Immobilization of L-aspartate oxidase from Sulfolobus tokodaii as a biocatalyst for resolution of aspartate solutions

Article abstract of DOI:10.1039/c4cy00968a

L-Aspartate oxidase from the thermophilic archaebacterium Sulfolobus tokodaii (StLASPO) catalyzes the stereoselective oxidative deamination of L-aspartate to yield oxaloacetate, ammonia and hydrogen peroxide. The recombinant flavoenzyme shows distinctive

Full text of DOI:10.1039/c4cy00968a

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Immobilization of L-aspartate oxidase from
Sulfolobus tokodaii as a biocatalyst for resolution
Cite this: DOI: 10.1039/c4cy00968a
of aspartate solutions
Paola D'Arrigo, abc Chiara Allegretti,a Andrea Fiorati,a Luciano Piubelli,bd
*
Elena Rosini,bd Davide Tessaro,ab Mattia Valentinobc and Loredano Pollegionibd
L-Aspartate oxidase from the thermophilic archaebacterium Sulfolobus tokodaii (StLASPO) catalyzes
the stereoselective oxidative deamination of L-aspartate to yield oxaloacetate, ammonia and hydrogen
peroxide. The recombinant flavoenzyme shows distinctive features that make it attractive for biotechnological
applications (it is highly thermostable, it is stable in a broad pH range, it tightly binds the FAD cofactor
and it shows a low Km for dioxygen). In order to set up an efficient and economically feasible bioconversion
process, in this work, we investigated the immobilization of this novel biocatalyst. The best results in terms
of immobilization yield have been obtained when StLASPO was immobilized on the amino
support Relizyme™ HA403/S R after activation with glutaraldehyde and on the epoxy support
SEPABEADS® EC-EP/S (in both cases, through the free amino groups of the enzyme), as well as prepared
as cross-linked enzyme aggregates (CLEA). By using the Relizyme™ HA403/S R support, as well as by the
CLEA preparation procedure, full immobilization in terms of enzymatic activity was obtained. Immobilized
StLASPO was used for the resolution of racemic solutions of 50 mM D,L-aspartate achieving full resolution
in <1 hour. Indeed, the Relizyme-StLASPO immobilized enzyme (the one showing the highest immobilization
yield) was efficiently reused in 5 cycles keeping full oxidation of L-aspartate in ≤2 hours. In a
semi-preparative scale reaction, 66 mg of enantiomerically pure D-aspartate were produced in 1 day
using 20 units of enzyme.
Received 25th July 2014,
Accepted 5th October 2014
DOI: 10.1039/c4cy00968a
www.rsc.org/catalysis
be exploited for the production of optically pure amino acids
by resolution of D,L-racemic mixtures. Enantiomerically pure
1. Introduction
L-Amino acid oxidase (LAAO, EC 1.4.3.2), a flavoenzyme
containing non-covalently bound flavin adenine dinucleotide,
catalyzes the stereospecific oxidative deamination of L-amino
acids to α-keto acids, ammonia and hydrogen peroxide
(Fig. 1). LAAOs purified from snake venoms are the best-
studied members of this family of enzymes; several LAAOs
from bacterial and fungal sources have also been reported
(for a review, see ref. 1). In analogy to the well-known biotech-
nological applications of D-amino acid oxidase (DAAO, EC
1.4.3.3),2,3 LAAOs have important potential biotechnological
applications, in particular, as part of biosensors and as cata-
lysts in biotransformations.4 DAAO and LAAO activities can
proteinogenic and synthetic amino acids are of increasing
interest for the fine chemical and pharmaceutical industries:
unnatural α-amino acids (such as naphthyl and fluorinated
substituted amino acids, i.e. D-Phe for the antibiotics ampicil-
lin and amoxicillin) are used in drug discovery for the synthe-
sis of combinatorial libraries and for the de novo synthesis of
peptides and are also used as additives in the food industry
and as valuable pharmaceuticals (e.g. D- and L-DOPA and
D-2-naphtylalanine). Unluckily, the use of LAAO was ham-
pered by the difficulty to produce suitable amounts of this
enzyme from both the original and the recombinant sources.1
The conversion of N-ε-CBZ-L-lysine to CBZ-L-oxylysine, a pre-
cursor of the anti-hypertensive drug ceronapril, represents the
first attempt to use microbial LAAO activity (whole cells
of Providencia alcalifaciens).5 More recently, Rhodococcus sp.
AIU Z-35-1 LAAO has been employed for the resolution of
D,L-mixtures of citrulline, Gln, homoserine, N-ε-acety-Lys and
Arg,6 and crude extracts of A. fumigatus have been used for
the racemic resolution of Ala, Phe and Tyr: nevertheless, an
optically pure product was obtained only for D-Ala.7 LAAO from
the bacterium R. opacus DSM 43250 was also used for the
a Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”,
Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy.
