Characterization of a novel amine transaminase from Halomonas elongata

Article abstract of DOI:10.1016/j.molcatb.2015.07.009

Chiral amines are indispensable building blocks in the production of biologically active compounds. They are fundamental for the pharmaceutical industry, both as active molecules themselves and as chiral auxiliaries in asymmetric synthesis; however, the available synthetic strategies often present disadvantages. ω-Transaminases (ω-TAs) appear as an attractive alternative by driving the stereoselective amination of prochiral ketones. HEWT is a novel amine transaminase from the moderate halophilic bacterium, Halomonas elongata DSM 2581, which is highly (S)-selective, being able to fully convert (S)-1-phenylethylamine to acetophenone and showing no activity with the corresponding (R)-1-phenylethylamine. HEWT has a broad substrate scope, active with a range of amino donors and acceptors, and naturally accepts isopropylamine (IPA) as amino donor in asymmetric synthesis providing a 41% conversion of pyruvate in 24 h at 37°C starting with 1:1 molar ratio between the reagents. HEWT also accepts ortho-xylylenediamine as amino donor in for amine synthesis, in particular, with benzaldehyde yielding high conversions between 90 and 95%. The enzyme is also tolerant to the presence of cosolvents up to 20% making it a promising candidate for industrial applications.

Full text of DOI:10.1016/j.molcatb.2015.07.009

Accepted Manuscript
Title: Characterization of a novel amine transaminase from
Halomonas elongata
Author: Lorenzo Cerioli Matteo Planchestainer Jennifer
Cassidy Davide Tessaro Francesca Paradisi
PII:
S1381-1177(15)30031-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.07.009
MOLCAB 3200
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Journal of Molecular Catalysis B: Enzymatic
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Revised date:
Accepted date:
5-5-2015
14-7-2015
15-7-2015
Please cite this article as: Lorenzo Cerioli, Matteo Planchestainer, Jennifer
Cassidy, Davide Tessaro, Francesca Paradisi, Characterization of a novel amine
transaminase from Halomonas elongata, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2015.07.009
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Characterization of a novel amine transaminase from Halomonas elongata
Lorenzo Cerioli,a, b, # Matteo Planchestainer,a, # Jennifer Cassidy,a Davide Tessaro,b, c Francesca Paradisi a, *
a
UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland, Fax:
(+) 353-1-716 2501
b
Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Piazza
Leonardo da Vinci 32, 20133 Milano, Italy
c
Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “The Protein Factory”, Politecnico di Milano
and Università degli Studi dell’Insubria, Via Mancinelli 7, 20131 Milano, Italy
# Lorenzo Cerioli and Matteo Planchestainer contributed equally to the paper
E-mail
Lorenzo Cerioli: lorenzo.cerioli@polimi.it
Jennifer Cassidy: jennifer.cassidy.1@ucdconnect.ie
Matteo Planchestainer: matteo.planchestainer@ucdconnect.ie
Davide Tessaro: davide.tessaro@polimi.it
Corresponding author
Francesca Paradisi
E-mail: francesca.paradisi@ucd.ie
Telephone: (+) 353-1-716 2967
Fax: (+) 353-1-716 2501
Address: UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland

Abstract
Chiral amines are indispensable building blocks in the production of biologically active compounds. They are
fundamental for the pharmaceutical industry, both as active molecules themselves and as chiral auxiliaries in
asymmetric synthesis; however, the available synthetic strategies often present disadvantages. -Transaminases (-
TAs) appear as an attractive alternative by driving the stereoselective amination of prochiral ketones. HEWT is a
novel amine transaminase from the moderate halophilic bacterium, Halomonas elongata DSM 2581, which is highly
(S)-selective, being able to fully convert (S)-1-phenylethylamine to acetophenone and showing no activity with the
corresponding (R)-1-phenylethylamine. HEWT has a broad substrate scope, active with a range of amino donors and
acceptors, and naturally accepts isopropylamine (IPA) as amino donor in asymmetric synthesis providing a 41%
conversion of pyruvate in 24 h at 37 °C starting with 1:1 molar ratio between the reagents. HEWT also accepts
ortho-xylylenediamine as amino donor in for amine synthesis, in particular, with benzaldehyde yielding high
conversions between 90-95%. The enzyme is also tolerant to the presence of cosolvents up to 20% making it a
promising candidate for industrial applications.
Keywords: Amine transaminase • Biocatalysis • Asymmetric synthesis • Isopropylamine • o-Xylylenediamine
Graphical Abstract

 Introduction
Classification of transaminases have recently been reviewed [1]. Amine transaminases are most well know for
asymmetric chiral amine synthesis and some are also able to convert ω-amino acids [2]. They do not require the
presence of carboxylic moieties in the substrate to convert chiral amines. Transaminases are a family of enzymes
with high potential in biotechnological applications. They can be very useful for the enantioselective production of a
series of compounds with high value such as chiral amines and enantiopure amino alcohols which find use in many
chemical fields; above all, for the synthesis of biologically active compounds (Scheme 1) [3, 4]. The synthesis of
enantiopure amines by enzymatically catalyzed reactions presents several advantages as an alternative to traditional
approaches such as mild reaction conditions, high stereoselectivity, fewer synthetic steps, potential total substrate
conversion and no environmental issues unlike in the case of transition metal catalysts [5, 6]. However to be
recognized as economically viable, the catalyst must have a high space-time yield, which refers to the amount of
product produced per quantity of catalyst per unit time [7].
Scheme 1. Asymmetric synthesis by -transaminase
Within the class of -transaminases, amine transaminases play a central role in the biocatalytic preparation of
enantiopure amines. Some transaminases displayed high turnover rate, high stability, broad substrate specificity and
no requirement for external cofactors [8, 9]. Issues such as unfavourable equilibrium constant, inability to accept
larger molecules, substrate and product inhibition and reduced stability at extreme pHs, temperature and organic
solvents are often still challenging but by protein engineering several aspects can be addressed. For instance, the
remarkable work by Savile et al. led to an engineered version of ATA-117, an (R)-selective variant of the -
transaminase from Arthrobacter sp. which was able to accept the very bulky substrate precursor of sitagliptin [10].
Recently, an alternative substrate to the use of an excess of the amino donor to shift the equilibrium of the reaction
has been reported by O’Reilly [11]. Still, it is apparent that the pool of enzymes effectively suitable for these
biotransformations is limited and there is scope to investigate new sources and novel enzymes [12].
As part of a larger project with the aim to identify new biocatalysts with particular characteristics such as high
temperature resistance, organic solvent compatibility and uncommon substrate specificity, we focused our interest
on enzymes from extremophiles. The adaptation process for extremophiles has affected the features of their enzymes
providing them with improvements in respect to their mesophilic counterparts [13]. In particular, we have developed
an interest in halophilic organisms and their enzymes as they often have a remarkable tolerance to organic solvents
[14-16].

