One-pot conversion of cephalosporin C to 7-aminocephalosporanic acid in the absence of hydrogen peroxide

Article abstract of DOI:10.1002/adsc.200505099

The main drawback in the production of 7-aminocephalosporanic acid (7-ACA) at the industrial level is the inactivation of the enzymes implicated in the process due to the presence of hydrogen peroxide during the reaction. As an alternative, we have developed the conversion of cephalosporin C to 7-ACA in a single reactor without the presence of hydrogen peroxide during the reaction, achieving more than 80% yield. In order to develop this process, D-amino acid oxidase (DAAO) was co-immobilized with catalase (CAT), which is able to fully eliminate in situ the hydrogen peroxide formed by the neighbouring DAAO molecules. Thus, the product of the reaction is only α-ketoadipyl-7-ACA. This system prevents the inactivation of the oxidase by hydrogen peroxide, solving the main problem of the enzymatic process. Moreover, we have found that α-ketoadipyl-7-ACA is recognized as a substrate by glutaryl acylase (GAC) and hydrolyzed as long as glutaric acid is absent from the reaction medium (because it is able to inhibit the hydrolysis). The low stability of α-ketoadipyl-7-ACA justifies the use of a single reactor, in which glutaryl acylase is already present when this substrate is generated. Thus, the whole process may (and must) be performed in a single step, and in the absence of hydrogen peroxide that could affect the stabilities of the involved enzymes.

Full text of DOI:10.1002/adsc.200505099

FULL PAPERS
DOI: 10.1002/adsc.200505099
One-Pot Conversion of Cephalosporin C to
7-Aminocephalosporanic Acid in the Absence of Hydrogen
Peroxide
Fernando Lopez-Gallego, Lorena Batencor, Aurelio Hidalgo, Cesar Mateo,
Roberto Fernandez-Lafuente,* Jose M. Guisan*
´
´
Departamento de Biocatalisis, Instituto de Catalisis, CSIC, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid,
Spain
Fax: (þ34)-91-585-4760, e-mail: rfl@icp.csic.es
Received February 28, 2005; Accepted: May 16, 2005; Published online: September 26, 2005
Abstract: The main drawback in the production of 7- main problem of the enzymatic process. Moreover, we
aminocephalosporanic acid (7-ACA) at the industrial have found that a-ketoadipyl-7-ACA is recognized as
level is the inactivation of the enzymes implicated in a substrate by glutaryl acylase (GAC) and hydrolyzed
the process due to the presence of hydrogen peroxide as long as glutaric acid is absent from the reaction me-
during the reaction. As an alternative, we have devel- dium (because it is able to inhibit the hydrolysis). The
oped the conversion of cephalosporin C to 7-ACA in low stability of a-ketoadipyl-7-ACA justifies the use
a single reactor without the presence of hydrogen per- of a single reactor, in which glutaryl acylase is already
oxide during the reaction, achieving more than 80% present when this substrate is generated. Thus, the
yield. In order to develop this process, d-amino acid whole process may (and must) be performed in a sin-
oxidase (DAAO) was co-immobilized with catalase gle step, and in the absence of hydrogen peroxide that
(CAT), which is able to fully eliminate in situ the hy- could affect the stabilities of the involved enzymes.
drogen peroxide formed by the neighbouring DAAO
molecules. Thus, the product of the reaction is only a- Keywords: d-amino acid oxidase; 7-aminocephalo-
ketoadipyl-7-ACA. This system prevents the inactiva- sporanic acid; catalase; co-immobilized enzymes; glu-
tion of the oxidase by hydrogen peroxide, solving the tarayl acylase; hydrogen peroxide
Introduction
boxy-5-oxopentanamido)-cephalosporanic acid (a-ke-
toadipyl-7-ACA) and hydrogen peroxide, this last prod-
7-Aminocephalosporanic acid (7-ACA) is a key inter- uct is able to carry out the decarboxylation of a-keto-
mediate for the production of many semi-synthetic adipyl-7-ACA producing glutaryl-7-aminocephalospor-
cephalosporin antibiotics. Chemical deacylation of anic acid (GL-7-ACA) spontaneously.[3a] Secondly the
cephalosporin C (CPC),[1] a fermentation product, is GL-7-ACA, formed in the previous step, is hydrolyzed
the primary method used to produce 7-ACA industrial- to 7-ACA and glutaric acid by an enzyme named glutar-
ly. This production method is economically feasible us- yl acylase (GAC) (Scheme 1).[4]
ing currently established processes; however, there are
The principal drawback of this process for the produc-
concerns with environmental and safety issues due to tion of 7-ACA is the presence of hydrogen peroxide dur-
the large quantities of hazardous chemicals used. To ing the reaction, because hydrogen peroxide can inacti-
overcome these problems, efforts are being made to de- vate the enzymes employed in the reaction, especially
velop an efficient, entirely enzymatic process for the DAAO.[5]
conversion of CPC to 7-ACA.
