Biocatalytic transamination with near-stoichiometric inexpensive amine donors mediated by bifunctional mono- and di-amine transaminases

Article abstract of DOI:10.1039/c6gc02102f

The discovery and characterisation of enzymes with both monoamine and diamine transaminase activity is reported, allowing conversion of a wide range of target ketone substrates with just a small excess of amine donor. The diamine co-substrates (putrescine, cadaverine or spermidine) are bio-derived and the enzyme system results in very little waste, making it a greener strategy for the production of valuable amine fine chemicals and pharmaceuticals.

Full text of DOI:10.1039/c6gc02102f

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Slabu, N. J. Weise, C. Iglesias, F. Parmeggiani, R. Lloyd and N. J. Turner, Green Chem., 2016, DOI:
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DOI: 10.1039/C6GC02102F
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Biocatalytic transamination with near-stoichiometric inexpensive
amine donors mediated by bifunctional mono- and di-amine
transaminases§
Received 00th January 20xx,
Accepted 00th January 20xx
James L. Galman,a Iustina Slabu,a Nicholas J. Weise,a Cesar Iglesias,b Fabio Parmeggiani,a
DOI: 10.1039/x0xx00000x
Richard C. Lloyd,c Nicholas J. Turner*,a
www.rsc.org/
The discovery and characterisation of enzymes with both integrated into modern industrial practice. The broad
monoamine and diamine transaminase activity is reported, substrate tolerance of this class of enzymes is one of their
allowing conversion of a wide range of target ketone substrates most appealing features in synthetic applications. A prominent
with just a small excess of amine donor. The diamine co- example of this is the commercial manufacture of sitagliptin –
substrates (putrescine, cadaverine or spermidine) are bio-derived probably one of the most cited success stories of industrial
and the enzyme system results in very little waste, making it a biocatalysis.3
greener strategy for the production of valuable amine fine For these reactions, the thermodynamic equilibria are often
chemicals and pharmaceuticals.
unfavourable and hence different methods for shifting the
equilibrium have been sought to address this long-standing
challenge (Scheme 1).4 One of the earliest approaches, still
heavily relied on, was the coupling of the enzymatic reaction
with a secondary concomitant irreversible reaction (e.g.,
conversion of pyruvate by-product to L-lactate with lactate
dehydrogenase, paired with an oxidoreductase to recycle
NAD(P)H, Scheme 1a).5,6 Whilst producing naturally-
degradable biochemicals as by-products, this method has the
obvious disadvantage of requiring two additional enzymes and
an expensive cofactor to achieve high conversion of the
starting ketone.
Biocatalytic strategies for the synthesis of high added-value
optically pure chiral intermediates provide extraordinary
benefits to the pharmaceutical and agrochemical industries,
due to the excellent activity, stereo-/regioselectivity and
stability of many biocatalysts, often unmatched by their
corresponding chemical equivalents.1 There are obvious
sustainability benefits to such approaches, including
renewable resourcing of recombinant biocatalysts, solvent-
and metal-free chemical transformations and ambient
temperature/pressure of operation. In spite of this highly cited
“greenness”, the concept of any given biocatalytic
transformation being effectively environmentally benign has
been questioned in several instances.2 Many widely used
biotransformation protocols require, for example, expensive
cofactor supplementation and/or large excesses of co-
substrate to combat poor atom efficiency and unfavourable
equilibria.
The asymmetric amination of ketones to enantiomerically
enriched amines mediated by ω-amine transaminases (ω-
ATAs, E.C. 2.6.1.18) is undoubtedly one of the most studied
and exploited biotransformations which has become
a
Manchester Institute of Biotechnology (MIB), School of Chemistry, The University
of Manchester, Manchester, United Kingdom.
Facultad de Quimica, Universidad de la República, Montevideo, Uruguay.
Dr. Reddy's Laboratories, Chirotech Technology Centre, Cambridge, United
b
c
Kingdom.
* Corresponding author: nicholas.turner@manchester.ac.uk
Electronic Supplementary Information (ESI) available: complete experimental
section, molecular biology protocols, analytical methods, product characterisation.
See DOI: 10.1039/x0xx00000x
Scheme 1. Different approaches to shifting the thermodynamic equilibrium of ω-ATA
transaminations.
§ This manuscript is dedicated to Professor Romas Kazlauskas on the occasion of
his 60th birthday.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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On the other hand, for industrial scale application, the transaminase toolbox, we undertook a searVcihew Afroticrle Onnelinwe
DOI: 10.1039/C6GC02102F
approach of choice is the use of a sacrificial amine donor in candidate enzymes able to accept diamine donors.
large excess to drive the equilibrium towards the desired As a promising starting point for the development of an
product. Isopropylamine is widely used for this purpose, enzyme with broader substrate range we identified the
because of its effectiveness and availability.7 This approach has putrescine transaminase gene spuC, part of the polyamine
proven very cost-efficient in many cases, however, a 100-fold uptake and utilization pathway in Pseudomonas aeruginosa
excess (or higher) is required to drive equilibrium. Also, in PAO1.13 The spu (spermidine utilisation) operon, consisting of
order to improve the yields, the removal of the volatile 9 genes, was previously characterised as responsible for
acetone waste product by evaporation or stripping needs to be spermidine transport and the catabolic route of putrescine in
implemented using specialised temperature platforms and the arginine decarboxylase pathway. Genes with analogous
heat-stable enzymes.8
functions are found in E. coli such as the complementary ygjG
More recently, the use of synthetic diamines such as o- gene encoding for putrescine transaminase that suggests a
xylylenediamine9 and but-2-ene-1,4-diamine10 has been the common pathway between the two microorganisms.14
focal point to displace the equilibria of ω-ATA reactions by To probe the evolutionary relationships between known
spontaneous cyclisation and subsequent ring aromatisation of putrescine transaminases and well-characterised ω-TAs, a
the aminocarbonyl by-product (Scheme 1c). However, these multiple sequence alignment was performed, from which a
donors are usually expensive, highly toxic and often form cladogram could be constructed to infer the order of
difficult to remove polymers following aromatisation, adding divergence events (Figure 1). Interestingly, it was found that
to downstream processing costs.
sequences with reported diamine transaminase activity
In contrast, biogenic terminal diamines present themselves as clustered within the ω-ATA sequences as polyphyletic groups.
renewably-sourced alternative amino donors. In addition to SpuC from P. aeruginosa (PA-SpuC) was revealed to have high
their simple bio-based production and relatively low cost, sequence identity with the well-studied Cv-ATA from
upon transamination they are converted into reactive amino Chromobacterium violaceum (55%)15,16 and only 24% identity
aldehydes which spontaneously convert to cyclic imines, thus with the more functionally similar E. coli YgjG protein.14 This
