Enzymatic Synthesis of Aspartame

Aspartame is a low-calorie sweetener whose apparent sweetness is

150- 200 times that of sucrose. It is prepared by condensation of Laspartic acid and the methyl ester of L-phenylalanine (two amino
acids).
Its sweet taste depends on:
L-conformation of the two constituent amino acids
presence of the methyl ester
correct coupling of the amino acids.

H2N

NH

NH
H
O

Ph

Ph

CO2H

CO2Me

H2N

H
H

CO2H

CO2Me

-L-aspartyl-L-phenylalanine methyl ester -L-aspartyl-L-phenylalanine methyl ester

-aspartame (APM)]

Sweet

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Bitter

J.S. Parent

Industrial Enzymatic Synthesis of Aspartame

The unique regio and stereoselectivity afforded by enzymes has been
exploited on an industrial scale Aspartame production.
The process employs a protease,
thermolysin, to catalyze the
condensation of the modified Asp
and Phe).
The forward reaction is written as:
CO2H
X

N
H

CO2H

Amine-protected (X)
L-aspartic acid
(Z-L-Asp)

H2N

NH
H
O

H2N

CO2Me

Methyl ester of
L-phenylanaline
(L-PM)

CO2Me

-L-aspartyl-L-phenylanaline methyl ester

-aspartame (APM)]

Ph

Ph

CO2H

thermolysin

CO2H
X

N
HH

NH
H
O
(APM)

Ph

+
CO2Me

Note however, that the synthesis reaction is equilibrium limited by the

reverse (hydrolysis) reaction for which proteases are known.
Furthermore, the equilibrium strongly favours hydrolysis.

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OH2

Structural Properties of Thermolysin

Thermolysin is a metalloenzyme
(316 amino acids) requiring a zinc
ion and four calcium ions to maintain
an active tertiary structure.
Two distinct hemispheres exist with
a zinc atom located at the bottom of
the cleft. Three residues (142, 146
and 166) serve as ligands for zinc.
Calcium is a structural element, and
is not believed to interact with the
substrate at the active site.
Open circles: -carbon positions
Stippled circle: zinc with its three protein
ligands as broken lines
Solid circles: four calcium atoms

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Chemical Properties of Thermolysin

Thermolysin is an extracellular enzyme produced by a bacterial
strain that can withstand high temperatures. Hence, themolysin has
a temperature stability that is superiour to most enzymes.
Thermolysin is classified as a protease, in that it catalyzes the
cleavage of the peptide bonds that constitute proteins.
The term endopeptidase applies, as the internal bonds in
polypeptides are susceptible to the action of thermolysin
The term neutral protease applies, as the pH optimum lies
about pH 7.5
The term metalloenzyme is appropriate, given the necessity
of zinc at the active site and the requirement for calcium to
maintain an active tertiary structure. Chelating agents
deactivate thermolysin.
Enzymes of this class demonstrate substrate specificity which
requires a hydrophobic amino acid such as phenylalanine as the
residue whose amido group is cleaved.

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Kinetics of the Aspartame Synthesis

The rate of APM production is firstorder with respect to the total
concentration of enzyme [Eo], and a
bell-shaped pH-rate profile with the
highest activity at pH 7.5 is observed.
Shown is a typical time course of the

thermolysin catalysed condensation of

N-benzyloxycarbonylaspartic acid with
phenylalanine methyl ester. Initial rate
measurements (from t=0 to t=10 min)
as a function of reagent concentrations
define the overall reaction kinetics.
[Z-L-Asp] = 1.82 x 10-2 M
[L-PM] =
3.64 x 10-2 M
[Eo] =
4.85 x I0-6 M
pH = 6.5; 0.364 M
T = 40C

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Influence of [PM] on the Condensation Rate

APM synthesis is first-order WRT
phenylalananine methyl ester,
with no apparent saturation
behaviour that is common in
enzyme-mediated reactions.
Note that the presence of D-PM
has no effect on the reaction
rate, and it is not found in the
product.
* [L-PM] =
[D-PM] =

1.82x10-2 M with
9.09x10-3 M

** [L-PM ] =
[D-PM] =

3.64 x10-2 M with

1.82 x10-2 M

L-PM

D,L-PM

[Z-L-Asp] = 1.82 x 10-2 M

[Eo] =
4.85 x I0-6 M
pH 6.5; 0.364 M; 40C

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J.S. Parent

Influence of [Z-L-Asp] on the Condensation Rate

A plot of [Z-L-Asp] against the
APM production rate shows
saturation of the rate, typical
Michaelis-Menten behaviour.
Rate retardation occurs in the
presence of Z-D-Asp,
indicating that the enantiomer
acts as a competitive inhibitor.
Hence only pure L-Asp can be
used in APM synthesis, while
racemic mixtures of D,L-PM
can be accommodated.

