disadvantage of the genetic engineering approach regards the biosynthesis of
peptides integrating unnatural amino acids. These unnatural amino acids are
important functional groups currently incorporated into some drugs and pharmacologic compounds.
3.4.3 Enzymatic Synthesis
Special attention was also given to the rational synthesis of bioactive peptides via
enzymatic process due to the high stereospecificity associated with enzymes, the
softness of reaction conditions, the minimal need of protection of the more sensitive
side-chain amino acids, and avoid the racemization of the final peptide in comparison with the chemical synthesis. Like chemical synthesis, enzymatic synthesis
of peptides can occur by extension of N- or C-terminal. However, enzymatic
synthesis is limited to the synthesis of peptide that not exceed peptide chains with
more than 10 amino acids [63].
The most commonly used proteolytic enzymes in this strategy of rational synthesis of peptides are proteases of the serine or cysteine group such as subtilisin,
trypsin, chymotrypsin, or papain. However, other hydrolases were also tested, such
as lipases and penicillin acylase [64]. Typically, enzymatic synthesis of peptides
catalyzes the formation of peptide bonds between amino acids, dipeptides, or small
peptide fragments. The first one functions as a donor of an acyl group (e.g., carboxyl) then localized in the part of the N-terminal. In turn, the second one works as
an amine group donor associated with the C-terminal of the peptide sequence.
Enzyme synthesis of peptides can occur using one of two distinct reaction strategies, i.e., enzyme synthesis under thermodynamic control or kinetic control [65].
The final product yield under thermodynamic control can be increased: (i) using
an excess of one of the reagents; (ii) removing one of the final products; (iii) adding
an organic solvent to alter the pka of the amine and carboxyl groups; (iv) using a
biphasic reaction system, and, more recently; (v) using ionic liquids or anhydrous
reaction medium containing a minimum concentration of water.
In the case of peptide synthesis under thermodynamic control occurs in an
aqueous reaction medium, then these conditions must favor the removal of the final
product (e.g., precipitation) with the consequent displacement of thermodynamic
equilibrium. Another strategy is the use of organic solvents that alter the dielectric
constant of the reaction medium by lowering the acidity of the carboxyl group and
reducing the alkaline strength of the amine group. This effect induces an increased
concentration of species with uncharged groups that are more reactive to be
involved in the formation of peptide bonds. On the other hand, the use of organic
solvents to alter the thermodynamic equilibrium affects the catalytic and stability of
the protease negatively. The main disadvantages of this specific strategy are the
requirement of a large amount of enzyme and low reaction rates.
In the strategy of enzymatic synthesis of peptides under kinetic control, one
activated (e.g., ester) amino acid or a small peptide binds with a second amino acid
or small peptide. In this reaction strategy, the synthesis velocity (S) is much higher
than the hydrolysis velocity (H), and the S/H ratio should be as high as possible.
The activated amino acid or small peptide is used since its conversion into the final
Enzymatic Production of Bioactive Peptides from Whey Proteins …
485
peptides integrating unnatural amino acids. These unnatural amino acids are
important functional groups currently incorporated into some drugs and pharmacologic compounds.
3.4.3 Enzymatic Synthesis
Special attention was also given to the rational synthesis of bioactive peptides via
enzymatic process due to the high stereospecificity associated with enzymes, the
softness of reaction conditions, the minimal need of protection of the more sensitive
side-chain amino acids, and avoid the racemization of the final peptide in comparison with the chemical synthesis. Like chemical synthesis, enzymatic synthesis
of peptides can occur by extension of N- or C-terminal. However, enzymatic
synthesis is limited to the synthesis of peptide that not exceed peptide chains with
more than 10 amino acids [63].
The most commonly used proteolytic enzymes in this strategy of rational synthesis of peptides are proteases of the serine or cysteine group such as subtilisin,
trypsin, chymotrypsin, or papain. However, other hydrolases were also tested, such
as lipases and penicillin acylase [64]. Typically, enzymatic synthesis of peptides
catalyzes the formation of peptide bonds between amino acids, dipeptides, or small
peptide fragments. The first one functions as a donor of an acyl group (e.g., carboxyl) then localized in the part of the N-terminal. In turn, the second one works as
an amine group donor associated with the C-terminal of the peptide sequence.
Enzyme synthesis of peptides can occur using one of two distinct reaction strategies, i.e., enzyme synthesis under thermodynamic control or kinetic control [65].
The final product yield under thermodynamic control can be increased: (i) using
an excess of one of the reagents; (ii) removing one of the final products; (iii) adding
an organic solvent to alter the pka of the amine and carboxyl groups; (iv) using a
biphasic reaction system, and, more recently; (v) using ionic liquids or anhydrous
reaction medium containing a minimum concentration of water.
In the case of peptide synthesis under thermodynamic control occurs in an
aqueous reaction medium, then these conditions must favor the removal of the final
product (e.g., precipitation) with the consequent displacement of thermodynamic
equilibrium. Another strategy is the use of organic solvents that alter the dielectric
constant of the reaction medium by lowering the acidity of the carboxyl group and
reducing the alkaline strength of the amine group. This effect induces an increased
concentration of species with uncharged groups that are more reactive to be
involved in the formation of peptide bonds. On the other hand, the use of organic
solvents to alter the thermodynamic equilibrium affects the catalytic and stability of
the protease negatively. The main disadvantages of this specific strategy are the
requirement of a large amount of enzyme and low reaction rates.
In the strategy of enzymatic synthesis of peptides under kinetic control, one
activated (e.g., ester) amino acid or a small peptide binds with a second amino acid
or small peptide. In this reaction strategy, the synthesis velocity (S) is much higher
than the hydrolysis velocity (H), and the S/H ratio should be as high as possible.
The activated amino acid or small peptide is used since its conversion into the final
Enzymatic Production of Bioactive Peptides from Whey Proteins …
485