driving the equilibrium towards amination of ketones. indicated two separate acquisitions of such activity in this
Enzymes active on such compounds, known as α,ω-diamine family, with one convergence (Pa-SpuC) occurring relatively
transaminases (α,ω-DTAs), have exploited these advantages recently in evolutionary history compared to the other. As
for the synthesis of N-heterocycles precursors.11 However, previous reports with the more ancient putrescine
these enzymes were found to have strict preference for transaminases (Ec-, Bme- and Bmy-YgjG) revealed minimal
pyruvate over other ketones, making them unsuitable for the acceptance of monoamine substrates,11 it is probable that any
synthesis of a broad range of amines. Nevertheless, if the ancestral ω-TA function of these enzyme has been lost. Due to
substrate specificity could be addressed, biogenic diamines the higher sequence similarity between the Pa-SpuC and
would offer a distinct approach to equilibrium issues (Scheme characterised ω-ATA biocatalysts, this enzyme was chosen for
1d), intrinsically “greener” than previous ones, since near- investigation of potential latent ω-ATA side-activity. Previous
stoichiometric loadings of sacrificial diamines could be applied, experiments carried out with Pa-SpuC revealed that, although
in principle.
it was likely to be involved in the catabolism of diamines,13
Herein, we report the discovery and characterisation of a minimal activity was observed with putrescine and derived
panel of bifunctional α,ω-DTA/ω-ATA enzymes that readily amides.17 In light of this, a selection of predicted orthologues
accept cheap and easily-accessible mono-/diamine donors as were mined from publically-available sequence data, cloned
well as possessing broad ketone acceptor scope, allowing an and expressed to access the natural diversity of SpuC enzymes
equilibrium shift to reach theoretical yield of 100% aminated with respect to activity and potential monoamine/diamine
product with almost stoichiometric donor loadings.
acceptance.
A Basic Local Alignment Search Tool (BLAST) analysis of the
characterised spuC gene from the pathogenic P. aeruginosa
PAO1 strain revealed other homologous genes from
Results and discussion
The lack of activity of commonly used ω-ATAs towards simple
aliphatic diamines such as putrescine (1,4-diaminobutane, 1a),
cadaverine (1,5-diaminopentane, 1b) and spermidine (1,8-
diamino-4-azaoctane, 1c) has been previously reported.10,11
Indeed, the only relevant example is a single commercial
transaminase (ATA256) recently found through extensive
ARS-ATA (ω-ATA)
Cv-ATA (ω-ATA)
Pa-SpuC (α,ω-DTA)
Vf-ATA (ω-ATA)
Ad-ATA (ω-ATA)
Bme-YgjG (α,ω-DTA)
Bmy-YgjG (α,ω-DTA)
Ec-YgjG (α,ω-DTA)
ATA-117 (ω-ATA)
screening of
a large biocatalyst panel from Codexis,
highlighting the rarity of such activity.12 This transaminase was
shown to have activity with a narrow range of ketone
acceptors, and its commercial nature precludes evolutionary,
sequence and structural insight into its substrate scope or
optimisation through engineering. To address this gap in the
Figure 1. Cladogram showing evolutionary relationships between various wild-
type transaminase enzymes as inferred from amino acid sequence identity.
2 | J. Name., 2012, 00, 1-3
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Pseudomonas species, in particular P. putida (Pp-spuC), P. promiscuous activity with industrially relevant monoamines
View Article Online
chlororaphis subsp. aureofaciens (Pc-spuC) and P. fluorescens (S)-methylbenzylamine 1d, isopropylaminDeOI1: 1e0.a10n3d9/bCu6GtyCla0m210in2eF
(Pf-spuC) all with an aligned protein sequence identity of 69% 1f, which have been extensively studied with ω-ATAs, but none
(UNIPROT). The three putative spuC genes were cloned from has been reported to have high activity with biogenic amines.
our in-house NCIMB culture collection and subcloned into Further studies on the diaminoalkane series was in close
apET-28b vector followed by overexpression in E. coli agreement with Cv-ATA as the carbon backbone increased in
BL21(DE3) cells. All three enzymes were successfully size (>C6), in contrast to the diminished activity presented in
overproduced and purified as recombinant N-terminal His6- Ec-YgjG (see ESI). This pattern is in part due to the narrow
tagged protein for screening against a representative panel of hydrophobic channel approaching the enzyme active site
diamine and monoamine donors.
elucidated from recent crystal structures.19
In order to determine the activity of the SpuC enzymes, an Encouraged by these preliminary studies, we turned to the
adapted version of the previously described L-amino acid investigation of non-keto acid prochiral acceptors for
dehydrogenase colorimetric assay18 was employed, using preparative applications. As a model keto acceptor for reaction
pyruvate as the amino acceptor, L-alanine dehydrogenase optimisation we chose o-fluoroacetophenone 4a, since the
from Bacillus megaterium to regenerate pyruvate with chiral amine (S)-6a produced after transamination can be
concomitant reduction of NAD+, and the phenazine- coupled to rhodanine scaffolds resulting in heterocycle
tetrazolium system (MPMS+INT) for colour development analogues (e.g., 7) with a range of pharmacological activities
20,21
(Scheme 2). The results for amines 1a-f are summarised in such as the clinical treatment of type II diabetes mellitus.
Table 1 (additional mono- and diamines were tested, and Biotransformations were conducted using our SpuC enzymes
kinetic constants were determined, see ESI).
using different amounts of the diamine donors 1a-c. The best
conversions (Table 2) were obtained with Pp-SpuC (that also
showed the highest specific activity for 1a-b before), although
the values for Pf-SpuC and Pc-SpuC were very similar.
Table 2. Testing of different diamine loadings in the transamination of 4a
mediated by Pp-SpuC.
Scheme 2. Activity assay used for the characterisation of ω-ATAs and α,ω-DTAs.
Diamine
1a
1b
1c
C
(%)a
84
91
87
ee
C
(%)a
95
96
95
ee
C
(%)a
83
78
73
ee
(%)a
>99
>99
>99
86
Equiv.
1.5
5
10
25
(%)a
>99
>99
>99
>99
(%)a
>99
>99
>99
>99
Table 1. Specific activities of the novel SpuC enzymes against amine donors 1a-f
compared with Ec-YgjG and Cv-ATA.
,
Specific activity (U/mg)a
77
83
63
Subs.
1a
Pp-SpuC
1.3
Pf-SpuC
0.4
Pc-SpuC
1.4
Ec-YgjG
2.7
Cv-ATA
n.d.
Expt. Cond.
:
5 mM 4a, 1 mM PLP, 1% v/v DMSO, 2 mg mL–1 purified SpuC, 50 mM
HEPES buffer, pH 9.0, 40°C, 250 rpm, 24 h. a: measured by HPLC on a chiral
stationary phase. C = conversion. ee = enantiomeric excess.
1b
1c
1d
1e
27.3
15.7
23.2
20.4
16.5
16.5
16.5
25.7
18.5
19.3
15.1
21.5
30.4
18.7
13.5
2.7
2.1
n.d.
0.05
n.d.
7.7
n.d.
28.9
5.6
14.1
Remarkably, high conversions were observed at near-
stoichiometric amounts of diamine donors 1a-1c, indicating an
efficient displacement of the equilibrium via in situ cyclisation,
providing a greener substitute than 50 equivalents of volatile
isopropylamine. The transamination of 1a-b gave ω-
aminoaldehydes which underwent spontaneous cyclisation to
1-pyrroline 5a and 1-piperideine 5b, respectively (as confirmed
by GC-MS). Biotransformations were conducted at pH 9.0 and
1f
Expt. Cond.
:
5 mM 1a-1f, 1 mM
2
, 1 mM PLP, 0.2 mg mL–1 purified ATA, 100 mM
sodium phosphate buffer, pH 8.0, 30°C. a: one unit of enzyme activity was
defined as the amount of enzyme that produced 1 µmol of alanine per min at
30°C. n.d.: not determined (too low activity).
Our initial findings showed that all the recombinant SpuC
proteins displayed high activity with the biogenic diamine
donors 1a-1c of interest with comparable specific activities
