Overexpression and export of Vibrio anguillarum metalloprotease in Escherichia coil

Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, hetemlogous expression of the empA gene encoding metallopmtease and export of the recombinant metallopmtease 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 Nterminal pmpoptide, a mature region and a C-terminal pmpoptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metallopmtease as the mature pmtease was further confirmed by SDS-PAGE and im- munoblotting. It was found that recombinant metallopmtease with the EmpA activity and antigenicity was exported into the poriplasm of Escherichia 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.

海洋酵母自溶突变株生产单细胞蛋白及其组分分析

热敏感突变株Z114容易在低渗透压和非耐受温度下将胞内蛋白释放到细胞外部。以雪莲果的浸出物为营养基质,优化主要营养成分。在自然pH,装样量50mL,转速170r/min,接种量10%,温度28℃,180.0g/L雪莲果提取物,25.0g/L豆饼粉水解液,15.0g/L硫酸铵条件下,培养45h后,其细胞内粗蛋白含量达到干细胞质量的59.1%。在37℃热处理条件下,细胞内61%的粗蛋白释放到细胞外部。生长在雪莲果提取物的高产蛋白突变株的必需氨基酸及其它营养物质仍保持较高水平,说明雪莲果提取物可作为热敏突变株生长培养基,进行蛋白质合成与生产。

Enhanced Carotenoid Production by a Mutant of the Marine Yeast Rhodotorula sp. hidai

After a serial of UV, EMS and NTG mutagenesis, a mutant named MM of the red marine yeast strain Rhodotorula sp. hidai was obtained. The mutant MM could produce 603.93 μg g-1 of carotenoid within 5 days in the medium containing 4.0 g sucrose, 1.5 g yeast extract, 0.1 g MgSO4, and 100 mL of sea water, with pH 6.0 and at 30 ℃, while only 213.18 μg g-1 of carotenoid was pro-duced by the wild type under the same conditions.

Amylase Production by the Marine Yeast Aureobasidium pullulans N13d

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pa-cific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identi-fication methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 ℃, aeration rate 6 Lmin-1 and agitation speed 250 rmin-1. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.

Characterization of a Mutant of Alteromonas aurantia A18 and Its Application in Mariculture

Mutant J61321 with enhanced siderophore production of Alteromonas aurantia AI8 was obtained after a series of chemical-physical mutageneses. It was found that the antibacterial activity against Vibrio anguillarum W-1 and siderophore production of the mutant were higher than those by the original strain A 18 which had been used in mariculture. The results of the specific assay（Csaky and Arnow methods） of siderophore showed that the sidrophore with hydroxamate group was produced by mutant J61321 and the original strain A 18, respectively, while the siderophore with catechol group was yielded by strain W-1 （Aibrio anguillarum）. Meanwhile, the siderophore yield, antibacterial activity and anti-chelator activity of strain J61321 were higher than those of its parent strain A 18.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

Occurrence and Diversity of Marine Yeasts in Antarctica Environments

A total of 28 yeast strains were obtained from the sea sediment of Antarctica.According to the results of routine identi-fication and molecular characterization,the strains belonged to species of Yarrowia lipolytica,Debaryomyces hansenii,Rhodotorula slooffiae,Rhodotorula mucilaginosa,Sporidiobolus salmonicolor,Aureobasidium pullulans,Mrakia frigida and Guehomyces pullu-lans,respectively.The Antarctica yeasts have wide potential applications in biotechnology,for some of them can produce b-galactosidase and killer toxins.

Occurrence and Diversity of Candida Genus in Marine Environments

A total of 317 yeast isolates from seawater, sediments, mud of salterns, guts of marine fishes and marine algae were obtained. The results of routine identification and molecular characterization showed that six isolates among these marine yeasts belonged to Candida genus as Candida intermedia for YA01a, Candida parapsilosis for 3eA2, Candida quercitrusa for JHSb, Can- dia rugosa for wlS, Candida zevlanoides for TJY 13a, and Candida membranifaciens for W 14-3. Isolates YA01 a （Candida intermedia）, wl8 （Candida rugosa）, 3eA2 （Candida parapsilosis）, and JHSb （Candida quercitrusa） were found producing cell-bound lipase, while isolate Wl4-3 （Candida membran（faciens） producing riboflavin. These marine yeast Candida spp. seem to have wide potential applications in bioteehnology.

短梗霉普鲁兰多糖合成和调控的研究进展

普鲁兰多糖是一种由短梗霉属真菌合成的胞外多糖。虽然普鲁兰多糖在生物技术的各个领域有着广泛的应用,但其生物合成与调控的具体机制有待进一步研究。近年来,研究人员利用分子生物学方法对普鲁兰多糖的合成与调控的分子机制进行阐明,发现以跨膜蛋白AmAgs2为关键多糖合成酶的合成途径,并揭示cAMP-Proteinkinase A(cAMP-PKA)、Target of rapamycin1(TORC1)、High osmotic glycerol 1(HOG1)和Sucrose nonfermentable 1(Snf1)信号通路参与普鲁兰多糖合成的调控。因此,本文对近年来该领域的研究进行综述,为真菌胞外多糖合成调控的机制提供研究参考,并为高产普鲁兰多糖细胞工厂的构建提供理论支持。