Metagenomic discovery of a novel transaminase for valorization of monoaromatic compounds

Article abstract of DOI:10.1039/c8ra02764a

The profitability of next-generation biorefineries is acutely contingent on the discovery and utilization of biocatalysts that can valorize lignin. To this end, the metabolic catalogues of diverse microbiota have been mined previously using functional metagenomics in order to identify biocatalysts that can selectively degrade lignin into monoaromatic compounds. Herein, we have further improved the valorization factor of biorefining by deploying functional metagenomics toward the identification of a novel transaminase that can selectively functionalize lignin-derived monoaromatics to produce value-added feedstocks for pharmaceutical synthesis. We implemented a high-throughput colorimetric assay using o-xylylenediamine as the amino donor and successfully identified a transaminase that utilizes the canonical cofactor, pyridoxal 5′-phosphate, to aminate as many as 14 monoaromatic aldehydes and ketones. We subsequently identified the optimal conditions for enzyme activity towards the most favoured amino acceptor, benzaldehyde, including temperature, pH and choice of co-solvent. We also evaluated the specificity of the enzyme towards a variety of amino donors, as well as the optimal concentration of the most favoured amino donor. Significantly, the novel enzyme is markedly smaller than typical transaminases, and it is stably expressed in E. coli without any modifications to its amino acid sequence. Finally, we developed and implemented a computational methodology to assess the activity of the novel transaminase. The methodology is generalizable for assessing any transaminase and facilitates in silico screening of enzyme-substrate combinations in order to develop efficient biocatalytic routes to value-added amines. The computational pipeline is an ideal complement to metagenomics and opens new possibilities for biocatalyst discovery.

Full text of DOI:10.1039/c8ra02764a

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Metagenomic discovery of a novel transaminase for
valorization of monoaromatic compounds†
Cite this: RSC Adv., 2018, 8, 22490
ab
Steven J. Hallam and Vikramaditya G. Yadav *ad
c
Sandip V. Pawar,
The profitability of next-generation biorefineries is acutely contingent on the discovery and utilization of
biocatalysts that can valorize lignin. To this end, the metabolic catalogues of diverse microbiota have been
mined previously using functional metagenomics in order to identify biocatalysts that can selectively degrade
lignin into monoaromatic compounds. Herein, we have further improved the valorization factor of biorefining
by deploying functional metagenomics toward the identification of a novel transaminase that can selectively
functionalize lignin-derived monoaromatics to produce value-added feedstocks for pharmaceutical synthesis.
We implemented a high-throughput colorimetric assay using o-xylylenediamine as the amino donor and
0
successfully identified a transaminase that utilizes the canonical cofactor, pyridoxal 5 -phosphate, to aminate
as many as 14 monoaromatic aldehydes and ketones. We subsequently identified the optimal conditions for
enzyme activity towards the most favoured amino acceptor, benzaldehyde, including temperature, pH and
choice of co-solvent. We also evaluated the specificity of the enzyme towards a variety of amino donors, as
well as the optimal concentration of the most favoured amino donor. Significantly, the novel enzyme is
markedly smaller than typical transaminases, and it is stably expressed in E. coli without any modifications to
its amino acid sequence. Finally, we developed and implemented a computational methodology to assess the
activity of the novel transaminase. The methodology is generalizable for assessing any transaminase and
facilitates in silico screening of enzyme–substrate combinations in order to develop efficient biocatalytic
routes to value-added amines. The computational pipeline is an ideal complement to metagenomics and
opens new possibilities for biocatalyst discovery.
