472466 Improving the Activity of a Thermophilic Lipase By Increasing the Flexibility Proximal to the Active Site

Friday, November 18, 2016: 1:06 PM
Continental 9 (Hilton San Francisco Union Square)
Weigao Wang1, Siva Dasetty1, Sapna Sarupria1 and Mark A. Blenner2, (1)Chemical and Biomolecular Engineering, Clemson University, Clemson, SC, (2)Chemical & Biomolecular Engineering, Clemson University, Clemson, SC

Lipases are long chain ester hydrolases used across multiple industries including the detergent, food, flavor, pharmaceutical, fine chemicals, agrochemical, biosensor, bioremediation and cosmetic fields. Cold active lipases are frequently sought after for their selectivity and efficient catalysis as noted by high kcat values. Even though cold-active lipases are widely used in these fields, the vast majority of naturally evolved psychrophilic lipases are too thermolabile for most applications at ambient and moderate temperatures. To take advantage of the intrinsically efficient catalysis of psychrophiles, we must have methods to increase their thermostability; however, prior efforts to improve the thermostability of psychrophilc lipases through rational engineering and directed evolution resulted in weakened cold-activity.

The thermodynamic parameters of reactions catalyzed by psychrophilic enzymes show that they are characterized by either lower transition state free energy values or smaller temperature dependence of activity than their thermophilic counterparts. The latter is achieved by the disruption of fewer enthalpy-driven interactions during the activation process. Therefore, localized increases in flexibility near the active site are responsible for the higher catalytic reaction rates of psychrophiles. Recently, the cold-activity of some thermophilic enzymes has been improved successfully by incorporating the structural elements of their psychrophilic counterparts, which shows that cold activity and thermostability are not mutually exclusive. In this study, we use site directed mutagenesis to increase the flexibility proximal to the active sites of lipase from Geobacillus thermocatenulatus by analysis of lipase from psychrophilic Photobacterium lipolyticum. Additionally, we compare the thermostability and kinetic parameters between the wild type lipase and the mutated one. In our talk, we will present these results discussing the structural differences and how the activity of thermophilic enzyme is affected.

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See more of this Session: Protein Structure, Function, and Stability
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division