Supplementary material for
P-LinK: A Method for Generating Multicomponent Cytochrome P450 Fusions with Variable
Linker Length
Ketaki D. Belsare1, Anna Joëlle Ruff1, Ronny Martinez1, Amol V. Shivange2, Hemanshu Mundhada3, Dirk
Holtmann4, Jens Schrader4 and Ulrich Schwaneberg1*
1
Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany
Department of Chemical Engineering, University of California, Santa Barbara, California 93106, USA
3
Technical University of Denmark, Novo Nordisk Foundation Center for Biosustainability, Fremtidsvej 3,
DK-2970 Hørsholm, Denmark
4
Biochemical Engineering Group, DECHEMA Research Institute, 60486, Frankfurt am Main, Germany
2
Protein expression in microtiter plates
Microtiter plates containing individual clones of CinA-CinC linker library of variable length from 1 to 16
amino acids were duplicated using a 96-well pin replicator to preculture flat bottom microtiter plates
(Grierner Bio-one, GmbH, Frickenhausen, Germany) containing 150 µl of LBkan per well (17 µM
kanamycin). The latter were incubated in a microtiter plate shaker (37 °C, 16 h, 900 rpm, 70 % humidity;
Multitron II, Infors GmbH, Einsbach, Germany). Expression of CinA-CinC linker library was carried out in 2
ml deep well plates (Polypropylene plates, Brand GmbH, Wertheim, Germany) containing TB media (600
µl; supplemented with 17 µM kanamycin, 0.1 mM trace element solution, 0.1 mM thiamine and 0.5 mM
δ-aminolevulinic acid). Expression cultures were inoculated from overnight grown precultures (6 µl).
Expression was carried out first at 37 °C in a microtiterplate shaker (900 rpm, 70 % humidity; Multitron
II, Infors GmbH, Einsbach, Germany) until an OD600nm of 0.6 was reached, induced with isopropyl-thio-βgalactopyranoside (0.1 mM) and incubated further for 18 h at 27 °C (900 rpm, 70 % humidity; Multitron
II, Infors GmbH, Einsbach, Germany). Expression cultures were harvested by centrifugation (3200 g, 4 °C
for 20 min) and cell pellets were frozen over night (-20°C). Cell pellets were resuspended in 200 µl lysisbuffer (50 mM KHxPO4, pH 7.4) containing 1.5 mg/ml lysozyme (≥ 40,000 U/mg) (Sigma Aldrich,
1
Taufkrichen, Germany) and cell lysis was subsequently performed in a microtiter plate shaker (37 °C, 1 h;
900 rpm, 70 % humidity; Multitron II, Infors GmbH, Einsbach, Germany). Clear supernatants were
obtained by centrifugation (3200 g, 4 °C for 20 min) and used subsequently for SDS-PAGE analysis, CObinding measurements, and 1,8-cineole conversions.
Protein expression in flasks (CinA, CinC and CinA-CinC fusion protein)
Preculture was grown in LBkan medium (4 ml, 37 °C, 16 h, 250 rpm, 17 µM kanamycin). Expression culture
(400 ml, TB media supplemented with 17 µM kanamycin, 0.1 mM trace element solution, 0.1 mM
thiamine and 0.5 mM δ-aminolevulinic acid (except for CinC)) was inoculated with 1% preculture and
incubated first at 37 °C (250 rpm, 70 % humidity, Infors HT, Minitron, Einsbach, Germany) until an
OD600nm of 0.6 was reached, induced with isopropyl-thio-β-galactopyranoside (0.1 mM) and incubated
further for 18 h at 27 °C (250 rpm, 70 % humidity, Infors HT, Minitron, Einsbach, Germany). Expression
cultures were harvested by centrifugation (3200 g, 4 °C for 20 min) and cell pellets were frozen overnight
(-20°C). All following steps were performed on ice unless otherwise specified. Cell pellets from 400 ml
expression culture were resuspended in 25 ml of cold (4 °C) loading buffer (50 mM Tris-HCl buffer, pH 7.8
for CinA and CinA-CinC fusion proteins; 50 mM Tris-HCl, 50 mM KCl pH 7.4 for CinC). Resusupension was
performed by sonication (40 % amplitude, 30 sec x 4 cycles; SONICS Vibra cell VCX-130, Zinsser Analytics,
Frankfurt, Germany) followed by cell lysis using high pressure homogenation (3 cycles; 1500 bar
EmulsiFlux C3, Avestin Europe GmbH). Disrupted cells were centrifiged (3200 g, 4 °C for 30 min) and the
supernatant obtained was further filtered through a polyvinylidene difluoride filter (0.45 µm; Ministart
RC 25 disposable syringe filters; Sartorius, Hamburg, Germany). The cleared lysate was used for
purification.
