Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades

Article abstract of DOI:10.1002/cssc.201802580

Plant-derived carbohydrates are an abundant renewable resource. Transformation of carbohydrates into new products, including amine-functionalized building blocks for biomaterials applications, can lower reliance on fossil resources. Herein, biocatalytic production routes to amino carbohydrates, including oligosaccharides, are demonstrated. In each case, two-step biocatalysis was performed to functionalize d-galactose-containing carbohydrates by employing the galactose oxidase from Fusarium graminearum or a pyranose dehydrogenase from Agaricus bisporus followed by the ω-transaminase from Chromobacterium violaceum (Cvi-ω-TA). Formation of 6-amino-6-deoxy-d-galactose, 2-amino-2-deoxy-d-galactose, and 2-amino-2-deoxy-6-aldo-d-galactose was confirmed by mass spectrometry. The activity of Cvi-ω-TA was highest towards 6-aldo-d-galactose, for which the highest yield of 6-amino-6-deoxy-d-galactose (67 %) was achieved in reactions permitting simultaneous oxidation of d-galactose and transamination of the resulting 6-aldo-d-galactose.

Full text of DOI:10.1002/cssc.201802580

Accepted Article
Title: Biocatalytic production of amino-carbohydrates through
oxidoreductase and transaminase cascades
Authors: Ville Aumala, Filip Mollerup, Edita Jurak, Fabian Blume,
Johanna Karppi, Antti Koistinen, Eva Schuiten, Moritz Voss,
Uwe Bornscheuer, Jan Deska, and Emma Master
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10.1002/cssc.201802580
ChemSusChem
FULL PAPER
Biocatalytic production of amino-carbohydrates through
oxidoreductase and transaminase cascades
Ville Aumala[a], Filip Mollerup[a], Edita Jurak[b], Fabian Blume[c], Johanna Karppi[a], Antti E. Koistinen[a],
Eva Schuiten[d], Moritz Voß[d], Uwe Bornscheuer[d], Jan Deska[c], Emma R. Master[a,e]
*
key building blocks in the synthesis of different types of
polymers[6,7]. For example, diacids, diamines and AB monomers
(e.g. molecules containing both the carboxylic acid and amino
groups) are required for polyamide synthesis[8], and diacids and
Abstract: Plant-derived carbohydrates constitute an abundant
renewable resource. Transformation of carbohydrates into new
products, including amine-functionalized building blocks for
biomaterial applications, can lower reliance on fossil resources.
Herein, we demonstrate biocatalytic production routes to amino-
carbohydrates, including oligosaccharides. In each case, we
performed a two-step biocatalysis to functionalized D-galactose-
containing carbohydrates, which employed either the galactose
oxidase from Fusarium graminearum or a pyranose dehydrogenase
from Agaricus bisporus followed by the w-transaminase from
Chromobacterium violaceum (Cvi-w-TA). Formation of 6-amino-6-
deoxy-D-galactose, 2-amino-2-deoxy-D-galactose and 2-amino-2-
deoxy-6-aldo-D-galactose was confirmed by mass spectrometry. Cvi-
w-TA activity was highest towards 6-aldo-D-galactose, where highest
yield of 6-amino-6-deoxy-D-galactose (67%) was achieved in
reactions permitting simultaneous oxidation of D-galactose and
transamination of the resulting 6-aldo-D-galactose.
diols for polyester synthesis[9–13]
.
An emerging area of research aims to utilize the versatility and
the ensuing useful properties of the native structures of plant
based carbohydrates instead of degrading the structures to
monomers[14–17]
.
Bifunctional carbohydrates (e.g., diacids,
diamines or AB monomers) from native carbohydrate structures
are among the desired products. Besides enzymatic synthesis of
amino-sugars from activated sugar nucleotides and sugar
phosphates[18–20], biocatalytic cascades to amino-carbohydrates
from biomass-derived, native carbohydrates are still missing. If
established, these pathways would create a new class of
telechelic, amino-functionalized building blocks that retain
inherent attributes of native carbohydrate structures (e.g.,
biocompatibility, hydrophilicity), while being primed for assembly
(e.g., through stable amide linkages) with other building blocks or
polymers with complementary functionalities (e.g. carboxyl
Introduction
groups)[15,21,22]
.
Given their wide availability and structural versatility, plant-
derived carbohydrates represent an important raw material for the
production of new bio-based products that reduce reliance on
petroleum. To date, most applications of plant carbohydrates
begin by deconstructing corresponding polysaccharides to
Existing chemical pathways for carbohydrate amination include
applications of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) to
oxidize primary hydroxyl groups[23], and sodium periodate to
oxidize secondary hydroxyl groups[24], followed by subsequent
monomers for fermentation to fuels and platform chemicals[1–5]
.
reductive amination to assemble the corresponding amines[25]
.
Bifunctional molecules containing a terminal acid and an amine
functionality are among the desired products, because they are
These routes, however, often require toxic transition metal
catalysts, produce volatile organic compounds, and result in
decreased polymer chain length[26]. As a gentle alternative to
chemical oxidation procedures, the galactose oxidase from
Fusarium graminearum (FgrGaOx, UniProt: A0A2H3HJK8) has
been used to introduce an aldehyde functionality at the C6
position of D-galactose in D-galactose-containing oligo- and
polysaccharides [15,27,28]. Specificity of FgrGaOx towards the C6
position of D-galactose has been well documented[29], and the
resulting oxidized positions have served as sites for further
derivatization, including reductive amination, crosslinking through
acetal formation, and phosphorylation[15,30,31]. Our group recently
described the application of the oligosaccharide oxidase from
Sarocladium strictum to permit amide bond formation with
clickable monomers, leading to telechelic molecules from xylo-
oligosaccharides that are primed for reassembly through copper-
catalyzed azide-alkyne cycloaddition[22]. Thus, while chemo-
enzymatic routes to aminated carbohydrates have been
demonstrated, fully biocatalytic cascades for amination of non-
activated carbohydrates are unprecedented yet highly desirable
in order to simplify reaction pathways, increase sustainability, and
to achieve higher control over reaction products.
