A novel immobilized chloroperoxidase biocatalyst with improved stability for the oxidation of amino alcohols to amino aldehydes

Article abstract of DOI:10.1016/j.molcatb.2012.04.010

Chloroperoxidase from Caldariomyces fumago (CPO, EC 1.11.1.10) is one of the most promising of the heme peroxidase enzymes for synthetic applications. Since the synthetic use of CPO suffers severely from its rapid deactivation in the presence of peroxides, the immobilization of this enzyme was studied as a possibility for stability improvement. Three methods of immobilization were considered using monoaminoethyl-N-aminoethyl (MANA) agarose gels: ionic adsorption, covalent attachment via carbodiimide coupled activation and covalent attachment of oxidized CPO. The most successful results led to almost complete immobilization with retained activities of around 51% for the two methods of covalent attachment and 77% for the ionic adsorption of CPO on MANA. Besides, all of the immobilized enzyme systems showed drastically improved stability toward presence of peroxide; CPO immobilized on MANA through carbodiimide coupled method resulted to be the most stable one with an increase in apparent half-life time of more than 500-fold that of the soluble enzyme. CPO immobilized by this method was compared to the soluble enzyme as catalyst for Cbz-ethanolamine oxidation to Cbz-glycinal using tert-butyl hydroperoxide (t-BuOOH) as an oxidant. Despite the lower reaction rate, the reaction catalyzed by immobilized CPO reached higher Cbz-glycinal yield with almost three-fold lower activity loss.

Full text of DOI:10.1016/j.molcatb.2012.04.010

Journal of Molecular Catalysis B: Enzymatic 84 (2012) 144–151
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
A novel immobilized chloroperoxidase biocatalyst with improved stability for the
oxidation of amino alcohols to amino aldehydes
Milja Pesˇic´, Carmen López∗, Gregorio Álvaro, Josep López-Santín
Department of Chemical Engineering, Applied Biocatalysis Unit associated to IQAC (UAB-CSIC), School of Engineering, Universitat Autònoma de Barcelona, 08193-Bellaterra (Cerdanyola
del Vallès), Catalunya, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 15 April 2012
Chloroperoxidase from Caldariomyces fumago (CPO, EC 1.11.1.10) is one of the most promising of the
heme peroxidase enzymes for synthetic applications. Since the synthetic use of CPO suffers severely
from its rapid deactivation in the presence of peroxides, the immobilization of this enzyme was stud-
ied as a possibility for stability improvement. Three methods of immobilization were considered using
monoaminoethyl-N-aminoethyl (MANA) agarose gels: ionic adsorption, covalent attachment via car-
bodiimide coupled activation and covalent attachment of oxidized CPO. The most successful results led
to almost complete immobilization with retained activities of around 51% for the two methods of covalent
attachment and 77% for the ionic adsorption of CPO on MANA. Besides, all of the immobilized enzyme
systems showed drastically improved stability toward presence of peroxide; CPO immobilized on MANA
through carbodiimide coupled method resulted to be the most stable one with an increase in apparent
half-life time of more than 500-fold that of the soluble enzyme. CPO immobilized by this method was
compared to the soluble enzyme as catalyst for Cbz-ethanolamine oxidation to Cbz-glycinal using tert-
butyl hydroperoxide (t-BuOOH) as an oxidant. Despite the lower reaction rate, the reaction catalyzed by
immobilized CPO reached higher Cbz-glycinal yield with almost three-fold lower activity loss.
© 2012 Elsevier B.V. All rights reserved.
Keywords:
Chloroperoxidase (CPO)
Enzymatic immobilization
MANA-agarose
Ionic adsorption
Covalent attachment
1. Introduction
acids usually constitute one of the major fractions of surface groups
on proteins [3]. Monoaminoethyl-N-aminoethyl (MANA) agarose
Immobilization of enzymes has been developed rapidly in the
past 40 years since its benefits were evaluated and utilized with
success in biotechnological, medical and analytical applications.
The chemical, biochemical, mechanical and kinetic properties of
the immobilized enzyme depend both on the properties of the
enzyme and the support used for immobilization, while the appar-
ent activity retention upon the process depends mostly on the
support characteristics, the enzyme-support interaction and the
amount of soluble enzyme used. The measured enzymatic activity
after immobilization may be reduced by the binding procedure, and
also by mass transfer effect as a result of diminished availability of
enzyme molecules within pores or from slowly diffusing substrate
molecules. Nevertheless, this drawback can be compensated by a
stability improvement of the immobilized enzymes under working
conditions compared to the soluble enzyme, resulting in overall
benefit [1,2].
gels containing very low pK primary amino groups are very suit-
able supports for immobilization of enzymes at low pH values [3].
The method proposed consists in ionic adsorption of the enzymes
to the support in a low ionic-strength media. Further, by applying
a mild coupling method for the activation of carboxyl groups of
the enzyme using carbodiimide, ionic adsorption is transformed to
covalent attachment.
Another possibility for the successful and simple application of
these primary amino supports is the immobilization of glycopro-
teins via their sugar moieties, previously oxidized with periodate
resulting in a high density of aldehyde groups. These aldehydes can
further react with amino groups from the resins, yielding Schiff base
bonds [4,5].
Haloperoxidases are peroxidases capable to halogenate sub-
strates in the presence of halide and peroxide [6,7]. Heme-thiolate
haloperoxidases are the most versatile biocatalysts of the heme-
protein family and for a long time, only one enzyme of this type
– the chloroperoxidase (CPO; EC 1.11.1.10.) from the ascomycete
Caldariomyces fumago – was known [8,9]. CPO was firstly isolated
and characterized over 40 years ago [10]. Molecular weight of this
enzyme is 42,000 Da. CPO is rich in aspartic acid, glutamic acid,
serine and proline; these four amino acids constitute about 45%
The methods of immobilization via carboxyl groups on the pro-
tein surface may be very suitable since the aspartic and glutamic
∗
Corresponding author. Tel.: +34 93 5812694; fax: +34 93 581 2013.
E-mail address: carmen.lopez@uab.es (C. López).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.04.010

M. Pesˇi´c et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 144–151
145
of the total amino acid content. It has 321 amino acids with pre-
dominantly acidic residues and a pI in a range of 3.2–4.0. CPO is
a glycoprotein; 25–30% of the molecule is carbohydrate, in which
major constituents are glucosamine and arabinose.
