6. Applications of synthetic peptides

The Peptide synthesis (Figure 9) holds varied applications including


#### 7. Proteases

For about a century, the enzymes that play the central role in the degradation of proteins by hydrolysing peptide bonds have been known as 'proteases' and the term protease is therefore equivalent to 'peptide hydrolase'. They hold first place in the

N-end of the developing peptide chain to frame the beginning peptide security. To affirm total coupling a test is performed called Kaiser Test (Figure 7).

Diagram of peptide synthesis. Peptide bond formation between the deprotected N-terminus of the first amino acid and the activated C-terminus of the incoming amino acid. This cycle of deprotection and coupling is

immediately after coupling to allow the next incoming amino acid to bind to the growing peptide chain is called deprotection. Boc is removed using moderately strong acid such as trifluoroacetic acid (TFA) while Fmoc is a base-labile protecting

The synthetic peptide purification is by compounds like water, anisol or thiol derivatives are added in excess during the deprotection step to react with any of

Diagram of peptide cleavage after synthesis. The remaining N-terminal protecting groups, all side-chain protecting groups and the C-terminal protecting group or solid support are removed by strong acid treatment

Removal of specific protecting groups from the newly added amino acid

4.Deblocking the amino group of amino acid

repeated until the full-length peptide is formed.

Figure 7.

Peptide Synthesis

Figure 8.

70

after peptide synthesis is completed.

group that is removed with a mild base such as piperidine.

4. Deblocking the carbonyl group of amino acid

world market of enzymes, estimated at US\$3 billion [5]. Proteases are distributed widely in different parts of the biological sources. In occurrence of proteases, plant kingdom occupies the highest rank (43.85%) followed by bacteria (18.09%), fungi (15.08%), animals (11.15%), algae (7.42%) and viruses (4.41%). According to


Figure 11. Plant proteases.

Figure 12.

Figure 13.

proteases.

73

Amino acid sequence around the active site cysteine and histidine residues (in bold) of some plant cysteine

Determination of Substrate Specificity of the Purified Novel Plant Cysteine Protease Solanain…

DOI: http://dx.doi.org/10.5772/intechopen.90184

Distribution of proteases in plant parts.

#### Table 2.

List of plant cysteine proteases and their sources.

Figure 10. Distribution of proteases.

Determination of Substrate Specificity of the Purified Novel Plant Cysteine Protease Solanain… DOI: http://dx.doi.org/10.5772/intechopen.90184

Figure 12. Distribution of proteases in plant parts.



Figure 13.

Amino acid sequence around the active site cysteine and histidine residues (in bold) of some plant cysteine proteases.

world market of enzymes, estimated at US\$3 billion [5]. Proteases are distributed widely in different parts of the biological sources. In occurrence of proteases, plant kingdom occupies the highest rank (43.85%) followed by bacteria (18.09%), fungi (15.08%), animals (11.15%), algae (7.42%) and viruses (4.41%). According to

[9, 11, 19]

Khan and Polgar, 1983

Sugiura and Sasaki, 1974 Devaraj et al., 2008

Baines and Brocklechurt, 1982

Greenberg and Winnick, 1940 Morcelle et al., 2004 Jaffe, 1943 Singh, 2008 Yadav et al., 2006 Rudenskaya et al., 1998

Messing and Santoro, 1960 Agundis et al., 1977

Daley and Vines, 1978 Yamaguchi et al., 1982 Yamaguchi et al., 1982

[12]

Kunimitsu and Yasunobu, 1970, Robinson, 1975;

Lynn, 1979; Lynn and Yaguchi, 1979; Polgar, 1981;

Castaneda-Agullo et al., 1942; Soriano et al., 1975 Sgarbieri et al., 1964; Kramer and Whitaker, 1964;

Cooreman et al., 1976; Murachi 1970; 1976

Arcus, 1959; McDowall, 1973; Brocklehurst et al., 1981 Murachi, 1970; 1976; Heinicke and Gortner, 1957

