) was obtained form the A403 /A280 absorbance ratio [16]. Protein content (milligram of protein per millilitre) was determined by the colorimetric method according to Lowry using bovine serum albumin (BSA) as standard [18]. 2.3. CPO activity determination For the determination of peroxidase activity 2,4dichlorophenol (2,4-DCP) was used as substrate in the presence of 4-aminoantipyrine (4-AA). Absorbance increase of the coloured derivative was followed at 510 nm (s = 7100 M−1 cm−1 ) at pH 6.0 in absence of chloride ions [17]. One unit of peroxidase activity was defined by the amount (micromoles per millilitre) of the red oxidised derivative per minute. 2.4. Determination of the reagents molar excesses To optimise reaction conditions for the GA, EDAC and HMDA reactions, three molar excesses (10, 100 and 1000) were evaluated, based on the molar concentration of the surface target amino acid residues per molecule of CPO (3,9,16). In all cases, a CPO preparation with Rz 1.4, 4.2 kU/mL 1,1-dimethyl-4-chloro-3,5-cyclohexandione (MCD) [1] and 3.46 mg/mL was used. 2.5. CPO chemical modification reactions In all cases reaction mixtures presented CPO: reagents molar excesses of 10, 100 or 1000. GA cross-linking reac- The determination was performed according to the TNBS method [19]. Solutions of the native CPO, or its modified preparations, presenting the same protein concentration (mg/mL) were added to a mixture of 0.1% trinitrobenzensulphonic acid (TNBS) in 100 mM bicarbonate buffer pH 8.5 (1:1 ratio), previously equilibrated at room temperature. The reaction mixture was incubated at 40 ◦ C and stirred for 2 h. After this time interval 1.0 mL of 10% SDS solution, in the same buffer, was added to prevent precipitation prior to the addition of 1.0 mL of hydrochloric acid (1.0N) to stop the reaction. The concentration of sulphonated products was determined at A335 against a blank containing Milli-Q water instead of protein. Results were expressed as percentage of modified amino-groups. In the case of HMDA and EDCA the modification degree of the derivatives was expressed by the enhancement in the number of free amino groups. 2.7. Determination of hydrophobicity change The native or modified CPO preparations were added to a mixture of phenyl-sepharose CL-4B suspension and 1.7 M ammonium sulphate in 10 mM potassium phosphate buffer pH 6.0 (1:1 ratio), previously equilibrated at room temperature. After 5 min of gentle stirring, the mixture was centrifuged at 3500 rpm for the separation of the phases and measurement of CPO activity in each phase. The partition coefficient was estimated from the activity ratio of the organic phase (phenyl-sepharose) and that of the aqueous phase (ammonium sulphate). 2.8. Effect of pH and temperature on enzyme activity and stability The pH activity profile of the native and modified CPO was determined in the pH range of 2.0–8.0 using sodium potassium phosphate buffers 100 mM at room temperature. For the pH stability experiments enzyme preparations were pre-incubated for 0.5–1.0 h at room temperature in the same C.E. La Rotta Hernandez et al. / Enzyme and Microbial Technology 37 (2005) 582–588 585 pH range. The temperature profiles were determined within the range 20–80 ◦ C at pH 6.0. For the stability experiments enzyme preparations were pre-incubated in the same temperature range for 0.5–1 h, followed by activity determination using 2,4-DCP method. 2.9. Effect of organic media on enzyme activity and stability This parameter was evaluated using several reaction mixtures (binary systems) containing an increasing concentration of tert-butanol, up to 80% (v/v) in 100 mM potassium phosphate buffer, pH 6.0, and room temperature. Subsequent to incubating the reaction mixtures for 1, 2 and 4 h the residual peroxidase activities were evaluated. 2.10. Reagents 1,1-Dimethyl-4-chloro-3,5-cyclohexanedione (MCD), 2,4-dichlorophenol, 4-aminoantipyrine (4-AA), 1-ethyl3-(3-dimetylaminopropyl) carbodiimide HCl ultra-pure (EDAC), glutaraldehyde solution at 25% (GA), polyethylene glycol 1500, sodium dodecyl sulphate (SDS), 5 M 2,4,6-trinitrobenzen sulphonic acid solution (TNBS), phenyl-sepharose CL-4B (affinity chromatography media), 9-anthraldehyde (9A) and ammonium sulphate were purchased from Sigma-Aldrich (St. Louis, USA). Sodium cyanoborohydride was purchased by Fluka Chemie GmbH (Buchs, Switzerland). Hexamethylendiamine (HMDA), synthesis grade, was purchased from Riedel de Haën (Hannover, Germany). 2.11. Analytical equipment Spectrophotometers Shimadzu Multispect 1501 and Shimadzu UV160A with temperature-controlled cell (Shimadzu Co., Japan) were used for the enzymatic assays. 3. Results and discussion C. fumago chloroperoxidase (CPO) was treated with sodium cyanoborohydride and 9-antraldehyde (9A) for reductive alkylation and with 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDAC) or with hexamethylendiamine (HMDA) for amidation. Furthermore, native CPO and amidated derivatives were cross-linked with glutaraldehyde (GA). Fig. 1. Effect of molar excess of the modifier agents on CPO activity. (A) Single treatments: ( ) CPO–9A; ( ) CPO–GA; (