Sunshine Chemlab, Inc (Downingtown, PA, USA). All the other
reagents and solvents used were obtained from diverse commercial
suppliers and were of the highest available purity and of analytical
grade.
CPO uses peroxides as electron acceptors and does not require
any cofactor for its activity. Nevertheless, a main shortcoming of
CPO and all heme-containing peroxidases is their low operational
stability, which is caused by the rapid deactivation by peroxides,
despite its catalase activity. Deactivation involves destruction of
the heme group, but its precise mechanism is not clear yet [11].
CPO exists in two active forms: the acidic form (pH < 3)
which catalyses halide-dependent reactions, and the neutral form
(pH 5–6) which catalyses halide-independent reactions [7,12,13].
Within the group of halide-independent reactions CPO has been
demonstrated to be able to catalyze the oxidation of primary
alcohols to the corresponding aldehydes [14,15]. Among these
compounds, amino aldehydes have special interest as they can
act as substrates of aldolases in the aldol addition of dihydroxy-
acetone phosphate (DHAP), obtaining aminopolyols. In turn, these
aminopolyols can be directly cyclated or isomerized to obtain
iminocyclitols, which are inhibitors of glycosidases and glycosyl-
transferases with a great therapeutic potential [16].
2.2. Chloroperoxidase activity assay
The spectroscopic assay for the measurement of CPO activ-
ity was based on the decrease in absorbance of a solution of
monochlorodimedone, MCD (ε278 nm = 12,200 M−1 cm−1) as it was
converted to dichlorodimedone, DCD (ε278 nm = 120 M−1 cm−1), by
CPO according to the method described by Hager et al. [29]. The
assay mixture contained 100 mM potassium phosphate buffer pH
2.75, 20 mM KCl, 2 mM hydrogen peroxide and 0.16 mM MCD. The
absorbance was measured at 25 ◦C at a wavelength of 278 nm in
UV-Visible Spectrophotometer Cary 50 (Varian, Palo Alto, CA, USA)
using quartz cuvettes. One unit of CPO was defined as the enzyme
activity required to catalyze the conversion of 1 ␮mol of MCD to
DCD per minute at 25 ◦C. In order to measure the immobilized
enzyme activity in the suspension samples, magnetic stirring was
applied to the spectrophotometric cuvette. No interference of the
resin was observed in the assay.
Several methods for CPO immobilization were reported. Immo-
bilization of CPO on silica-based materials was demonstrated to
increase its pH, oxidizing agent and temperature tolerance [17–19].
Besides, CPO was immobilized on mesoporous materials by adsorp-
tion [20,21] or covalent attachment [22,23], on aminopropyl-glass
beads [24], talc [25] and celite [26] by adsorption, and on magnetic
beads [27] and chitosan membranes [28] by covalent attachment.
Generally, the main disadvantage of all the adsorption methods is
the leaching of the enzyme from the support. All of the reported
methods based on covalent attachment involved the formation
of the bonds between various functional groups on the support
surface and amino groups from the lysine residues of CPO. The pres-
ence of only four lysine residues in the CPO amino acid sequence
is the most probable reason for the relatively low immobilization
yields achieved using this kind of supports.
2.3. Stability of soluble CPO
Effects of pH on CPO stability were studied over the pH range
5.0–10.0. The reaction media (10 mL of selected buffer – sodium
phosphate or bicarbonate, depending on the pH – in a concen-
tration of 50 mM) containing 20 U of enzyme were incubated at
room temperature on a roller. Effect of sodium cyanoborohydride
on CPO stability was analyzed using molar excesses of 1:10, 1:100,
1:1000 and 1:10,000 (CPO:cyanoborohydride). The reaction media
containing 2 U/mL of enzyme and 100 mM phosphate buffer pH 6.0
were incubated at 4 ◦C in a roller. In both cases, after different times
of incubation aliquots of enzyme solution were withdrawn and
enzymatic activity was measured using the previously described
assay.
In the present work, CPO was proposed to be immobilized on
MANA-gels applying three different methods: ionic adsorption,
covalent attachment using carbodiimide and covalent attachment
of oxidized enzyme. Application of these immobilization methods
allowed the formation of covalent bonds between the amino groups
of the support and either the most abundant amino acid residues of
CPO (Asp and Glu) or its sugar moiety. Furthermore, reaction of oxi-
dation of amino alcohols catalyzed by immobilized and soluble CPO
was performed. Since it was previously shown that branched-chain
alcohols were not substrates for CPO [14], ethanolamine containing
a protective carbobenzoxy (Cbz) group was selected as substrate
for the synthesis of the corresponding protected amino aldehyde
Cbz-glycinal.
2.4. Preparation of MANA-agarose support
The amino-agarose support was prepared from glyoxyl-agarose
gels as previously described by Fernandez-Lafuente et al. [3].
Glyoxyl-agarose gels were prepared by etherification of 10%
crosslinked agarose gels with glycidol, and further periodate oxi-
dation of the resulting glyceril-agarose [30]. For that, 15 mL of
agarose gels were washed thoroughly with distilled water and the
gel was suspended in 11.6 mL of a solution that contained NaOH,
NaBH4 and glycidol, in final concentrations of 0.32 N, 5.38 mg/mL
and 2.04 M, respectively. The reaction was left on mild agitation
in the rotary evaporator for approximately 19 h at room tem-
perature (20–25 ◦C). Once the reaction was finished, agarose was
vacuum-filtered and washed with distilled water (around 10 suc-
cessive washings) in order to eliminate the rest of reagents. The
next step was the oxidation of the resulting glyceryl-agarose with
an excess of sodium periodate (300 ␮mol NaIO4/mL agarose), dur-
ing approximately 30–45 min. The formation of aldehyde groups
was calculated from the consumed NaIO4, which was evaluated
by colorimetry, detecting the iodine liberated from KI by oxida-
tion with remained NaIO4 in the presence of NaHCO3. The values
of absorbances were measured using Spectrophotometer Cary 50
(Varian, Palo Alto, CA, USA). This support presented 264 ␮mol of
aldehyde groups per mL of agarose. After the oxidation, glyoxyl-
agarose was vacuum-filtered and washed with distilled water.
