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.

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.

Cyanothece sp. 113, a unicellular, aerobic, diazotrophic and photosynthetic marine cyanobacterium, produced 22.34 g/l of exopolysaccharidein 11 days at 29℃, aeration rate of 7.0 l/min and continuous illumination with 4300 lux. After purification, the spectra of UV, IR, 1H NMR, 13C NMR and GC–MS analysis showed that the purified exopolysaccharide was a-D-1,6-homoglucan. This is first report describing linear a-D-1,6-homoglucan exopolysaccharide produced by marine cyanobacteria.

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.

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.