An efficient enzymatic synthesis of 5-aminovaleric acid

Article abstract of DOI:10.1016/j.molcatb.2009.12.006

The title compound was prepared enzymatically from l-lysine in an excellent yield and under buffer-free conditions. l-Lysine was oxidized by the action of l-lysine α-oxidase from Trichoderma viride followed by spontaneous oxidative decarboxylation of the intermediate 6-amino-2-oxocaproic acid in the reaction medium. l-Lysine α-oxidase was immobilized on an epoxy-activated solid support (Sepabeads EC-EP) and the activity of both solution-based and immobilized enzyme in this reaction was determined.

Full text of DOI:10.1016/j.molcatb.2009.12.006

Journal of Molecular Catalysis B: Enzymatic 65 (2010) 58–62
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
An efficient enzymatic synthesis of 5-aminovaleric acid
a
b,d
c
Aliaksei V. Pukin , Carmen G. Boeriu , Elinor L. Scott ,
c
a,∗
Johan P.M. Sanders , Maurice C.R. Franssen
a
Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB, Wageningen, The Netherlands
Agrotechnology and Food Sciences Group, Wageningen UR, Wageningen, The Netherlands
Valorisation of Plant Production Chains, Wageningen University, Wageningen, The Netherlands
University “A. Vlaicu”, Arad, Romania
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 14 December 2009
The title compound was prepared enzymatically from l-lysine in an excellent yield and under buffer-free
conditions. l-Lysine was oxidized by the action of l-lysine ␣-oxidase from Trichoderma viride followed by
spontaneous oxidative decarboxylation of the intermediate 6-amino-2-oxocaproic acid in the reaction
medium. l-Lysine ␣-oxidase was immobilized on an epoxy-activated solid support (Sepabeads EC-EP)
and the activity of both solution-based and immobilized enzyme in this reaction was determined.
Keywords:
l-Lysine ␣-oxidase
Immobilization
©
2009 Elsevier B.V. All rights reserved.
5
-Aminovaleric acid
NMR
Biocatalysis
1
. Introduction
application in industrial synthesis of chemicals [6]. In the develop-
ment of such environmentally benign production processes, lysine
attracts attention as a suitable precursor in view of the availability
of this amino acid from fermentation [7] or from lysine enriched
plants. Thus, the accumulation of lysine in potato has been well
investigated and described [8]. In the starting process of lysine
transformations, l-lysine ␣-oxidase can be used.
Polymeric substances are essential in modern society. With their
applications in textiles, coatings, adhesives and plastics as well as
their use in biomedical applications, synthetic polymers play an
important role in our daily life. The building blocks for polymer
production are highly dependent on fossil resources. Thus, hydro-
carbon monomers like ethylene, propylene, and styrene are yielded
from the refinement of crude oil, while nitrogen-containing build-
ing blocks like e.g. acrylamide, acrylonitrile and caprolactam are
obtained by using ammonia (derivatives) at high temperatures and
pressures in reactions with naphtha derived hydrocarbons, which
is extremely energy-demanding [1,2]. Allied with this and other
environmental concerns, there is a need to search for alternative
sources of nitrogen-containing bulk chemicals. Plants are particu-
larly attractive in this respect, as many products formed in plants
l-Lysine ␣-oxidase (LysOx), a flavin-containing enzyme from
the wheat fungus Trichoderma viride, catalyses the oxidation of
the ␣-carbon atom of lysine (1, Scheme 1) [9]. For the oxidation
of one mole of 1, one mole of molecular oxygen is required. The
formal product of this reaction is 6-amino-2-ketocaproic acid (2)
[9], which is formed along with equimolar amounts of ammo-
nia and hydrogen peroxide. If the reaction is conducted in the
presence of catalase to remove H O , the final product repre-
2
2
1
sents ꢀ -piperideine-2-carboxylate (3, Scheme 1A) as a result of
the spontaneous intramolecular cyclization of the intermediate. In
the absence of catalase, the action of the enzymatically generated
H O on the intermediate leads to the oxidative decarboxylation
(
e.g. amino acids) already contain nitrogen.
In the course of our investigations of the use of plant mate-
rials as a feedstock for the bulk chemical industry [3], we have
been involved in the application of enzymes for the conversion of
amino acids into nitrogen-containing chemicals [4,5]. Enzymatic
processes are environmentally friendly, and due to their intrin-
sic and selective catalytic properties, enzymes have found wide
2
2
of the latter to form 5-aminovaleric acid (4) as the final product
(Scheme 1B).