within the different Pseudomonas species. Despite a 2-fold
increase in activity of Ec-YgjG with putrescine 1a, Pp-SpuC
exhibited a 10-fold greater activity with cadaverine 1b, Pc-
SpuC with spermidine 1c, with little or no activity displayed
with Cv-ATA. Surprisingly, the SpuC enzymes also gave
1
it was previously shown via H NMR that the imine monomers
are stable at conditions between pH 7-13.22 Amino donor 1c
,
instead, afforded the corresponding amino-imine that
spontaneously cyclises a second time to yield the fused bicyclic
structure 5c under biotransformation conditions. Interestingly,
increasing the concentration of 1c not only did not improve
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
the conversion to 6a, it was even detrimental, possibly due to
inhibition by the N-heterocycle side-product. The optical purity
of the product was almost invariably >99% with all three
enzymes tested.
In order to test the feasibility of larger scale processes we also
performed a preparative biotransformation with 4a and 2
View Article Online
DOI: 10.1039/C6GC02102F
Table 3. Amination of ketones 4a-n with diamine donors 1a-c (only the best
conversion obtained among the three SpuC enzymes is shown).
Diamine 1a
C
Diamine 1b
C
Diamine 1c
ee
(%)a
>99
>99
>99
75
>99
>99
>99
>99
>99
>99
>99
>99
>99
92
ee
C
(%)a
97
93
96
47
30
74
86
99
14
38
81
91
55
91
ee
Subs.
SpuC
SpuC
SpuC
(%)a
92
(%)a
97
(%)a
>99
>99
>99
74
(%)a
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
95
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
Pf
Pc
Pf
Pf
Pf
Pc
Pp
Pc
Pp
Pc
Pp
Pp
Pp
Pp
Pc
Pc
Pc
Pc
Pp
Pc
Pp
Pc
Pc
Pc
Pc
Pp
Pp
Pf
Pc
Pc
Pc
Pp
Pc
Pc
Pc
Pc
Pf
Pc
Pf
Pf
Pp
Pp
89b
75
93
equivalents of 1b, purifying the product by column
91
chromatography after extraction. The procedure afforded (S)-
6a with an isolated yield of 79% and >99% ee.
34b
12b
35b
63b
99b
12
72b
42b
62b
80b
93
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
Finally, to prove the wide applicability of this procedure, we
also tested a panel of prochiral ketones 4a-n that afford
synthetically useful chiral amines of interest for the production
of fine chemicals and pharmaceuticals (Scheme 3). Even
though the monomeric imines 5a and 5b are known to
oligomerise in aqueous environment,12 the corresponding
products (as well as the bicyclic product 5c) are water soluble
and do not interfere with the standard work-up. Therefore, the
chiral amine products of the transamination 6a-n can be easily
recovered by extraction (together with residual ketone) for
HPLC analysis. The highest conversions obtained for each
substrate among the three SpuC enzymes are listed in Table 3.
The (S)-enantiomer of the product was formed with all
substrate/enzymes pairs, most often with perfect
35
37b
92
80b
87
79
96
62b
99b
95
99b
Expt. Cond.
:
5 mM 4a-n, 1.5 equiv. 1a-c, 1mM PLP, 1% v/v DMSO, 2 mg mL–1
purified SpuC, 50 mM HEPES buffer, pH 9.0, 40°C, 250 rpm, 24 h. a: determined
by HPLC on a chiral stationary phase. b: in these cases 5. equiv. of amine donor
were used to increase the conversion values. C = conversion. ee = enantiomeric
excess.
For non-activated ketone substrates (4d-g, j and n) the
conversions appeared considerably lower, therefore we tested
also a slightly higher loading of amine donors 1a-b (5 equiv.),
leading to modest to excellent conversions. Interestingly,
however, with spermidine 1c comparable conversions were
reliably obtained at near-stoichiometric amounts (1.5 equiv.)
compared to the better amino donor 1b (5 equiv.). This effect
can be rationalised by the higher stability of the bicyclic amine
enantioselectivity. Only substrates 4d, i, k and n afforded in
some cases slightly lower ee values (56-96%), however, at least
one of the three SpuC enzymes tested provided >99% ee. This
emphasises the importance of screening several different
members of the family for each application.
product 5c
.
A few examples of the pharmaceutical relevance of the chiral
amine products thus obtained are several N-Methyl-D-
aspartate (NMDA) glycine-site antagonists used in the
treatment neuropathic pain (i.e., Parkinson’s disease),
synthesised from (S)-6l-m 25
,
or the analogues of the
antiarrythmic mexiletine obtained from (S)-6k 26
.
All these
compounds were obtained in almost quantitative conversion
and excellent enantioselectivity (Table 3).
Conclusions
In summary, we report the identification, heterologous
production and characterisation of SpuC orthologues, a class of
transaminases that uniquely allow the conversion of numerous
ketone substrates at the expense of natural diamines. The
enzymes were found to afford high conversion of a broad
range of substrates with a modest excess of amine donor,
giving good to excellent enantiomeric excess values for
pharmaceutically relevant chiral amines.
The application of these bifunctional SpuC enzymes effectively
combines the appealing substrate breadth of traditional ω-
ATAs with the equilibrium shifting potential of diamine donor
acceptance by α,ω-DTAs. This shift, requiring just 1.5-5.0
equivalents of bio-derived putrescine or cadaverine,
represents a clean, ‘green’ and effective synthetic strategy, as
opposed to the widespread use of a 50-fold excess of
conventional, often poorly-accepted monoamines. The
Scheme 3. Panel of aromatic and aliphatic ketones tested with SpuC enzymes.
The sterically hindered ortho-substituted ketone substrates 4a-
c
afforded high conversions with high enantioselectivities
(>99%). This high conversion is presumably due to a stabilising
interaction between the amine formed and the halogen atom
at the ortho-position, that drives the transamination reaction
more readily,23 than solely based on the electrostatic
activation of the halogen substituent (as evident from the
contrasting low conversion attained for the para-fluoro
derivative 4i and the high conversion for the para-nitro
derivative 4h). Likewise, a similar stabilising effect of the
intramolecular H-bond with the newly formed amine and
vicinal oxygen atom of 4k has been suggested,24 that would
account for the good conversion (41-59%) of this substrate.
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Berglund, M. Höhne and U. T. Bornscheuer, BioVteiecwhAnrtoicll.e AOdnlvin.e,
method also mitigates the need for expensive cofactor
supplementation and complex regeneration systems often
employed in conjunction.
The approaches developed in this work are particularly
relevant to recent advances in metabolic engineering of host
strains for enhanced fermentation of putrescine,27 potentially
enabling the creation of an integrated microbial cell catalyst
for chiral amine synthesis.
2015, 33, 566–604.
DOI: 10.1039/C6GC02102F
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10 L. Martínez-Montero, V. Gotor, V. Gotor-Fernández and I.
Lavandera, Adv. Synth. Catal., 2016, 358, 1618–1624.
11 I. Slabu, J. L. Galman, N. J. Weise, R. C. Lloyd and N. J. Turner,
ChemCatChem, 2016, 8, 1038–1042.
12 A. Gomm, W. Lewis, A. P. Green, E. O’Reilly, Chem. Eur. J.
2016, in press, DOI: 10.1002/chem.201603188.
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184, 3765–3773.
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Ptitsyn, FEBS Lett., 2005, 579, 4107–4112.
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This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Green Chemistry
Page 6 of 19
Novel
α,ω-DTAs
α,ω-DTAs
ω-ATAs
ω-ATAs
bifunctional TAs
Bifunctional TAs
ygjG
CvATA
YgjG
Cv-ATA
SpuC
SpuC
Table of contents text:
Bifunctional transaminases enable a strategy for the production of
chiral amines using small excesses of diamine donors.

