**5. Heterologous production of bioactive angiotensin I-converting enzyme inhibitory (ACEI) peptides**

In the broad range of known bioactive peptides, angiotensin I-converting enzyme inhibitory (ACEI) peptides derived from food proteins have attracted particular attention and have been studied the most comprehensively for their ability to prevent hypertension [95]. In this chapter we will further focus on the possibility to genetically engineer crop plants to produce and deliver antihypertensive ACEI peptides, therefore creating alternative sources to fight hypertension and prevent cardiovascular disease.

#### **5.1 Cardiovascular disease and the renin-angiotensin system**

Cardiovascular disease (CVD) has been recognized as the leading cause of death in developed countries. Hypertension or high blood pressure is one of the major independent risk factors for CVD [96]. States of CVD include conditions such as coronary heart disease, peripheral artery disease, and stroke. Hypertension is a condition defined by a blood pressure measurement of 140/90 mmHg or above and is thought to affect up to 30% of the worldwide adult population [95]. The kininnitric oxide (KNO) system and the renin-angiotensin system (RAS), **Figure 1**, play a crucial role in the control of hypertension by the action of angiotensin I-converting key enzyme (EC 3.4.15.1; ACE) [96–99].

Several synthetic ACE inhibitors such as captopril, enalapril, and lisinopril have been prescribed for the treatment of hypertension, congestive heart failure, and diabetic neuropathy [100]. However, their consumption is associated with various side effects including cough, skin rashes, hypotension, loss of taste, angioedema, reduced renal function, and fetal abnormalities [95]. The side effects associated to synthetic ACE inhibitors and the high prevalence of hypertension have led scientists to search for natural and safer therapies. Interestingly, the study of ACEI peptides has revealed that they do not have significant effects on blood pressure in normotensive subjects, suggesting a convenient mechanism that avoids acute hypotensive effects. Based on this finding, it is hypothesized that ACEI peptides could be used in initial treatment of mildly hypertensive individuals or even as supplemental treatments [101].

#### **5.2 Antihypertensive ACEI peptides**

So far, several ACEI peptides have been identified in food proteins, mainly in milk, eggs, and plants, currently constituting the most well-known class of bioactive peptides [102–104]. These peptides are inactive within the sequence of parent proteins, but they can be released by enzymatic proteolysis in vivo or

**61**

production.

**Figure 1.**

production of ACEI peptides.

*adapted from Erdmann et al. [96]).*

*Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants…*

in vitro, for example, during gastrointestinal digestion or during food processing. A common feature shared by the majority of ACEI peptides is the generally short sequence, i.e., 2–12 amino acids in length. However, some larger inhibitory sequences have been identified in milk fermented with *Enterococcus faecalis* [105] and *Lactobacillus casei* Shirota [106], in koumiss [107], tuna [108], bonito [109], and rotifer [110]. Studies have also indicated that binding to ACE is strongly influenced by the substrate's C-terminal tripeptide sequence. Hydrophobic amino acid residues with aromatic or branched side chains at each of the C-terminal tripeptide positions are common features among potent inhibitors. The presence of hydrophobic Pro residues at one or more positions in the C-terminal tripeptide region seems to positively influence a peptide's ACE-inhibitory activity [95]. In general, the peptides showing higher activity against ACE have Tyr, Phe, Trp, or Pro at their C-terminus [95]. The peptides TQVY from rice [111], MRW from spinach [112], and YKYY from wakame [113] are some examples of this principle. **Table 1** reviews some examples of ACEI activities of plant origin, whose peptides responsible for such activity may be potential sources for the heterologous

*The kinin-nitric oxide (KNO) system and the renin-angiotensin system (RAS). The left side (KNO system) shows the mechanism of the action of ACEI on ACE that cleaves bradykinin, a nonapeptide acting as vasodilatory hormone, and causes the formation of an inactive heptapeptide. In the right side (RAS system), the inhibition of ACE activity plays an important physiological role in regulation of blood pressure by inhibiting the conversion of the hormone angiotensin I to angiotensin II, a potent vasoconstrictor (figure* 

The most common method to produce and identify ACEI peptides is through enzymatic hydrolysis of food proteins with gastrointestinal enzymes such as pepsin and trypsin or with commercial proteases such as Alcalase™ [127]. ACEI peptides have also been produced with *Lactobacillus, Lactococcus lactis*, and *E. faecalis* strains during milk fermentation [105, 106]. Nevertheless, there are problems associated to this type of industrial production of ACEI peptides, including the difficulty to isolate the peptide of interest from the complex mixture of compounds produced by enzymatic hydrolysis, the high cost, low recovery, and the low bioavailability. These disadvantages denote the need to develop new and alternative approaches for their

*DOI: http://dx.doi.org/10.5772/intechopen.84419*

*Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants… DOI: http://dx.doi.org/10.5772/intechopen.84419*

#### **Figure 1.**

*Genetic Engineering - A Glimpse of Techniques and Applications*

trations conducting to higher protein volumes [7].

**enzyme inhibitory (ACEI) peptides**

key enzyme (EC 3.4.15.1; ACE) [96–99].

