Development of a high throughput screening tool for biotransformations utilising a thermophilic l-aminoacylase enzyme

Article abstract of DOI:10.1016/j.molcatb.2009.12.013

Micro-reactors containing a monolith-immobilised thermophilic l-aminoacylase, from Thermococcus litoralis, have been developed for use in biotransformation reactions and a study has been carried out to investigate the stereospecificity and stability of the immobilised enzyme. The potential to use the developed micro-reactors as a tool for rapid screening of enzyme specificity was demonstrated, confirming that the l-aminoacylase showed a similar substrate specificity to that previously reported of the free enzyme. From this baseline, the technique was employed as a tool to evaluate potential unreported substrates with N-benzoyl- (l-threonine, l-leucine and l-arginine) and N-acetyl- (d,l-serine, d,l-leucine, l-tyrosine and l-lysine) protecting groups. The order of preferred substrates was found to be Phe > Thr > Leu > Arg for N-benzoyl substrates and Phe ? Ser > Leu > Met > Tyr > Trp for N-acetyl substrates. It was found that by using the micro-reactor a significantly smaller quantity of enzyme and substrates was required. It was shown that the micro-reactors were still operational in the presence of selected organic solvents, such as ethanol, methanol, acetone, dimethylformamide (DMF) and dimethylsulfoxide (DMSO). The results indicated that a combination of a small amount of an appropriate solvent (5% DMSO) and a higher reaction temperature could be employed in biotransformations where substrate solubility was an issue.

Full text of DOI:10.1016/j.molcatb.2009.12.013

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 81–86
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Development of a high throughput screening tool for biotransformations utilising
a thermophilic l-aminoacylase enzyme
B. Ngamsoma,1, A.M. Hickeyb, G.M. Greenwaya,∗, J.A. Littlechildb, P. Wattsa,1, C. Wilesa,1
a Department of Chemistry, The University of Hull, Cottingham Road, Hull HU6 7RX, UK
b Henry Wellcome Building for Biocatalysis, School of Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Micro-reactors containing a monolith-immobilised thermophilic l-aminoacylase, from Thermococcus
litoralis, have been developed for use in biotransformation reactions and a study has been carried out to
investigate the stereospecificity and stability of the immobilised enzyme. The potential to use the devel-
oped micro-reactors as a tool for rapid screening of enzyme specificity was demonstrated, confirming that
the l-aminoacylase showed a similar substrate specificity to that previously reported of the free enzyme.
From this baseline, the technique was employed as a tool to evaluate potential unreported substrates
with N-benzoyl- (l-threonine, l-leucine and l-arginine) and N-acetyl- (d,l-serine, d,l-leucine, l-tyrosine
and l-lysine) protecting groups. The order of preferred substrates was found to be Phe > Thr > Leu > Arg
for N-benzoyl substrates and Phe ꢀ Ser > Leu > Met > Tyr > Trp for N-acetyl substrates.
It was found that by using the micro-reactor a significantly smaller quantity of enzyme and substrates
was required. It was shown that the micro-reactors were still operational in the presence of selected
organic solvents, such as ethanol, methanol, acetone, dimethylformamide (DMF) and dimethylsulfoxide
(DMSO). The results indicated that a combination of a small amount of an appropriate solvent (5% DMSO)
and a higher reaction temperature could be employed in biotransformations where substrate solubility
was an issue.
Received 17 August 2009
Received in revised form
23 November 2009
Accepted 14 December 2009
Available online 24 December 2009
Keywords:
Thermophilic enzyme
l-Aminoacylase
High throughput screening
Biotransformations
Micro-reactor
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
organic solvents than their mesophilic counterparts. Although the
thermophilic enzymes have optimal activity at elevated temper-
Enzymes are continuing to be exploited for industrial purposes
[1,2] and in particular, this area has received attention from phar-
maceutical companies for the production of high value chemicals
and new drug intermediates [3]. The use of enzyme catalysts allows
high synthetic performance under mild conditions, minimising
problems such as isomerisation, racemisation and epimerisation;
which can reduce the quantity of side products that are generated
during the process. In addition, enzyme reactions are stereoselec-
tive and therefore can produce optically pure products. The number
of new pharmaceutical compounds on the market that are chiral is
expected to increase to 70% by 2010 [4].
