Cell-free biocatalytic syntheses of l-pipecolic acid: A dual strategy approach and process intensification in flow

Article abstract of DOI:10.1039/d0gc01817a

As an alternative to the traditional chemical synthesis or in vivo production of l-pipecolic acid, we have developed two ex vivo strategies using purified and immobilised enzymes for the production of this key building block. Firstly, a transaminase capable of lysine ?-deamination was coupled with a novel pyrroline-5-carboxylate reductase, yielding 60% conversion at the 50 mM scale with free enzymes and in situ recycling of the cofactor. A second, simpler, redox neutral system was then constructed by combining the pyrroline-5-carboxylate reductase with a lysine-6-dehydrogenase. This bienzymatic system, with catalytic amount of free cofactor yielded >99% of pipecolic acid in batch and, following co-immobilisation of both enzymes, it was applied as a packed-bed reactor in continuous flow achieving again a molar conversion of >99% with 30 min residence time, and a space-time yield up to 2.5 g L-1 h-1. The sustainability of the system was further improved by a catch-and-release strategy to purify the product, and recovery and recycling of the cofactor.

Full text of DOI:10.1039/d0gc01817a

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DOI: 10.1039/D0GC01817A
ARTICLE
Cell-free biocatalytic syntheses of L-pipecolic acid: a dual
strategy approach and process intensification in flow
David Roura Padrosaa,b,†, Ana I. Benítez-Mateosa,b,†, Liam Calvey , and Francesca
b
Received 00th January 20xx,
Accepted 00th January 20xx
a,b,
Paradisi
*
DOI: 10.1039/x0xx00000x
As an alternative to the traditional chemical synthesis or in-vivo production of L-Pipecolic acid, we have
developed two ex-vivo strategies using purified and immobilised enzymes for the production of this key
building block. Firstly, a transaminase capable of lysine ε-deamination was coupled with a novel pyrroline-
5-carboxylate reductase, yielding 60% conversion at the 50 mM scale with free enzymes and in-situ
recycling of the cofactor. A second, simpler, redox neutral system was then constructed by combining the
pyrroline-5-carboxylate reductase with a lysine-6-dehydrogenase. This bienzymatic system, with catalytic
amount of free cofactor yielded >99% of pipecolic acid in batch and, following co-immobilisation of both
enzymes, it was applied as a packed-bed reactor in continuous flow achieving again a molar conversion of
>
99% with 30 min residence time, and a space-time yield up to 2.5 g/L/h. The sustainability of the system
was further improved by a catch-and-release strategy to purify the product, and recovery and recycling of
the cofactor.
2
photocatalysis using a TiO catalyst allowed the production of
racemic and L-PA with moderate enantiomeric excess but still
4
A.
Introduction
0% final yield.17
Pipecolic acid (PA) is a natural non-proteinogenic α-amino acid
found in a plethora of biologically active molecules1,2. It is
incorporated in natural products with anticancer or antibiotic
activity (Swainsonine, Virginiamycin S1, and Rapamycin) 3–6 and
it serves as a precursor to some simple manmade bioactive
molecules, such as amide anaesthetic drugs (Mepivacaine and
N
OH OH
H
H
N
NH
O
N
OH
O
N
Ropivacaine
Mepivacaine
Swainsonine
7
Ropivacaine) , (Figure 1A). In addition, pipecolic acid can be used
OH
O
as chiral organocatalyst in stereoselective Mannich reactions,8,9
N
as
a
building block in peptidic catalysts,10 as well as
N
O
O
O
O
O
O
diketopiperzine scaffolds.11
N
O
O
O
O
HO
O
HN
O
Despite its simplicity, L-PA as an optically pure molecule, is
orders of magnitude more expensive than the homologous five-
member ring L-proline, naturally impacting on the cost of the final
drugs. Its preparation by available chemical methods normally
involves harsh conditions and hazardous reagents despite the
most recent efforts to move towards greener strategies.12–14
Moreover, classical synthetic approaches require multi step
synthesis, with protection and deprotection steps, which affect the
overall yield. Ginesta et al, developed a 5 step synthesis of
pipecolic acid in very good enantiomeric excess but with only
O
O
HN
O
N
O
HN
HO
Virginiamycin S1
N
OHO
H
O
Rapamycin
B.