[a]
V. Aumala, F. Mollerup, Dr. J. Karppi, A.E. Koistinen, Prof. E.R.
Master
Department of Bioproducts and Biosystems
Aalto University
Kemistintie 1, 02150, Espoo, Finland
E-mail: emma.master@utoronto.ca
Dr. E. Jurak
Department of Aquatic Biotechnology and Bioproduct Engineering
University of Groningen
Nijenborgh 4, 9747 AG, Groningen, The Netherlands
F. Blume, Prof. J. Deska,
Department of Chemistry and Materials Science
Aalto University
Kemistintie 1, 02150, Espoo, Finland
E. Schuiten, M. Voß, Prof. U. Bornscheuer
Department of Biotechnology and Enzyme Catalysis
Greifswald University
Felix-Hausdorff-Straße 4, 17487 Greifswald, Germany
Prof. Emma R. Master
[b]
[c]
[d]
[e]
Department of Chemical Engineering and Applied Chemistry,
University of Toronto, 200 College Street, Toronto, Ontario, M5S
3E5, Canada
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cssc.201802580
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Transaminases are pyridoxal 5’-phosphate (PLP) dependent
enzymes that catalyze the transfer of an amino group from
primary amines acting as the amine donor to carbonyls acting as
amine acceptor[32]. Briefly, transaminases operate via a ping-
pong, bi-bi reaction, where the first half-reaction comprises the
transfer of the amino group from the amine donor to the PLP
cofactor. The deaminated amine donor (i.e. the respective
ketone/aldehyde product) is then released, leaving the cofactor
as pyridoxamine 5’-phosphate (PMP). In the second half reaction,
the amino group is transferred from enzyme-bound PMP to the
acceptor, regenerating the PLP cofactor and completing the
transamination cycle. Transaminases have been functionally
classified as a-transaminases (a-TAs) and w-transaminases (w-
TAs), based on amine donor and acceptor specificity. Whereas a-
TAs transfer amino groups from the a-carbon of amino acids to a-
keto acids, w-TAs are more versatile as they do not require a
carboxylate group in the amine acceptor and can donate the
amino group to a-keto acids as well as other ketones and
aldehydes[32–34]. In addition to substrate versatility, no requirement
for cofactor regeneration or nucleotide sugars as substrates are
distinguishing advantages of w-TAs relative to other types of
enzymes potentially capable of producing carbohydrate amines
(e.g. reductive aminases)[35–37]. So-called sugar transaminases
have been shown to accept nucleotide sugars as amine donors
and acceptors[19,20]; however, these are not suitable for amination
of plant-based carbohydrates.
ketone-functionalized carbohydrates, which are also potential
substrates for w-TAs[53–55]
.
Scheme 1. Biocatalytic cascades to aminated carbohydrates. (A) Oxidation of
a D-galactosyl subunit on a carbohydrate molecule to 6-aldo-D-galactosyl (2b-
6b; Table S1 for 3b-6b structures) by FgrGaOx and the subsequent amination
of the aldehyde group to 6-amino-6-deoxy-D-galactosyl (2c-6c) by the Cvi-w-TA.
(B) Oxidation of D-galactose (2a) to 2-keto-D-galactose (2d) by AbiPDH1[53], and
the subsequent amination of the ketone 2d to the amine 2e by the Cvi-w-TA.
(C) Oxidation of 2e by FgrGaOx to the bifunctional intermediate 2f, and the
putative amination of the aldehyde group to the diamine 2g by the Cvi-w-TA. R
= remaining oligosaccharide. Note: whereas the a-configuration of galactose is
drawn, both a- and b-isomers occur. The conformation of the C-2 amino group
in reaction product 2e is unknown. Chiral (S)-(−)-PEA was used instead of a
Amines in general are greatly underrepresented in renewable
biomass, when compared to the frequent need of amines in
chemical and polymer synthesis[38,39]. This makes biocatalytic
production of amines from alcohols highly desirable, yet
challenging, because no known single enzyme can catalyze such
a transformation, and the chemical or chemo-enzymatic methods
involving oxidation and reductive amination often involve toxic
chemicals and require complicated synthetic procedures[40–42]. To
date, w-TAs have been studied extensively for asymmetric
synthesis of pharmaceuticals, which has been summarized in
several excellent reviews[41,43–45]. In that context, enzymatic
pathways from primary and secondary alcohols to the
racemic mixture due to the strict stereoselectivity of the Cvi-w-TA[56]
.
Abbreviations: AbiPDH1, pyranose dehydrogenase from Agaricus bisporus;
Cvi-w-TA, w-TA from Chromobacterium violaceum; FgrGaOx, galactose
oxidase from Fusarium graminearum; HRP, horseradish peroxidase from
horseradish; ScoSLAC, small laccase from Streptomyces coelicolor.
corresponding
dehydrogenases coupled with an w-TA have been
demonstrated[46–48]
By contrast, application of w-TAs for
bioproduct development from renewable biomass has only been
investigated in a few studies.[49,50]. For example, Lerchner et al.
