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

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℃.

Vibrio anguillarum metalloprotease is an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum. It is synthesized from the empA gene a 611- residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, heterologous expression of the empA gene encoding metalloprotease and export of the recombinant metalloprotease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide (611 amino acids) consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. The empA gene inserted in plasmid pBAD24 was overexpressed in Top10 strain of E. Coli after arabinose induction. The 36kDa polypeptide munoblotting. It was found that recombinant metalloprotease with the EmpA activity and antigenicity was exported into the periplasm of Esherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. Coli are similar to those in V. anguillarum.

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.

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.