E-mail: paola.d'arrigo@polimi.it; Fax: +39 02 23993180; Tel: +39 02 23993075
b The Protein Factory, Politecnico di Milano, ICRM CNR Milano, and Università
degli Studi dell'Insubria, Via Mancinelli 7, 20131 Milano, Italy
c CNR – Istituto di Chimica del Riconoscimento Molecolare (ICRM),
Via Mancinelli 7, 20131 Milano, Italy
d Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi
dell’Insubria, Via J.H. Dunant 3, 21100 Varese, Italy
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Fig. 1 Kinetic resolution of D,L-aspartic acid. (A) Reaction catalyzed by StLASPO on D,L-aspartic acid: oxidation of the L-isomer yields pure
D-aspartate and oxaloacetate IJ2-oxosuccinate). Catalase is used frequently to eliminate hydrogen peroxide, thus halving the overall amount of oxygen
consumed by the oxidase. (B) Chemo-enzymatic process for D-aspartate quantitative obtainment starting from racemic D,L-aspartic acid.
racemic resolution of
7
mM D,L-Phe and D,L-Leu and
thermophilic archaea Sulfolobus tokodaii (StLASPO) was
efficiently produced as a recombinant protein in E. coli in
the active form as holoenzyme (up to 9% of the total proteins
in the crude extract and up to 13.5 mg L−1 in the fermenta-
tion broth).11,12 This monomeric (52 kDa) recombinant
flavoenzyme shows the classical properties of FAD-containing
oxidases but also possesses distinctive features that make it
attractive for biotechnological applications: i) high thermal
stability (it is fully stable up to 80 °C) and high temperature
optimum, ii) stable activity in a broad range of pH (7.0–10.0),
iii) weak inhibition by the product (oxaloacetate) and by the
D-isomer of aspartate, and iv) tight binding of the FAD
cofactor. Indeed, steady-state measurements highlighted a
low Km,O2 (0.3 mM); this property significantly distinguishes
StLASPO from the E. coli counterpart.13,14
3,4-dihydroxyphenyl-L-alanine (L-DOPA), the precursor of the
melanine pigment, of all cathecolamine neurotransmitters,
and of hormones and one of the main drugs for treatment of
Parkinson's disease. By employing a batch biotransformation
based on a membrane reactor, complete oxidation of L-DOPA
was reached for three consecutive cycles performed by adding
fresh L-DOPA to the same reaction mixture without discharge
of the products.8 Altogether, LAAOs have not been extensively
exploited for biotechnological applications because of the dif-
ficulties in their expression as recombinant proteins.1 In
order to obtain substantial amounts of a catalyst, as well as
to improve its biochemical properties by protein engineering,
the recombinant production of the protein of interest is
essential.9,10
A
suitable alternative to LAAO is represented by
Altogether, StLASPO represents an appropriate novel bio-
catalyst for the production of D-aspartate: a very low
amount of StLASPO (9 U) allowed the resolution of a racemic
mixture of D,L-aspartate (final ee > 99.5%).12 In fact, a
straightforward enzymatic method to convert an α-amino
acid to the corresponding α-keto acid is based on the use of
a highly enantioselective amino acid oxidase: the enzyme spe-
cifically oxidizes one enantiomer only based on its enantio-
selectivity (e.g., the L-form – (S) enantiomer – is oxidised
when LAAO is employed) to the imino acid that rapidly deam-
inates to the corresponding α-keto acid; see the reaction
L-aspartate oxidase (LASPO, EC 1.4.3.16) which catalyzes the
oxidative deamination of L-aspartate and L-asparagine. From
a structural point of view, LASPO is substantially different
from the “classical” LAAOs: it belongs to the succinate dehy-
drogenase/fumarate reductase family of flavoproteins and is
not evolutionarily related to the classical LAAO members.1
Each LASPO monomer is formed by a single polypeptide
chain (comprising 472 residues) that folds into three dis-
tinct domains: a FAD-binding domain, a capping domain,
and a helical domain.1,11 In particular, LASPO from the
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scheme in Fig. 1A. A full deracemization is then obtained
using non-selective inorganic reducing agents (i.e. sodium
borane) that convert the α-imino acid intermediate into the
D,L-amino acid racemate:15 after a few cycles of enzymatic oxi-
dation of the L-isomer and chemical reduction of the imino
acid, the D-amino acid only accumulates (Fig. 1B). Aspartate
is used in the pharmaceutical industry, for parenteral nutri-
tion, as food additive and in sweetener manufacturing.
Immobilized enzymes are currently the subject of consid-
erable interest because they facilitate industrial applications
by improving reusability and, in turn, by reducing the costs
associated with production and purification of the enzyme.16
For a review on the properties of immobilized thermophilic
enzymes, see ref. 17. It should be pointed out that the
enzyme reuse is often an essential prerequisite in order to
establish an economically viable enzyme-catalyzed process.
