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Production and Characterization of Medium-Sized and Short Antioxidant Peptides from Soy Flour-Simulated Gastrointestinal Hydrolysate.

ABSTRACT: Soybeans (Glycine max) are an excellent source of dietary proteins and peptides with potential biological activities, such as antihypertensive, anti-cholesterol, and antioxidant activity; moreover, they could prevent cancer. Also, soy contains all the essential amino acids for nutrition; therefore, it represents an alternative to animal proteins. The goal of this paper was the comprehensive characterization of medium-sized and short peptides (two to four amino acids) obtained from simulated gastrointestinal digestion. Two different analytical approaches were employed for peptide characterization, namely a common peptidomic analysis for medium-sized peptides and a suspect screening analysis for short peptides, employing an inclusion list of exact m/z values of all possible amino acid combinations. Moreover, fractionation by preparative reversed-phase liquid chromatography was employed to simplify the starting protein hydrolysate. Six fractions were collected and tested for antioxidative activity by an innovative antioxidant assay on human gastric adenocarcinoma AGS cell lines. The two most active fractions (2 and 3) were then characterized by a peptidomic approach and database search, as well as by a suspect screening approach, in order to identify potential antioxidant amino acid sequences. Some of the peptides identified in these two fractions have been already reported in the literature for their antioxidant activity.

SUBMITTER: Cavaliere C

PROVIDER: S-EPMC8148578 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPeptides have recently become attractive for therapeutic applications. However, efficient production of medium- to large-sized peptides (30-100 amino acids [aa]) remains challenging both by recombinant and chemical synthesis. We previously reported the formation of active enzyme aggregates in Escherichia coli cells induced by the short β-structured peptide ELK16 (LELELKLKLELELKLK) and developed a streamlined protein expression and purification approach. In this approach, a cleavable self-aggregating tag (cSAT) consisting of an intein molecule and ELK16 was used to release the recombinant peptides with reasonable purity from active aggregates.ResultsIn this work, we extended the cSAT approach to a generalized expression and purification solution for a set of medium- to large-sized peptides with important therapeutic uses, including human glucagon-like peptide 1 (31 aa), B-type natriuretic peptide (32 aa), exendin 4 (39 aa), chemokine (C-C motif) ligand 5 (also known as RANTES, 66 aa), stromal cell-derived factor 1α (67 aa), insulin-like growth factor 1 (70 aa), and leptin (146 aa). After intein-mediated cleavage, the soluble peptides were released directly into the supernatant while insoluble peptides could be refolded and purified by reverse phase high-performance liquid chromatography. Additionally, an N-terminal thioredoxin tag was added upstream of the target peptides, which can be removed by enterokinase cleavage, generating native N-terminus for target peptides. Final yields of the peptides ranged from 0.1 to 1.8 μg/mg wet cell weight at laboratory scale.ConclusionsThe approach described in this study provides a fast and efficient route to express and purify peptides that are difficult or expensive to produce by chemical synthesis or by ordinary recombinant methods. It is particularly well suited for large peptides, peptides likely to be degraded, and peptides that have toxic effects on the host. It can greatly reduce the cost and time of downstream processing, and thus may be useful for both industrial manufacture and laboratory applications.

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Production and Characterization of Medium-Sized and Short Antioxidant Peptides from Soy Flour-Simulated Gastrointestinal Hydrolysate.

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