STUDY OF ACETONITRILE EFFECT IN STABILITY AND MOBILITY OF LIPMNK LID BY MOLECULAR DYNAMIC APPROACH

• Main Author: Herasari, Dian
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/72275
• Institution: Institut Teknologi Bandung

Lipase is one of the enzymes belonging to the class of hydrolase that play a role in the hydrolyzing of lipid. Lipase is widely applied in various industrial fields, including in the food industry, detergent industry, waste processing industry, textile industry, the pharmaceutical industry and also in the pulp or paper industry. With its very wide and diverse applications, the production of lipase must not only be obtained in large quantities but also must have high catalytic efficiency and stability. From several studies, it is known that the solvent component can affect lipase activity. One of the components that significantly affect lipase activity is the presence of organic solvents as water-soluble co-solvents. Some lipases increase in activity after organic solvents are added with certain polarities and compositions. One of the organic solvents that can significantly increase lipase activity is acetonitrile. In this study, we will study the effect of acetonitrile addition on the stability and activity of lipase at the atomic level by molecular dynamics (MD) simulation approach. Lipase used in this study was lipMNK derived from thermophilic bacteria Geobacillus uzenencis isolated from Crater Manuk, West Java. This enzyme has determined its amino acid sequence and is known to have 416 amino acids comprising 28 amino acids signals peptide and 388 amino acids from the lipase structure gene. Lip-MNK has optimum activity at pH 7 and temperature 85oC, with 1,23 U/mg activity. To study the effect of acetonitrile on lipMNK stability was performed by observing the change of conformation of this enzyme at various concentrations of acetonitrile. To study the effect of acetonitrile on lipMNK activity was done by observing the mobility of lid lipMNK on various concentrations of acetonitrile. The first step of MD is preparation of the initial structure of lipMNK. This stage is done through structural prediction using comparative modeling method using lipase mold structure with the highest level of identity. For lid mobility studies, two initial structures were required, ie closed lid conformation (c-lipMNK) and open lid conformation (o-lipMNK). To predict the structure of c-lipMNK and v o-lipMNK each using lipase L1 (1KU0) and BTL2 (2W22) as a mold. The predicted results show the lid on the c-lipMNK and o-lipMNK models each composed of two ?-helices consisting of: (1) residues 175 to 195 - called lid A and (2) residues 221 to 230 - are called lid B. RMSD analysis and visualization of lipMNK structure of MD results over 100 ns at various acetonitrile howed that this enzyme had a wide tolerance range. The enzyme structure appears stable in the range of acetonitrile concentration up to 70%, where the RMSD value in this state is about 3 - 4 Å. A conformational damage is observed at concentrations above this limit, with RMSD > 4 Å. To observe the effect of acetonitrile on lid mobility, lid movement were observed from closed to open state and vice versa on various concentrations of acetonitrile. Visualization of MD structure product and lid displacement measurements showed that no lid opening movement at 0% acetonitrile concentration. The lid closing movement is observed at acetonitrile concentrations of 20%, 40%, 80%, 90%, and 100%. At 60% and 70% concentrations of acetonitrile are not observed lid closing movement because acetonitrile is not well distributed. At 80% and 90% concentration of acetonitrile observed lid closing movement, but seen other parts outside the lid (segment ARC, 196 – 220 residues) unfolded. This unfolding state is indicated by a fairly high RMSD (100ns 0% ascetonitrile as reference) value (5 Å). The lid closing movement is observed at 20% acetonitrile concentration. In these circumstances the lid displacement was 13 Å and 3.81 Å respectively for lid A and lid B, with RMSD (100 ns simulation at each acetonitrile concentration) of 2,095 and 0.894, respectively. The lid displacement increased significantly compared to that at 0% acetonitrile, each of 48.15 times for lid A and 12.29 times for lid B. Interlide angle observation and measurement also showed that at the beginning of the opening mechanism lid c-lipMNK, both lid segments both lid A and lid B do not move in parallel. This suggests both segments of the lid move independently of each other to lead to the opening of the lid. In addition to observing the lid opening movement from closed state, in this study also conducted MD to observe the lid closing movement of the initial open state. The observation results showed 100% concentration of acetonitrile not seen lid closing movement. The lid closing movement begins to appear after the concentration of acetonitrile 80% (water 20%). Compared to the lid opening movement which starts at 80 ns simulation, the lid closing movement observed faster which starts at 40 ns simulation. The most significant lid closing movement, which are seen by lid displacemnt, are observed at a 20% acetonitrile concentration almost with that at 0% acetonitrile. This suggests the presence of 20% acetonitrile does not inhibit the lid closing movement. At 20% acetonitrile concentration lid displacements are 5.09 Å and 13.63 Å respectively for lid A and lid B. This lid displacement increased significantly compared to that at 100% acetonitrile, respectively 26.79 times for lid A and 97.36 times for lid B. To further evaluate the lid closing pattern, the distance between representative residue in each lid and one residue selected at non lid region as the reference was measured. The data indicates the segment lid B initiates the closing motion of lid o-lipMNK until the simulation time of 90 ns for the next two lids to move toward the closed state almost in parallel. Further analysis of MD results for mobility of lid lipMNK was found to be important interactions that should be maintained in the lid area, Asp178 - Lys229 salt bridge on the interlid, and two Asp182 - Arg179 salt bridges, Glu189 - Lys185 on intralid A, and one bridge salt Glu226 - Lys229 in intra-lid B. Intralid interaction B is maintained at conformation changes c-lipMNK to o-lipMNK. The interlid and intralid interactions A are present only in c-lip-MNK conformations and are lost in o-lipMNK. Mutations in Lys229Gln, Arg179Gln and Lys185Gln, both single mutation and triple mutation can accelerate the lid opening movement, but cause unfolding in other segments of c-lipMNK. This suggests that a salt bridge involving all three residues has an important role in maintaining the stability of the lipMNK structure. These results indicate that lid integrity is not only important for the stability of the lid but is also important for the overall stability of the c-lip-MNK structure. From the results of the MD study above, it can be concluded that the concentration of 20% acetonitrile is the best concentration in terms of stability and lipMNK activity. The salt bridge in the lid section is a crucial interaction for the stability and mobility of lipid lipids.