The possible alterations in enzyme properties related to the
effect of the immobilization procedure on the protein struc-
ture, as well as the impact of partition effects and diffusional
limitations, have been reported in detail in ref. 18.
In this work, StLASPO was immobilized on various
matrices and the immobilized recombinant flavoenzyme has
been employed in an efficient kinetic resolution of a racemic
mixture of D,L-aspartate. Since StLASPO is a very stable
flavoenzyme (see above), this investigation aims to
improve the recovery/reuse of the biocatalyst. The solid sup-
ports which have been evaluated are SEPABEADS® EC-EP/A,
SEPABEADS® EC-EP/S, Relizyme™ EP403, Relizyme™ EP113,
Relizyme™ HA403/S R, Relizyme™ HA113, and Eupergit® C.
The best results in terms of immobilization yield and volu-
metric activity (U per g support) have been obtained with the
amino support Relizyme™ HA403/S R and with the epoxy
support SEPABEADS® EC-EP/S, the former showing a higher
L-aspartate conversion yield when reused for five times. Also
cross-linked enzyme aggregates (CLEA)19,20 were prepared
from StLASPO by using glutaraldehyde as a cross-linking
agent: the CLEA-StLASPO preparation also exhibited high
activity and stability.
The activity of the final solution was 2 U mL−1, corresponding
to 1.3 U per mg of protein.
2.3. Activity assays
The StLASPO activity was assayed by measuring the initial rate
of production of hydrogen peroxide with a coupled peroxidase/
dye assay. The colored product produced by the horseradish
peroxidase from H2O2 and 4-aminoantipyrine was detected
spectrophotometrically at 505 nm (ε = 6.58 mM−1 cm−1) at
37 °C.12 One unit (U) is defined as the amount of enzyme that
catalyses the degradation of 1 μmol of L-aspartate per minute.
The standard assay mixture contained 10 mM L-aspartate
in 50 mM sodium pyrophosphate buffer (pH 8.0), 1.5 mM
4-aminoantipyrine, 10 mM phenol, 20 μM FAD, 2.5 U of
horseradish peroxidase, and 30 μg (≈0.04 U) of StLASPO in a
total volume of 1 mL.
For the assessment of pH dependence of the rate of oxida-
tion of L-aspartate by the various StLASPO preparations, the
reaction was performed at a pH value of 8.0, 9.0 or 10.0 using
the “magic buffer” composed of 33 mM Tris–HCl, 33 mM
Na2CO3, and 33 mM H3PO4 (final concentration).21
For the assessment of the effect of hydrogen peroxide on
StLASPO stability, a 1.5 mg mL−1 enzyme preparation was
mixed in 20 mM Tris–HCl at pH 7.2 and 10% glycerol with 1
or 10 mM H2O2 at 50 °C (and as a control in the absence of
H2O2). Aliquots were withdrawn at different times (up to
96 hours) and assayed with the oxygen consumption method
on 10 mM L-aspartate as a substrate (at 37 °C):22 residual
activity was expressed as percentage of the one determined
before incubation.
2.4. StLASPO immobilized on Relizyme™ HA403/S R resin
The Relizyme™ HA 403/S R resin (200 mg) was treated with
10 mL of 0.125% glutaraldehyde solution in water for 2.5 h
in a rotating device at 18 °C. The glutaraldehyde solution was
then removed by centrifugation, and the resin was washed
three times with 0.1 M phosphate buffer at pH 7.5. The
buffer was then removed, and 1 mL of dialyzed StLASPO
(2 U) and 1 mL of 0.1 M phosphate buffer at pH 7.5 were
added to the activated support. The mixture was incubated in
a rotatory shaker for 24 h at 18 °C: the residual enzymatic
activity in the solution was assayed by the coupled peroxidase/
dye assay and was close to zero. The resin was then washed
with 50 mM phosphate buffer at pH 7.5 and stored at 4 °C
in the same buffer. The immobilized enzyme showed an
activity of ≈10 U per g matrix corresponding to 100% immo-
bilization yield.
2. Experimental
2.1. Chemicals
All chemicals were purchased from Sigma-Aldrich. All
solvents were of analytical grade. Catalase from beef liver
(20 mg mL−1, 65 kU per mg protein) and horseradish peroxi-
dase were purchased from Roche. Relizyme™ HA403/S R and
SEPABEADS EC-EP/S supports were supplied by Resindion
(Milano, Italy).