In this study we identified, cloned, and expressed a putative amine transaminase (HEWT) from the genome of the
bacterium Halomonas elongata; this is a moderate halophile which evolved an organic-osmolyte strategy to
overcome the high osmotic pressure of its natural environment [17]. H. elongata does not exhibit the extensive
adaptation of the intracellular macromolecules present in archaea halophiles, which preferentially evolved to
maintain their proper osmotic pressure by the accumulation of high cytoplasmic concentration of potassium chloride
[18]. On the contrary, H. elongata preserves an appropriate cytoplasm osmotic pressure by accumulation and/or
biosynthesis of organic solute.
Since H. elongata is a haloadapted bacterium, it allows for heterologous expression of its proteins inside a common
mesophilic host like Escherichia coli hence avoiding less conventional expression systems required for true
halophilic proteins [19, 20]. H. elongata aminotransferase sequence presents an identity of 38% with the -TA from
Vibrio fluvialis and 55% with -TA from Chromobacterium violaceum. The former is the most investigated (and
commercially available) -TA [21]. The latter was identified in a precedent study for its 38% sequence identity with
the V. fluvialis enzyme and shares with it the substrates preference for aromatic amines [3]. Moreover, the -TA
from C. violaceum presents an uncommon selectivity for TAs, exploiting the amination on ,_-dihydroxyketones
which are not usually accepted as substrates [22, 23].
 Materials and methods

spuC gene and HEWT bioinformatic analysis
The gene named spuC (HELO_1904) was identified inside the genome of the moderate halophilic bacterium
Halomonas elongata DSM 2581. The genome of this bacterium was obtained from DSMZ (Braunschweig,
Germany). Conserved domain analysis was performed on the amino acid sequence of HEWT using the NCBI
conserved domain database (CDD) (http://www.ncbi.nlm.nih.gov/cdd/). A specific hit was obtained with the
OAT_like, acetyl ornithine aminotransferase family a member of the pyridoxal phosphate (PLP)-dependent aspartate
aminotransferase superfamily (fold I). The amino acid sequence of HEWT was subsequently submitted to the web
based server Phyre2 (Protein Homology/AnalogY Recognition Engine) for protein structure prediction [24]. The
sequence of the H. elongata aminotransferase showed a sequence identity of 55% with 100% of query cover for C.
violaceum CV2025 -TA and a sequence identity of 38% with 100% of query cover for V. fluvialis js17 -TA.
From the sequence alignment was possible to identify the three residues considered to be responsible for the
transaminase catalytic activity: D255 Aspartic acid: salt bridge/H-bond to N1 of pyridoxal 5_-phosphate, K284
Lysine: Schiff base with pyridoxal 5_-phosphate, R412 Arginine: salt bridge/H-bond with -carboxylate group of
substrate [25-27].

Molecular biology tools, bacteria, enzymes and chemicals

Phusion™ DNA polymerase was purchased from Finnzymes (Espoo, Finland). PCR primers production and protein
sequencing were performed by Eurofins MWG Operon (Ebersberg, Germany). Molecular weight marker
HyperLadder™ I was purchased from Bioline (London, UK). Agarose Gel DNA Extraction Kit, High Pure PCR
Product Purification Kit and PD-10 Columns were purchased from Roche (Mannheim, Germany). Plasmid
extraction kit GeneJET™ Plasmid Miniprep Kit was purchased from Thermo Fisher Scientific GmbH (Dreieich,
Germany). T4 DNA Ligase, restriction enzymes EcoRI and BamRI and broad range protein marker P7702 (2-212
kDa) were purchased from New England Biolabs (Beverly, MA, USA). The pRSET B vector was purchased from
Invitrogen (Carlsbad, CA, USA). E. coli XL10-Gold® Ultracompetent cells were purchased from Stratagene (San
Diego, USA). E. coli BL21(DE3) competent cells were purchased from Novagen (Darmstadt, Germany). Ampicillin
sodium salt was purchased from Molekula Ltd. (Gillingham, UK). Millex®HV 0.45 μm PVDF filters were
purchased from Millipore Ltd. (London, UK). His-tagged protein purification columns Ni-NTA Superflow
Cartridges were purchased from QIAGEN Gmbh (Hilden, Germany). Stain/DeStain-Xpress™ protein detection kit
was provided by Enzolve Technologies Ltd. Enzolve Technologies Ltd. (Dublin, Ireland). All chemical reagents,
unless stated otherwise, were purchased as analytical grade from Sigma-Aldrich Chemie Gmbh (Munich, Germany).