(2013) showed the two-step conversion of isosorbide to the
corresponding diamine using an alcohol dehydrogenase and an
w-TA[49,51]. In another, more recent study, Dunbabin et al. (2017)
amines, utilizing
oxidases or
alcohol
The w-TA from Chromobacterium violaceum (Cvi-w-TA, UniProt:
Q7NWG4) is recognized as having a broad substrate range, and
has activity towards hydroxylated aldehydes such as D-erythrose
(1), glycolaldehyde and glyceraldehyde[47,48,56]. In the present
study, Cvi-w-TA was investigated for its potential to aminate aldol-
and keto-carbohydrates initially formed through oxidation by
FgrGaOx or the pyranose dehydrogenase from Agaricus bisporus
(AbiPDH1, UniProt: Q3L1D1)[53], respectively. Cvi-w-TA activity
on oxidized carbohydrates was also compared against the M1
variant of the w-TA from Vibrio fluvialis (Vfl-w-TA) (Uniprot:
F2XBU9) engineered by the Bornscheuer group for improved
preference towards substrates other than pyruvate, and generally
improved activity in the neutral pH range[57]. Our analysis
demonstrates biocatalytic cascades to aminated cyclic
carbohydrates, including oligosaccharides (Scheme 1).
Corresponding pathways generate a new class of renewable
.
demonstrated
transaminase-catalyzed
production
of
furfurylamines from furfurals[52]. On the other hand, the ability of
FgrGaOx to oxidize C-6 hydroxyls on galactose-containing mono-
oligo- and polysaccharide substrates has been shown to be an
efficient
way
to
produce
aldehyde-functionalized
carbohydrates[27,28]
,
which might be accepted by w-TAs.
Moreover, carbohydrate oxidoreductases with different regio- and
substrate specificities beyond the oxidation of the primary C-6
hydroxyl group (e.g., pyranose dehydrogenases) can help extend
the range of available carbonyl-containing carbohydrates to
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10.1002/cssc.201802580
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telechelic molecules that were missing from the arsenal of
building blocks to new bio-based polymers.
TA in an attempt to produce the amine 2e. In the case of each
sequential reaction, a near quantitative yield for the oxidation of
2a was confirmed by TLC before initiating the transaminase
reaction (data not shown).
Results and Discussion
Mass spectrometric ESI-Q-TOF analysis verified the enzymatic
production of both amines 2c and 2e. The masses of protonated
and sodiated ion adducts of amines 2c (Figure 1A) and 2e (Figure
1B), as well as the expected isotopes, were all found in
corresponding reactions, confirming their production through the
oxidoreductase-transaminase cascade reactions.
Activity of Cvi- -TA on oxidized D-galactose and D-
w
galactosamine
The yields of FgrGaOx and AbiPDH1 produced in Pichia pastoris
were 108 mg/L and 3.9 mg/L, respectively, whereas the yields of
Cvi-w-TA and Vfl-w-TA M1 produced in E. coli were 115 mg/L and
98 mg/L, respectively (Figure S1 in Supporting Information).
These values are in the same range as previous reports
Having confirmed the production of 2e, we ventured to produce
the diamine 2g, which is expected to permit carbohydrate
coupling through imine bond formation[61]. The activity of FgrGaOx
on 50 mM 2e was determined with the ABTS assay to be 50.1 ±
4.9 U/mg enzyme, which is about 10% of FgrGaOx activity on 2a.
Formation of the corresponding bifunctional intermediate product
2f was confirmed by ESI-Q-TOF (Figure 1C), and near complete
conversion in the subsequent transaminase reaction was verified
by HPAEC (Figure S3 in Supporting Information). Despite this,
diamine 2g was not detected by mass spectrometry, possibly due
to the formation of unknown adducts or side reactions. While
depletion of intermediate 2f through formation of imine derivatives
can not be ruled out, Cvi-w-TA accepted 2f as a substrate as
measured using the acetophenone assay (Table 1).
describing the recombinant production of these enzymes[58,59]
.
Activity of Cvi-w-TA on the oxidized carbohydrates produced by
FgrGaOx or AbiPDH1, and on pyruvate and D-erythrose (1), was
measured using the acetophenone assay[60]. To our delight, Cvi-
w-TA exhibited significant activity on the aldehyde 2b (160 ± 1
U/g), which, while lower than Cvi-w-TA activity measured on
pyruvate (2995 ± 90 U/g), was in the same order of magnitude as
that on 1 (a preferred substrate of Cvi-w-TA) (Table 1; Scheme
1)[56]. Importantly, hydrate and oxidized derivatives of 2b will form
in the reaction mixture, effectively lowering the concentration of
the intermediate aldehyde 2b in reactions containing D-galactose
(2a), compared to pyruvate and 1[28]. AbiPDH1 was previously
shown to primarily target the C2 position of 2a[53], suggesting that
2-ketogalactose (2d) served as the main substrate for subsequent
transamination by Cvi-w-TA. Although the activity of Cvi-w-TA on
2d was about 30% of that measured using 2b (Table 1), Cvi-w-TA
also showed activity on this sugar substrate yielding the amine 2e
in the multi-enzyme cascade (Figure 1B). It is worth mentioning
that, despite a minor fraction of 2a expected to be in the open
chain conformation, transamination of the C1 aldehyde was not
observed through the acetophenone assay, nor were the
corresponding products detected by HPAEC-PAD or mass
spectrometry. Activity of Vfl-w-TA M1 towards the FgrGaOx and
AbiPDH1 products was tested, but was found to be less than 10%
of that of Cvi-w-TA, which is why experiments with Vfl-w-TA M1
were not continued.
Table 1. Colorimetric activity assay of Cvi-w-TA on selected substrates.
