Full Papers
doi.org/10.1002/cctc.202100163
ChemCatChem
agar with 100 μg/mL ampicillin for all constructs, except for
transformations using pET29a or pKK32 vectors, here 50 μg/mL
transparent or amber colored microcentrifuge tubes in a thermo-
mixer (comfort 5335r, Eppendorf, Hamburg, Germany). Product
formation was detected via HPLC analysis. The enzymatic reaction
in samples taken during the initial rate experiments was quenched
1:20 (v/v) in a mixture of 45% acetonitrile, 55% H2O and 0.1%
trifluoroacetic acid (v/v). The samples were analyzed by reversed
phase chromatography using an Ultimate 3000 HPLC system
(Thermo Fisher Scientific, Waltham, MA, USA) equipped with a
diode array detector and a LiChrospher® 100 RP-18 column (Merck,
Darmstadt, Germany). The analysis was carried out isocratically with
1
2
3
4
5
6
7
8
9
°
kanamycin was used, and incubated overnight at 37 C.
Production and purification of enzymes
For all enzymes, the production was performed under normal light
and under dark conditions. For normal light conditions, windows
were left uncovered and the electric light was switched on but no
specific or targeted illumination occurred. In dark conditions,
illumination was avoided as much as possible. Flasks and solutions
were wrapped in aluminum foil, windows were covered, and the
electric light was switched off. The target enzymes were produced
in shaking flasks with a filling volume of up to 15%. A single colony
from the respective overnight plate was transferred to 50 mL LB
medium (with 100 μg/mL ampicillin for all constructs, except for
cells transformed with pET29a or pKK32 vectors, here 50 μg/mL
kanamycin was used) and the precultures were cultivated overnight
°
a flow rate of 1.5 mL/min at 25 C for 10 min using a solvent
mixture of 45% acetonitrile, 55% H2O and 0.1% trifluoroacetic acid
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
(v/v).
A
10 μL sample was injected and the absorption of
acetophenone (6) was detected at 254 nm with a retention time of
4.3 min. A slope was generated from the resulting peak areas,
corresponding to the relative activity of the respective ATA. For
specific activity determination, the concentration of the formed
acetophenone (6) was calculated from the peak areas using a
calibration curve (Figure SI.1). The activity is given in UmLÀ 1, which
is defined as the amount of enzyme in 1 mL reaction solution,
which catalyzes the formation of 1 μmol acetophenone (6) per
minute. The error bars in the respective diagrams give information
about the standard deviation of technical triplicates.
°
at 37 C and 150 rpm (Infors HT, Bottmingen, Switzerland). For the
main culture, auto induction (AI) medium was used, which was
°
°
incubated for 48 h at 37 C (decreased to 20 C after 2 h) and
150 rpm (Infors HT, Bottmingen, Switzerland). Cells were harvested
by centrifugation (Avanti J-20 XP, Rotor JLA-8.1000, Beckman
°
Coulter, Brea, CA, USA, 30 min, 8,000 rpm, 4 C) and the resulting
cell pellets were frozen. For the purification, frozen cells containing
the respective enzymes were thawed and resuspended (30% (w/v))
in TRIS buffer (50 mM, pH 7.5, 0.2 mM PLP) with 1 mg/mL lysozyme
(Merck, Darmstadt, Germany) and 10 units/mL benzonase (Merck,
Darmstadt, Germany) for 30 min on ice. The cells were disrupted by
ultrasonication (Digital Sonifier 450, Emerson Electric Co., Ferguson,
MO, USA) on ice for a total sonication time of 5 min (2 s pulse, 8 s
pause) at 60% intensity. After centrifugation (Avanti J-20 XP, Rotor
LDC initial rate reactions and analytics
The relative LDC activities in different light setups were measured
via a newly developed colorimetric assay using the indicator 4-
nitrophenol for the decarboxylation reaction from L-lysine (8) to
cadaverine (9; manuscript in preparation). Illumination of enzyme
and PLP solutions was done as described previously using ten times
concentrated stock solutions. The corresponding illumination setup
is depicted in Figure SI.6.A–B. Colorimetric measurements and initial
