Production of Bioactive Peptides from RuBisCO Protein Fractions of Pumpkin Leaves through In Vitro Enzymatic Hydrolysis and In Silico Method
Year: 2024
Type: conference-paper
Abstract: Recently, growing concerns about public health impacts of the long-term intake of the synthetic antioxidants have driven the demand for natural and sustainable plant antioxidants. Side-streams from the oil processing industry like pumpkin leaves appear to be highly promising source of bioactive peptides because of high content of protein and large availability in oil industry. The soluble protein fraction of leaf proteins, commonly called white protein, is primarily composed of the photosynthetic enzyme RuBisCO (ribulose-1,5-bisfosfat-caroboxylase/oxygenase). As the main enzyme in plant photosynthesis and the most abundant protein on Earth, RuBisCO presents an attractive and sustainable source of bioactive peptides. This research serves as a proof of concept that protein isolate from green pumpkin leaves can be efficiently used as a substrate for producing bioactive peptides, using the endopeptidase from Bacillus licheniformis (Alcalase or Subtilisin; EC 3.4.21.62). The in silico hydrolysis process was performed using different RuBisCO fractions available in the UniProtKB/Swiss-Prot and TrEMBL protein sequence databases, including large and small subunits, and native RuBisCO protein. Selected protein sequences were subjected to the action of endopeptidase in the BIOPEPUWM peptide database, where predictions of the biological activity of the resulting peptides were also made. PeptideRanker software was used to analyze the peptides' theoretical bioactivity, identifying those with high bioactivity (>0.5). In the study, in vitro production of bioactive peptides was also performed. The specificity of the endopeptidase for hydrolysis was monitored over time in a batch reactor at enzyme to substrate ratios of 1-8%, and quantified by the degree of hydrolysis (0-15%), protein yield, and the peptide ability to inhibit the ABTS•+ radicals and to chelate Fe2+ ions (>50% and 30%, respectively, at a protein concentration of 1 mg/cm3). High enzyme specificity towards the substrate was noted in the first 75 min (12%), as well as a positive correlation between the antioxidant activity and the degree of hydrolysis, which distinguished this hydrolysate for subsequent characterization in terms of peptide fraction sizes. Peptide fractions 1- 3 and < 1 kDa were found to have significantly greater bioactivity. Considering the results of both enzymatic hydrolysis approaches, this work provides theoretical and practical guidance in the development of antioxidant functional food – peptides.
Authors: Knežević-Jugović, Zorica, Jovanović, Jelena, Simović, Ružica, Korićanac, Marija, Jugović, Branimir, Antov, Mirjana
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7051897314