Then, 15 g of glyoxyl-agarose were suspended in 135 mL of 0.1 M
bicarbonate buffer at pH 10.0 with 2 M ethylenediamine. The sus-
pension was gently stirred on a roller for 2 h. Finally, 1.5 g of NaBH4
2. Materials and methods
2.1. Materials
CPO from C. fumago was obtained from Chirazyme Labs
(Greenville, NC, USA) as a solution of partially purified enzyme
(11.6 mg protein/mL), with
a specific activity of 1400 U/mg.
Agarose 10% crosslinked beads (10 BCL) was purchased from
Iberagar (Coina, Portugal). Monochlorodimedone (1,1-dimethyl-
4-chloro-3,5-cyclohexanedione), Cbz-ethanolamine (benzyl-N-(2-
hydroxyethyl)-carbamate), Cbz-glycine, tert-butyl hydroperoxide
(70% (w/w) in water) and N-ethyl-Nꢀ-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDAC) were purchased from Sigma
Aldrich (St. Louis, MO, USA). Cbz-glycinal was obtained from

146
M. Pesˇi´c et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 144–151
(10 mg/mL) was added to the suspension and it was left on mild
agitation on a roller for other 2 h in order to finish the reduc-
tion. The reduced gels were vacuum filtered and washed with
150 mL of 0.1 M acetate at pH 5.0 containing 1 M NaCl, with 150 mL
0.1 M bicarbonate at pH 10.0 containing 1 M NaCl, and finally, with
750 mL of distilled water.
as samples of blank, were withdrawn periodically and enzymatic
activity was tested. The retained activities were calculated as the
difference between activity of the suspension and the supernatant
in relation to the initial activity of the process. The immobilization
yields were calculated as the differences between the initial activity
and the supernatant activity at the end of the process related to the
initial activity. Finally, at the end of the immobilization, the immo-
bilized enzyme preparation was vacuum-filtered and its residual
activity was measured.
2.5. Periodate oxidation of CPO
CPO solution was previously dialyzed against 0.05 M phosphate
buffer pH 5.0 at 4 ◦C, in order to minimize the presence of impurities
that could interfere in the oxidation process. Oxidation of CPO was
carried out with an excess of sodium periodate (125 mM) in 0.05 M
phosphate buffer pH 5.0 at 4 ◦C in the dark. Aliquots were taken
periodically to determine the periodate consumption. The quantity
of consumed NaIO4 was evaluated by the same colorimetric method
that was described for preparation of the glyoxyl agarose support.
Once the oxidation was finished, the oxidized enzyme solution was
dialyzed against 0.05 M phosphate buffer pH 5.0 at 4 ◦C.
2.10. Studies of diffusion limitations of immobilized CPO
preparations
The significance of diffusion limitations for different enzy-
matic loads was studied for the preparation obtained by coupling
oxidized CPO on MANA-agarose gels. CPO activities used in the
different experiments were: 1, 5, 10, 20, 50 and 100 U per mL of
MANA-agarose. Enzyme was left to bind to the support under mild
agitation on a roller at room temperature, and when no activity was
detected in the supernatant, the residual activity of the suspension
was measured using the standard assay for CPO.
2.6. Immobilization of CPO by adsorption on MANA-agarose gels
1 mL (1.07 g) of highly derivatized MANA-agarose gel was sus-
pended in 9 mL of buffer (sodium acetate, sodium phosphate, HEPES
or MES) of known concentration (10, 25, 50 or 100 mM) and pH
value (5.0 or 6.0). Once mixed and before adding the enzyme, read-
justing the pH value was necessary since the presence of high amine
groups density on the support causes an increase of pH. Then,
between 10 and 10,000 U of native CPO were added and left to
immobilize on the support under mild agitation on a roller at room
temperature.
2.11. Peroxide dependent inactivation of immobilized CPO
preparations
The immobilized enzymes, each one prepared by adding 20 U of
CPO per mL of MANA-agarose, were washed with 100 mM acetate
buffer and mixed in v:v ratio of 1:10 with the medium containing
100 mM acetate buffer at pH 5.0 in a volume of 10 mL. In case of
soluble enzyme, 20 U of CPO were added to the same volume of
medium. The process started after addition of a 5-␮L pulse of a
solution containing 1.67 M of tert-butyl hydroperoxide. t-BuOOH
pulses were added at a rate of 3 mM/h (18.02 × 10−6 L/h) using a
single-syringe automatic microburette (Crison Instruments, Spain).
Aliquots were withdrawn periodically and the enzymatic activity
was measured in both the suspension and the supernatant, in order
to determine the possible leaching of the enzyme from the support
at these conditions.
2.7. Immobilization of CPO by covalent attachment with EDAC on
MANA-agarose gels
For the multipoint covalent attachment of CPO on MANA-
agarose, the first step was the ionic adsorption of the enzyme on
the support for 15 min, until no activity was detected in the super-
natant. At this moment, different amounts of carbodiimide (final
concentrations of 25 or 50 mM) in buffer (sodium phosphate or
MES) were added and the enzymatic preparations were left on
roller agitation during different intervals of time (2–3 h). Finally,
NaCl was added in final concentration of 1 M in order to eliminate
the non-covalently bound enzyme and left on mild agitation during
1 h.
2.12. Oxidation of Cbz-ethanolamine to Cbz-glycinal catalyzed by
soluble and immobilized CPO
2.12.1. Reaction with soluble CPO
2.8. Immobilization of oxidized CPO by covalent attachment on
MANA-agarose gels
5000 U of CPO were introduced in a 10 mL reaction medium
containing 20 mM Cbz-ethanolamine and 100 mM acetate buffer
at pH 5.0. The reaction started after addition of a 5-␮L pulse
of a solution containing 1.67 M of tert-butyl hydroperoxide. t-
BuOOH pulses were added at a rate of 3 mM/h (18.02 × 10−6 L/h)
using a single-syringe automatic microburette (Crison Instruments,
Spain). Samples were taken periodically and analyzed by liquid
chromatography in order to quantify Cbz-ethanolamine and Cbz-
glycinal concentrations.
1 mL (1.07 g) of highly derivatized MANA-agarose gel was sus-
pended in 9 mL of sodium phosphate at 25 mM and pH value of 5.0
or 6.0. Once the support mixed with the buffer, readjustment of
the pH was necessary. Then, between 10 and 10,000 U of oxidized
CPO and NaCNBH3 in molar excess of 1:100 were added and left
to immobilize on the support on mild agitation on a roller at room
temperature. When no activity was detected in the supernatant,
solid NaCl was added (1 M) and left on mild agitation on a roller
during 1–2 h.