2.5. StLASPO immobilized on SEPABEADS® EC-EP/S resin
2.2. Enzyme preparation
The dialyzed StLASPO preparation (0.5 mL, 1 U) was added to
60 mg of SEPABEADS® EC-EP/S resin and to 0.3 mL of
1.25 M potassium phosphate buffer at pH 8.0. The mixture
was incubated in a rotatory shaker for 18 h at 25 °C: at the
end of incubation, approximately 50% of the enzymatic activ-
ity was present in solution suggesting that half of the added
StLASPO was overexpressed in E. coli cells and was purified
to >95% purity by the procedure described in ref. 12. The
purified StLASPO was stored in 20 mM Tris–HCl buffer at
pH 7.5 and 10% glycerol. Before use, it was dialyzed against
0.05 M sodium phosphate buffer, pH 7.5, at 4 °C for 24 h.
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enzyme has been immobilized. The immobilized StLASPO
was stably bound to the support: no enzyme was released
when the SEPABEADS-StLASPO support was incubated with
20 mM potassium phosphate at pH 8.0 and left rotating for
an additional hour. The immobilized enzyme showed an
activity of 8.3 U per g dry matrix.
Concerning the CLEA-StLASPO preparation, 1.1 U of
immobilized enzyme were added to 0.5 mL of a 50 mM
D,L-aspartate solution in “magic buffer” and adjusted to the
desired pH (8.0, 9.0 or 10.0) by adding 1 M NaOH.
In the case of the recycling of immobilized StLASPO, at
the end of each cycle, the matrices were collected by centrifu-
gation, washed several times with water and stored in the
starting buffer.
2.6. Precipitation of StLASPO as CLEA
2.9. Chiral HPLC analysis of aspartate racemic mixtures
The precipitation of StLASPO and the CLEA formation were
conducted by adding 4.5 mL of precipitating solution
(i.e. 60% w/v ammonium sulphate in 0.1 M sodium phosphate
buffer at pH 7.5) and 20 μL (0.11 mmol) of glutaraldehyde
(50% w/v in water, 5.6 M, d 1.208 g mL−1) (final 0.4% (v/v)
corresponding to 200 μmol) to 0.5 mL of dialyzed enzyme
solution (2 U mL−1, 14.8 nmol).23,24 The mixture was incu-
bated on a rotatory shaker for 2.5 h at room temperature: no
residual enzymatic activity was detected in the solution.
Then, the mixture was centrifuged and the residue was
washed twice with 0.1 M phosphate buffer at pH 7.5,
centrifuged and decanted. The CLEA-StLASPO was stored at
4 °C in 0.05 M phosphate buffer at pH 7.5. The CLEA-StLASPO
showed an activity of 4 U per g wet CLEA.
The OPA-NAc reagent was prepared by dissolving 4 mg of
ortho-phthalaldehyde (OPA) in 300 μL of methanol, followed
by the addition of 250 μL of 0.4 M borate buffer at pH 9.4. To
this mixture, 15 μL of 1 M N-acetylcysteine (NAc) in 1 M
NaOH were added, and then the solution was diluted with
435 μL of distilled water. At fixed time intervals, 10 μL ali-
quots were withdrawn from the reaction mixture and diluted
with 40 μL of distilled water. 10 μL of this solution were
derivatized with 25 μL of OPA-NAc reagent as described in
ref. 25, diluted with 350 μL of HPLC eluent and analyzed by
HPLC chromatography in a Zorbax SB-Aq column (150 × 4.6,
5 μm, Agilent Technologies; eluent: 100 mM sodium ace-
tate buffer, pH 5.2, isocratic flux of 0.6 mL min−1; detection
at 340 nm). The separation allowed us to distinguish and
quantify the L- and D-isomers of aspartate and thus to calcu-
late the enantiomeric excess (ee); Rt (D-aspartate): 7.5 min, Rt
(L-aspartate): 8.8 min.
2.7. Resolution of D,L-aspartate by free StLASPO
The setup of the experimental conditions to resolve a 50 mM
D,L-aspartate solution by using free StLASPO was performed
by adding 0.3 U of StLASPO to the reaction medium at
pH 10.0 (final volume 1.0 mL). The reaction was carried out
in the 25–80 °C temperature range under shaking (using a
thermomixer set at 600 rpm). Samples were collected at
different time intervals for D- and L-aspartate concentration
measurements. Subsequently, the same reaction was performed
under the same conditions at 50 °C at different pH values
(in the 8–13 range).
3. Results
3.1. Bioconversion of D,L-aspartate using free StLASPO
A previous study established that for free StLASPO the
highest activity was achieved at 65 °C and at pH 10.5.12 In
order to identify the best conditions for the resolution of a
50 mM solution of D,L-aspartate, we analyzed the effect of
temperature on the conversion performed at pH 10.0.
Full resolution was reached in that case in 4 h in the
37–70 °C range using 0.3 U StLASPO mL−1: the fastest conver-
sion was apparent at 50 °C (see the time course of conversion
in Fig. 2a).
2.8. Resolution of D,L-aspartate by immobilized StLASPO
All reactions were performed at 70 °C in a thermomixer set to
600 rpm; the addition of 2 μL of catalase did not affect the
time course of conversion.
