An alginate lyase encoding gene sagl from Flavobacterium sp. H63 was codon optimized and recombinantly expressed at high level in P.pastoris through high cell-density fermentation. The highest yield of recombinant enzyme of sagl (rSAGL) in yeast culture supernatant reached 226.4 μg/mL (915.5 U/mL). This was the highest yield record of recombinant expression of alginate lyase so far. The rSAGL was confirmed as a partially glycosylated protein through EndoH digestion. The optimal reaction temperature and pH of this enzyme were 45 °C and 7.5; 80 mM K⁺ ions could improve the catalytic activity of the enzyme by 244% at most. rSAGL was a thermal stable enzyme with T50¹⁵ of 57–58 °C and T50³⁰ of 53–54 °C. Its thermal stability was better than any known alginate lyase. In 100 mM phosphate buffer of pH 6.0, rSAGL could retain 98.8% of the initial activity after incubation at 50 °C for 2 h. Furthermore, it could retain 61.6% of the initial activity after 48 h. The specific activity of the purified rSAGL produced by P. pastoris attained 4044 U/mg protein, which was the second highest record of alginate lyase so far. When the crude enzyme of the rSAGL was directly used in transformation of sodium alginate with 40 g/L, 97.2% of the substrate was transformed to di, tri, tetra brown alginate oligosaccharide after 32 h of incubation at 50 °C, and the final concentration of reducing sugar in mixture reached 9.51 g/L. This is the first report of high-level expression of thermally stable alginate lyase using P. pastoris system.

A novel exopolysaccharide from marine-derived red yeast Rhodotorula mucilaginosa strain YL-1 was produced and characterized. The highest yield of polysaccharide reached 15.1 g/L after medium and culture parameter optimization. This exopolysaccharide, composed of four neural monosaccharides including glucose, mannose, galactose and fucose, had an average molecular weight of 1200 KDa. It had good immunomodulatory activity on RAW256.7 cell lines. ELISA (enzyme linked immunosorbent assay) and Q-PCR (quantitative real-time PCR) results showed that the cell was stimulated to express more IL-6, IL-18, IL-1β and TNFα cytokines than the control group. This is the first report of an exopolysaccharide with immunomodulatory activity from marine-derived Rhodotorula mucilaginosa.

A GH49 dextranase gene DexKQ was cloned from marine bacteria Arthrobacter oxydans KQ11. It was recombinantly expressed using an Escherichia coli system. Recombinant DexKQ dextranase of 66 kDa exhibited the highest catalytic activity at pH 9.0 and 55 °C. kcat/Km of recombinant DexKQ at the optimum condition reached 3.03 s⁻¹ μM⁻¹, which was six times that of commercial dextranase (0.5 s⁻¹ μM⁻¹). DexKQ possessed a Km value of 67.99 µM against dextran T70 substrate with 70 kDa molecular weight. Thin-layer chromatography (TLC) analysis showed that main hydrolysis end products were isomalto-oligosaccharide (IMO) including isomaltotetraose, isomaltopantose, and isomaltohexaose. When compared with glucose, IMO could significantly improve growth of Bifidobacterium longum and Lactobacillus rhamnosus and inhibit growth of Escherichia coli and Staphylococcus aureus. This is the first report of dextranase from marine bacteria concerning recombinant expression and application in isomalto-oligosaccharide preparation.