separation by enzymatically catalyzed reactions chapter 10
TRANSCRIPT
SEPARATION BY ENZYMATICALLY
CATALYZED REACTIONS
Chapter 10
r-1-Phenylethylacetate
r,s-1-Phenylethanol
VinylacetateCatalyst: Novozym 435 (Lipase)
s-1-Phenylethanol
Carbon dioxide, supercritical
Carbon dioxide +
r-1-Phenylethylacetate
s-1-Phenylethanol
Carbon dioxide
Reactive separation
Enzyme Catalysis
Kinetics: Influence of Substrates and Enzymes
[E]+ [S] [ES] [P][E]+k3k1
k2
Michealis-Menten-mechanism
Reaction of 2nd order with an equilibrium in front. Free enzyme and substrate are in equilibrium with the enzyme-substrate-complex.
[E] Enzyme concentration,[S] Substrate concentration,[ES] Concentration of enzyme-substrate-complex,[P] Product concentration.
Enzymes in supercritical carbon dioxide
The use of enzymes in non-aqueous media has several reasons:
Hydrolysis is avoided,
Solubility of organic molecules is better, resulting in higher yields,
In supercritical carbon dioxide the removal of reactands is possible during the reaction.
Randolph investigated the phospholipase (EC 3.1.3.1) catalyzed hydrolysis of Di-Natrium-p-nitrophenylphosphate in supercritical CO2.
Enzymes in supercritical carbon dioxide: Example
O2N P
O
ONa
ONa 2H2O O2N P
O
OH
OH 2NaOH+ +Phosphatase
Hydrolysis of Di-Natrium-p-nitrophenylphosphate in supercritical CO2 (p = 10 MPa, T = 308 K).
Influence of water on stability and activity
For most enzymes:
activity increases with increasing water content.
part of the water is fixed to the protein by hydrogen bonding as a water shell.
enzymes can be active without water in organic media.
additional amount of water should be kept low.
conformation of the enzyme seems not to depend on acidity.
1,E-01 1,E+01 1,E+03 1,E+05 1,E+07
Lysozym
Penicillinase
Lactat-Dehydrogenase
Carboanhydrase
Alkohol-Dehydrogenase
Turnover number [s-1]
Supercritical CO 2
Comparison of contact rate of several enzymes in aqueous solution and of alcohol dehydrogenase in scCO2
Activity in water and in sc-CO2
Hrnjez et al.: regio specific and stereo specific activity of the lipase-catalyzed esterification of chiral dioles with anhydrous butyric acid.
OH
OH
OH
O
O
O
H3C
H3C
O
O
+ Lipase- AcOH
OH
OH
O
OOHO
H3C
H3C
O
O
+ Lipase- AcOH
Lipase-catalyzed esterification of chiral dioles with anhydrous butyric acid. (p = 3.5 - 20 MPa, T = 40 °C).
Specific enzymes and substrates
Lipases
Lipases split fats into fatty acids and glycerol
Lipase* Lipase*-Substrat
Lipase Product
+ Substrate
Surface
HydrophobicMedium
HydrophilicMedium
Activation of lipases by the surface between hydrophobic and hydrophilic medium.
Enrichment of enantiomers by lipase catalysis
Enantio selective synthesis catalyzed by enzymes is due to the faster reaction of one enantiomer.
Surface
Enzyme* (S)-Substrate Enzyme + (-)-Enantiomer
Enzyme* (R)-Substrate Enzyme+ (+)-Enantiomer
Enzyme Enzyme*
kcat
k-1k1(R)
k'cat
k'catk'1(S)
kp
kd
k'r
kr
Scheme of a catalyzed enantio-selective transesterification
Enantioselectivity
B
AM
'E
M
cat
cat
Kk
Kk
kcat/KM apparent equilibrium constant for the 2nd order reaction at infinitesimal substrate concentration, kcat and KM represent the turnover number and the Michaelis-Menten constant.
If the Michaelis-Menten-constant is equal for both substrates:
B
A
'E
cat
cat
k
k
Indices A and B stand for the enantiomers.
For irreversible reactions the enantioselectivity E can be derived from yield (U) and the enantiomeric excess (ee).
