Racemization Mandelic Acid

Investigation of an integrated
racemization/separation process for the production

of enantiomerically pure mandelic acid
Garca-Rivera M.1, Wrzosek K.2, Renken A.1, SeidelMorgenstein A.2,3
1EPFL
Swiss Federal Institute of Technology, Lausanne/Switzerland

Planck Institute for Dynamics of Complex Technical Systems, Magdeburg/Germany
3Chair for Process Systems Engineering, Otto-von-Guericke University Magdeburg/Germany
2Max
MAX-PLANCK-INSTITUT
DYNAMIK KOMPLEXER
TECHNISCHER SYSTEME
MAGDEBURG
Max Planck Institute Magdeburg
Integrated racemization-separation process for enantiopure mandelic acid
June 2015
Contents
1. Introduction
2. Integrated racemization-separation process
3. Enzyme production and immobilization

4. Enzymatic activity
5. Storage stability
6. Racemization-chromatography compatibility
7. Feasibility for crystallization coupling
8. Summary
June 2015
1. Introduction
Fine-chemicals industry
Limited quantities vs bulk chemicals
High-value products
Selectivity, separation, purification
High production costs
(S) enantiomer
(R) enantiomer
Enantiomer formulations
Food, agrochemicals, pharmaceuticals
Syntheses precursors: flavourings, essences, drugs
Single enantiomers enantiomerically pure compounds
Vanillin
Sertraline
OH
OH
Antiseptics
Skin treatments
Mandelic acid
and derivatives
Penicillin
Cephalosporin
June 2015
1. Introduction
Enantiopure compounds production[1,2]
Pure enantiomers
from
Chiral or achiral
compounds
Racemates
through
through
Enantioselective
synthesis
Resolution
techniques
Asymmetric catalysts
Non-synthetic
Integrated processes
Synthetic
Biocatalysts
Chiral auxiliaries
Crystallization
Classical resolution 50% yield
Chiral pool
Chromatography
Kinetic resolution
L-L extraction
Dynamic kinetic resolution
>50% yield
100% yield
Membrane separation
June 2015

Integrated processes[3]
Racemization
Overcome 50% yield 100%

Racemate
Chromatography
Cost-effective methods
Pure enantiomer
Racemization, chromatography and

crystallization
Racemization
Versatility and diverse operation configurations

Racemate
Crystallization
Pure enantiomer
R+S
S or R
Racemization
R+S
S
S
Racemate
R+S
Chromatography
Crystallization
Pure enantiomer
June 2015

(R)
(R)
(S)
(R/S)
Racemization
Chiral chromatography
Chromatography
Mandelate racemase[4]
Chirobiotic T
Well studied and characterized enzyme
Highly enantioselective
Broad spectrum of mandelic acid

derivatives as substrate
Variety of enantiomers
Mild reaction conditions
Over-expressed in E. coli
Encoded in pET-52b(+)
plasmid1
Teicoplanin
1
Dalhousie University, Halifax, Canada
June 2015

Transformation1
Single colony
Pre-culture
Stable cell line

Stored - 20C
Streaking
LB2 medium
1% ampicillin
LB medium
1% ampicillin
T = 37C
First culture
Main culture
Minimum medium
Microelements
T = 37C
Minimum medium
Microelements
T = 37C
5 mL
Fermentation
Extraction
IPTG3 induction
Cell disruption
Clarification
T = 37 18 C
60 mL
250 mL
Immobilization
Storage
Free enzyme
Immobilized enzyme
Extract
Adsorbent
5L
1
Wet carrier
Analysis and Redesign of Biological Networks, MPI Magdeburg

Lysogeny medium
3 Isopropyl-b-D-1-thiogalactopyranoside
2
Eluent
June 2015

Eupergit CM
Epoxide acrylic beads
..
Particle size: 50 300 mm
Mandelate
racemase
Eupergit CM
Covalent multipoint attachment
Immobilization conditions:
1 M HEPES1 pH = 8.2
Incubation: 70 h
Low stirring
Immobilized mandelate
racemase
Immobilization efficiency:
Immobilized protein recovery:
100 * (1 1
Remaining protein in eluent

Total available protein
) = 47%
Activity recovery:
100 * (
Activity in total mass of wet carrier

Activity in total volume of extract
) = 49%
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
June 2015
Enzymatic activity, in U1
Potential action catalytic activity
Determined T, C and pH
U=
Amount of substrate converted

time
mmolsubstrate
min
Specific activity, in U/mgprotein

Free enzyme (extract)
U/mgprotein =
Protein concentration

time * mass of protein
mmolsubstrate
min * mgprotein
Specific activity, in U/gcarrier