Concerning the effect of pH, under similar experimental
conditions and at 50 °C, the fastest resolution apparently
occurred in the 10–11 pH range (see Fig. 2b). These results
show that free StLASPO can be used as a biocatalyst in
kinetic resolution of racemic solution of aspartate: the main
drawback is represented by the impossibility to reuse the
enzyme for additional cycles, and this has a negative impact
on the cost of the overall process.
To test the sensibility of StLASPO to hydrogen peroxide,
free StLASPO was incubated at pH 7.2 and 50 °C with 0, 1 or
10 mM hydrogen peroxide and the enzymatic activity on
L-aspartate was assayed with the oxygen consumption
method. As shown in Fig. 3, StLASPO stability is not affected
Concerning the SEPABEADS-StLASPO matrix, the resolution
of D,L-aspartate was carried out by adding 0.5 mL of the
racemic 50 mM amino acid solution (in water, prepared and
adjusted to pH 10.0 with 1 M NaOH, final concentration of the
amino acid 37.7 mM) and 200 μL of water to the immobilized
enzyme (60 mg of support containing ≈0.5 U of StLASPO).
In the case of Relizyme-StLASPO, 0.5 mL of a 50 mM
D,L-aspartic solution (adjusted to pH 10.0) and 200 μL of
water were added to the immobilized enzyme (≈2 U). For
the scaling-up of the reaction, 2 g of Relizyme-StLASPO
(corresponding to 20 U) were added to 5 mL of 200 mM
D,L-aspartic acid and 5 mL of water at pH 10.0, under constant
air flux and stirring.
by hydrogen peroxide: after 96 h of incubation, 82
1%
of the initial enzymatic activity is maintained in the presence
of 1 or 10 mM H2O2 vs. 86 3% in the absence of hydrogen
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Fig. 3 Effect of hydrogen peroxide on the stability of free StLASPO.
Conditions: StLASPO (1.5 mg mL−1) was incubated in 20 mM Tris–HCl
buffer, pH 7.2, and 10% glycerol at 50 °C in the presence of 1 mM (■)
or 10 mM (♦) hydrogen peroxide. As a control, the activity in the
absence of H2O2 is also reported (●). Aliquots were withdrawn at
different times and assayed with the oxygen consumption method on
10 mM L-aspartate as a substrate (at 37 °C): residual activity was
expressed as percentage of the one determined before incubation. The
values are the average of at least three measurements; error bars are
not shown since error was <5%.
on the two solid supports named Relizyme™ HA403/S R
and SEPABEADS® EC-EP/S that resulted in higher StLASPO
immobilization yields in preliminary experiments. These
supports are composed of a rigid methacrylic polymer matrix
with high physical and chemical stability given by
intense cross-linking. They also exhibit the same particle size
(100 to 300 nm).
Fig.
2 (a) Effect of temperature on the kinetic resolution of
D,L-aspartate by free StLASPO. Conditions: 1 mL of 50 mM D,L-aspartic
acid at pH 10.0, 0.3 U of StLASPO, at 700 rpm. Symbols used: (●) 25 °C,
(○) 37 °C, (■) 50 °C, (□) 60 °C, (▲) 70 °C, and (▼) 80 °C. The values are
the average of at least three measurements; error bars are not shown
since error was <5%. (b) Effect of pH on the kinetic resolution of
D,L-aspartate. Conditions: 1 mL of 50 mM D,L-aspartate solution
adjusted at different pH values, 0.3 U of StLASPO, 50 °C, at 700 rpm.
Symbols used: (●) pH 8.0, (○) pH 9.0, (■) pH 10.0, (□) pH 11.0,
(▲) pH 12.0, and (▼) pH 13.0. The values are the average of at least
three measurements; error bars are not shown since error was <5%.
3.2.1 StLASPO immobilized on Relizyme™ HA403/S R.
Immobilized StLASPO was used as a biocatalyst for the
resolution of the racemic solution of the amino acid
aspartate. Relizyme™ HA403/S R is a rigid methacrylic
polymer matrix (particle size of 100–300 μm with a mean
diameter of 40–60 nm) which is functionalized with amino
groups. A pre-activation step is necessary using a bifunctional
coupling agent such as glutaraldehyde. As stated in ref. 26,
the final effect of glutaraldehyde treatment will be a mixture
of cross-linking and chemical modifications. Then, the cova-
lent binding between the enzyme and the derivatized support
is performed. Briefly, pure StLASPO was immobilized on acti-
vated Relizyme™ HA 403/S R support at pH 7.5 in 100 mM
potassium phosphate buffer: the immobilized enzyme
showed an activity of ≈10 U per g matrix corresponding to
100% immobilization yield. These conditions should favor
ionic exchange of StLASPO prior to covalent immobilization.