Substrate
Structure
Activity ± SD (U/g)[a]
700 ± 20
D-Erythrose (1)
6-Aldo-D-galactose
(2b)
160 ± 1
45 ± 1
2-Keto-D-galactose
(2d)
2-Amino-2-deoxy-6-
aldo-D-galactose[b]
(2f)
60 ± 3
[a] Reaction conditions: V = 200 µL, 10 mM amine acceptor substrate, 10
mM 1-PEA, 20 µM PLP, 30 µg (2.9 µM) Cvi-w-TA in 50 mM HEPES-NaOH
buffer (pH 7.5) at 37 °C and 700 rpm. [b] Activity on 2f was measured at an
amine acceptor concentration of 5 mM due to the high background
absorbance of the substrate at 245 nm. All measurements were conducted
in triplicate at minimum.
The purified FgrGaOx and Cvi-w-TA, or AbiPDH1 and Cvi-w-TA,
were then tested in combination to establish a two-step pathway
to amino-carbohydrates. Specifically, 2a was oxidized to the
aldehyde 2b by FgrGaOx and then treated with Cvi-w-TA in an
attempt to produce the amine 2c. Alternatively, D-galactose was
oxidized to ketone 2d by AbiPDH1 and then treated with Cvi-w-
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Figure 1. ESI-Q-TOF mass spectra of conversion of A) D-galactose (2a) to 6-amino-6-deoxy-D-galactose (2c) expected from sequential action of FgrGaOx and Cvi-
w-TA; B) D-galactose (2a) to 2-amino-2-deoxy-D-galactose (2e) expected from sequential action of AbiPDH1 and Cvi-w-TA; C) the expected 2-amino-2-deoxy-D-
galactose (2e) from (B) to 2-amino-2-deoxy-6-aldo-D-galactose (2f) through action of FgrGaOx. Similar spectra were collected from each of the three reaction
replicates.
Quantification of 6-amino-6-deoxy-D-galactose from D-
galactose
The comparison of different oxidized forms of 2a showed that
highest Cvi-w-TA activity was obtained using the aldehyde 2b
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(Table 1). Therefore, the sequential, two-step enzymatic
conversion of 2a to the amine 2c was followed by HPAEC to
quantify product formation (Figure 2).
µM to 1 mM only moderately increased product yields in
sequential reactions (Table 2) Because isopropylamine (IPA) is
the preferred amine donor used by industry to push reaction
equilibria towards the aminated product due to its cheap cost and
facilitated acetone by-product removal [63], IPA was tested herein
as a means to further increase the formation of 2c from 2b.
However, substituting 1-PEA for IPA resulted in undetectable
product formation (data not shown), consistent with the
As previously reported[28,29,62],oxidation of 2a by FgrGaOx
generated several different derivatives of the aldehyde group,
including the hydrate (geminal diol) and the corresponding uronic
acid due to further oxidation (data not shown). Accordingly, 2a
and the chemically synthesized 2c were used to generate
standard curves to calculate substrate depletion in the oxidation
reaction and product formation in the amination reaction (Figure
S2 in Supporting Information). Nearly all (95 ± 2 mol%) of 2a was
depleted in the sequential oxidation and amination reaction,
where the formation of 2c from 2a was 18 ± 2 mol% prior to any
optimization. Notably, calculating the formation of 2c based on the
consumption of 2a inevitably underestimates the efficiency of the
amination step as side reactions can occur after formation of the
2b intermediate[28], and so not all of this intermediate is available
for the desired transamination step. In an attempt to reduce the
formation of side products from 2b by reducing the time 2b
remains in aqueous solution, the sequential, two-step enzymatic
reactions were instead performed simultaneously. Remarkably,
performing the oxidization and transamination steps
simultaneously increased the formation of 2c by nearly 2.5 times
comparatively high sensitivity of Cvi-w-TA to IPA[64]
.
Table 2. Influence of reaction set-up on the formation of 2-amino-2-deoxy-D-
galactose (2c) from D-galactose (2a)
Amine
Donor
PLP
Concentration
% mol of product (2c) formation
Sequential
Reaction[a]
Simultaneous
Reaction[b]
1-PEA
20 µM
18 %[c]
N/A[d]
1-PEA
L-Ala
1 mM
1 mM
27 %
67 %
2.5 %
6.5 %
(Table 2).
A similar relative increase from sequential to
Reaction conditions: 50 mM HEPES buffer containing 10 mM D-galactose
(2a), 10 mM amine donor (1-PEA or L-Ala), 20 µM or 1 mM PLP. Enzyme
concentrations were 0.44 µM FgrGaOx, 0.53 µM catalase, 0.12 µM HRP,
simultaneous reaction was observed when using L-alanine as the
amine donor, but the yields obtained using L-alanine were roughly
10 times lower than when using 1-PEA as amine donor. This was
consistent with the unfavorable equilibrium for this reaction [47,48,
63]. Also notable, increasing PLP cofactor concentration from 20
and 2.9 µM Cvi-w-TA. [a] Sequential reactions proceeded for 4 h + 1.5 h for
[b]
the oxidation and transamination steps, respectively.
Simultaneous
reactions proceeded for 5.5 h. Product (2c) formation was quantified by
HPAEC-PAD. [d] Data not available.
