SYNTHESIS OF IMMOBILIZED THERMOSTABLE LIPASE AS A BIOCATALYST IN TRANSESTERIFICATION REACTIONS

• Main Author: Haryati, Titin
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/70561
• Institution: Institut Teknologi Bandung

Bacterial lipases are the most widely used (more than 50%) as catalysts in the industry. Bacterial lipase has a better stability and a higher catalytic activity. The use of lipases as biocatalysts in industry requires solvent-tolerant lipases, since most industrial-scale synthesis processes involve the use of organic solvents. Furthermore, lipases which have organic solvent tolerance characteristics generally have a positive correlation with the thermostability properties. Both of these characteristics are important for the use of lipase as an industrial biocatalyst. As a result, the discovery of new lipases with thermostable properties and tolerance to organic solvents is very important for further research and development. The realization of lipase as a catalyst requires a immobilized lipase because it is stable, easily separated from the reaction mixture, and can be used repeatedly. However, immobilization can cause a decrease in enzymatic activity, so it is necessary to choose the proper immobilization method that can increase enzymatic activity and stability. This research, in general, aims to obtain local isolates of thermostable lipases that are tolerant to organic solvents, obtain detailed information on lipase characterization through experimentation and computational analysis, obtain lipases immobilized on the NiSiO3 NSs matrix, and obtain information on lipase characterization as a biocatalyst in biodiesel synthesis. Lk2 and Lk3 are recombinant thermostable lipases which are highly expressed in Escherichia coli hosts. Cell lysis using ultrasonication was unable to obtain soluble enzymes. Therefore, the thermolysis method was used with the addition of SDS to obtain soluble lipase. In this research, cell lysis optimisation was carried out using PBS buffer pH 8 with the addition of SDS concentration variations and lysis temperature variations. The optimal thermolysis conditions were obtained at a 50°C lysis temperature and a 0.1% SDS concentration. Under these optimum conditions, were obtained soluble and active Lk2 and Lk3 crude extracts. The crude extracts Lk2 and Lk3 have lipolysis activity. A complete characterization of lipases is needed to obtain information on the activity of lipases and their use as a biocatalyst. Lipase Lk2 and Lk3 were characterized for their transesterification activity using a variety of methyl ester (C12-18) and para-nitrophenol (pNP) substrates. The characterization performed included the effect of variations in substrate, temperature, solvent, metal ions, and EDTA on transesterification activity and enzyme kinetics determination. A computational study was conducted to confirm the experimental data.Lk2 prefers methyl oleate (C18:1), while Lk3 prefers methyl linoleate (C18:2). This preference has been supported by docking experiments, which discovered that the optimal substrate lipase has a stronger affinity and a closer hydrogen bond between the active site and the substrate. The transesterification activity in Lk2 is greater than in Lk3. This may be explained using catalytic pocket analysis. Lk2 has a larger catalytic pocket than Lk3. Enzyme kinetics calculations also show that the value of kcat/KM Lk2 is 5.7 times that of Lk3 on methyl oleate substrate. Both lipases are active in non-polar organic solvents (n-hexane). According to the molecular simulation analysis, the enzyme-solvent interaction in n-hexane was found to be weakerer than that of acetone or acetonitrile. Lk3 is more thermostable than Lk3. Computational analysis revealed that Lk3 had more hydrophobic clusters and more hydrogen bonds than Lk2. Hydrophobic clusters, hydrogen bonds and salt bridges describe the structural stability of proteins and are linked to their thermostability characteristics. The activities of both enzymes are activated with Fe3+ ions, but are inhibited with Ca2+ ions. Analysis of the histidine loop around the catalytic triad at Lk2 and Lk3 indicates that there is a distortion in the amino acid residues that bind the Ca2+ ions. This causes a change in the conformation of the catalytic triad and a reduction in lipase activity. Deactivation of Lk2 and Lk3 by Ca2+ ions is in contrast to lipases in the I.1 family, which are known as Ca2+ dependent enzymes. These results suggest that Lk2 and Lk3 are new members of the lipase I.1 family. Immobilization of Lk2 and Lk3 was carried out using a NiSiO3 NS matrix. The resulting NiSiO3 NSs matrix is spherical with an average diameter of 270 nm. Immobilized lipases have different substrate preferences from their free lipases. The optimal activity of immobilised lipase was observed on the C16 substrate. In the temperature range of 40 - 60 °C, immobilized lipase has higher activity than free lipase. Both stable immobilized lipases were used for up to 5 repetitions and 70% of the initial activity was maintained in the 10th repetition. The optimal conditions for biodiesel synthesis are as follows: optimal enzyme weight is 0.5 g (12.5% by weight of catalyst/reaction volume), oil:methanol mole ratio is 1:12 in Lk2 and 1:18 in Lk3, optimal pH is 10, water:oil weight ratio is 4%. The highest biodiesel conversion percentages were respectively 49.8% for Lk2 and 62.7% for Lk3 obtained during 6 hours of reaction. The success of obtaining an adequate immobilization method and the use of lipase in the synthesis of organic compounds indicate that both enzymes can be developed as industrial biocatalysts