Enzymes are however often unstable under the necessary
operating conditions of the commercial process and enzymatic
processes can be more expensive to develop than those using tra-
ditional synthetic chemistry. Enzymes isolated from thermophilic
organisms have evolved to operate optimally at high tempera-
tures and are inherently more stable to elevated temperatures and
atures they are also active at lower temperatures which allows a
broad operating window.
The l-aminoacylases are important enzymes for industrial
applications since they can be used to resolve a racemic mix-
ture of N-acyl amino acids to produce a range of l-amino acids
and amino acid analogues. We have chosen to use a thermostable
l-aminoacylase from the thermophilic archaeon Thermococcus
litoralis which we have previously cloned, sequenced and over-
expressed in Escherichia coli [5]. The purified enzyme has been
characterised and found to have optimal activity at 85 ◦C in Tris–HCl
buffer at pH 8.0 [5]. It has been shown to exhibit most speci-
ficity towards substrates containing N-benzoyl or N-chloroacetyl
protected amino acids (Scheme 1). This is unlike the more com-
mon commercially available ‘Amano’ aminoacylase which has
preferential selectivity for acetyl > chloroacetyl ꢀ Boc > benzoyl.
It is also has different substrate specificity to other ther-
mophilic l-aminoacylases from Pyrococcus furiosus [6] and the
related Pyrococcus horikoshii [7]. The Thermococcus l-aminoacylase
is
a homotetramer of 43 kDa monomers, and has an 82%
sequence identity to the aminoacylase from P. horikoshii and 45%
sequence identity to a carboxypeptidase from Sulfolobus solfatar-
icus. Aminoacylase inhibitors, such as mono-tert-butyl malonate,
∗
Corresponding author. Tel.: +44 01482 466369; fax: +44 01482 466410.
E-mail address: g.m.greenway@hull.ac.uk (G.M. Greenway).
Tel.: +44 01482 466369; fax: +44 01482 466410.
1
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.12.013

82
B. Ngamsom et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 81–86
Scheme 1. Reaction catalysed by l-aminoacylase; R represents the N-protecting group [2,3].
have only a slight effect on the activity of the Thermococcus
enzyme [5].
the thermophilic l-aminoacylase from T. litoralis was immobilised
onto polymer monoliths formed inside micro-channels, which has
allowed screening of the immobilised enzyme for activity towards
a range of potential substrates with different protecting groups
(benzoyl-, chloroacetyl-, acetyl-, Cbz- and Boc-) and a variety of
amino acids (Phe, Met, Thr, Tyr, Trp, Leu, Ser and Arg), under a range
of conditions.
Previous work has been successful in immobilising the T. litoralis
enzyme [8]. A column bioreactor containing the recombinant l-
aminoacylase immobilised onto Sepharose beads was constructed
with the substrate, N-acetyl-dl-Trp, continuously flowing at 60 ◦C
for 10 days. Using a flow system has the added advantage that any
problems with substrate and product inhibition are eliminated.
Microfluidic systems were first developed for analytical chem-
istry where they are commonly referred to as micro-total analytical
systems (␮TAS) [9]. More recently micro-reactors, having reac-
tion channels between 200 and 1000 ␮m, have been used for fine
chemical synthesis. It is now well established that chemical reac-
tions conducted in such devices generate products in higher yield,
purity and selectivity when compared to batch reactors [10,11]. The
advantages of micro-reactors for enzymatic reactions have been
reviewed by Miyazaki and Maeda [12].
