An Engineered Alcohol Oxidase for the Oxidation of Primary Alcohols

Article abstract of DOI:10.1002/cbic.201800556

Structure-guided directed evolution of choline oxidase has been carried out by using the oxidation of hexan-1-ol to hexanal as the target reaction. A six-amino-acid variant was identified with a 20-fold increased k_cat compared to that of the wild-type enzyme. This variant enabled the oxidation of 10 mm hexanol to hexanal in less than 24 h with 100 % conversion. Furthermore, this variant showed a marked increase in thermostability with a corresponding increase in T_m of 20 °C. Improved solvent tolerance was demonstrated with organic solvents including ethyl acetate, heptane and cyclohexane, thereby enabling improved conversions to the aldehyde by up to 30 % above conversion for the solvent-free system. Despite the evolution of choline oxidase towards hexan-1-ol, this new variant also showed increased specific activities (by up to 100-fold) for around 50 primary aliphatic, unsaturated, branched, cyclic, benzylic and halogenated alcohols.

Full text of DOI:10.1002/cbic.201800556

Accepted Article
Title: An engineered alcohol oxidase for the oxidation of primary
alcohols
Authors: Nicholas John Turner, Rachel S. Heath, William R.
Birmingham, Matthew P. Thompson, Andreas Taglieber, and
Laurent Daviet
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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To be cited as: ChemBioChem 10.1002/cbic.201800556
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ChemBioChem
10.1002/cbic.201800556
FULL PAPER
An engineered alcohol oxidase for the oxidation of primary
alcohols
[
a]
[a]
[a]
[b]
Rachel S. Heath, William R. Birmingham, Matthew P. Thompson, Andreas Taglieber, Laurent
[
b]
[a]
Daviet and Nicholas J. Turner *
[
4]
Abstract: Structure guided directed evolution of choline oxidase has
been carried out using the oxidation of 1-hexanol to hexanal as the
target reaction. A six amino acid variant was identified with an
increased kcat of 20 fold compared to the wild-type enzyme. This
variant enabled the oxidation of 10 mM hexanol to hexanal in less
than 24 h with 100% conversion. Furthermore, this variant showed a
marked increase in thermostability with a corresponding increase in
other electron mediators as a cocatalyst. ADHs have been
+
used as biocatalysts but require a NAD(P) /cofactor recycling
system and the equilibrium is unfavourable for preparation of the
aldehyde. Use of an NAD(P)H oxidase (NOX) to recycle the
[
5,6]
cofactor alleviates these issues.
Alcohol oxidases meanwhile
simply use oxygen (air) for cofactor recycling. Importantly, the
reaction is irreversible, though hydrogen peroxide is generated
as the by-product. If necessary, peroxide may be removed using
catalase.
m
T of 20 degrees. Improved solvent tolerance was demonstrated
with organic solvents including ethyl acetate, heptane and
cyclohexane, thereby enabling improved conversions to the
aldehyde by up to 30% above conversion for the solvent free system. Alcohol oxidases contain either a Cu or flavin adenine di-
Despite evolution of choline oxidase towards 1-hexanol, this new
variant also showed increased specific activities (by up to 100-fold)
for ca. 50 primary aliphatic, unsaturated, branched, cyclic, benzylic
and halogenated alcohols.
nucleotide (FAD) cofactor and although the family of enzymes
[
7]
covers a wide substrate scope, each individual alcohol oxidase
has a relatively narrow substrate range. Previously, enzyme
engineering of alcohol oxidases has focused on the Cu
containing galactose oxidase with examples of engineering for
stability, and switching the activity away from galactose and
[
8,9]
towards benzylic alcohols.
Recently however, alcohol
Introduction
oxidases have begun to attract more attention: a fungal alcohol
[
10]
oxidase was engineered towards activity with glycerol, and 5-
hydroxymethylfurfural oxidase (HMF oxidase) has been
engineered for enhanced enhanced catalytic activity, improved
overoxidation to the acid, thermostability and activity (albeit low)
Aldehydes are versatile intermediates in the synthesis of fine
chemicals and pharmaceuticals and are components of flavours,
fragrances and food ingredients. Oxidation of primary alcohols to
aldehydes is commonly employed, although typical chemical
reagents are toxic heavy metals or highly reactive oxidants and
the process generates large amounts of waste. Increasingly
there is a need for catalytic methods that use mild oxidizing
agents, with minimal waste, especially using oxygen or peroxide
as the primary oxidant. Examples include use of porphyrin
derivatives with hydrogen peroxide or tungsten, vanadium or
molybdate catalysts, nitroxyl radical based systems (TEMPO)
and N-oxyl adamantane derivatives in the presence of sodium
[11–13]
towards a secondary alcohol.