O
 - Deamination
OH
OH
N
O
O
P2C
H2N
OH
OH
Reduction
NH
L-PA
O
NH2
40% overall yield.15 More recently, two single-step strategies
~
L-Lys

- Deamination
N
have been reported. In the first one, the direct synthesis of
derivatives of pipecolic acid was achieved by stereoselective aldol
reaction, but again low yields were obtained (30%).16 Alternatively,
P6C
Figure 1. A. Chemical structures of L-PA containing drugs (L-PA identified in
red). B. General reaction scheme of the biosynthetic pathway for the production
of L-PA.
a.Department of Chemistry and Biochemistry University of Bern,
Freiestrasse 3, Bern (Switzerland)
School of Chemistry, University of Nottingham, University Park, NG7 2RD,
Nottingham (UK)
As an alternative, new approaches mimicking the natural
pathways have gained momentum in the last years. PA pathways
have been studied for years now in plants and animals, due to
their relationship with lysine metabolism defects. The conversion
from L-lysine to PA normally occurs in a two-step process, where
b.
†
These authors contributed equally to the work.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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L-lysine is deaminated either in the α or ε-position by a lysine the substrate and the formation of the correspondingVieawmAirntiecl.e OOnnlinae
dehydrogenase or lysine oxidase, leaving the corresponding 10 mM scale, HeWT was capable Do Of I: 1d 0e . 1a 0m3 9i n/ Da t0 i nG gC 01 b8 o1 7t Ah
aldehyde which spontaneously cyclises in water. The resulting enantiomers of lysine with moderate yields (~50%), suggesting
1
1
product (Δ -piperidine-2-carboxylic acid (P2C) or Δ -piperidine-6- that the ε-amino group rather than the α-position, is deaminated.
carboxylic acid (P6C)) is then reduced by a specific reductase to In fact, the deamination of the α-amino group would require HeWT
yield PA (Figure 1B). Some cell-free examples using these to accommodate double-bulky primary amines in the active site,
combinations of enzymes have been reported. 18–20 While in the ε- which for this enzyme has never been observed.
deamination the stereochemistry of the intermediate (at the -
carbon) is preserved throughout, in the α-deamination path, the To further shift the equilibrium of the first reaction and to complete
chirality is lost and reintroduced in the reduction step by the cascade, a new pyrroline-5-carboxylate reductase was also
stereospecific imine reductases.
identified in the genome of Halomonas elongata. The protein,
In addition to the two-step reaction catalysed by two different named He-P5C, was successfully cloned and expressed (ESI
enzymes, a lysine cyclodeaminase, has been recently shown to section 1, Figure S1), and had activity with P6C. While previously
perform both the deamination and the subsequent reduction of the reported bacterial P5C reductases showed a higher specificity for
cyclic intermediate, but in this case the reaction is very slow, NADPH,33,34 He-P5C exhibited dual cofactor affinity, with a K
of
M
achieving 90% conversion on a 5 mM scale in 60 hours.20–24 In- 0.013 ± 0.004 for NADH and 0.17 ± 0.03 for NADPH (Figure S2),
vivo, the combination of a lysine-6-dehydrogenase (Lys6DH) with suggesting that, in fact, the preferred electron donor is NADH. For
a pyrroline-5-carboxylate reductase (P5C) has been successfully our purpose, the dual affinity for both cofactors offers flexibility on
implemented in Corynebacterium glutamicum, with a productivity the choice of cofactor regeneration system, which is required to
of 15 g/L over 70h fermentations.25 In another example of whole make the overall reaction sustainable.