Due to its high selectivity towards the oxidation of l-lysine,
LysOx has attracted a great deal of attention, mainly as an
instrument for the determination of l-lysine in various biological
materials. A number of tools based on immobilized LysOx have
been designed for this purpose [10–18], and the physicochemi-
cal and biological properties of this enzyme have been reviewed
∗ Corresponding author. Tel.: +31 317 482976; fax: +31 317 484914.
E-mail addresses: aliaksei.pukin@wur.nl (A.V. Pukin), carmen.boeriu@wur.nl
[
19]. However, the reported applications of LysOx in synthesis are
(
C.G. Boeriu), elinor.scott@wur.nl (E.L. Scott), johan.sanders@wur.nl (J.P.M. Sanders),
maurice.franssen@wur.nl (M.C.R. Franssen).
scarce and limited to its use as a part of the enzymatic systems for
1
381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.12.006

A.V. Pukin et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 58–62
59
Scheme 1. Oxidation of lysine by l-lysine ␣-oxidase in the presence (A) and absence (B) of catalase.
The use of 1H NMR to determine the relative amounts of the
starting lysine and the product in the reaction mixture is partic-
ularly attractive, given the simplicity of the 1H NMR spectra of
both compounds and only moderate overlapping of the multiplets
belonging to 1 and 4, especially at the lower pH values.
the synthesis of l-pipecolic acid [20,21]. To the best of our knowl-
edge, there is no report describing the isolation of either of the
compounds 3 or 4 from the reaction mixtures followed by proper
characterisation of these products, and to date, not much attention
has been paid to the reaction in the absence of catalase (Scheme 1B).
In our view, however, application of LysOx to the production of
Moreover, contrary to the HPLC-based quantification of amino
acids, neither additional functionalization, nor laborious sample
preparation is needed prior to the measurement. In addition, the
amounts of starting material used in preparative reactions do allow
5
-aminovaleric acid (4) according to this scheme could be bene-
ficial over the methods currently employed for the synthesis of 4
22–28], as an environmentally friendly route that uses a biolog-
[
1
ically derived starting compound. 5-Aminovaleric acid may serve
inter alia as the precursor of valerolactam, a potential building block
for the production of nylon 5.
for H NMR analysis of the reaction mixtures. Unfortunately, the
necessity of oxygen for the enzyme functioning precludes from con-
ducting the experiment outright in an NMR tube, so samples taken
at the fixed time points of the reaction should be analyzed offline.
It is important to stop the reaction immediately after sampling,
in order to prevent any change in the composition of the reaction
components prior (during) the NMR measurement. To this end, a
Thus, herein we describe the use of l-lysine ␣-oxidase (LysOx)
on a preparative scale for the oxidation of lysine in the absence
of catalase to produce 5-aminovaleric acid. An assay for LysOx for
this reaction was developed and used in optimizing the reaction
conditions. In addition, LysOx was immobilized onto an epoxy-
activated support Sepabeads EC-EP and the performance of thus
obtained enzyme preparation was compared to that of the solu-
ble enzyme. Finally, we report on an efficient enzymatic synthesis
of 5-aminovaleric acid with the use of the immobilized LysOx on
a preparative scale and under buffer-free conditions, which allow
for simplified product isolation.
solution of CF COOH can be used.
3
Conducting the reaction in D O allows avoiding the suppres-
2
sion of water 1H signal that dominates NMR spectra of samples in
H O and thus ensures more precise internal normalization. In this
2
case, however, the formation of 5-amino-2,2-dideuterovaleric acid
should be taken into account. In the spectrum of this compound, the
multiplet at 2.35–2.25 ppm does not appear, and the integration of
the signals in this region should not be included in the calculation
of degree of conversion.
2
. Results and discussion
Based on these considerations, we performed the assay by deter-
mining relative concentrations of lysine and 5-aminovaleric acid
2.1. Development of an NMR-based assay for l-lysine ˛-oxidase
1
from H NMR spectra of the samples taken at given time points from
the reaction mixtures containing initially 60 mM of lysine (Fig. 1).
The taken samples were immediately added to a 4% (v/v) solu-
tion of CF COOH in D O. The calculated percentages were plotted
against time, and the activity of the LysOx was determined as a
function of the slope of the resulting graph (Fig. 2).