2.12.2. Reaction with immobilized CPO
2.9. Monitoring of immobilization process
CPO immobilized by covalent attachment on MANA-gels in the
presence of EDAC was prepared by mixing 10,000 U of CPO per mL
of MANA-agarose. The immobilized biocatalyst was washed with
100 mM acetate buffer and added in ratio 1:10 (v:v) to a 10 mL reac-
tion medium containing 20 mM Cbz-ethanolamine and 100 mM
acetate buffer at pH 5.0. The reactions were performed and moni-
tored in the same way as for the soluble enzyme.
All the immobilization processes were carried out under mild
magnetic stirring conditions and at room temperature. A blank con-
taining water instead of the agarose gel was followed in parallel
in the three procedures. In order to study the time course of the
immobilization, aliquots of the supernatant and suspension, as well

M. Pesˇi´c et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 144–151
147
MANA-agarose gels present very low pK value and thus are suit-
able for the enzyme immobilization at low pH. Several options for
the immobilization of CPO on this support were considered: simple
ionic adsorption, covalent attachment of CPO through its carboxyl
groups via carbodiimide coupled method and finally, direct cova-
lent immobilization of CPO through its glycosidic chains previously
oxidized with periodate.
3.2.1. Ionic adsorption of CPO on MANA-agarose gels
Immobilization by adsorption is the simplest immobilization
method and consists in reversible surface interactions between
the enzyme and the support [2,31]. In general, adsorption of CPO
on MANA-agarose support occurs via electrostatic interactions
between the external charges on the enzyme and the support,
and can be performed at pH values in the range of 4–6 [3]. The
parameters studied in order to optimize the conditions for the
immobilization were pH value, incubation time, and type and con-
centration of the buffer (Table 1).
Depending on the chemical structure of the available surfaces
for adsorption and the pI of CPO (around 4.0) alteration in the
pH of the medium will have an influence on the support-protein
interactions. The pH 5.0 was chosen as optimal, as it favored both
positive charge of the support which has pK value 6.8 and negative
charge of the enzyme and did not affect the stability of the soluble
chloroperoxidase. Accordingly, higher retained enzymatic activi-
ties were achieved at pH 5.0 using sodium acetate and phosphate
buffers comparing to those at pH 6.0 using MES and HEPES buffers.
Influence of the incubation time was studied at pH 5.0 in sodium
acetate at different buffer concentrations. In general, slight increase
of the immobilization yield was observed at longer incubation
times, but it was not always advantageous respect to the retained
enzymatic activity, as the activity of the suspension decreased with
time.
Adsorption performed in sodium phosphate buffer was more
successful than the one in sodium acetate buffer using the same
conditions, and immobilization processes required much less time.
Since low concentrations of the buffers (between 10 and 100 mM)
were used in all cases to ensure low ionic strength conditions and
permit the ionic interactions between the enzyme and the support,
no significant differences in terms of immobilization yields were
observed within that range of buffer concentrations. However,
concerning activity retentions, higher values were obtained when
using 50 mM phosphate buffer comparing to the ones obtained with
10 or 25 mM, probably due to more effective buffering when higher
concentrations were applied.
Fig. 1. Effect of pH on the activity of soluble CPO after different times of incubation:
5 min (᭹), 1 h (ꢁ), and 3 days (ꢀ). Soluble CPO was incubated at 25 ◦C in 50 mM
sodium phosphate buffer at pH values: 5.0, 6.0, 6.5, 7.0, 7.5 or sodium bicarbonate
buffer at pH value 10.0. The initial CPO activities were around 2 U/mL and were set
to 100% at t = 0.
2.13. Cbz-ethanolamine, Cbz-glycinal and Cbz-glycine
quantification
Concentrations of Cbz-ethanolamine, Cbz-glycinal and Cbz-
glycine were analyzed by HPLC in
a Dionex UltiMate 3000
with UltiMate 3000 Variable Wavelenght Detection (Sunnyvale,
USA) employing a reversed-phase column X Bridge C18, 5 ␮m,
4.6 mm × 250 mm from Waters (Wexford, Ireland) at 30 ◦C. The
solvent system consisted of solvent A, composed of 0.1% v/v tri-
fluoroacetic acid (TFA) in H2O, and solvent B, composed of 0.095%
v/v TFA in H2O/CH3CN 1:4. The solvents were eluted at a flow rate
of 1 mL/min using a gradient from 20% B to 36% B over 24 min and
peaks were detected at 200 nm. Quantitative analysis of products
was performed from peak areas by the external standard method by
means of a prior calibration with samples of known concentration.
3. Results and discussion
3.1. pH stability of chloroperoxidase
One of the most limiting factors when choosing an immobi-
lization method is the commonly narrow interval of pH in which
the enzymatic stability is preserved. Therefore, in order to prevent
CPO inactivation during the immobilization, the pH stability of the
enzyme must be evaluated. Although it is known that its stability
decreases when increasing pH, it might be possible to maintain it
in sufficiently high level during the time necessary to complete the
immobilization.
3.2.2. Multipoint covalent attachment of CPO on MANA-agarose
gels
Once the CPO enzyme was ionically adsorbed to MANA-agarose,
carbodiimide (EDAC) was added to the suspension as an activat-
ing agent of the carboxylic groups enabling the transformation of
adsorption to more stable covalent attachment.
The effect of pH value on the stability of CPO is shown in Fig. 1.
CPO resulted to be extremely instable at alkaline pH values, and it
showed slightly slower inactivation at neutral pH value. Even so,
at pH 7 the instability of the enzyme was still too rapid having in
mind the time required to perform the immobilization processes.
Lowering further the pH to slightly acidic values, the stability of
CPO importantly increased so that at pH 6.5 it retained about 70%
of initial activity after 3 days of incubation, while at both pHs 6.0
and 5.0 CPO retained more than 95%.
Even though sodium phosphate buffer at 50 mM and pH 5.0
was the most appropriate one for the ionic adsorption of CPO to
MANA-agarose gels, the use of this buffer for the carbodiimide
coupled transformation of ionic interaction to covalent bonds is
limited by the instability of EDAC in this buffer [32]. The presence
of inorganic phosphate was shown to have a very strong influence
on decomposition of EDAC and its half-life decreases rapidly with
the increase of phosphate concentration and decrease of pH value.