The use of micro-reactors containing an immobilised enzyme
offers an opportunity to minimise the quantities of enzyme, sub-
strate and other reagents required, therefore reducing the costs
when compared to a batch reaction. Several approaches have been
taken towards immobilising enzymes in micro-reactors; the ini-
tial approach tended to be to immobilise the enzyme onto silica or
polymer microparticles which were then packed within the micro-
reactor [13]. A recent development by us has been to pack the
reaction channel with an immobilised gamma lactamase catalyst
and to produce an efficient micro-reactor [14]. The immobilisation
of the enzyme onto the micro-reactor wall, can overcome prob-
lems associated with high back pressures, but the enzyme loading
is lower [15]. The use of membranes within channels to immo-
bilise enzymes has also been investigated, however this is difficult
to achieve [16].
2. Materials and methods
Chemicals were purchased from the sources indicated
and were used as supplied; ethylene dimethacrylate, EDMA
(98%,
Sigma–Aldrich),
Sigma–Aldrich),
glycidylmethacrylate,
GMA
(97%,
3-
2,2-dimethoxy-2-phenyl-acetophenone,
(trimethoxysilyl)propyl methacrylate (98%, Sigma–Aldrich),
DMPA (99%, Sigma–Aldrich), tris(hydroxymethyl) aminomethane,
Tris–buffer (Sigma–Aldrich), dodecanol (99%, Sigma–Aldrich),
cyclohexanol (99%, Sigma–Aldrich), N-benzoyl-l-phenylalanine
(Novabiochem),
l-phenylalanine
(99%,
Sigma–Aldrich),
N-acetyl-l-phenylalanine (98%, Sigma–Aldrich), N-chloroacetyl-
l-phenylalanine (98%, Sigma–Aldrich), N-acetyl-l-tryptophan
(98%, Sigma–Aldrich), N-benzoyl-l-leucine (98%, Sigma–Aldrich),
N-benzoyl-l-threonine (98%, Sigma–Aldrich), N-acetyl-l-tyrosine
(98%,
Sigma–Aldrich),
N-benzoyl-d,l-phenylalanine
(98%,
Sigma–Aldrich), N-t-Boc-l-phenylalanine (Sigma–Aldrich), N-
CBZ-l-phenylalanine (99%, Sigma–Aldrich), N-benzoyl-l-arginine
(99%, Alfa Aesar), N-acetyl-l-methionine (99%, Sigma–Aldrich),
N-acetyl-l-serine (99%, Sigma–Aldrich), N-acetyl-d,l-leucine (98%,
Sigma–Aldrich), N-acetyl-l-lysine (98%, Sigma–Aldrich). Ethanol
and acetonitrile were HPLC grade and purchased from Fisher
Scientific.
In comparison to these techniques, monoliths are well-ordered
macroporous materials with low flow resistance, high reaction
efficiency and good flow-through properties. They can be formed
by in situ polymerisation making them ideal for use as compo-
nents in micro-reaction systems. The application of monoliths in
enzyme immobilisation in micro-reactors has proved to be suc-
cessful for glucose oxidase [17] and a protease [18], where the
monolith was prepared from a mixture of tetramethoxysilane and
methyltrimethoxysilane.
The only reported use of an industrially important acylase
enzyme within a micro-reaction system was for an analytical appli-
cation in which the optical resolution of racemic amino acids was
performed [19]. The work reported focused mainly on the extrac-
tion of the product l-amino acids from uncleaved d,l-substrates
using an in-line aqueous extraction. The enzymatic reaction was
carried out on acetyl-d,l-amino acids at 40 ◦C and using a flow
rate of 0.5 ␮l min−1. Although employing cross-linked polymerised
aminoacylase (Amano) with a polylysine matrix on the surface of
the micro-channels prevented high back pressure, a reduced sur-
face to volume ratio results compared to immobilisation onto a
monolith.