Page 7 of 19
Green Chemistry
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DOI: 10.1039/C6GC02102F
ELECTRONIC SUPPLEMENTARY INFORMATION (ESI)
Biocatalytic transamination with near-stoichiometric
inexpensive amine donors mediated by bifunctional
mono- and di-amine transaminases
James L. Galman,a Iustina Slabu,a Nicholas J. Weise,a Cesar Iglesias,b
Fabio Parmeggiani,a Richard C. Lloyd,c Nicholas J. Turner*,a
a
Manchester Institute of Biotechnology (MIB), School of Chemistry, The University of Manchester,
Manchester, United Kingdom.
Facultad de Quimica, Universidad de la República, Montevideo, Uruguay.
Dr. Reddy's Laboratories, Chirotech Technology Centre, Cambridge, United Kingdom.
b
c
Table of contents
I. General methods and materials .....................................................................................................................2
1.1 Materials.....................................................................................................................................................2
II. Molecular biology protocols ...........................................................................................................................3
2.1 Cloning of spuC genes from Pseudomonas strains: ...........................................................................3
III. Expression and purification of HisTag recombinant proteins...................................................................4
3.1 Protein expression protocols...................................................................................................................4
3.2 Protein purification....................................................................................................................................4
3.3 Protein determination and SDSꢀPAGE analysis ..................................................................................5
Table S3. Purification of recombinant Pp_spuc, Pf_spuC and Pc_spuC expressed in E.coli. ............6
IV. Spectrophotometric activity analysis and determination of kinetic constants .......................................7
4.1 LꢀAlanine dehydrogenase (LꢀAlaDh) activity assay screen................................................................7
4.2 Determination of kinetic parameters for substrates 1aꢀk ....................................................................8
V. Biotransformations........................................................................................................................................10
5.1 Analytical scale........................................................................................................................................10
5.2 Preparative scale ....................................................................................................................................10
5.3 GCꢀassay conditions .............................................................................................................................. 12
5.4 HPLC assay conditions..........................................................................................................................12
S1

