Projects per year
Abstract
Increased use of lipases is seen across a broad range of industries, often as green alternatives to conventional chemistry. Lipases naturally catalyse the hydrolysis of triglycerides in the presence of water. Substituting water with an organic solvent can shift the equilibrium towards condensation reactions, such as esterification and amidation. This thesis explores lipase performance in organic media at varying water activities using an interdisciplinary approach of enzyme kinetics assays and molecular dynamics (MD) simulations. Three lipases were included in the study, namely Candida antarctica lipase B (CALB), Rhizomucor miehei lipase (RML), and Thermomyces lanuginosus lipase (TLL). One of the most studied lipases is CALB, but the knowledge of its activity in low-water environments is still limited, and the lipase was, therefore, chosen as the primary lipase for this work. The thesis is composed of three main parts, each dedicated to a specific research goal.
In the first part, an optimised drying protocol for aqueous lipase solutions was designed, where the initial water activity (aw) could be controlled using saturated salt solutions. The work on the protocol revealed that continuous stirring and volume correction after the pre-equilibration step is essential to ensure reproducible and consistent enzyme kinetics data. The activity profiles of soluble and immobilised CALB in three organic solvents and at increasing aw are compared. It was subsequently tested if the drying protocol could be used on a fatty acid photodecarboxylase.
In the second part, the activity profile of CALB was investigated with the hypothesis that water cluster formation close to CALB’s active site may govern the lipase’s activity. It has previously been suggested that the water cluster is facilitated by two acidic residues (Glu188 and Asp223) and that the cluster can sterically block the entrance to the active site when it grows in size at high aw resulting in lower enzyme activity. MD simulations and an enzyme kinetics assay were used to study native CALB and two mutants (CALBE188Q+D223N and CALBE188Q+D223L). The study revealed that the specific point mutations diminish the negative effect of high aw on CALB’s activity, indicating that the residues facilitate the water cluster formation. Interestingly, lid movements and active site geometry also influenced the lipase’s activity in hexane at varying aw. It was tested if the water cluster formation of CALB could be detected with attenuated total reflection and transmission infrared spectroscopy.
In the third and last part, the study was extended to an additional two solvents (acetonitrile and methyl tert-butyl ether) and the two lipases, RML and TLL. This study aimed to see how CALB’s activity profile differed from those of other lipases and if lipase performance could be explained and predicted in the organic solvents at different aw. The study revealed that lipases show higher activity in nonpolar solvent than in polar solvents. In addition, the MD simulations indicate that while CALB’s performance is controlled by water cluster formation, the same is not true for RML and TLL. RML and TLL’s activity seems to be governed by the stability of their lipase-substrate complex and the geometry of their active sites.
Overall, the research in this Ph.D. thesis paves the way for a deeper understanding of lipase performance in organic solvents. The thesis reveals that the topic of lipase performance in organic media is a complex issue and that no single parameter can explain nor predict lipase activity. However, a deeper understanding of the topic can be achieved when multiple criteria are considered simultaneously.
In the first part, an optimised drying protocol for aqueous lipase solutions was designed, where the initial water activity (aw) could be controlled using saturated salt solutions. The work on the protocol revealed that continuous stirring and volume correction after the pre-equilibration step is essential to ensure reproducible and consistent enzyme kinetics data. The activity profiles of soluble and immobilised CALB in three organic solvents and at increasing aw are compared. It was subsequently tested if the drying protocol could be used on a fatty acid photodecarboxylase.
In the second part, the activity profile of CALB was investigated with the hypothesis that water cluster formation close to CALB’s active site may govern the lipase’s activity. It has previously been suggested that the water cluster is facilitated by two acidic residues (Glu188 and Asp223) and that the cluster can sterically block the entrance to the active site when it grows in size at high aw resulting in lower enzyme activity. MD simulations and an enzyme kinetics assay were used to study native CALB and two mutants (CALBE188Q+D223N and CALBE188Q+D223L). The study revealed that the specific point mutations diminish the negative effect of high aw on CALB’s activity, indicating that the residues facilitate the water cluster formation. Interestingly, lid movements and active site geometry also influenced the lipase’s activity in hexane at varying aw. It was tested if the water cluster formation of CALB could be detected with attenuated total reflection and transmission infrared spectroscopy.
In the third and last part, the study was extended to an additional two solvents (acetonitrile and methyl tert-butyl ether) and the two lipases, RML and TLL. This study aimed to see how CALB’s activity profile differed from those of other lipases and if lipase performance could be explained and predicted in the organic solvents at different aw. The study revealed that lipases show higher activity in nonpolar solvent than in polar solvents. In addition, the MD simulations indicate that while CALB’s performance is controlled by water cluster formation, the same is not true for RML and TLL. RML and TLL’s activity seems to be governed by the stability of their lipase-substrate complex and the geometry of their active sites.
Overall, the research in this Ph.D. thesis paves the way for a deeper understanding of lipase performance in organic solvents. The thesis reveals that the topic of lipase performance in organic media is a complex issue and that no single parameter can explain nor predict lipase activity. However, a deeper understanding of the topic can be achieved when multiple criteria are considered simultaneously.
Original language | English |
---|
Publisher | DTU Chemistry |
---|---|
Number of pages | 158 |
Publication status | Published - 2024 |
Fingerprint
Dive into the research topics of 'Computational screening tool for lipase performance in organic media'. Together they form a unique fingerprint.Projects
- 1 Finished
-
Computional Screening Tool for Lipase Reactions in Organic Media
Tjørnelund, H. D. (PhD Student), Peters, G. H. J. (Main Supervisor), Brask, J. (Supervisor), Larsen, R. W. (Supervisor), Woodley, J. (Supervisor), Ipsen, J. H. (Examiner) & Pleiss, J. (Examiner)
01/10/2020 → 07/05/2024
Project: PhD