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-
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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
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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
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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.
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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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