Green Chemistry
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DOI: 10.1039/C6GC02102F
I. General methods and materials
1.1 Materials
Commercially available reagents were used throughout without further purification. Putrescine and all
other reagents were purchased from Sigma Aldrich (St Louis, MO, USA) or Acros including anhydrous
solvents. Restriction enzymes, T4 ligase, taq polymerase, dNTPs and broad protein marker (2ꢀ212
kDa) were purchased from New England Biolabs (Ipswich, MA, USA). Escherichia coli DH5α and
BL21 (DE3) cells were purchased from New England Biolabs (Ipswich, MA, USA). Expression vector
pETꢀ28b was purchased from Novagen (Darmstadt, Germany) and was used for gene expression.
Chromobacterium violaceum (CvꢀTA) was purchased from LG standards (ATCC 12472, NCIMB
9178). Pseudomonas florescens (Asc no. 13500), Pseudomonas chlororaphis subsp. aureofaciens
30-84, (Asc. no 9392), Pseudomonas putida NBRC 14161 (Asc no. 9494), and Bacillus megaterium
(Asc. No 9376) were purchased from the NCIMB culture collection (Aberdeen, UK).
S2

Page 9 of 19
Green Chemistry
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DOI: 10.1039/C6GC02102F
II. Molecular biology protocols
2.1 Cloning of spuC genes from Pseudomonas strains:
The coding region of the spuC gene was amplified by colony PCR from Pseudomonas putida NBRC
14161, Pseudomonas chlororaphis subsp. aureofaciens 30-84, and Pseudomonas fluorescens using
the primers detailed in Table S1.
Table S1. Primers for cloning the spuC gene from Pseudomonas putida NBRC 14161, Pseudomonas
chlororaphis subsp. aureofaciens 30-84, and Pseudomonas fluorescens.
PfꢀspuC
(5’ꢀCAGCCATATGATGACCCGCAATAACCCGCAAACCCGTGAAꢀ3’)
(5’ꢀCACCTCGAGTTAGCCTTGCAACGCACTGAGCGTCAGGTCCꢀ3’)
PpꢀspuC (5’ꢀ AGTCGACATATGAGCACCAACAACCCGCAAACCCGꢀ3’)
(5’ꢀ ATTCGGCTCGAGCTACCGAATCGCCTCAAGGGTCꢀ3’)
PcꢀspuC FW: (5’ꢀ GCAAGCATATGAYCAGCAACAAYCCGCAAACCCGTGꢀ3’)
RV: (5’ꢀ GCAAGCTCGAGTTAGCCCTGYAAYGCACTCARSGTCꢀ3’)
The NdeI and XhoI restriction sites are underlined. The following PCR protocol was used: 5 min
denaturation at 95 °C and then 30 cycles of 30 s denaturation at 95 °C, 30 s annealing at 58 °C and
90 s elongation at 68 °C with a 5 min final extension time at 68 °C. The PCR product was cloned into
ZERO Blunt TOPO PCR cloning vector (K2830ꢀ20) following the manufacture’s protocol.
The alanine dehydrogenase (Aldh) gene from Bacillus megaterium was amplified using forward (5’ꢀ
GCAGCCATATGATTATTGGCGTACCAAAAGAAATCꢀ3’) and reverse (5’ꢀGCAAGCTCGAGTTAGATꢀ
AGAAGCTAATTCTTTTTCAAGAGꢀ3’) primers (XhoI/NdeI restriction sites underlined) and was
amplified and cloned into TOPO PCR cloning vector as above.
PfꢀspuC and Aldh genes were subcloned into pETꢀ28b expression vector containing an Nꢀterminal
His6 tag with a thrombin linker. PcꢀspuC and PpꢀspuC, was subcloned into pETꢀ22b expression vector
containing a Cꢀterminal His tag with a thrombin linker. The inserted genes were inꢀframe downstream
from the ribosome binding site as confirmed via DNA sequencing (Eurofins).
Table S2. Sequenceꢀidentity matrix heat map of the transaminases used to draw the cladogram in
Figure 1.
ARS-
ATA
Bme-
YgjG
Bmy-
YgjG
[%]
ATA-117
Ec-YgjG
AdATA
VfATA
CvATA
PaSpuC
ATA-117
ARS-ATA
Ec-YgjG
Bme-YgjG
Bmy-YgjG
AdATA
100.0
16.7
17.0
18.1
17.7
16.4
15.0
16.1
15.5
16.7
100.0
25.6
27.8
29.2
25.8
22.9
25.7
28.3
17.0
25.6
100.0
61.5
60.0
28.8
25.0
26.7
28.3
18.1
27.8
61.5
100.0
85.8
29.8
26.0
27.9
28.4
17.7
29.2
60.0
85.8
100.0
30.3
24.6
25.9
28.7
16.4
25.8
28.8
29.8
30.3
100.0
28.9
32.8
35.9
15.0
22.9
25.0
26.0
24.6
28.9
100.0
37.3
38.3
16.1
25.7
26.7
27.9
25.9
32.8
37.3
100.0
59.6
15.5
28.3
28.3
28.4
28.7
35.9
38.3
59.6
100.0
VfATA
CvATA
PaSpuC
S3

Green Chemistry
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DOI: 10.1039/C6GC02102F
III. Expression and purification of HisTag recombinant proteins
3.1 Protein expression protocols
The spuC genes from Pseudomonas putida NBRC 1416 and Pseudomonas chlororaphis subsp.
aureofaciens 30-84, were transformed into E. coli BL21 (DE3) for yielding E. coli BL21 (pETꢀ22b(+)4–
Pp_spuC) and E. coli BL21 (pETꢀ22b(+)4–Pc_spuC). Similarly, the spuC gene from Pseudomonas
fluorescens F113 in pETꢀ28b(+) expression vector and the ald gene from Bacillus megaterium DSM
319 in pETꢀ22b(+)ꢀ4 expression vector were used to yield E. coli BL21 (pETꢀ28b(+)–Pf_spuC) and E.
coli BL21 (pETꢀ22b(+)4–Bm_AlaDh), respectively. The freshlyꢀprepared strains were cultivated in 600
mL of the LB medium supplemented with 50 ꢁg/mL antibiotic (ampicillin or kanamycin) in 1ꢀL
Erlenmeyer flasks at a rotary shaking rate of 220 rpm at 37°C. The recombinant protein expression
was induced by adding isopropyl bꢀDꢀ1ꢀthiogalactopyranoside (IPTG) (0.2 mM, final) when A600
reached 0.6 ꢀ 0.8. The cell cultures were incubated at 18 °C for 16 h. The cells were harvested by
centrifugation (4°C, 3,250xg, 20 min).
3.2 Protein purification
For purification purposes, cell pellets were resuspended (1g of wet cell paste/10 mL) in HEPES buffer
(100mM, 1mM PLP, 5mM imidazole, pH 8). The cell pellets were lysed in an iced bath by ultraꢀ
sonication by Soniprep 150 (20 cycles of 20s on/20s off). After centrifugation (4⁰C, 16,000xg, 20 min)
the supernatant was used for protein purification manually or using an AKTA Pure system. Crude
extract was then loaded onto a 5 mL HisTrap column. Purification was achieved manually or
automated using an AKTA Pure system, using deꢀgassed HEPES buffer 1 (100 mM, 1mM PLP,
30mM imidazole, pH 8.0) and an elution buffer of HEPES buffer 2 (100 mM, 1mM PLP, 300mM
imidazole, pH 8.0).
Figure S1. Representative SDSꢀPAGE analysis for the purification of the recombinant PpꢀSpuC
protein on AKTA (A2ꢀA5 and B6: flowꢀthrough of crude extract during column loading; B5 and B4:
fractions of PpꢀSpuC protein post HisTag purification; Mark12™ Unstained Standard protein ladder).
S4

