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.