Pure Z-L-Asp

9.1x10-3 M Z-D-Asp added

[L-PM] = 3.64 x 10-2 M

[Eo] =
4.85 x I0-6 M
pH 6.5; 0.364 M; 40C

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J.S. Parent

Proposed Reaction Mechanism

Competitive inhibitors reduce the rate of product formation through
binding the enzyme in an inactive form.
Often these inhibitors are structurally similar to the substrate,
and therefore are capable of binding at the active site
Enzyme-bound inhibitor either lacks a needed functional group
or is held in an unsuitable position for reaction.
We have seen an example of this behaviour in aspartame production,
where the enantiomer of L-Asp inhibited the reaction. A plausible
mechanism for this inhibition is shown below:

+ Z-L-Asp

k1

Z-L-Asp k-1

Z-L-Asp*E

+ L-PM
k2 r.d.s.

Note thatZ-D-Asp
Z-D-Asp
+ Z-D-Asp binds thermolysin in an

k3 thereby reducing the active

k-3inactive state,
enzyme concentration and lowering the
reaction rate.
Z-D-Asp*E

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Z-APM

Competitive Inhibition by Z-D-Asp

From this proposed mechanism we can derive a rate expression
that accounts for competitive inhibition.
r1:

Z-L-Asp

r3:
r2:

Z-D-Asp

Z-L-Asp*E

E
L-PM

k1

k-1
k3
k-3
k2
r.d.s.

Z-L-Asp*E

Z-D-Asp*E
ZAPM

Assigning r2 as the rate determining step of the process, we find

the reaction velocity is:

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k 2 [E]T [ ZLAsp][LPM]

[ ZDAsp]
[ ZLAsp]
K1 1
K3

J.S. Parent

Validating the Proposed Reaction Scheme

Although more sophisticated regression techniques are available,
the simplest means of testing the model is to linearize the rate
expression
k 2 [E]T [ ZLAsp][LPM]
r

[ ZDAsp]
[ ZLAsp]
K1 1
K3

by inverting it:

[ ZDAsp]

K 1 1
K3
1
1
1

r
k 2 [E] T [LPM] [ ZLAsp] k 2 [E] T [LPM]
A plot of 1/rate versus 1/[Z-L-Asp] should be linear, with a slope of
K1(1+[Z-DAsp]/K3)/(k2[E]T[L-PM]) and an intercept 1/(k 2[E]T[L-PM])
This is commonly referred to as a Lineweaver-Burk plot
It is necessary that the data fit the rate expression, but it is not
sufficient proof that the mechanism is correct
From the slope, intercept, [E] T and [L-PM], numerical estimates
of K1 and k2 can be derived.

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Lineweaver-Burk Plot of the Kinetic Data

Plotting the inverse of the APM production
rate (moleL-1s-1) against 1/[Z-L-Asp]
reveals a linear relationship

[ ZDAsp]

K 1 1
K3
1
1
1

r
k 2 [E] T [LPM] [ ZLAsp] k 2 [E] T [LPM]

The proposed mechanism is consistent

with the kinetic data, and may be correct.
From the slopes and intercepts,
k2 = 2.65 L mole-1 s-1
K1 = 1.03x10-2 mole L-1
K3 = 2.35x10-2 mole L-1
Line A: no Z-D-Asp;
Line B: [Z-D-Asp]=9.09x10-3 M
[L-PM] = 1.82 x 10-2 M
[Eo] =
4.85 x 10-6 M
pH 6.5; 0.364 M; 40C

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Isolation of the Aspartame Product

Proteases are recognized as catalysts for peptide bond cleavage, and
using them to catalyze the reverse condensation reaction can be
problematic.
CO2H
X

N
H

CO2H

Amine-protected (X)
L-aspartic acid
(Z-L-Asp)

Ph

H2N

CO2Me

Methyl ester of
L-phenylalanine
(L-PM)

thermolysin

CO2H
X

N
HH

NH
H
O
(APM)

Ph

+
CO2Me

a APMaH2O
The equilibrium constant derived from K

APM
aLAspaLPM
the Gibbs energies of the reaction
components is quite small, making the
[ APM] APM [H2O] H2O
conversion of a standard batch reaction

[LAsp] LAsp [LPM] LPM

equilibrium limited.

