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。

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.

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.

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadexi G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45℃, respectively. The enzyme was activated by Cu2+ (at a concentration of 1.0 mM) and Mn2+ and inhibited by Hg2+, Fe2+, Fe3+, Zn2+, and Co2+. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 17–10-phenanthroline, and iodoacetic acid. The Km and Vmax values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 mmol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggestthat the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.

Marine yeast strain 1, isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the medium. This marine yeast was identiWed as a strain of Pichia guilliermondii according to the results of routine yeast identiWcation and molecular methods. The crude inulinase produced by this marine yeast worked optimally at pH 6.0 and 60