cell production, Cheng et al. achieved a higher yield (46.7 g/L)
The cascade reaction was then assayed on a 10 mM scale
over 36h fermentations by creating and artificial pathway, co- using only HeWT and He-P5C with, initially, equimolar amount of
expressing 4 recombinant proteins in E. coli.26 However, many cofactor and no recycling system. The presence of the second
biocatalytic methods
requirements, and chemical methods offering higher yields are deamination (while yielding L-PA), indicating an effect on the
often preferred.27
overall reaction equilibrium (Figure S3). From the different tested
are still far from meeting industrial enzyme, He-P5C, improved significantly the yield of lysine
Interestingly, from an enzymatic perspective, even though the conditions, the best results were obtained with benzaldehyde and
first reaction in the synthesis of PA from L-lysine consists of a pyruvate as amino acceptors, with ~60% and ~40% L-lysine
deamination, no transaminase based approach has been deamination yields respectively (Figure S4A). Only with pyruvate,
described to date, albeit lysine-ε-aminotransferases have been equimolar amount of L-PA is formed, as He-P5C was found to be
identified in bacteria, fungi and animals, and even some ω- inactivated by benzaldehyde, benzylamine and DMSO (Figure
transaminases have shown activity with lysine.28–30 Recently, S5). When using D-lysine, the aldehyde intermediate was formed
lysine has been suggested as the amino donor in whole cells in a similar yield to L-lysine but no production of D-pipecolic acid
expressing an S-selective transaminase.31
was observed, confirming the high stereoselectivity of He-P5C
previously observed for similar reductases.35–37 These
Here, we describe the successful application in batch of a conversions were maintained when scaling up L-lysine to 50 mM
transaminase-based synthesis of -PA and derivates, using (Figure S4B). However, the depletion of D-lysine was significantly
HeWT,32 in combination with
a
newly cloned pyrroline-5- lower at higher scale, possibly indicating an inhibition effect of the
carboxylate reductase (He-P5C), both from the halotolerant substrate.
organism Halomonas elongata. As an alternative, to simplify the
To construct
cascade and move towards redox neutral approach we dehydrogenase from Bacillus megaterium with dual cofactor
developed an additional cascade using lysine-6-dehydrogenase specificity, but preference for NADP (Bm-GDH)
Gs-Lys6DH) and He-P5C. The resulting system could be used in dependant formate dehydrogenase from Candida boidinii (Cb-
a
more cost-efficient cascade,
a
glucose
a
+
38,39
and a NAD-
(
batch with both free and co-immobilised enzymes, as well as with FDH)40 were trialled in combination with the two enzymes, 50 mM
a concurrent immobilisation of the cofactor to avoid the need of L-lysine and just 0.1 eq of the suitable cofactor. While with
external addition of NAD(H). Finally, the latter system was BmGDH the efficiency of the second step yielded ~50% of the
implemented as a packed bed reactor in a continuous flow system final product, with Cb-FDH total conversion of the intermediate
with catalytic amount of free cofactor. Here, the purification of L- was observed (Table S1). Further improvement was obtained in
PA and the reuse of the cofactor was accomplished in continuous the first step of the reaction (lysine deamination) by increasing the
by using a scavenger column.
pH of the reaction from 8 to 10 (Figure S6). At higher pH,
significant production of L-PA was observed only with Cb-FDH as
recycling system, while Bm-GDH was rapidly inactivated.38
Results and discussion
Multi-enzymatic cascade combining HeWT and He-P5C
With fully optimised conditions, the cascade was expanded to
two additional substrates (Table 1). The system produced almost
equimolar amount of the two six member rings, with respect to the
cyclic imine intermediate. In the case of L-ornithine, the substrate
was fully deaminated but only 27.5 mM (55%) of the
corresponding cyclic amine (L-proline) was formed, probably due
to the reduced activity of He-P5C over long periods (Figure S7).
Despite the efficiency of the second step, the equilibrium of the
deamination was only marginally improved (Figure S8).
While the transaminase from Halomonas elongata has been
extensively studied and displayed a broad substrate scope,32 its
ability to deaminate lysine at the ε-position was unknown. Initial
studies on HeWT mediated deamination of lysine were performed
using pyruvate, glyoxylate and benzaldehyde as amino acceptors.
The reactions were followed by monitoring both the depletion of
2
| J. Name., 2012, 00, 1-3
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Table 1. Synthesis of L-PA and derivates. The reaction was performed with 50
mM of each substrate, 2 mg/mL HeWT, 2 mg/mL of He-P5C and 2 mg/mL Cb-
FDH, 1 mM NAD+ in 50 mM carbonate buffer pH 10 at 37ºC.