The aim of this work is to apply structure guided directed
evolution to an alcohol oxidase in order to generate an alcohol
oxidase with a wider substrate scope for the oxidation of primary
alcohols (Figure 1).
[1,2]
hypochlorite or air.
Supported nanoparticles of noble metals
have been used to catalyze the oxidation of alcohols under an
[
3]
applied pressure of oxygen.
Alternatively biocatalysts can be used: specifically three different
classes of enzyme, namely laccases, alcohol dehydrogenases
(
ADHs), and alcohol oxidases. Laccases are Cu containing
Figure 1. Aim of this work: use structure guided mutagenesis to
evolve choline oxidase into a broader spectrum alcohol oxidase.
enzymes of largely fungal origin that reduce oxygen to water
alongside oxidation of phenolic substrates but require TEMPO or
The FAD containing choline oxidase from Arthrobacter
cholorphenolicus (AcCO) was chosen as our target enzyme for
protein engineering (Figure 1) because: i) initial screening of the
wildtype (WT) enzyme showed activity (albeit low in many
cases) towards a panel of primary alcohols including hexanol
and benzyl alcohol (Table 3, 1 and 37) (previous reports showed
that the substrate scope was limited to choline and
[
[
a]
b]
Dr. R. S. Heath, Dr. W. R. Birmingham, Dr. M. P. Thompson, Prof.
N. J. Turner
School of Chemistry, University of Manchester, Manchester Institute
of Biotechnology, 131 Princess Street, Manchester, M1 7DN, United
Kingdom
E-mail: nicholas.turner@manchester.ac.uk
Dr. A. Taglieber, Dr. L. Daviet
Firmenich SA, Route des Jeunes 1, P.O. Box 239, Genève 8 CH-
[
14,15]
analogues)
, ii) the enzyme can be heterologously expressed
1211, Switzerland
in E. coli and is thus easily manipulated (many other alcohol
oxidases require eukaryotic expression systems) and iii) the
crystal structure of choline oxidase from Arthrobacter globiformis
Supporting information for this article is given via a link at the end of
the document.
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ChemBioChem
10.1002/cbic.201800556
FULL PAPER
(
AgCO) (to which AcCO has a high sequence identity) is known,
glycine betaine is shown in purple. Positions of libraries made in
active site and access channel of AcCO (for which residue
number also applies) are indicated.
thus allowing structure guided evolution.
Our chosen target substrate for evolution of choline oxidase was
1
-hexanol. Previous examples of hexanol oxidation by wild-type
The most active variants resulting from screening these initial
libraries with 1-hexanol were S101A, M359R and V355T/F357R.
Kinetic parameters of these variants, and those in which the
mutations were combined (from here on abbreviated by the
alcohol oxidases are limited. Aliphatic alcohols are oxidized by
long chain alcohol oxidases and short chain alcohol oxidases
but either activity towards hexanol is low, or the substrate scope
is small, or these enzymes require the use of eukaryotic
3
number of mutations e.g. S101A/V355T/F357R = AcCO ), were
[
16–20]
expression systems.
For example, the alcohol oxidase from
determined and compared to the wildtype (WT) enzyme for both
the natural substrate choline and 1-hexanol. With the exception
of M359R, the mutations were found to decrease the kcat and
Pichia pastoris has high activity towards short chain aliphatic
alcohols (methanol, ethanol) but activity decreases with
increasing chain length (though oxidation of longer chains is
k
cat/K
M
towards choline compared to WT whilst simultaneously
[
21,22]
possible in biphasic systems).
A
galactose oxidase
enhancing activity towards 1-hexanol (Tables 2).