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Green Chemistry
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DOI: 10.1039/C6GC02102F
The column was initially washed with 5 column volumes HEPES buffer 1. The protein was then eluted
with 10 column volumes of 100% elution buffer (50 mM HEPES, 1mM PLP, pH 9.0). 5 mL fractions
were collected during the elution phase and a protein gel was run to identify fractions containing the
transaminase protein, before these were pooled together. The protein solutions were concentrated in
an Amicon® Ultraꢀ15 10K centrifugal filter device and the purified enzyme was flash frozen and stored
at –80°C. The purity was analysed by SDS/PAGE and the protein was more than 95% pure.
IustinaHis TrapPp4x10m Lshorter001:10_U V1_280nm
Ius tinaH isT rapPp4x10mLs horter001:10_Conc
IustinaHis TrapPp4x 10mLs horter001:10_Fractions
m AU
5000
4000
3000
2000
1000
0
F2
0.0
A2
A3
A4
A5
A6 F 2
20.0
B6
B5
B4
B3
B2
B1
C1
W aste
ml
10.0
3 0.0
40.0
50.0
Figure S2. Representative chromatogram for purification of the His6ꢀtagged PpꢀSpuC
recombinant protein by metal ion affinity chromatography. Absorbance at 280 nm is shown in
blue and the imidazole concentration of the buffer used in green (maximal value: 300 mM).
The fractions collected (5mL each) are shown in red.
3.3 Protein determination and SDS-PAGE analysis
Concentrations of soluble protein was analysed by SDSꢀPAGE with 15% resolving gel and 5%
stacking gel in a Trisꢀglycine buffer system. The gel was stained with BioꢀRad Coomassie Blue 250 kit
(BioꢀRad lab, Munich, Germany), with bovine serum albumin (BSA) as a standard protein.
S5

Green Chemistry
Page 12 of 19
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DOI: 10.1039/C6GC02102F
Figure S3. SDSꢀPAGE analysis of the three spuC recombinant proteins and Cv_TA (lane 1: Marker
12 ; lane 2: Pp_spuC; lane 3: Pf_spuC; lane 4: Pc_spuC; lane 5: Cv_TA)
Table S3. Purification of recombinant Pp_spuc, Pf_spuC and Pc_spuC expressed in E.coli.
Recombinant
protein
Culture medium
volume (L)
Cell pellet
(g)
Yield pure
protein (g)
Purity
(%)
Pp_spuC
Pf_spuC
Pc_spuC
0.6
0.6
0.6
5.8
6
0.200
0.250
0.390
98
94
95
5
S6

Page 13 of 19
Green Chemistry
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DOI: 10.1039/C6GC02102F
IV. Spectrophotometric activity analysis and determination of kinetic constants
4.1 L-Alanine dehydrogenase (L-AlaDh) activity assay screen
The transaminase activity was determined on a Tecan Sunrise™ plate reader, using a modified
alanine dehydrogenase/ transaminase assay method. The spectrophotometric assay monitors the
reduction of the tetrazolium salt INT by NADH, using 1ꢀmethoxy PMS as catalyst (λ= 503nm, ε=
5286.2 Mꢀ1cmꢀ1).
Solution 1 (doubleꢀfold concentrated stock) was made up of the following: 2 mM nicotinamide adenine
dinucleotide (NAD+, 100 mM in water), 0.6 mM 2ꢀ(4ꢀiodophenyl)ꢀ3ꢀ(4ꢀnitrophenyl)ꢀ5ꢀphenylꢀ2Hꢀ
tetrazolium (INT, 60 mM in DMSO), 0.01 mM 1ꢀmethoxyꢀ5ꢀmethylphenazinium methyl sulfate (1ꢀ
methoxy PMS, 1 mM in water), 0.05 mg/mL alanine dehydrogenase from B. megaterium (Bm_AlaDh)
in 100 mM sodium phosphate buffer pH 8.
The 96ꢀwell substrate microplates contained 5 mM amine substrate (doubleꢀfold concentrated, in 100
mM sodium phosphate buffer, pH 8) and 0.2 mg/mL transaminase (doubleꢀfold concentrated purified
enzyme in 100 mM HEPES buffer with 1 mM PLP, pH 8). 100 ꢁL of solution 1 were added to the
substrate microplate and the assay was initiated by addition of 2 mM sodium pyruvate (10 mM in
water, doubleꢀfold concentrated).
Experiments were run on a spectrophotometer at 37 ºC with the measured activity at a wavelength of
503 nm and the absorbance taken every 13 seconds for 60 minutes. One Unit (U) is defined by one
ꢁmole of product per minute.
Specific activities against substrates 1a-k are shown in Table S4.
Table S4. Specific activities for substrates 1a-k determined using the LꢀAlaDhꢀTA assay.
Specific activity U/mg
substrate
Pp_spuC Pf_spuC Pc_spuC Ec_yjgj Cv_TA
1,4ꢀdiaminobutane
1,5ꢀdiaminopentane
spermidine
1.3
27.3
15.7
23.2
20.4
16.5
14.9
15.2
7.5
0.4
16.5
16.5
25.7
18.5
19.3
18.1
24.7
2.7
1.4
15.1
21.5
30.4
18.7
13.5
13.5
14.7
2.3
2.7
2.7
2.1
nd
nd
7.7
nd
(S)ꢀmethylbenzylamine
isopropylamine
28.9
5.6
0.05
nd
butylamine
14.1
10.9
18.4
4.4
1,6ꢀdiaminohexane
1,7ꢀdiaminoheptane
1,8ꢀdiaminooctane
1,9ꢀdiaminononane
1,10ꢀdiaminodecane
0.6
0.45
0.33
nd
13
4.4
3.1
4.6
2.1
3.8
1.7
nd
2.3
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Green Chemistry
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DOI: 10.1039/C6GC02102F
4.2 Determination of kinetic parameters for substrates 1a-k
Kinetic parameters (Tables S5ꢀS7) were deduced by nonꢀlinear leastꢀsquare regression
analysis based on MichaelisꢀMenten kinetics.
7
Ec_yjgj M
Ec_yjgj C
Pp_spuC M
Pp_spuC C
Pf_spuC M
6
5
4
3
2
Pf_spuC C
1
Pc_spuC M
0
Pc_spuC C
0
50
100
150
200
Substrate concentration (mM)
Figure S4. Determination of kinetic constants for Ec_ygjg, Pp_spuC, Pf_spuC and Pc_spuC for
spermidine substrate using the LꢀAladh/TA liquid phase assay. The measured (M, dotted line) and the
calculated (C, solid line) rates of reactions for each protein are plotted against the substrate
concentration. The kinetics parameters are determined using nonꢀlinear leastꢀsquares regression
method.
Table S5. Values of Km for substrates 1a-k determined using the LꢀAlaDhꢀTA assay.
Km (mM)
substrate
Pp_spuC Pf_spuC Pc_spuC Ec_yjgj Cv_TA
1,4ꢀdiaminobutane
1,5ꢀdiaminopentane
1,6ꢀdiaminohexane
1,7ꢀdiaminoheptane
1,8ꢀdiaminooctane
1,9ꢀdiaminononane
1,10ꢀdiaminodecane
spermidine
62.34
25
52.99
32.38
2.31
1.02
0.39
2.13
2.14
16.95
4.64
6.05
59.87
22.99
4.05
0.64
0.52
1.64
0.17
25.00
3.81
5.86
0.47
0.34
2.54
0.44
0.53
nd
nd
29.9
nd
Nd
48.04
3.92
1.09
0.73
2.11
0.34
nd
1.41
1.06
0.59
3.48
0.91
20.82
1.25
5.86
(S)ꢀmethylbenzylamine
butylamine
1.06
10.19
nd
S8