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Figure 2. Staggered HPAEC-PAD chromatograms tracking the conversion of 2a to 2c. A) 2a and 2c standards in ddH2O; B) FgrGaOx treatment of 20 mM 2a in
ddH2O (4 h at 30 °C, 700 rpm); C) Control experiment: incubation of the oxidation products (i.e. B) under the conditions of the transaminase reaction but without the
addition of transaminase, D) Cvi-w-TA treatment of 10 mM oxidation products containing the aldehyde 2b (i.e. B) (1.5 h at 37 °C, 700 rpm in 10 mM 1-PEA, 20 µM
PLP, 50 mM HEPES-NaOH (pH 7.5)). Prior to analysis, samples were diluted so that the total of the concentrations of 2a, along with oxidation and amination
products, were 90 µg/mL. 1 = amine 2c (tR = 4.7 ± 0.1 min); 2 = 2a in A (tR =10.3 ± 0.1 min) and overlapping peaks of D-galactose oxidation products in B, C and D;
3 = HEPES; 4 = side products formed in the transaminase reaction; 5 = derivatives formed during the oxidation reaction (tR between 19.5-33.0 min).
We demonstrate the application of two different, fully biocatalytic,
Activity of Cvi- -TA on selected oxidized 6-aldo-D-
w
galactosyl-containing carbohydrates
cascades that employ carbohydrate oxidoreductases to transform
specific hydroxyl groups to carbonyls, and Cvi-w-TA to introduce
amine functionalities at the oxidized positions of the substrates.
The pathways produced amino-galactoses with two different
regioselectivities: (1) the combination of FgrGaOx and Cvi-w-TA
yielded galactose derivatives aminated at the C-6 position, while
(2) the combination of AbPDH1 with Cvi-w-TA yielded galactose
derivatives aminated at the C-2 position. Production of 6-amino-
In addition to monosaccharide substrates, Cvi-w-TA was tested
on a series of D-galactose containing oligosaccharides, each first
oxidized using FgrGaOx. As done for 2a, near quantitative
oxidation of each oligosaccharide was confirmed by TLC (data not
shown) and subsequent Cvi-w-TA activity on each oxidized
carbohydrate was measured with the acetophenone assay using
1-PEA as the amine donor.
6-deoxy-D-galactosyl-containing
oligosaccharides
through
pathway 1 was also detected using the acetophenone activity
assay. Notably, a multi-step synthetic route was required to
synthesise the aminogalactose derivative used as an analytical
standard in the current study, further highlighting the benefits of
the biocatalytic approach. Steps taken to maximize product
formation included (1) establishing a simultaneous oxidation plus
transaminase reaction to the aminated carbohydrate, (2)
increasing PLP concentration, and (3) testing of different amine
donors. Greatest gains in product (2c) formation were achieved
when performing the oxidation and transamination steps
simultaneously rather than sequentially, consistent with reduced
formation of undesired side-products from the aldehyde
intermediate (2b)[28]. This work takes the first step in unlocking the
potential of w-TAs for carbohydrate functionalization, and
therefore expands the pool of building blocks available for new
bio-based materials.
Cvi-w-TA activity was detected using all tested oxidized
oligosaccharides generated using FgrGaO, including aldo-
melibiose (20 ± 4 U/mg), aldo-lactose (50 ± 6 U/g), aldo-raffinose
(50 ± 3 U/g) and aldo-xyloglucan oligosaccharides (32 ± 4 U/g).
While mass spectrometry options must be optimized to
unequivocally confirm the identity of resulting products, substrate
docking studies showed that all investigated saccharides dock
similarly, with the catalytically relevant aldehyde group orientated
towards the catalytically active exocyclic amino group of PMP
(Figure S4 in Supporting Information). Notably, comparison with
the docked aldo-xyloglucan oligosaccharide and the crystal
structure of the Vfl-w-TA (PDB ID: 4E3Q) shows the beneficial
architecture of the active site of Cvi-w-TA towards
oligosaccharides, since the access to the active site of Cvi-w-TA
is more exposed (Figure S5 in Supporting Information).
Experimental Section
Conclusions
Materials
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Yeast extract, yeast nitrogen base and peptone were purchased
from Lab M Ltd. (UK). D-galactose, lactose, melibiose and
raffinose were of analytical grade and purchased from Sigma
A pET29a+ plasmid containing the Cvi-w-TA gene (GenBank:
AAQ59697.1) obtained from GenScript, and the pET24b plasmid
encoding Vfl-w-TA M1[57], were transformed into chemically
competent E. coli BL21. Selected E. coli transformants containing
each plasmid were grown at 37 °C, 220 rpm in shake flasks
containing 250 mL LB medium supplemented with 50 µg/ml
kanamycin and 30 µg/ml chloramphenicol. When the OD600
reached 0.8–1, the E. coli transformant was induced to express
the protein of interest by addition of 1 mM isopropyl-b-D-
thiogalactopyranoside (IPTG). After 15 h of induction at 30 °C, the
cells were harvested by centrifugation (5,000 g and 4 °C for 45
min). For each production, the cell pellet was suspended in 50 mL
50 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM
PLP and lysed using an Emulsiflex-C3 French press (Avestin Inc.,
Canada) at 10,000 PSI for 20 min.
Aldrich.
Xyloglucan
oligosaccharides
(hepta+octa+nona
saccharides) were purchased from Megazyme (O-XGHON; Lot
number 20509). 1,2,3,4-Di-O-isopropylidene-a-D-galactose, used
for preparing the synthetic 6-amino-6-deoxy-D-galactose used as
a standard for product quantification, was purchased from Alfa
Aesar. All other chemicals were reagent grade and obtained from
Sigma Aldrich (Germany) and were used without further
purification unless otherwise specified.