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Isolation of the Aspartame Product

Luckily APM forms, via its free side-chain carboxylic acid, a
sparingly soluble addition compound with excess PM.
The synthesis can be driven using LeChataliers Principle by
removal of the precipitation of the product.
Once isolated from the enzyme, hydrolysis of Z-APM is no
longer a concern, excess PM can be removed and the
product can be deprotected to yield aspartame.
Ph
H2N

CO2H
X

N
H

CO2H

Amine-protected (X)
L-aspartic acid

CO2H
H

CO2Me

Methyl ester of
L-phenylalanine

thermolysin

N
HH

NH
H
O

H2N

J.S. Parent

CO2Me

Ph

Ph
H CO2Me
Methyl ester of
D-phenylalanine

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Ph

H2N

CO2Me

L-L dipeptide deposits

as an addition compd.

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Effects of Immobilization on Enzyme Stability and Use

Design of enzymatic processes requires knowledge of:
reactant and product selectivity
thermodynamic equilibria that may limit product yield
reaction rate as a function of process conditions ([Enzyme],
[substrate(s)], [Inhibitors], temperature, pH, )
Two design issues that we have not considered are:
enzyme stability
efficiency losses associated with the use of homogeneous
(soluble) catalysts
Immobilization of an enzyme allows
it to be retained in a continuous reactor,
but its initial activity and its stability
directly influence its usefulness
in industrial applications.

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Enzyme Stability
Although enzyme storage stability is important, it is the operational
stability of an enzyme that governs its reactor performance.
Operation stability is a complex function of temperature, pH,
[substrate] and the presence of destabilizing agents.
Generally, the rate of free enzyme deactivation is first order with a
deactivation constant, kd:
6.0

d[E]T
k d [E]T
dt
Integrating this expression
yields the concentration of
active enzyme as a function
of time:

[E]T [E]T,o e

k dt

[Enzyme] *1E6 M

5.0
No decay

4.0

kd = 6E-6 s-1

3.0

kd = 3E-5 s-1

2.0
1.0
0.0
0

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20

40

60

Time (hours)

15

80

100

Effect of Thermolysin Instability on APM Production

Recall the rate expression developed for APM synthesis by
thermolysin:

d[ ZAPM] k 2 [E]T [ ZLAsp][LPM]

dt
K1 [ ZLAsp]
If thermolysin deactivation were adequately described as a first
order process, the observed reaction rate would have an explicit
time dependence, as shown below:

d[ ZAPM] k 2 [ ZLAsp][LPM]

[E]T,o e k dt
dt
K1 [ ZLAsp]
where [E]T,o represents the initial enzyme concentration and k d is the
deactivation rate constant.
The conversion versus time profile for aspartame synthesis by a
batch process can be developed from this expression by integration.

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Effect of Thermolysin Instability on APM Production

The evolution of [L-Asp] and conversion with time for a batch
process is shown below.
Depending on the relative rates of reaction and enzyme
deactivation, the ultimate conversion can be strongly affected
APM Synthesis by Thermolysin

APM Synthesis by Thermolysin

Batch Process at 40C

0.020
0.018

kd = 0 s-1

kd = 3E-5 s-1

0.014
0.012
0.010

kd = 6E-6 s-1

0.008
0.006

kd = 0 s-1

0.004

L-Asp Conversion

0.80

0.016
[L-Asp]: M

Batch Process at 40C

0.90
0.70
0.50
0.40
0.30
0.10

0.000

0.00
20

40
60
Time (hours)

80

[LPM]o 0.0182 M
[LAsp]o 0.0182 M
k2
2.65 M-1s-1 K1
0.0103 M-1s-1
[E]o
4.85E-06 M

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J.S. Parent

kd = 3E-5 s-1

0.20

0.002
0

kd = 6E-6 s-1

0.60

20

40
60
Time (hours)

80

[LPM]o 0.0182 M
[LAsp]o 0.0182 M
k2
2.65 M-1s-1 K1
0.0103 M-1s-1
[E]o
4.85E-06 M

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100

Effect of Immobilization on Operational Stability

Given that activity of enzymes is dictated by structure and
conformation, the environmental change resulting from immobilization
affects not only maximum activity, but the stability of the enzyme
preparation.
The factors that inactivate enzymes are not systematically
understood, and depend on the intrinsic nature of the enzyme,
the method of immobilization, and the reaction conditions
employed.
In general, immobilized enzyme preparations demonstrate better
stability.
Note that the immobilized
preparation is often more
stable than the soluble
enzyme and displays a
period during which no
enzyme activity appears to
be lost.