Many attempts have been carried out to avoid the in-
The direct conversion of CPC to 7-ACAin one-step by activation of DAAO and GAC by hydrogen peroxide
only one enzyme has been previously reported.[2] Never- through different immobilization strategies but with
theless, the conversion rate of this enzyme is too low for only moderate success.[6] The complete elimination of
an industrial application. Nowadays, the unique enzy- hydrogen peroxide from the reaction medium would
matic route industrially applied for the conversion of be the best solution for this problem. To achieve this
CPC to 7-ACA consists in a two-pot process.[3] Firstly, goal, we propose catalase (CAT) as a suitable alterna-
the CPC suffers an oxidative deamination catalyzed by tive. CATis an enzyme able to decompose the hydrogen
d-amino acid oxidase (DAAO), rendering 7-b-(5-car- peroxide into molecular oxygen and water.[7] The system
1804
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2005, 347, 1804 – 1810

One-Pot Conversion of Cephalosporin C to 7-Aminocephalosporanic Acid
FULL PAPERS
Figure 1. Inactivation of DAAO (top) and GAC (bottom) by
hydrogen peroxide. 4 IU DAAO immobilized on glyoxyl
agarose and 12 IU/mL PEI-GAC-glut derivative were sepa-
rately incubated with and without 10 mM hydrogen peroxide
Scheme 1. Different routes for the conversion of CPC to 7-
ACA.
*
at pH 7 and 258C. : enzyme without hydrogen peroxide and
&
: enzyme with hydrogen peroxide.
DAAO/CAT has been used to produce phenylpyruvic tor, DAAO immobilized on glyoxyl agarose carried out
acid, where co-immobilization of both enzymes was nec- the conversion of CPC to GL-7-ACA under very mild
essary to obtain only the desired product.[8] This exam- reaction conditions (pH 7.5 and 258C). This reaction
ple also illustrated that in this way, hydrogen peroxide was completed in 200 min with less than 1% CPC re-
could not inactivate the enzymes present in the reaction maining at this point. Table 1 shows that the GL-7-
system.
ACA yield was 81%. Moreover, approximately 10%
In this work, we have designed a one-pot process for a-ketoadipyl-7-ACA was formed during the reaction.
converting CPC to 7-ACA via a trienzymatic route This 10% a-ketoadipyl-7-ACA means that not all the hy-
(DAAO, CATand GAC) where the oxidative deamina- drogen peroxide was used to oxidatively decarboxylate
tion is carried out by DAAO yielding a-ketoadipyl-7- a-ketoadipyl-7-ACA to GL-7-ACA (Scheme 1A), and
ACA and hydrogen peroxide as products, the hydrogen therefore, it can inactivate DAAO or any other enzyme
peroxide is immediately decomposed by CAT co-immo- present in the reactor. Figure 1 shows the susceptibility
bilized near DAAO, and the a-ketoadipyl-7-ACA can of DAAO (top) and GAC (bottom) to hydrogen perox-
then be hydrolyzed to 7-ACA (final product) by GAC ide, mainly DAAO which, in presence of 10 mM hydro-
(Scheme 1). Therefore, this process could be carried gen peroxide, only retained 20% of its initial activity after
out in one reactor (one-pot) because hydrogen peroxide 24 h of incubation in presence of hydrogen peroxide.
is not present in the reaction medium.