1
&
homologue from Colletotrichum graminicola demonstrated
excellent kinetic parameters for the oxidation of C3-C7 primary
alcohols but conversions in biotransformations did not exceed
Biotransformations performed with 10 mM hexanol correlated
with the kinetic data: WT enzyme gave <50% conversion after
24h whereas AcCO
variants in between (Figure SI3). The kcat/K
not significantly different (see SI for calculations) for
V355T/F357R, AcCO , AcCO and AcCO (see below) but it
4
gave 100% conversion, with the other
[
23]
<30%.
M
for 1-hexanol was
3
4
6
seems that kcat is the major factor influencing conversion.
Results and Discussion
Table 1. Kinetic parameters of choline oxidase variants with choline.
Activity towards 1-hexanol: The active site and access channel
-
1
of AgCO were identified from the crystal structure as key areas
Variant
kcat (min )
KM (mM)
kcat/K
M
-1
[24]
(
min
for mutagenesis to alter the substrate scope (Figure 2).
-1
mM )
Saturation libraries (ie. covering all possible natural amino acids)
were thus made at these sites. (AcCO has 91% sequence
identity to AgCO thus the AgCO structure was used as a
homology model for AcCO). To allow for cooperativity, libraries
contained pairs of proximal residues; three or more positions
increases screening effort above our current feasibility. A
colony-based solid-phase screen in which the by-product
hydrogen peroxide is detected using a horse-radish peroxidase
wt
872 ± 15
541 ± 16
271 ± 8
0.61 ± 0.04
2.01 ± 0.18
3.3 ± 0.3
1430 ± 92
M359R
269 ± 25
82.2 ±6.7
2.0 ± 5.8
3.0 ± 1.6
0.5 ± 0.2
0.3 ± 0.1
S101A
V355T/F357R
S101A/V355T/F357R (AcCO
11.5 ± 0.9
10.2 ± 0.3
0.9 ± 0.3
1.2 ± 0.1
5.8 ± 1.2
3
)
3.41 ± 1.8
1.8 ± 0.6
S101A/V355T/F357R/M359R
(AcCO
S101A/D250G/F253R/V355T/
F357R/M359R (AcCO
(
HRP) and a dye was used to screen libraries W61/M62, S101,
4
)
[
25]
4.7 ± 1.4
V355/F357, D358/M359 and Y465/H466.
Colonies which
6
)
changed colour were picked and the DNA sequenced in order to
determine the causative mutation(s).
Table 2. Kinetic parameters of choline oxidase variants with choline.
-
1
Variant
k
cat (min )
K
M
(mM)
k
cat/K
M
-
1
(min
-
1
mM )
wt
2.8 ± 0.6
2.2 ± 0.1
5.9 ± 0.1
9.9 ± 0.4
10.1 ± 0.5
20.6 ± 0.8
59.2 ± 2.4
6.14 ± 1.80
3.49 ± 0.50
0.67 ± 0.04
0.39 ± 0.06
0.43 ± 0.04
0.84 ± 0.09
3.16 ± 0.40
-1
0.5 ± 0.2
M359R
0.6 ± 0.1
8.8 ± 0.5
25.4 ± 0.4
23.5 ± 2.5
24.5 ± 2.8
18.7 ± 2.5
S101A
V355T/F357R
S101A/V355T/F357R (AcCO
3
)
S101A/V355T/F357R/M359R
AcCO
S101A/D250G/F253R/V355T/
F357R/M359R (AcCO
(
4
)
6
)
-
1
Assay conditions: 30 °C, 0.1 mg ml HRP, 0.7 mg mL ABTS, air-saturated
1
00 mM potassium phosphate buffer, pH 8.0. The increase in absorbance at
20 nm was followed. Detailed method can be found in the Supporting
4
Information.
Figure 2. Structure of AgCO active site and entrance channel.
(
PDB 4mJW) The FAD cofactor is in yellow and natural product
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Stability: The stability of the WT enzyme was measured at
different temperatures (by incubating purified enzyme at a
defined temperature for 3 hours before measuring initial rates).
Up to 30 °C the enzyme remained stable, however at >40 °C,
the remaining activity was negligible (Figure SI1). A further
1
00
wt
AcCO6
9
8
0
0
70
60
experiment determined the T
m
to be 32 °C (Figure 3 and SI2).