Half-lives of EDAC in 100 mM and 10 mM phosphate buffer at pH
5.0 were 0.16 h and 1.43 h, respectively. Therefore, EDAC had to be
added in excess and phosphate buffer used at low concentration.
Experimentally, the benefit of EDAC addition, i.e. the covalent
bond success, was analyzed by testing the CPO desorption from
the support – after immobilization – by addition of 1 M NaCl. From
3.2. Immobilization of CPO
Considering the results about pH stability of CPO, the immo-
bilization of this enzyme was performed at acidic pH values.

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M. Pesˇi´c et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 144–151
Table 1
Immobilization yield and retained activity of CPO immobilized by adsorption on MANA-agarose. Experiments were performed by adding 20 U/mL of support in a ratio 1:10
(support volume: total volume) at 20 ◦C.
Buffer
pH
5.0
Ionic strength (mM)
Adsorption time (min)
Immobilization yielda (%)
Retained activityb (%)
30
60
45
90
15
45
45
90
67.5
67.9
74.9
80.2
75.7
81.4
70.0
68.0
58.4
40.2
49.3
51.4
41.4
41.5
40.4
35.5
10
25
Sodium acetate
50
100
10
25
50
15
15
15
93.8
92.8
91.6
50.5
57.9
76.6
Sodium phosphate
MES
5.0
6.0
6.0
25
25
10
25
69.3
92.8
29.8
9.3
HEPES
a
The immobilization yields were calculated as the differences between the initial activity and the supernatant activity at the end of the process related to the initial
activity.
b
The retained activities were calculated as the difference between activity of the suspension and the supernatant in relation to the initial activity.
Table 2
Immobilization yield, retained activity and desorption of CPO covalently immobilized on MANA-agarose using EDAC. Experiments were performed by adding 20 U/mL of
support in a ratio 1:10 (support volume: total volume) at 20 ◦C.
Buffer
pH
Ionic strength
(mM)
EDAC incubation
time (h)
EDAC concentration
(mM)
Immobilization
yield (%)
Retained
activity (%)
Desorptionb
(%)
2
2
3
25
50
50
83.4
88.4
94.0
32.9
44.9
54.5
10.9
7.4
1.0
Sodium phosphate
5
10
Sodium phosphate/MESa
5/6
25/25
2.5
25
98.7
31.3
0.0
a
Buffer for ionic adsorption/buffer for covalent attachment with EDAC.
Desorption was analyzed by adding 1 M NaCl at the end of the EDAC incubation time.
b
the immobilization performed in 10 mM phosphate buffer at pH
5.0 (Table 2) it can be seen that by increasing EDAC concentration
from 25 mM to 50 mM and incubating it for 2 h, the desorption
from the support was reduced from 10.9 to 7.4% respect to the
overall initially added activity. The possibility of desorption was
almost completely eliminated by prolonging the incubation time
to 3 h employing 50 mM EDAC (Table 2).
3.2.3. Multipoint covalent attachment of oxidized CPO on
MANA-agarose gels via its sugar moiety
Carbohydrate moieties of CPO can be oxidized to form aldehyde
groups that can further react with amino groups of MANA-agarose
support. However, during the oxidation of the glycoproteins,
Other buffers were tested for the adsorption and posterior cova-
lent attachment of CPO in MANA-agarose. It was described that
EDAC remains stable in 50 mM MES buffer at pH 7.0 (half-life of
37 h), while at lower pH it decomposes more rapidly (at pH 6.0 and
pH 5.0 its half-life has the value of 20 and 3.9 h respectively) [32].
As the buffer MES at pH 6.0 enabled low retained activities for the
ionic adsorption of CPO (Table 1), the performance of the immobi-
lization in two separated steps for ionic and covalent attachment
was considered. In the first one the enzyme was left to adsorb ioni-
cally to the support in 25 mM phosphate buffer at pH 5.0. Then, the
immobilized enzyme preparation was vacuum-filtered and added
to 25 mM MES buffer at pH 6.0. As at these conditions EDAC is quite
stable, 25 mM were enough to enable covalent attachment as any
desorption was observed. However, the final retained activity was
31.3% and the major part of activity loss, around 42%, took place in
the moment of change of the buffer between the two steps.
Observing the time course of the immobilization, the major
loss of enzymatic activity occurs during the ionic adsorption step
(Fig. 2). In parallel with the immobilization, a blank with soluble
enzyme at identical conditions but in absence of the support was
performed. While the soluble enzyme suffered a deactivation in
presence of EDAC, the enzyme in the immobilization suspension
showed more stability toward the same concentration of EDAC.
Furthermore, enzymatic retentions were a bit higher when using
higher EDAC concentrations. These two facts point out an increase
of enzymatic stability once the enzyme is covalently immobilized.
Fig. 2. Covalent immobilization of CPO on MANA-agarose in 10 mM phosphate
buffer pH 5.0 at room temperature (20 ◦C) using 50 mM EDAC. (I) Ionic adsorption.
(II) Carbodiimide coupled formation of covalent bonds between the carboxyl groups
of the enzyme and the amino groups of the support. (III) Checking of possible des-
orption of the enzyme from the support by adding 1 M NaCl. Time course of the
immobilization was determined measuring the activities for the suspension (᭹),
the supernatant (ꢁ) and the blank (ꢀ).

M. Pesˇi´c et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 144–151
149
Fig. 4. Effect of the initially added enzymatic activity on the apparently retained
activities for the enzymatic preparation of oxidized CPO which was covalently
immobilized on MANA-agarose. Experiments were performed using an immobi-
lized enzyme volume: total volume ratio of 1:10, in 25 mM phosphate buffer pH 5.0
at room temperature.
Fig. 3. Covalent immobilization of oxidized CPO on MANA-agarose with NaCNBH3
added in molar excess 1:100 respect to CPO, in 25 mM phosphate buffer pH 5.0 at
room temperature. Time course of the immobilization was determined measuring
the activities in the suspension (᭹), the supernatant (ꢁ) and blank (ꢀ).
very low values of pH, close to 1, spontaneous breaking of bonds is
unlikely and, consequently, reduction of bonds is not required.
certain amino acid residues may be oxidized as well, especially
cysteine, cystine, methionine, tryptophan, tyrosine, histidine and
N-terminal serine and threonine [33]. Whenever these residues are
essential for the activity of the enzyme, their oxidation can lead to
enzymatic inactivation. The periodate oxidation of CPO was per-
formed in an excess concentration of sodium periodate during 24 h,
after which no further consumption of periodate was observed.
