Heterologous Expression and Functional Analysis of <i>Exiguobacterium</i> Algin Lyase Gene by <i>Pichia Pastoris</i>

Algin is the most abundant substance in algae. Alginate lyase degrades algin, producing algin monosaccharides, disaccharides, and oligosaccharides, which are widely used in bioenergy, food, medicine, and other fields. In this study, an Exiguobacterium strain isolated from rotten kelp exhibited a robust ability to degrade algae. Sequencing of this strain revealed the presence of three different types of alginate lyase. However, the expression of these three genes in Escherichia coli showed lower alginate lyase activity compared to the original strain. After codon optimization, the gene with the highest activity of the three was successfully expressed in Pichia pastoris to produce recombinant EbAlg664. In 5L high-density fermentation, the activity of the recombinant enzyme reached 1306 U/mg protein, 3.9 times that of the original Exiguobacterium strain. Enzymatic analysis revealed that the optimal temperature and pH range of recombinant EbAlg664 were narrower compared to the original strain. Furthermore, the presence of Cu2+ and Co2+ enhanced enzymatic activity, whereas Mg2+ and Fe3+ inhibited the recombinant alginate lyase. This study provides a theoretical and practical foundation for the industrial-scale production of engineered Pichia pastoris with high alginate lyase activity.