Construction of the expression vector
The spuC gene was amplified by PCR from genomic DNA of H. elongata with Phusion DNA polymerase with the
oligonucleotide primers FWD-spuC (5’-AAAGGATCCGATGCAAACCCAAGACT ATCAGG-3’) and REW-spuC
(5’-AAAGAATTCTCATGCGGTTGG CTCCTCTTGC-3’). The primers were designed to incorporate BamHI and
EcoRI restriction sites, respectively (underlined). The manufacturer’s protocol for Phusion DNA polymerase PCR in
50 µL reaction was used. PCR was carried out with 1 U of enzyme in presence of H. elongata genome (2 ng/µL)
and the two primers (0.5 µM each) with the following program: 3 min of initial denaturation at 95 °C and 35 cycles
of 30 s denaturation at 94 °C, 30 s annealing at 68 °C and 50 s extension 72 °C. The 35 cycles were followed by 5
min of final elongation at 72 °C. The gel-purified PCR product was digested with EcoRI and BamHI for 16 h
following the manufacturer’s guidelines and cloned into digested pRSET B and used to transform E. coli XL10-
Gold® Ultracompetent cells. The construct was verified by sequencing and the obtained plasmid was named
pHESPUC.

Expression of the spuC gene in E. coli
E. coli BL21(DE3) competent cells were transformed with pHESPUC plasmid. Expression was tested both in LB
broth and ZYM-5052 auto-induction medium.
With the first method, cells were grown in 300 mL LB broth in the presence of ampicillin 100 µg/mL at 37 °C
shaking at 200 rpm starting with a 3 mL inoculum of seed culture previously grown overnight. The culture was
grown until an OD600 of around 0.6 was reached and then the expression was induced with isopropyl -D-1-

thiogalactopyranoside (IPTG) 1 mM and the culture was allowed to grow at 37 °C, shaking at 200 rpm. After 20 h
the cells were harvested, weighed and then frozen at -20 °C.
With the second method, cells were grown in 300 mL of ZYM-5052 medium for auto-induction [28] (N-Z-Amine
1%, yeast extract 0.5%, KH2PO4 50 mM, Na2HPO4 50 mM, (NH4)2SO4 25 mM, NaSO4 5 mM, MgSO4 2 mM,
Glycerol 0.5%, Glucose 0.05%, -Lactose 0.2%, 1000x trace elements 0.2%) in presence of ampicillin 100 µg/mL
at 37 °C shaking at 200 rpm. The cells were harvested when the OD600 of the culture stopped increasing, weighed
and then frozen at -20 °C.
The pellet was thawed and resuspended in the lysis buffer containing potassium phosphate buffer pH 8.0 (50 mM)
and pyridoxal 5′-phosphate (PLP) (0.1 mM) employing 7.5 mL per gram of pellet. Cells were disrupted by
sonication at 4 °C using Sonics VCX 130 with 10 cycles of 30 s of sonication and 30 s of cooling. After
centrifugation (38,000×g, 4 °C, 45 min), the supernatant was clarified by filtration (0.45 μm filter) before
chromatography and the activity of the raw extract was tested with the spectrophotometric enzymatic assay.

Purification
Chromatography was performed using an ÄKTA Purifier (GE Healthcare, Little Chalfont, UK). The filtered cellular
extract was taken up in 0.5 M NaCl and loaded into a 5 mL column of Ni-NTA Superflow pre-charged with NiSO4
(0.1 M). The column was washed at a flow rate of 5 mL/min with at least 10 column volumes of phosphate buffer
(pH 8, 50 mM, containing 100 mM NaCl, 0.1 mM PLP and 30 mM imidazole), and the enzyme was eluted with
phosphate buffer (pH 8, 50 mM, containing 100 mM NaCl, 0.1 mM PLP and 300 mM imidazole) after an
intermediate step with the 10% of elution buffer to remove non-specifically bound proteins. The fractions containing
the enzyme were desalted via size exclusion chromatography with a 20 mM phosphate buffer, pH 8, containing 0.1
mM PLP. The purified enzymes were conserved at 4 °C for short term storage or -20 °C for long term storage. The
enzymatic activity was tested following the spectrophotometric enzymatic assays (see below) and the obtained pure
enzyme was named with the acronym HEWT (Halomonas elongata -transaminase).

Size Exclusion Chromatography
The molecular mass of the native HEWT was determined by size exclusion chromatography on a Superdex 200 high
resolution 10/30 column with a total bed volume of 24 mL (GE Healthcare) equilibrated with 20 mM sodium
phosphate buffer, pH 8.0, containing 150 mM NaCl. Purified enzyme solution (100 µL) was injected and the
experiment was run at a flow rate of 0.2 mL/min. Fractions were collected and those corresponding to the eluted
peak were assayed for activity to confirm the protein was still correctly folded. Protein molecular weight markers
(Sigma-Aldrich) used to prepare the calibration curve were β-amylase (200 kDa), yeast alcohol dehydrogenase (150
kDa) and bovine serum albumin (66 kDa), with Blue Dextran (2,000 kDa) being used to indicate the void volume.
The samples for the calibration curve were run in 50 mM sodium phosphate buffer, pH 8.0.


Protein determination and SDS-PAGE analysis
The concentration of the purified protein was determined spectrophotometrically by UV absorption at 280 nm using
urea 6 M as denaturing agent. The extinction coefficient 60850 M-1 cm-1, at 280 nm, measured in water, was
estimated by ExPASy ProtParam tool, accessible from the ExPASy website (www.expasy.ch) [29]. SDS-PAGE was
carried out employing a 12% polyacrylamide gels, stained with Coomassie Brilliant Blue R250 [30] and a broad
range protein marker (2–212 kDa) was used for determination of relative molecular weight.