) CPO–EDAC; ( ) CPO–HMDA. (B) Double treatment () CPO–GA–GA; (
) CPO–EDAC–GA; ( ) CPO–HMDA–GA. the augment in the molar excess ranging from an almost unnoticeable loss for the molar excess 1:10 to a decrease, in the range of 75–91% for the molar excess 1:1000, also observed elsewhere [9]. It was prominent; however, the activity loss of the CPO–9A derivative (91%) inferring that the extreme reductive conditions associated to pH increase by excess of cyanoborohydride were extremely damaging to the enzyme structure. The use of the molar excess 1:100 resulted in an activity loss within the range of 10–17% (CPO–9A 10.5%, CPO–HMDA and CPO–EDAC, 11.2% and 13.4%, respectively and CPO–GA of 16.6%). This ratio was used in further experiments. In control experiments, performed in the absence of the modifier agents, an activity loss of 5% of the enzyme was observed. Native CPO, CPO–EDAC and CPO–HMDA derivatives were also treated with the cross-linking agent glutaraldehyde (GA), using the molar excess of 1:100. According to results presented in Fig. 1B, it was observed, as expected, an additional activity decrease that amounted to 25%. Since the results obtained with the CPO–GA and –EDAC derivatives, using the commercial CPO and the enzyme preparation from our laboratory, were similar, either biocatalysts were used in the continuation of our experiments. 3.1. Optimisation of the reagents molar excesses 3.2. Degree of modification Three molar excesses (1:10; 1:100; 1:100) of the different reagents in respect to CPO were studied aiming at the synthesis of a more stable CPO biocatalysts. Results for the cross-linking, amidation and alkylation treatments are shown in Fig. 1A. Activity loss increased in response to The extension of the amino acids residues modification in the CPO molecule, for each treatment, was evaluated by the titration of free amino groups using TNBS. According to data presented in Fig. 2, the treatment with 9A or GA resulted in a 586 C.E. La Rotta Hernandez et al. / Enzyme and Microbial Technology 37 (2005) 582–588 Fig. 2. Modification degrees of CPO amino acid residues using a 1:100 molar excess. Black columns represent the increase and the white ones the decrease in free amino groups. modification degree lower than 10% of the CPO free amino groups whereas for the amidated plus GA-treated derivatives, the degree of modification reached 24%. The CPO–HMDA and CPO–EDAC derivatives presented an increase of the titrated amino groups around 10% in comparison to the value obtained for native CPO. The lowest degree of modification, around 5%, was observed for the CPO–9A derivative. This could be related to the molecular size and low solubility of 9A in aqueous media. The possibility of a misleading result due to the interference of cyanide was avoided using controls for the reaction mixtures. 3.3. Effect of the chemical modification on the solubility and hydrophobicity of the CPO derivatives A general decrease in the solubility of the CPO derivatives was observed within the range of 20–40% in comparison to the native CPO (Fig. 3A). The derivatives that presented the highest aqueous solubility (CPO–EDAC and –HMDA) also showed the lowest increase in hydrophobicity (Fig. 3B). The highest hydrophobicity was presented by the CPO–9A derivative, as expected, whose partition coefficient was 4.0fold higher in comparison to the native CPO, in despite of its low modification degree of around 5%. This result is promising considering the use of this modified biocatalyst for organic synthesis with low water solubility substrates. Interesting results were also observed for the CPO–GA derivative Fig. 3. Effect of the modifier agent using a molar excess of 1:100 on the solubility (A) and hydrophobicity (B) ( ) CPO–9A; ( ) CPO–GA; (
) PO–EDAC; ( ) CPO–HMDA. whose partition coefficient was 2.3-fold higher than that of CPO, which could be related to the formation of CPO aggregates [12]. 3.4. CPO derivatives activity and stability in the presence of tert-butanol Although 5% tert-butanol did not affect the native CPO activity, it was observed a gradual activity decrease, that reached 30%, in response to solvent concentrations up to 60%. The majority of the derivatives behaved similarly to the native CPO, although the overall activities in the presence of 60% tert-butanol were higher in comparison to the native Fig. 4. Effect of tert-butanol concentration on the activity of modified and native CPO and its derivatives. C.E. La Rotta Hernandez et al. / Enzyme and Microbial Technology 37 (2005) 582–588 587 Fig. 5. Effect of tert-butanol concentration on the stability of modified and native CPO after an incubation period of 4 h: () native CPO; ( ) CPO–EDAC; () CPO–GA; (
