Selective aerobic oxidation reactions using a combination of photocatalytic water oxidation and ...

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SUPPLEMENTARY INFOARMrtAiTcIlOeNs
https://doi.org/10.1038/s41929-017-0001-5
In the format provided by the authors and unedited.
Selective aerobic oxidation reactions using a
combination of photocatalytic water oxidation and
enzymatic oxyfunctionalizations
Wuyuan Zhang   1, Elena Fernández-Fueyo1, Yan Ni1, Morten van Schie1, Jenö Gacs1, Rokus Renirie2,
Ron Wever2, Francesco G. Mutti   2, Dörte Rother3, Miguel Alcalde4 and Frank Hollmann   1*
1Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. 2Van’t Hoff Institute for Molecular Sciences (HIMS), Faculty of
Science, University of Amsterdam, Amsterdam, The Netherlands. 3Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich
GmbH, Jülich, Germany. 4Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain. *e-mail: [email protected]
NAtuRE CAtAlYSiS | www.nature.com/natcatal
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Supplementary Methods
Materials used in this study
All chemicals were purchased from Sigma-Aldrich, Fluka, Acros or Alfa-Aesar with the highest purity
available and used without further treatment. Titanium (IV) oxide (rutile or anatase phase) was
bought from Sigma-Aldrich (The Netherlands) and used as received. Note that rutile TiO was
2
synthesised through a gas phase method (as confirmed by the supplier). Anatase TiO used in this
2
study contains mixed phases of rutile and anatase (9 : 91). Gold(III) chloride (64.4% minimum) was
brought from Alfa-Aesar. Water-18O (97 atom % 18O) was brought from Sigma-Aldrich (The
Netherlands).
Synthesis and characterisation of the photocatalysts
Au-TiO was produced following a previously published procedure.1, 2 In order to deposit Au
2
nanoparticles onto the surface of TiO , the so-called deposition-precipitation method was carried out
2
as follows: an aqueous solution of AuCl (5 mM, slightly yellow) was firstly heated to 70 °C. The pH of
3
this solution was adjusted to 7.2 by using NaOH (0.1 M solution). Then 11 mL of above Au-solution
was added into 97 mL of MilliQ water (preincubated at 70 °C). After 10 minutes stirring, 1.0 g of TiO
2
particles was added and the mixture was stirred for 1h at 70 °C. After cooling to room temperature,
the Au-TiO nanoparticles were centrifuged (6000 rpm for 15 min), washed three times with MillQ
2
water and dried at 70 °C overnight. In order to prepare Au-TiO doped with varied Au-content, the
2
concentration of AuCl solution was varied. To obtain larger Au-particle size on TiO surface, the dried
3 2
Au-TiO sample was calcined at 600 °C in air for 2h with a heating rate of 5 °C min-1.
2
g-C N was produced following a previously published procedure.3 The urea precursor was calcined
3 4
at 600 °C in air for 4h with a heating rate of 5 °C min-1. After cooling to room temperature, a
yellowish powder was obtained.
Au-BiVO was produced following a previously published procedure.4 The Au-deposited BiVO was
4 4
calcined at 400 °C in air for 2 hours with a heating rate of 5 °C min-1.
Carbon nanodots were produced following a previously published procedure.5 After thermolysing
citric acid (100 g) at 180 °C for 40 hours with a heating rate of 5 °C min-1, NaOH (5 M) was added to
neutralize the solution. A brown-orange power was obtained via freeze-drying.
The structures of the photocatalysts were characterised by a Bruker D8 Advance X-ray diffractometer
using Co-Kα radiation (λ = 1.789 Å) at 35 kV and 40 mA. The data were collected from 2θ = 5.0°-80°
with a step size of 0.020° and a counting time of 0.5 s per step. The particle size and morphology
were analysed by using Philips CM30T TEM.
The mass fraction of anatase and rutile of the TiO was calculated according to the following
2
equation:6
1
𝑋 = ×100%
𝑅 𝐼
1+1.26 𝐴
𝐼
𝑅
where I and I are the intensities of anatase (101) and rutile (110) diffraction peak, respectively.
A R
The XRD measurement proved that the commercial anatase TiO used herein is a mixture of rutile
2
and anatase (named as anatase TiO ), the mass fraction of each phase is 8.9 and 91.1%, respectively.
2
The rutile TiO is nearly pure (named as rutile TiO ). Aiming at visible-light driven catalytic reactions,
2 2
four different samples of anatase Au-TiO in terms of Au-doping content and Au-particle size are
2
prepared (Supplementary Table 1).
Supplementary Table 1. Information of anatase Au-TiO photocatalyst.
2
Sample name Au-content, wt% Au-particle size, nm Note
Au -TiO 0 / Non-doped and non-calcined.
0 2
Au -TiO 0.6 2.8 non-calcined.
0.6 2
Au -TiO 1.2 2.8 non-calcined.
1.2 2
Au -TiO 1.8 2.8 non-calcined.
1.8 2
Au -TiO 1.2 7.9 Calcined at 600 oC in air for 2h
1.2 2
)
1
0
1 A: anatase
(
A
R: rutile
commercial TiO2
)
0
Au-TiO2 )011(R )101(R)400(A 02(A )112(R)501(A )512(A Au-TiO2
TiO
2
10 20 30 40 50 60 70 80 90
2 Theta (degree)

