Preserve enzymatic activity in deep eutectic solvents by deciphering biophysical design principles

• Applicant: Professor Dr. Ulrich Schwaneberg
• Subject Area: Biological Process Engineering
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 556217041

Deep eutectic solvents (DES) are promising solvents for the development of environmentally friendly industrial processes using renewable resources. This is primarily attributed to their dissolution properties, their biodegradability, and their often renewable (‘green’) origin. The Decode project aims to understand the molecular reasons behind the DES-enzyme interaction and to discover general design principles for DES-tolerance of enzymes. In the Decode project an ‘in house’ variant library of the Bacillus subtilis Lipase A (BSLA; 181*20= 3620 variants), which contains all the natural diversity of BSLA with one amino acid exchange, will be screened against seven DESs. Through the use of seven DES we will systematically analyze the influence of different hydrogen bond acceptors/donors on the DES-resistance of the BSLA. Statistical analysis of the screening data will reveal key positions for DES-resistance and how the chemical nature of each substitution influences the resistance against DES. Molecular dynamic (MD) simulations will be performed to reveal molecular interactions responsible for DES-resistance. Combining the statistical analysis and the MD-simulations general principles of DES-resistance of BSLA will be formulated. To our knowledge, this is the first systematic study to investigate the influence of full natural diversity on DES-resistance. The transferability of the general principles for DES-resistance of BSLA will be tested with the well-studied cutinase-like PETase TfCut2, which has a similar α/β hydrolase protein fold. Applying the general principles and supported by MD simulations, 200 possibly beneficial TfCut2 variants will be identified and characterized. The data will be used to train the machine learning model MERGE. Following, MERGE can suggest novel beneficial TfCut2 variants. After characterizing these variants, the transferability of the general principles to other α/β hydrolase enzymes will be evaluated. In case of success, the DES-resistance principles can be used for a broad range of α/β hydrolase fold enzymes and computer-based methods for the identification of enzyme variants with improved DES-resistance could be established. These methods would decrease the work effort to identify enzyme variants with improved DES-resistance. Within the Decode project we envision to identify general biophysical principles of DES-resistance of α/β hydrolase fold enzymes and thereby advance molecular understanding as well as computational methodologies for DES-resistance analysis. Very likely, the postulated principles can be transferred to other enzymes with α/β hydrolase fold. With these enzymes and with DES as solvent, efficient and sustainable catalytic routes from biomass (e.g. lignin) or synthetic polymers, with lowered CO2 footprints, compared to many processes using organic solvents, could be established.

• DFG Programme: Research Grants