Protein purification
2
Purification of CinA-CinC fusion proteins and CinA was performed using ÄKTA prime plus protein
purification system (GE Healthcare, München, Germany) on preparative LC columns (internal diameter
15 mm, length 125 mm; KRONLAB Glass Columns, TAC 15/125 PE5-AB-2, YMC Europe GmbH, Dinslaken)
by anion exchange chromatography using Toyopearl 650S-DEAE matrix (Tosoh Biosciences, Tokyo, Japan)
(bed size: 15 mm x 110 mm). Column was equilibrated in loading buffer (20 CVs; 50 mM Tris-HCl buffer,
pH 7.8, flow rate 3 ml/min). The filtered and clarified lysate (25 ml) was loaded on the column (flow rate
1 ml/ min) and unbound protein was washed out with loading buffer (20 CVs, flow rate 1.5 ml/min).
Protein was eluted by salt gradient (1 M NaCl, 50 mM Tris-HCl buffer, pH 7.8, gradient from 0% to 40 % in
200 ml, flow rate 1.5 ml/min). Purity was checked by SDS-PAGE (12 % SDS-gel; 20 mA, 50 W, 80 min;
Mini-Protean Tetra Cell, Bio-Rad Laboratories GmbH, München Germany). Expected sizes for CinA is 45
kDa and for CinA-CinC fusion protein 61 kDa. Purified protein was concentrated using 10K Amicon Ultra-4
filters (Darmstadt, Germany) and subsequently desalted using PD-10 desalting columns with 50 mM
KHxPO4 buffer (Matrix: SephadexTM G- 25- 5 ml; GE-healthcare, USA). Next, the desalted protein was
lyophilized for 18 h (Christ Alpha 1-2 LD Plus; Omnilab laborzentrum GmbH & Co. KG, Germany) and
stored at -20 °C.
Purification of CinC was performed by anion exchange chromatography using Toyopearl 650S-DEAE
matrix (Tosoh Biosciences, Tokyo, Japan) (bed size: 15 mm x 110 mm) like described for CinA with the
following modifications. Column was equilibrated in loading buffer (20 CVs; 50 mM Tris-HCl, 50 mM KCl
pH 7.4, flow rate 3ml/min). Subsequently, the filtered and clarified lysate (25 ml) was loaded on the
column (flow rate 1 ml/min) and unbound protein was washed out with loading buffer (20 CVs, flow rate
1.5 ml/min). CinC was eluted using salt gradient (1 M KCl, 50 mM Tris-HCl, pH 7.4, gradient from 0% to 40
% in 200 ml, flow rate 1.5 ml/min). Purified CinC was concentrated using 3K Amicon Ultra-4 filters
(Darmstadt, Germany) and subsequently purified by a second size exclusion chromatrography step using
G-Sephadex matrix (bed volume: 15 mm x 110 mm, pore size: 100-300 µM; Sigma Aldrich, Taufkrichen,
3
Germany) using a preparative LC column (internal diameter 15 mm, length 125 mm; KRONLAB Glass
Columns, TAC 15/125 PE5-AB-2, YMC Europe GmbH, Dinslaken). The column was equilibrated (20 CVs, 50
mM Tris- HCL, pH 7.4, flow rate 3 ml/min) and pooled fractions containing CinC obtained after first
purification step were loaded (flow rate 1 ml/min). CinC was eluted in buffer (50 mM Tris-HCl, pH 7.4;
flow rate 1.5 ml/min, fraction size 1 ml). Purity was checked by SDS-PAGE like described above for CinA.