After that, the reaction products were offered to GAC
in a second pot. This hydrolysis reaction was carried out
by PEI-GAC-glut derivative, again under very mild con-
ditions (pH 8 and 258C). Table 1 shows that the 2nd reac-
tion yielded 78% 7-ACA with only 3% of GL-7-ACA
not hydrolyzed in 30 minutes, (very likelydue tothermo-
dynamic reasons). Most important was that all the initial
a-ketoadipyl-7-ACA formed in the first reaction was
not hydrolyzed by GAC.
Results and Discussion
Conversion of CPC to 7-ACA in a Two Reactor
Process with a Bienzymatic System
As described in Scheme 1A the classical reaction has to
This is the reason why a-ketoadipyl-7-ACA was not
be performed in two different reactors. In the first reac- considered to be a substrate of GAC, in many instances
Adv. Synth. Catal. 2005, 347, 1804 – 1810
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1805

Fernando Lopez-Gallego et al.
FULL PAPERS
Table 1. Results from the two-pot reaction with a bienzymatic system, single cycle.
Time [min]
Reactors
Conversion [%]
Yield [%]
CPC
0
–
Glutaryl-7-ACA
a-ketoadipyl-7-ACA
10.3ꢀ0.4
7-ACA
–
78.2ꢀ4.5
Side products
4.5ꢀ0.2
200
30
Reactor 1
Reactor 2
81ꢀ4
2.8ꢀ0.1
9.5ꢀ0.5
3.6ꢀ0.2
Table 2. Effect of the DAAO:CATb ratio on the production
of GL-7-ACA.
Ratio [mg DAAO:mg CAT]
% Glutaryl-7-ACA
1: 0.1
1: 1
1: 10
20ꢀ1
0.60ꢀ0.03
0
GL-7-ACA was not produced, which indicates that hy-
drogen peroxide was not present in the reaction me-
dium. With lower ratios than 1:10 (DAAO: CATb) in
the co-immobilized derivative, the full elimination of
hydrogen peroxide was not achieved because GL-7-
ACA appeared in the reaction medium.
Figure 2. Effect of glutaric acid on the a-ketoadipyl-7-ACA
amidase and GL-7-ACA amidase activities. ^: GL-7-ACA
amidase activity and : a-ketoadipyl-7-ACA amidase activity.
&
Further details are described in the Experimental Section.
Therefore, the optimal co-immobilized DAAO/CATb
derivative was achieved when the amount by weight of
the first products need to be treated with the addition of CATb was 10-fold higher than the amount by weight
hydrogen peroxide in a third pot to have 100% glutaryl- of DAAO.
7-ACA.[5a,9] This solution would be very positive in terms
of 7-ACA yield; however the process would be very
more complex and expensive due to the use of an addi- Kinetic Parameters of PEI-GAC-glut with Different
tional reactor.
Substrates
However, we found that this is due to the accumula-
tion of glutaric acid formed by the more favourable hy- In order to develop a process without hydrogen perox-
drolysis of GL-7-ACA. Figure 2 shows that the glutaric ide, the oxidative deamination of CPC catalyzed by
acid induced a higher inhibition in the a-ketoadipyl-7- the co-immobilized DAAO/CATb derivative must pro-
ACA activity than in the GL-7-ACA activity (Figure 2). duce only a-ketoadipyl-7-ACA. In this way, the kinetic
Therefore, the main drawback of this process is the parameters of the GAC derivative (PEI-GAC-glut)
presence of hydrogen peroxide during the reaction, with a-ketoadipyl-7-ACA as substrate were analyzed
which may inactivate the enzymes employed in the sys- (Table 3).
tem.
The GAC derivative carried out the hydrolysis of GL-
7-ACA in a more favourable way than the hydrolysis of
a-ketoadipyl-7-ACA, because the Km for GL-7-ACA
Co-Immobilization of DAAO and CATb to Carry Out was 4-fold lower than Km for a-ketoadipyl-7-ACA.
the Oxidative Deamination of CPC and the
Decomposition of Hydrogen Peroxide in situ
However, the hydrolysis of a-ketoadipyl-7-ACA was
possible, although the hydrolysis rate for this substrate
was lower than for GL-7-ACA.