To increase the reusability of the biocatalysts, iVmiemw oArbticilliesOantiloinne
of both Gs-Lys6DH and He-P5C was D aO cI :h 1i e0 .v1 e0 d3 9/ Do n0 G Cv 0a 1r 8i o1 u7 As
supports. Although the immobilisation yield of Gs-Lys6DH
reached >90 % with all the supports tested, the best results in
terms of recovered activity (immobilised specific activity [U/mg] /
free specific activity [U/mg]) x 100) were obtained with the most
hydrophilic matrix (Table S2). Specifically, more than 90% of
recovered activity was achieved when using agarose microbeads
as support. In contrast, the activity of He-P5C dropped
dramatically upon immobilisation. To better understand the
factors governing such loss of activity, a model of the enzyme
structure was studied (Figure S9). The distortion of the He-P5C
dimeric structure induced by covalent bonding could explain its
low recovered activity, since the catalytic site is located at the
interface of the two monomers. It has been previously reported
that subunit dissociation of immobilised enzymes can be
prevented by pre- or post-immobilisation polymer coating, such
as with poliethyleneimine (PEI).42,43 In our case, the addition of
PEI during the immobilisation of He-P5C increased the recovered
activity up to 21% (Figure S10 and S11).
Substrate
L-lysine
Imine intermediate (%)
Cyclic amine (%)
57 ± 2
60 ± 2
55 ± 3
99 ± 1
L-lysine ethyl ester
L-ornithine
49 ± 3
55 ± 2
Redox neutral cascade: coupling Gs-Lys6DH and He-P5C
To overcome the limitation of the transaminase-based cascade, a
NAD-dependent lysine dehydrogenase from Geobacillus
stearothermophilus (Gs-Lys6DH) was selected instead to perform
the first catalytic step due to its high reported activity and
excellent stability.41 As the He-P5C has a higher preference for
NADH, the complementarity of the two enzymes is optimal, with
Gs-Lys6DH requiring the oxidised form for the deamination step
and He-P5C the reduced one for P6C reduction, establishing a
closed cofactor regeneration system.
Self-sufficient biocatalytic module with co-immobilised NAD+
Previous work reported
a strategy for the reversible co-
immobilisation of cofactors through amine groups on the support
to retain the cofactor within their microenvironment.44–46 This
approach would be ideally suited to this system allowing for the
reuse and recycle of the cofactors directly on the resin avoiding
the need for external supply. In our case however, despite several
attempts (ESI section 5), only marginal success was obtained.
Likely, the requirement for high ionic strength in the buffer to
maintain He-P5C activity causes the lixiviation of the of the
cofactor over time. On the other hand, when low ionic strength is
used, while the cofactor is retained, a rapid loss of catalytic
efficiency of the system is observed (Figure S12 and S13).
The cofactor recycling capability of the cascade was tested at
+
increasing concentration of L-lysine keeping NAD at 1 mM (Table
2). Complete conversion was observed both at 10 and 50 mM
substrate concentration in 24h, while on a 100 mM scale 62% of
L-lysine was converted during the first 24h with no further
increase.
Table 2. Conversion to L-PA from L-lysine. The reaction was performed with
varying amounts of substrate with 0.5 mg/mL Gs-Lys6DH, 0.5 mg/mL of He-P5C
Flow biocatalysis: Process intensification
and 1 mM of NAD+ in 100 mM phosphate buffer pH 8 at 37 ºC.
While cofactor co-immobilisation on the resin could not be
exploited, the immobilised bi-enzymatic system with catalytic
amounts of cofactor showed excellent performance in batch and
the enzyme mediated synthesis of L-PA on a 10 mM scale was
moved to a continuous flow PBR (packed-bed reactor) (Table 3).
The immobilised biocatalyst was packed into a 2 mL-column
which was fed with L-lysine. The optimization of the flow
conditions was achieved yielding complete conversion at 37 ºC
using just 0.1 equivalent of cofactor and 30 minutes residence
time (Table S4). While in batch conditions the turnover number
Conversion to L-PA
Substrate
concentration (mM)
(
TN) of each free enzyme was 0.04 μmol/min/mg, under optimised
flow conditions it increased to 0.33 μmol/min/mg which is almost a
0-fold increase. Moreover, the stability of the biocatalyst was
4h
24h
1
sustained for more than 24 hours continuous operation at different
flow conditions retaining more than 95% of its initial activity.
