The marine yeast strain G7a isolated from sediment of China South Sea was found to secrete a large amount of inulinase into the medium. This marine yeast strain was identified to be a strain of Cryptococcus aureus according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast showed the highest activity at pH 5.0 and 50 8C. The optimal medium for inulinase production was artificial seawater containing inulin 4.0% (w/v), K2HPO4 0.3% (w/v), yeast extract 0.5% (w/v), KCl 0.5% (w/v), CaCl2 0.12% (w/v), NaCl 4.0% (w/v) and MgCl2

基于酵母表面展示技术的基因工程活载体疫苗，在鱼类疫苗的研究中至今未见报道。海水鱼类病原菌哈维氏弧菌的溶血素是其主要的毒力因子之一，为制备以酒精酵母为载体的基因工程疫苗，参照其Genebank中基因序列设计一对引物，PCR扩增得到预期长度的产物，双酶切插入用于酒精酵母表面展示的穿梭质粒载体pYD1，转化大肠杆菌TOP10，提取阳性质粒转化酒精酵母菌株EBY100，诱导表达后，用免疫荧光和流式细胞仪检测外源蛋白的表达，结果测得最佳诱导时间为36-48小时，诱导培养后约30.0%左右的酵母细胞表达外源蛋白；同时以EBY100和转空质粒的酵母菌株为对照，测得诱导培养后的阳性酵母转化株的细胞对牙鲆鱼血细胞具有溶血活性，以此酵母活细胞分设高中低三个深度组，腹腔注射养殖牙鲆幼鱼，检测其毒性，结果表明此重组酵母对牙鲆是安全的。为下一步鱼用活载体疫苗免疫效果的研究奠定了基础。

A yeast strain, Aureobasidium pullulans, which could produce the high yield of protease was isolated from sediment of saltern in Qingdao, China. Maximum production of enzyme (623.1 U/mg protein; 7.2U/ml) was obtained in a medium containing 2.5 g soluble starch and 2.0 g NaNO3, 100ml seawater, initial pH 6.0, after fermentation at 24.5℃ for 30 h. The protease had the highest activity at pH 9.0 and 45℃.

Glucose repression occurs in many yeast species and some filamentous fungi, and it represses the expression and secretion of many intracellular and extracellular proteins. In recent years, it has been found that many biochemical reactions in yeast cells are mediated by phosphatidylinositol (PI)-type signaling pathway. However, little is known about the relationships between PI-type signaling and glucose repression, gene expression and invertase secretion in yeasts. Many evidences in our previous studies showed that glucose repression, invertase secretion, gene expression and cell growth were mediated by inositol and PI in Saccharomyces and Schizosaccharomyces. The elucidation of the new regulatory mechanisms of protein secretion, gene expression and glucose repression would be an entirely new aspect of inositol and PI-type signaling regulation in yeasts.

The extracellular zinc-metalloprotease of Vibrio anguillarum is a secreted virulence factor. It is synthesized from the empA gene as a 611-residue preproprotease and processed to the active mature protease (EmpA) with concomitant secretion via the type II secretion pathway. Active EmpA has been found only in the V. anguillarum culture supernatant and the process of the activation seems to vary depending on strains analyzed. To better understand the mechanism of EmpA export and processing, the empA gene was cloned and expressed in Escherichia coli strains. Expression of empA did not have toxic effect on bacterial growth. Rupturing E. coli TOP10 cells by heating in gel-loading buffer resulted in activation of EmpA and severe proteolysis of the samples. In contrast, the same treatment of the E. Coli MC4100A strain did not lead to the general proteolysis. In this strain, EmpA was exported into the periplasm via the Sec pathway. The periplasmic EmpA was detected in two active conformations. Therefore, in E. coli processing of EmpA precursor to an active enzyme did not require secretion to the media and the help of other V. anguillarum protein. Like in V. anguillarum, heterologous expression of empA in E. coli showed strain-specific activation process.

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.