Thus, the full elimination of hydrogen peroxide was nec-
essary for a second reason: to increase enzyme stability
and to prevent inhibition of the a-ketoadipyl-7-ACA ac- Conversion of CPC to 7-ACA in Two Reactors via a
tivity. In order to eliminate the hydrogen peroxide dur- Trienzymatic System
ing the reaction, the co-immobilization of DAAO and
CATb was suggested as a solution. GL-7-ACA was The conversion of CPC to 7-ACAwithout hydrogen per-
used as an indicator of the hydrogen peroxide forma- oxide was performed using the co-immobilized DAAO/
tion, in order to evaluate different ratios of co-immobi- CATb derivative and, later on, the a-ketoadipyl-7-ACA
lized DAAO and CATb. Table 2 shows that with a 1:10 hydrolysis using PEI-GAC-glut. When the conversion
(DAAO:CATb) ratio in the co-immobilized derivative, of CPC to 7-ACA was carried out in two reactors, the
1806
asc.wiley-vch.de
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2005, 347, 1804 – 1810

One-Pot Conversion of Cephalosporin C to 7-Aminocephalosporanic Acid
FULL PAPERS
Table 3. Kinetics parameters of GAC with GL-7-ACA and a-ketoadipyl-7-ACA.
Kinetic parameters
Glutaryl amidase activity
a-ketoadipyl amidase activity
Km [mM]
1.06ꢀ0.05
87.8ꢀ0.7
82.8ꢀ0.4
4.9ꢀ0.3
2.8ꢀ0.5
0.56ꢀ0.02
Kcat [minꢁ1
]
Kcat/Km [minꢁ1 ꢂmMꢁ1
]
Table 4. Results from the two-pot reaction with a trienzymatic system, single cycle.
Time [min]
Reactors
Conversion [%]
Yield [%]
CPC
Glutaryl-7-ACA
a-ketoadipyl-7-ACA
7-ACA
Side products
200
133
Reactor 1
Reactor 2
0
–
0
0
54.3ꢀ1.1
–
13.3ꢀ0.4
0.20ꢀ0.01
48ꢀ2
9.19ꢀ0.46
Figure 3. Decomposition of a-ketoadipyl-7-ACA in the reac-
tion conditions. 4 mL of a-ketoadipyl-7-ACA were incubated
at pH 7 and 258C. The reduction of the peak area corre-
sponding to a-ketoadipyl-7-ACA was monitored by HPLC
as described in the Experimental Section.
Figure 4. Reaction course of the conversion of cephalosporin
C into 7-aminocephalosporanic acid using the trienzymatic,
one-pot process. The reaction was carried out as described
&
*
in the Experimental Section; : 7-ACA. : CPC. ~: a-keto-
*
adipyl-7-ACA; : side products and ^: GL-7-ACA.
a-ketoadipyl-7-ACAyield was not high, although GL-7- In this system, CPC is converted to a-ketoadypyl-7-
ACAwas not observed (Table 4). This makes that the fi- ACA and hydrogen peroxide by DAAO, and the hydro-
nal 7-ACAyield was much lower (47%) than in the con- gen peroxide is decomposed in situ by CATb. Moreover,
ventional process. This fact could be explained because a-ketoadipyl-7-ACA is in situ hydrolyzed to 7-ACA by
the a-ketoadipyl-7-ACAwas quite unstable and rapidly the GAC derivative, avoiding its accumulation (Scheme
decomposed into unknown products. Figure 3 shows 1B).
that 80% of the initial a-ketoadipyl-7-ACA decom-
The reaction course of the one-pot system (Figure 4)
posed after 18 h under the reaction conditions (pH 7.5 shows that 80% of 7-ACA was achieved in180 minutes.
and 258C). In this way, this process did not look industri- Moreover, only 2.5% of a-ketoadypyl-7-ACA was not
ally interesting.
hydrolyzed.
Direct Conversion of CPC to 7-ACA in only One
Reactor (One-Pot Process) via a Trienzymatic System
Conclusion
The inactivation of the enzymes, which carry out the
A possible solution to the previous problems is to avoid conversion CPC to 7-ACA (primarily DAAO) by hy-
the accumulation of a-ketoadipyl-7-ACA by hydrolyz- drogen peroxide is the main disadvantage of the conven-
ing it quickly after it is produced. The development of tional process used industrially for the production of the
this system involves only one reactor with two different antibiotic core 7-ACA. In this work, we have developed
enzymatic derivatives (DAAO and CATb co-immobi- a one-pot system that produces 7-ACA from CPC with
lized on glyoxyl agarose and PEI-GAC-glut derivative). three immobilized enzymes (DAAO/CATb and
Adv. Synth. Catal. 2005, 347, 1804 – 1810
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1807

Fernando Lopez-Gallego et al.