Focused on the route leading to the formation of 5-aminovaleric
acid, we had to develop an assay for LysOx in this specific reac-
tion, in view of inapplicability of the described assays hereto. Thus,
in the reported procedures the enzyme is assayed by the reaction
of the formal ␣-keto acid product (2, Scheme 1) with 3-methyl-
-benzothiazolone hydrazone hydrochloride or by determining
-piperideine-2-carboxylate with o-aminobenzaldehyde [9], as
3
2
Fig. 2 clearly shows, that the use of D O instead of H O in the
2
2
2
ꢀ
reaction has no effect on the enzyme activity.
1
well as by reacting the oxidized product generated from lysine with
semicarbazone [29]. However, in the absence of catalase the prod-
uct of initial oxidation of the ␣-aminogroup of lysine does not lead
to the compounds determined in conventional assay procedures.
Therefore, for the quantification of product formation, a method
that differs principally from those used in traditional endpoint and
continuous LysOx assays has to be applied.
2.2. Optimization of the reaction conditions: effect of the nature
of the buffer and temperature
In order to establish general principles for the reaction on a
preparative scale, we first examined the oxidation of lysine by
LysOx in a catalase-free reaction under conditions that are tradi-

6
0
A.V. Pukin et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 58–62
Fig. 1. NMR spectra of the reaction components for the conversion of lysine into
-aminovaleric acid by l-lysine ␣-oxidase in D2O. A—lysine, B–D—progress of the
Fig. 3. Activities of l-lysine ␣-oxidase in different media.
5
reaction (conversion of lysine 11% for B, 37% for C, and 69% for D), E—product.
solutions allows simplifying the product isolation, as an easily
removable compound (NH HCO ) or the substrate itself are used as
4
3
tionally used for the assessment of the oxidative activity of this
enzyme towards lysine in a reaction in the presence of catalase.
Thus, a 70 mM phosphate buffer at pH 7.4 was used as a reac-
the buffer basis. The activities of the LysOx in all the studied media
were determined and compared (Fig. 3). From this comparison, the
conclusion can be drawn that the aqueous solution of a mixture
of lysine free base and lysine monohydrochloride at pH 7.4 is the
buffer of choice for the reaction under study.
◦
tion medium and the reaction was conducted aerobically at 37 C
with initial lysine monohydrochloride concentration of 60 mM. The
progress of the reaction was followed by NMR (vide supra) and the
incubation was stopped when all the starting material was con-
verted. The activity of LysOx in this reaction was found to be 8
times lower as compared to that determined in the standard assay
for this enzyme in the presence of catalase. Whether this can be
◦
Increasing the temperature from 37 to 50 C had little to no effect
on the reaction rate, therefore in the subsequent experiments the
◦
reactions were conducted at 37 C.
2.3. Immobilization of l-lysine-˛-oxidase
attributed to the detrimental action of H O remains unclear, as all
2
2
the formed hydrogen peroxide is consumed in the oxidative decar-
boxylation reaction according to the reaction scheme. It should be
noted, that in spite of the observed activity difference, LysOx proved
to be very stable at the applied conditions. Thus, even after 5 days
of incubation, it showed oxidative activity towards lysine.
However, the isolation of 5-aminovaleric acid from the
phosphate buffered reaction mixture proved to be challenging,
prompting us to search for the alternative reaction media. In sub-
In order to further facilitate the product isolation, we set out to
catalyse the reaction by immobilized LysOx. Immobilization would
also allow for an increased stability and reusability of the enzyme.
Among the solid supports used for enzyme immobilization, the
epoxy-activated ones have recently received an increasing atten-
tion owing to their ability to react with different functional groups
on the enzyme surface to form extremely strong linkages with min-
imal chemical modification of the protein. In view of this and other
properties, an epoxy-activated support Sepabeads EC-EP [4,30–32]
was used in the present study.
Immobilization of LysOx on Sepabeads EC-EP was achieved by
incubation of the enzyme solution in phosphate buffered saline
with the solid support at room temperature for 36 h. High ionic
strength was applied to ensure the hydrophobic adsorption of the
protein onto the surface of the fairly hydrophobic Sepabeads. The
adsorption is the first step in the two-step sequence of immobi-
lization [31], and is followed by the covalent attachment of the
adsorbed enzyme to the epoxy groups on the support surface.