Spectrophotometric enzymatic assays
A kinetic assay derived from Schatzle, et al. [31] was used as standard enzymatic assay using pyruvate as the amino
acceptor except where stated otherwise. The reactions was carried out at 25 °C employing a reaction mixture
−)
−)
containing 1 mL phosphate buffer (50 mM, pH 8), 2.5 mM (S)-( -1-phenylethylamine ((S)-( --
methylbenzylamine), 2.5 mM pyruvate, 0.25% DMSO, 0.1 mM PLP and an appropriate amount of enzyme. These
parameters were modified in order to investigate the behaviour of the enzyme in different reaction conditions.
Modifications to the standard assay are indicated when applied. The activity was estimated following the production
of acetophenone during the first three minutes of reaction at 245 nm using a Varian Cary 50 Scan UV–Visible
spectrophotometer equipped with a Cary single cell Peltier temperature controller.
In the study of the effect of the pH on enzyme activity and stability of the enzyme, instead of the phosphate buffer, a
universal buffer was employed. This system contains 25 mM citric acid, 25 mM KH2PO4, 25 mM Tris, 12.5 mM
Na2B4O7, and 25 mM KCl [32, 33].
The effect of additives on the transaminase was checked by incubating the enzyme at 25 °C in presence of the
indicated concentration of each chemical. For the test on enzyme stability at different additive concentrations the
solutions of additives were prepared in phosphate buffer pH 8, 0.5 M and the pH was adjusted to pH 8 before use
−)
(Figure 4.A). For the effect of interaction between (S)-( -1-phenylethylamine (5 mM) and pyridoxal 5′-phosphate
(5 mM) on the enzyme stability phosphate buffer pH 8, 0.05 M was employed, (Figure 4.B). The residual activity
was measured in both cases with the standard spectrophotometric assay.
The effect of cosolvents on HEWT activity and stability was studied storing the enzyme in the presence of either 10
or 20% (v/v) of cosolvent at 4 °C for 22 h. Before and after incubation the residual activity was determined with the
standard spectrophotometric enzymatic assay in presence of the corresponding amount of co-solvents.

Enzymatic reaction
The enzymatic reactions were carried out at 37 °C in pH 8.0 potassium phosphate buffer 100 mM and 15% v/v
purified enzyme solution corresponding to 0.89 U/mL for the screening of the amino donors, and 0.71 U/mL for the
screening of the amino acceptor reactions. The reaction mixture contained 10 mM enantiopure amino donor (20 mM
if racemic), 10 mM amino acceptor and 0.05 mg/mL of HEWT in a reaction volume of 200 µL. For the

determination of enantioselectivity, reactions were carried out at 37 °C in pH 8.0 potassium phosphate buffer 100
mM in 1 mL, containing 20 mM rac-1-phenylethylamine, 10 mM pyruvate, 0.1 mM PLP with 0.1 mg/mL of
HEWT. As a control, the reactions were set up as previously described but without the addition of HEWT, in which
case only starting material was detected. The activity of the enzyme in the various reaction conditions was
established employing (S)-(−)-1-phenylethylamine as amino donor and pyruvate as acceptor. One enzymatic unit
was defined as the amount of enzyme that converts 1 µmol of (S)-(−)-1-phenylethylamine in 1 minute. The initial
rate was determined by monitoring the decrease of the concentration of (S)-(−)-1-phenylethylamine during the first
three minutes. Frozen aliquots of the enzyme at -20 °C were used. H. elongata -transaminase was stable for several
months without evident loss of activity. Data were obtained by averaging the measurements on three independent
samples.


Analytical methods
Spectrophotometric analysis
For the amino donor, final conversion and the relative initial rate were estimated spectrophotometrically employing
a Varian Cary 50 Scan UV–Visible spectrophotometer equipped with a Cary single cell Peltier temperature
controller. The concentration of pyruvate was determined after derivatization with o-phenylenediamine at a molar
ratio of 20:1 respect to the initial concentration of the ketoacid in 80% acetic acid at 50 °C for 10 minutes, followed
by 1:10 dilution, and by measuring the adsorption at 337 nm of the corresponding quinoxaline derivative . The
calibration curve of the quinoxaline derivative was determined at seven different concentrations ranging from 0 to
0.1 mM and for each reaction run, a sample was taken every 15-30 seconds to generate time points. Each run was
performed in triplicate [34]. In this concentration range, a linear correlation between absorbance and concentration
was observed. Errors are reported as standard deviations, where < 10% was deemed acceptable. The kinetic catalytic
constants Vmax and Km were measured at pH 8.0 and 25 °C using the two spectrophotometric coupled assays as
described above in the presence of 0.25% DMSO, 0.1 mM PLP and an appropriate amount of enzyme (0.1 mg/mL).
−)
Reactions were carried out at 5 mM pyruvate and various concentrations (0.5 to 10 mM) of (S)-( -1-
−)
phenylethylamine, at 5 mM (S)-( -1-phenylethylamine and various concentrations (0.05 to 5 mM) of pyruvate.
Concentrations higher than 5 mM of (S)-(−)-1-phenylethylamine gave significant interference with the UV detection
signal and for this reason it was kept below saturation conditions. The initial-velocity data were fitted to the
Michaelis-Menten equation using SigmaPlot software (Version 11.0) [35].