Received 30th March 2018
Accepted 12th June 2018
DOI: 10.1039/c8ra02764a
rsc.li/rsc-advances
products that can be manufactured from a resource, the greater is
its valorization factor; and lignin's poor valorizability can be
1
. Introduction
Biorening is loosely dened as the separation, isolation and attributed to its recalcitrance to most biocatalytic or thermocata-
5,6
conversion of cellulose, hemicellulose and lignin from lignocellu- lytic transformations. The inability to efficiently valorize lignin
1
losic biomass into fuels, chemicals, materials and energy. A represents a lost opportunity for biorening, especially since lignin
2
3
number of biocatalytic and thermocatalytic processes have been has a higher carbon and lower oxygen content compared to both,
7
developed for biorening. However, regardless of the mode of cellulose and hemicellulose. In particular, lignin is arguably one
conversion employed, biomass comprising a higher content of of the most attractive feedstocks for the manufacture of aromatic
cellulose and hemicellulose is generally preferred as a feedstock specialty and ne chemicals that are, among other applications,
4
compared to biomass with a higher proportion of lignin. The widely used in pharmaceutical synthesis. Although these chem-
degree to which a resource can be valorized (which we dene as the icals are presently derived from petroleum, they can be sourced
valorization factor) is a function of the ratio of the value of all from lignin at higher atom economies. The development of
products that can be manufactured from the resource to the value conversion platforms that can efficiently valorize lignin is highly
of the resource. Typically, the greater the number of useful desired, particularly biocatalytic processes that are greener, emit
2
lesser CO and are more energy efficient than thermocatalytic
8–13
alternatives. To this end, functional metagenomics is poised to
be a major driver of the discovery of superior biocatalysts for the
valorization of lignin. Functional metagenomics provides reliable
and easy access to the entire catalogue of microbial biocatalytic
transformations without the need to culture any microorganisms
a
Department of Chemical & Biological Engineering, The University of British Columbia,
Vancouver, BC, Canada. E-mail: vikramaditya.yadav@ubc.ca
b
University Institute of Pharmaceutical Sciences, Punjab University, Chandigarh,
Punjab, India
c
Department of Microbiology and Immunology, The University of British Columbia,
14,15
in the laboratory.
Vancouver, BC, Canada
d
School of Biomedical Engineering, The University of British Columbia, Vancouver, BC,
We had previously reported the identication of 147 novel,
Canada
whole-cell biocatalysts that selectively degrade lignin using
functional metagenomics. We employed a whole-cell, GFP-
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra02764a
16
1
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based biosensor to screen 42 520 fosmid clones comprising the
environmental DNA of archaea and bacteria from coal beds.
Each fosmid ranged between 30- to 50-kilo base pairs in length,
and the clones selectively degrade lignin into vanillin and
syringaldehyde. Herein, we further improve the valorization
factor of lignin use by re-deploying functional metagenomics to
discover a novel transaminase that catalyzes the asymmetric
synthesis of chiral amines from the monoaromatic degradation
products of lignin. It is estimated that roughly 40% of all new
chemical entities (NCEs) approved by the US Food & Drug
1
7
Administration (FDA) contain chiral amines building blocks
that are the products of enzymes such as imine reductase,
18
Scheme 1 Overview of the colorimetric assay for high-throughput
screening of TAs.
19
20
monoamine oxidase, ammonia lyases, amine dehydroge-
21
22,23
nase, and transaminases.
Of these, transaminases (TAs)
ꢀ
1
5
5 mL LB medium supplemented with 12.5 mg mL of chlor-
are especially promising for biocatalytic aminations since they
can convert monoaromatic aldehydes and ketones into their
corresponding amines at high yields and selectivities under
mild, aqueous reaction conditions. In fact, several chemo-
enzymatic schemes employing TAs for the syntheses of enan-
tiopure (R)- or (S)-pharmaceutical intermediates have been re-
ꢀ
1
amphenicol and 100 mg mL L-arabinose. Chloramphenicol is
the antibiotic selection marker whereas arabinose ensures that
a dened number of copies of the fosmid are maintained within
the bacteria. All solvents and chemicals used in this study were
purchased from Sigma Aldrich, Alfa Aesar and Santa Cruz
Biotechnologies. The microcultures were allowed to grow at
24
2
5–30
ported in recent years.
Amination by a canonical TA
ꢁ
0
37 C for 24 hours, aer which they were fed 2 mM of PLP,
5 mM of o-xylylenediamine and 5 mM of the amino acceptor,
commences with the binding of the cofactor pyridoxal 5 -
phosphate (PLP) to a lysine residue within the enzyme's active
site, which generates an internal (or enzyme-bound) aldi-
2
ꢁ
benzaldehyde (4), and then incubated at 30 C for an additional
48 hours. At the end of the 48 hour incubation, the plates were
imaged from above and the RGB values of the pixel corre-
sponding to the centre of each well were extracted using MAT-
LAB. Eight 384-well plates totalling 3072 fosmid clones were
screened in this manner and we successfully identied three
primary hits. Hit verication subsequently winnowed the
number of conrmed TAs to a single candidate, S2A24, which
translates to a hit rate of 0.03% (on a clonal basis) or 1 per 122.9-
mega base pairs. This hit rate is signicantly higher than the
median rate that is observed for functional metagenomic
31,32
mine.