Although this system is efficient with a catalytic amount of
>
99 ± 3
>99 ± 2
1
0 mM
0 mM
2
6 ± 2
>99 ± 5
62 ± 3
5
+
NAD , the implementation of cofactor-dependent enzymes is still
limited on a larger scale in flow, as the costly cofactors are lost
downstream.47,48 In order to maximize the efficiency of the system,
we applied an in-line catch-and-release strategy enabling the
separation of the L-PA product from the cofactor (Table 3 and
Figure S14). Using a single scavenger column packed with
1
2 ± 4
100 mM
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Amberlyst® A26, the L-PA produced continuously over 180 min reported so far. In batch, co-immobilisation of the coVifeawcAtortricloenOntlhinee
was purified with a 97% yield, while the cofactor was efficiently same microbead was also achieved. How eDv Oe Ir :,1 t0h . 1e 0 3l o9 w/ D s0 tGa Cb 0i l 1i t8y 1 7oA f
+
trapped by the A26 resin. The NAD could be easily recovered by the enzymes in lower ionic strength buffers which are required to
flushing the column with a slightly acidic buffered solution (pH 6) retain the cofactor on the solid phase, hampered its application.
achieving a recovery yield of more than 87% (Figure S15). As an alternative, a catch-and-release strategy was introduced to
Afterwards, the cofactor solution was recirculated into the system simultaneously separate and reuse the cofactor and efficiently
and re-used for a new flow reaction obtaining similar conversions. purify the target product.
Finally, the A26 column was regenerated with 1M NaOH for 10
min and reused in a new catch-and-release reaction, enhancing
the overall sustainability of the system.
Experimental Section
Materials
Table 3. Flow-assisted production of L-Pipecolic acid with in-line recovery and
reuse of cofactor. The reactions were performed with 10 mM L-Lysine in 50 mM
phosphate buffer pH 8.
6BCL Agarose was acquired from Agarose Beads Technologies.
Polyethyleneimine branched 270,000 Da (PEI270), L-lysine hydrochloride,
D-lysine hydrochloride, L-lysine ethyl ester dihydrochloride, L-ornithine, L-
pipecolinic acid, pyridoxal 5’-phosphate monohydrate were purchased from
Sigma Aldrich. Polyethyleneimine 50% aq. solution branched 60,000 Da
0
.1 M phosphate pH 6
2
mg/g imm. Gs-Lys6DH
mg/g imm. He-P5C
2
Amberlyst® A26
(
PEI60) and Amberlyst® A26 were acquired from Thermo Fisher Scientific.
L-pipecolic acid
All the methacrylate-based supports (EC-EP/S, EP403/S, EC-HFA/S,
HFA403/S, EP113/S resins) were donated by Resindion. Nicotinamide
37 ºC
L-lysine
NAD+
NAD+
+
adenine dinucleotide (NAD ) was purchased from Apollo Scientific. All
other reagents were of analytical grade unless otherwise specified.
PBR
L-PA production
99 %
2.8 mg
Scavenger column
Protein expression and purification
+
L-PA purification
97.3 %
NAD Recovery
The plasmid harbouring He-P5C was transformed into E. coli BL21(DE3)
Star from Thermo Fisher Scientific. 1 L flasks containing 300 mL of Terrific
Broth media were inoculated with 1 mL of an overnight culture in LB and
left growing at 37 ºC and 180 rpm until OD600 reached 0.7-0.9. At that
point, the expression was induced with 1 mM IPTG and the cultures left to
grow for 16 h at 30 ºC and 180 rpm. For Gs-Lys6DH, a single colony of E.
