Detection and characterisation of a new metallocarboxypeptidase inhibitor from Solanum tuberosum cv. Desirèe using proteomic techniques

Obregón, Walter David; Ghiano, Natalia; Tellechea, Mariana Edith; Cisneros, José Sebastián; Lazza, Cristian Martin; López, L. Madero; Avilés, Francesc X.

doi:10.1016/j.foodchem.2011.08.030

Cited by 13 publications

(13 citation statements)

References 15 publications

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“…Then, the clarified crude extract was subjected to thermal treatment at 60, 70, 85, and 100 °C for 60 min. In previous studies, it was demonstrated that MCPIs are heat-stable molecules due to the highly stable knottin scaffold [ 37 ]. After incubation at the indicated temperatures and centrifugation of the samples, the analysis of the soluble fraction using Bradford revealed an important reduction in the total protein content in all the heat-treated conditions (60 °C treatment = 112 μg·mL −1 , 70 °C treatment = 88 μg·mL −1 , 85 °C treatment = 92 μg·mL −1 , 100 °C treatment = 82 μg·mL −1 ) in comparison with the soluble fraction of the untreated crude extract (CE = 789 μg·mL −1 ).…”

Section: Resultsmentioning

confidence: 99%

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña

Cotabarren

Tellechea

Tanco

et al. 2018

IJMS

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Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.

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Section: Resultsmentioning

confidence: 99%

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña

Cotabarren

Tellechea

Tanco

et al. 2018

IJMS

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“…The mass spectra obtained after tryptic digestion (peptide mass fingerprint) of inhibitor isolated from Solanum tuberosum cv. Desirée analysed with the 'MASCOT search tool' also did not match any of the inhibitors of other plants [30].…”

Section: Discussionmentioning

confidence: 99%

Trypsin Inhibitor from Edible Mushroom Pleurotus floridanus Active against Proteases of Microbial Origin

Ali

Sapna

Mol

et al. 2014

Appl Biochem Biotechnol

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Protease inhibitors can be versatile tools mainly in the fields of medicine, agriculture and food preservative applications. Fungi have been recognized as sources of protease inhibitors, although there are only few such reports on mushrooms. This work reports the purification and characterization of a trypsin inhibitor from the fruiting body of edible mushroom Pleurotus floridanus (PfTI) and its effect on the activity of microbial proteases. The protease inhibitor was purified up to 35-fold by DEAE-Sepharose ion exchange column, trypsin-Sepharose column and Sephadex G100 column. The isoelectric point of the inhibitor was 4.4, and its molecular mass was calculated as 37 kDa by SDS-PAGE and 38.3 kDa by MALDI-TOF. Inhibitory activity confirmation was by dot-blot analysis and zymographic activity staining. The specificity of the inhibitor toward trypsin was with Ki of 1.043 × 10(-10) M. The inhibitor was thermostable up to 90 °C with maximal stability at 30 °C, active over a pH range of 4-10 against proteases from Aspergillus oryzae, Bacillus licheniformis, Bacillus sp. and Bacillus amyloliquefaciens. Results indicate the possibility of utilization of protease inhibitor from P. floridanus against serine proteases.

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“…Glyoxyl-agarose gel was prepared by etherification of agarose 10 BLC (Hispanagar) with glycidol and further oxidation of the resulting glyceryl-agarose gel with periodates [33]. The immobilization process was carried out according to the protocol used by Guisan [23] with modifications [34]. For this purpose, 10 g of the activated support were suspended in 25 mL of 0.1 M sodium bicarbonate buffer (pH 10) containing an appropriate amount of araujiain so as to get a ratio of 35 mg enzyme/g carrier.…”

Section: Immobilization Of Araujiainmentioning

confidence: 99%

A Highly Stable Biocatalyst Obtained from Covalent Immobilization of a Non-Commercial Cysteine Phytoprotease

Ceccacci¹

2015

J Bioprocess Biotech

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In this work, araujiain (enzymatic preparation obtained from the latex of Araujia hortorum fruits) was successfully immobilized on glyoxyl-agarose via multipoint covalent attachment. Thus, good efficiency of immobilization and high operational stability of immobilized enzyme were obtained. The activity of araujiain at alkaline pH was significantly improved after immobilization. In addition, immobilized araujiain also showed high activity and good stability, without significant loss in its activity, at temperatures between 37 and 60°C and in the presence of immiscible organic solvents. Immobilized araujiain also showed good performance in a mixture of 50% ethyl acetate in buffer, used for peptide synthesis, with better results than when the free enzyme was used as catalyst. These results indicate that immobilized araujiain via multipoint covalent attachment can be highly stabilized and this method might be used for practical applications of araujiain in hydrolytic and synthetic processes.

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Detection and characterisation of a new metallocarboxypeptidase inhibitor from Solanum tuberosum cv. Desirèe using proteomic techniques

Cited by 13 publications

References 15 publications

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña

Trypsin Inhibitor from Edible Mushroom Pleurotus floridanus Active against Proteases of Microbial Origin

A Highly Stable Biocatalyst Obtained from Covalent Immobilization of a Non-Commercial Cysteine Phytoprotease

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