HPLC analysis
The relative initial reaction rate and the final conversion of the different amino acceptor were determined employing
a Shimadzu HPLC instrument equipped with a Supelcosil LC-18-T (250mm x 4.6 mm, 5 µm particle size; Supelco,
Sigma-Aldrich, Germany). The substrates were detected at 210 nm after an isocratic run with 25% acetonitrile/ 75%
trifluoroacetic acid (TFA) 0.1% v/v at 25 °C with a flow rate of 1 mL/min. The depletion of aromatic amines,
aldehydes and the formation of acetophenone were evaluated by calibration curve. The samples were injected after a

dilution of 1:50 with TFA 0.2% in the quenching step (the reactions were monitored at 1, 2, 3, 5 and 10 minutes by
quenching an aliquot of the mix, in triplicates, generating 5 time-points per slope); at this concentration (≤ 0.2 mM)
only the aromatic compounds are detectable at 210 nm between 5 and 25 minutes [36]. The retention times in
minutes are: acetophenone (16.10), benzaldehyde (14.42), benzylamine (7.23), cinmamaldehyde (16.93), (S)-(−)-1-
phenylethylamine (7.45), phenylacetaldehyde (10.94), 2-phenethylamine (13.80), 2-phenylpropionaldehyde
(13.31), vanillin (5.73). All samples were quenched with TFA 0.2% and then centrifuged before the HPLC analysis.
To facilitate the enantiomeric separation by chiral HPLC analysis, rac-1-phenylethylamine was derivatized to the
corresponding amides by adapting the method reported by Andrade et al. and Codex® ATA Screening Kit [37].
Addition of 100 µL 5 M NaOH to the sample was followed by extraction in 900 µL methyl-tert-butyl ether (MTBE)
and then by addition of 5 µL triethylamine and 5 µL acetic anhydride. For the determination of enantioseletivity, a
normal-phase HPLC method was performed using an Agilent Technologies 1200 equipped with a Chiralpak® IC
(250mm x 4.6 mm, 5 µm particle size). Employing this method, product and starting materials were separated with
−)
retention times in minutes: acetophenone (7.14), derivatized (R)-(+)-1-phenylethylamine (16.72) and (S)-( -1-
phenylethylamine (19.14). Detection at 254 nm after an isocratic run 95:05 Heptane:Ethanol at 25 °C with a flow
rate of 1 mL/min.
 Results and Discussion
The spuC gene was identified as a putative aminotransferase from H. elongata. The gene was cloned into the vector
pRSET B, expressed in E. coli BL21(DE3) and purified by metal affinity chromatography. The His-tagged enzyme
was characterized at different temperatures, pHs, salt concentrations and in the presence of different cosolvents and
additives. The substrate specificity of the transaminase was studied against a library of amino donors and amino
acceptors and compared with the -TA from V. fluvialis and C. violaceum.

Purification and molecular mass of HEWT
The expression of the his-tagged transaminase was performed and compared in two different media: LB broth with
IPTG and ZYM-5052 auto-induction medium and purified by immobilized metal affinity chromatography (IMAC).
In both cases, the enzyme was obtained in the pure form (Figure 1). The protein was expressed at higher
concentration in auto induction medium (Purified protein specific activity 4.51 U/mg - Volumetric yield 40.6 mg/L)
in comparison to the LB/IPTG broth (Purified protein specific activity 2.67 U/mg - Volumetric yield 10.9 mg/L).
The enzyme was named with the acronym HEWT (Halomonas elongata -transaminase). The molecular mass of
native HEWT was estimated to be 109 kDa from the calibration curve, which indicated a dimeric quaternary
structure (theoretical value 108 kDa).
Figure 1.

SDS-PAGE of H. elongata transaminase purification fractions. Lanes: 1. Protein marker (2-212 kDa), 2-5. Four
different purification fractions containing different concentrations of the enzyme obtained with auto induction
medium, 6. Cell-free extract, 7. Affinity column flow-through.

Optimal operation pH and temperature
The effect of pH on HEWT activity are reported in Figure 2. HEWT activity reached the maximum at pH 10, but the
catalyst was more stable at lower pHs. The optimal balance between activity and stability was found around pH 8-9.
Figure 2.
●) and stability (○). HEWT preparation was diluted 1:10 with a universal buffer
pH effect on enzyme activity (
solution at the desired pH and stored at 4 °C for 30 minutes. After incubation the residual activity was determined
with the spectrophotometric enzyme assay carried out at each test conditions and 100 % corresponds to 2.58 U/mL.
Every reaction was performed in three replicates and the results are reported as the average of the data obtained.
The effect of temperature on HEWT activity is displayed in Figure 3.A. Thermal stability was tested after incubation
of the enzyme at variable temperatures as reported in Figure 3.B. The highest activity was reached at 50 °C and the
stability is well preserved for temperatures up to 35 °C over 24 hours.
Figure 3.
Temperature effect on enzyme activity (A) and stability (B). The activity was determined with the
spectrophotometric enzyme assay performed at the indicated temperature. For the stability test, the enzyme was
stored in phosphate buffer 50 mM pH 8 in the presence of pyridoxal 5′-phosphate (PLP) 1 mM. The analysis of the
residual activity was carried out at 25 °C and 100 % corresponds to 3.05 U/mL. Every reaction was performed in
three replicates and the results are reported as the average of the data obtained.

Effect of additives on enzyme stability
The stability of HEWT in the presence of organic molecules involved in the standard reaction was determined. The
enzyme was stored with different concentrations of (S)-(−)-1-phenylethylamine, pyruvate and pyridoxal 5′-
phosphate (PLP) at 25 °C and the activity was monitored over time under standard conditions (Figure 4.A). An
increase in the enzyme stability was found at increasing pyruvate concentrations, while increasing the amine
concentration resulted in a progressive inactivation of the catalyst. The destabilizing effect of (S)-(−)-1-
phenylethylamine is, in any case, significantly lower with respect to what reported for V. fluvialis -TA: in that case
the enzyme was completely inactivated when the concentration of the amine was higher than 5 mM [25]. The
−)
simultaneous presence of PLP with (S)-( -1-phenylethylamine alleviates the inactivating effect of the aromatic
amine (Fig. 4.B).

