Enzyme Technology
Practical examples of the use of enzymes 'in reverse'
It has long been known that if proteases are supplied with high
concentrations of soluble proteins, peptides or amino acids, polymers
(plasteins) are produced with apparently random, if rather
hydrophobic, structures. This reaction has been used, for example, to produce
bland-tasting, colourless plasteins from brightly coloured, unpleasant tasting,
algal biomass and for the introduction of extra methionine into low quality soy
protein. However in general, the non-specific use of proteases in the synthesis
of new structures has not found commercial use. However, proteases have come
into use as alternatives to chemical methods for the synthesis of peptides of
known and predetermined structure because their specificity allows reactions to
proceed stereospecifically and without costly protection of side-chains.
A method
for the synthesis of the high intensity sweetener aspartame exemplifies the
power of proteases (in this case, thermolysin). Aspartame is the dipeptide of L-aspartic
acid with the methyl ester of L-phenylalanine (a-L-aspartyl-L-
phenylalanyl-O-methyl ester). The chemical synthesis of aspartame requires
protection of both the b-carboxyl group and the a-amino group of
the L-aspartic acid. Even then, it produces aspartame in low yield and at high
cost. If the b-carboxyl group is not protected, a cost saving is
achieved but about 30% of the b-isomer is formed and has, subsequently,
to be removed. When thermolysin is used to catalyse aspartame production the
regiospecificity of the enzyme eliminates the need to protect this
b-carboxyl group but the a-amino group must still be protected
(usually by means of reaction with benzyl chloroformate to form the
benzyloxycarbonyl derivative, i.e., BOC-L-aspartic acid) to prevent the synthesis
of poly-L-aspartic acid. More economical racemic amino acids can also be used as
only the desired isomer of aspartame will be formed.
If stoichiometric quantities
of BOC-L-aspartic acid and L-phenylalanine methyl ester are reacted in the
presence of thermolysin, an equilibrium reaction mixture is produced giving
relatively small yields of BOC-aspartame. However, if two equivalents of the
phenylalanine methyl ester are used, an insoluble addition complex forms in high
yield at concentrations above 1 M. The loss of product from the liquid phase due
to this precipitation greatly increases the overall yield of this process.
Later, the BOC-aspartame may be released from this adduct by simply altering the
pH. The stereospecificity of the thermolysin determines that only the L-isomer
of phenylalanine methyl ester reacts but the addition product is formed equally
well from both the D- and L-isomers. This fortuitous state of affairs allows the
use of racemic phenylalanine methyl ester, the L-isomer being converted to the
aspartame derivative and the D-isomer forming the insoluble complex shifting the
equilibrium to product formation. D-phenylalanine ethyl ester released from the
addition complex may be isomerised enzymically to reform the racemic mixture.
The BOC-aspartame may be deprotected by a simple hydrogenation process to form
aspartame.
[7.8]
Immobilised thermolysin cannot be used in this process
as it has been found to co-precipitate with the insoluble adduct. However, this
may be circumvented by its use within a liquid/liquid biphasic system.
Table 7.2 The specificity of
some specific industrial proteases, involving acyl intermediates.
a AA represents any amino acid
residue and Z represents amino acid residues, esters or amides. The cleavage
sites () are those preferred by the pure enzyme; crude preparations may have
much broader specificities.
The synthesis of aspartame is a very simple example of how
proteases may be used in peptide synthesis. Most proteases show specificity in
their cleavage sites (Table 7.2) and may be used to synthesise specific peptide
linkages. Factors that favour peptide synthesis are correct choice of pH, the
selection of protecting residues for amino and carboxyl groups that favour
product precipitation and the use of liquid/liquid biphasic systems, all of
which act by controlling the equilibrium of the reaction. An alternative
strategy is kinetically controlled synthesis where the rate of peptide product
synthesis (kP) is high compared with the rate of peptide hydrolysis
(kH). This may be ensured by providing an amino acid or peptide which
is a more powerful nucleophile than water in accepting a peptide unit from an
enzyme-peptide intermediate. This kinetically controlled reaction may be
represented as
[7.9]
where X represents an alcohol, amine or
other activating group, i.e., the reactant is an ester, amide (peptide) or
activated carboxylic acid. The relative rate of peptide formation compared with
hydrolysis depends on the ratio kP/kH, the ratio of the
Km values for water and amine, and the relative concentrations of the
(unprotonated) amine and water (see equation 1.91). Thus, where necessary, the
reaction yield may be improved by lowering the water activity. It has been found
that the yields may be increased by reducing the temperature to 4°C,
perhaps by a disproportionate effect on the Km values. The amine and
enzyme concentrations should be as high as possible for such kinetically
controlled reactions and only those enzymes which utilise a covalently-linked
enzyme-peptide intermediates can be used (see Table 7.2). Also, the reaction is
stopped well before equilibrium is reached as, under such thermodynamic control,
the product peptide will be converted back though the enzyme intermediate to the
carboxylic acid, a process made almost irreversible by its ionisation in
solutions of pH above its pKa, as shown in reaction scheme [7.10].
[7.10]
Peptides may be lengthened either by the
addition of single amino acid residues or by the condensation of peptide
fragments. The size of the peptide required as the final product may determine
the type of synthesis used; condensation of fragments may be performed in
kinetically controlled processes whereas stepwise elongation is best achieved
using biphasic solid/liquid (i.e., precipitation) or liquid/liquid
thermodynamically controlled processes.
In general, few proteases are required
for such synthetic purposes. As they are quite costly, especially at the high
activities necessary for kinetic control, they may be immobilised in order to
enable their repeated use. Many examples of enzymic peptide synthesis may be
sited. The conversion of porcine insulin to human insulin requires the
replacement of the C-terminal alanine (b30) residue by a
threonine. This can be achieved by a single transpeptidation step catalysed by
trypsin using an carboxyl-protected threonine in an aqueous solution with an
organic co-solvent. The protective group can be simply removed later by mild
hydrolysis and the product purified by silica gel chromatography (see scheme [7.11]).
[7.11]
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This page was established in 2004 and last updated by Martin
Chaplin on
10 February, 2017
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