Upon oxidation, CPO retained around 54% of its initial activity.
Immobilization by multipoint covalent attachment of oxidized
CPO on MANA-agarose consisted in two steps. Firstly, oxidized CPO
solution and MANA-support were incubated in the presence of
sodium cyanoborohydride, which enabled the reduction of Schiff
base bonds formed between the enzyme and the support yield-
ing more stable secondary amine bonds. Sodium cyanoborohydride
was selected as a reducing agent due to the possibility to perform
the reduction step at acidic pH values [34]. As explained before,
once immobilization was completed, 1 M NaCl was added in order
to determine the possible desorption of the enzyme from the sup-
port.
First of all, the stability of CPO was evaluated in the presence
of sodium cyanoborohydride, in order to determine the adequate
concentration to prevent its inactivation during the immobilization
process. This reducing agent did not cause any inactivation of CPO
after 2 h of incubation when used in molar excess of 1:10 and 1:100
(CPO:cyanoborohydride). However retained activities decreased in
38.2 and 92.6% for molar excess of 1:1000 and 1:10,000. Therefore,
a cyanoborohydride molar excess of 1:100 was selected for the sta-
bilization of Schiff base bonds formed during the immobilization of
oxidized CPO on MANA-agarose.
3.3. Diffusion limitations of the immobilized CPO preparations
Kinetics of reactions catalyzed by immobilized enzymes may
differ significantly from those using soluble enzymes, as the reac-
tion rate strongly depends on the physical processes of substrates
and products transport through the interior of the beads. Substrate
diffusion inside the porous medium proceeds simultaneously with
the chemical reaction and its effect becomes more significant for
relatively high enzyme concentrations in the solid phase [35,36].
The possible significance of diffusion limitations for CPO
immobilized on MANA-agarose was studied for the reaction cor-
responding to the enzymatic assay using monochlorodimedone
(MCD) as substrate. Oxidized CPO covalently immobilized on
MANA-agarose was studied as model immobilized enzyme. The
percentage of apparent retained activity of the immobilized prepa-
ration respect to the initially added activity was measured for added
activities within the range of 1–100 U/mL of support. In all cases
the immobilization yield was close to 100%. It is well known that
in the absence of diffusion limitations and when saturation is not
occurring, the percentage of the retained activity upon the immo-
bilization does not depend on the activity offered. However, for
the immobilized system corresponding to oxidized CPO on MANA-
agarose the diffusion limitations were present for loads over 5 U
added per mL of support, under which the measured retained activ-
ity remained unchanged (Fig. 4). Therefore, it can be concluded that
approximately 33% of the CPO activity loss was due to the immobi-
lization process itself, while the rest of the apparent activity loss can
be attributed to the presence of diffusion limitations. Hence, when
catalyzing the chlorination of the natural substrate, MCD, enzy-
matic loads lower than 5 U per mL of the support are required to
eliminate the limitations by diffusion. Nevertheless, for any other
reaction with lower reaction rate than that one with MCD, diffu-
sion limitations will appear at higher enzymatic loads or even will
not appear. Therefore, the real influence of the diffusion limitations
on CPO-MANA catalyzed oxidation of Cbz-ethanolamine will be
evaluated by comparison of the target reaction performance using
soluble and immobilized CPO.
Complete immobilization of oxidized CPO on MANA-agarose
occurred after only 30 min of incubation of the enzyme and the
support together with reducing agent (Table 3 and Fig. 3). Similar
retentions of activity were obtained in all the cases studied; the one
obtained at pH 5.0 and 4 ◦C being slightly higher than others (51.4%).
Almost no desorption was detected in the experiments after the
addition of 1 M NaCl, even when no sodium cyanoborohydride was
used for the reduction of Schiff base linkages. This was probably due
to a very high number of available groups on the enzyme molecule
able to react with the groups on the support, forming a very stable
multipoint linkage. As the reversibility of the Schiff base occurs at

150
M. Pesˇi´c et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 144–151
Table 3
Immobilization yield, retained activity and desorption of oxidized CPO covalently immobilized on MANA-agarose. Experiments were performed by adding 20 U/mL of support
in a ratio 1:10 (support volume: total volume) using 25 mM sodium phosphate buffer at 20 ◦C.
Molar excess of NaCNBH3
pH
T (◦C)
Immobilization yielda (%)
Retained activitya (%)
Desorptionb (%)
1:100
1:100
1:100
No NaCNBH3 added
6
5
5
5
4
4
21
21
96.2
97.8
99.1
97.5
46.2
51.4
48.0
47.0
1.3
0.4
0.0
0.3
a
Values obtained after 30 min of covalent attachment.
Desorption was analyzed by adding 1 M NaCl at the end of the EDAC incubation time.
b
3.4. Stability of the immobilized CPO preparations
the enzyme from the support was not observed in these conditions
for none of the three preparations tested.
As it was mentioned before, the main limitation for the synthetic
application of peroxidases is their fast inactivation in the presence
of peroxides. In order to minimize this inactivation, reactions are
usually performed under a continuous addition of peroxide, and the
addition rate is selected looking for a compromise between reaction
rate and activity preservation [37]. Considering our target reaction,
i.e. the oxidation of the amino alcohol Cbz-ethanolamine to Cbz-
glycinal catalyzed by CPO, tert-butyl hydroperoxide was selected as
oxidant instead of the most commonly applied hydrogen peroxide,
due to the high catalase activity of CPO toward H2O2 [38].
The stabilities of the immobilized CPO preparations on MANA-
agarose were studied at the conditions in which reaction of interest
takes place. Since mass-transfer-controlled reactions appear to be
much less sensitive to the decay of enzyme activity [39], low enzy-
matic loads were used (20 U/mL of MANA) avoiding high diffusion
limitations. The activities of soluble and immobilized enzymes
were measured under addition of t-BuOOH at the rate of 3 mM/h.
