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.

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。

Under the optimal conditions, 10 U/ml of glucoamylase was produced by the marine yeast Aureobasidium pullulans N13d. It was noticed that the crude glucoamylase actively hydrolyzed potato starch granules, but poorly digested raw corn starch and sweet potato starch, resulting in conversion of 68.5, 19 and 22% of them into glucose within 6 h of incubation in the presence of 40 g/l of potato starch granules and 20 U/ml of the crude enzyme. When potato starch granules concentration was increased from 10 to 80 g/l, hydrolysis extent was decreased from 85.6 to 60%, while potato starch granules concentration was increased from 80 to 360 g/l, hydrolysis extent was decreased from 60 to 56%. Ratio of hydrolysis extent of potato starch granules to hydrolysis extent of gelatinized potato starch was 86.0% and the hydrolysis extent of potato starch granules by action of the crude glucoamylase (1.0 U/ml) was 18.5% within 30 min at 60 8C. Only glucose was detected during the hydrolysis, indicating that the crude enzyme could hydrolyze both a-1,4 and a-1,6 linkages of starch molecule in the potato starch.

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.

Yeast strain Y68, producing a large amount of pullulan, was isolated from the leaves collected in the south of China. This strain was identified to be Rhodotorula bacarum by BIOLOG analysis and routine yeast identification. This is the first time to report that pullulan was produced by R. bacarum. The optimal medium for pullulan production by this strain was 8.0% (w/v) glucose, 2.0% (w/v) soybean cake hydrolysate, 0.5% (w/v) K2HPO4, 0.1% (w/v) NaCl, 0.02% (w/v) MgSO4·7H2O, 0.06% (w/v) (NH4)2SO4, pH 7.0. The optimal cultivation conditions for pullulan production by this strain in 300-ml shake flask containing 50 ml of medium were observed at 28℃ and with 180 rpm. Under these conditions, 5.9% (w/v) pullulan was produced within 60 h. This was the highest pullulan yield produced by yeasts obtained so far. No pigment in the medium was observed during the fermentation, suggesting that strain Y68 was a non-pigmented yeast strain.