The expected size of CinC is 16 kDa. Additionally, the purity was monitored by UV spectroscopy at 456
nm. Purified CinC was subsequently desalted in a third purification step using PD-10 desalting columns
with 50 mM KHxPO4 buffer (Matrix: SephadexTM G- 25- 5 ml; GE-healthcare, USA). Finally, the desalted
protein was lyophilized for 16 h (Christ Alpha 1-2 LD Plus; Omnilab laborzentrum GmbH & Co. KG,
Germany) and stored at -20 °C.
Expression and Purification of Fpr
Expression of E.coli flavodoxin reductase (Fpr) was performed as described above for expression of CinA
and CinC with the following modification. The expression culture (400 ml, TB media supplemented with
17 µM kanamycin, 0.1 mM trace element solution, 1 mM MgCl2, 5 mM NaCl) was incubated at 33 °C (250
rpm, 70 % humidity, Infors HT, Minitron, Einsbach, Germany) until an OD600nm of 0.6 was reached,
induced with isopropyl-thio-β-galactopyranoside (0.1 mM) and incubated for further 4 h at 33 °C (250
rpm, 70 % humidity, Infors HT, Minitron, Einsbach, Germany). Expression culture was harvested like
described above for Cin A and pellet of expressed Fpr was resuspended in 25 ml of cold (4 °C) loading
buffer (50 mM Tris- HCl, 200 mM NaCl, 1 mM dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride, pH
7.4). The clear cell lysate was prepared as described above for CinA.
Purification of Fpr was performed as previously reported with the following modification to the protocol
of Hawkes et al (1,2). Fpr was purified by two anion exchange chromatography steps. In the first step,
using Toyopearl 650S-DEAE matrix (Tosoh Biosciences, Tokyo, Japan, bed size: 15 mm x 110 mm) and
ÄKTA prime plus protein purification system (GE Healthcare, München, Germany) on a preparative LC
4
columns (internal diameter 15 mm, length 125 mm; KRONLAB Glass Columns, TAC 15/125 PE5-AB-2;
YMC Europe GmbH, Dinslaken). Column matrix was equilibrated in loading buffer (20 CVs; 50 mM TrisHCl; pH 7.4, flow rate 3 ml/min). The filtered and clarified lysate (25 ml) was loaded on the column (flow
rate 1 ml/min) and unbound protein was washed out with loading buffer (20 CVs, flow rate 1.5 ml/min).
Protein was eluted by salt gradient in elution buffer (1 M NaCl, 50 mM Tris-HCl, pH 7.4, gradient from 0%
to 40 % in 200 ml, flow rate 1.5 ml/min). Purity was checked by SDS-PAGE after each chromatography
step (12 % SDS-gel; 20 mA, 50 W, 75 min; Mini-Protean Tetra Cell, Bio-Rad Laboratories GmbH, München
Germany). Expected size for Fpr is 29 kDa. Fractions containing Fpr were pooled and purified by a second
chromatography step using Toyopearl HW-55F column (Tosoh Biosciences LLC, Tokyo, Japan; bed
volume: 15 mm x 100 mm) and LC columns (internal diameter 15 mm, length 125 mm; KRONLAB Glass
Columns, TAC 15/125 PE5-AB-2, YMC Europe GmbH, Dinslaken). Column was equilibrated in loading
buffer (20 CVs; 50 mM Tris-HCl; pH 7.4, flow rate 3 ml/min). Pooled fractions containing Fpr obtained
after first purification step were loaded on the column (flow rate 1 ml/min) and unbound protein was
washed out using loading buffer (20 CVs, 50 mM Tris-HCl; pH 7.4, flow rate 1.5 ml/min). Fpr was eluted
by salt gradient (1 M NaCl, 50 mM Tris-HCl, pH 7.4, gradient 0 to 40 % in 200 ml, flow rate 1.5 ml/min).
Purity was checked by SDS-PAGE after each chromatography step (12 % SDS-gel; 20 mA, 50 W, 75 min;
Mini-Protean Tetra Cell, Bio-Rad Laboratories GmbH, München Germany). Expected size for Fpr is 29
kDa. Purified Fpr was subsequently desalted in a third purification step using PD-10 desalting column
with 50 mM KHxPO4 buffer (Matrix: SephadexTM G- 25- 5 ml; GE-healthcare, USA). Finally, the desalted
protein was lyophilized for 18 h (Christ Alpha 1-2 LD Plus ; Omnilab laborzentrum GmbH & Co. KG,
Germany) and stored at -20 °C.