Supplementary Figure 1. XRD patterns of anatase TiO and anatase Au -TiO nanoparticles.
2 1.2 2

)0 A: antase
1
1(R )1 R: rutile
1) 12
10 (R
rutile Au-TiO )101(A R( )002(R)111(R )012(R )022(R )200(R)013(R )103(R)211(R
2
rutile TiO
2
10 20 30 40 50 60 70 80 90
2 Theta ( degree)

Supplementary Figure 2. XRD patterns of rutile TiO and rutile Au -TiO nanoparticles.
2 1.2 2
Supplementary Figure 3. (HR)TEM of antase Au -TiO with Au-particle size 2.8 nm (a, non-calcined) and 7.9
1.2 2
nm (b-d, calcined at 600 oC in air for 4h).

Supplementary Figure 4. TEM of rutile Au -TiO with average Au-particle size 3.0 nm.
1.2 2
Biocatalysts preparation
Preparation of rAaeUPO: the recombinant evolved unspecific peroxygenase mutant from Agrocybe
aegerita (rAaeUPO) was overproduced in Pichia pastoris and purified as described previously.7, 8 The
culture broth with P. pastoris cells containing rAaeUPO was clarified by centrifugation at 8000 rpm
for 2 hours at 4 ◦C. The supernatant was filtered through a 20 µm filter and kept at -80°C. rAaeUPO
activity was determined to be 652 ± 5 U mg-1 (ABTS assay, pH 5.0 in NaPi buffer). One unit of the
enzyme activity was defined as the amount of the enzyme catalysing the oxidation of 1 µmol of ABTS
per minute.
Protein purification: the supernatant was concentrated (Amicon 10-kDa-cut-off) and dialysed against
100 mM sodium phosphate, pH 7. rAaeUPO was purified using an NGC Chromatography system
(Biorad), in one single step. The separation was performed on a Q Sepharose FF 30-mL cartridge with
a flow rate of 5 mL min-1. After 90 mL, the retained protein was eluted with a 0–50 % NaCl gradient
in 450 mL, followed by 50–100 % gradient in 50 mL and 100 % NaCl in 75 mL. Peroxidase activity was
followed by ABTS oxidation in the presence of H O , and the appropriate fractions were pooled,
2 2
concentrated and dialysed against 100 mM sodium phosphate buffer (pH 7). The purification of
rAaeUPO was confirmed by sodium dodecyl sulfate (SDS)–PAGE in 12% gels stained with Coomassie
brilliant blue R-250 (Sigma).

1 2

99

66

45

30

Supplementary Figure 5. SDS-gel analysis of the purified rAaeUPO. Lane 1: protein standards (99 kDa, 66 kDa,
45 kDa and 30 kDa), lane 2 purified rAaeUPO (M =45 kDa).
w
Concentration of rAaeUPO: the concentration of rAaeUPO was determined using the molar
extinction coefficient of 115 mM-1 cm-1 at 420 nm. Absorption spectrum in the UV/Vis range was
recorded in a Biomate5 (Thermo) spectrophotometer (2).

Supplementary Figure 6. UV/Vis spectrum of purified rAaeUPO, with a Reinheitszahl (Rz: A /A ) value of 1.6.
420 280

Immobilisation of rAaeUPO: to immobilise the rAaeUPO, our previous procedure9 was used:
RelizymeTM HA 403/M resin (1 g) was treated with 50 mL of 0.125% glutaraldehyde solution in water
for 2.5 h in a shaking device at 16 °C. The glutaraldehyde solution was then removed by
centrifugation, and the resin was washed three times with 0.1 M phosphate buffer at pH 7. The
buffer was then removed, and 3 mL of pure rAaeUPO (1.35 mg) and 1 mL of 0.1 M phosphate buffer
at pH 7 were added to the activated support. The mixture was incubated in a shaker for 24 h at 16 °C.
The residual enzymatic activity in the solution was monitored by using the ABTS oxidation assay. The
resin was then washed with 50 mM phosphate buffer at pH 7, dried and stored at 4 °C.
Supplementary Figure 7. Schematic procedures of rAaeUPO immobilisation (upper) and spatial separation
between TiO and (immobilised rAaeUPO).
2

