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.

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.

In this study, Schizosaccharomyces pombe, a natural inositol auxotroph, was transformed with the two plasmids, pADH-INO and pSPIN-22, which contain a Pichia pastoris INO1 gene encoding inositol-phosphate synthase. The two transformants named Sch. p944 and Sch. p1025 were obtained, respectively. The results show that the plasmids in the cells could replicate autonomously and confer inositol prototrophy to S. pombe, but the rate of inositol synthesis and the amount of inositol synthesized in Sch. p944 were faster and more than those in Sch. p1025. Plasmid loss experiments show that the rate of plasmid loss in Sch. p944 was lower than that in Sch. p1025. More mRNA encoding inositol synthase in Sch. p944 was detected than that in Sch. p1025.We found that the highest specific invertase activity occurred when the Sch. p944 was grown in the synthetic medium containing 1.0% (w/v) glucose, whereas repression of invertase secretion occurred when Sch. p1025 was cultivated in the same medium containing glucose more than 0.2% (w/v). It was also found that more mRNA encoding secreted invertase and more phosphatidylinositol (PI) and less phosphatidylserine (PS) contents existed in Sch. p944 than those in Sch. p1025. The results show that the increase in produced inositol in the cells could lead to the increase of PI and the decrease of PS. These results also mean that PI may be involved in derepression of invertase secretion at transcriptional level.

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℃.