Isolation, purification and characterization of the antibacterial, antihypertensive and antioxidative properties of the bioactive peptides present in the major storage protein of pigeon pea (Cajanus cajan) seeds

Date

10-2012

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Mary Ann O. Torio

Abstract

Bioactive peptides were isolated from the purified major globulin protein of Pigeon pea or "kadyos"seeds, known as cajanin. In silico analysis of the complete amino acid sequence of the target protein was done to assess the presence of bioactive peptides. Isolation and purification of the protein was done to assess the presence of biactive peptides. Isolation and purification of the protein was done through extraction using 3.5% saline solution, followed by ammonium sulfate fractionation. The 90% ammonium sulfate fraction was further purified via dialysis and Gel Filtration Chromatographic (GFC) analysis. Protein digestion was done using three enzymes, trypsin, chymotrypsin, pepsin, and combinations of each, at specified intervals: 0, 3, 5, 10, 20, 60 minutes and 24 hours. The degree of digestion each enzymes was assessed through Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and densitometric analysis. The 24-hour digest using the enzymes combination pepsin-chymotrypsin-trypsin (PCT), which showed the highest degree of digestion, was subjected to further analysis. The PCT digest showed the higest % ACE-inhibition (87.50%). The target peptides were isolated through HPLC analysis and the two fractions collected were subjected to bioactivity assays. The % H2O2-scavenging activities of the fractions were higher (Fraction 1: 1.47%; Fraction 2: 1.51%) than the PCT digest (1.25%). Same results were obtained for the Total Antioxidant Capacity (TAC) assays, where 0.005 mg/ml each of HPLC fraction 1, HPLC fraction 2 and the PCT digest gave 0.000880 mg/ml, 0.00110 mg/ml and 0.00035 mg/ml Ascorbic acid equivalents, respectively. The IC50 value for the ACE-inhibitory activity of the HPLC fractions were lower (Fraction 1: 0.00535 mg/ml; Fraction 2: 0.00432 mg/ml) than that of the PCT digest (0.0323 mg/ml). Thin-Layer Chromatography was done to identify the possible peptide sequence of each fraction: Fraction 1: RA, RR, DA; Fraction 2: LK/KL and KLS.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2012 C42 /B73

Document Type

Thesis

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