AcCO performed marginally better after incubation at 40 °C,
4
50
40
30
retaining 10% activity (Figure SI1). In an attempt to improve the
thermostability we targeted residues that had high B factors for
mutatagenesis. Examination of the B factor (which reflects the
[
26]
20
flexibility) of each residue using BFITTER
high B factors. Saturated libraries at these positions (F253,
R241/R242 and D238/A239) were constructed in AcCO as well
identified five with
10
0
4
as a random (error prone) library over residues 225-265. The
libraries were screened after incubation at 50 °C for three hours
using the previously described colony based screen. The
positive hits D250G and F253R were combined with AcCO
4
to
S101A/D250G/F253R/V355T/F357R/M359R
maintained close to 50% activity after
Solvent overlay
give
AcCO
AcCO
). AcCO
6
Figure 4. Effect of solvent overlays on conversion of 1-hexanol
to hexanal using WT and AcCO . Conditions: 50 mM 1-hexanol,
mg mL enzyme, 50% v/v air-saturated 100 mM potassium
(
6
6
incubation for three hours at 40 °C, a five-fold improvement over
AcCO (Figure SI1). We then measured the T of AcCO to be
2 °C (Figure 3 and SI2), thus showing a twenty degree
increase in the thermostability of the six-point variant above the
WT. AcCO also displayed higher kcat towards hexanol, 3-fold
greater than AcCO (Table 2) and in biotransformations showed
a 20% higher conversion after 1h (Figure SI4).
-
1
1
4
m
6
phosphate buffer, pH 8.0, 50% solvent v/v, 30 °C, 200 rpm, 24h.
Conversion measured by GC-FID. Full experimental details can
be found in the Supporting Information.
5
6
4
6
Substrate scope: To determine the substrate scope of AcCO we
examined the activity with a panel of 50 primary alcohols and
compared the results to the WT enzyme (Table 3). For most
1
00
wt
90
80
70
60
50
40
30
20
10
0
substrates the activity of AcCO
improvements ranging from ca. two-fold (9) to >100 fold (44, 45).
Amino alcohols were the only substrates where AcCO showed
6
was higher than the WT with
AcCO6
6
marked lower activities compared to the WT, perhaps this is not
surprising since we have intentionally mutated activity away from
the natural substrate
–
the amino alcohol choline.
Biotransformations with AcCO
6
were then applied to alcohols 1,
7, 8, 9, 10, 11, 28, 29, 30, 32, 34, 35, 36, 38, 39, 40, and 42 on a
0 mM scale (Figure 5), thus covering a range of saturated,
unsaturated, aliphatic, cyclic and benzylic alcohols. For all
alcohols tested, AcCO showed improved activity relative to the
WT. Conversions were improved by >50% compared to WT for
, 28, 29, 30, 35, 40 and 42. Saturated alcohols with C6-C10
chain lengths and unsaturated alcohols were excellent
substrates. For cyclic alcohols, AcCO gave some of the most
improved conversions compared to WT.
1
6
0
20
40
60
80
1
Temperature / °C
6
Figure 3. Shift in temperature stability of AcCO compared to
6
WT. Enzyme was incubated for 15 minutes at the given
temperature before the initial rate was measured at 420 nm.
-1
-1
Conditions: 5 mM hexanol, 30 °C, 0.1 mg ml HRP, 0.7 mg mL
ABTS, air-saturated 100 mM potassium phosphate buffer, pH
.0.
Preparative oxidation of hexanol (100 mg) with AcCO
00% conversion to hexanal with an isolated yield of 72%. WT
choline oxidase catalyzes over oxidation to the carboxylic acid
and hence this side-activity was checked for AcCO (see SI).
6
gave
1
8
6
The effect of organic solvent was also examined using WT and
AcCO with 50 mM hexanol (Figure 4). Remarkably, three
solvents increased the conversion achieved by AcCO by up to
0%. For the WT, all solvents tested inhibited conversion; in
Hexanol over-oxidation was negligible (<1%) but with cinnamyl
alcohol (40), 20% conversion to carboxylic acid was observed.
Due to the solvent tolerance of the six-point variant, further
oxidation could be minimized by use of a biphasic system in
which the aldehyde partitions to the organic phase preventing
6
6
3
many cases conversion was negligible. Thus the mutations
responsible for improving temperature stability also enhanced
tolerance for organic solvents, a phenomenon that has been
[28]
gem-diol formation necessary for over-oxidation.
