AbstractThe potential of engineered enzymes in practical applications is often constrained by limitations in their expression levels, thermal stability, and the diversity and magnitude of catalytic activities.De-novoenzyme design, though exciting, is challenged by the complex nature of enzymatic catalysis. An alternative promising approach involves expanding the capabilities of existing natural enzymes to enable functionality across new substrates and operational parameters. To this end we introduce CoSaNN (Conformation Sampling using Neural Network), a novel strategy for enzyme design that utilizes advances in deep learning for structure prediction and sequence optimization. By controlling enzyme conformations, we can expand the chemical space beyond the reach of simple mutagenesis. CoSaNN uses a context-dependent approach that accurately generates novel enzyme designs by considering non-linear relationships in both sequence and structure space. Additionally, we have further developed SolvIT, a graph neural network trained to predict protein solubility inE.Coli, as an additional optimization layer for producing highly expressed enzymes. Through this approach, we have engineered novel enzymes exhibiting superior expression levels, with 54% of our designs expressed in E.Coli, and increased thermal stability with more than 30% of our designs having a higher Tm than the template enzyme. Furthermore, our research underscores the transformative potential of AI in protein design, adeptly capturing high order interactions and preserving allosteric mechanisms in extensively modified enzymes. These advancements pave the way for the creation of diverse, functional, and robust enzymes, thereby opening new avenues for targeted biotechnological applications.