Production and purification of Fusarium graminearum
galactose oxidase (FgrGaOx) and Agaricus bisporus
pyranose dehydrogenase (AbiPDH1)
F.
graminearum
(FgrGaOx;
D-galactose:
oxygen
6-
Immediately after cell lysis, the lysates were clarified by
centrifugation at 15,000 g, 4 °C for 45 min. The supernatants each
containing the soluble protein were filtered through Sterivex-GP
0.22 µm PES filter units (Millipore). Cvi-w-TA and Vfl-w-TA M1,
oxidoreductase, EC 1.1.13.9, CAZy family AA5_2) and A.
bisporus pyranose dehydrogenase (AbiPDH1; pyranose:acceptor
oxidoreductase, EC 1.1.99.29 CAZy family AA3_2) were
heterologously expressed in Pichia pastoris KM71H. Genes
(GenBank accession number: AH005781.2 coding FgrGaOx;
KM851045 AbiPDH1) with C-terminal 6xHis-tags were obtained
from GenScript (New Jersey, US) subcloned into pPICZaA or
pPICZB vectors, respectively. Both enzymes were produced in
shake flask cultivations, as previously described[69]. Briefly,
precultures were grown in up to 750 mL of buffered glycerol-
complex medium (BMGY; 100 mM potassium phosphate buffer,
pH 6.0, 2% (w/v) peptone, 1% (w/v) yeast extract, 4 × 10-5% (w/v)
biotin, 1% (v/v) glycerol) at 30 °C, 220 rpm. Methanol induction
was performed over 4 days at 25 °C, 220 rpm, in buffered
methanol-complex medium (BMMY with 0.5% (v/v) methanol),
where 0.5% (v/v) methanol was added every 24 h to replenish the
inducer.
each containing
a C-terminal His tag, were purified to
homogeneity using Ni-NTA resin as described above, except this
time 0.1 mM PLP was added to the binding buffer and elution
buffer. Purified fractions were then exchanged to 50 mM
phosphate buffer (pH 7.5) with 0.1 mM PLP using a 10 kDa
Vivaspin 20 spin column (Sartorius AG, Germany). Cvi-w-TA was
concentrated to 9.5 mg/mL and Vfl-w-TA M1 to 3.3 mg/mL, and
then stored at −80 °C until further use. Protein concentration was
measured using the Bradford method (Bio-Rad Laboratories, US),
and protein purity was assessed by SDS-PAGE (Figure S1 in
Supporting Information).
Galactose oxidase assay
The activity of FgrGaOx was measured using the chromogenic
ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))
assay, originally described by Baron et al.[70], with modifications.
The standard reaction mixture (final volume: 200 µL) contained
270 µg (31 µM, assuming purity) horseradish peroxidase (HRP,
from horseradish, Sigma-Aldrich, Germany), 2 mM ABTS, and 50
mM 2a in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazine-
ethanesulfonic acid) buffer (pH 7.5); reactions were initiated by
adding 5 µL of appropriately diluted enzyme sample to obtain
initial rates of reaction. Absorbance was measured at 420 nm at
30 °C for 20 min in an Eon microplate reader (BioTek™, US).
Activity values were calculated based on the extinction coefficient
(36,000 M-1cm-1 at 420 nm)[71]. Each reaction was performed in
triplicate, at minimum.
After induction and spinning down the cells, the supernatant was
recovered, adjusted to pH 7.4 and filtered through a Sterivex-GP
0.45 µm PES filter unit (Millipore, Germany). The filtrate was
loaded directly to 6 mL Ni-NTA resin (Qiagen, Germany)
equilibrated in binding buffer (50 mM sodium phosphate buffer,
pH 7.4, 500 mM NaCl, 20 mM imidazole) and packed in a XK-
16/10 column (GE Life Sciences, Germany). Bound protein was
eluted using a linear gradient of 0-100% Ni-NTA elution buffer (50
mM sodium phosphate, pH 7.4 with 500 mM imidazole, 500 mM
NaCl). Purified fractions were then exchanged to 50 mM
phosphate buffer (pH 7.5) using a 10 or 30 kDa Vivaspin 20 spin
column (Sartorius AG, Germany).
FgrGaOx and AbiPDH1 were concentrated to 13.5 mg/mL and 1.8
mg/mL, respectively, and stored at −80 °C until further use.
Protein concentration was measured using the Bradford method
(Bio-Rad Laboratories, US), and protein purity was assessed by
SDS-PAGE (Figure S1 in Supporting Information).
Enzymatic production of oxidized carbohydrates
Enzymatic oxidation of D-galactosyl-containing substrates using
FgrGaOx (reaction volume 3 or 5 mL) was performed in 15 mL
Cellstar tubes (Greiner BioOne) containing 20 mM D-galactose-
equivalent of substrates (i.e., D-galactose, melibiose, lactose,
raffinose, or xyloglucan oligosaccharides), 150 µg (0.44 µM)
Production and purification of the
-TA M1)
-TAs (Cvi- -TA and Vfl-
w w
w
This article is protected by copyright. All rights reserved.

10.1002/cssc.201802580
ChemSusChem
FULL PAPER
FgrGaOx, 160 µg (0.53 µM, assuming purity and based on
molecular weight of the catalase monomer) catalase (from bovine
liver, Sigma-Aldrich, Germany) and 27 µg (0.12 µM) HRP in 50
mM HEPES-NaOH (pH 7.5). Catalase was used as the primary
means of removing hydrogen peroxide in the cascade, while HRP
was included due to its known ability to activate FgrGaOx[72]. The
effect is based on the ability of the horseradish peroxidase to
maintain the copper radical in the active site of FgrGaOx in the
correct oxidation state (II)[73]. The enzyme concentrations were
chosen based on previous experience to achieve maximum
Enzyme concentrations were 0.44 µM FgrGaOx, 0.53 µM
catalase, 0.12 µM HRP, and 2.9 µM Cvi-w-TA. For each
simultaneous oxidation-transamination reaction, a sequential
oxidation-transamination reaction was performed in otherwise
identical conditions, but by first running the oxidation reaction for
4 h, and then initiating the transaminase reaction by addition of
PLP, 1-PEA and Cvi-w-TA, and allowing the transamination
reaction proceed for 1.5 h. Product formation was quantified by
HPAEC.
oxidation of the galactosyl residues in each substrate[27,28]
.