The enzyme is covalently bound to the matrix since no pro-
tein was released when the Relizyme-StLASPO resin was
boiled for 3 minutes – as assessed by SDS-PAGE analysis
performed on the soluble fraction obtained after centrifuga-
tion of the heated resin.
peroxide. Because of the insensitivity of StLASPO to hydrogen
peroxide, there is no need to add catalase during the bio-
conversion. Indeed, catalase shows a very low residual
activity when incubated at pH 10.0 and 70 °C for 30 minutes
(<2% of the activity assayed at neutral pH and room
temperature).
3.2. Bioconversion of D,L-aspartate using
immobilized StLASPO
Then, immobilization of StLASPO has been investigated in
order to increase the competitiveness of the resolution pro-
cess. With the SEPABEADS® EC-EP/A, Relizyme™ EP403,
Relizyme™ EP113, Relizyme™ HA113, and Eupergit® C
supports, StLASPO was only partially immobilized, ranging
from 8% with Relizyme™ HA113 to 29% with SEPABEADS
EC-EP/A. On the other hand, we have focused our attention
The resolution of 50 mM D,L-aspartate was carried out at pH
10.0 and 70 °C using the immobilized StLASPO (≈2 U). Full
oxidation of L-aspartate was obtained in ≤1 hour (ee ≈ 100%)
for three sequential cycles, while for the following cycles a
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longer reaction time was required (Table 1). This result
points to partial inactivation of the immobilized StLASPO
when reused for more than 3 times since no StLASPO was
released from the immobilized matrix (as demonstrated by
SDS-PAGE analysis).
In order to evaluate the influence of D,L-aspartate concen-
tration on the enzymatic resolution, the reaction was carried
out by adding the StLASPO immobilized on Relizyme™
HA403/S R resin (2 U) to a solution containing 0.5 mL of
D,L-aspartate at different concentrations (from 50 to 500 mM)
at pH 10.0 and 200 μL of water. As reported in Table 2, in the
presence of 200 mM D,L-amino acid (final concentration
142 mM), 6.6 mg of L-aspartate were fully converted by 2 U of
enzyme in 4 h and at 70 °C. The observation that at
500 mM D,L-aspartate concentration the reaction stopped at
21% conversion points to an inhibition of StLASPO activity
by the racemic amino acid solution.
3.2.2 StLASPO immobilized on SEPABEADS® EC-EP/S
resin. SEPABEADS® EC-EP/S is a methacrylate hydrophobic
support functionalized with epoxide groups (131 μmol
oxirane content per g wet) which react efficiently with the
amino groups of the protein and with a median pore
diameter of 10–20 nm and a particle size of 100–300 μm.
Immobilization was performed at high ionic strength to
allow protein adsorption so that covalent attachment can
then take place.16 Each StLASPO protomer contains 28 lysine
residues: analysis of the 3D structure (pdb code 2E5V) shows
that three of them (namely K24, K147 and K396) show a very
high degree of accessibility. One unit of pure StLASPO was
immobilized on 60 mg of SEPABEADS® EC-EP/S matrix: the
flavoenzyme was immobilized with a ≈50% yield in terms
of activity using 20 mM potassium phosphate buffer at
pH 8.0. The covalent linkage between the resin and the
enzyme has been verified by the boiling procedure as stated
above for the Relizyme support: also in this case, no protein
was released.
3.2.3. StLASPO immobilized as CLEA. The cross-linked
enzyme aggregates (CLEAs) technology is applicable to a wide
variety of enzymes as a mean for improving the performance
of enzymes under operating conditions.27–29 It is a very useful
method in biocatalysis because it combines purification and
immobilization of enzymes into a single operation. Cross-
linked StLASPO aggregates were prepared using a two-step
procedure: aggregation by precipitation with ammonium sul-
phate and cross-linking with glutaraldehyde.23,24 Different
cross-linker/precipitation solution ratios have been used: 0.4%
(v/v, glutaraldehyde solution/total precipitation solution) was
chosen to stabilize towards leaching since the enzyme was fully
active even after different bioconversion cycles (see Table 4). In
fact, the CLEA-StLASPO preparations obtained using a 0.2% or
0.6% ratio lost a large part of the initial activity after one cycle
of bioconversion. About 1.1 units of pure StLASPO were precipi-
tated as CLEA at pH 7.5: the enzymatic activity was 4 U per g
wet CLEA. The stability of the CLEA-StLASPO aggregate was
tested by the boiling procedure (as stated above): no free
protein was released from the preparation.
The resolution of 50 mM D,L-aspartate was carried out at
70 °C using the CLEA-StLASPO preparation, in “magic buffer”
adjusted to the desired pH (8.0, 9.0 or 10.0) by adding 1 M
NaOH. In the first cycle, a full conversion was reached at all
pH values tested and in a comparatively shorter time at the
highest pH. Indeed, at pH 9.0, complete oxidation of
L-aspartate was obtained in ≤4 hours for 5 cycles.