2.1. Preparation of monolithic micro-channels
The borosilicate glass micro-reactor used in this study
was prepared ‘in house’ using standard fabrication tech-
niques [22] and had channel dimensions of 100 ␮m depth,
300 ␮m width and 15 mm length. Poly(glycidylmethacrylate-
co-ethylenedimethacrylate) monoliths were formed inside the
micro-reactor channels using photoinitiation (Fig. 1) [23]. Fig. 1a
shows where the monoliths were formed within the micro-
channels and Fig. 1(b) is a schematic illustrating how the system
was utilised for biotransformation reactions. The channel surfaces
of the micro-reactor were pre-treated to enable covalent attach-
ment of the monolith to the channel walls. This was achieved
by firstly treating the channels with 0.2 M sodium hydroxide for
1 h, washing them with water and then filling them with 0.2 M
hydrochloric acid for 1 h followed by a water and ethanol wash.
The channels were subsequently filled with the silanising agent, 3-
(trimethoxysilyl)propyl methacrylate (20% in ethanol, adjusted to
pH 5 with acetic acid) and left for 1 h. After this the channels were
dried with a stream of nitrogen.
To prepare the monolith, two portions of the monomer solution
consisting of GMA and EDMA (3:1) (structures of both monomers
are provided in Fig. 1) were mixed with one portion of the porogenic
mixture containing 4:1 cyclohexanol:dodecanol, into which 1 wt%
of the initiator (DMPA), with respect to total weight of monomers,
was dissolved. The mixture was then sonicated to ensure thorough
Recent developments involving monolith production over the
past decade suggests that by careful selection of monomer and
porogenic mixtures a higher degree of functionalisation and min-
imal back pressures can be achieved [20,21]. Based on this, the
present study aimed to develop a micro-reaction system, where

B. Ngamsom et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 81–86
83
Fig. 1. (a) The monolithic micro-reactor developed for enzyme immobilisation in this work and (b) schematic illustrating how the system was utilised for biotransformation
reactions where all four reactor channels were simultaneously used.
mixing and purged with nitrogen for 30 min. The micro-channels
were then filled with the polymerisation solution and the areas
where monolith formation was not desired were covered with an
opaque tape. Polymerisation was initiated by placing the micro-
reactors under a UV lamp (365 nm) and irradiated for 30 min. The
epoxy derived monoliths were then washed with methanol and
dried with nitrogen prior to use. Although straight reaction chan-
nels were used for this particular investigation, it must be noted
that this protocol can be readily adapted for any channel pattern
including those containing serpentines.
where the result gave 100% conversion, the results were identi-
cal; error bars are shown for the other experiments (flow rates
>5 ␮l min−1) however in all cases the error was <2%.
The enzyme specificity towards other substrates was
also screened; N-acetyl-l-phenylalanine, N-chloroacetyl-l-
phenylalanine, N-CBZ-l-phenylalanine, N-Boc-l-phenylalanine,
N-acetyl-l-tryptophan, N-benzoyl-l-leucine, N-acetyl-l-tyrosine,
N-benzoyl-l-threonine,
methionine, N-acetyl-d,l-serine,
N-acetyl-d,l-leucine,
N-acetyl-l-
and
N-acetyl-l-lysine
N-benzoyl-l-arginine. The reactions were carried out using
10 mM of each substrate in 100 mM Tris–HCl pH 8.0 buffer and
run at a flow rate of 1 ␮l min−1 at room temperature. Although the
enzyme’s optimum temperature was found to be 85 ◦C, the pre-
liminary experiment conducted in the developed micro-reactor
suggested that the immobilised enzyme, with the monolithic
micro-channels, could readily convert the most specific substrate
(N-benzoyl-l-phenylalanine) (high enzyme activity could be
obtained) at room temperature and a flow rate of 1 ␮l min−1, see
Section 3.1. Experiments on substrate screening were therefore
carried out at these reaction conditions.
2.2. Preparation of l-aminoacylase (E.C. 3.5.1.14)
A starter culture of E. coli over-expressing the T. litoralis
l-aminoacylase was grown overnight in Luria Bertani broth, con-
taining ampicillin (100 mg l−1), at 37 ◦C and at 200 rpm. This culture
was used to inoculate 1 l flasks, containing media as above which
were incubated at 37 ◦C and at 200 rpm. Gene expression was
induced by the addition of isopropyl-beta-d-thiogalactopyranoside
(IPTG) and the cells were harvested 16 h after induction, by cen-
trifugation at 18,000 × g for 30 min at 4 ◦C. A cell-free enzyme
extract was prepared by resuspending the cell paste (10%, w/v) in
100 mM Tris–HCl pH 8.0 buffer. The cell suspension was then sub-
mitted to four cycles of 30 s pulses of sonication. The cell debris was
removed by centrifugation at 12,000 × g for 15 min at 4 ◦C and this
cell-free supernatant was used for all enzyme investigations.
