separation by enzymatically catalyzed reactions

50
SEPARATION BY ENZYMATICALLY CATALYZED REACTIONS Chapter 10

Upload: palmer

Post on 14-Jan-2016

61 views

Category:

Documents


0 download

DESCRIPTION

Chapter 10. SEPARATION BY ENZYMATICALLY CATALYZED REACTIONS. Reactive separation. Carbon dioxide + r-1-Phenylethylacetate. r,s-1-Phenylethanol. s-1-Phenylethanol. r-1-Phenylethylacetate. Catalyst: Novozym 435 (Lipase). Vinylacetate. Carbon dioxide, supercritical. s-1-Phenylethanol. - PowerPoint PPT Presentation

TRANSCRIPT

Page 1: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

SEPARATION BY ENZYMATICALLY

CATALYZED REACTIONS

Chapter 10

Page 2: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 3: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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.

Page 4: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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.

Page 5: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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).

Page 6: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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.

Page 7: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 8: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 9: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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.

Page 10: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 11: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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.

Page 12: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 13: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

0

20

40

60

80

100

120

0 50 100 150

Yield [%]

1000

100

10ee

Change of enantiomeric excess with product formation.

Enantioselectivity

E =

Page 14: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 15: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 16: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 17: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

be

rsch

[%

]

x

0,0

0,1

0,5

1,0

5,0

K

Page 18: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

Separation by enymatically catalyzed reactions:

Examples and Experiments

Page 19: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 20: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

(±) 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

Page 21: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 22: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 23: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

Enantiomer separation by chiral Gas chromatography

Competitive Process: Chromatography

COOC2H5

CH3

H3C

CH3H

Educt Product

COOC2H5

CH3

H3C

HH3C

Page 24: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 25: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 26: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 27: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 28: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 29: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 30: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 31: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 32: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

Structure of the enzyme Lipase Arctica candida

Page 33: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 34: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

1-Phenylethanol -

vinylacetate - CO2

Novozym 435

Initial substrate concentration: 0,5 M

Reference: 1g immob. Enzyme, 100 oC

Effect of temperature on reaction rate

Page 35: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 36: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 37: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

Enantiomeric excess ee [%] of 1-phenylethanol () and ibuprofen () reaction respectively enantiomeric ratio E of ibuprofen ().

Enantiomeric excess

Page 38: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 39: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

-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

Page 40: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 41: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

Rührkessel- autokla

Festbettreaktor

FestbettEnzym

Kreislaufpumpe

Substrate

CO

2

Reactor types

Stirred tank reactor

Fixed bed

tubular reactor

Cycle pump

Fixed bed

CO2

CO2

Substrate

Page 42: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 43: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 44: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 45: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 46: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 47: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 48: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 49: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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

Page 50: SEPARATION BY ENZYMATICALLY  CATALYZED REACTIONS

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)