FULL PAPERS
GAC). The process was performed without hydrogen Methods
peroxide in the reaction medium, due to its in situ de-
All experiments were performed at least in triplicate and the
results are presented as the mean value. Experimental error
was never over 5%.
composition by CATb co-immobilized with DAAO.
This fact is the great advantage of this process in compar-
ison with the conventional two-pot system.
Moreover, another problem in the two-step system
was the accumulation of a-ketoadipyl-7-ACA during
the 1st reaction. This product could not be hydrolyzed
in the 2nd reaction because of the presence of glutaric
acid, a potent inhibitor of the hydrolysis of a-ketoadip-
yl-7-ACA, formed by the hydrolysis of GL-7-ACA
(main product of the first reaction). This problem would
not occur in the one-pot system, since GL-7-ACA was
not produced due to the absence of hydrogen peroxide
which could carry out the decarboxylation of a-keto-
adipyl to GL-7-ACA.
Preparation of Glutaryl-7-ACA Acylase
The commercial preparation of GAwas diluted (1/5) in 25 mM
potassium phosphate buffer solution pH 7.0 and then dialyzed
three times against 100 volumes of 5 mM potassium phosphate
buffer, pH 7. The dialyzed enzyme was then centrifuged
(12000 rpm for 30 minutes at 48C) and the supernatant (con-
taining 16 IU/mL) was used as the enzymatic preparation for
further experiments. More than 90% of initial activity was re-
covered after this process.
Hence, the direct conversion of CPC to 7-ACA via a
trienzymatic system in only one reactor, presented two
important advantages with respect to the conventional
two-pot enzymatic route. However, this one-pot system
may be improved if GAC hydrolysis rate with a-keto-
Determination of Catalase Activity
Catalase activity was determined spectrophotometrically by
monitoring the decomposition of H2O2, through the measure-
adipyl-7-ACA is improved. The solution to this problem ment of the change in the absorbance at 240 nm.[13] 2.9 mL of
H2O2, 0.14% (w/v) in 50 mM sodium phosphate buffer
pH 7.0 were incubated with 0.2 mL of enzyme solution. All
the measurements were carried out at 258C.
One catalase unit was defined as the amount of enzyme that
decomposes 1 micromol of H2O2 under the previously de-
scribed conditions.
is the search for an enzyme with better properties to-
wards the hydrolysis of a-ketoadipyl-7-ACA than the
GAC used in this work. This aim could be achieved
with the new techniques of molecular biology as direct-
ed evolution. In this direction, new mutants of GAC
have been made for carrying out the adipyl-7ADCA hy-
drolysis (its side chain is very similar to that of a-keto-
adipyl-7-ACA) in a very successful way.[10]
d-Amino Acid Oxidase Activity Assay
DAAO activity in its soluble form was analyzed spectrophoto-
metrically using cephalosporin C as substrate measuring the in-
crement of absorbance at 445 nm promoted by coupling the ox-
idative deamination of the substrate with the reaction between
the hydrogen peroxide and o-phenylenediamine catalyzed by
peroxidase.[13] The reaction mixture consisted of 1.5 mL of
25 mg/mL cephalosporin C solution in 100 mM potassium
phosphate at pH 7.5, 0.5 mL of 1.85 mM o-phenylenediamine
in distilled water and 0.1 mL of a 1 mg/mL peroxidase solution
in 50 mM potassium phosphate at pH 7.5. The reagents were
preincubated at 258C. The reaction was initiated by adding a
maximum of 0.1 units of DAAO.