3. Results and discussion
3.1. Development of a micro-reactor for immobilised
thermophilic l-aminoacylase from T. litoralis
The development of a reproducible monolith was challenging
as there were several criteria that had to be considered. In the first
instance the surface of the monolith needs to have an appropri-
ate functional group onto which the enzyme could be anchored;
an epoxy group (using glycidyl methacrylate as the monomer and
ethylene dimethacrylate as the cross-linking agent) was found to
enable efficient enzyme immobilisation to be achieved. The mono-
lith was also required to have a high surface area for efficient
immobilisation but, at the same time to have a high porosity to
prevent the build-up of back pressure, especially during enzyme
introduction; furthermore it needs to be formed in a reproducible
manner. Attempts to make highly porous monoliths with low back
pressure were made by (a) adjusting the monomer to porogenic
mixture ratios and (b) varying the alcohol (1-propanol, 1-butanol,
heptanol, octanol, decanol, dodecanol and cyclohexanol) employed
in the porogenic mixture (results not shown here). The monoliths
with least back pressure were prepared using cyclohexanol and
dodecanol (4:1) as the porogenic mixture.
The SEM images (Fig. 2) show the internal morphology of a
prepared monolith which demonstrates that the porous polymer
was homogeneous across the entire monolith. The absence of voids
between the channel wall and the monolith also indicates that the
monoliths were covalently bound to the surface of the glass chan-
nels. It is important that the monolith is bound to the walls so that
it would not be pumped out of the micro-reactor during the flow
reaction and/or the chemicals would not by-pass the immobilised
enzyme.
2.3. Enzyme immobilisation and evaluation of immobilised
enzymatic activity
The cell-free supernatant containing recombinant l-
aminoacylase (E.C. 3.5.1.14) from the thermophilic archaeon,
T. litoralis, was immobilised onto the monolithic channels via the
primary amine group of the enzyme within the micro-reactors by
passing a solution of l-aminoacylase in Tris–HCl buffer (1/100, v/v)
through the channels using a syringe pump (Bioanalytical Systems
Inc., MD-1001) at a flow rate of 1 ␮l min−1 for 3 h. Any unbound
enzyme was then washed out of the micro-channels with Tris–HCl,
pH 8.0 buffer for 30 min prior to use. Substrates were screened by
dissolving them in 100 mM Tris–HCl buffer pH 8.0. The substrate
solutions were charged into a 1 ml syringe and supplied to the
monolithic micro-reactor using a microfluidic pump. The activity of
the immobilised enzyme to each substrate was investigated at flow
rates of 1–8 ␮l min−1, at both room temperature and 50 ◦C. The
initial biotransformations used 10 mM N-benzoyl-l-phenylalanine
as the model substrate and the reaction products analysed by HPLC
equipped with a 4.6 × 150 mm C18 BDS Hypersil column (Thermo
Electron Corporation, UK) using 35% acetonitrile in water as the
mobile phase at a flow rate of 2 ml min−1 at ambient temperature;
the product and substrate peaks were detected by UV adsorption
at 210 nm. Activities towards the N-protected amino acids were
also determined by a modified Cd/Ninhydrin method previously
reported [5]. The experiments were conducted in duplicate and

84
B. Ngamsom et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 81–86
Fig. 2. SEM images of the synthesised monoliths formed within micro-reactors.
Prior to immobilisation of the enzyme, flow studies were con-
increase in flow rate. One long term goal is therefore to prepare
more porous monoliths to enable higher flow rates to be used which
would allow a higher product throughput to be achieved. It must
be noted however that under the aforementioned conditions all
monoliths prepared were found to be reproducible with respect to
flow rate and substrate conversion.
