Ingegnerizzazione di Bacillus thermocatenulatus lipase (BTL2) per lavorare in modo efficiente in solvente organico adatto ad applicazioni industriali.

Responsabili di progetto

Alessandro Venturini, Osman Ugur Sezerman

Accordo

TURCHIA - TUBITAK - Scientific and Technological Research Council of Turkey

Bando

CNR-TUBITAK 2016-2017

Dipartimento

Scienze chimiche e tecnologie dei materiali

Area tematica

Scienze chimiche e tecnologie dei materiali

Stato del progetto

Nuovo

Proposta di ricerca

The technological utility of enzymes, which have a remarkable potential as practical catalysts [1], can be greatly enhanced by using them in organic solvents rather than in water. In fact, use of enzymes in their natural, aqueous reaction media, limits their industrial exploitation by a variety of considerations:
i) most of the reactive compounds are insoluble in water;
ii) water frequently gives rise to unwanted side reactions;
iii) water degrades common organic reagents;
iv) some products may be quite labile in an aqueous environment;
v) thermodynamic equilibria of many processes are not favored in water.
Enzyme-catalyzed reactions in organic solvents, and even in supercritical fluids and in gas phase, have found numerous potential applications, some of which are already commercialized.
Lipases, triacylglycerol ester hydrolase [2], catalyze hydrolytic and synthetic reactions and are a group of enzymes capable of performing reactions in both, aqueous and organic solvents. The reaction mechanism of lipases in organic media is different from that in aqueous media. In water, lipases carry out hydrolysis reaction, whereas, changing reaction environment from water to organic solvent, the hydrolysis reaction is suppressed and esterification and trans esterification reactions become favored. With the change in the type of reaction, usage of lipase in organic media offers us new possibilities in biocatalysis approach. In addition, lipase, in this environment, increases its activity and stability, regiospecificity and stereoselectivity and solubility of the substrate. Moreover it becomes easier products recovery and ability to shift the reaction equilibrium toward synthetic direction. Lipases have many potential applications in the field of biotechnology such as the production of biofuels, organic synthetic compounds, detergents, perfumes, cosmetics, enantiopure pharmaceuticals, medical diagnostics foods and feeds. Enzyme selection for each of these applications depends on its specificity and stability in different solvent systems. Although their usage in organic solvents offer new possibilities in biocatalysis approach, the catalytic activity of lipases in organic solvents is lower than in aqueous systems because of diffusional limitations, changes in protein flexibility and destabilization of enzyme. To overcome these limitations, it is essential to add small amount of water around lipase in organic media. With the addition of small amount of water, the solvent polarity is adjusted and the required stability and flexibility of lipase in non-aqueous media are provided. Anyway, the loss of activity in organic solvents, which is mainly due to the enhanced rigidity of hydrophobic lipases, is still a major problem. Therefore enhancing the activity of lipases in organic liquids is an important field of research.
In this work we propose to perform engineering of lipase enzymes to understand their activation mechanism in organic media through molecular dynamic simulations and to improve their catalytic efficiency in non-aqueous solvent with in silico mutagenesis
Methodology
Computational:
This part of the project is divided in two sections:
i) Structural and Thermal Stability of Lipase in organic solvents with different percentages of water. Molecular Dynamics simulations will be carried out at different temperatures to determine the structural properties and activity of the enzymes.[3]. Analysis of structure conservation and protein flexibility at different temperatures will provide guidelines to design new point mutations in lipase for better activity in organic solvents. Simulations will be performed in organic solvents with different percentages of water.
ii) A clustering algorithm will be developed to identify clustered regions of the lipase in organic solvents even at elevated temperatures. These sites will be further studied as potential mutation sites to reduce packing and increase flexibility to enhance the activity
iii) Alanine scanning of the residues that were selected in step ii) will be carried out using FoldX algorithm to determine crucial sites for packing and flexibility. Voronia 1.0 program will be used to check whether point mutations will lead to any cavities or not. Analysis of these runs will reveal crucial sites for mutations. All possible mutations will be carried out at crucial sites and their impact will be studied using molecular dynamics simulations.
Experimental part.
Selected mutations will be transferred to BTL via site directed mutagenesis and characterization will be performed against several industrially important substrates in organic liquid. The steps to be carried out are:
o Site-directed mutagenesis will be performed to insert the desired properties to lipase. Mutant lipases will be selected through plate assays and the best mutants will be expressed in E.coli.
o After expression of mutant lipases in E.coli, the purification will be performed through affinity chromatography method. The specific enzyme activity of purified lipases will be determined toward soluble substrates in organic media. pH and thermal stability analysis of mutant lipases will be also performed in organic media.
o Enzyme activity essays against several industrially important substrates will be carried out in organic solvents.
References
1) A.M.Klibanov, "Improving Enzymes by using them in organic solvents" Nature, 2001, 409, 241-246.
2) C. Carrasco-Lopez, C. Godoy, Blanca de las Rivas, G. Fernandez-Lorente, J. M. Palomo, J. M: Guisàn, R. Fernàndez-LaFuente, M. Martìnez-Ripoll and J. A. Hermoso "Activation of bacterial Thermoalkalophilic Lipases Is Spurred by Dramatic Structural Rearrangements" Journal of Biological Chemistry, 2009, 284, 4365-4372.
3) G.Bayram Akcapinar, A. Venturini, P.L. Martelli, R. Casadio and U. O. Sezerman, "Modulating the thermostability of Endoglucanase I from Trichodermareesei using computational approaches" Prot. Eng. Design and Selection, 2015, 20, 127-135.

Obiettivi della ricerca

- To study the structural properties of lipase enzymes in organic and mixed water/organic solvents.
- To develop robust and selective chemical protocol for establishing enzyme mutations.
- To generate new fundamental knowledge in enzyme technology.

Ultimo aggiornamento: 09/08/2025