The amino donor binds to the internal aldimine
thereaer and molecular rearrangements ensue, which
sequentially generate an external (or dissociated) aldimine,
a quinonoid, a ketamine, and, nally, pyridoxamine phosphate
(PMP) and the oxo product. The latter is released from the active
site and is replaced with the amino acceptor, which then reacts
with PMP to produce the desired amino product and re-
generate PLP.
A variety of high-throughput, enzyme-coupled assays have
33–37
been employed previously to identify TAs.
These assays
39
screening. We later picked the S2A24 clone from the original
library and extracted its fosmid using a GeneJET plasmid min-
iprep kit. Sequencing and annotation of the fosmid conrmed
the presence of a novel TA and 37 other unique ORFs. The 371-
amino acid sequence of the novel enzyme is roughly 15%
shorter than and <30% similar to canonical TAs expressed by
employ other enzymes in addition to the TA to transform ami-
nation intermediates into products that can be detected using
rapid analytical procedures such as spectrophotometry.
Enzyme-coupled assays are quite sensitive but typically neces-
sitate extensive and expensive optimization before they can be
38
deployed for metagenomic screening. As a consequence, we
employed a colorimetric assay utilizing o-xylylenediamine (1) as
the amino donor to screen novel TAs expressed by an in-house
library of E. coli fosmid clones that exhibit activity towards
40
41
Pseudomonas putida, Vibrio uvialis and Chromobacterium
35
violaceum, as well as another TA, pQR1108, that was discov-
24
ered recently (the multiple sequence alignment has been
summarized in Fig. S3 in the ESI or ESI package†). Nevertheless,
the active site and folds of the novel TA aligns quite well with
those of previously discovered candidates (Fig. 1).
22
monoaromatic degradation products of lignin. Each fosmid
once again ranged between 30- to 50-kilo base pairs in length.
The reaction between o-xylylenediamine and the internal aldi-
mine within the active site of a likely TA generates 1H-isoindole
We subsequently synthesized the gene that encodes
a version of the novel TA bearing a N-terminal polyhistidine tag.
Its codons were optimized for expression in E. coli and the gene
was later cloned into a high-copy pET-15b plasmid under the
control of a T7 transcriptional promoter. Additional details
about the molecular cloning have been provided in the ESI.†
The resulting plasmid is labelled as pET-TA. This plasmid was
then transformed into E. coli BL21 (DE3) using the heat shock
(2) and PMP. PMP remains in the active site and subsequently
reacts with the amino acceptor. However, 1H-isoindole is
released into solution, where it tautomerizes to 2H-isoindole
(3). The latter then rapidly polymerizes to yield a black precip-
itate (Scheme 1).
2. Results & discussion
method, which involves temporarily exposing the bacteria to
ꢁ
4
2 C to permeabilize their cell walls in order to facilitate the
The E. coli fosmid clones were assessed for desired TA activity in
84-well plates. The microcultures were initially propagated in
42
delivery of the plasmid into the cells. The transformed cells
3
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Fig. 2 Readout of the 96-well plate o-xylylenediamine assay for the
novel TA. Clone A: E. coli transformed with pET-TA but not fed with
benzaldehyde; clone B: E. coli transformed with pET-TA; clone C: cell
lysate of E. coli transformed with pET-TA; clone D: E. coli transformed
with S2A24 fosmid; clone E: cell lysate of E. coli transformed with
S2A24 fosmid. With the exception of clone A, which was not provided
with any benzaldehyde, all wells are fed with 5 mM benzaldehyde and
Fig. 1 Structural alignment between the model of the novel TA
(
prepared by homology modeling using I-TASSER) and the TAs
expressed by Pseudomonas putida (PDB code: 3A8U), Vibrio fluvialis
PDB code: 3NUI) and Chromobacterium violaceum (PDB code:
A6R).