Determination of Protein concentration
5
Concentration of active P450 in purified CinA and CinA-CinC fusion proteins was determined by CO
differential spectroscopy as described (3). Concentration of CinC and E.coli flavodoxin reductase (Fpr)
was determined as reported previously (4,5).
Activity assay, coupling and gas chromatographic analysis
Activity of CinA- linker-CinC fusion protein library was determined in 96-well microtiter plate format
using crude cell lysate (prepared as mentioned in the section protein expression in microtiter plates) and
automated TLC for semi-quantifying 2-β-hydroxy-1,8-cineole production. E. coli Fpr was supplemented in
all reaction mixtures and NADPH was used as reduction equivalent. The final reaction mixtures (50 mM
KHxPO4, pH 7.4; 0.25 ml MTP format) contained CinA-linker-CinC fusion protein variant (1 µM), Fpr (1
µM), 1,8-cineole (6 mM) and catalase (1500 U; Catalase from bovine liver, Sigma Aldrich, Taufkrichen,
Germany). Reactions were initiated by supplementing NADPH (0.4 mM final concentration) and
incubated for 2 h at 30° C in microtiter plate shaker (900 rpm, 70 % humidity; Multitron II, Infors GmbH,
Einsbach, Germany). Reaction product was extracted with 250 µl ethyl acetate containing 800 µM
thymol as internal standard, dried over MgSO4, and 40 µl of extracted product was spotted on a precoated Silica gel 60 F 254 plates (20 cm x 10 cm; Merck KGaA, Darmstadt, Germany) using an automatic
TLC sampler (CAMAG automated TLC sampler 4, Camag GmbH, Berlin Germany). A hexane/ethyl acetate
(17:3) mixture was employed for TLC separation and a p-anisaldehyde/glacial acetic acid/91%H2SO4
(0.5:60:0.5) mixture was used for 2-β-hydroxy-1,8-cineole visualization after heat incubation (heat gun;
100-105° C). Densitometry evaluation of the TLC plates was carried out at a wavelength of 634 nm (TLC
scanner 3, CAMAG GmbH; Berlin, Germany) and data analysis was performed with WinCATS software.
The 32 most active variants and 15 inactive variants were selected for sequencing. Active CinA-CinC
fusion variants (linker length > 5) were used for further activity measurements.
6
Activity measurements for purified CinA-linker-CinC fusion proteins (1 ml scale; 50 mM KHxPO4, pH 7.4;
CinA-linker-CinC fusion protein variant (1 µM), Fpr (1 µM), 1,8-cineole (6 mM), catalase (1500 U), NADPH
(0.4 mM)) were monitored by spectrophotometric measurement of NADPH depletion at 340 nm
(Spectrophotometer Varian Cary 50 UV, Agilent Technologies, Darmstadt, Germany). The reaction
product was extracted with ethyl acetate (1 ml) containing 800 µM thymol as internal standard and dried
over MgSO4. Subsequently, the product amount was determined by Gas Chromatography (GC-2010,
Schimadzu, Duisburg, Germany; column: 30 m x 0.25 µm x 0.25 mm, FS Supreme-5 ms from CSChromatographic Service, GmbH Langerwehe, Germany) with the following temperature program: from
80 °C to 150 °C with 5 °C /min (Injection volume: 1 µl, Split ratio: 20, carrier gas: nitrogen). The retention
time of 2-β-hydroxy-1,8-cineole was determined to be 9.3 min.
Coupling efficiency of CinA-CinC fusion proteins was determined by the following procedure. Reactions
(1 ml scale; 50 mM KHxPO4, pH 7.4) contained CinA-CinC fusion proteins (1 µM), E.coli Fpr (1 µM), 1,8cineole (6 mM), catalase (1500 U) and were initiated by addition of NADPH (0.4 mM). NADPH depletion
was monitored by spectrophotometric measurement at 340 nm (Spectrophotometer Varian Cary 50 UV,
Agilent Technologies, Darmstadt, Germany). Reaction was stopped at 700 sec by addition of extraction
solvent (1 ml) containing 800 µM thymol as internal standard and product concentration was
determined by GC. The coupling efficiency (in percentage) was calculated as the ratio of NADPH used for
substrate oxidation to the total amount of NADPH consumed by P450 in the reaction.
