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The Extraction and Purification of Bromelain

Presenter: Natali Dallos

Co-Presenter(s):
Chad Henry

Presenter Status: Undergraduate student

Academic Year: 22-23

Semester: Spring

Faculty Mentor: Monica Lares

Department: Chemistry

Funding Source/Sponsor: Class Project

Screenshot URL: https://drive.google.com/uc?id=1r_KDLbf7XNKNmjYm1z5ph7H-T1JI_seF

Abstract:
This project explores the processes of extracting and purifying the enzyme bromelain from the fruit of a pineapple. Bromelain is commonly used as a meat tenderizer and is available in stores as a dietary supplement. Bromelain is a proteolytic enzyme that has cysteine residue active sites that forms a catalytic triad between cysteine, histidine, and asparagine as the mechanism of action. In order to isolate this enzyme, the fruit was crushed and filtered with a cheesecloth. To further extract the protein, a series of centrifugations were performed, each time both pellet and supernatant were collected. Following this, the first purification step was carried out by an ammonium sulfate precipitation of the supernatant. This is a method of salting out, where the target enzyme precipitates out under high salt concentrations and is then resuspended in a buffer. Upon completion of the ammonium sulfate precipitation, dialysis was performed to remove the salts. The buffer was changed multiple times as the dialysis ran for a few days. Additionally, the second purification step was done utilizing an anion exchange column. Due to the nature of fruit bromelain, choosing an anion exchange column meant the protein would stick to the column when a neutral buffer is washed through. Knowing that the pI of the enzyme is low, this led to the use of a mildly acidic buffer to reach the pI so the protein would no longer carry a net charge and elute. To confirm the extraction and purification steps were a success, three assays were done in order to determine the presence, amount, and activity of our desired enzyme. SDS-PAGE is run to separate the enzymes based on their molecular weight, which helps determine if the bromelain is present as the theoretical weight is known. The Lowry assay is used to determine the concentration of bromelain exhibited by a color change of the sample solution in proportion with the protein concentration. Beer’s law is how the concentration is quantified. Lastly, the casein assay is used to determine the proteolytic activity of bromelain, whether the integrity of the protein has been disrupted during the previous processes. This casein assay is done before and after running the sample through the anion exchange column. In this method, casein is the substrate that bromelain will digest, thus releasing tyrosine which interacts with Folin’s reagent to produce a color change. To quantify, the absorbance values generated by the activity of bromelain is compared to an L-tyrosine standard curve to correlate the changes in absorbance with the amount of tyrosine.