Immobilized enzyme
Wet carrier
1
U/gcarrier =

time * mass of wet carrier
mmolsubstrate
min * gcarrier
U is the amount of enzyme that produces certain amount of enzymatic activity
June 2015
Non-stirred batch
3500
4 mL substrate
40 mL free enzyme
Polarimeter
Free enzyme (extract)

Continuous measurement of rotation angle
every second (high time resolution)
Substrate consumption
(mmol/mgprotein)
Polarimetry method
3000
2500
2000
1500
1000
initial rate enzymatic activity
500
0
10
15
20
25
Time (min)
Reaction: free enzyme (polarimetry method)

Initial concentration: 10 g/L (R)-mandelic acid
HPLC method
8 mL substrate
60 mg wet carrier
Stirred batch
Immobilized enzyme (wet carrier)
Sampling at 2, 4, 6, 8, 10 and 12 min
Reaction stopped by filtering and heating up to

90 C
Substrate consumption
(mmol/gcarrier)
3500
3000
2500
2000
1500
1000
initial rate enzymatic activity
500
0
0
Analytical HPLC measurement: Chirobiotic T
10
15
Time (min)
Reaction: immobilized enzyme (HPLC method)

Initial concentration: 10 g/L (R)-mandelic acid
June 2015
10
Enzyme kinetics
E+S
EX+
ES
EP
E+P
350
E+S
k1
k-1
Reaction rate:
ES
v=
k2
E+P
d[P]
= k2 [ES]
dt
Initial velocity [U/g]
Michaelis-Menten kinetics
250
200
150
100
50
0
0
Steady-state:
d[ES]
= k1 [E][S] k-1 [ES] k2 [ES] = 0
dt
[E] is constant
ES is continuously formed and disrupted
Rate-limiting step is the catalytic step
S flows through E to form P in a steady stream
Zero order
First order
300
50
100
150
200
250
300
350
Substrate concentration [mM]
Immobilized enzyme (polarimetry method)

Substrate: (R)-mandelic acid
June 2015
11
Racemization mechanism[5,6]
Hydrophobic cavity
Glu 317
Glu 317
dimer[4]
Glu 317
Lys 164
Lys 164
Lys 164
+
OH
OH
O
O
HN
HN
2+
Mg
N:
H
O
H
His 297
Asn 197
His 297
NH
Lys 166
NH3
O
2+
Mg
O
H
HN
OH
Mg
NH
O
H
Asn 197
+
NH3
(R)-(-)-mandelic
acid
NH3
NH3
His 297
Asn 197
H
: NH2
NH3
Lys 166
Lys 166
(R)-(-)-mandelic acid
Planar enolate
(S)-(+)-mandelic acid
complex
intermediate
complex
OH
2+
(S)-(+)-mandelic
acid
June 2015
12
Michaelis-Menten equation
v=
Vmax [S]
v: initial reaction rate (enzymatic activity)
k-1 + k2
Km =
k1
Km + [S]
[S]: substrate concentration

Vmax: maximum rate at maximum substrate concentration
Km: Michaelis-Menten parameter
Immobilized enzyme kinetics

(measured by HPLC method)
High standard error
350
Acceptable fit correlation value

Relatively large Km and Vmax compared
against previous results[7]:
Vmax= 307.9 4.54 U/gcarrier
Km = 11.29 0.948 mM
300
v (U/gcarrier)
More measurements required
Plateau at 10 g/L
400
250
200
150
Vmax= 376.40 35.82 U/gcarrier
100
Km = 29.44 9.53 mM
50
R2 = 0.9436
0
0
50
100
150
200
250
300
350
[S] (mM)
June 2015
13
4. Enzyme storage stability