Confirmation of the oxidation and amination products by ESI-
Q-TOF mass spectrometry
Reaction mixtures were incubated for 4 h at 30 °C, with shaking
(700 rpm). Conversion of each oxidation reaction was evaluated
by TLC (data not shown) on Macherey-Nagel pre-coated silica gel
plates (TLC Silica gel 60 F254). AbiPDH1 oxidation of D-galactose
was performed as described by Sygmund and coworkers[74], with
minor modification. Specifically, reaction mixtures contained 58
The following samples were analyzed by direct injection ESI-Q-
TOF (Agilent 6530 Q-TOF, Singapore): 1) 100 ppm 2a, 2) product
from FgrGaOx oxidation of 2a containing up to 100 ppm 2b, 3)
product of the amination reaction containing up to 100 ppm of the
prospected amine 2c formed in the reaction (reaction conditions
specified above). The product compounds were not isolated prior
to analysis. Prior to dilution, samples 2) and 3) were desalted with
AG 2-X8 anion exchange resin (BioRad, US), and proteins
removed by filtration using a Sartorius Vivaspin 500 spin column
(10,000 kDa cutoff). Electrospray ionization was performed in
positive mode, and nitrogen gas was used as both the nebulizing
and drying gas. The following ionization parameters were used:
the drying gas temperature was 250 °C, the drying gas flow was
3 L min-1, the capillary voltage was 3,500 V, nebulizer pressure
was 15 PSIG. Samples were injected directly to the ion source by
the infusion pump at a flow rate of 250 µL min-1 by elution with
0.1% formic acid in 50% acetonitrile. Agilent Masshunter
Qualitative Analysis (version B.07.00.Ink) was used for the data
analysis. All samples were prepared and analyzed in triplicate.
µg (3.61 µM) of AbiPDH1, 50 mM D-galactose,
5 mM
benzoquinone in ddH2O. The small laccase (ScoSLAC; 260 µg,
32.5 µM) from Streptomyces coelicolor was used to recycle the
electron acceptor (benzoquinone) as shown in Scheme 1[75,76]
.
w
-TA reactions and activity assays
After verifying that the oxidation reaction had reached maximum
conversion (evaluated by TLC, data not shown), the activity of the
purified Cvi-w-TA towards oxidized carbohydrates was measured
using chromogenic detection of acetophenone from 1-
phenylethylamine (1-PEA)[60]. Activity of Vfl-w-TA M1 towards
aldehyde 2b was also measured. Briefly, transaminase reactions
(200 µL final volume) were carried out at 37 °C in 96-well
microtiter plates (96-Well UV Microplate, Thermo Scientific, US)
incubated in a plate reader (Biotek Eon, US). The reaction mixture
contained 10 mM 1-PEA (i.e. amine donor substrate), up to 10
mM 2b or 6-aldo-D-galactosyl groups in oligosaccharide
substrates, 20 µM PLP, and 30 µg (2.9 µM) of purified Cvi-w-TA
or Vfl-w-TA M1. The reaction was buffered with 50 mM HEPES-
NaOH (pH 7.5). In sequential reactions, products of the oxidation
reactions were formed over 4 h as described above, and then
directly used as substrates in the transaminase reaction.
Reactions (200 µL) substituting the oxidized carbohydrate with 10
mM pyruvate and containing 30 ng w-TA served as positive
controls, whereas reactions substituting w-TA with ddH2O served
as negative controls. Initial rates were determined by colorimetric
detection of acetophenone over 30 min at 245 nm[60]. Reactions
were then transferred to a Thermomixer (Eppendorf, Germany) to
continue incubation at 37 °C, 700 rpm for another 1 h prior to
product measurement by HPAEC and ESI-Q-TOF mass
spectrometry as described below. All reactions were performed in
triplicate at minimum.
Synthesis
of
6-amino-6-deoxy-D-galactopyranose
trifluoroacetate salt (2c*TFA)
Commercially available reagents were used without further
purification. Column chromatography over silica gel was
performed with Merck Millipore 60, 40-60 µm, 240-400 mesh silica
gel. Reactions were monitored by TLC. Visualization of the TLC
plates was achieved by UV light or staining with a basic potassium
permanganate solution. 1H and 13C NMR spectra were recorded
on a Bruker AV-400 (Germany) instrument at 20 °C (Supporting
Information, section 1).
1,2,3,4-Di-O-isopropylidene-a-D-galactopyranose (1.5 g, 5.7
mmol) was dissolved in EtOAc (38 mL) and IBX (4.8 g, 17 mmol)
was added. After complete oxidation, monitored by TLC, yielding
1,2,3,4-Di-O-isopropylidene-a-D-galactohexodialdo-1,5-
pyranose[77], the precipitate was removed by filtration and the
crude reaction mixture was concentrated under reduced
pressure. The crude product was purified by column
chromatography (SiO2, cyclohexane/EtOAc; 85/15 to 70:30)
affording the product as clear viscous oil (1.00 g, 3.87 mmol,
67%). Rf = 0.51 (n-hexane/EtOAc 2/1).
Reactions (500 µL) permitting simultaneous oxidation of
galactose (2a) to the aldehyde (2b) and transamination of 2b to
the corresponding amine (2c) were performed for 5.5 h at 30 °C
and 700 rpm in 50 mM HEPES-NaOH (pH 7.0), and contained 10
mM 2a, 1 mM PLP, and 10 mM 1-PEA or 10 mM L-alanine.