After immobilization, the resulting enzyme preparation was fil-
tered, washed subsequently with phosphate buffer and water (to
ensure the desorption of any possible non-covalently attached pro-
tein) and the enzyme activity in the supernatant and washings
was determined. The remaining enzyme activity in the supernatant
was 2%, while the washings showed no activity at all. This allows
assigning the immobilization yield at 98%.
sequent experiments, a solution of NH HCO3 at pH 7.8 (containing
4
6
0 mM of lysine monohydrochloride), a 60 mM aqueous solution
of the lysine free base, and a solution representing a mixture of
lysine monohydrochloride and lysine free base at pH 7.4 and the
total lysine concentration of 60 mM were used. Application of these
2.4. An assay for the immobilized enzyme
The immobilized LysOx was assayed analogously to the enzyme
in solution. Though stopping of the reaction in a taken sample was
not necessary in this case, it still was inserted in the 4% (v/v) solu-
tion of CF COOH in D O prior to measurement, in view of a better
Fig. 2. Progress of the l-lysine ␣-oxidase-mediated oxidation of l-lysine in H2O and
3
2
D2O.
resolution of the multiplets belonging to different reaction com-

A.V. Pukin et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 58–62
61
onto a solid support and the resulting enzyme preparation showed
no loss of activity as compared to the enzyme in solution. The oper-
ational stability of the immobilized LysOx decreases after the first
batch reaction but remains stable during further reactions. This
indicates that a continuous process may be the best system for
this enzyme. The immobilized enzyme was successfully used in the
preparative synthesis of 5-aminovaleric acid.
4. Experimental
4.1. General information
l-Lysine ␣-oxidase from T. viride, l-lysine monohydrochloride
and l-lysine monohydrate were purchased from Sigma. Sepabeads
EC-EP were kindly donated by Dr. Paolo Caimi from Resindion
S.R.L. (Mitsubishi Chemical, Milan, Italy). All other chemicals were
obtained from Sigma or Fluka and were analytical or higher grade
and were used as received.
Fig. 4. Comparison of the activities of the immobilized and soluble l-lysine ␣-
oxidase.
NMR spectra were recorded on a 400 MHz Bruker Avance
machine.
ponents at lower pH. Similarly to the soluble enzyme, for all the
studied reaction media the immobilized LysOx showed maximum
activity in the aqueous solution of a mixture of lysine free base and
lysine monohydrochloride at pH 7.4.
Surprisingly, the activity of the immobilized enzyme was found
to be at least equal to that of the enzyme in solution at the otherwise
identical conditions (Fig. 4).
4.2. Solutions
A stock solution of l-lysine oxidase was prepared by dissolv-
ing the solid preparation obtained from Sigma in water to reach
a protein concentration of 0.43 mg/ml. The lysine solutions used
in this study were prepared as follows. Solution A: 0.06 M solu-
tion of lysine monohydrochloride in a 0.07 M phosphate buffer (pH
◦
Apparently the immobilization provides a certain stabilization
7.4 at 37 C). Solution B: 0.06 M solution of lysine monohydrate in
of the enzyme towards inter alia presence of H O2 and the pH fluc-
water. Solution C: 0.06 M solution of lysine monohydrochloride in
a 0.1 M ammonium bicarbonate solution (pH 7.8 at 37 C). Solution
D: a 0.06 M solution of lysine monohydrochloride was titrated with
a 0.06 M solution of lysine monohydrate to pH 7.4 at 37 C. Solu-
2
◦
tuations induced by the constant formation of ammonia and CO2
in the progress of the reaction without catalase.
◦
However, the reusability of the immobilized enzyme was found
◦
to be only modest, as determined for the reaction at 37 C in a mix-
tion E: a 0.06 M solution of lysine monohydrochloride in D O was
2
ture of lysine free base and lysine monohydrochloride at pH 7.4.
Thus, when the immobilized enzyme was filtered off upon the com-
plete conversion of the substrate, washed with phosphate buffered
saline and water, and then used for the second time at the identical
conditions, its activity in the second run consisted only 50% of that
in the first cycle (data not shown). The supernatant obtained after
filtration of the reaction mixture of the first run did not show any
enzyme activity. This allows ruling out the possibility of the protein
desorption during the reaction. Surprisingly, in the third cycle the
activity of the immobilized enzyme did not drop further, and was
equal to that in the second run (data not shown).
titrated with a 0.06 M solution of lysine monohydrate in D O to pD
7.4 at 37 C.
