The use of papaya leaves to extractpapain enzyme was evaluated for different extraction technique is studied byNordin (2010), which mainly used grinding method, enzyme-assisted extractionand ultrasonication. Enzyme concentration was then determined by measuring theamount of amino acid released in the reaction of protease assay. Thecombination of grinding method, enzyme-assisted extraction was proven to givehighest yield of papain by 36.75% when compared to grinding alone. Then furtherpurification of the extracted papain was done by using ammonium sulfateprecipitation. The optimized concentration of ammonium sulfate saturation wasachieved at 55% concentration in order to fully precipitate the protein.

Thecombination of grinding, enzyme-assisted extraction and ultrasonication can beconsidered as a potential extraction technique to extract papain as it is moredelicate towards protein extraction. Furthermore, the investigation on othersource of papain such as from the petioles, stalks and stem could also be done.In the study on extraction of papainfrom papaya latex conducted by Rodriguez, (2011), papain was obtained bysolubilizing latex and later dilution on alcohol (ethanol, 96% v/v) until ithad 10% alcohol concentration. The impurities precipitated were eliminated byfiltration on diatomaceous earth. The filtrated liquid was added to ethanol (96%v/v) in two proportions latex: alcohol, 1:2.1 or 1:3, to obtain a precipitatewhich was recovered by vacuum filtration using Wathman paper N°1. Finally, thesolid was dried out using two different methods: (1) Vacuum drying (at 50 °C,Lab live duo-vac oven, Lab Line Instruments) or (2) Drying by refractancewindow (MCD Technologies, Inc.

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) using 95 °C water as heating medium. The enzymewas grinded to get a fine powder. Enzyme ActionThe enzymatic activity of papain can bedetermined using benzoyl-l-arginineethyl-ester (BAEE) as the substrate. BAEEwas dissolved in 0.1 m pH 6.1 papain solutions which contains 1mm EDTA, then5mM L-cysteine was added. The reaction rate was determined by UV absorbance at258nm with a UV-Vis spectrophotometer (Miyamoto, Watanabe, & Ishihara,2004).

One unit enzyme activity was defined as the activity that hydrolysed1µmole/min of BAEE at 37º C (Arnon, 1970). Furthermore the enzyme activitycould also be measured by HPLC method by terminating the activity with theaddition of acetic acid. The reaction is 5 then filtered and the filtrate canbe further analysed by HPLC. The amount of product will be linearly related tothe peak height and the rate of enzyme reaction can be accurately determinedfrom the peak height of benzoyl arginine (Chen, Wu, & Wang, 1982). Asimpler method used casein as a substrate and the reaction will be terminatedby trichloroacetic acid. This colorimetric method also used FolinCiocalteu’sreagent. (Nordin, 2010)

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