HL1 gene encoding haemolysin from Vibrio harveyi SF-1 was expressed in yeast cells and the expressed haemolysin was displayed on the cell surface. After induction for 36 h in galactose-containing medium, one-third of the cells contained the displayed protein and the displayed cells had haemolytic activity on erythrocytes from flounder. The double diffusion agar analysis showed that the sera from the flounder immunized with the displayed yeast cells having the haemolytic activity could form precipitate with the purified haemolysin. ELISA analysis indicated that immunization times had great influence on increased production of the specific antibody against haemolysin in turbot immunized with the displayed yeast cells having the haemolytic activity. After the challenge with V. harveyi SF-1, it was found that earlier protection in flounder and significant protection in turbot, both of which were immunized with the displayed yeast cells having the haemolytic activity, were achieved. These results suggested that the displayed yeast cells with the haemolytic activity could be used as potential live vaccine in marine fish.

Trehalose accumulation from starch by Saccharomycopsis fibuligera sdu was examined in 300-ml shaken flask culture and Biostat B2 2-1 fermentation. In the 300-ml flask, 16.5% (w/w) trehalose accumulated in the yeast cells (cell dry weight) was observed with 100-ml medium shaken at 200 rpm for 50 h at 30

The full length empA gene encoding Vibrio anguillarum metalloprotease was amplified by PCR and fused to the expression vector pBAD24. The carboxy-terminal 6xHis-tagged recombinant metalloprotein (rEmpA) was expressed from plasmid pBADVAP6his in E. coli TOP10 and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed a molecular mass of the mature rEmpA predicted to be 36 kDa. The optimal temperature and pH for the purified rEmpA were 37℃ and 8.0, respectively. The enzyme was stable below 30℃ and between pH 5.0 and 8.0, respectively. The results show that Ca2+, Na+ and Mg2+ had an activating effect on the enzyme while Zn2+ and Cu2+ acted as inhibitors of the enzyme. The purified rEmpA was characterized as a zinc metalloprotease as it was inhibited by zinc- and metal-specific inhibitors, such as 1,10-phenanthroline, EDTA and EGTA. The results indicate that some characteristics of EmpA from marine V. anguillarum had been modified after expression and processing in the engineered E. Coli. The purified rEmpA showed degradation activity towards various kinds of proteins, indicating its potential role in pathogenesis.

A pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculatus was identified to be Metschnikowia bicuspidate according to the results of routine yeast identification and 18S rDNA and ITS sequences. After screening of more than 300 yeast strains from different sources in marine environments, it was found that strain YF07b had the highest ability to produce killer toxin against the pathogenic yeast. Strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The optimal conditions for killer toxin production by strain YF07b were the production medium with 2.0% NaCl, pH 4.5, cultivation temperature of 20℃ and the optimal conditions for action of the crude killer toxin against the pathogenic yeast were the assay medium with 6.0% NaCl, pH 4.5 and temperature 15℃.

Total 427 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish, and marine algae were obtained. After inulinase activity of the yeast cultures was estimated, we found that four strains (OUC1, G7a, OUC2, and G7a1) of the marine yeasts grown in the medium with inulin could secrete a large amount of inulinase into the medium. The results of routine identification and molecular methods show that they belong to Pichia guilliermondii OUC1, Cryptococcus aureus G7a, Yarrowia lipolytica OUC2, and Debaryomyces hansenii G7a1, respectively. The optimal pHs of inulinase activity produced by them were 6.0, 5.0, 5.0, and 5.0, respectively, while the optimal temperatures of inulinase activity produced by them were 60-, 50-, 60-, and 50-C, respectively. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis by the crude inulinase produced by P. guilliermondii OUC1, indicating that the crude inulinase had a high exoinulinase activity while a large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase produced both by C. aureus G7a and D. hansenii G7a1. However, no monosaccharides and disaccharides were detected after inulin hydrolysis by the crude inulinase produced by Y. lipolytica OUC2, suggesting that the crude inulinase had no exoinulinase activity.