Latex peptidases produce peptides capable of delaying fungal growth in bread.

Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera, Cryptostegia grandiflora, and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1: 25 enzyme:  substrate ratio) inhibited the growth of Aspergillus niger, A. chevalieri, Trichoderma reesei, Pythium oligandrum, Penicillium sp., and Lasiodiplodia sp. by 60-90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin.

Structural and enzymatic characterization of Peruvianin­I, the first germin-like protein with proteolytic activity.

The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60 °C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. These structural peculiarities make Peruvianin­I an interesting model for further understanding of the action of laticifer fluids in plant defense.

Identification and characterization of two germin-like proteins with oxalate oxidase activity from Calotropis procera latex.

Germin-like proteins (GLPs) have been identified in several plant tissues. However, only one work describes GLP in latex fluids. Therefore, the goal of this study was to investigate GLPs in latex and get new insights concerning the structural and functional aspects of these proteins. Two complete sequences with high identity (>50%) with other GLPs, termed CpGLP1 and CpGLP2, were obtained and consecutively presented 216 and 206 amino acid residues, corresponding to molecular masses of 22.7 and 21.7kDa, pI 6.8 and 6.5. The three-dimensional models revealed overall folding similar to those reported for other plant GLPs. Both deduced sequences were grouped into the GER 2 subfamily. Molecular docking studies indicated a putative binding site consisting of three highly conserved histidines and a glutamate residue, which interacted with oxalate. This interaction was later supported by enzymatic assays. Superoxide dismutase (common activity in GLPs) was not detected for CpGLP1 and CpGLP2 by zymogram. The two proteins were detected in the latex, but not in non-germinated or germinated seeds and calli. These results give additional support that germin-like proteins are broadly distributed in plants and they are tissue-specific. This particularity deserves further studies to better understand their functions in latex.