Substrat
Substrat
eeU
eeU
11ln
11lnE
Produkt
Produkt
eeU
eeU
11ln
11lnE
SR
SR
cc
ccee
ProduktSubstrat
Substrat
cc
cU
1
U Yield [%],eeProdukt Enantiomeric excess of products [%],eeSubstrat Enantiomeric exc. of substrate [%],cProdkukt Product concentration [mol / l],cSubstrat Substrate concentration [mol / l],cR Concentration R-enantiomer [mol / l],cS Concentration S-enantiomer [mol / l],E Enantioselectivity.
Enantioselectivity
0
20
40
60
80
100
120
0 50 100 150
Yield [%]
1000
100
10ee
Change of enantiomeric excess with product formation.
Enantioselectivity
E =
0
20
40
60
80
100
120
0 50 100 150
Yield [%]
1000
100
10ee
The highest enantiomeric excess is achieved (with high enantioselectivity) at 50 %.
Enantioselectivity
E
If the reaction proceeds, ester and alcohol react reversibly into the initial substrates (educts).
r
cat
r
cat
k
k
k
kK
'
'
kcat and kr are the rate constants of forward and backward reaction.
U1U11ln
U1U11lnE
Substrat
Substrat
eeK
eeK
odukt
odukt
eeUK
eeUK
Pr
Pr
111ln
111lnE
Enantioselectivity
ee
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100
Yield [%]
ee 0,00,10,51,05,0
K
Variation of enantiomeric excess of the remaining substrate with increasing K (K = 0; 0,1; 0,5; 1; 5) for E = 100.
Enantioselectivity
Variation of enantiomeric excess with increasing K (K = 0; 0,1; 0,5; 1; 5) for E = 100. With the formation of products, enantiomeric excess drops rapidly.
Enantioselectivity
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100
Umsatz [%]
En
an
tiom
ere
nü
be
rsch
uß
[%
]
x
0,0
0,1
0,5
1,0
5,0
K
Separation by enymatically catalyzed reactions:
Examples and Experiments
Enrichment by enzyme catalyzed interesterification in supercritical carbon dioxide
Substrates
(+)-epi-Methyljasmonate is responsible for theintensive jasmin fragrance. ()-Methyljasmonate /()-epi-Methyljasmonate ratio 4:1
R-/S-Ibuprofenmethylester
COOC2H5
CH3
H3C
HH3C
(S)-(+)-Ibuprofen is a nonsteroidalanti-inflammatory drug.
()-epi-Methyljasmonate
O
OO CH3
Racemic mixtures of Ibuprofen a. epi-Methyljasmonate
(±) R COOCH3 (+) R COOC2H5 (-) R COOC2H5+
Reaction scheme
Lipase: Novozym 435 (strain of Candida antarctica), 1-2 % w/w H2O
Solvent: Supercritical CO2, Hexane
Conditions: 100 - 200 bar, 40 - 60 °C
High pressure cell
P: up to 4000 bar
T: 150 °C.
0.1 mmol ester, 0.4 mmol alcohol 15 mg immobilized lipase
Test cell
Conversion of Racemic Ibuprofenmethylester With Alcohols in n-Hexane
p = 100 bar, T = 50 °C; Catalyst: Lipase Novozym 435
Enantiomeric Excess: Ibuprofen
0
10
20
30
40
50
60
0 20 40 60 80Conversion [%]
Ena
ntio
mer
ic E
xces
s [%
] EthanolPropanolButanol
E 3.5K 0.4
product
educt
Enantiomer separation by chiral Gas chromatography
Competitive Process: Chromatography
COOC2H5
CH3
H3C
CH3H
Educt Product
COOC2H5
CH3
H3C
HH3C
Enantioselectivity and Equilibriumconstants
are affected by different Alcohols
The enantioselectivity E increases with theequilibrium constant K.
The difference in the reaction rate decreases.
E K v
vR
S
max,
max,
Ethanol 3.5 0.35 4.3
(1)-Butanol 6.0 0.4 3.4
(2)-Methoxyethanol 7.0 1.0 2.6
Influence of various reaction partners
51
45
42
0
10
20
30
40
50
60
23 % Conversion
100 bar
150 bar
200 bar
Enantiomeric Excess [%]
Interesterification of ibuprofen methylester with ethanol (T = 50 °C)
Enantiomeric excess: Ibuprofen
0
2
4
6
8
10
12
14
16
18
0 500 1000 1500Time [min]
Con
vers
ion
[%]
1-2 % w/w5-6 % w/w6-7 % w/w
Reaction rate independent of Enzyme water content
Influence of water
0
10
20
30
40
50
60
70
0 5 10 15 20 25Conversion [%]
Ena
ntio
mer
ic E
xces
s [%
]
epi-Ethyl-
epi-Methyl-
epi-Methoxy-
epi-Methyl-
EthanolE 7K 0.2
(2)-MethoxyethanolE 8K 1.8
Enrichment of (+)-epi-Methyljasmonate in SC-CO2
Below 10 % percent yield the enzyme catalyzed reaction is irreversible. (R)-Ibuprofenmethylester is preferably converted.