3.3. Resolution on a semi-preparative scale of D,L-aspartate
The immobilized StLASPO on Relizyme™ HA403/S R resin
was incubated under mechanical stirring with 100 mM
D,L-aspartic acid (10 mL final volume) at pH 10.0 and 70 °C
under constant air stream. The oxidation of L-isomer of
aspartate was complete after 24 hours of incubation: note-
worthily, the immobilized StLASPO preparation was reused
with no lack of activity. In particular, the scale-up of the enzy-
matic reaction catalyzed by Relizyme-StLASPO produced
66 mg of optically pure D-aspartate in 24 hours using 20 units
of StLASPO per cycle.
The resolution of 50 mM D,L-aspartate was carried out at
pH 10.0 and 70 °C with the immobilized enzyme: this reac-
tion yielded full oxidative deamination of the L-isomer of
aspartate into 2-oxosuccinic acid in 4 hours for 3 cycles. For
the following cycles, the full resolution required a longer
time, up to 10 hours in the fifth cycle (Table 3).
4. Conclusions
The L-amino acid oxidase class of flavoproteins comprises a
group of enzymes sharing similar functional and structural
properties that can be divided into two groups:1 LAAOs
possessing a broad substrate specificity and LAAOs which oxi-
dize one or a narrow group of substrates, such as LASPO,
L-lysine oxidase, L-tryptophan oxidase, etc. Recombinant
StLASPO shows distinctive features making it attractive for
biotechnological applications:11,12 a) it is a highly stable
flavoenzyme: it is thermostable (i.e. fully stable up to 80 °C),
shows a high temperature optimum, and is stable in a broad
range of pH; b) it presents suitable kinetic properties: it is
weakly inhibited by the product 2-oxosuccinic acid and by
the D-isomer of aspartate, Km for dioxygen is pretty low, and
the FAD cofactor is stably bound. Indeed, we now
Table 1 Results of kinetic resolution of 50 mM D,L-aspartate solution
(0.5 mL) using 2 U of StLASPO immobilized on Relizyme™ HA403/S R
support at pH 10.0 and 70 °C
Cycle number
Time (h)
eea (%)
1
2
3
4
1
1
1
1
2
1
2
>99.5
>99.5
>99.5
87.0
>99.5
77.0
5
>99.5
a
ee values calculated using the HPLC analysis.
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Table 2 Effect of D,L-aspartate concentration on the resolution by StLASPO (2 U) immobilized on Relizyme™ HA403/S R support at pH 10.0 and 70 °C
L-Aspartate conversion (ee)
Time (h)
ijD,L-Asp]solution
(mM)
0.5
1
2
3
4
5
7
21
42
35
70
110
140
180
360
>99.5%
62%
12%
—
>99.5%
36%
—
—
>99.5%
—
—
—
67%
—
—
—
>99.5%
53%
17%
—
—
—
—
69%
21%
—
—
—
—
84%
21%
—
—
—
—
—
21%
—
—
—
—
—
21%
Table 3 Results of kinetic resolution of a 50 mM D,L-aspartate (0.5 mL)
solution using 0.5 U of StLASPO immobilized on SEPABEADS® EC-EP/S
support at pH 10 and 70 °C
In order to facilitate the recovery of the reaction products
and the reuse of the biocatalyst, immobilization experiments
of pure, recombinant StLASPO have been carried out. The
best results in terms of immobilization yield have been
obtained with the amino support Relizyme™ HA403/S R
(100% of StLASPO activity) and in terms of volumetric activity
(U per g support) and recycling with both the Relizyme™
HA403/S R and the epoxy support SEPABEADS® EC-EP/S.
Because of activation of the Relizyme™ HA403/S R support
with glutaraldehyde, both matrices reacted with the free
amino groups of the flavoenzyme.
Cycle number
1
Time (h)
eea (%)
3
4
2
4
2
4
2
4
6
2
4
6
8
10
68
>99.5
66
>99.5
65
>99.5
59
81
>99.5
28
45
73
2
3
4
StLASPO was also used as CLEA form: the immobilization
via precipitation and cross-linking of enzyme molecules with
a bifunctional cross-linking agent is a carrier-free method
and the resulting biocatalyst is permanently insoluble and
essentially maintains 100% of its activity for 5 cycles, with
complete conversion in <8 hours. The volume ratio of cross-
linker to enzymatic precipitation solution of 0.4% was chosen
to stabilize towards leaching. Such a value was considered a
moderate concentration of glutaraldehyde: it is sufficient to
ensure the activation of most amino groups with one mole-
cule of glutaraldehyde. Indeed, the high recovery in terms of
enzymatic activity of CLEA-StLASPO excludes loss of enzyme
flexibility due to an excessive cross-linking, as instead we
observed at 0.6% volume ratio. Glutaraldehyde is a widely
used cross-linker whose reaction with amino groups is
still not fully elucidated.26 The optimization of StLASPO
immobilization will require further investigations of the
glutaraldehyde-induced production of intra- and inter-
molecular enzyme cross-links and a stricter control of the
immobilization process. The time course of the CLEA-catalyzed
reaction was similar to that obtained with the flavoenzyme
immobilized on SEPABEADS, even if slower compared to the
Relizyme-immobilized StLASPO (compare Tables 1, 3 and 4).