2
◦
4.3. l-Lysine ˛-oxidase activity assay
Activity of the l-lysine oxidase was determined by measuring
the change in concentrations of lysine and 5-aminovaleric acid
in the course of the reaction. In a typical experiment, 2 ml of a
0.06 M solution of lysine (system A, B, C, D or E, vide supra) was
◦
pre-incubated at 37 C for 5 min. The reaction was started by the
addition of an appropriate aliquot of the stock solution of l-lysine
◦
oxidase and the whole was incubated at 37 C (slow rotation of the
2
.5. Synthesis of 5-aminovaleric acid
mixture on a rotary evaporator) under a gentle flow of air. At given
time intervals, 150 ␮l samples were taken followed by immediate
inserting thereof into 300 ␮l of 4% (v/v) solution of CF COOH in D O
With the optimum conditions for enzyme functioning in
3
2
hand, we performed a preparative reaction to synthesize 5-
aminovaleric acid. Thus, the reaction mixture consisted of 50 ml
of an aqueous solution of a mixture of lysine free base and
lysine monohydrochloride at pH 7.4 with lysine concentration of
to quench the reaction. Percentages of l-lysine and 5-aminovaleric
acid were determined from a 1H NMR spectrum of the resulting
solution by comparison the sum of the integral values of the multi-
plets in 1.65–1.42 ppm region with the sum of the integral values of
all the multiplets in the spectrum (when the reaction is performed
1
20 mM, and 3 U of immobilized LysOx. The mixture was incubated
◦
aerobically for 5 days at 37 C after which NMR showed the com-
plete conversion of the starting lysine. Filtering off the enzyme
preparation and lyophilization of the reaction mixture allowed
the target 5-aminovaleric acid in an excellent isolated yield of
in D O, the region of 2.35–2.25 ppm should not be integrated). The
2
calculated percentages were plotted against time. The activity of
LysOx was determined as a function of the slope of the resulting
graph.
9
5%.
4.4. Immobilization of l-lysine ˛-oxidase on Sepabeads EC-EP
3
. Conclusions
A total of 0.5 g of wet support was suspended in 2 ml of the
We have studied the reaction of the lysine oxidation catalysed
enzyme solution (with a protein concentration of 0.025 mg/ml) in
a 0.1 M phosphate buffer at pH 8 (the ionic strength of the buffer
was adjusted to 5 M with NaCl). The mixture was incubated at 25 C
by l-lysine ␣-oxidase from T. viride in the absence of catalase. An
NMR-based assay was developed and applied in the optimization
of reaction conditions. The enzyme was successfully immobilized
◦
(slow rotation of the mixture on a rotary evaporator) for 36 h after

6
2
A.V. Pukin et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 58–62
which the beads were filtered, and the activity of the supernatant
was checked. The immobilized enzyme was washed by suspending
it in 2 ml of a 0.01 M phosphate buffer at pH 8, followed by stirring
air. The progress of the reaction was followed by NMR. The enzyme
was filtered off, and the supernatant was lyophilized to give 0.67 g
(5.7 mmol, 95% yield) of 5-aminovaleric acid as a white solid.
◦
at 25 C for 10 min and filtering. This procedure was repeated twice,
and finally the beads with the immobilized enzyme were washed
on a glass filter with an excess of distilled water and stored as wet
preparation at 4 C.
Acknowledgment
◦
The authors thank NWO (The Dutch Organisation for Scientific
Research) for financial support (project no. 700.56.041).
4.5. Activity assay of the immobilized l-lysine oxidase
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◦
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Lopez-Gallego, O. Abian, J.M. Palomo, L. Betancor, B.C.C. Pessela, J.M. Guisan, R.
Fernandez-Lafuente, Biomacromolecules 4 (2003) 772–777.
4
.7. Synthesis of 5-aminovaleric acid
[
[
The reaction mixture representing a suspension of the immobi-
lized LysOx (3 U) in 50 ml of a lysine solution (solution D, 0.12 M,
32] R. Torres, C. Mateo, M. Fuentes, J.M. Palomo, C. Ortiz, R. Fernandez-Lafuente,
J.M. Guisan, Biotechnol. Prog. 18 (2002) 1221–1226.
◦
containing 0.88 g (6 mmol) of lysine) was incubated at 37 C for 5
days by slow rotation on a rotary evaporation under a gentle flow of