coli BL21 (DE3) Star cells previously transformed with the corresponding
plasmid, were inoculated in autoinduction media ZYP-5052. The cells were
left to grow at 37 ºC and 180 rpm for 20 h. Cells were collected by
centrifugation at 4500 rpm and resuspended in phosphate buffer (50 mM),
NaCl (0.3 M), imidazole (30 mM) pH 8. Cells were disrupted by sonication
>
87.4 %
1
12.5 mg
4.3 mg
Conclusion
Greener strategies for the synthesis of important pharmaceutical
building blocks, such as L-pipecolic acid, are essential to progress with pulses of 5 seconds ON and 5 seconds off at 40% amplitude for 8
towards a sustainable society. In this sense, the use of enzymes min. The insoluble fraction was separated by centrifugation at 14500 rpm
and specially of immobilised biocatalysts is gaining momentum. In for 45 min. The supernatant, after filtration with 0.45 µm filters, was loaded
this work, immobilised cell-free biocatalysts have been used for into a Ni-NTA column using an AKTA-pure FPLC. The protein was eluted
the first time to produce L-pipecolic acid using cheap L-lysine as with phosphate buffer (50 mM), NaCl (0.3 M) and imidazole (300 mM) pH
substrate. The final product is 2000-fold more expensive with 8. He-P5C was dialysed twice against 50 mM phosphate buffer pH 8 while
respect to the current cost of the starting material.49 Therefore, the Gs-Lys6DH was dialysed also two times against phosphate buffer (10 mM)
implementation of cost-efficient approaches towards the synthesis pH 8 containing DTT (0.1 mM) and glycerol (100 g/L) as described
of such a high value chemical, which could be produced on- before.41
demand with high degree of purity, on a simple and portable
Activity assay
system such as a 2mL reactor, appears extremely timely. Both a
transaminase (HeWT) and
a
lysine-6-dehydrogenase (Gs- For Gs-Lys6DH, HeWT and Cb-FDH, the activity assays were performed
Lys6DH) have been proved to efficiently perform the specific ε-
deamination of the substrate. For the reduction of the resulting
cyclic imine, a novel P5C from Halomonas elongata has been
identified as an excellent biocatalyst due to its catalytic activity
and dual cofactor specificity (accepting both NADH and NADPH).
as indicated previously.32,41,50 He-P5C activity assay was performed with L-
Thiazolidine-4-carboxylic acid (10 mM), NAD+ (1 mM) in phosphate buffer
(50 mM) pH 8 at 25ºC. One unit of enzymatic activity was defined as the
reduction of 1 nmol of cofactor per minute and mg of enzyme. For the
immobilised enzyme, the activity was measured using the same conditions
M
Remarkably, the calculated K for NADH is the lowest reported to
date for this type of enzymes. The promiscuous cofactor affinity as the free enzyme. Typically, from 20 to 50 mg of biocatalyst were added
offers not only flexibility on the choice of recycling system, but to a 5 mL reaction pot. At regular interval of times during 10 min, a sample
also impacts on its stability and cost-efficiency. NAD(H) is nearly
0 times less expensive than NADP(H) and has been shown to be
more stable. Moreover, when He-P5C was immobilised together
with Gs-Lys6DH, a redox-neutral cascade was achieved and
successfully applied as packed-bed reactor in flow. The calculated
space-time yield for this system is 2.5 g/L/h which is the highest
was taken to measure the absorbance at 340 nm. In the case of the
immobilised enzyme, the specific activity of the biocatalyst (U/g) was
defined as the reduction or consumption of 1 nmol of the cofactor per
minute and g of biocatalyst.
2
4
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The continuous flow reactions were performed by using VaiewRA2r+ti/cRle4Onflloinwe
Enzyme biotransformation
reactor from Vapourtec equipped with an Omnifit glass column (6.6 mm i.d
DOI: 10.1039/D0GC01817A
Batch reactions with pure soluble enzymes were performed at 37 ºC with a
suitable amount of enzyme in 0.5 mL of reaction mix. Buffers, substrate
and enzyme concentrations were varied as described in this manuscript.
The reaction was monitored by HPLC. To allow for the detection of both
substrates and products, FMOC derivatization was used. In short, to 100
µL of sample with a maximum concentration of substrate (10 mM), 200 µL
of borate buffer (0.1 M) pH 9 were added followed by the addition of 400
µL of FMOC (15 mM) in acetonitrile. The reaction was left for 5 min to
proceed and then 100 µL of the mix were added to 450 µL of 0.1% HCl
and 450 µL of acetonitrile for injection. The samples were analysed by
HPLC (Dionex UltiMate 3000, Waters X-Bridge C18 (3.5 µm, 2.1x100 mm),
x 100 mm lenght) and filled with the biocatalyst (2 – 2.5 g). A first washing
step with buffer was performed at flow rate of 0.2 mL/min to equilibrate the
column. Then, a substrate solution containing L-lysine (10 mM) and NAD+
(
at the specified concentration) in potassium phosphate buffer (50 mM) at
pH 8.0 was pumped towards the column PBR with the biocatalyst (PBR
volume: 1.6 - 2 mL) at 0.1 mL/min for two column volumes. Afterwards, the
flow rate was varied in order to obtain the desired residence time of the
reaction. The resulting flow product was analysed by HPLC following the
protocol describe above. In case of co-immobilised cofactor, the substrate
solution was prepared in phosphate buffer (10 mM) at pH 8.0.