Preparation of CiVCPO: for heterologous expression and purification of CiVCPO a slightly modified
literature procedure was used.10 A 2 L culture of Escherichia coli transformant (E. coli TOP10
(Invitrogen) with the construct pBAD-VCPO) was grown at 37 °C in LB medium supplemented with
100 µg/mL ampicillin to an OD 600 nm of 0.6-0.8. Protein expression was induced after cooling the
fermentation broth to 20 °C and addition of 0.02 % L arabinose, followed by another 72 hours of
incubation. The expression of CiVCPO in E. coli yielded an enzyme content of 15 mg·L-1 culture. Cells
were harvested by centrifugation at 8000 rpm for 10 min at 4 °C (4.3 xg). The cells were re-
suspended to 1 g mL-1 in 50 mM Tris/H SO , pH 8.1 fortified with protease inhibitors, lysozyme (2 mg
2 4
mL-1) and DNaseI. Cells were lysed using a Cell disruptor and debris was removed by centrifugation at
15000 rpm for 1 h at 4 °C. After centrifugation an equal volume of isopropyl alcohol was added to the
supernatant to precipitate nucleic acids and unstable proteins. After centrifugation (30 min at 15000
rpm), the clear supernatant was applied to a DEAE Sephacel column (Amersham Pharmacia Biotech)
(5mL min-1) equilibrated with 50 mM Tris/H SO pH 8.1. After washing of the column with 2 volumes
2 4
of 50 mM Tris/ H SO , pH 8.1, and 2 volumes of 0.1 M NaCl in 50 mM Tris/H SO , pH 8.1, the enzyme
2 4 2 4
was eluted with 1 M NaCl in 50 mM Tris/HCl, pH 8.1. Finally the pure apoenzyme was dialysed
against 100 µM orthovanadate in 50 mM Tris H SO , pH 8.1 to obtain the reconstituted holoenzyme.
2 4
As illustrated below, SDS-PAGE monitoring of the purification process showed that the whole soluble
fraction from the E. coli cultures (lane 4) was considerably enriched in the CiVCPO (67.5 kDa) band,
incubation and centrifugation with isopropanol (lane 3) partially removed undesired proteins and
finally after DEAE chromatography (lanes 5 and 6) protein was ≥ 90 % pure. Protein concentration
was estimated by the BSA assay and CiVCPO activity was determined to be 120 U mg-1. One unit of
the enzyme activity was defined as the amount of the enzyme that catalyses the bromination of 1
mol MCD per min at pH 5 and 30 °C (using a saturating concentration of bromide (0.5 mM) in 0.1 M
citrate (pH 5) after the addition of 10 mM of H O ).
2 2
Photoenzymatic reactions using rAaeUPO: photochemical enzymatic reactions using rAaeUPO were
performed at 30 °C in 1.0 mL of sodium phosphate buffer (NaPi, pH 7.0, 60 mM). Unless mentioned
otherwise, 5.0 mg of photocatalyst was firstly suspended in 900 uL of NaPi buffer under sonication (5
min in an ultrasonication bath), 350 nM of rAaeAPO and 15 mM of substrates (final concentration)
were then added to the suspension. The volume of the reaction mixture was adjusted to 1.0 mL in a
final step. The reaction vial was closed, and exposed to visible light (Philips 7748XHP 150 W, white
light bulb) under gentle stirring. The homemade experimental setup is shown in Supplementary
Figure 7. The distance between the reaction vial and bulb is 3.6 cm. At intervals, aliquots were
withdrawn, extracted with ethyl acetate (containing 5 mM of 1-octanol/dodecane as internal
reference) and analysed by Gas Chromatography.
Photoenzymatic reactions using CiVCPO: the reaction conditions for the bromination of thymol were
adapted from our previous work.10 In a typical reaction condition, 5.0 mg of Au-TiO was added into
2
900 uL of NaPi buffer and sonicated for 5 min, then 50 nM of CiVCPO, 3 mM of thymol, 2 eq. of Br-,
and 50 µM of Na VO were added. The reaction volume was adjusted to 1.0 mL and the mixture was
3 4
irradiated under visible light with gentle stirring. The products were analysed by Gas
Chromatography.
Supplementary Figure 8. Image of homemade photocatalytic setup.

2400
2000
)
2
m 1600
c

/

W
µ 1200
(

y
t
is
n 800
e
t
n
I
400
0
300 400 500 600 700 800
Wavelenght (nm)

Supplementary Figure 9. Light intensity of homemade light-setup. The measurement was performed with a
distance of 45 cm between the calibrated spectrophotometer and light bulb.