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DOI: 10.1039/C6GC02102F
Table S6. Values of Vmax for substrates 1a-k determined using the LꢀAlaDhꢀTA assay.
Vmax (mM/min)
substrate
Pp_spuC Pf_spuC Pc_spuC Ec_yjgj Cv_TA
nd
1,4ꢀdiaminobutane
1,5ꢀdiaminopentane
spermidine
(S)ꢀmethylbenzylamine
butylamine
1,6ꢀdiaminohexane
1,7ꢀdiaminoheptane
1,8ꢀdiaminooctane
1,9ꢀdiaminononane
1,10ꢀdiaminodecane
0.13
2.73
1.57
2.32
1.65
1.49
1.52
0.75
1.3
0.04
1.65
1.65
2.57
1.93
1.81
2.47
0.27
0.44
0.38
0.14
1.51
2.15
3.04
1.35
1.35
1.47
0.23
0.31
0.17
0.27
0.27
0.77
nd
2.1
nd
nd
2.89
1.41
1.09
1.84
0.44
0.46
0.23
0.06
0.05
0.03
nd
0.21
nd
Table S7. Values of Kcat for substrates 1a-k determined using the LꢀAlaDhꢀTA assay.
Kcat (sꢀ1)
substrate
Pp_spuC Pf_spuC Pc_spuC Ec_yjgj Cv_TA
2.33
2.33
18.17
nd
nd
6.68
nd
25.09
12.24
9.46
15.97
3.82
3.99
1.99
1,4ꢀdiaminobutane
1,5ꢀdiaminopentane
spermidine
(S)ꢀmethylbenzylamine
butylamine
1,6ꢀdiaminohexane
1,7ꢀdiaminoheptane
1,8ꢀdiaminooctane
1,9ꢀdiaminononane
1,10ꢀdiaminodecane
1.11
23.15
13.32
19.68
13.99
12.64
12.89
6.36
0.34
14.38
14.38
22.41
16.82
15.77
21.53
2.35
1.19
12.86
18.31
25.89
1.50
11.49
12.51
1.95
nd
0.51
0.38
0.28
nd
11.03
1.78
3.83
2.64
nd
3.31
1.44
S9

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DOI: 10.1039/C6GC02102F
V. Biotransformations
5.1 Analytical scale
All biotransformations were carried out in 2 mL Eppendorf tubes, in a volume of 0.5 mL. To a mixture
of amine substrate (25 mM from a 500 mM stock in 50 mM HEPES buffer, 1mM PLP, pH 9) and
ketone (5 mM from a 500 mM stock solution in DMSO or MeOH), was added the enzyme ( 2mg/mL,
purified or as cellꢀfree lysate, prepared as described). The reactions were placed in a shaking
incubator at 30°C and 250 rpm for 12 hours. The reactions were quenched by addition of 10 M NaOH
(100ꢁL), followed by extraction with methyl tertꢀbutyl ether (300 ꢁL). The organic phase was dried on
MgSO4 and analysed on normal phase chiral HPLC or GCꢀFID. For the GCꢀFID analysis, the samples
were derivatized to the corresponding acetamides by addition of 30 ꢁL Et3N and 20 ꢁL Ac2O.
5.2 Preparative scale
In a 250 mL conical flask, the amine (20 mM, 2 eq) was dissolved in 50 mM HEPES buffer, containing
1mM PLP, pH 9 and the transaminase, Pp_spuC (60 mg from a stock of 20 mg/mL in buffer prepared
as above) was added. The pH of the mixture was adjusted to 9 and the ketone (10 mM, 1eq) in
DMSO (2.5% v/v) was added. The reaction was placed in a shaking incubator, 200 rpm, 30°C for 24h
and the progress was followed by HPLC, with samples prepared as described in the analytical scale
procedure. Samples were taken every hour for an initial period of 5 hours, followed by sampling at
19h, 21h and 29 h, respectively. The time course of the reaction is shown in Figure S5.
120
100
80
60
40
20
0
amine
ketone
0
10
20
30
Time (h)
Figure S5. Kinetics of the transamination of 2ꢀfluoroacetophenone (10 mM) by Pp_spuC, using
cadaverine 1b as amino donor (20 mM, 2 eq). The formation of the (S)ꢀ2ꢀfluoromethylbenzylamine is
shown in solid blue line, whilst the concentration of the 2ꢀfluoroacetophenone is shown in red dotted
line.
S10