**5.2 Antihypertensive ACEI peptides**

cardiovascular disease.

easily distribute medicines to those in need. Furthermore, the optimization of plant's expression level can also ease downstream processing, with higher protein concen-

Finally, several purification strategies have been investigated to separate target transgenic proteins from host plant proteins, which are tailored for each individual protein based on its solubility, size, pI, charge, hydrophobicity, or affinity to specific ligands, and the parallel characteristics of plant host proteins. Chromatographic methods, such as affinity chromatography, have been the most extensively used. However, recently increasing attention is being paid to nonchromatographic methods to provide alternatives for large-scale production [22].

**5. Heterologous production of bioactive angiotensin I-converting** 

**5.1 Cardiovascular disease and the renin-angiotensin system**

In the broad range of known bioactive peptides, angiotensin I-converting enzyme inhibitory (ACEI) peptides derived from food proteins have attracted particular attention and have been studied the most comprehensively for their ability to prevent hypertension [95]. In this chapter we will further focus on the possibility to genetically engineer crop plants to produce and deliver antihypertensive ACEI peptides, therefore creating alternative sources to fight hypertension and prevent

Cardiovascular disease (CVD) has been recognized as the leading cause of death in developed countries. Hypertension or high blood pressure is one of the major independent risk factors for CVD [96]. States of CVD include conditions such as coronary heart disease, peripheral artery disease, and stroke. Hypertension is a condition defined by a blood pressure measurement of 140/90 mmHg or above and is thought to affect up to 30% of the worldwide adult population [95]. The kininnitric oxide (KNO) system and the renin-angiotensin system (RAS), **Figure 1**, play a crucial role in the control of hypertension by the action of angiotensin I-converting

Several synthetic ACE inhibitors such as captopril, enalapril, and lisinopril have been prescribed for the treatment of hypertension, congestive heart failure, and diabetic neuropathy [100]. However, their consumption is associated with various side effects including cough, skin rashes, hypotension, loss of taste, angioedema, reduced renal function, and fetal abnormalities [95]. The side effects associated to synthetic ACE inhibitors and the high prevalence of hypertension have led scientists to search for natural and safer therapies. Interestingly, the study of ACEI peptides has revealed that they do not have significant effects on blood pressure in normotensive subjects, suggesting a convenient mechanism that avoids acute hypotensive effects. Based on this finding, it is hypothesized that ACEI peptides could be used in initial treatment of mildly hypertensive individuals or even as supplemental

So far, several ACEI peptides have been identified in food proteins, mainly in milk, eggs, and plants, currently constituting the most well-known class of bioactive peptides [102–104]. These peptides are inactive within the sequence of parent proteins, but they can be released by enzymatic proteolysis in vivo or

**60**

treatments [101].

*The kinin-nitric oxide (KNO) system and the renin-angiotensin system (RAS). The left side (KNO system) shows the mechanism of the action of ACEI on ACE that cleaves bradykinin, a nonapeptide acting as vasodilatory hormone, and causes the formation of an inactive heptapeptide. In the right side (RAS system), the inhibition of ACE activity plays an important physiological role in regulation of blood pressure by inhibiting the conversion of the hormone angiotensin I to angiotensin II, a potent vasoconstrictor (figure adapted from Erdmann et al. [96]).*

in vitro, for example, during gastrointestinal digestion or during food processing. A common feature shared by the majority of ACEI peptides is the generally short sequence, i.e., 2–12 amino acids in length. However, some larger inhibitory sequences have been identified in milk fermented with *Enterococcus faecalis* [105] and *Lactobacillus casei* Shirota [106], in koumiss [107], tuna [108], bonito [109], and rotifer [110]. Studies have also indicated that binding to ACE is strongly influenced by the substrate's C-terminal tripeptide sequence. Hydrophobic amino acid residues with aromatic or branched side chains at each of the C-terminal tripeptide positions are common features among potent inhibitors. The presence of hydrophobic Pro residues at one or more positions in the C-terminal tripeptide region seems to positively influence a peptide's ACE-inhibitory activity [95]. In general, the peptides showing higher activity against ACE have Tyr, Phe, Trp, or Pro at their C-terminus [95]. The peptides TQVY from rice [111], MRW from spinach [112], and YKYY from wakame [113] are some examples of this principle. **Table 1** reviews some examples of ACEI activities of plant origin, whose peptides responsible for such activity may be potential sources for the heterologous production of ACEI peptides.

The most common method to produce and identify ACEI peptides is through enzymatic hydrolysis of food proteins with gastrointestinal enzymes such as pepsin and trypsin or with commercial proteases such as Alcalase™ [127]. ACEI peptides have also been produced with *Lactobacillus, Lactococcus lactis*, and *E. faecalis* strains during milk fermentation [105, 106]. Nevertheless, there are problems associated to this type of industrial production of ACEI peptides, including the difficulty to isolate the peptide of interest from the complex mixture of compounds produced by enzymatic hydrolysis, the high cost, low recovery, and the low bioavailability. These disadvantages denote the need to develop new and alternative approaches for their production.

#### *Genetic Engineering - A Glimpse of Techniques and Applications*


#### **Table 1.**

*Examples of ACEI peptide activity from different plant origin.*
