Lipases are biological catalysts that catalyze the hydrolysis of triacylgylcerols (glycerols and fatty acids) and possess catalytic properties of the degradation of lipids. Lipases are found in plants, animals, microorganisms, and more recently bacteria and fungi. COLD ACTIVATED LIPASES Wider oxyanion hole, increased flexibility, and weakening of hydrophobic clusters. Enzyme activity is particularly promoted by higher temperatures and the aqueous environment. The M37 lipase from the Psychrophilic Photobacterium lipolyticum is an enzyme that can function in cold water conditions from 0 to 30°C. The Photobacterium lypolyticum species was discovered from a sediment sample in the Yellow Sea, which allowed for the isolation of M37 lipase. Surprisingly, the M37 lipase retains its stability in methanol environments and therefore has been used for biodiesel production. The lipase characterization, reactivity, and stability have been previously studied experimentally, but a structural explanation of lipase function in a methanol environment and at cold temperatures is missing. Through the use of molecular dynamics (MD) simulations in water, methanol, and water/methanol environments, the structure and dynamics of this lipase enzyme were analyzed to further understand its functionality so that the enzyme can be better optimized for use in industrial applications. Molecular Dynamics Simulations of M37 Lipase from Psychrophilic Photobacterium lipolyticum in Water and Methanol Rhiannon Jacobs and Harish Vashisth Department of Chemical Engineering, University of New Hampshire, Durham, NH Biodiesel/biofuels production Medical/pharmaceutical Synthesis of fine chemicals Food Industry Environmental applications Structure file obtained from Protein Data Bank (2ORY) determined from crystalline structure. Protein structure visualized in VMD and solvated in water using the solvate function. The water box converted to methanol box. NAMD used to run simulations. 1.Proteins could be solvated in both water and methanol solutions; but methanol and water boxes proved difficult to simulate. 2.Further quantitative analysis will provide optimum conditions for the enzyme activity. 3.Data is consistent with previous simulation on enzyme. 4. RMSD values consistent with values for short simulations on small proteins. Molecular dynamics are a computer based approach to statistical mechanics which allows for an estimation of equilibrium and dynamic properties of a complex system that cannot be done analytically. Molecular Dynamics is an approach to evolve positions of a system of particles in time, where particles interact with each other under a complex potential function. MD simulations are run on the principle of classical mechanics; where F=ma. This equation is then applied amongst the varying forces. MD displays the atoms continuously interacting with each other. Software: Visualization software is Visual Molecular Dynamics (VMD) which displays, animates, and analyzes biomolecular systems using 3D graphics. Simulation software is NAMD software which is distinctly designed for high performance simulation of biomolecular systems. MOLECULAR DYNAMICS METHODS [1] Hasan, Fariha, Aamer Ali Shah, and Abdul Hameed. "Industrial Applications of Microbial Lipases." Enzyme and Microbial Technology 39.2 (2006): Web. [2] Joseph, Babu, Pramod W. Ramteke, and George Thomas. "Cold Active Microbial Lipases: Some Hot Issues and Recent Developments." Biotechnology Advances 26.5 (2008): Web. [3] Jung, Suk-Kyeong, Dae Gwin Jeong, Mi Sook Lee, Jung-Kee Lee, and Hyung- Kwoun Kim. "Structural Basis for the Cold Adaptation of Psychrophilic M37 Lipase from Photobacterium Lipolyticum." Proteins(2008): Web. [4] Yang, Kyung Seok, Jung-Hoon Sohn, and Hyung Kwoun Kim. "Catalytic Properties of a Lipase from Photobacterium Lipolyticum for Biodiesel Production Containing a High Methanol Concentration." Journal of Bioscience and Bioengineering (2009): Web. CONCLUSIONS APPLICATIONS REFERENCES ABSTRACT ACKNOWLEDGEMENTS LIPASES SYSTEMS Harish Vashisth, Ph.D, Faculty Mentor University of New Hampshire Dept. of Chemical Engineering Gregory Samuel [Summer Teacher Researcher] Figure 1: Lid domain of the lipase used in simulation highlighted in blue. Catalytic triad (SER 174 ASP 236 HIS 312) highlighted in red. Figure 2: Lipase solvated in water box (left) lid domain (blue) and catalytic triad (red) highlighted. Figure 3: Lipase solvated in methanol box (right) lid domain (red) and catalytic triad (yellow) highlighted. Figure 5: Snapshots of simulation run at 288 K for 8 nanoseconds in water. t=0 nst=3 nst=6 nst=8 ns Figure 4: RMSD versus simulation time (ns) for different temperatures: 288 K (red), 298 K (green) and 313 K (blue). RESULTS