[
27]
observed with other enzymes.
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FULL PAPER
Table 3. Specific activities of WT and AcCO
6
Table 3 cont.
[
a]
WT
AcCO
mU/mg
6
fold
[b]
Structure
AcCO
6
fold
WT
mU/mg
increase
Structure
mU/mg
mU/mg
increase
1
2
3
4
5
6
7
8
9
20
0
571
29
3
7
8
1.7
115
68
0.53
1.2
4.4
246
558
234
38
N/A
N/A
29
0
3
n.t.
1.7
N/A
0.15
16
31
42
18
16
17
2.5
104
69
46
28
15
39
45
636
14
18
4
0
1
1.5
3.7
8.2
56
5.5
15
5.6
2.1
1.6
0.88
0.96
7.7
5.6
2.6
3.6
4
26
1
0
1
2
3
4
5
15
4
2
3
0.25
6.2
25
1
1
1
1
1
2.4
803
384
118
100
4
0.07
2.8
5.8
82.9
125
44
351
4
5
6
2.4
0
319
2.4
133
N/A
1
6
7
8.6
0
16
15
1.9
1
N/A
4
1
1
2
2
8
9
0
1
318
151
98
52
16
2.6
0
0.16
0.1
4
4
4
5
7
8
9
0
0.64
17
51
80
7
0.03
N/A
122
655
16
62
10.6
16.6
22
23
24
25
10623
91
0.009
N/A
6.2
33
549
34
0
-
1
-1
Assay conditions: 30 °C, 0.1 mg ml HRP, 0.7 mg mL ABTS, 5 mM
substrate, air-saturated 100 mM potassium phosphate buffer, pH 8.0. The
increase in absorbance at 420 nm was followed. Further details can be found
in the Supporting Information.
1.1
6.8
76
1.3
58
2
6
7
1.6
14
47
597
39
29
43
53
100
9
8
7
6
5
4
0
0
0
0
0
0
2
2
8
9
0.74
2
3
91
30
N/A
4.5
30
[
b]
n.d.
n.d.
536
31
119
30
20
3
2
3
23
10
643
523
28
52
3
1
0
34
36
118
3.3
0
1
7
8
9 10 11 28 29 30 32 34 35 36 38 39 40 42
Substrate
3
5
6
45
96
84
85
1.9
0.9
wt
AcCO6
Figure 5. Conversions of 10 mM alcohol to aldehyde by WT
choline oxidase and AcCO . Substrates as in Table 3. Columns
represent an average of minimum of three replicates.
Conversion analysed by GC-FID Conditions: 24 hrs, 30 °C, 200
3
6
[
a] in SA from WT to AcCO
6
, [b] n.d. = not determined (color change pre
a
enzyme), n.t. = not tested, N/A =not applicable
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-1
rpm,
phosphate buffer.
1
mg mL enzyme, 100 mM pH 8.0 air-saturated Experimental Section
Full experimental details can be found in the Supporting Information.
Mutations identified from screening libraries of residues thought
to be important for substrate scope led to the identification of
4 M
AcCO , a variant with 50 fold improvement in kcat/K toward
Acknowledgements
hexanol compared to WT. This variant also has much reduced
activity towards choline. Analagous to the results here, the
S101A mutation in AgCO had also previously been shown to
The research leading to these results has received funding from
the European Union’s Seventh Framework Programme for
research, technological development and demonstration under
grant agreement no. 613849 supporting the project BIOOX.
[
29]
reduce activity towards choline.
Mutations remote from the active site have previously been
shown to increase the activity of enzymes by lowering the free
energy of the catalytic conformation of the enzyme, as well as
Keywords: enzyme engineering • oxidase • thermostability •
alcohol • aldehyde
[30,31]
increasing thermostability and/or solvent tolerance.
mutations at D250 and F253 combined with AcCO to form the
variant AcCO which possessed improved stability at higher
temperatures including a 20 degree higher T than the WT and
also showed a 3-fold increase in activity (kcat) compared to
AcCO . Residues D250 and F253 form part of a loop comprising
amino acids 250-255 which by X-ray crystallography appear in
Two
4
[
1]
M. Shibuya, K. Furukawa, Y. Yamamoto, Synlett 2017, 28, 1554–
1557.
