title: Development and Screening of promiscuous aldolases
Creators: name: Glesner, Tobias
Publisher: name: UNSPECIFIED
Year: 2024

description: Aldolases are enzymes that play a crucial role in the metabolism of carbohydrates. These 
enzymes belong to the class of lyases and exhibit catalytic activity, especially in the Aldol 
reaction, where aldehydes or ketones react with an electrophile to form an aldol bond.
Despite impressive achievements in the development of aldolases, their application has been 
highly specific, such as in the synthesis of building blocks for statin synthesis, as seen in the 
case of DERA. The significant restriction regarding accepted donors for the aldol reaction in 
aldolases has limited their widespread use in industrial applications.
Geometric analysis of the active center based on existing crystal structures allows for the 
expansion of aldolase catalytic activity through mutagenesis. The goal is to enlarge the existing 
space in the active center. In this study, EcDERA enzyme was used as the starting aldolase, 
which already carries a single-point mutation (F200I) on the surface. Enzyme libraries with up 
to three additional point mutations in the active center were generated based on the analysis 
of existing crystal structures. The introduced mutations, all located in the active center, aim 
to expand the available space by introducing "space-saving" mutations with smaller amino 
acids.
Wild-type EcDERA accepts acetaldehyde, acetone, propanal, and fluoroacetone as donors, 
demonstrating a relaxed donor spectrum compared to other known aldolases. The 
introduction of mutations in the active center aims to enable the acceptance of cyclic ketones 
such as cyclobutanone, cyclopentanone, and cyclohexanone. Additionally, other ketones like 
chloroacetone, butanone, 1-hydroxybutanone, and many others were tested for activity. The 
hypothesis that cyclic ketones could be accepted was confirmed. Some enzymes from the 
mutagenic EcDERA library were able to convert cyclopentanone, but none of the enzyme 
variants showed activity for cyclobutanone and cyclohexanone.
Due to the limited success of the mutagenic EcDERA library and the availability of 
metagenomic enzyme libraries DERA and FSA from our partner Prozomix®, these were used 
to find active enzymes for the selection of donors. The use of metagenomic enzyme libraries 
expanded the known donor spectrum for DERA by three donors. New, previously unknown 
activities for DERA enzymes included i-valeraldehyde as an acceptor with butanone, 
methoxyacetone, and chloroacetone. Notably, an aliphatic acceptor was used, unlike the 
examples presented in the literature, mainly generated with D-glyceraldehyde-3-phosphate, 
where the natural acceptor compensates for the lower activity with unnatural donors. Thus, 
the results represent a significant step towards the universal application of aldolases for the 
production of fine chemicals.
The third chapter of this thesis deals with the effects of inorganic salts on enzyme stability. In 
1888, Franz Hofmeister published the results of his study "Zur Lehre der Wirkung der Salze" 
on the salting-out behavior of protein-containing solutions under the influence of inorganic 
salts. For his experiment, Hofmeister used separated liquid from beaten eggs, which settled 
overnight. After adding concentrated salt solutions, he observed different time spans until the 
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first signs of precipitated protein appeared in the form of a whitish cloudiness. His results led 
to the still-relevant "Lyotropic Series" or colloquially known as the Hofmeister series.
For this study, the influence of inorganic salts and other compounds on the thermostability of 
three example enzymes was analyzed. The enzymes used include lysozyme from Gallus gallus, 
alcohol dehydrogenase from Saccharomyces cerevisiae, and FSA from Escherichia coli. 
NanoDSF equipment was used to determine the melting point of enzymes in aqueous solution 
for assessing thermostability. The methodology allows determining the melting point of a total 
of 48 aqueous protein samples based on intrinsic tryptophane fluorescence within an hour, 
depending on the temperature gradients used. The literature standard for the temperature 
gradient is 1°C/minute, and the Prometheus device from NanoTemper can heat the sample 
up to 115°C, making the methodology suitable for high-throughput screenings of 
(hyper)thermophilic enzyme libraries.
The results of this study confirm the Hofmeister series from 1888. With high salt 
concentrations, the trend of the Hofmeister series was observed for all tested enzymes. 
Particularly, the destabilizing portion of the series exhibited consistent behavior with 
Hofmeister's results