PLP is the prosthetic group of the enzyme and is necessary for enzymatic activity, but its concentration affects the
enzyme stability. It is well known that PLP usually presents a destabilizing effect on -TAs at concentrations higher
than 1 mM [25], therefore it is commonly used within 1 and 0.1 mM in enzymatic reactions and storage buffer [38,
39]. As shown, the enzyme at concentrations greater than 5 mM of cofactor loses the activity significantly faster
than 0.1 mM, confirming the destabilization effect (Figure 4.A).
Figure 4.
Enzyme stability at different additive concentrations (A). Stability interaction of pyridoxal 5′-phosphate and (S)-(−)-
1-phenylethylamine (PEA), both 5 mM, on the enzyme (B). Every reaction was performed in three replicates and the
results are reported as the average of the data obtained.

Effect of salt concentration and co-solvents on enzyme stability
The addition of a co-solvent to the reaction mixture was expected to affect both enzyme activity and stability. The
effects of a selection of common water-miscible organic solvent were investigated (Table 1). In all cases, a decrease
in enzyme activity was observed. However, the transaminase stability was unaffected by organic solvents. Hence,
this enzyme is suitable for enantioselective amination of ketones and aldehydes where the presence of a co-solvent is
necessary due to the poor solubility of the substrate in water.
The effect of different salt concentrations on enzyme stability and activity was also investigated. In all conditions,
enzyme activity decreased with the increase of salt concentration, when compared to the enzyme at pH 8.0 in 50
mM phosphate buffer (Table 2). On the other hand, the salt presence shows from little to no effect on the stability of
the enzyme.
Table 1.
Co-solvent effect on HEWT activity and stability. The activities are reported as relative values expressed respect to
the activity of the reaction carried out with no co-solvent. Every reaction was performed in three replicates and the
results are reported as the average of the data obtained.
Table 2.
Effect of the salt concentration on enzyme activity and stability. Incubation was carried out at the indicated salt
concentration for 1.5 h and 24 h at 4 °C. The residual activity of the enzymatic solution was determined by the
standard spectrophotometric assay carried out in presence of the salt concentration indicated and expressed in
function of the activity under standard conditions. Every reaction was performed in three replicates and the results
are reported as the average of the data obtained.

Amino donor spectra

We investigated also a range of amino donors (Table 3). Interestingly, this enzyme accepts isopropylamine as amino
donor with a relative initial rate 3-4 times higher respect to C. violaceum and V. fluvialis -TAs with 41% of final
conversion. This is a desirable substrate for -transaminases and the ability to accept efficiently this compound as
amino donor in an aqueous media is not common for -TAs [40]. The -TA from V. fluvialis, for example, had to
be engineered in order to efficiently accept aliphatic amines like isopropylamine [5]. This donor has been
increasingly used due the fact that it is cost-effective, easily obtainable and the generated acetone which can be
easily removed thus shifting the equilibrium to the products side [41]. Alternative methods used to overcome the
low equilibrium constant of the transaminase reaction require the presence of NADH/NAD+ and other two enzymes
in addition of -TA, and this affects the overall cost of the process. On the other hand, employing isopropylamine
the equilibrium can be shifted simply acting on reaction conditions such as substrate concentration, temperature and
pressure [10, 42-44].
HEWT shares a similar characteristic with C. violaceum and V. fluvialis, as it does not accept -alanine and L-serine
as substrates. HEWT lacks the particular preference showed by the C. violaceum -TA for the racemic form of 1-
phenylethylamine and presents a more common behavior exhibiting higher activity on the (S) enantiomer. HEWT is
highly (S)-selective, being able to fully convert (S)-1-phenylethylamine to acetophenone and showing no detectable
activity with the opposite enantiomer; a resolution of the racemate stops at 50% of conversion permitting the
recovery of (R)-1-phenylethylamine with e.e.>99% (E>100). This high stereoselectivity prompted further
investigations into the asymmetric synthesis of amines; moreover, its ability to process ,_-dihydroxyketones
could be extended to chiral amino alcohols [45, 46].
Table 3
HEWT specificity on different amino donor are reported in comparison with the data relative to C. violaceum -TA
[3] and V. fluvialis -TA [47] obtained in similar conditions. The enzymatic reactions and the initial reaction rates
and final conversions with HEWT are reported in the experimental section. Every reaction was performed in three
replicates and the results are reported as the average of the data obtained. The detection limit of pyruvate after
derivatization was 5%.

Amino acceptor spectrum
A library of compounds was selected in order to investigate the characteristics of the new amine transaminase in
comparison to the two well-known structural homologues from C. violaceum and V. fluvialis [47] (Table 4). In spite
of a higher sequence identity with the former, HEWT shows initial rates that are more comparable with the V.
fluvialis -TA activity. Higher initial reaction rates were measured for pyruvate, propanal and phenylacetaldehyde.
Similarly to the other two transaminases, HEWT is inactive or shows a very low reaction rate with D-fructose, L-
ribulose, 3-hydroxy-2-butanone and -ketoglutarate. The absence of activity with -ketoglutarate and glutamic acid,