In these conditions, soluble CPO lost 96.2% of its activity after only
2 h of incubation (Fig. 5). All of the immobilized enzymes showed
significantly improved stability in these conditions, especially CPO
immobilized by covalent attachment coupled with EDAC which
appeared to retain almost 90% of its activity after 48 h in these
conditions. This improvement was reflected in a sharp increase of
apparent half-life times, which rose from 0.33 h for soluble CPO to
47.4, 175.7 and 103.6 h for the enzymes immobilized by adsorp-
tion, by covalent attachment with EDAC and for oxidized enzyme
immobilized by covalent attachment, respectively. Desorption of
3.5. Oxidation of Cbz-ethanolamine to Cbz-glycinal catalyzed by
immobilized CPO
Encouraged by the results of the improved stability of immo-
bilized enzymes, the most stable one, i.e. CPO immobilized by
covalent attachment on MANA-agarose using EDAC, was applied as
biocatalyst and compared to the soluble enzyme. The oxidation of
Cbz-ethanolamine to yield the aldehyde Cbz-glycinal was selected
as target reaction. Some experimental conditions, such as continu-
ous addition of tert-butyl hydroperoxide at a rate of 3 mM/h and a
high enzymatic activity of 500 U/mL, were determined by previous
assays.
One of the main constraints when trying to oxidize alcohols
to aldehydes is the easy further oxidation of aldehydes to acids.
The selection of both the enzyme and the reaction conditions is
essential to avoid this further oxidation. When catalyzing the oxi-
dation of Cbz-ethanolamine to Cbz-glycinal using CPO immobilized
on MANA-agarose with EDAC, around 44.7% of Cbz-glycinal and
17.8% of amino acid Cbz-glycine were obtained (Table 4 and Fig. 6).
The yield of Cbz-glycinal reached its maximum at 30 h. After this
time the accumulation of amino aldehyde accelerates its further
conversion to amino acid, which becomes more pronounced with
the reaction time, resulting in slightly lower yield of Cbz-glycinal at
48 h than the corresponding one at 30 h. However, the reaction cat-
alyzed by soluble CPO reached a maximum of 39.1% of Cbz-glycinal,
Fig. 5. Stability of soluble and immobilized CPO under addition of 3 mM/h of t-
BuOOH in acetate buffer 100 mM pH 5. Soluble CPO (ꢁ), CPO immobilized by
adsorption on MANA-agarose (᭹), CPO covalently immobilized on MANA-agarose
using EDAC (ꢂ) and oxidized CPO covalently immobilized on MANA-agarose (ꢀ).The
initial CPO activities were around 2 U/mL and were set to 100% at t = 0.
Fig. 6. Profile for the oxidation of Cbz-ethanolamine (ꢁ) to Cbz-glycinal (ꢀ) and
Cbz-glycine (᭹) catalyzed by CPO covalently immobilized on MANA-agarose with
EDAC, using tert-butyl hydroperoxide as oxidant at the rate of 3 mM/h. The reaction
medium contained 30 mM of Cbz-ethanolamine and 500 U/mL of CPO in 100 mM
acetate buffer pH 5.0 and was performed at room temperature (20–23 ◦C).

M. Pesˇi´c et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 144–151
151
Table 4
Oxidation of Cbz-ethanolamine to Cbz-glycinal catalyzed by soluble CPO and CPO covalently immobilized on MANA-agarose with EDAC, using tert-butyl hydroperoxide as
oxidant at the rate of 3 mM/h. The reaction medium contained 20 mM of Cbz-ethanolamine and 500 U/mL of CPO in 100 mM acetate buffer pH 5.0. All the experiments were
performed at room temperature (20–23 ◦C).
Enzyme
Reaction
time (h)
Activity
loss (%)
Cbz-ethanolamine
conversion (%)
Cbz-glycinal
yield (%)
Cbz-glycine
yield (%)
Initial reaction rate
(mM Cbz-glycinal/h)
Soluble CPO
8
94.2
47.9
39.1
8.8
1.40
30
48
34.6
53.8
59.1
59.9
44.7
36.6
17.8
21.8
0.55
Immobilized CPO
whereas much less Cbz-glycine was obtained, as a result of the
practically total enzymatic activity loss after 8 h of reaction.
Concerning reaction rates, a value of around 1.4 mM/h was
obtained when using soluble enzyme, which is around 2.5-fold
higher than the value achieved with the immobilized CPO. The
main reason could be the high limitations by diffusion of the
substrate to reach the immobilized enzyme that is situated inside
the pores of the support. These limitations had been observed when
using the conversion of monocholorodimedone to dichlorodime-
done as activity test for activities over 5 U/mL support. Despite the
oxidation of Cbz-ethanolamine to Cbz-glycinal is much slower, the
limitations were also observed due to the high enzymatic load of
the support.
Although the reaction catalyzed by the immobilized CPO
resulted to be slower, the enzyme was active for a much longer
period of time than in the reaction with soluble enzyme. The immo-
bilized CPO withstood the continuous addition of peroxide at the
rate of 3 mM/h during 48 h with 53.8% of apparent CPO activity loss,
while the soluble enzyme lost 94.2% of its activity after only 8 h. At
the moment when no further consumption of Cbz-ethanolamine
was detected, conversions of 59.9 and 47.9% of Cbz-ethanolamine
were reached for immobilized and soluble enzyme, respectively.
This fact highlights one of the benefits of the immobilized enzymes,
which is the higher stability against addition of peroxide. This
advantage can be further exploited in future works, by coupling this
reaction with the reaction catalyzed by DHAP dependent aldolases,
in which once formed, amino aldehyde would be immediately con-
sumed as a substrate for the aldol addition of DHAP. This could
possibly result in accelerated reaction of Cbz-ethanolamine oxi-
dation and also could avoid or minimize the further oxidation of
Cbz-glycinal to Cbz-glycine. Another important benefit from the
immobilization itself and the improved stability of the immobi-
lized enzymes is the possibility of enzyme recovery and reuse, as
the residual activity at the end of one reaction was high enough to
be applied to another enzymatic reaction.
Acknowledgements
This work has been supported by the Spanish MICINN (project
CTQ2008-00578) and by Generalitat de Catalunya (research group
2009SGR281). The Department of Chemical Engineering of UAB
constitutes the Biochemical Engineering Unit of the Reference Net-
work in Biotechnology of the Generalitat de Catalunya (XRB).
References
[1] L. Cao, Curr. Opin. Chem. Biol. 9 (2005) 217–226.