As the idea of enzyme immobilisation is to generate enzymes
with higher operational stability and improve the storage life-
time, the immobilised enzyme was subsequently subjected to
a stability test both during continuous use and when stored in
buffer at 4 ◦C. When the enzyme was immobilised onto the mono-
lithic micro-reactor and operated at room temperature it produced
reproducible substrate conversion over 120 h (continuous use),
with no loss in activity. When the micro-reactor was operated at a
ducted and it was found that flow rates up to 8 ␮l min−1 could be
utilised within these systems however applying higher flow rates
led to high back pressure, resulting leaks at the inter-connections.
Once the monolith was successfully produced, and evaluated
as described, the thermophilic enzyme l-aminoacylase was then
immobilised in situ. As described previously this was achieved
by pumping the enzyme through the micro-reactor channels and
allowing it to bind to the monolith via reaction between the amino
groups of the enzyme and the epoxy terminal groups on the sur-
face of the monolith. It should be emphasised that it was not
possible to measure the enzyme loading because the amount of
enzyme was too small to quantify and therefore the activity of the
enzyme on the monolith was monitored indirectly via the conver-
sion of the substrate into the desired product. The success of this
methodology was initially investigated by measuring its ability to
convert the model substrate, 10 mM N-benzoyl-l-phenylalanine to
l-phenylalanine. The reaction was carried out in Tris–HCl pH 8.0
buffer where highest activity was observed for the free enzyme
[5] and the enzyme activity was investigated at flow rates of
1–8 ␮l min−1 (Fig. 3). Although this enzyme is known to have opti-
mal activity at 85 ◦C [5,8], it was found that complete substrate
conversion could be obtained in the micro-reactor at room temper-
ature (≤4 ␮l min−1); at higher flow rates the substrate conversion
decreased. Since the enzyme is thermophilic, the reaction temper-
ature can be increased above ambient conditions. The operating
window in the micro-reactor was therefore investigated by increas-
ing the reaction temperature to 50 ◦C and increasing the flow rate
up to 8 ␮l min−1; the highest flow rate possible in the monolith
without having back pressure problems. At this higher tempera-
ture, 100% conversion of the substrate was obtained despite the
Fig. 3. Effect of flow rate on the activity of the enzyme in converting 10 mM. N-
Benzoyl-l-phenylalanine in 100 mM Tris–HCl buffer pH 8.0 at room temperature
and 50 ◦C.

B. Ngamsom et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 81–86
85
Scheme 2. l-Aminoacylase catalyses the conversion of N-benzoyl-l-phenylalanine to l-phenylalanine while it is inactive in cleaving the d-form.
higher temperature (50 ◦C) it showed no activity loss after 50 h (the
reaction was terminated after this point), confirming the enzymes
stability at this temperature. These results indicate that the contin-
uous reaction system consisting of the enzyme immobilised onto
the monolithic micro-channels is a suitable tool for further devel-
opment.
ity to convert the potential substrates was measured at room
temperature, Table 1.
At 1 ␮l min−1, N-benzoyl-l-Phe, N-chloroacetyl-l-Phe and N-
acetyl-l-Phe were readily hydrolysed by the enzyme (100%
conversion), whereas lower conversions were found for Boc-
and Cbz-protecting groups; 40.9 and 30.9%, respectively. To
study in more detail, the enzyme specificity towards benzoyl-
, chloroacetyl- and acetyl-groups the reaction flow rate was
3.2. Utilising the developed micro-reactor for substrate screening
increased to 2 ␮l min−1
. At this increased flow rate, the
enzyme remained very active towards N-benzoyl- (100% con-
version) but lower substrate conversions were obtained for
N-chloroacetyl- (90.1%) and N-acetyl- (66.1%). The order of pre-
ferred N-protecting groups can therefore be summarised as
follows: benzoyl > chloroacetyl > acetyl > Boc > Cbz. Generally, the
immobilised l-aminoacylase behaved similarly to the free l-
aminoacylase but it differed in that it was more active towards
Boc- than CBZ-protecting groups. This may be due to the fact that
the CBZ-group is more sterically demanding than the Boc-group,
preventing it from accessing all of the immobilised enzyme.