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immobilized
enzymes
free (soluble)
enzymes

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Classification of Immobilization Methods for Enzymes

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Immobilization by Entrapment
Gel entrapment places the enzyme within the interstitial
spaces of crosslinked, water-insoluble polymer gels.
Polyacrylamide gels:

Polysaccharides: The solubility of alginate and -Carrageenan varies with

the cation, allowing these soluble polymers to be crosslinked upon the
addition of CaCl2 and KCl, respectively.
Variations of pore size result in enzyme leakage, even after washing. The
effect of initiator used in polyacrylamide gels can be problematic.

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Immobilization by Entrapment
Microencapsulation encloses enzymes within spherical,
semi-permeable membranes of 1-100 m diameter.
Urethane prepolymers, when mixed with an aqueous
enzyme solution crosslink via urea bonds to generate membranes of
varying hydrophilicity.
Alternatively, photocrosslinkable resins
can be gelled by
UV-irradiation.

Advantage of Entrapment
Enzymes are immobilized without a chemical or structural
modification. A very general technique.
Disadvantage of Entrapment
High molecular weight substrates have limited diffusivity, and
cannot be treated with entrapped enzymes.

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Immobilization by Carrier Binding

Attachment of an enzyme to an insoluble carrier creates an active
surface catalyst. Modes of surface attachment classify carrier
methods into physical adsorption, ionic binding and covalent
binding.
Physical Adsorption: Enzymes can be bound to carriers
by physical interaction such as hydrogen bonding and/or
van der Waals forces.
the enzyme structure is unmodified
carriers include chitosan, acrylamide polymers
and silica-alumina
binding strength is usually weak and affected by temperature
and the concentration of reactants.
Ionic Binding: Stronger enzyme-carrier binding is obtained with
solid supports containing ion-exchange residues.
cellulose, glass-fibre paper, polystyrene sulfonate
pH and ionic strength effects can be significant

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Immobilization by Carrier Binding

Covalent attachment of soluble enzymes to an insoluble support is
the most common immobilization technique.
Amino acid residues not involved in the active site can be
used fix the enzyme to a solid carrier
Advantages:
1. Minimal enzyme leaching from the support results
in stable productivity
2. Surface placement permits enzyme contact with
large substrates
Disadvantages:
1. Partial modification of residues that constitute the active site
decreases activity
2. Immobilization conditions can be difficult to optimize (often done
in the presence of a competitive inhibitor)

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Most Convenient Residues for Covalent Binding

Amino acid residues with
polar and reactive functional
groups are best for covalent
binding, given that they are
most often found on the
surface of the enzyme.
Shown are the most
convenient residues in
descending order. The
average percent
composition of proteins
(reactive residues only) is
shown, along with the
number of potential binding
reactions in which the
amino acids partake.

Abundance(%)Reactions
CH2

+
NH3

CH2 SH
CH2

OH

HN

CH2
O
CH2 C O

7.0

27

Cysteine (Cys)

3.4

31

Tyrosine (Tyr)

3.4

16

Histidine (His)

2.2

13

Aspartic Acid (Asp) 4.8

Glutamic Acid (Glu) 4.8

3.8

1.2

O
CH2 CH2 C O
CH2

CH2

NH C NH2 Arginine (Arg)

+NH2
N
H

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Lysine (Lys)

J.S. Parent

Tryptophan (Trp)

24

Covalent Attachment Techniques

Cyanogen bromide activates supports with vicinal hydroxyl groups
(polysaccharides, glass beads) to yield reactive imidocarbonate
derivatives:

Diazonium derivatives of supports having aromatic amino groups are

activated for enzyme immobilization:

Under the action of condensing agents (Woodwards reagent K),

carboxyl or amino groups of supports and amino acid residues can be
condensed to yield peptide linkages.
Other methods include diazo coupling, alkylation, etc.

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Immobilization by Crosslinking
Bi- or multi-functional compounds serve as reagents for
intermolecular crosslinking of enzymes, creating insoluble
aggregates that are effective heterogeneous catalysts.
Reagents commonly have two identical functional groups
which react with specific amino acid residues.
Common reagents include glutaraldehyde,

and diisocyanates,

Involvement of the active site in crosslinking can lead to great

reductions in activity, and the gelatinous nature of the product can
complicate processing.

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Effects of Enzyme Immobilization on Activity

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Selecting an Immobilization Technique

It is well recognized that no one method can be regarded as the
universal method for all applications or all enzymes. Consider,
widely different chemical characteristics of enzymes
different properties of substrates and products
range of potential processes employed

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