[2] W. Tischer, F. Wedekind, in: W.D. Fessner (Ed.), Biocatalysis – From Discovery
to Application, Springer, Berlin, 1999, pp. 95–126.
[3] R. Fernandez-Lafuente, C.M. Rosell, V. Rodriguez, C. Santana, G. Soler, A. Bastida,
J.M. Guisán, Enzyme Microb. Technol. 15 (1993) 546–550.
[4] Z. Knezevic, N. Milosavic, D. Bezbradica, Z.P.R. Jakovljevic, Biochem. Eng. J. 30
(2006) 269–278.
[5] G. Fleminger, E. Hadas, T. Wolf, B. Solomon, Appl. Biochem. Biotechnol. 23
(1990) 123–137.
[6] F. van De Velde, F. van Rantwijk, R.A. Sheldon, TRENDS Biotechnol. 19 (2001)
73–80.
[7] M.P.J. van Deurzen, F. van Rantwijk, R.A. Sheldon, Tetrahedron 53 (1997)
13183–13220.
[8] M. Hofrichter, R. Ullrich, Appl. Microb. Technol. 71 (2006) 276–288.
[9] S.G. Burton, TRENDS Biotechnol. 21 (2003) 543–549.
[10] D.R. Morris, L.P. Hager, J. Biol. Chem. 241 (1966) 1763–1768.
[11] M. Ayala, C.V. Batista, R. Vazquez-Duhalt, J. Biol. Inorg. Chem. 16 (2011) 63–68.
[12] J.A. Thomas, D.R. Morris, L.P. Hager, J. Biol. Chem. 245 (1970) 3129–3134.
[13] S. Colonna, N. Gaggero, C. Richelmi, P. Pasta, TRENDS Biotechnol. 17 (1999)
163–168.
[14] E. Kiljunen, L.T. Kanerva, J. Mol. Catal. B: Enzym. 9 (2000) 163–172.
[15] J. Geigert, D.J. Dalietos, S.L. Neidleman, T.D. Lee, J. Wadsworth, Biochem. Bio-
phys. Res. Commun. 114 (1983) 1104–1108.
[16] P. Clapes, W.D. Fessner, G.A. Sprenger, A.K. Samland, Curr. Opin. Chem. Biol. 14
(2010) 154–167.
[17] A. Petri, T. Gambicorti, P. Salvadori, J. Mol. Catal. B: Enzym. 27 (2004) 103–106.
[18] C. Montiel, E. Terres, J.M. Dominguez, J. Aburto, J. Mol. Catal. B: Enzym. 48 (2007)
90–98.
[19] P. Toti, A. Petri, T. Gambicorti, A.M. Osman, C. Bauer, J. Mol. Catal. B: Enzym. 38
(2006) 65–72.
[20] S. Hudson, J. Cooney, B.K. Hodnett, E. Magner, Chem. Mater. 19 (2007)
2049–2055.
[21] Y.J. Han, J.T. Watson, G.D. Stucky, A. Butler, J. Mol. Catal. B: Enzym. 17 (2002)
1–8.
[22] D. Jung, C. Streb, M. Hartmann, Int. J. Mol. Sci. 11 (2010) 762–778.
[23] J. Aburto, M. Ayala, I. Bustos-Jaimes, C. Montiel, E. Terres, J.M. Dominguez, E.
Torres, Micropor. Mesopor. Mater. 83 (2005) 193–200.
[24] T.A. Kadima, M.A. Pickard, Appl. Environ. Microb. 56 (1990) 3473–3477.
[25] S. Aoun, C. Chebli, M. Baloulene, Enzyme Microb. Technol. 23 (1998) 380–385.
[26] M. Andersson, B.K. Samra, H. Holmberg, P. Adlercreutz, Biocatal. Biotransform.
17 (1999) 293–303.
4. Conclusions
Three possibilities were studied for CPO immobilization on an
aminated support, based on adsorption and covalent attachment.
While covalent attachment required oxidation of the enzyme or the
presence of coupling or reducing agents, adsorption was performed
in milder conditions, and higher retained activities were obtained.
However, covalently immobilized enzymes gained much more sta-
bility against tert-butyl hydroperoxide addition than adsorbed CPO,
and apparent half-life time was improved more than 500 fold when
using CPO immobilized on MANA-support with EDAC, compared to
the soluble enzyme. The immobilized preparations showed diffu-
sion limitations for the natural reaction of monochlorodimedone
chlorination, as well as for oxidation of Cbz-ethanolamine to Cbz-
glycinal. In spite of this, the covalently attached CPO using EDAC
was revealed as better catalyst than soluble CPO for the Cbz-glycinal
synthesis in terms of production yield and remained activity at the
end of the reaction.
[27] G. Bayramog˘lu, S. Kiralp, M. Yilmaz, L. Toppare, M.Y. Arıca, Biochem. Eng. J. 38
(2008) 180–188.
[28] L.H. Zhang, C.H. Bai, Y.S. Wang, Y.C. Jiang, M.C. Hu, S.N. Li, Q.G. Zhai, Biotechnol.
Lett. 31 (2009) 1269–1272.
[29] L.P. Hager, D.R. Morris, F.S. Brown, H. Eberwein, J. Biol. Chem. 241 (1966) 1769.
[30] J.M. Guisán, Enzyme Microb. Technol. 10 (1988) 375–382.
[31] S.F. D’Souza, Curr. Sci. 77 (1999) 69–79.
[32] M.A. Gilles, A.Q. Hudson, C.L. Borders, Anal. Biochem. 184 (1990) 244–248.
[33] J.R. Clamp, L. Hough, Biochem. J. 94 (1965) 17.
[34] C.F. Lane, Synthesis 3 (1975) 135–146.
[35] C. Horvath, J.M. Engasser, Biotechnol. Bioeng. 16 (1974) 909–923.
[36] J.M. Engasser, C. Horvath, J. Theor. Biol. 42 (1973) 137–155.
[37] I. Mielgo, C. Lopez, M.T. Moreira, G. Feijoo, J.M. Lema, Biotechnol. Progr. 19
(2003) 325–331.
[38] K.M. Manoj, L.P. Hager, Biochim. Biophys. Acta 1547 (2001) 408–417.
[39] W. Tischer, V. Kasche, TRENDS Biotechnol. 17 (1999) 326–335.