10. Materials and methods

#### 10.1 Preparation of peptide solutions

First step is preparation of 10 mM peptide solution having tryptophan, tyrosine and phenylalanine. Peptides were solubilized using dilute acid, the assay mixture contained higher buffer concentration viz. 0.15 M.

#### 10.1.1 Solanain action on peptides

Peptide was incubated by solanain in 0.05 M Phosphate buffer (pH 7.0), 10 mm β-mercaptoethanol, 2 mM EDTA, 2.5 mm amide and 0.5 mg to 1.0 mg/ml catalyst protein. Incubation was administered for 48 h at 30°C. The reaction was detected by sampling the digestible mixture on aid. For every digestion mixture, a bearing while not substrate or without catalyst was analysed. Skinny layer activity was performed as delineated by Renderath (1963).

#### 10.1.2 Preparation of colloid silica gel G plates

About 25 weight unit of colloid Silica Gel G was mixed with 50 cm3 of H2O and stirred smartly during a closed round shape flask till completely distributed. The suspension was transferred into Stahl's mechanical spreader adjusted to 250 μ

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

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

### 10.1.3 Developing the chromatographic plate

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.

### 11. Results and discussion

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;

8. Screening of plant lattices for novel plant latex cysteine protease

economical production of commercially useful industrial proteases from novel sources. A number of plant lattices belonging to different plant families have been collected in and around Visakhapatnam and screened for protease activity. Their activities were assayed immediately and were stored in ice for further investigations. A novel plant latex cysteine protease namely Vallaris solanacea was identified in Biodiversity Park, Visakhapatnam which showed maximum protease activity. Preliminary studies on protease activity from the latex of Vallaris solanacea were carried out. The protease was purified and characterized. Specificity studies towards syn-

thetic peptide and ester substrates by the protease purified solanain.

9. Substrate specificity of the purified solanain from the latex of

Plant lattices are rich source of proteases. Latex of Vallaris solanacea also showed high protease activity. The cysteine protease solanain was purified by ammonium sulphate precipitation followed by DEAE-cellulose ion exchange and gel chromatography. The purpose of the present study is determination of specificity of purified solanain towards synthetic peptide and ester substrates. Extensive and more systematic studies [13–16] have been made on papain, ficin [17] and bromelain [18].

First step is preparation of 10 mM peptide solution having tryptophan, tyrosine and phenylalanine. Peptides were solubilized using dilute acid, the assay mixture

Peptide was incubated by solanain in 0.05 M Phosphate buffer (pH 7.0), 10 mm β-mercaptoethanol, 2 mM EDTA, 2.5 mm amide and 0.5 mg to 1.0 mg/ml catalyst protein. Incubation was administered for 48 h at 30°C. The reaction was detected by sampling the digestible mixture on aid. For every digestion mixture, a bearing while not substrate or without catalyst was analysed. Skinny layer activity was performed

About 25 weight unit of colloid Silica Gel G was mixed with 50 cm3 of H2O and stirred smartly during a closed round shape flask till completely distributed. The suspension was transferred into Stahl's mechanical spreader adjusted to 250 μ

Taking into consideration, the increasing demand for proteases and the need for

Figures 10–13).

Peptide Synthesis

Vallaris solanacea

10. Materials and methods

10.1.1 Solanain action on peptides

as delineated by Renderath (1963).

74

10.1.2 Preparation of colloid silica gel G plates

10.1 Preparation of peptide solutions

contained higher buffer concentration viz. 0.15 M.

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


#### Table 3.

Reaction of di- and tripeptides by refined Solanain.


#### Table 4.

Hydrolysis of various synthetic ester substrates with purified solanain.

hydrophobic residues (peptides half dozen to 9) with a large aspect chain e.g., leucine, essential amino acid, essential amino acid and essential amino acid. However, if the organic compound contributory the group of the bond was aromatic the bond was not hydrolysed although the C-terminal amino acid was deliquescent. Neither L-Trp-Gly nor L-Tyr-Gly was hydrolysed. It seems that the presence of a deliquescent organic compound at the N-terminal finish is not needed for the protein.

In Table 3, the results of the speed measurements given for 10 parts as Vo/Eo wherever Vo is initial rate and Eo is concentration of the protein in moles. The concentration of substrate used was one.56 10-4 M. If the metric linear unit values of the p-nitrophenyl esters of CBZ-amino acids were of constant magnitude as for different thiol proteases like papain [9], ficin [17], bromelain [18] and ananain [12] then Vo/Eo values obtained for Glycine and amino acid were love the Kcat values of the corresponding esters of different proteases.

The refined solanain of Vallaris family Solanaceae showed close to identical specificity towards all the substrates.

Solanain, differs from the amino alkanoic acid proteases in having low amidase activity with BAPNA and conjointly did not show any esterase activity with BAEE. This finding suggests considerably low specificity for essential amino acid residues. It differs from enzyme, ficin and bromelain during this facet. By showing broad specificity, Solanain resembled enzyme [11], ficin [14, 17] and bromelain [18] that change a spread of amide bonds. All these, however, showed a preference for basic amino acids. The solanain of Vallaris potato family hydrolysed leucyl bonds with efficiency and during this respect resembled ficin [17] and differed from bromelain [18] (Table 4).

Author details

Silpa Somavarapu

77

Andhra Pradesh, India

provided the original work is properly cited.

Department of Food Technology, Vikrama Simhapuri University, Nellore,

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

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

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: silpasomavarapu.1981@gmail.com

#### 12. Conclusion

Substrate specificity studies showed solanain exhibited broad specificity. It showed peptidase activity, amidase activity. The enzyme was capable of catalysing the hydrolysis of p-nitrophenyl esters of amino acids. It exhibited difference in specificity towards simple peptide substrates.

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