rate reactions were performed using a UV-Vis spectrophotometer
°
JA-25.50, Beckman Coulter, Brea, CA, USA, 45 min, 20,000 rpm, 4 C),
the supernatant was applied to a Ni-NTA Superflow resin (Qiagen,
Hilden, Germany), pre-equilibrated with TRIS buffer (50 mM, pH 7.5,
0.2 mM PLP) using an ÄKTA pure chromatography system (GE
Healthcare, Bosten, MA, USA). After a washing step with an
appropriate washing buffer (50 mM TRIS buffer, pH 7.5, 0.2 mM PLP,
25 mM imidazole), the His6-tagged proteins were eluted (50 mM
TRIS buffer, pH 7.5, 0.2 mM PLP, 300 mM imidazole). Relevant
protein samples were pooled and desalted on a HiTrapTM Sephadex
G-25 resin (GE Healthcare, Bosten, MA, USA), pre-equilibrated with
TRIS buffer (10 mM, pH 7.5, 0.2 mM PLP). For enzymatic reactions
with the CvATA and VfATA in potassium phosphate buffer,
potassium phosphate buffer was used instead of TRIS buffer during
the production and purification process. The protein concentration
of the pooled relevant samples was adjusted to 1 mg/mL with
water. After freezing the protein solutions overnight, it was
submitted to lyophilization (Alpha 1-4 LD Plus, Martin Christ
Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany).
°
(UV-1800, Shimadzu, Kyōto, Japan) in a 1.5 mL cuvette at 20 C,
measuring the absorbance at 400 nm in 10 s intervals. Before the
enzymatic reactions in 1 mL scale were started, 10 mM L-lysine (8)
and 0.1 mM PLP were pre-incubated in 25 mM HEPES buffer
(pH 6.0) for 4 min. Then, 0.1 mM 4-nitrophenol was added and the
initial rate measurement was started by addition of 0.05 mg/mL
EcLDC or 0.025 mg/mL SrLDC. After manual stirring for a couple of
seconds, a blank measurement was performed and then the
photometric measurement was started, which lasted 30 min. The
error bars in the respective diagrams give information about the
standard deviation of the technical triplicates.
Blue light inactivation of the CvATA
Blue light experiments with the CvATA were performed for initial
rate reactions as described above. A reaction solution containing
0.03 mg/mL enzyme was incubated for 10 min in an amber colored
microcentrifuge tube in a thermomixer (comfort 5335r, Eppendorf,
ATA initial rate reactions and analytics
°
Hamburg, Germany; 600 rpm, 22 C), before the solution was
The ATA activities were tested in different light setups, which were
explained above. Illumination of enzyme and PLP solutions
occurred using ten times concentrated stock solutions. The trans-
formations were performed in 1 mL scale in 100 mM HEPES buffer,
TRIS buffer or potassium phosphate buffer (pH 7.5) containing
0.1 mM PLP, 30 mM (rac)-α-MBA (4) and 60 mM pyruvate (5). For an
optimal initial rate measurement, the enzyme concentrations were
set to 0.5 mg/mL for CvATA, 0.05 mg/mL for VfATA, 0.1 mg/mL for
BmATA and 1 mg/mL for AsATAmut11. The protein concentrations
were determined according to Bradford[54] after diluting relevant
samples 1:100 (v/v) in the respective buffer. Initial rate reactions
subjected to blue light illumination for 25 min. Inactivation experi-
ments were carried out in a stirred 2 mL glass vessel (600 rpm),
blue LED strips (60 LEDs, 450 nm, X105-0200, revoART GmbH,
Markkleeberg, Germany; ~12 mW/cm2) were used for illumination.
The corresponding blue light setup is depicted in Figure SI.6.E–F.
Light intensities at 450 nm were determined with a distance of
1 cm from the light source using an energy meter PM100D
equipped with a S302 C sensor (Thorlabs, Newton, NJ, USA). As the
energy meter shows sensitivity not only for light but as well for
temperature changes, the measured light intensities might be error
prone. The reaction setup was cooled with ice to compensate for a
temperature increase mediated by the LED strips. The temperature
°
were incubated for a duration of 30 min at 20 C and 600 rpm in
ChemCatChem 2021, 13, 1–10
8
© 2021 The Authors. ChemCatChem published by Wiley-VCH GmbH
��
These are not the final page numbers!