Additionally, substrate specificity was investigated for different
amino acids. Having found that the most specific protecting group
for the thermophilic l-aminoacylase was benzoyl-, other amino
acids with this protecting group which have not been reported
before were chosen for the investigation, i.e. threonine, leucine
and arginine. The order of preferred amino acids was found to be
Phe > Thr > Leu > Arg. The results confirm the enzymes preference
for Phe over Arg regardless of the protecting group of the substrates
(either acetyl or benzoyl; much higher activity of Phe over Arg was
reported when the study was carried out using N-acetyl protecting
group on both amino acids) [5]. It is interesting to note that despite
poor activity towards N-acetyl-l-arginine, the enzymes ability to
convert N-benzoyl-l-arginine was moderate (43.6%).
Substrates containing other amino acids with N-acetyl-
protecting group were also examined both on previously reported
substrates (Phe, Met and Trp) and other new substrates (Ser,
Leu and Tyr). Substrate screening over serine and leucine were
performed using the racemic form, where only 50% maximum
substrate conversion could be expected. The order of preference
was found to be Phe ꢀ Ser > Leu > Met > Tyr > Trp; the enzyme was
nevertheless found to be inactive towards lysine. The immo-
bilised enzyme behaved in a similar manner to the free enzyme
towards Phe, Met and Trp. Generally, the enzyme appears to
prefer hydrophobic and uncharged amino acids, with a similar
trend observed when taking N-benzoyl- or N-acetyl substrates into
consideration. For instance, among the N-benzoyl substrates, the
enzyme was least specific towards arginine which is hydrophilic,
polar and charged at the operating pH 8.0. Additionally, the
enzyme was incapable of cleaving N-acetyl-l-lysine which is also
hydrophilic, polar and charged at the operating pH.
Having developed a successful micro-reaction system where the
thermophilic l-aminoacylase from T. litoralis was immobilised onto
the monolithic micro-channels and 100% conversion of 10 mM N-
benzoyl-l-phenylalanine to l-phenylalanine was achieved at room
temperature, the next step was to investigate the use of the micro-
reactor as a screening tool for other substrates. The first experiment
looked at the enzyme stereospecificity following its immobilisation
onto the monolithic micro-channels, using a racemic substrate of
10 mM N-benzoyl-d,l-phenylalanine. In this biotransformation the
enzyme only cleaves the l-form while leaving d-form unreacted,
Scheme 2. The maximum substrate conversion was found to be 50%
as predicted (Table 1) confirming the enzyme’s stereospecificity.
To confirm that the immobilised enzyme was working in a
reliable manner within the micro-reactor environment, substrate
screening was initially carried out on a set of substrates previously
studied [5,8]. In the first instance the investigation was focused on
determining the most preferred N-protecting group for the amino
acid, this protecting group was subsequently used to study what
other amino acids could be converted by the enzyme.
In previously published work, the enzyme has shown greater
specificity towards benzoyl- and chloroacetyl-, rather than acetyl-
or other N-protecting groups [5]. In order to determine whether
the immobilised enzyme retained its selectivity towards potential
N-protecting groups, it was tested with substrates containing the
same amino acid residue, Phe, with different N-protecting groups
(benzoyl-, chloroacetyl-, acetyl-, Boc- and Cbz-). The enzymes abil-
Table 1
Activity of immobilised l-aminoacylase towards its potential substrates, recorded
using the micro-reactor operated at room temperature with a flow rate of 1 ␮l min−1
.
All substrates were screened at the concentration of 10 mM.