The enantiomeric excess increases with decreasing pressure and decreasing temperature.
The enzyme specificity is enhanced by introducing polar functional groups into the acyl acceptor. Optimum water content is 1-2 % w/w. Addition of water to Novozym 435 does not accelerate the reaction rate. (+)-epi-Methyljasmonate is preferably converted.
The enzyme specificity in hexane is similar compared to that
in supercritical carbon dioxide.
Some conclusions
Lipase-catalysed kinetic resolution of racemates at temperatures from 40°C to 160°C
in supercritical CO2
The enzyme: Novozym 435, EC 3.1.1.3 from Candida antarctica B, 7000 PLU/g (activity
expressed in propyl laurate units base on a batch synthesis assay), water content 1-2% w/w,
Example: Phenylethanol
C*
C*
CH
CH
CH H C
OH
OHO
3
3
O
O
3 3
CH
OOC
CH
3
2+
+
1-Phenylethanol (R,S) Vinylacetate
1-Phenylethylacetate Vinylalcohol Acetaldehyde
Reaction Scheme
s-1-Phenylethylacetate
r,s-1-Phenylethanol
VinylacetateCatalyst: Novozym 435 (Lipase)
s-1-Phenylethanol
Carbon dioxide, supercritical
Carbon dioxide +
r-1-Phenylethylacetate
s-1-Phenylethanol
Carbon dioxide
Reactive separation
Structure of the enzyme Lipase Arctica candida
0 2 4 6 8 10 12 140
10
20
30
40
50 P=15MPa
95°C 136°C
Um
satz
bez
ogen
auf
Rac
emat
(%
)
Reaktionszeit (h)
Conversion of (R,S)-1-phenylethanol at 95°C () and 136°C () for phenylethanol esterification at 15 MPa.
Reaction time [h]
Yie
ld r
el. T
o ra
cem
ate
[%]
Separation of r,s-1-phenylethanol
1-Phenylethanol -
vinylacetate - CO2
Novozym 435
Initial substrate concentration: 0,5 M
Reference: 1g immob. Enzyme, 100 oC
Effect of temperature on reaction rate
20 40 60 80 100 120 140 160
0,1
0,2
0,3
5
10
15
20
25
30
35
40
1-Phenylethanol r0
Ibuprofen r(0-0,5h)
Rea
ktio
nsg
esch
win
dig
keit
[mm
ol h
-1 g
-1 im
mb.
Enz
ym]
Temperatur [°C]
Comparison of reaction rate
8 10 12 14 16 18 20 22 24 26 28 30 320
20
40
60
80
100
120
140
160
180
200
220
1 Phase2-PhasenT=const.=90°C
exp2 exp1
v 0 R
acem
atab
bau
(mm
ol m
g-1 h
-1)
Druck (MPa)
Pressure dependence of the enzyme activity at 60°C of 1-phenylethanol reaction.
Pressure dependence
Pressure [MPa]
Yie
ld r
acem
ate
conv
ersi
on m
mol
mg-1
h-1
Enantiomeric excess ee [%] of 1-phenylethanol () and ibuprofen () reaction respectively enantiomeric ratio E of ibuprofen ().
Enantiomeric excess
00:00 00:10 00:20 00:30 00:40
0
5
10
15
20
25
30
35
Substratmenge (mmol)
0,2
0,1
0,3
0,4
0,6
1,0
2,0
Um
satz
(%
)
Zeit (hh:mm)
Yield of the reaction of 1-phenylethanol with vinyl acetate in n-hexane in dependence on substrate quantity. Solvent: 4 ml
Reaction in n-hexane
-0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2
0
100
200
300
400
500
Km=0,279 mmol
rmax=413,6 mmol mg-1 h
-1
r 0 R
acem
atab
bau
(mm
ol m
g-1 h
-1)
Substratmenge nio (mmol)
Initial reaction rate in dependence on substrate quantity. Non linear fit to Michaelis-Menten eq.