Free enzyme storage
Immobilized enzyme storage
(measured by polarimetry method)
(measured by HPLC method)
250 mM HEPES buffer
Storage temperature: 4 C
Different temperatures
Typical buffer:
150 mM Tris HCl 0.05% sodium azide
Reaction buffer:
50 mM HEPES 3.3 mM MgCl2 pH = 7.5
Activity retention = 100 * (
Activity at time t
Initial activity
Reaction:
10 g/L (R)-mandelic acid + reaction buffer
Dry form:
Lyophilized
120
120
80
Activity retention [%]
Activity retention [%]
- 20 C
100
4 C
60
40
20
100
80
60
40
20
25 C
0
0
0
10
20
30
40
50
Storage time [days]
60
70
10
20
30
40
50
60
Storage time [days]
June 2015
14
5. Racemization-chromatography compatibility
Mobile phase
(R)
Suitable for subsequent

racemization
(R)
(S)
(R/S)
Racemization
Chromatography
20 mM HEPES 3.3 mM MgCl2

pH = 6.8
20% methanol
Operation conditions
Response variables
P < 100 bar, T = 25 C
(R) and (S) concentration
Flow rate retention time
Racemization and resolution yields
Concentration (R)-mandelic acid
Optimal settings max productivity
June 2015
15
Solvent, pH and temperature

dependency
Determination of solubility curves and
MSZ for crystallization process design
Concentration (%)
Solubility of mandelic acid

Nucleation border
Solubility in water
Solubility in mobile phase
MSZW: Metastable zone width

Temperature (C)
Solubility measurements
Isothermal mode
Addition method
Final concentration:
20 mM HEPES
Solvent: mobile phase

pH correction until 6.8
Final volume: 12 mL
3.3 mM MgCl2
20% MeOH
Final pH : 6.8
June 2015
16

MeOH: 0.2Vtotal
Buffer: 0.4 Vtotal
H2O: 0.1 Vtotal
pH=6.8
Iterative method
Fix a Vtotal
Add a known mass

of mandelic acid
Stirring until
dissolved
Add a known volume of
NaOH 10 N
Yes
V<Vtotal
No
No
Fix a new Vtotal
V=Vtotal
Yes
No
Completely
dissolved
Yes
T = 24 C
Fixed volume
Fixed buffer concentration
pH correction
Increasing (R)-mandelic acid mass
Increasing 10 N NaOH volume
First hints
pH 6.8
C (%w)
No
Add a known volume of

NaOH 10 N
Solubility in water
23%
Yes
Solubility in mobile phase
9.9%
Adjust final pH = 6.8
24 C
T (C)
June 2015
17
7. Summary
Enzyme immobilization and characterization are comparable

with previous experiences results are repeatable
Enzyme is stable either in free or immobilized form under
tested storage conditions
Procedures from previous experiences will allow
investigate optimal settings maximum productivity
to
First hints of solubility in mobile phase will allow further

crystallization strategy
June 2015
18
References
1. Todd, M.H., Separation of enantiomers. Synthetic methods. 2014: Wiley-VCH.

2. Lorenz, H. and A. Seidel-Morgenstern, Processes To Separate Enantiomers. Angewandte Chemie
International Edition, 2014. 53(5): p. 1218-1250.
3. Kaspereit, M., S. Swernath, and A. Kienle, Evaluation of Competing Process Concepts for the
Production of Pure Enantiomers. Organic Process Research & Development, 2012. 16(2): p. 353363.
4. Narmandakh, A. and S.L. Bearne, Purification of recombinant mandelate racemase: Improved
catalytic activity. Protein Expression and Purification, 2010. 69(1): p. 39-46.
5. Bearne, S.L. and R.J. Spiteri, Reduction of intrinsic kinetic and thermodynamic barriers for enzymecatalysed proton transfers from carbon acid substrates. Journal of Theoretical Biology, 2005. 233(4):
p. 563-571.
6. Nagar, M., et al., Potent Inhibition of Mandelate Racemase by a Fluorinated Substrate-Product

Analogue with a Novel Binding Mode. Biochemistry, 2014. 53(7): p. 1169-1178.
7. Wrzosek, K., K. Bettenbrock, and A. Seidel-Morgenstern, Immobilized mandelate racemase
performance for dynamic resolution of mandelic acid, in 3rd Multistep Enzyme Catalyzed Processes
2014: Madrid, Spain.
June 2015
19
Immobilized enzyme production

Global mass balance
+
5 L culture
227 mg protein
=
13.6 g adsorbent
Minimum medium: 5L
Ampicillin: 1:100 dil
Glucose: 0.5%
Thiamine: 5 mg/L
40 g wet carrier
3x weight of dry adsorbent