2
mM PLP. The volume of each reaction mixture is 200 mL and the plate
(
4
ꢁ
is maintained at 30 C and pH 7.5. For reaction conditions 1–3, 5 mM of
o-xylylenediamine was supplied to the well. For reaction conditions
4
–6, 25 mM of o-xylylenediamine was supplied to the well. The raw
ꢀ1
were cultured on agar plates supplemented with 100 mg mL of
image of the plate is included in the ESI.†
ampicillin, and a single colony was later picked and cultured
ꢁ
overnight at 37 C in 5 mL of LB medium also containing 100 mg
ꢀ1
concentration of o-xylylenediamine, which can be attributed to
resistance to transport of the amino donor to the intracellular
reaction milieu. Neither control exhibited any TA activity.
Although the colour of these reaction mixtures changed, this
mL of ampicillin. This overnight culture was subsequently
used as the inoculum for a 10 mL culture in the same
ampicillin-supplemented LB medium. The latter culture was
ꢁ
propagated at 37 C until it attained an optical density of 0.6,
22,38
shi in coloration is attributable to the formation of PMP.
following which isopropyl b-D-1-thiogalactopyranoside (IPTG)
We also veried the results of the colorimetric assay using
HPLC. We added 500 mL of methanol to each mixture to quench
the reaction. The samples were then centrifuged and the
supernatant was injected into a Water Atlantis C18 column (5
mm, 4.6 mm ꢂ 250 mm). A gradient of 10 mM ammonium
acetate and acetonitrile (ACN) was employed to resolve the
species. The gradient commences with 60% ammonium acetate
and 40% ACN, transitions to 100% ACN aer 10 minutes, and
was added to the medium and the incubation temperature was
lowered to 25 C. The concentration of IPTG in the medium was
ꢁ
0.5 mM and the cultures were propagated overnight. The cells
were then harvested by centrifugation and lysed, and the TA was
puried using Qiagen Ni-NTA spin columns. SDS-PAGE
conrmed high expression of the enzyme by cultures of the
transformed cells that are induced with IPTG (Fig. S2 in the
ESI†).

nally reverts back to 60% ammonium acetate and 40% ACN 20
We repeated the o-xylylenediamine assay for 200 mL cultures
and lysates of E. coli cells that have been transformed with pET-
TA and the S2A24 fosmid. The reactions were conducted in
triplicate in a 96-well plate. The culturing and reaction condi-
tions, with the exception of the concentration of o-xylylenedi-
amine, were identical to those of the 384-well plates described
earlier. o-Xylylenediamine was supplied at a concentration of
either 5 mM or 25 mM. Cultures of E. coli cells transformed with
either an empty pET-15b vector or the pET-TA plasmid, but
neither of which were supplied with benzaldehyde, were
employed as the controls. We observed that the wells harbour-
ing the cell lysates exhibited signicantly darker coloration
compared to the other wells (Fig. 2). In fact, the lysates of the E.
coli cells that had been transformed with pET-TA produced the
darkest colour at the end of the 48 hours. We also detected TA
activity when the concentration of o-xylylenediamine was
reduced from 25 mM to 5 mM. Moreover, the activity is indis-
tinguishable for lysates of the pET-TA cultures and only slightly
higher for lysates of the S2A24 clone. However, amination by
whole-cell cultures of E. coli transformed with pET-TA and the
S2A24 fosmid exhibit marked differences in response to the
minutes into the run. Under these conditions, benzylamine, a-
methylbenzylamine, benzaldehyde and acetophenone elute at
3.6, 4.2, 7.7 and 8.1 minutes, respectively, and the concentra-
tions of benzylamine in the samples align well with the readouts
of the colorimetric screen.