) CPO–HMDA; () CPO–GA–GA; (䊉) CPO–EDAC–GA and () CPO–HMDA–GA. enzyme. The derivative CPO–9A showed to be particularly resistant to the presence of 60% tert-butanol (Fig. 4). Native and derivatized CPO showed to be quite stable upon incubation up to 2 h in all the binary systems tested (data not shown), although activity loss was observed after 4 h of incubation in mixtures containing tert-butanol above 20% (v/v), with exception of the CPO–GA was stable in tert-butanol concentrations up to 60% (v/v) as shown in Fig. 5. 3.5. Effect of pH on the activity- and stability-modified CPO at 30 ◦ C were very similar in all cases. Two activity peaks, at pH values 6.0 and 3.0 were observed in the case of the native CPO, in accordance to previous reports for MCDO, phenolic compounds and tetrametylparaphenylendienamine (TMPD) oxidation [1,6,13]. Native and CPO derivatives showed a substantial loss of peroxidase activity at the extreme pH values 2.0 and 8.0. Concerning pH stability, incubation for 2 h in the pH range studied resulted in a similar profile for the native and derivatized enzyme, with exception of pH 7 and 8 where the modified enzymes showed a higher residual activity with emphasis to the CPO–HDMA derivative (Fig. 6B). Fig. 6A shows the effect of pH on the peroxidase activity of native and derivatized CPO. Profiles for 2,4-DCP oxidation Fig. 6. The effect of the pH on the activity of CPO and its derivatives. pH profiles for (A) activity and (B) pH stability after 60 min of incubation: () native CPO; ( ) CPO–EDAC; () CPO–GA; (
) CPO–HMDA; () CPO–GA–GA; (䊉) CPO–EDAC–GA and () CPO–HMDA–GA. Fig. 7. The effect of the temperature on the activity of CPO and its derivatives. Temperature profiles for (A) activity and (B) thermal stability after 120 min of incubation. () Native CPO; ( ) CPO–EDAC; () CPO–GA; (
) CPO–HMDA; () CPO–GA-GA; (䊉) CPO–EDAC-GA and () CPO–HMDA-GA. 588 C.E. La Rotta Hernandez et al. / Enzyme and Microbial Technology 37 (2005) 582–588 3.6. Effect of temperature on the activity and stability of modified CPO According to the data presented in Fig. 7A, the effect of the temperature on the activity of the native and modified CPO were quite similar in the temperature range studied (20–80 ◦ C) although slightly higher activity was observed at 30 ◦ C for CPO–HMDA–GA. This derivative also presented a higher temperature stability as after incubation at 40 and 60 ◦ C it showed a residual peroxidase activity of 75 and 40%, respectively, in comparison to the native CPO activity of 35% and lower than 5%, respectively. It is worth noticing that the CPO–HMDA derivative also showed the highest increment in free amino groups, which favoured a more effective crosslinking upon the GA treatment (Fig. 2). In general, all the modified biocatalysts showed to be more stable towards temperature in comparison to the native biocatalyst. 4. Conclusions This study evaluated the effectiveness of amidation, reductive alkylation and cross-linking reactions for the stabilisation of CPO towards temperature, pH and organic media. Upon the use of the optimised molar excess, reagents:CPO of 1:100 the most important catalytic improvements of the CPO derivatives were as follows: CPO–9A presented a partition coefficient 4.0-fold higher in comparison to the native CPO and also showed the highest activity in reaction mixtures containing tert-butanol 60% (v/v). However the CPO–GA derivative showed to be the more stable after incubation during 4 h under these conditions. Although the temperature and pH profiles of the native and modified CPO were quite similar in the temperature and pH range studied (20–80 ◦ C and pH 2–8) with highest activity at pH 6.0 and 30 ◦ C, slightly higher activities were observed at 30 ◦ C for the CPO–HMDA–GA derivative. CPO–EDAC and –HMDA and all GA cross-linked derivatives presented 40% residual activity after incubation for 120 min at 60 ◦ C in pH 6.0, and during 60 min at 30 ◦ C in pH 7.0, conditions that completely inactivated the native CPO. All in all the highest efficiency and stability in organic media was observed for CPO–9A and –GA, respectively. The biocatalyst CPO–HMDA–GA was the best performer considering pH and temperature activity and stability. Acknowledgements We are grateful for the financial support from the following institutions: The Brazilian Research Council (CNPq), The International Büro des BMBF (Germany) and the Brazilian Petroleum Agency (ANP). 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