At present, there have been many molecular modifications for alginate, mainly focused on site directed mutagenesis and domain recombination. For example, Lu et al. obtained a mutant with a 75.80% increase in enzyme activity compared to the wild type through site mutagenesis[8]. Zhang et al. obtained a mutant with enzyme activity 2.35 times higher than the wild-type through domain recombination[9]. However, the use of error prone PCR for the modification of alginate enzymes is still limited. Li et al. used error prone PCR technology to increase the activity of alginate, causing enzyme activity to be 2.41 times higher than before [10]. Our experiments have confirmed the significant potential of error prone PCR to modify alginate enzyme activity, which can be applied to improve alginate enzyme activity, increase alginate oligosaccharide production, and meet industrial needs.

Alginate oligosaccharides have extremely high value, and their main production method is still the degradation of alginate by alginate enzymes. Wild type enzymes often have low activity. However, there are still not many highly active alginate enzymes that have been artificially modified. This experiment is a beneficial attempt. We hope to use error prone PCR technology to modify and obtain more highly active alginate enzymes in the future.

In the experiment, we noticed some issues that need to be studied and improved. Alginate cannot self color and lacks the necessary identification markers for high-throughput screening. The traditional DNS method is slightly cumbersome, and the method we use to detect the amount of product using the value of OD₂₃₅ is relatively simple. However, the value of OD₂₃₅ is difficult to accurately quantify the product. It can only be used for comparison, indicating that the activity of the mutant is higher than that of the wild type. In addition, this method may be affected by some interfering factors. 

Due to the reaction characteristics, the electrophoretic bands of error-prone PCR products are not very bright, and sometimes there was not enough product which can be recovered. Our strategy is to conduct PCR reactions on multiple tubes simultaneously. 

During the dry lab, site-directed mutagenesis yielded the R30W variant, which demonstrated enhanced enzymatic activity relative to the 5ZU5 construct. This increase in activity may be attributed to the fact that, despite the molecular docking scores remaining invariant between R30W and 5ZU5, the number of binding sites with sodium alginate for R30W has been augmented. An increased number of binding sites could potentially amplify the intermolecular interactions between the protein and its ligand, culminating in a heightened binding affinity and, consequently, an elevation in the protein's bioactivity. 

We also encountered the issue of inconsistent standards for the activity of alginate enzymes. We attempt to construct a model that can predict the enzyme activity of a specific sequence encoding alginate enzymes. However, the inconsistent enzyme activity measurement standards in the papers have posed difficulties for our attempts to construct models. We hope to develop a unified and easily measurable standard to measure the activity of alginate enzymes.