7
Figure 1. Coupling efficiency of CinA-CinC fusion proteins generated by P-Link versus linker length from 016 amino acids and unfused CinA+CinC+Fpr. The coupling efficiency (in percentage) was calculated as the
ratio of NADPH used for substrate oxidation to the total amount of NADPH consumed by P450 in the
reaction.
Correlation between TLC assays and GC-FID measurements
TLC screening was used as a pre-screen to differentiate between active and inactive clones using crude
cell lysates and semi-quantify (in µM, with 50 µM approximation) the amount of 2-β-hydroxy-1,8-cineole
formed. In the following experiments, GC-FID was used to accurately quantify the amount of 2-βhydroxy-1,8-cineole formed by each linker construct using purified enzyme. The correlation between TLC
assays using crude cell lysates and GC analysis using purified fusion enzymes is shown in Figure 2.
8
Product [µM]
Linker
Length
GC-FID
TLC
6
88.34
100
7
102.03
100
8
116.12
100
9
140
150
10
176.67
200
11
12
13
14
15
154.26
128.17
95.54
105.61
105.49
150
150
100
100
100
16
105.64
100
Figure 2. Correlation between thin layer chromatography using crude extracts (lysate from 96-well
microtiter plate expression) and GC analysis of reactions by purified fusion enzymes (P450 amount
normalized to 1 µM).
The screening using TLC was performed in a 96-well microtiter plate without normalizing the amount of
P450 in the reaction whereas in case of purified enzymes, reactions were performed using normalized
amount of P450 (1 µM). Figure 1, table on the right shows the amount of 2-β-hydroxy-1,8-cineole as
calculated by TLC and GC-FID. Quantification using TLC assay could determine the amount of 2- βhydroxy-1,8-cineole only in steps of 50 µM and therefore it was necessary to use GC-FID for precise
quantification of 2- β-hydroxy-1,8-cineole. As seen in figure 2, a good correlation was observed between
detection of 2-β-hydroxy-1, 8-cineole using the TLC in the screening assay and GC-FID for mutant
characterization.
1
2
3 4
5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 3. An exemplary TLC plate as obtained with automated TLC in the screening assay. The samples
were spotted with Automatic TLC sampler 4, developed using Automatic developing chamber (ADC 2 in
n-hexane: ethyl acetate (17:3); stained with p-anisaldehyde: glacial acetic acid: concentrated H2SO4
9
(0.5:60:05) and scanned using TLC scanner 3. Lane 1-12: samples applied. Blue stop represent 2-βhydroxy-1, 8-cineole and orange spot represent thymol.
Table 2. Oligonucleotides used in the study
Primer
Name
Purpose
Sequence
FA1
RA1
FC1
RC1
FP1
RP1
cinA amplification
cinA amplification
cinC amplification
cinC amplification
Vector amplification
Vector amplification
RL1
FL1
FL2
FL3
FL4
FL5
R.P. for linkers 1-5
F.P. for Linker 1
F.P. for Linker 2
F.P. for Linker 3
F.P. for Linker 4
F.P. for Linker 5
ttcgctcagacgTTTGCCTTTCGGAAAAATAATC
cgtctgagcgaaCCTATGGGTAATGCCCTGATTTTATATGGCAC
cgtctgagcgaaCCTTCTATGGGTAATGCCCTGATTTTATATGGCAC
cgtctgagcgaaCCTTCTCCAATGGGTAATGCCCTGATTTTATATGGCAC
cgtctgagcgaaCCTTCTCCATCTATGGGTAATGCCCTGATTTTATATGGC
cgtctgagcgaaCCTTCTCCATCTACTATGGGTAATGCCCTGATTTTATATGGC
RL2
FL6
FL7
FL8
FL9
FL10
FL11
R.P. for linkers 6-11
F.P. for Linker 6
