OPTIMASI EKSPRESI DAN KARAKTERISASI ENZIM SUKROSA ISOMERASE (PalI) MUTAN DARI Klebsiella pneumoniae AxMC

• Main Author: Octavianus Soetrisno Tji, Tommy
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/39814
• Institution: Institut Teknologi Bandung

Palatinose (isomaltulose) is an isomer of sucrose with lower glycemic index so it is suitable to be used in sports drink. Isomaltulose is produced by utilizing sucrose isomerase enzyme (palI) which can be found in bacteria, one of them is Klebsiella pneumoniae AxMC. This enzyme produces isomaltulose and trehalulose. In proceeding experiment, heterologous expression of this enzyme in E. coli has been done successfully and it was found that 339RLDRDS334 motif ini this enzyme determine its product specifity. Mutation on protein motif 339RLDRDS334 become 339RLDRYP334 which is motif found highly efficient isomaltulose synthase from Pantoea dispersa UQ68J. Characterization of mutant enzyme is required to confirm the effect of mutation. This aim of this research was determine harvesting time of bacterial cells and optimum IPTG concentration to express native and mutant sucrose isomerase, the optimum pH and temperature of those enzymes originated from various fractions of cells, and the effect of the mutation on specific activity of the enzyme. In this research, two types of BL21 (DE3) E. coli clones are used which are pTXB-palI clone and pTXB-mutant palI clone. These clones have been confirmed in this research. Cell harvesting time determination was done from growth curve of the clones. Optimization of palI gene expression was done with IPTG induction on bacterial colony incubated in 37°C, 200 rpm shaker incubator for 4 hours. The IPTG concentrations used are 0, 200, 400, 600, 800, 1000 ?M. In this research, three enzyme fractions were produced which are membrane, cytoplasmic, and unlysed cell fraction. Membrane fraction and cytoplasmic fraction are obtained through sonication of induced clones. Every enzyme fraction’s specific activity was determined using DNS reagent and Bradford test. Determination of optimum pH and temperature was done through reacting enzyme extract with its substrate in various pH and temperature. This research concludes each clone containing wildtype and mutant sucrose isomerase could be harvested optimally at the same time which is when the culture reach OD600 of 0,6. The optimal IPTG concentration to express wildtype palI in membrane, cytoplasmic, cell fraction respectively are 200, 600, 200 ?M, while the concentration needed to express mutant palI in three fractions are the same which is 400 ?M. Highest specific enzymatic activity were found in membrane fraction for both types of enzyme. The optimum pH and temperature of the native enzyme are different from its mutated type which are respectfully 5,5 and 40°C for the mutant enzyme, while also 6,0 and 45°C for the native enzyme. Specific activity of mutant enzyme is lower than native enzyme’s in every experimented cell fraction and condition. Substitution of amino acid in 339RLDRDS344 motif to 339RLDRYP344 motif resulted in reduction of the enzyme’s optimum specific activity by 64.39 %, 17.13%, 31.76% respectfully in membrane, cytoplasmic, and cell fraction.