New small proteinase inhibitors from Capsicum annuum seeds: Characterization, stability, spectroscopic analysis and a cDNA cloning

Abstract

Recent results from our laboratory have previously shown the purification of a small serine proteinase inhibitor (PI), named CaTI1, from Capsicum annuum seeds. This work demonstrated the characterization of CaTI now named CaTI1, and the identification of two other small serine PIs, named CaTI2 and CaTI3, also present in these seeds. CaTI1 presented molecular mass of 6 kDa and pI value of ∼9.0. CaTI1 inhibited both trypsin and chymotrypsin with inhibition constants (Ki and Ki') of 14 and 2.8 nM for trypsin and 4.3 and 0.58 nM for chymotrypsin, respectively. Circular dichroism analysis suggested the predominance of both disordered and β-strands regions in the secondary structure. CaTI1 presented striking physico-chemical stability. In an attempt to get the entire sequence of CaTI1 we found another PI called CaTI2. The discussion of this finding is in the main text. A degenerate primer was designed based on the sequence of trypsin inhibitor CaTI1 in an attempt to achieve the cloning of this PI. Surprisingly, the alignment of the predicted peptide derived from the cDNA with the protein database showed similarity with other C. annuun PIs, and thus it was called CaTI3.

Biochemical analysis of antimicrobial peptides in two different Capsicum genotypes after fruit infection by Colletotrichum gloeosporioides

“…Protease inhibitors were also detected by zymography (Figure 4B), and the results corroborate those from the trypsin inhibition assays (Figure 4A). Ribeiro et al [20] identified and purified a protein with trypsin inhibitory activity, called CaTI , from the C. annuum seed. This protein had the ability to permeabilize the plasma membrane of Saccharomyces cerevisia e and Candida albicans cells after 24 h of growth.…”

“…To stop the reaction, 100 μl of acetic acid was added. Afterward, photometric reading of the treatments was performed on the basis of released p -nitroanilide at wavelength of 405 nm [20].…”

Antimicrobial peptides from Capsicum chinense fruits: agronomic alternatives against phytopathogenic fungi

“…Recent studies have shown that some pepper species have peptides with strong antimicrobial activity against bacteria [ 11 ], yeasts [ 12 ], filamentous fungi [ 13 ] and have inhibitory activity against α-amylases [ 14 ] and proteases [ 15 ]. The J1-1/GST defensin of Capsicum showed strong inhibitory activity against fungi of the genus Colletotrichum , which cause anthracnose, an infectious disease that despite the efforts to control it, remains an endemic disease, resulting in large reductions in annual yields worldwide [ 16 ].…”

Application and bioactive properties of CaTI, a trypsin inhibitor from Capsicum annuum seeds: membrane permeabilization, oxidative stress and intracellular target in phytopathogenic fungi cells

“…The reaction was interrupted by adding 100 µL of 30% acetic acid (v/v). The substrate hydrolysis was quantified by measuring the absorbance of released p ‐nitroaniline at 405 nm …”