Structural and functional characterization of yellow field pea (Pisum sativum) soluble protein aggregates and cholinesterase-inhibitory peptides
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Date
2023-03-07
Authors
Asen, Nancy D.
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Abstract
This thesis determined the structure - function properties of soluble protein aggregates and
acetylcholinesterase and butyrylcholinesterase-inhibitory peptides (CEI) prepared from yellow pea
protein (YFP) respectively. To obtain the soluble aggregates, a commercial YFP protein concentrate
(PPC) was heated at different pH values and the supernatant was sequentially passed through 30 and 50
kDa molecular weight cut-off membranes to collect the <30, 30 – 50, and >50 kDa fractions. The >50
kDa fractions had high protein contents (>60%) among the fractions and were collected to determine their
physicochemical properties and functionality in food systems. The morphological structure of the protein
samples and emulsion microstructures showed that heat treatment of the samples at different pH values
produced different protein aggregates under varying processing conditions. Treatments at pH 3.0
produced aggregates with better solubility at acidic pH as well as superior emulsifying and foaming
properties. Secondly, the CEI peptides were prepared by enzymatic digestion of PPC into protein
hydrolysates using six proteases. Peptide fractions from the digests were obtained by membrane
ultrafiltration (UF) and reverse-phase HPLC separation, which was followed by mass spectroscopy where
the mass to charge ratios were obtained to identify the peptide sequence and de novo synthesis was
carried out. Antioxidant properties as well as cholinesterase inhibitory activities of the hydrolysates,
peptide fractions, and synthesized peptides were determined. Hydrolysates obtained from alcalase,
flavourzyme and pepsin exhibited significantly (p < 0.05) higher (20-30% increases) CEI and radical
scavenging activities than the other hydrolysates and the potency increased (>10%) after UF or HPLC
separation. The interaction between the peptide and cholinesterases was confirmed using intrinsic
fluorescence and circular dichroic spectra. Mixed mode inhibition was exhibited by most of the peptides
against the cholinesterases and molecular docking assay revealed that most peptides were bound to
cholinesterases through the catalytic anionic site or the peripheral anionic site with low docking energy
scores. The structure-function relationship as determined by PLS modeling showed that the most potent
CEI peptides mostly contained polar, slightly polar, and neutral amino acids like aspartic acid, threonine,
alanine, proline, valine, arginine, histidine, and leucine.
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Keywords
Yellow field pea, Protein, Functional properties, Heat, Ultrafiltration, aggregates, Alzheimer's disease, Cholinesterase, Peptides, Inhibition, QSAR, Molecular docking