Purification and characterization of a proteolytic enzyme from tiger prawn (Penaeus monodon) hepatopancreas

Date

4-1999

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Veronica C. Sabularse

Co-adviser

Jamela R. Revilleza

Committee Member

Maxima E. Flavier

Abstract

Tiger prawn hepatopancreas was homogenized in Tris-HCI buffer containing CaCI, and the extract collected after filtration. The crude extract was analyzed and found to have proteolytic activity of 0.0251 U/mg protein. Resolution of protein components and thus, purification was carried out via two schemes: hydrophobic interaction chromatography (H1C) of crude extract using phenyl sepharose as column material and sequential ammonium sulfate (AS) precipitation and HIC of corresponding protease active AS fractions. When the crude extract was directly subjected to H1C, protease activity was localized in the unbound fraction resulting in an 11-fold purification. On the other hand, differential precipitation concentrated the protease activity in the 40-60% ASP (precipitate or cut) and 60-80% ASP. These suggests the presence of protease isozymes from the sample. Subsequent HIC resulted in the collection of a protease active unbound fraction from the 60-80% ASP with a purification fold of 9.1. However, there was not significant improvement on the degree of purification when AS precipitation was used as an initial step. Thus, HIC could be used as a one-step protocol to purify a protease from Tiger prawn hepatopancreas. The purified enzyme from the crude unbound protein and all fractions with activity were subjected to denaturing gel electrophoresis to monitor the progress of purification. Single bands obtained were an indication that the enzyme was putatively purified. The estimated molecular weight of the single subunit enzyme using gel filtration chromatography and SDS-PAGE was 53.7 kD.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 1999 C4 H84

Document Type

Thesis

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