Experimental Section
Materials
Cephalosporin C (CPC), and glutaryl-7-aminocephalosporan-
ic acid (GL-7-ACA), were kindly donated by Bioferma Murcia
S. A. Glutaryl acylase from Pseudomonas sp. (GAC) was ob-
tained from Roche (Basel, Switzerland) and d-amino acid ox-
idase from Trigonopsis variabilis (DAAO) was obtained from
Recordati (Milan, Italy). Catalase from bovine liver (CATb)
was purchased from Fluka (Buch, Switzerland). Glyoxyl agar-
ose 4-BCL (20 mmoles of glyoxyl groups/g support) was pre-
pared according to previously described methods.[11] Polyethy-
leneimine 600 KDa-agarose 4BCL support was prepared ac-
cording to previously described methods.[12] Glutaric acid was
purchased from Sigma-Aldrich (St. Louis, MO, USA). Kroma-
sil C-8, (5 mm, 250ꢂ4.6 mm) column was purchased from Ana-
lisis vínicos (Tomelloso, Spain). Ammonium acetate (HPLC
grade) was purchased from Panreac (Barcelona, Spain), aceto-
nitrile was purchased from Scharlau (Barcelona, Spain). All
other reagents were of analytical grade.
One DAAO unit is defined as the amount of enzyme able to
oxidize one micromole of cephalosporin C per minute in the
previously described conditions.
Glutaryl Acylase Activity Determination
The activity of both immobilized and soluble enzyme was
measured as follows: the initial reaction rates were measured
with a pH-STAT using an automatic titrator (Crison micro
TT 2050) to determine the amount of glutaric acid formed.
The assays were carried out by adding 0.1 mL of soluble en-
zyme or suspension of immobilized enzyme to 10 mL of a
10 mM solution of GL-7-ACA 0.1 M potassium phosphate buf-
fer pH 7.5, and titrating the reaction mixture with 25 mM
NaOH at 258C.
1808
asc.wiley-vch.de
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2005, 347, 1804 – 1810

One-Pot Conversion of Cephalosporin C to 7-Aminocephalosporanic Acid
FULL PAPERS
One unit of glutaryl 7-ACA acylase activity was defined as
the amount of enzyme that is necessary to produce 1 micromol
of glutaric acid per minute in the previously described condi-
tions.
with 25 mM potassium phosphate buffer at pH 7, resuspended
in the same buffer and further incubated at 258C for 20 h to
have an intense support-enzyme cross-linking.[14] The final de-
rivative, termed PEI-GAC-glut, was stored at 48C until further
use.
Product Analysis by HPLC
Kinetic Constants of GAC for GL-7-ACA and a-
Ketoadipyl-7-ACA
The products of the reactions were determined through HPLC
with a Kromasil C8 (5 mm, 250ꢂ4.6 mm) column, mobile
phase 20 mM ammonium acetate pH 5.2; acetonitrile (95:5
v/v). The retention times of the different products were: 4.1 mi-
nutes for CPC, 4.9 minutes for 7-ACA, 6.7 minutes for a-keto-
adipyl-7-ACA and 12.3 minutes for GL-7-ACA. Other minor
peaks were considered as side products.
Km, Kcat and Km/Kcat were determined for the GL-7-ACA
and a-ketoadipyl-7-ACA activities of the PEI-GAC-glut de-
rivative. The GL-7-ACA activity was determined with an auto-
matic titrator as previously described, with different concen-
trations of GL-7-ACA (0.5–5 mM). The a-ketoadipyl-7-
ACA activity was determined by incubating 0.1 g of PEI-
GAC glut with 1.5 mL of a-ketoadipy-7-ACA solution at dif-
ferent concentrations and 258C (1.75–13.8 mM) prepared in
0.1 M potassium phosphate buffer at pH 7. Supernatant sam-
ples were withdrawn at different times and analyzed by
HPLC as described above. The Michaelis constant was deter-
mined for each substrate from Lineweaver–Burk plots.
Immobilization of DAAO on Glyoxyl Agarose
10 g of glyoxyl agarose 4BCL (20 mmoles of glyoxyl groups/g
support) were added to 20 mL of a 1 mg/mL DAAO solution
in 25 mM sodium carbonate at pH 10.00. The suspension was
gently stirred at 48C. Periodically, samples of the supernatants
were withdrawn and analyzed for enzyme activity and protein
concentration. After 15 hours of gently stirring at 48C, 1 mg of
solid NaBH4 was added per mL of suspension and 30 min later
the reduced derivative was washed with 25 mM potassium
phosphate buffer at pH 7.0. Finally, the derivative was stored
at 48C until further use.