Substrate quantity
Rea
cted
qua
ntity
Reaction in n-hexane
0 200 400 600 800 10000
50
100
150
200
250
300
350
400
450
linear: Km=0,30265 mmol r
max=428,6 mmol mg-1 h-1
Exp.Dec: Km=0,2963 mmol r
max=443,4 mmol mg-1 h-1
Eadie-Hofstee-Diagramm (Hexanreihe)
r0lin r0Exp Data2r0li Data2r0Ex
r 0 (m
mol
mg-1
h-1)
r0 / n
io (mg
-1 h
-1)
Eadie-Hofstee-diagram
Reaction in n-hexane
Rührkessel- autokla
Festbettreaktor
FestbettEnzym
Kreislaufpumpe
Substrate
CO
2
Reactor types
Stirred tank reactor
Fixed bed
tubular reactor
Cycle pump
Fixed bed
CO2
CO2
Substrate
40 50 60 70 80 90 1000
10
20
30
40
50
60
70
80 v0 (exp) K-Anlage v0 (lin) B-Anlage
v 0' R
acem
atab
bau
(mm
ol m
g-1 m
l-1 h
-1)
Temperatur (°C)
Reaction in n-hexane
Comparison of stirred tank and tubular fixed bed reactor
tubular fixed bed reactor
stirred tank reactor
el.
M
PI
PIPI
10
12
8
9 11
1 4
2 3
5
6
7
1: CO2-feed, 2: CO2-cooler, 3: CO2 -pump, 4: inlet valve, 5: outlet valve, 6: spindle press, 7: reactor valve, 8: reactor pressure gauge, 9: injection valve, 10: view cell, 11: sample valve, 12: stirrer
Flow scheme of batch apparatus
Ibuprofenveresterung: Umsatz-Zeit-Kurve
0
10
20
30
40
50
60
70
80
90
100
00:00 01:00 02:00 03:00 04:00 05:00 06:00
Reaktionszeit [hh:mm]
Um
satz
[%
]
Umsatz gesamt [%]
Umsatz R-Form
Umsatz S-Form
T=70°C, P=15 MPa
Yield Ibuprofen Esterification in sc-CO2
Time
Yie
ld
Ibuprofenveresterung: Umsatz-Enantiomerenreinheit
0
10
20
30
40
50
60
0 10 20 30 40 50 60 70 80
Umsatz U [%]
En
anti
om
eren
rein
hei
t ee
[%
]
Enantiomeric excess (ee) vs yield
Yield
Reaktionsgeschwindigkeit zu verschiedenen Reaktionszeiten
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
50 60 70 80 90 100 110 120
Temperatur [°C]
Rea
ktio
nsg
esch
win
dig
keit
[m
mo
l/h
/g]
P=15 MPa
r (t=0,5h)
r (t=1h)
r (t=2h)
r (t=3h)
Temperature dependence of reaction rate
Enantioselektivität und Reaktionsgeschwindigkeit
0
10
20
30
40
50
60
70
50 60 70 80 90 100 110 120
Temperatur T [°C]
En
anti
om
eren
üb
ersc
hu
ß e
e [%
]
Enantiomerenüberschuß ee
Enatiomerenindex E
Temperature dependence of enantioselectivity
Fischölethylester in 2-Propanol
0
10
20
30
40
50
60
70
00:00 01:00 02:00 03:00
Reaktionszeit [hh:mm]
rel.
Ant
eil [
%]
C20:5 (EPA)
C20:5*
DHA
DPA
DHA *
DPA *
Esterification of FAEE
Novozym 435in
2-propanol
Fischöl Glycerin in Hexan
0
10
20
30
40
50
60
70
80
00:00 01:00 02:00 03:00 04:00 05:00
Reaktionszeit [hh:mm]
rel.
Ant
eil [
%] DHA
DPA
22:1 w-11
Transesterification with glycerol in n-hexane
Enzyme: Lipase Novozym 435
Fischöl Glycerin in Hexan (Blindprobe ohne Enzym)
0
10
20
30
40
50
60
70
80
00:00 02:00 04:00 06:00
Reaktionszeit [hh:mm]
rel.
Ant
eil [
%]
C20:5 (EPA)
DHA
DPA
22:1 w-11
Transesterification with glycerol in n-hexane (no enzyme)