Cofactor monitoring during enzymatic reactions by means of Fluorescence Spectroscopy
Fluorescence Spectroscopy
A. Pashkova, L. Greiner, J. Schrader, J. Z. Bloh
e‐mail: [email protected]
Funded by: Deutsche Forschungsgemeinschaft (DFG)
Period: 01.09.2014 ‐ 31.01.2017
Motivation and project aims
Nicotinamide cofactors
The nicotine amide dinucleotide cofactors NADP+/NADPH and NAD+/NADH are of
paramount importance for the industrial utilisation of oxidoreductases. For the
rational use a knowledge gap persists between predictions based on initial rate
measurements and their application in processes. The goal of the project is the inline
quantification of cofactors under conditions as close as possible to process
conditions by application of fluorescence spectrometry allowing monitoring at µmol‐
1 concentrations which are common in their application.
Oxidised form
Oxidised form
25000
Reduced form
Reduced form
15000
FS Intensity / a.u.
20000
10000
5000
No absorption at 340 nm NADPH stability under UV irradiation
Absorption at 340 nm (Emission/ Fluorescence) at 460 nm)
0
300
400
500
600
700
800
900
1000
Wavelength / nm
65
NADPH half life experiments
were performed in a batch
set‐up with the possibility to
saturate the NADPH/Buffer
solution with either N2 or O2.
NADPH h lf lif / h
NADPH half life / h
O2 saturation / Sampling interval 1 h
60
Sampling
interval
1 s
300 s (5 min)
3600 s (1 h)
55
50
NADPH signal
non linear curve fit
Experimental conditions
T = 25°C / V = 100 ml
100 mM ADA Buffer (N‐(2‐
Acetamido)‐iminodiacetic
acid)
20 mM MgCl2
40
ExpDec1
25
Model
Equation
Reduced Chi‐Sqr
Adj. R‐Square
20
c(NADPH)
y0
c(NADPH)
A1
62,85997
0,4057
c(NADPH)
t1
32,99695
0,30966
35
30
y = A1*exp
1,42094
C (NADPH) / µM
45
The instrument
tec5 Spectrometer with Helma® Fluorescence Probe
100 % lamp intensity
120 ms irradiation with 1s 300s or
120 ms irradiation with 1s, 300s or 3600s sampling interval
NADPH Fluorescence spectrum
NADPH
Fluorescence spectrum
C(NADPH) = 100 µM
0,99485
Value
15
Result C0 = A1 = 63 µM
k = 1/t1 = 0,030 h‐1
t1/2 = ln2/k t1/2 ln2/k = 23,1 h
23,1 h
10
5
0
0
20
40
60
Standard Error
0
80
 irradiation frequency is the main factor for NADPH degradation
 presence of O2 does not seem to have an effect on degradation
d
d ti
 sampling interval of 300s has no adverse effects
0
100
O2
saturation
14.4
23.9
23.1
N2
saturation
16.4
23.9
23.7
120
time / h
Enzymatic process
Model reaction: Continuous enzymatic synthesis of (R)‐2‐octanol from 2‐octanone with enzyme coupled cofactor regeneration Product
OH
Enzymes
Concentration profiles
Substrate
H+
O
NADP+
HO
O
O
OH
HO
OH
GDH
HO
OH
HO
Hy
dr
ol
ys
is
OH
OH
D-(+)-Gluconic acid
(GDL)
 glucose dehydrogenase
(GDH)
D-Glucose
OH
OH
Reaction medium
HO
O
O
N
O
HO
lactone
NH2
OH
OH
Enzyme coupled
cofactor regeneration
OH
OH
Gluconic acid
O
2,0
ADA‐buffer
40
1,8
35
1,6
30
1,4
change
pump flow back to
change
pump flow to
1,2
1,0
7.6 ml h
4 ml h
‐1
25
‐1
20
Fluorescent NADPH
species
 alcohol dehydrogenase
from Lactobacillus brevis
(LbADH)
(R)-2-Octanol
0,8
15
0,6
2-octanone
2-octanol
NADPH
0,4
0,2
10
5
0
0,0
0
20
40
60
80
100
120
140
160
180
reaction time / h
Reaction figures (after 190 h run time)
95
C(NADP+) = 0.050 mM
C(glucose) = 100 mM
C (LbADH) = 33 mg L‐1
C (GDH) = 113 mg L‐1
1.8
15
V (EMR) = 15 mL
dV/dt = 4 ml h‐1
Summary
 based on batch experiments for NADPH stability, the optimal conditions for its detection via a fluorescence probe have been identified
 „in‐line“ monitoring of µM NADPH concentrations under process conditions has been demonstrated for up to 200 hours
90
= 3,75 h
pH = 7
T = 25
T 25°C
C
80
70
60
50
1.9
Experimental conditions
C(2‐octanone) = 1 mM
C (ADA) = 100 mM
C (MgCl
( g 2)) = 10 mM
100
+
X2‐octanone
/ %
TONLbADH
/ 105
TONGDH
/ 104
STY
/ mg L‐1d‐1
Conversion plot
Conversion of 2-octanone and NADP / %
Experimental set‐up
40
change
pump flow to
30
7.6 ml h
change
pump flow back to
‐1
4 ml h
‐1
20
2‐octanone
10
NADP
0
0
20
40
60
80
100
120
140
160
+
180
reaction time / h
Outlook
 verification of NADPH concentration in regular time intervals via „off‐line“ measurement with a Fluoresence Multiplate Reader
 optimisation of reaction conditions for run times of up to 1000 hours  test of a different enzymatic system relying on NADP+/NADPH as a cofactor pair
C(NADPH) / µM
LbADH
2-Octanone
C(2-octanone) and C(2-octanol) / m
mM
O