This article is protected by copyright. All rights reserved.

10.1002/cssc.201802580
ChemSusChem
FULL PAPER
not contain charged residues[80,81]. The docking ligands 2b (6-
aldo-D-galactose), and the corresponding 6-aldo-D-galactosyl-
containing aldo-lactose and aldo-raffinose were constructed using
the built-in oligosaccharide building tool of YASARA, using the b-
D-conformation of each sugar. The docking ligand aldo-melibiose
was constructed by oxidizing the corresponding melibiose
structure (PubChem Identifier: CID 11458). The XLLG molecule
(Figure S6) was extracted as a ligand from the crystal structure
PDB ID: 2VH9 and subsequently oxidized to obtain aldo-XLLG.
All ligands were energy minimized prior to the docking
experiments. The docking was performed with YASARA using the
dock_runensemble.mcr macro utilizing the VINA docking method
with appropriate simulation cells covering the active site of the
receptor. The flexible R416 residue was fixed in place. Plausible
docking results were selected by evaluating orientation of the
substrate to the PMP cofactor in the active site. In addition to the
location and orientation of the substrate, binding energies and
dissociation constants reported by YASARA were also
considered (Table S2 in Supporting Information). Figures of the
dockings were created using PyMOL (The PyMOL Molecular
Graphics System, Version 2.2.0 Schrödinger, LLC).
1,2,3,4-Di-O-isopropylidene-a-D-galactohexodialdo-1,5-
pyranose (250 mg, 0.97 mmol) was dissolved in a solution of
methanol (5 mL) containing ammonium acetate (800 mg, 10.4
mmol). After 15 min, NaCNBH3 (100 mg, 1.6 mmol) was added.
The reaction mixture was stirred for 24 h at room temperature,
after which all volatiles were removed under reduced pressure.
The residue was redissolved in water (15 mL) and extracted with
EtOAc (10 mL). This organic layer was discarded. The aqueous
phase was brought to a basic level (pH > 12) by addition of solid
sodium hydroxide. The aqueous layer was extracted with EtOAc
(3 x 10 mL), the combined organic layers were dried over
anhydrous MgSO4 and the volatiles were removed under reduced
pressure.
The
product,
6-amino-6-deoxy-1,2,3,4-di-O-
isopropylidene-a-D-galactopyranose, was obtained as clear
viscous oil (180 mg, 0.69 mmol, 71%).
The protected amino sugar (35 mg, 0.14 mmol) was dissolved in
deuterium oxide (0.7 mL) and trifluoroacetic acid (11 µL, 0.14
mmol) was added. After stirring for 24 h at room temperature, 1H
NMR analysis confirmed completion of the deprotection and
quantitative formation of the trifluoroacetate salt of 6-amino-6-
deoxy-D-galactopyranose. The thus obtained solution of the
synthetic reference of 2c (0.2 M, 0.14 mmol, 99%) was used
without further manipulation as stock solution. (Supporting
Information, section 1).
Acknowledgements
This study was financially supported by the Academy of Finland
(decision numbers 308996, 252183 and 298250), and the
European Research Council (ERC) Consolidator Grant to ERM
(BHIVE – 648925). UB thanks the German Research Foundation
(DFG, Grant No. Bo1862/16-1) and the EU (Horizon2020, Grant
No. 722610) for funding.
Quantification of reaction yields with high performance
anion exchange chromatography coupled with a pulsed
amperometric detector (HPAEC-PAD)
Amination reactions were performed as described above. Prior to
analysis, the reaction samples for HPAEC-PAD were diluted with
ddH2O to 50-100 ppm of carbohydrate. Eluents used were
100 mM NaOH (A) and 100 mM NaOH with 1 M NaOAc (B). The
chromatographic runs were performed using
a Dionex™
Keywords: Carbohydrate amination, Omega-transaminase,
Galactose oxidase, Pyranose dehydrogenase, Enzyme
Cascades.
CarboPac™ PA1 IC column and with a flowrate of 0.6 mL min-1,
where 100% eluent A was used for the first 5 min followed by 0-
100% eluent B over the next 50 min. Thermo Scientific™
Dionex™ Chromeleon™
7 Chromatography Data System
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NH2
O
galactose oxidase
catalase, peroxidase
HO
HO
HO
HO
ω-transaminase
Two-step biocatalysis
utilizing two carbohydrate
oxidoreductases, galactose
oxidase or pyranose
Ville Aumala, Dr. Filip
Mollerup, Dr. Edita Jurak,
Dr. Fabian Blume, Dr.
Johanna Karppi, Antti E.
Koistinen, Eva Schuiten,
Moritz Voß, Prof. Uwe
Bornscheuer, Prof. Jan
Deska, Prof. Emma R.
Master*
O
O
OH
OH
HO
HO
O2
H2O
dehydrogenase, with an w-
transaminase for the
functionalization of D-
galactose-containing
carbohydrates to amino-
carbohydrates. Production
of amine-functionalized
building blocks for
OH
HO
HO
NH2
O
O
Ph
Ph
OH
HO
H2O
O2
OH
O
OH
O
Page numbers: 1 – 10
HO
HO
HO
HO
pyranose dehydrogenase
laccase
Biocatalytic production of
amino-carbohydrates
ω-transaminase
OH
OH
O
H2N
biomaterial applications
Layout 2:
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Author(s), Corresponding Author(s)*
((Insert TOC Graphic here; max. width: 11.5 cm; max. height: 2.5 cm))
Page No. – Page No.
Title
Text for Table of Contents
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