0.8 mL/min, measuring at 210 nm, 250 nm and 265 nm) using a gradient
method from 40:60 to 95:5 (H2O:MeCN 0.1%TFA) over 4 minutes with a
flow rate of 0.8 mL/min. The retention times of the different compounds
were: lysine-FMOC 4.11 min, lysine ethyl esther-FMOC 4.02 min,
ornithine-FMOC 4.00, proline-FMOC 1.98 min, pipecolic acid-FMOC 3.15
min, alanine-FMOC 1.58 min. Molar conversions were calculated through
a standard curve of the product or the substrate.
In-line purification of L-PA and NAD+
Under optimised reaction conditions (substrate solution: NAD+ (1 mM), L-
lysine (10 mM) in phosphate buffer (50 mM) at pH 8; Tº: 37ºC; R.T.: 30
min; molar conversion: >99%), a scavenger column (1 - 1.7 mL) containing
Amberlyst® A26 was connected to the PBR to perform the in-line
purification of L-PA, retaining the cofactor into the column (Figure S12 and
S13). Then, a washing step with phosphate buffer (50 mM) pH 8.0 was
performed for 3 column volumes at R.T. 30 min. Finally, the cofactor was
Enzyme immobilisation
The immobilisation of the enzymes, either in methacrylic or agarose resins,
was performed as described previously.51 In short, 1 g of resin was treated
with 2 mL of modification buffer (sodium borate (0.1 M) and iminodiacetic
acid (2 M) in phosphate buffer (50 mM) pH 8.5) under gentle shaking for 2
h at room temperature. The resin was then filtered, washed three times
with distilled water and mixed with 5 mL of the metal solution (1 M sodium
chloride and 5 mg/mL of cobalt chloride in 50 mM phosphate buffer pH 6)
for another 2 h. Following the same procedure, the sample was washed 3
times with distilled water and then 2 mL of the protein solution were added.
desorbed from the scavenger column and recovered by flushing
phosphate buffer solution (100 mM) at pH 6 for 10 column volumes at R.T.
0 min. The NAD+ recovered from both the washing and the desorption
a
1
steps was collected and reused in a new flow reaction by mixing with a
more concentrated solution of L-lysine. L-PA was quantified by HPLC as
+
described above. NAD was detected by measuring the absorbance at 260
The sample was kept under agitation during
4 h. When He-P5C
nm in the EPOCH microplate reader (Agilent).
immobilisation was carried out with the addition of PEI, 4 mL of enzyme
solution containing PEI (2 g) were used. The support was then filtered and
washed thoroughly with desorption buffer ( EDTA (50 mM) and NaCl (0.5
M) in phosphate buffer (20 mM) pH 7.4) and washed with distilled water.
Finally, 4 mL of blocking buffer (glycine (3 M) in phosphate buffer (50 mM)
pH 8.5) were added and the suspension was left under agitation for 20
hours (in case of further PEI-coating of immobilised biocatalysts, blocking
step was avoided). The beads were washed, collected and conserved in
an appropriate volume of storage buffer at 4ºC.
Conflicts of interests
The authors declare no conflicts of interest.
Acknowledgements
This project was supported by the Welcome Prime Award (F.P.) from the
University of Nottingham. The authors wish to thank Resindion S.R.L. for
donating the EC-EP/S, EP403/S, EC-HFA/S, HFA403/S, EP113/S resins.
Co-immobilisation of NAD+
The previously immobilised biocatalyst was coated with PEI as previously
described.44 Briefly, 10 mL of a solution containing PEI (100 mg) in
Notes and references
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suspension was incubated overnight. Then, the suspension was thoroughly
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