Inhibition of the GL-7-ACA and a-Ketoadipyl
Activities of the PEI-GAC-glut Derivative by Glutaric
Acid
The inhibition by glutaric acid on the GL-7-ACA activity was
determined measuring the hydrolysis of GL-7-ACA with an
automatic titrator as previously described in the presence of
different glutaric acid concentrations (0–50 mM).
The inhibition of glutaric acid on the a-ketoadipyl-7-ACA
activity was determined by measuring the formation of 7-
ACA via HPLC as described above, in a suspension of 0.1 g
of derivative with 1.6 mL of 13.8 mM a-ketoadipyl-7-ACA
and glutaric acid at different concentrations (0–40 mM) at
pH 7.5 and 258C.
Co-Immobilization of DAAO and CATb on Glyoxyl
Agarose
100 mL of a CATb solution at different concentrations (1 mg/
mL, 0.1 mg/mL or 0.01 mg/mL) were added to 10 g of unre-
duced, immobilized DAAO (2 mg DAAO/g support) on glyox-
yl agarose to permit the co-immobilization of CATb. The sus-
pension was gently stirred at 258C. Periodically, samples of
the supernatants were withdrawn and analyzed for enzyme ac-
tivity and protein concentration. After 3 h of gentle shaking at
258C, 1 mg of solid NaBH4 was added per mL of suspension
and 30 min later the reduced derivative was washed with
25 mM potassium phosphate buffer at pH 7.0. The derivative
was stored at 48C until further use.
Conversion of CPC to 7-ACA in Two Reactors (Two-
Pot Process)
1st Reactor. Conversion of CPC to GL-7-ACA or a-Ketoadipyl-
7-ACA: 0.2 g of immobilized DAAO or co-immobilized
DAAO-CATb on glyoxyl agarose were added to 4 mL of
40 mM CPC in potassium phosphate buffer at pH 7.5 and
258C to convert the CPC to GL-7-ACA or a-ketoadipyl-7-
ACA, respectively. Samples were withdrawn from the superna-
tant at different times and analyzed by HPLC as described
above. When all CPC was completely converted, the suspen-
sion was filtered and the supernatant was recovered for further
experiments
Covalent Immobilization of GAC Adsorbed on PEI
through Glutaraldehyde Treatment of the Adsorbed
Protein
10 g of PEI-agarose were suspended in 60 mL of GAC solution
prepared as previously described. The suspension was gently
shaken at 258C. Periodically, samples of the supernatants
were withdrawn and analyzed for enzyme activity and protein
concentration. When the immobilization was completed, the
suspension was filtered and suspended in 40 mL of a 0.5% glu-
taraldehyde solution in 25 mM potassium phosphate buffer at
pH 7. The suspension was left under mild stirring at 258C dur-
ing 1 h. This step permits the activation of primary amino
groups of the support as well as the enzyme with one molecule
of glutaraldehyde.[12] Then the suspension was filtered, washed
2nd Reactor. Conversion of GL-7-ACA or a-Ketoadipyl-7-
ACA to 7-ACA: 4 mL of GL-7-ACA or a-ketoadipyl-7-ACA
obtained as described above were added to 1.5 g of PEI-
GAC-glut derivative (60 mg GAC/g support) and the suspen-
sion pH was increased to 8.0. The suspension was gently shaken
and the pH was maintained at 8.0 by continuous addition of 4M
NaOH. Periodically, samples were withdrawn and analyzed via
HPLC as described above.
Adv. Synth. Catal. 2005, 347, 1804 – 1810
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1809

Fernando Lopez-Gallego et al.
FULL PAPERS
Baker, Y. Q. Shen, B. L. Wong, R. Noiles, C. W. Rausch,
Biotech. Bioeng.1995, 46, 510–513; c) A. Parmar, H. Ku-
mar, S. S. Marwaha, J. F. Kennedy, Crit. Rev. Biotech.
1998, 18, 1–12.