Family, species Name, EC-number Reference(s)

3.4.99.23 Indicain Milin Taraxilisin Fruit bromelain, 3.4.22.4 Pinguinain, 3.4.99.18 Hemisphaericin Actindin, 3.4.22.14 Stem bromelain, 3.4.22.4 Ananain Leaf proteases ………… …………

Papain, 3.4.22.2 Chymopapain, 3.4.22.6 Papayapeptidase-I (A and B) Mexicain, 3.4.99.14 Ficin, 3.4.22.3 Protease Asclepain, 3.4.22.7 Funastrain c II Tabernamontanain,

In latices (I) Caricaceae 1. Carica papaya 2. Pileus mexicanus (II) Moraceae 3. Ficus carica 4. Ficus racemosa (III) Asclepiadaceae 5. Asclepias speciosa 6. Funastrum clausum (IV) Apocynaceae 7. Tabernaemontana grandiflora (V) Urticaceae 8. Morus indica (VI) Euphorbiaceae 9. Euphorbia milii (VII) Asteraceae 10. Taraxacum officinale In fruits (IX) Bromeliaceae 30. Ananas comosus 31. Bromelia penguin 32. Bromelia hemisph (X) Actinidiaceae 33. Actinidia chinensis In vegetative organs (XI) Bromeliaceae 34. Ananas comosus (XII) Zingiberaceae 35. Zingiber officinale

Peptide Synthesis

36. Miut

List of plant cysteine proteases and their sources.

Table 2.

Figure 10.

72

Distribution of proteases.

Barrett and McDonald [6], plant proteases are classified into serine, cysteine, aspartic and metalloproteases. Cysteine proteases (EC 3.4.2.2) are found in bacteria [7], eukaryotic microorganisms [8], plants [9] and animals. Cysteine proteases are represented by 70 families belonging to 12 different classes [10] (Table 2; Figures 10–13).

thickness, victimization the spreader; layers were ready on 20 20 cm glass plates. The gel was allowed to dry for a couple of minutes and so activated by drying in

Determination of Substrate Specificity of the Purified Novel Plant Cysteine Protease Solanain…

About 5–10 μl of the sample was loaded. Solvent used was 4:1:1 butanol: carboxylic acid: water (v/v/v). After development the plates were removed, dried and detected by spraying with 0.2% ninhydrin in butanol: ethanoic acid (95, 5 v/v) mixture. Rf values of the spots were calculated. Amino acids were identified.

Studies on substrate specificity were done and results were tabulated. Solanain was capable of hydrolysing peptide bonds involving the amino groups of hydrophilic amino acid residues (peptides 1 to 5) and incapable of peptide bonds involving the groups of deliquescent organic compound residues (peptides one to 5) and incapable of hydrolysing amide bonds wherever amino group was given by a

S. No Peptides Protease activity

 Gly – Gly ++ Gly – L - α- Ala + Gly – L – Asn +++ Gly – DL – Asn + Gly – D – Asn +++ Gly – L – Leu Gly – L - β- phe Gly – L – Trp 9 L – Ala – L – Met L – Leu– L – Met +++ L – Trp – Gly L – Trp – L – Tyr L – Tyr – Gly

 N – Z – L – Glu – L – phe +++ N – Z – L – Glu – L – Tyr ++ N – Z – L – Ileu – L – Met N – Z – L – Met – Gly – OEt\* ++++ Hippuryl – L – Arg ++

19 Gly – Gly – Gly 20 L-Glu-L-Val-L-Phe 21 L – Leu- Gly – Gly +

\*Both ester bond and the peptide bond of N – Z – L – Met – Gly – OEt were hydrolysed.

Reaction of di- and tripeptides by refined Solanain.

associate degree kitchen appliance at 1100°C for half-hour.

10.1.3 Developing the chromatographic plate

DOI: http://dx.doi.org/10.5772/intechopen.90184

11. Results and discussion

Simple dipeptides

N-aryl-dipeptides

Tripeptides

Table 3.

75