Substrate
Substrate conversion (%)
N-Benzoyl-d,l-Phe
N-Benzoyl-l-Phe
N-Chloroacetyl-l-Phe
N-Acetyl-l-Phe
N-t-BOC-l-Phe
N-CBZ-l-Phe
50.0
100, 100.0a
100, 90.1a
100, 66.1a
40.9
30.9
N-Benzoyl-l-Thr
N-Benzoyl-l-Leu
N-Acetyl-d,l-Ser
N-Benzoyl-l-Arg
N-Acetyl-d,l-Leu
N-Acetyl-l-Met
N-Acetyl-l-Tyr
N-Acetyl-l-Trp
N-Acetyl-l-Lys
68.3
52.2
25.6b
43.6
After being employed to screen various substrates, the micro-
reactor was again subjected to the most preferred substrate, N-
benzoyl-l-Phe, in order to check whether the enzyme was still fully
active; as expected its ability to convert the preferred substrate to
l-phenylalanine was found to be retained.
20.5b
38.7
33.3
7.0
0
In summary, the use of an immobilised enzyme within mono-
lithic micro-channels has proved to allow rapid screening in a
a
Substrate conversions observed at 2 ␮l min−1
Substrate conversions out of a maximum of 50%.
.
b

86
B. Ngamsom et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 81–86
4. Conclusions
A micro-reactor containing polymer monoliths formed inside
the micro-channels was developed for biotransformation reac-
tions catalysed by the thermophilic l-aminoacylase from T. litoralis.
The micro-reactor proved to be a useful tool for high through-
put screening of potential substrates, where individual substrates
could be evaluated in approximately 10 min after reactor charac-
terisation. This robust system was reliable (as we determined that
enzyme activity was retained for 50 h even at 50 ◦C) and it also
reduced enzyme consumption with high stability under both oper-
ational and storage conditions. Additionally, it could be operated
at room temperature to yield similar enzyme activities for some
preferred substrates (N-benzoyl-l-Phe and N-chloroacetyl-l-Phe).
The operating temperature could also be increased for less pre-
ferred substrates to gain higher enzyme activities. Clearly using the
reactor at room temperature was advantageous as we had demon-
strated that the activity was retained for a longer period of time.
The stability of the enzyme micro-reactor system as well as the
enzyme activity in the appropriate solvent to aid substrate solubil-
ity suggested the potential use of this system for high throughput
biotransformations.
Fig. 4. Effect of solvents on substrate conversion; the reaction was performed with
10 mM N-benzoyl-l-phenylalanine at room temperature for a period of 1 h using a
flow rate of 1 ␮l min−1
.
reliable manner compared to utilising the free enzyme in a conven-
tional batch system. The micro-reactor experiments were analysed
every hour in order to evaluate the reaction efficiency over time,
however in screening experiments it was possible to conduct
individual experiments within 10 min, the limitation being the sen-
sitivity of the analytical method used for quantification. The major
advantages include the lower quantity of reagent required, the re-
useable nature of the enzyme in the micro-reactor and the milder
reaction conditions introduced by the use of micro-reactors.
The system developed was also useful for screening new poten-
tial substrates with a N-benzoyl-protecting group which was not
the most specific substrate for l-aminoacylase from other sources.
Further system development will be directed towards improv-
ing the porosity of the monoliths, with a view to a reduction in
back pressure, since this should allow higher flow rates and subse-
quently a higher throughput to be attained.
3.3. Use of the developed micro-reactor system in organic
solvents
Acknowledgement
Many biotransformation reactions need to be carried out in
organic solvents due to the insolubility of the substrates being
used and where inactivation is observed for homogeneous biocat-
alysts, enzyme immobilisation onto solid supports often helps to
stabilise enzymes for use under these conditions. Rapid inactivation
of l-aminoacylase from T. litoralis in the presence of 50% organic
solvents at 60 ◦C was previously reported [5] and the presence of
10% N,N-dimethylformamide (DMF) was also found to have caused
more than a 70% decrease in stability of aminoacylase from hog
kidney after 50 min at 25 ◦C [24].
This work was financially supported by Engineering and Physi-
cal Sciences Research Council (EPSRC), UK.
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