STUDY ON STABILITY AND ACTIVITY OF PAPANDAYAN LIPASE AT VARIOUS TEMPERATURE AND ORGANIC SOLVENT IN FREE AND IMMOBILIZED STATE

• Main Author: Tri Wahyudi, Setyanto
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34132
• Institution: Institut Teknologi Bandung

Thermostable lipase gen has been cloned from local isolate PPD2 derived from local hot spring in Papandayan Crater, Garut, West Java. The gene has been expressed successfully in E.coli. The protein was named as lipase ITB2.1. From the perspective of industry, the ability of enzyme to adapt to the extreme environment has been geiven a chance to use the enzyme as biocatalysts in industrial processes. From point of view a researcher, the character of thermostable enzyme became interesting to be investigated. The conclusion of many studies showed that the decisive factor in the stability of the enzyme for adapting to extreme environments will be differed from enzymes to enzymes. To probe the determinants of the stability of lipase ITB2.1 further research is needed. This study has been conducted to probe the stability and the activity of free and immobilized lipase ITB2.1 in water and organic solvent based on experiment and computational analysis. Computational approach through the study of molecular dynamics simulations has been carried out at 300, 350, 400, 450 and 500K. Simulations was performed in water box, on a two-state, APO and HOLO enzyme. The HOLO enzyme of lipase ITB2.1 was indicated by the present of Zn2+ and Ca2+ ions, which is bound to the metal ion binding domain. In addition simulating on water, also performed simulations in organic solvents, such as acetonitrile and n-hexane. The free lipase at 70°C and pH 9.0 in aqueous solvent showed 737.56 U/mg lypolitic activity. The stability was performed by incubating the enzyme in a waterbath with temperature 70°C for 30 hours withe the interval time of 5 hours. The results of the stability test showed that the enzyme still has 50% after incubation for 4 hours. The activity test in organic solvents showed the enzyme activity in line with the decrease of solvent polarity. Lipase activity 930.28 U/mg enzyme in n-hexane, and decreased activity becomes 796.53 U/mg enzyme in acetonitrile. To perform an immobilized of lipase ITB2.1 required pendukunging materials. The material used was hydroxyapatite (HAP) which is synthesized by precipitation method. Basic materials synthesis derived from the shells of chicken eggs. HAP was used directly for enzyme immobilization. Enzyme binding capacity was obtained as follows, 10.17 µg/mg, 41.03 µg/mg and 75.21 µg/mg enzyme per solid pendukung by varying the amount of enzyme used for immobilization. Activity assay for immobilized lipase showed decrease compared to that the free lipase. Immobilized lipase activity in water solvent, acetonitrile and n-hexane are is 99.04; 97.43 and 55.56 U/mg enzyme respectivuly The stability test of the immobilized lipase in solvent showed a decrease in lipase activity by 50% of the initial activity after incubation for 1 hour. For thermal stability study through molecular dynamics simulation method 3 dimensional structure of lipase ITB2.1 obtained from modeling through MODELLER program was used. The result of the model showed that a 3D model of the lipase ITB2.1 was very good quality model. RMSD and SASA of APO and HOLO enzyme showed strong influenced by Ca2+ and Zn2+ in maintaining the enzyme. . In APO and HOLO state, there are two pairs of salt bridges are strongly suspected as determinant residues for the stability of the lipase, Asp222-Lys185 and Asp178-Lys229. The radius gyration of ITB2.1 lipase when simulated in the solvent n-hexane showed higher shrinkage than those in water and acetonitrile. Furthermore the value of SASA polar in n-hexane was smaller compared to the water an acetonitrile Based on the data obtained the enzyme showed high activity at high temperature and organics solven. Hydroxyapatite material is not suitable when used directly for immobilization in this condition. It is indicated by decreasing activity of the immobilized enzyme. Molecular dynamics simulation studies proved the effect of metal ions on ITB2.1 lipase and salt bridges Asp222-Lys185, and Asp178- Lys229 are key residues and factors which affecting the stability of the protein structure. Stability mechanism in organic solvents maybe caused by making polar residues on the surface flat the molecule, to prevent the hydrophobic core more exposed