TY  - GEN
AV  - public
A1  - Kling, Christopher
CY  - Heidelberg
TI  - A Molecular Flip: How Polyols Affect the Stability and Activity of an Industrial Relevant Subtilisin Protease in Liquid Formulations
Y1  - 2024///
N2  - Due to their natural origin, enzymes are highly adapted to the physiological conditions in living systems. Thus, their application in industrial processes is often hindered by insufficient stability. In addition to the targeted modification of the enzyme structure, immobilization on surfaces, or encapsulation, targeted adjustment of the surrounding medium can contribute to increased enzyme stability. Consequently, osmolytes such as polyols are added to liquid formulations after fermentation, in downstream processing or before storage. This is to ensure the highest level of stability and prolonged activity.
Storage stability is monitored by determining the active enzyme content at several points in time. This allows the effect of different additives to be compared with each other. In this context, the MIRA infrared spectrometer was validated as an alternative to conventional photometric assays and analytical methods for determining the enzyme/solvent concentration. MIRA combines classical IR spectroscopy and AI-based approaches for the reproducible quantification of analytes in aqueous environments. By using multivariate data analysis and the developed workflow to reduce the influence of the solvent signal by using difference spectra and external parameter orthogonalization (EPO), PCR/PLS models with a so far unmatched accuracy could be generated based on different subtilisin protease/endocellulase formulations with 1,2-propanediol. The determination of enzyme/solvent concentration in liquid formulations by means of IR spectroscopy is a completely new approach that has several advantages over conventional methods. These include a missing need for sample processing, the speed of the method and the high information content of a measurement, which in combination with modern data processing makes additional analytical methods obsolete.

Since differences in storage tests, dependent on the enzyme and formulation, only become apparent after days, weeks or months, the determination of thermal stability by differential scanning calorimetry (DSC) has established itself as an alternative. However, the thermal stability in formulations, especially for proteases, can only be partially matched with storage test results. The reason for this discrepancy was to be investigated further using DSC measurements, molecular dynamic (MD) simulations and long-term storage test studies. In this context, the influence of polyols (including glycerol, sorbitol and 1,2-propanediol) and their concentration (10-50 wt%) on the thermal and long-term stability of a subtilisin protease was evaluated. The determined Tm values of the DSC measurements showed a strong linear correlation with molecular descriptors of the MD simulations. In contrast, no correlation could be determined between DSC and storage test results.
While a possible influence of the polyols on the number of bound calcium ions and thus on enzyme stability/activity was not confirmed, an inhibitory effect of smaller/hydrophobic polyols on Km and Vmax in the presence of the artificial protease substrate Suc-AAPF-pNA could be demonstrated by the characterization of enzyme kinetics. SDS-PAGE analyses further allowed to determine the contribution of autoproteolysis to the total loss of active enzyme in the storage test samples and thus to demonstrate the interaction between thermal unfolding and autoproteolysis. Based on the MD trajectories, a flipping of the catalytic histidine side chain and an associated increase in the distance between the catalytic histidine N?2 and serine O? depending on the size and hydrophilicity/hydrophobicity of the polyols could be determined as a possible cause. Consequently, it could be demonstrated, for the first time, that polyols, in addition to their partial stabilizing effect on the enzyme structure, can have an influence on the catalytic activity or autolysis of proteases. Since this effect is not evident in DSC measurements, the results obtained provide new insights into the discrepancy between DSC and storage test results. Based on this, new approaches in the field of enzyme stabilization and furthermore new ideas for the targeted adaptation of the enzyme structure to different formulation environments arise.
ID  - heidok33518
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/33518/
ER  -