6
m
[2]
L. Tebben, A. Studer, Angew. Chemie Int. Ed. 2011, 50, 5034–
5068.
4
[3]
R. A. Sheldon, Catal. Today 2015, 247, 4–13.
L. Martínez-Montero, V. Gotor, V. Gotor-Fernández, I. Lavandera,
Green Chem. 2017, 19, 474–480.
[24]
two different conformations (“open” and “closed”).
In the
[
[
4]
5]
closed conformation, the side chain of F253 (from the other
subunit) sterically hinders access to the channel to the active
site. Mutation of these residues may affect the position of the
loop and hence substrate access to the active site. Further
increase in activity will likely require more mutations that are
P. Könst, H. Merkens, S. Kara, S. Kochius, A. Vogel, R. Zuhse, D.
Holtmann, I. W. C. E. Arends, F. Hollmann, Angew. Chemie Int. Ed.
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012, 51, 9914–9917.
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0.1002/cbic.201800421.
[
[
[
6]
7]
8]
[
32]
distal to the active site and will be predicted computationally.
1
M. Pickl, M. Fuchs, S. M. Glueck, K. Faber, Appl. Microbiol.
Biotechnol. 2015, 99, 6617–6642.
The solvent tolerance of AcCO was also significantly enhanced
6
compared to WT and in several cases the conversion to product
was increased compared to no solvent overlay. Such was the
solvent tolerance of the six-point variant that it enabled us to
perform (continuous) flow reactions (using immobilized enzyme)
L. Sun, I. P. Petrounia, M. Yagasaki, G. Bandara, F. H. Arnold,
Protein Eng. Des. Sel. 2001, 14, 699–704.
[9]
F. Escalettes, N. J. Turner, ChemBioChem 2008, 9, 857–860.
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acs.biochem.8b00918.
[
33]
for the oxidation of 1-hexanol in pure organic solvent.
For
[10]
aldehydes that are sensitive to over-oxidation to the acid, this
route, with its absence of water, provides a solution.
[
[
[
11]
12]
13]
W. P. Dijkman, C. Binda, M. W. Fraaije, A. Mattevi, ACS Catal.
6
Although AcCO was identified by screening with 1-hexanol,
pleasingly it demonstrates a broader substrate scope with
2015, 5, 1833–1839.
C. Martin, A. Ovalle Maqueo, H. J. Wijma, M. W. Fraaije, Biotechnol.
Biofuels 2018, 11, 56.
oxidation of other primary alcohols including 31, 37, 38 and 50
[
11,34–36]
which are natural substrates of other alcohol oxidases.
M. Pickl, C. Winkler, S. Glueck, M. Fraaije, K. Faber, M. Pickl, C. K.
Winkler, S. M. Glueck, M. W. Fraaije, K. Faber, Molecules 2017, 22,
2
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004, 430, 264–273.
S. Ikuta, S. Imamura, H. Misaki, Y. Horiuti, J. Biochem. 1977, 82,
741–1749.
Conclusions
[
[
[
[
14]
15]
16]
17]
2
By examination of the crystal structure of choline oxidase, key
sites for mutagenesis to both alter substrate scope and increase
thermostability were determined. A six-point variant was evolved
which was more active on our substrates of interest as well as
demonstrating markedly improved thermostability and solvent
tolerance compared to the wildtype. The enzyme could be used
in biotransformations for the oxidation of primary alcohols to
aldehydes with full conversion in many cases.
1
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Alcohol oxidases are advantageous in
that they require no additional cofactor
or cofactor recycling systems, simply
requiring molecular oxygen for
Rachel S. Heath, William R.
Birmingham, Matthew P. Thompson,
Andreas Taglieber, Laurent Daviet and
Nicholas J. Turner*
catalysis. Their use is currently limited
by activity and substrate scope. An
alcohol oxidase was evolved to
produce an enzyme with a broad
scope for primary alcohol oxidations.
Further improvements were made to
enzyme stability and demonstrated by
its increased thermostability and
solvent tolerance.
Page No. – Page No.
An engineered alcohol oxidase for
the oxidation of primary alcohols
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Author(s), Corresponding Author(s)*
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Title
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