F.P. for Linker 7
F.P. for Linker 8
F.P. for Linker 9
F.P. for Linker 10
F.P. for Linker 11
agtagatggagaAGGTTCGCTCAGACGTTTGCCTTTC
tctccatctactGACATGGGTAATGCCCTGATTTTATATGGCAC
tctccatctactGACCAAATGGGTAATGCCCTGATTTTATATGGCAC
tctccatctactGACCAATCCATGGGTAATGCCCTGATTTTATATGGCAC
tctccatctactGACCAATCCCCTATGGGTAATGCCCTGATTTTATATGGCAC
tctccatctactGACCAATCCCCTTCTATGGGTAATGCCCTGATTTTATATG
tctccatctactGACCAATCCCCTTCTACTATGGGTAATGCCCTGATTTTATATG
RL3
FL12
FL13
FL14
FL15
FL16
R.P. for linkers 12-16
F.P. for Linker 12
F.P. for Linker 13
F.P. for Linker 14
F.P. for Linker 15
F.P. for Linker 16
agtagaaggggaTTGGTCAGTAGATGGAGAAGGTTCGCTCAGACGTTTGCC
tccccttctactGGAATGGGTAATGCCCTGATTTTATATGGCAC
tccccttctactGGAGACATGGGTAATGCCCTGATTTTATATGGCAC
tccccttctactGGAGACGCTATGGGTAATGCCCTGATTTTATATGGCAC
tccccttctactGGAGACGCTGTTATGGGTAATGCCCTGATTTTATATG
tccccttctactGGAGACGCTGTTGCTATGGGTAATGCCCTGATTTTATATG
gactcactatagGGGAATTGTGAGCGG
ttcgctcagacgTTTGCCTTTCGG
cgtctgagcgaaATGGGTAATGCCCTGATTTTATATGGC
cgggctttgttagCAGCCGGATCTCAG
ctaacaaagcccgAAAGGAAGCTGAGTTG
ctatagtgagtcGTATTAATTTCGAACATGTGAGC
*Underlined bold letters represents phosphorothioated nucleotides
10
Table 3. Conversion of 1, 8-Cineole was performed using CinA-CinC fusion system supplemented with E.coli Fpr (1
µM) and NADPH (0.4 mM) as a reduction equivalent. Control reactions were performed using unfused CinA and
CinC domains and empty vector pALXtreme-1a. (n.c. = no conversion detected)
Construct/Enzyme
CinA-CinC fusion
CinA
CinC
CinA+ CinC
Empty vector
Active/Inactive
Amount of 2-β-hydroxy,1,8cineole obtained (µM)
Coupling (%)
Inactive
Inactive
Inactive
Active
Inactive
n.c.
n.c.
n.c.
315±13.88
n.c.
n.c.
n.c.
n.c.
79±3.47
n.c.
Table 4. The secondary structure predictions of the linkers using the Chou- Fasman parameters
(http://www.biogem.org/tool/chou-fasman/). The percentage possibility for secondary structure of each linker
sequence has been classified in Helix, Sheet and Turn.
Linker
BM3 linker
First eight amino acids of P450 BM3
linker
Last eight amino acids of P450 BM3
linker
Last eight amino acids of designed
linker
New designed linker
Sequence
% possibility of sequence to form
Helix
62.5
Sheet
0.0
Turn
25.0
0.00
0.00
37.5
AKKVRKKA
87.5
0.00
25.0
PSTGDAVA
0.00
0.00
12.5
PSPSTDQSPST
GDAVA
0.0
0.0
25.0
PSPSTDQSAKK
VRKKA
PSPSTDQS
References
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of Cindoxin, an Unusual Fmn‐Containing Cytochrome P450 Redox Partner. ChemBioChem
11:1107-1114.
2.Jenkins, C.M. and M.R. Waterman. 1998. NADPH-flavodoxin reductase and flavodoxin from
Escherichia coli: characteristics as a soluble microsomal P450 reductase. Biochemistry 37:61066113.
3.Omura, T. and R. Sato. 1964. The carbon monoxide-binding pigment of liver microsomes. I. Evidence
for its hemoprotein nature. J biol Chem 239:2370-2378.
4.Aliverti, A., B. Curti, and M.A. Vanoni. 1999. Identifying and quantitating FAD and FMN in simple and
in iron-sulfur-containing flavoproteins, p. 9-23. Flavoprotein protocols. Springer.
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5.Macheroux, P. 1999. UV-visible spectroscopy as a tool to study flavoproteins, p. 1-7. Flavoprotein
protocols. Springer.
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