Immobilized protein recovery: 47%
Activity recovery: 49%
IPTG: 200 mM
Protease inhibitor: 100 mL/gbiomass
June 2015
20
Solubility measurement
Stock buffer:
Volume
buffer (mL)
Volume
H2O (mL)
Volume MeOH
(mL)
Mass (R)mandelic acid (g)
Total volume 10 N
NaOH (mL)
pH
8.25 Mm MgCl2
1.5
6.80
pH = 6.8
1.5
1.20
2.50
1.5
1.20
600
5.50
1.5
1.20
650
11.00
1.5
1.32
650
5.24
1.5
1.32
790
8.18
1.5
1.61
790
5.25
1.5
1.61
840
6.94
1.5
1.75
860
5.50
1.5
1.75
890
7.70
1.5
1.90
950
5.11
1.5
1.90
1040
6.18
4.8
1.5
2.4
2.65
1340
4.98
4.8
1.5
2.4
2.65
1422
6.80
4.8
1.5
2.4
3.05
1672
6.37
4.8
1.5
2.4
3.05
1680
6.80
50 mM HEPES
Total volume: 10 mL
Temperature: 24 C
Vtotal > 10 mL
Vtotal = 12 mL
June 2015
21
Culture in 5-L bioreactor
Cell growth
SDS-PAGE analysis
3.5
40
35
3
Harvest
30
Temperature
decrease to avoid
inclusion bodies
Temperature setting to
control the growth rate
25
OD420
20
1.5
15
Temperature [C]
2.5
1
10
0.5
Induction with
IPTG 200 mM
Cell growth
Temperature
0
0
10
11
12
13
14
15
16
17
Time [h]

June 2015
22
Results repeatability
Enzyme form
Measurement
method
Free
Immobilized
Polarimetry
HPLC
Enzymatic activity
(initial)
100 U/mgprotein
Previous results
107.3 U/mgprotein
After extraction with 1M HEPES
385 U/mgprotein
48 h stored at 250 mM HEPES
305 U/gcarrier
Previous results
309.78 U/gcarrier
Immobilization 1
308.35 U/gcarrier
Immobilization 2
Conditions
Reaction conditions: 10 g/L (R)-mandelic acid 50 mM HEPES 3.3 mM MgCl2 pH=7.5

June 2015
23

Racemization Mandelic Acid

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Racemization Mandelic Acid

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Investigation of an integrated

racemization/separation process for the production

Swiss Federal Institute of Technology, Lausanne/Switzerland

Max Planck Institute Magdeburg

Integrated racemization-separation process for enantiopure mandelic acid

3. Enzyme production and immobilization

Max Planck Institute Magdeburg

Integrated racemization-separation process for enantiopure mandelic acid

Integrated racemization-separation process for enantiopure mandelic acid

Classical resolution 50% yield

Dynamic kinetic resolution

Max Planck Institute Magdeburg

Integrated racemization-separation process for enantiopure mandelic acid

2. Integrated racemization-separation process

Overcome 50% yield 100%

Racemization, chromatography and

Versatility and diverse operation configurations

Max Planck Institute Magdeburg

Integrated racemization-separation process for enantiopure mandelic acid

2. Integrated racemization-separation process

Well studied and characterized enzyme

Broad spectrum of mandelic acid

Mild reaction conditions

Dalhousie University, Halifax, Canada

Max Planck Institute Magdeburg

Integrated racemization-separation process for enantiopure mandelic acid

3. Enzyme production and immobilization

Stable cell line

Analysis and Redesign of Biological Networks, MPI Magdeburg

Max Planck Institute Magdeburg

Integrated racemization-separation process for enantiopure mandelic acid

3. Enzyme production and immobilization

Particle size: 50 300 mm

Covalent multipoint attachment

Remaining protein in eluent

Activity in total mass of wet carrier

Max Planck Institute Magdeburg

Integrated racemization-separation process for enantiopure mandelic acid

Amount of substrate converted

Specific activity, in U/mgprotein

Amount of substrate converted

Specific activity, in U/gcarrier

Amount of substrate converted

U is the amount of enzyme that produces certain amount of enzymatic activity

Max Planck Institute Magdeburg

Integrated racemization-separation process for enantiopure mandelic acid

Free enzyme (extract)

initial rate enzymatic activity

Reaction: free enzyme (polarimetry method)

Reaction stopped by filtering and heating up to

initial rate enzymatic activity

Analytical HPLC measurement: Chirobiotic T

Reaction: immobilized enzyme (HPLC method)

Max Planck Institute Magdeburg

Integrated racemization-separation process for enantiopure mandelic acid

Initial velocity [U/g]

ES is continuously formed and disrupted

Rate-limiting step is the catalytic step

S flows through E to form P in a steady stream

Max Planck Institute Magdeburg

Substrate concentration [mM]

Immobilized enzyme (polarimetry method)

Integrated racemization-separation process for enantiopure mandelic acid