Assessment of Enzyme Stability in Subsurface Sediments by Computational Methods

The microorganisms found in marine subseafloor sediment play a vital role in global carbon and nitrogen cycles, with an estimated 2.9×10²⁹ cells, accounting for about 0.6% of Earth’s total living biomass. These microbes grow at a very slow rate, with carbon turnover occurring over the course of years to thousands of years, about six orders of magnitude slower than sulfate reducing bacteria in pure culture. These slow metabolic rates suggest that the enzymes they produce must also have extended lifespans in order to be effective over such long periods of time. As a result, these enzymes are likely to be highly stable. The central question of my thesis is: "Do subsurface microbes have more stable proteins compared to other organisms?". I tested this hypothesis by examining various Carbohydrate-Active enzyme (CAZy) families within sediment samples obtained from the Baltic Sea at two distinct depths (25 cm and 15 meters below the seafloor). previous research suggested a distinction in the stabilities of proteins belonging to two distinct families, GH29 and GH109.

I investigated the stability of proteins by using Gibbs free energy of folding ∆G_fold. Some prokaryotic proteins intended for export have an N-terminal signal peptide that directs them to the secretion pathway. Having this signal peptide signifies a substantial environmental change, therefore I conducted a comparative analysis of protein pairs, ensuring both pairs either contained signal peptides or lacked them, revealing a significant stability difference, with higher stability noted at greater depth.

Also, the stability is notably higher in protein pairs lacking signal peptides in deep samples compared to surface samples, predominantly among pairs from the glycoside hydrolase (GH) family. Contrary to our expectations, our investigation revealed no significant difference in the stability of extracellular proteins between deep and surface samples. This outcome does not align with our initial hypothesis, indicating that the environmental depth may impact the stability of these proteins. This suggests that the presence of a signal peptide may play a role in the environmental adaptability and stability of proteins, providing a specific area of interest for further research to understand the protein stability across different environments.