Indeed, the scale-up of the enzymatic reaction catalyzed by
Relizyme-StLASPO allowed us to produce 66 mg of D-aspartate
(with high ee) in 24 hours using 20 units of StLASPO per cycle.
Moreover, it has been previously demonstrated for D-amino
acid oxidase that the in situ chemical reduction of the imine
product allows us to converge to a full conversion into a
single enantiomer:30,31 a similar process is also feasible for
the reaction catalyzed by StLASPO on D,L-aspartate (Fig. 1B).
In conclusion, because of the very interesting properties of
this enzyme, we propose the immobilized StLASPO as an
5
80
>99.5
a
ee values calculated using the HPLC analysis.
Table 4 Results of kinetic resolution of a 50 mM D,L-aspartate (0.5 mL)
solution using 1.1 U of StLASPO precipitated as CLEA at different pH
values and 70 °C
eea (%)
Cycle
number
Reaction
time (h)
pH 8.0
pH 9.0
pH 10.0
1
2
3
4
5
2
4
6
2
4
6
2
4
6
2
4
6
2
4
6
7
49
87
>99.5
49
97
>99.5
69
73
>99.5
88
>99.5
32
>99.5
53
>99.5
63
>99.5
66
>99.5
69
>99.5
64
>99.5
54
>99.5
42
68
>99.5
40
69
91
>99.5
91
>99.5
95
>99.5
a
ee values calculated using the HPLC analysis.
demonstrate that StLASPO is not sensitive to hydrogen perox-
ide (Fig. 3): accordingly, the use of catalase during bioconver-
sions is not required.
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Catalysis Science & Technology
attractive tool for biotechnological applications and, together
with the set-up of an efficient expression system in E. coli,12
as a complete biocatalytic process for the synthesis of fairly
high amounts of enantiopure D-aspartate.
9 T. Johannes, M. R. Simurdiak and H. Zhao, Biocatalysis.
Encyclopedia of Chemical Processing, ed. Taylor & Francis,
2006, pp. 101–110.
10 T. Davids, M. Schmidt, D. Bottcher and U. T. Bornscheuer,
Strategies for the discovery and engineering of enzymes for
biocatalysis, Curr. Opin. Chem. Biol., 2013, 17, 215–220.
11 H. Sakuraba, K. Yoneda, I. Asai, H. Tsuge, N. Katunuma and
Abbreviations
T. Oshima, Structure of -aspartate oxidase from the hyper-
thermophilic archaeon Sulfolobus tokodaii, Biochim. Biophys.
Acta, 2008, 1784, 563–571.
L
CLEA
DAAO
LAAO
LASPO
cross-linked enzyme aggregates
D-amino acid oxidase (EC 1.4.3.3)
L-amino acid oxidase (EC 1.4.3.2)
L-aspartate oxidase (EC 1.4.3.16)
12 D. Bifulco, L. Pollegioni, D. Tessaro, S. Servi and G. Molla, A
thermostable -aspartate oxidase: a new tool for biotechno-
logical application, Appl. Microbiol. Biotechnol., 2013, 97(16),
7285–7295.
L
StLASPO Sulfolobus tokodaii L-aspartate oxidase
ee enantiomeric excess
13 M. Mortarino, A. Negri, G. Tedeschi, T. Simonic, S. Duga,
H. G. Gasses and S. Ronchi,
Escherichia coli. I. Characterization of coenzyme binding and
product inhibition, Eur. J. Biochem., 1996, 239, 418–426.
L
-aspartate oxidase from
Acknowledgements
This work was supported by grants from Fondo di Ateneo per
la Ricerca to L. Pollegioni and L. Piubelli. The authors
gratefully acknowledge PRIN 2010–2011 NANO Molecular
Technologies for Drug Delivery – NANOMED prot. 2010
FPTBSH and the support from Consorzio Interuniversitario
per le Biotecnologie (CIB).
14 G. Tedeschi, A. Negri, M. Mortarino, F. Ceciliani, T. Simonic,
L. Faotto and S. Ronchi,
coli. II. Interaction with C4 dicarboxylic acid and identification
of a novel -aspartate: fumarate oxidoreductase activity,
L
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L
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15 S. Servi, D. Tessaro and G. Pedrocchi-Fantoni, Chemo-
enzymatic deracemization methods for the preparation of
enantiopure non-natural α-amino acids, Coord. Chem. Rev.,
2008, 252, 715–726.
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