product is thermodynamically more favorable. The advantage of the kinetic control
strategy is to require a ratio enzyme to substrate lower than required in the thermodynamic control strategy. The efficiency of the peptide synthesis under kinetic
control is also profoundly affected by the pH value and ionic strength. Therefore,
the second peptide that is involved in the nucleophilic attack should be uncharged,
and hence it is vital to choose the pH in the reaction environment according to the
pka of the nucleophile amine group. The addition of co-solvents can also help to
dissolve the activated amino acid or small peptide and simultaneously minimize the
reaction rate of its hydrolysis and the final product.
3.4.4 Bioinformatic Tools on In Silico Design of Bioactive
Peptides
Ideally, the on in silico process of peptide synthesis should reduce the time required
for selecting the best hydrolysis conditions for various native proteins and multiple
known proteases based in databases like BIOPEP [66] and UniProtKB [67]. The
Bioinformatics tools like BIOPEP “enzyme action”, ExPASy Peptide Cutter [68],
and PoPS [69] algorithm are excellent strategies for enzymatic peptide synthesis.
However, the design of peptides based on in silico proteolysis tools cannot guarantee experimental reproducibility considering the complex nature of proteinprotease interactions. Furthermore, as the bioinformatics approaches currently used
do not consider the possibility of the existence of other peptide sequences than
those available in the “in silico hydrolysates”, and the lack of database update and
some attractive synthesis opportunities might be missed [70].
4 Bioactive Peptides Isolation and Purification Methods
The behavior and bioactive properties of an individual peptide present in a protein
hydrolysate may be significantly different compared to when it is isolated due to
antagonistic and or synergistic effects when present in a mixture of peptides. After
hydrolysis, the target peptides need to be concentrated and purified, which simultaneously facilitates its posterior identification. Peptides have similar physical–
chemical properties, but the specific side chains of amino acids composing their
primary structure allow their separation according to charge, size, solubility, and
hydropathy, i.e., the difference in hydrophobicity versus hydrophilicity of the
compound [71]. Peptide purification is a pre-requirement independent of the
methods used in its production, whether for specific applications in the food or
pharmaceutical industry. Of course, different degrees of purity are required,
inclusively higher than 95% for conventional pharmaceutical applications [72].
Selective precipitation, membrane filtration, and different chromatographic processes have been commonly used for peptide fractionation enrichment. Agyei et al.
[17] reviewed the methods for the extraction and purification of peptides.
486
A. F. A. Salvado et al.
strategy is to require a ratio enzyme to substrate lower than required in the thermodynamic control strategy. The efficiency of the peptide synthesis under kinetic
control is also profoundly affected by the pH value and ionic strength. Therefore,
the second peptide that is involved in the nucleophilic attack should be uncharged,
and hence it is vital to choose the pH in the reaction environment according to the
pka of the nucleophile amine group. The addition of co-solvents can also help to
dissolve the activated amino acid or small peptide and simultaneously minimize the
reaction rate of its hydrolysis and the final product.
3.4.4 Bioinformatic Tools on In Silico Design of Bioactive
Peptides
Ideally, the on in silico process of peptide synthesis should reduce the time required
for selecting the best hydrolysis conditions for various native proteins and multiple
known proteases based in databases like BIOPEP [66] and UniProtKB [67]. The
Bioinformatics tools like BIOPEP “enzyme action”, ExPASy Peptide Cutter [68],
and PoPS [69] algorithm are excellent strategies for enzymatic peptide synthesis.
However, the design of peptides based on in silico proteolysis tools cannot guarantee experimental reproducibility considering the complex nature of proteinprotease interactions. Furthermore, as the bioinformatics approaches currently used
do not consider the possibility of the existence of other peptide sequences than
those available in the “in silico hydrolysates”, and the lack of database update and
some attractive synthesis opportunities might be missed [70].
4 Bioactive Peptides Isolation and Purification Methods
The behavior and bioactive properties of an individual peptide present in a protein
hydrolysate may be significantly different compared to when it is isolated due to
antagonistic and or synergistic effects when present in a mixture of peptides. After
hydrolysis, the target peptides need to be concentrated and purified, which simultaneously facilitates its posterior identification. Peptides have similar physical–
chemical properties, but the specific side chains of amino acids composing their
primary structure allow their separation according to charge, size, solubility, and
hydropathy, i.e., the difference in hydrophobicity versus hydrophilicity of the
compound [71]. Peptide purification is a pre-requirement independent of the
methods used in its production, whether for specific applications in the food or
pharmaceutical industry. Of course, different degrees of purity are required,
inclusively higher than 95% for conventional pharmaceutical applications [72].
Selective precipitation, membrane filtration, and different chromatographic processes have been commonly used for peptide fractionation enrichment. Agyei et al.
[17] reviewed the methods for the extraction and purification of peptides.
486
A. F. A. Salvado et al.