We then assessed the propensity of the novel TA to employ
alternative amino donors. Each reaction mixture comprised
ꢀ
1
ꢃ1 mg mL of crude lysate of cultures of E. coli transformed
with the pET-TA plasmid, 2 mM of PLP, 25 mM of the amino
donor (Fig. 3) and 5 mM of benzaldehyde (dissolved in DMSO)
in 200 mL of a buffered solution of 100 mM potassium phos-
phate at pH 7.5. As before, the reactions were monitored
colorimetrically for 48 hours, aer which the mixtures were
analyzed for their benzylamine content using HPLC by
following the same methodology for sample preparation as
described earlier. Each reaction was performed and analyzed in
triplicate. We observed that the enzyme has a strong preference
for o-xylylenediamine. The conversion of benzaldehyde when o-
xylylenediamine is employed as the amino donor is 42%. In
contrast, the use of (S)-(ꢀ)-1-phenylethylamine (18) results in
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Fig. 4 Conversions of the amino acceptors considered in this study by
the novel TA. Reaction conditions: 5 mM of the individual amino
acceptors, 25 mM of o-xylylenediamine and 2 mM PLP in 100 mM
potassium phosphate solution buffered at pH 7.5 and maintained at
ꢁ
3
0 C.
reliable for the coloured substrates (species 6, 11–14 and 16).
This is a known limitation of the assay that needs improvement
in order to make it universally applicable for use in functional
metagenomic screening of TAs that can functionalize any
monoaromatic degradation product of lignin.
We evaluated the catalytic mechanism of the enzyme using
computational modeling. A homology model of the enzyme was
43
Fig. 3 Amino donors and acceptors evaluated in this study.
constructed using I-TASSER, and the structures of all mole-
cules that participate in the catalytic cycle were prepared in
44
conversion of 10%, and no benzylamine was formed when (R)- Marvin. We subsequently docked PLP into the active site of the
45
(
+)-1-phenylethylamine (19), L-alanine (20), D-alanine (21) and enzyme using AutoDock Vina. The input to the docking
isopropylamine (22) are used as amino donors. We also calculation comprises an initial set of atomic coordinates for
assessed the potential of the novel TA to aminate other mono- PLP, and the program outputs as many as 9 putative poses of
aromatic carbonyl compounds that are typically produced by PLP within the active site. A corresponding Gibbs free energy of
the breakdown of lignin. We used the same reaction conditions binding (DG) is also computed for each pose. In order to identify
as before, but replaced benzaldehyde with other amino accep- the pose that yields the global minimum for DG, we selected the
tors (Fig. 3). Aer 48 hours had elapsed, the mixtures were most stable pose and re-used its coordinates as the initial value
vortexed with an equal volume of ethyl acetate and the organic for a subsequent docking calculation. We iterated this meth-
phases were analyzed using GC-MS to determine conversion odology until an unchanging pose was outputted. The docked
and conrm 2H-isoindole formation. GC-MS analysis was per- pose of PLP (Fig. 5, Panel 1) aligns exceedingly well with the
formed using an Agilent J&W HP-5ms column (25 m length, 0.20 relative coordinates of PLP in the crystal structures of TAs
mm lm thickness, 0.25 mm inner diameter). Injector and expressed by Pseudomonas putida (PDB code: 3A8U), Vibrio u-
column conditions have been elaborated in the ESI.†
vialis (PDB code: 3NUI) and Chromobacterium violaceum (PDB
The TA successfully converted 42% of benzaldehyde, 27% of code: 4A6R), and is in close proximity to the K183 residue of the
acetophenone (9), 17% of 4-hydroxybenzaldehyde (5) and 7.5% enzyme. Additionally, the phosphate moiety of PLP is facing
of cinnamaldehyde (6) into their corresponding amines outward. This orientation of the phosphate moiety is an
(
Fig. 4). In contrast, the conversion of 4-hydroxy-3- important frame of reference for subsequent molecular events.
46
methoxybenzaldehyde (8), 3,4-dimethoxybenzaldehyde (15), We then used Maestro to generate an aldimine bond between
and 4-hydroxy-3-methoxyacetophenone (17) was less than 5%, the K183 residue and PLP, as well as add charges to individual
and the enzyme exhibited negligible activity towards the other atoms in the product. The energy of the structure was subse-
amino acceptors. Incidentally, we also monitored the reaction quently minimized in Maestro. The coordinates of all atoms
mixtures colorimetrically. Benzaldehyde and acetophenone with the exception of the lysine side chain and PLP were xed in
yielded the most intense colour, which is consistent with the the energy minimization (Fig. 5, Panel 2).
