Diffraction -based assay for evaluation of protease activity of commercial bromelain

Date

4-2013

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Milagros M. Peralta

Restrictions

Restricted: Not available to the general public and to those bound by the confidentiality agreement. Access is available only after consultation with author/thesis adviser.

Abstract

A diffraction-based assay for protease activity based on the affinity of the protease to a model substrate was evaluated as a rapid, alternative assay for determining protease activity relative to the conventional wet chemical assay using UV-Vis spectrophotometry. A glass slide was used as a fluid cell to which a dilute casein solution was applied in a definite pattern using polydimethylsiloxane (PDMS) stamp. The fluid cell was illuminated to generate a diffraction pattern. Partially purified protease was then applied and the resulting diffraction intensity of the third diffractive spot was determined as the voltage detected by the photodiode detector. Using this technique, the response of partially purified protease to patterned casein substrate was linear for concentrations from 0 – 20 mg/mL of casein substrate. Partially purified protease was extracted from the commercial bromelain. The precipitate from the 60% ammonium sulfate saturation cut (AS 60P) was designated as the partially purified protease and was used in monitoring and comparing the conventional assay with diffraction based assay for determining proteolytic activity. Results of proteolytic activity using both the UV-Vis spectrophotometric conventional assay and diffraction-based assay showed a linear relationship with casein substrate. In the conventional assay, proteolytic activity was determined and calculated in terms of amount of casein hydrolyzed by the partially purified protease from commercial bromelain based on the absorbance at 280 nm, while in the diffraction-based sensing assay proteolytic activity was also determined and calculated but in terms of change in voltage due to the specific interaction between the enzyme and its substrate, casein Based on the results of this study, it can be shown that diffraction sensing may be used as a simple, rapid, and cheap alternative method for the monitoring of protease-catalyzed reactions.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2013 C42 /A43

Document Type

Thesis

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