Experiment IX: Determination of Lipase Enzyme Activity

EXPERIMENT IX. DETERMINATION OF LIPASE ENZYME ACTIVITY Basic theory Enzymes are special proteins that function to catalyze biological reactions effective, precise and specific. The conventional names of enzymes are based on names the substrate it catalyzes by adding a suffix–ase, for example: urease catalyzes the hydrolysis of urea to ammonia and CO2. The structure of the enzyme is proteins, but there are many new enzymes that can work as catalysts if there is one or several other (non-protein) compounds mentionedcofactor. Cofactors can be metal ions or in the form of so-called organic compounds coenzyme,for example vitamins.So only the protein part calledapoenzyme,and the combination of apoenzymes with cofactors is calledholoenzyme. Enzyme activity is influenced by enzyme levels, substrate levels, pH and temperature, so the working conditions of enzymes need to be regulated so that they are at optimum conditions. The enzymes that work in the hydrolysis of fats and oils can be distinguished into two large groups, namely lipase enzymes and esterase enzymes. Both of them Involved in both fat metabolism and breakdown and breakdown processes fat. The difference between lipase and esterase lies mainly in the circumstances solution of the substrate. The lipase enzyme is more active in deep oil emulsion conditions water, while esterase is active both in solution and in emulsions with same speed. Based on the nomenclature of the International Union of Biochemistry, lipase enzyme functions to catalyze triglycerides into diglycerides and fatty acids, and It turns out that it can also further hydrolyze diglycerides into monoglycerides. Characteristics of the Lipase Enzyme: - Lipase activity depends on the type of substrate, and is also very dependent on pH and temperature. Pancreatic lipase, for example, has an optimum pH between 8.0 - 9.0. But it can decrease to 6.0 – 7.0 if the substrate is different. - Lipase activity also depends on the emulsifying compound used and the presence or absence of salt in the substrate. - The optimum temperature for lipase is generally around 30oC and 40oC. Although It has been found that lipase is still active at a temperature of – 29oC, esp on fish and shrimp that have not been frozen. - The presence of salt greatly influences the activity of the lipase enzyme, for example in NaCl salt concentration up to 7.0 mMol, lipase showed good activity maximum and will decrease if the salt concentration is increased. Salt Calcium also increases lipase activity and helps increase power heat resistant enzymes. - The lipase enzyme is found in various tissues, but the main source of lipase is used in the hydrolysis process is pancreatic tissue. Lipase too found in mammalian milk, can also be obtained from grains, microbes, nuts, cotton seeds and others. 2. Tools and materials TOOL : - Burette and stative - Erlenmeyer 250 mL - Test tube - Incubator 37oC - Water Bath MATERIAL : - 0.05M NaOH standard solution - 0.5% phenolphthalein solution in alcohol; digestive tablets (extract pancreas) - Making Oil Emulsion : Add 1 mL of olive oil to 5 mL 95% alcohol. Add the same volume of water, and shake. Add 10 drops of 0.04% phenol red indicator and add Na2CO3 0.1 M until the color turns pink. - Pancreatic Extract Suspension : Make a suspension of pancreatic extract from tablets digestion 50 mg/mL. The tablet layer is removed first. Procedure Prepare 4 test tubes and number them 1 to 4. Then add 2 mL of pancreatic extract suspension to each tube. On tube no. 1, the enzyme is killed by heating at a temperature of 100oC for 1 minute. Next, add 5 mL of oil emulsion and 1 mL of water to each tube the final volume was 8 mL. Incubate the mixture in all test tubes on temperature 37oC with time variations, namely, respectively 15, 30 and 45 minutes for tube no. 2 to 4, while tube no. 1 is considered 0 minute incubation due to enzymes turn off. At the end of incubation, each mixture was poured into 4 100 mL Erlenmeyer flask. Then added 20 mL of 95% alcohol and 10 drops phenolphthalein solution. Next, it was titrated with a standard 0.05 M NaOH solution until pink.