Fruit Bromelain Enzyme Poster

Extraction and Purification of Fruit Bromelain Enzyme from Pineapple (Ananas Comosus) Natalie Asemi, Sonia Gonzalez, Dr. Monica Lares#, Dr. Jennifer Whiles# Chemistry Department, Sonoma State University Background Research Aims Discussion What purification and extraction techniques will result in Fruit Bromelain Enzyme: A Cysteine Protease obtaining the purest and most active form of fruit Enzyme Activity Assay bromelain enzyme? v In our enzyme assay, casein solution was digested by the fruit bromelain, hydrolyzing peptide bonds and liberating tyrosine along v Extract fruit bromelain, utilizing reagents to minimize autodigestion with other amino acids, detected under the UV-Vis at 280 nm. v Purify the protein utilizing different biochemical techniques to remove v Activating agents like EDTA solution and L-cysteine, bind and hold complex groups and investigate if this increases proteolytic activity on to chelates and minerals, thus freeing our protein from being v Confirm the homogeneity of enzyme with assays and SDS-PAGE entrapped in casein aggregates and allow for it to maintain its native conformation and elicit its effects. Methods v 0.1 M potassium phosphate buffer was used to help preserve our Crude Extraction enzyme. • The pineapple fruit was cleaned, cut into small chunks, then homogenized in a v 5% trichloroacetic acid solution was used stop the assay’s reaction blender with ice and 20 ml of 0.1 M sodium phosphate buffer. to enable an accurate measurement without the protein The Mechanism of Action • The crude extract of fruit bromelain was then centrifuged at 6,000 rpm for 15 continuing to digest. minutes at 4°C. The sulfhydryl group on fruit bromelain is well conserved and is v Our UV-Vis spectrum showed increasing activity as the • The supernatant vacuum filtrated using Whatman paper, transferred in a vial, concentration of the enzyme increased. essential for its catalytic activity to hydrolyze peptide bonds. purged with nitrogen, and stored at 20°C. v Folin & Ciocalteu’s reagent was added to each test sample, Enzyme Activity Assay reacting with tyrosine, forming a blue chromophore. The increase •Bromelain can be assayed by measuring the proteolytic digestion of in color correlates to the amount of tyrosine liberated in the image Biomedical and Industrial Applications casein, expressed as CDU (casein digestion unit). of our enzyme assay solutions. Bromelain possesses multiple phyto-therapeutic actions and has many •CDU is the amount of enzyme that will liberate one µg of tyrosine after v Our standard curve showed that as the enzyme concentration one minute of digestion at 37°C, pH 7.0, at an absorbance of 280 nm industrial applications: increased, so did its activity with the slope to be found at .0024. •The more tyrosine liberated the higher the activity of the enzyme v Anti-inflammatory agent Extraction of Crude Fruit Bromelain Extract v Anti-tumor agent Results v The primary component of fruit bromelain is the sulfhydryl v Treatment of Dermatological disorders UV-Vis Spectrum, Fruit Bromelain Activity Against Casein proteolytic fraction. v Modulation of cytokines and immunity v In this study, we isolated fruit bromelain from the pineapple fruit v Meat tenderization in the food and beverage industry Enzyme Assay Solutions and called it our crude extract. v Pre-treatment of silk and wool v The fruit bromelain crude extract in this phase had undergone its first purification step through centrifugation at 6,000 rpm. While some positive effects of fruit bromelain have been reported, the v Stored in optimal conditions enables us to continue to purify our reason for this healing is not fully known and requiring the need for further protein in subsequent steps. investigation. Future Work Since the protein contains a concoction of carbohydrate groups such as phosphatases, glucosidases, peroxidases, cellulases, and glycoproteins, its is essential to find strategies that result in the highest purified bromelain: To Further Our Understanding of Fruit Bromelain: v Highest purified fruit bromelain removing the mixture of carbohydrate vContinue to purify fruit bromelain using salt precipitation, dialysis, anion- groups found on the protein exchange chromatography v Ease of purification Standard Graph Curve for Enzyme Activity Assay vEstimate protein content using a Bradford protein assay v Minimization of autodigestion during isolation Preparation of Crude Extract vConfirm the homogeneity of fruit bromelain using SDS-PAGE v Use of cost-effective methods to allow for large scale production A Major Cysteine Endopeptidase Acknowledgements v Fruit bromelain is more acidic in nature in contrast to stem bromelain Chemistry Department, Sonoma State University v In plant physiology, bromelain plays a specific role as a defense protein to protect the pineapple fruit throughout development, maturation, and References: 1. Gautam, S.S., S.K. Mishra, V. Dash, A.K. Goyal and G. Rath, 2010. Comparative study of extraction, purification and estimation of bromelain from stem and fruit of ripening process pineapple plant. Thai. J. Pharm. Sci., 34: 67-76. 2. Ota, S., Moore, S., & Stein, W. H. (1964). Preparation and chemical properties of purified stem and fruit bromelains. Biochemistry, 3(2), 180–185. v Fruit bromelain is one of the major endopeptidases found in pineapple 3. Ramli, Aizi Nor Mazila, et al. “Comparative Structural Analysis of Fruit and Stem Bromelain from Ananas Comosus.” Food Chemistry, vol. 266, 2018, pp. 183–191., doi:10.1016/j.foodchem.2018.05.125..

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