Alginate oligosaccharides have found extensive applications in the fields of food, healthcare, and biomedicine. Alginate lyase serves as the primary tool for the preparation of alginate oligosaccharides. Thus, the identification of alginate lyases with high enzymatic activity is of significant importance. However, the activity of wild-type alginate lyases is often insufficient for practical applications. Therefore, engineering alginate lyases at the molecular level to enhance their activity is a promising strategy.
Error-prone PCR is a widely utilized technique in directed evolution. By altering the reaction conditions, it increases the mutation rate during PCR, leading to the random incorporation of incorrect bases into the amplified genes at a certain frequency, thereby generating a population of randomly mutated DNA. Among these mutants, some may exhibit improved performance. Our objective is to carry out directional evolution based on the existing high-activity sequences and obtain sequences with higher enzyme activity. We employed error-prone PCR for the directed evolution of these genes, followed by the expression of alginate lyases in Escherichia coli. The produced enzyme solution will then be reacted with alginate. We measured the enzymatic activity of each mutant, with the aim of identifying mutants with enhanced activity that hold potential for industrial application. Additionally, from the point of view of computational simulation, the possibility of obtaining high activity enzyme was discussed. We found a positive mutant protein molecule R30W.
