ISOLASI, PEMURNIAN DAN KARAKTERISASI BETTA-AMILASE UBI JALAR (IPOMOEA BATATAS (L.) LAM)

• Main Author: Oktiarni, Dwita
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/71533
• Institution: Institut Teknologi Bandung

????-amylase (E.C 3.2.1.2) is an enzyme commonly found in plants and bacteria. The enzyme is an exo-acting carbohydrolase which hydrolyzes ????-1.4-glucosidic linkages of starch, removing maltose units from the non-reducing end of the polysaccharide chain, producing ????-maltosa and ????-limit dextrin as the final product. ????-amylase is widely distributed in the higher plants such as sweet potato. Like the other enzyme, the activity of ????-amylase is depend on the surrounding environment. Because of that, the crude extract of enzyme must be purified first from the other compound and when the cell is broken. This research methods consist of extraction and characterization of ????-amylase from sweet potatoes. The isolation and purification enzyme was conducted by fractionation with ammonium sulphate, FPLC and Native Gel Electrophoresis. The characterization consist of determination of pH and temperature optimum, KM, Vmax and Hill constant, effector affect of ????-amylase from sweet potato of Cilembu and Lampeneng. This enzyme was extracted from Cilembu and Lampeneng sweet potato in 0.05 M acetate buffer pH 4.8 and followed by ammonium sulphate fractionation. The fraction containing high of spesific activity (determined by Somogyi-Nelson and Lowry methods) was futher purified by FPLC (Fast Protein Liquid Chromatography). Fraction with high enzyme activity of Cilembu ????-amylase was fraction 60-70% (AC70) and 70-80% (AC80), with specific activity of 3.69 and 6.53 mg sugar.mg protein-1.minute-1, whereas specific activity of crude extract enzyme (AC) was 0.21 mg sugar.mg protein-1.minute-1. Then Lampeneng ????- amylase has fraction with high enzyme activity was fraction 60-70 % (AL70) and 70-80% (AL80) with specific activity of 3.27 and 6.77 mg sugar.mg protein- 1.minute-1, wherease specific activity of crude extract enzyme (AL) was 0.26 mg sugar.mg protein-1.minute-1. Fraction 60-70% (F70) and fraction 70-80% (F80) of ammonium sulphate purified enzyme, was futher purified by FPLC. Spesific activity of FPLC purified Cilembu ????-amylase were 5.22 and 9.85 mg sugar.mg protein-1.minute-1 while Lampeneng were 4.62 and 7.37 mg sugar.mg protein-1.minute-1. The purified Cilembu ????- amylase by FPLC toward fraction 60-70% (F70) and fraction 70-80% (F80) showed increasing in spesific activity the crude enzyme as much as 25 and 46 folds for FC70 and FC80 respectively, while those of Lampeneng showed 18 and 28 folds for FL70 and FL80, respectively. The purified Cilembu’s ????-amylase after Native Gel Electrophoresis showed increasing in spesific activity the crude enzyme as much as 58 folds while those of Lampeneng showed 37 folds. Spesific activity of Native Gel Electrophoresis purified Cilembu ????-amylase was 12.18 mg sugar.mg protein-1.minute-1 while Lampeneng’s of 9.57 mg sugar.mg protein-1.minute-1. The purified Cilembu’s ????-amylase from Native Gel Electrophoresis and Reverse Zymography showed that the enzyme-active had around 100 kDa and around 150 kDa from Cilembu and Lampeneng sweet potato, respectively. The determination of molecular weight and the molecular unit was conducted using SDS PAGE integrated with Native PAGE and Reverse Zymography. The study was showed that Cilembu’s ????-amylase has molecular weight of 100 kDa and Lampeneng’s ????- amylase has molecular weight of 158 kDa. Both enzyme consisted of more than one different molecular weight sub unit (heteromer). ????-amylase from Cilembu’s sweet potato consist of four molecular weight sub unit heteromer with three molecular weight sub unit of 26 kDa and one molecular weight sub unit of 31 kDa. Whereas ????-amylase from Lampeneng’s sweet potato consist of six molecular weight sub unit heteromer with four sub unit with molecular weight of 26 kDa, one sub unit molecular weight of 33 kDa and one sub unit molecular weight of 19 kDa. The characterization of purified ????-amylase from Native Gel Electrophoresis showed that Cilembu derived enzyme had optimum pH of 6.01, whereas the temperature was 70°C, with the KM app, Vmax app value and Hill constant were 0.0021 mg/mL, 10.83 mg sugar.minute-1 and 1.79, respectively. Whereas Lampeneng’s ????-amylase had pH and temperature optimum of 5.52 and 70°C, with the value of KM app, Vmax app and Hill constant were 0.012 mg/mL, 4.36 mg sugar.minute-1 and 1.07, respectively. Affect of effector showed that cation and anion can activates and inhibits the activity of enzyme. If Cl- as anion showed the positif effect of activity enzyme because they activates the activity of enzyme. Increasing of anion charge showed increasing positif effect, with optimum anion charge of -2. For K+ and Na+ as cation showed the positif effect of activity enzyme because they activates the activity of enzyme, wherease Cu2+ as cation showed the negative effect as negative effector of enzyme activity. From the data of characterization of enzyme above, it can be concluded that characteristic of ????-amylase from Cilembu sweet potato better than ????-amylase from Lampeneng sweet potato.