Abstract:Artemia salina is a small crustacean. The hatched larvae are high-quality bait for aquatic animals, and the remaining eggshells are treated as waste. However, Artemia salina eggshells are rich in protein and chitin, which have great potential for application. To prepare chitin oligosaccharides from Artemia salina eggshells, chitinase ChiB565, which is derived from Aeromonas veronii, and has a wide distribution of hydrolysates and contains chitin binding domain, is selected and recombinantly expressed in Pichia pastoris. The enzymatic properties are studied and high-density fermentation is carried out. A green process for preparation of chitin oligosaccharides by synergistic treatment of Artemia salina eggshells with neutral protease and recombinant chitinase ChiB565 is established. The results show that the highest enzyme activity of recombinant bacteria fermentation is 13.2 U/mL, which is 3.88 times that of shake flask fermentation. When Artemia salina eggshells are treated with neutral protease (30 mg/g substrate and 4 h) first to remove protein, and then treatd with recombinant ChiB565(40 U/g substrate and 5 h) to hydrolyze chitin, products are mainly chitobiose to chitotetraose, with a small amount of chitopentaose and chitohexaose. And the scavenging ability of the hydrolysate to DPPH and ABTS are 59.94% and 53.15%, respectively. The studies above illustrat that the preparation of chitin oligosaccharides by biological enzymatic method using Artemia salina eggshells as substrate has a higher recovery rate and a shorter time than traditional chemical methods. This research has laid an important foundation for the industrial preparation of chitin oligosaccharides.