confirms the nature of the enzyme as a -TA. As expected, the reactivity with aldehydes and -ketoacids is higher
with respect to the ketones due to the relative low electrophilicity of their carbonyl group compared to aldehydes
and ketoacids. Bulky aldehydes such as vanillin, cinnamaldehyde, and 2-phenylpropionaldehyde are well accepted
by the enzyme.
Of particular interest are the activities with 1,3-dihydroxyacetone and L-erythrulose. Usually, in fact, ,_-
dihydroxyketones are not accepted by TAs; two notable exceptions are the -TAs from C. violaceum [22, 23] and P.
aeruginosa [48]. The lack of activity with D-fructose and L-ribulose could be caused by the excessive bulk of such
substrates and impediment to be accommodated in the catalytic pocket. The final conversion was determined after
24 hours of reaction time, even though it is accepted that the reactions reaches the equilibrium within 2-3 hours.
Complete conversions were observed for pyruvate, glyoxylate and benzaldehyde. For other compounds, like
phenylacetaldehyde and propanal, despite a good initial rate, the reaction stopped before completion. This could be
due to several reasons such as product inhibition and the chemical equilibrium.
Table 4
HEWT specificity with different amino acceptors are reported in comparison with the data reported for C. violaceum
-TA [3] and for V. fluvialis -TA obtained in similar conditions. The enzymatic reactions with HEWT were
carried out as reported in materials and methods. Every reaction was performed in three replicates and results
−)
reported as the average value. The detection limit of (S)-( -1-phenylethylamine was 5%

Amine synthesis
Five amino acceptors were tested with isopropylamine and o-xylylenediamine to assess the synthetic scope of
HEWT. Although the equilibrium is unfavourable, increasing the amino donor equivalents or using o-
xylylenediamine shifts it towards the products. HEWT had moderate conversions across the range of amino
acceptors with isopropylamine (IPA) and o-xylylenediamine including acetophenone, cinnamaldehyde, vanillin and
2-phenylpropionaldehyde. Very efficient usage of isopropyl amine and o-xylylenediamine was found when
benzaldehyde was employed as acceptor with 90-95% conversion.
The enantiospecific reaction of HEWT with acetophenone and IPA was investigated by chiral HPLC. The (S)-
−)
enantiopreference was highlighted with an excellent ee > 99% towards the production of (S)-( -1-
phenylethylamine.
Table 5.
HEWT activity and conversion, exploiting isopropyl amine or o-xylylenediamine as amino donor. The reaction
mixture contained 10 mM amino donor, 10 mM amino acceptor and 0.05 mg/mL of HEWT in a reaction volume of
200 µL. Specific activity was determined using isopropyl amine as amino donor. Activity and conversion were
determined monitoring the aromatic signals as described in paragraph 2.10.2. Isopropylamine was also tested for
conversion only in excess with 20 eq. (200mM). Activity was assayed at 37 °C, while the conversion at 24 hours
was kept at room temperature.


Enzyme kinetics
The maximum velocity (Vmax) was 0.18 µM/s and the Michaelis-Menten constants (Km) were 2.57 mM and 0.56 mM
for (S)-(−)-1-phenylethylamine and pyruvate, respectively. The turnover number was also determined as kcat 0.094 s-
1 (figure 5). The cooperative profile found for (S)-(−)-1-phenylethylamine was outlined by the Hill coefficient of ca.
2 and confirmed by size exclusion chromatography that highlighted that HEWT is a dimeric protein in its active
state.
Figure 5
−)
Michaelis-Menten plot of the HEWT reaction for (S)-( -1-phenylethylamine (a) and pyruvate (b).
 Conclusions
This is the first study reported on an amine transaminases from the moderate halophile bacterium H. elongata.
HEWT tolerated 20% (v/v) co-solvents over 22 hours, the best solvents were MeOH (47%) and DMSO (27%).
Among the amino acceptors tested, benzaldehyde presented 60% conversion, shifting the equilibrium using IPA or
o-xylylenediamine, conversion increased up to 95%. Acceptance of isopropylamine as amino donor is an advantage
in asymmetric synthesis and a cost-effective benefit for industrial applications. The activity observed with L-
erythrulose and 1,3-dihydroxyacetone gives scope for further investigation into structural properties and as well as
reinforcing the potential for this enzyme for the application in the synthesis of chiral amino alcohols. With its high
enantioselectivity, large substrate spectra and stability in organic solvents, HEWT is a promising enzyme for
biotechnological applications in the production of chiral amines.
Acknowledgments
The authors wish to thank Science Foundation Ireland for funding through 11/RFP.1/BIC/3082 (Jennifer Cassidy)
and Synthesis and Solid State Pharmaceutical Centre code 12/RC/2275 (reagents and support). The authors wish to
thank also the Politecnico di Milano (Italy) for sponsoring Dr. Lorenzo Cerioli for this project, Dr. Elena Rosini and
Prof. Paola D’Arrigo for useful discussions. This project is also part of the COST Action CM1303 “SysBiocat”

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Table 1.
Relative activity (%)
Co-solvent ratio
Incubation time
No co-solvent
MeOH
10 %
20 %
0 h
22 h
100 ± 6
68 ± 2
42 ± 2
46 ± 4
36 ± 3
30 ± 1
n.d.
0 h
100 ± 3
48 ± 4
38 ± 2
23 ± 5
19 ± 1
16 ± 6
8 ± 1
0
22 h
100 ± 6
47 ± 4
27 ± 5
25 ± 2
20 ± 3
16 ± 1
n.d.
100 ± 3
78 ± 4
51 ± 1
40 ± 1
34 ± 6
28 ± 1
26 ± 3
17 ± 4
4 ± 5
DMSO
EtOH
tBuOH
iPrOH
ACN
nPrOH
n.d.
n.d.
THF
n.d.
0
n.d.
Not determined (n.d.)