Direct Conversion of CPC to 7-ACA in One Reactor
(One-Pot Process)
0.2 g of DAO-CATb (2 mg DAAO:20 mg CATb/g support) co-
immobilized on glyoxyl agarose plus 1.5 g of PEI-GAC-glut
derivative (60 mg GAC/g support) were added to 4 mL of
40 mM CPC in potassium phosphate buffer at pH 8 and
258C. The suspension was gently stirred and the pH was main-
tained at 8.0 by adding 4 M NaOH during all process. Periodi-
cally, samples were withdrawn and analyzed via HPLC as de-
scribed above.
[4] a) Y. Shibuya, K. Matsumoto, T. Fuzii, Agric. Biol.
Chem. 1981, 45, 1561–1567; b) K. Tsuzuki, K. Komatsu,
S. Ichikawa, Y. Shibuya, Nippon Nogeikogaku Kaishi
1989, 63, 1847–1853.
[5] a) E. S. Dey, S. Flygare, K. Mosbach, Appl. Biochem. Bi-
´
otechnol. 1991, 27, 239–250; b) I. De la Mata, F. Ramon,
´
´
V. Obregon, M. P. Castillon, C. Acebal, Enzyme Microb.
Technol. 2000, 27, 234–239.
´
[6] R. Fernandez-Lafuente, V. Rodríguez, C. Mateo, G. Fer-
Acknowledgements
´
nandez-Lorente, P. Arminsen, P. Sabuquillo, J. M. Gui-
´
san, J. Mol. Catal. B: Enzym. 1999, 7, 173–179.
This project has been funded bythe Spanish CICYT (PPQ 2002-
01231 A). We gratefully recognizea post-doctoral fellowship for
Dr Aurelio Hidalgo (Gobierno Vasco) and a pre-doctoral fel-
lowship for Fernando Lopez Gallego from Spanish MCYT.
[7] M. Zamocky, F. Koller, Prog. Biophys. Mol. Biol. 1999,
72, 19–66.
[8] R. Fernandez-Lafuente, V. Rodriguez, J. M. Guisan, En-
zyme Microb. Technol. 1998, 23, 28–33.
´
´
´
The help and suggestions by Angel Berenguer. MSc are grateful-
ly recognized.
[9] S. Cambiaghi, S. Tomaselli, R. Verga, European Patent
0,496,993, 1991.
[10] a) C. F. Sio, A. M. Riemens, J.-M. van der Laan, R. M. D.
Verhaert, W. J. Quax, Eur. J. Biochem. 2002, 269, 4495–
4504; b) L. G. Otten, C. F. Sio, J. Vrielink, R. H. Cool,
J. W. Quax, J. Biol. Chem. 2002, 277, 42121–42127.
References
[1] a) B. Fechtig, H. Peter, H. Bickel, E. Vischer, Helv.
Chim. Acta 1968, 51, 1109–1120; b) R. B. Morin, B. G.
Jackson, E. H. Flynn, R. W. Roeske, J. Am. Chem. Soc.
1969, 91, 1396–1400.
[2] a) A. Matsuda, K. Matsuyama, K. Yamamoto, S. Idhika-
wa, K. H. Komatsu, J. Bacteriol. 1987, 169, 5815–5820;
b) F. Reyes, M. J. Martinez, C. Alfonso, J. L. Copa-Pati-
no, J. Soliveri, J. Pharm. Pharmacol. 1990, 42, 128–131.
[3] a) A. Nikolov, B. Danielsson, Enzyme Microb Tech-
nol.1994, 16, 1037–1041; b) H. D. Conlon, J. Baqai, K.
´
[11] J. M. Guisan, Enzyme Microb. Tech. 1988. 10, 375–382.
[12] C. Mateo, O. Abian, R. Fernandez-Lafuente, J. M. Gui-
san, Biotechnol. Bioeng. 2000, 68, 98–105.
[13] H. Aebi, in: Methods of Enzymatic Analysis, 3rd edn.,
(Ed.: H. U. Bergmeyer), Verlag Chemie, Weinheim,
1974, pp. 672–684.
´
[14] F. Lopez-Gallego, L. Betancor, C. Mateo, A. Hidalgo, N.
´
Alonso-Morales, G. Dellamora-Ortiz, J. M. Guisan, R.
´
Fernandez-Lafuente, J. Biotechnol. submitted.
1810
asc.wiley-vch.de
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2005, 347, 1804 – 1810