Isolation, purification and characterization of the major storage protein from sweet potato (Ipomea batatas) with bioactive peptides exhibiting antihypertensive activity.

Date

10-2010

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Mary Ann O Torio

Abstract

SAMONTE, MA. LOURDES T. University of the Philippines-Los Banos. October 2010. ISOLATION, PURIFICATION AND CHARACTERIZATION OF THE MAJOR STORAGE PROTEIN IN SWEET POTATO (IPOMEA BATATAS) EXHIBITING ANTIHYPERTENSIVE ACTIVITY.

Adviser: Dr. Mary Ann 0. Torrio

Co-adviser: Dr. Roberta N. Garcia

ABSTRACT

In silico analysis using the sequence of the major storage protein (sporamin) of sweet potato revealed some sequences of bioactive peptides with antihypertensive activity. The major storage protein of sweet potato (lpomea batatas) was isolated using extracting buffer composed of 50 mM potassium phosphate buffer, pH 7.5 with 0.5 M sucrose, 10 mM MgCI, I% (%v/v) ascorbic acid and 24 grams of polyvinyl polypyrrolidine (PVPP). The total protein content using Bradford method was 0.607 mg/mL. The protein was further purified using gel filtration chromatography, ammonium sulfate precipitation and dialysis. The purified protein concentration was 0.490 mg/mL. The SDS-PAGE profile of the purified protein showed two major bands, one at 25 kDa with 79.61 % of total protein content of sweet potato tuberous roots while the other at 55 kDa exhibited 20.39 % of total protein content. The purified protein obtained was subjected to protein digestion using three proteases pepsin, chymotrypsin and thermolysin. Thermolysin was able to digest purified protein after 3 minutes, while pepsin and chymotrypsin completely hydrolyzed purified protein after 24 hours. Peptic, chymotryptic and thermolytic hydrolysates were found to inhibit ACE activity by 13.97, 10.92 and 77.73 %, respectively. The IC it values of of chymotryptic, therrnolytic and peptic hydrolysates were 0.45, 0.029, 0.40 tifv1 or mg/mL, respectively.

The protein hydrolysates were further subjected to reverse phase high performance liquid chromatography (104IPLC). Pepsin hydrolysis resulted to four fractions with IC ,L, values of 0.481. 0.467, 0.655 and 0.149 mg/inL. The fraction that exhibited the highest percent inhibitory activity (87.27 %) and lowest IC so value (0.149 ntg /mL) was characterized using thin layer chromatography. The possible identity of the fraction involves alanine, glycine and glutamic acid. Chymotrypsin digestion gave seven fractions with IC 5o values of 0.216, 0.570, 0.699, 0.430, 0.283, 0.970 and 0.301 mg/mL. The fraction that exhibited highest percent inhibition (92.18 %) and lowest IC m, value (0.216 mg/mL) composed of arginine and phenylalanine amino acid residues. Thermolysin digestion resulted to seven fractions which gave IC ,o values of 0.138, 0.121, 0.112, 0.258 0.083, 0.054 and 0.096 mg/mL. The fraction with the lowest IC re value composed of phenylalanine, glycine .d serine amino acid residues. Possible sequence identities of the peptides with antihypertensive activity from pepsin digestion were AGE, GE, and AG; from chymotrypsin digestion was RF; and from thermolysin digestion were FGS, PG and GS.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

Thesis

Document Type

Thesis

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