Page 17 of 19
Green Chemistry
Table S8. Amination of ketones 4aꢀm with diamine donors 1a-c
.
Diamine 1a
Diamine 1b
Diamine 1c
PpꢀSpuC
PfꢀSpuC
PcꢀSpuC
PpꢀSpuC
PfꢀSpuC
PcꢀSpuC
PpꢀSpuC
PfꢀSpuC
PcꢀSpuC
c
(%)a
84
ee
c
(%)a
92
ee
(%)a
>99
>99
>99
75
>99
>99
>99
>99
>99
>99
>99
>99
>99
c
ee
c
(%)a
95
ee
c
ee
(%)a
>99
>99
>99
90
>99
>99
>99
>99
>99
>99
>99
>99
>99
c
(%)a
97
93
91
72
34
62
56
93
35
80
87
5
ee
c
(%)a
83
67
79
47
13
8
62
55
13
28
88
65
55
ee
c
(%)a
92
65
81
41
13
27
13
63
14
12
81
91
11
ee
(%)a
>99
>99
>99
50
>99
>99
>99
>99
>99
>99
>99
>99
>99
c
(%)a
97
93
96
90
30
74
86
99
37
38
60
71
44
ee
Subs.
(%)a
>99
>99
>99
69
(%)a
92b
89b
88b
51b
13b
35b
85b
99b
13b
37b
35b
29b
62b
(%)a
>99
>99
96
(%)a
>99
>99
>99
>99
>99
>99
>99
>99
80
(%)a
95
(%)a
>99
>99
>99
74
(%)a
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
56
(%)a
>99
>99
>99
36
4a
4b
4c
4d
4e
4f
4g
4h
4i
80
68
68
88
65
83
67
75
37b
12b
5b
34b
12b
33b
5b
9
82
>99
85
>99
94
>99
>99
83
64b
42b
16b
80b
59
59b
25b
44b
46b
63
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
93
>99
>99
>99
>99
63b
41
57
12
11
28
13
4j
4k
4l
32b
92
27b
91
59b
99
>99
96
>99
>99
10b
98
79
58
77
49
96
95
58
73
>99
>99
4m
>99
57
Expt. Cond.: 5 mM 4a-n, 1.5 equiv. 1a-c, 1mM PLP, 1% v/v DMSO, 2 mg mL–1 purified SpuC, 50 mM HEPES buffer, pH 9.0, 40°C, 250 rpm, 24 h.
a: determined by HPLC on a chiral stationary phase. b: in these cases 5. equiv. of amine donor were used to increase the conversion values.
S11

Green Chemistry
Page 18 of 19
View Article Online
DOI: 10.1039/C6GC02102F
After 30 h, the reaction was stopped by addition of 10 M NaOH until the pH reached 12. The reaction
was filtered through a short pad of Celite and extracted with MTBE (3 x 10 mL); the combined
organics were washed with brine (1 x 20 mL) and dried on Na2SO4 and concentrated in vacuo to
afford the amine as a pale yellow oil (55 mg).
1H NMR (400 MHz, CDCl3) δ 7.41 (td, J = 7.7, 2.0 Hz, 1H), 7.25 – 7.17 (m, 1H), 7.12
(td, J =7.5, 1.3 Hz, 1H), 7.01 (ddd, J = 10.9, 8.1, 1.3 Hz, 1H), 4.39 (q, J = 6.7 Hz,
1H), 1.42 (d, J = 6.7 Hz, 3H)
5.3 GC-assay conditions
GCꢀMS method: Hewlett Packard HP 6890 equipped with a HPꢀ1MS column, a HP 5973 Mass
Selective Detector and an ATLAS GL FOCUS sampling robot; method: 90⁰C for 0 min, then 10⁰C
min1 to 200C, hold for 10 min. GC FID method: Agilent 6850 equipped with a Gerstel
Multipurposesampler MPS2L and an Agilent CHIRASILꢀDEX CB 25 m x 0.25 mm DF=0.25 column;
method: 120⁰C isothermal, 15 min. The amines were analyzed by GCꢀFID as their corresponding
acetamides, following derivatization as described previously.
5.4 HPLC assay conditions
Chiral normal phase HPLC was performed on an Agilent system (Santa Clara, CA, USA) equipped
with a G1379A degasser, G1312A binary pump, a G1367A well plate autosampler unit, a G1316A
temperature controlled column compartment and a G1315C diode array detector. CHIRALCEL® ODꢀH
Analytical (Daicel, Osaka, Japan), 250 mm length, 4.6 mm diameter, 5 ꢁm particle size) column was
used. The typical injection volume was 15 ꢁl and chromatograms were monitored at 265 nm. Solvent
mixtures are given in nꢀhexane/isopropanol ratios (+0.1% diethylamine v/v).
S12

Page 19 of 19
Green Chemistry
View Article Online
DOI: 10.1039/C6GC02102F
Table S9. Retention times for substrates and products
Ketone
Product
6a
Amine
(R)ꢀ 7.3 min, (S)ꢀ 7.81 min
(S)ꢀ 9.83 min
n-Hex/i-PrOH ratio
4a (4.59 min)
4b (6.93 min)
4c (5.82 min)
4d (8.44 min)
4e (5.93 min)
4f (5.57 min)
4g (5.81 min)
4h (11.37 min)
4i (5.53 min)
4j (9.05 min)
4k (10.5 min)
4l (4.92 min)
4m (4.96 min)
95:5
95:5
6b
6c
(R)ꢀ 7.3 min, (S)ꢀ9.07 min
(R)ꢀ 13.01 min, (S)ꢀ18.28 min
(S)ꢀ 11.09 min
95:5
6d
6e
95:5
95:5
6f
(S)ꢀ 9.41 mn
95:5
6g
6h
6i
(S)ꢀ 10.07 min
95:5
(S)ꢀ 14.89 min
90:10
95:5
(S)ꢀ 8.92 min
6j
(S)ꢀ 15.57 min
95:5
6k
(R)ꢀ 11.41 min, (S)ꢀ 17.87 min
(S)ꢀ 8.3 min
90:10
90:10
90:10
6l
6m
(S)ꢀ 11.45 min
S13