GC-MS results. However, the colorimetric assay was not as
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Fig. 5 Computational analysis of the catalytic mechanism of the newly discovered TA. We applied a variety of computational tools to simulate
the progression of the reaction within the active site of the enzyme. The Gibbs free energy of binding has also been estimated for each pose.
Panel 1: binding of PLP within the active site and the close proximity of the K183 residue; panel 2: reaction between the PLP and the K183 residue
to form the internal aldimine; panel 3: docking of o-xylylenediamine within the active site of the aldimine-functionalized TA; panel 4: docking of
(
S)-(ꢀ)-1-phenylethylamine within the active site of the aldimine-functionalized TA; panel 5: docking of (R)-(+)-1-phenylethylamine within the
active site of the aldimine-functionalized TA and confirmation of non-reactivity due to the large distance between its nitrogen atom and the
nitrogen atom in the aldimine bond; panel 6: formation of the ketamine intermediate on account of the reaction between o-xylylenediamine and
PLP; panel 7: formation of PMP in the active site; panel 8: docking of benzaldehyde within the active site of the TA alongside PMP; panel 9:
docking of acetophenone within the active site of the TA alongside PMP; panel 10: docking of 2,6-dimethyl-p-benzoquinone within the active
site of the TA alongside PMP.
We subsequently docked all the amino donors that were This prediction is consistent with the expected behaviour of the
studied previously into the new active site using the same iter- molecular in response to steric hindrances within the active
31,32
ative methodology described earlier. Of these, only o-xylylenedi- site,
as well as the pose of PMP when it has been docked
amine, and (R)-(+)-1- within the active site (Fig. 5, Panel 7). Also, the DDG between the
(S)-(ꢀ)-1-phenylethylamine
phenylethylamine successfully dock within the active site. interaction of o-xylylenediamine with the internal aldimine and
Moreover, the distances between the nitrogen atom in the aldi- the interaction of the enzyme with the ketamine intermediate is
mine bond and the nearest nitrogen atom of the amino donor higher than the corresponding interactions for (S)-(ꢀ)-1-phenyl-
that is a key participant in the sequence of steps that culminates ethylamine, which substantiates observed differences in the
with the formation of PMP and the oxo product are 4.9, 5.2 and conversions of the two amino donors.
˚
6
.1 A for o-xylylenediamine (Fig. 5, Panel 3), (S)-(ꢀ)-1-phenyleth-
Finally, we docked benzaldehyde (Fig. 5, Panel 8), acetophe-
ylamine (Fig. 5, Panel 4) and (R)-(+)-1-phenylethylamine (Fig. 5, none (Fig. 5, Panel 9) and 2,6-dimethyl-p-benzoquinone (14 and
Panel 5), respectively. This result aligns well with the observed Fig. 5, Panel 10) into the active site of the TA with PMP already
conversions of 42% and 10% for o-xylylenediamine and (S)-(ꢀ)-1- bound within. Interestingly, the docking calculation does not
phenylethylamine, respectively, and conrms the non-reactivity predict any discernible differences in reactivity of benzaldehyde
of (R)-(+)-1-phenylethylamine. We then docked the ketamine and acetophenone. In this context, we dene reactivity as the
intermediate of the reaction between PLP and o-xylylenediamine distance between the amino moiety of PMP and the carbonyl bond
within the active site of the unmodied TA (Fig. 5, Panel 6). The in the amino acceptors. However, the observed conversions of
calculation suggests that molecular rearrangements within the benzaldehyde and acetophenone by the enzyme are 42% and 27%,
intermediate cause it to ip its orientation within the active site respectively. Differences in conversion may arise due to an
in a manner such that its phosphate moiety is now facing inward. increased resistance to diffusion of acetophenone to the active site
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compared to benzaldehyde or a higher DDG between the interac- donor and we quantied the conversion of benzaldehyde to
tion of the enzyme with PMP and benzaldehyde and the interac- benzylamine as a function of temperature, pH, choice of co-
tion of the enzyme with PLP and benzylamine compared to solvent and concentration of the amino donor (Fig. 6). The co-
corresponding interactions for acetophenone. The docking calcu- solvent is utilized to dissolve benzaldehyde. All reactions
ꢀ1