Table 2.
Relative activity (%)
1.5 h
Incubation time
Phosphate buffer 50 mM
KCl 1 M
0 h
24 h
100 ± 0.8
57 ± 2.5
100 ± 1.5
55 ± 2.5
100 ± 4.7
50 ± 0.9
KCl 2 M
NaCl 1 M
NaCl 2 M
NaCl 3 M
49 ± 2.0
42 ± 0.8
27 ± 3.2
21 ± 0.5
46 ± 6.5
38 ± 0.4
27 ± 2.0
19 ± 2.2
36 ± 1.6
40 ± 1.4
29 ± 0.8
19 ± 0.4

Table 3.
H. elongata -TA
C. violaceum -TA[36]
V. fluvialis -TA[25]
Final conversion
Relative initial rate
(%)
Relative initial rate
(%)
Relative initial rate
(%)
(%)
Amines
rac-1-
Phenylethylamine
88 ± 1
97 ± 1
100 ± 6
100
100
(S)-(−)-1-
Phenylethylamine
106 ± 10
69.4
112.0
−)
(R)-( -1-
< 5
< 5
< 4
< 1
Phenylethylamine
Isopropylamine
41 ± 2
64 ± 1
< 5
15 ± 3
81 ± 5
< 5
< 4
133.9
< 4
3.6
115.6
< 1
Benzylamine
-Alanine
L-Alanine
D-Alanine
L-Serine
41.0 ± 1.1 a
66.2 ± 1.0 a
165.0
10.0
< 5 a
19.6 ± 0.2a
< 5 a
< 5 a
8.2 ± 0.7a
< 5 a
n.d.
9.0
< 1
6.4
< 1
n.d.
L-Glutamate
a) The reactivity of L-Alanine, D-Alanine, L-Serine and L-Glutamate was measured employing benzaldehyde as amino acceptor. The relative initial rate is reported
−)
compared to the rate of conversion of benzaldehyde with (S)-( -1-phenylethylamine (1.41 ± 0.04 U/mg). Initial reaction rates and final conversion with HEWT were
determined respectively by the concentration of benzaldehyde and benzylamine in the reaction measured by HPLC analysis. The detection limit of both the compound is
5 %.

Table 4.
V. fluvialis
-TA [25]
H. elongata -TA
C. violaceum -TA[36]
Final
Relative initial
Conversion at
Relative initial
Relative initial
conversion
(%)
rate
(%)
equilibrium
(%)
rate
(%)
rate
(%)
Ketoacids
Pyruvic acid
97.5 ± 2.5
39.7 ± 0.5
< 5
100 ± 2.2
14.8 ± 0.3
< 5
95-100
65
100
144.1
< 5
100
29.2
0.3
-Hydroxypyruvic acid
 -Ketoglutaric acid
Aldehydes
< 5
Glyoxylic acid
97.5 ± 2.5
22.5 ± 2.9
95-100
176.2
60.2
Acetaldehyde
Propanal
39.2 ± 2.8
55.1 ± 2.6
95.0 ± 5.0 a
33.0 ± 0.6
49.9 ± 4.6
20.4 ± 0.5 a
n.d.
60-70
90
n.d.
132.3
91.2
15.8
36.4
72.8
Benzaldehyde
Phenylacetaldehyde
48.1 ± 2.4 b
48.1 ± 6.2 b
68
107.0
68.5
2-Phenylpropionaldehyde
67.1 ± 1.1
21.2 ± 0.6
n.d.
n.d.
n.d.
45.5 ± 6.0 a
56.1 ± 1.6
4.3 ± 0.1a
5.5 ± 0.3
>60 d
>60 d
61.7
79.7
n.d.
Vanillin
(E)-Cinnamaldehyde
31.5
Ketones
Cyclohexanone
30.5 ± 1.7
< 5
16.2 ± 1.7
< 5
12.4 c
1.3 c
2.1 c
29.2
5.3
6.1
4.8
n.d.
3-Hydroxy-2-butanone
1,3-Dihydroxyacetone
35.3 ± 1.5
5.4 ± 0.0
3.5

L-Erythrulose
L-Ribulose
19.1 ± 3.8
< 5
15.9 ± 4.4
< 5
3.2 c
n.d.
13.0
n.d.
n.d.
n.d.
D-Fructose
< 5
< 5
n.d.
< 5
n.d.
Concentration of substrates was kept constant at 10 mM. a) Benzylamine and vanillin peak overlap the (S)-(−)-1-phenylethylamine in the HPLC analysis. The initial
reaction rate was determined by the acetophenone formation and the final conversion was estimated with the complete depletion of the benzaldehyde peak in 3 hours.
The conversion standard deviation of 5 % is assigned due to some degree of benzaldehyde evaporation during the reaction. b) Phenylacetaldehyde has a low water
solubility. The substrate was added in a methanol solution to guarantee the correct concentration of the substrate inside the reaction mixture. For this reason these
reactions was carried out in presence of 2.5 % of methanol. c) Conversion after one hour. d) The reactions were not run to equilibrium.

Table 5.
Isopropylamine
Conversion 1 eq. (%)
9.7 ± 0.4
o-Xylylenediamine
Conversion 1 eq. (%)
29.2 ± 0.2
Specific activity (U/mg)
n.d.a
Conversion 20 eq. (%)
13.0 ± 0.1
Acetophenone
Benzaldehyde
0.58 ± 0.01
0.07 ± 0.01
0.12 ± 0.01
0.24 ± 0.02
85.4 ± 2.2
95.3 ± 0.1
90.9 ± 2.0
Cinnemaldehyde
Vanillin
20.1 ± 0.7
21.8 ± 0.1
28.5 ± 0.1
10.2 ± 0.7
13.2 ± 0.4
15.6 ± 0.1
2-Phenylpropionaldehyde
16.0 ± 0.9
19.0 ± 2.0
22.7 ± 1.8
aActivity with acetophenone was not detectable (n.d.) under conditions tested.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5