Insight into the aggregation of lipase from Pseudomonas sp. using mutagenesis: protection of aggregation prone region by adoption of α-helix structure

Machine Learning Enables Selection of Epistatic Enzyme Mutants for Stability Against Unfolding and Detrimental Aggregation

“…Unfolding stability is an indicator of the robustness of the protein molecular structure, namely the intrinsic folding nature of a single molecule. Aggregation stability describes aggregation formation due to direct intermolecular interactions between native proteins, or between denatured proteins that have already undergone conformational changes [18–20,34,35] . Increased unfolding and aggregation stability are believed to enhance overall protein stability.…”

“…Although based on earlier computational design and directed evolution experiments to produce the learning dataset, our procedure nevertheless circumvents the need to generate and screen large libraries of enzyme variants, the traditional bottleneck of directed evolution [20–24] . Instead, ∼1.2×10 7 mutants were assessed computationally in one shot.…”

“…Aggregation stability describes aggregation formation due to direct intermolecular interactions between native proteins, or between denatured proteins that have already undergone conformational changes. [18][19][20]34,35] Increased unfolding and aggregation stability are believed to enhance overall protein stability. We therefore tested the stability of the randomly selected five variants based on UNcle stability platform and circular dichroism (CD) to confirm the prediction accuracy.…”

“…Undesired protein aggregation is a phenomenon prevalent in diseases such as Alzheimer and in biotechnological applications of enzymes as well. In the present ML study, we address the persisting question of how to prevent detrimental enzyme aggregation following unfolding, which causes a decrease or even shutdown in biocatalytic performance [18–20] . Unfortunately, some protein engineers do not realize that suboptimal enzyme activity might be due to detrimental aggregation.…”

“…In the present ML study, we address the persisting question of how to prevent detrimental enzyme aggregation following unfolding, which causes a decrease or even shutdown in biocatalytic performance. [18][19][20] Unfortunately, some protein engineers do not realize that suboptimal enzyme activity might be due to detrimental aggregation. In the present study, limonene epoxide hydrolase from Rhodococcus erythropolis (LEH), which acts via a one-step reaction mechanism in the hydrolysis of epoxides (Scheme 1), was chosen as the model enzyme.…”