lation also revealed that the distance between the amino moiety of comprised ꢃ1 mg mL of crude lysate of E. coli transformed
PMP and either carbonyl bond in 2,6-dimethyl-p-benzoquinone is with the pET-TA plasmid and 2 mM of PLP in a solution of
too high for a reaction to occur, which is conrmed by experi- 100 mM potassium phosphate buffer. Each reaction was
ments. This suggests that extensive functionalization of the undertaken in triplicate for 48 hours and had a nal volume of
aromatic ring of the amino acceptors introduces signicant steric 200 mL; and the benzylamine concentration was quantied
hindrances that greatly impact conversion. Nevertheless, the using HPLC. In order to assess the inuence of temperature on
congruency between experiments and simulation validates the enzymatic activity, we added 5 mM of benzaldehyde dissolved in
computational analysis that we have implemented to assess the DMSO and 25 mM of o-xylylenediamine to the reaction mixture
activity of TAs. The computational pipeline is an ideal complement and maintained the pH at 7.5. The reactions were then under-
to metagenomics and could be effectively deployed for large-scale taken at ve temperatures and we observed that the highest
ꢁ
in silico screening of enzyme–substrate combinations in order to conversion of benzaldehyde was obtained at 30 C (Fig. 6, Panel
develop efficient biocatalytic routes for amination of mono- A). In order to investigate the inuence of pH, we added 25 mM
aromatic derivatives of lignin for use as pharmaceutical of o-xylylenediamine and 5 mM of benzaldehyde dissolved in
ꢁ
intermediates.
DMSO to reaction mixtures maintained at 30 C and varied the
Lastly, we also identied the optimal conditions for activity pH between 6.5 and 9. We observed that the conversion peaked
of the novel TA towards the most favoured amino acceptor, at pH 7.5 (Fig. 6, Panel B).
benzaldehyde. o-Xylylenediamine was retained as the amino
ꢀ1
Fig. 6 Optimization of reaction conditions for cell lysates for E. coli transformed with pET-TA. All reactions comprised ꢃ1 mg mL of crude
lysate of E. coli transformed with the pET-TA plasmid and 2 mM of PLP in a solution of 100 mM potassium phosphate. Each reaction was
undertaken in triplicate for 48 hours and had a final volume of 200 mL; and the benzylamine concentration was quantified using HPLC. The
common reaction mixture was suitably modified to deduce the effect of temperature (panel A), pH (panel B), o-xylylenediamine (panel C) and
choice of co-solvent (panel D). The activity of the enzyme exhibits canonical behaviour in response to changes in temperature, pH and
concentration of the substrate. Each experiment was performed in triplicate and the shaded region denotes the standard error in the
measurements.
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Likewise, the impact of the concentration of o-xylylenedi-
amine on conversion was measured by varying its levels in
Acknowledgements
reaction mixtures comprising 5 mM of benzaldehyde in DMSO The authors would like to thank Joe Ho and Kateryna Ievdoky-
ꢁ
and maintained at pH 7.5 and 30 C. We observed that menko for their assistance with the experimentation. We thank
conversion of benzaldehyde exhibits a hyperbolic response to the Natural Sciences & Engineering Research Council (NSERC),
the concentration of o-xylylenediamine and plateaus near Canadian Foundation for Innovation and Mitacs for their
25 mM (Fig. 6, Panel C). The activity of the enzyme exhibits generous funding.
canonical behaviour in response to changes in temperature, pH
and concentration of the substrate. Finally, since lignin-derived
monoaromatic compounds are poorly soluble in aqueous
solutions, we also evaluated several co-solvents for their ability
to enhance conversion by improving solubility of benzaldehyde
in the reaction mixture. We prepared 10% (volumetric basis)
solutions of benzaldehyde in DMSO, acetonitrile (MeCN), iso-
propyl alcohol (IPA) and methanol (MeOH), and added them to
the reaction mixtures to attain a nal benzaldehyde concen-
tration of 5 mM. We also directly added benzaldehyde to the
reaction mixture without any co-solvent. To our surprise, we
observed that the reaction mixture without any co-solvents
attained a higher conversion than the mixture that was
supplied with benzaldehyde dissolved in IPA (Fig. 6, Panel D).
The use of DMSO yielded the highest conversion, followed by
MeOH and MeCN.
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