Rubroshiraia gen. nov., a second hypocrellin-producing genus in Shiraiaceae (Pleosporales).

Shiraiaceae is an important family in Pleosporales (Dothideomycetes), which includes medical fungi and plant pathogens. Two hypocrellin-producing taxa, Shiraia bambusicola and a novel genus Rubroshiraia gen. nov., typified by Rubroshiraia bambusae are treated in this article. Maximum likelihood analysis, generated via RAxML (GTR+G model), using a combined SSU, LSU, TEF1 and RPB2 sequence dataset, shows that Rubroshiraia is close to Shiraia and belongs to the family Shiraiaceae. Descriptions, illustrations and a taxonomic key are provided for the genera in Shiraiaceae. Rubroshiraia morphologically differs from Shiraia in having small and dark ascostromata and filiform ascospores. Production of the ascostromatal metabolites, hypocrellin A and B, were examined by HPLC and spectrophotometer. The content of hypocrellin A and B of specimen HKAS 102255 (R. bambusae) is twice that produced by HKAS 102253 (S. bambusicola). To clarify the relationship between R. bambusae and Hypocrella bambusae, type material of the latter was examined and provided the illustration.

[Expression of a thermostable a-amylase mutant into Escherichia coli and Pichia pastoris].

Alpha-amylase are of considerable commercial value. It can be produced by a wide variety of microorganisma. The alpha-amylase gene (amyE) from Bacillus licheniformis, which is widely used for the industrial hydrolysis of starch, was mutated (amyEM), then amplified by PCR and inserted into pBV220 and pPIC9k to obtain the recombinant vector pBV220-amyEM and pPIC9k-amyEM. These recombinant vectors were transformed into corresponding competent cell E. coli DH5alpha and P. pastoris GS115 respectively. The resulting recombinant strains, DH5alpha/pBV220-amyEM and GS115/ pPIC9k-amyEM, were then screened by measuring the enzymatic activity and SDS-PAGE. DH5alpha/pBV220-amyEM was induced by temperature and GS115/pPIC9k-amyEM by methanol. In contrast to the parent cells, the a-amylases were expressed in both the recombinant strains. In E. coli the molecular weight was approximately 55kDa; optimal temperature and pH of the recombinant a-amylase were 80 degrees C - 90 degrees C and 6.0 respectively. The recombinant amylase had high activity in pH 5.0 - 5.5 compared to wild type. In Pichia pastoris, the recombinant amylase was secreted to the medium; molecular weight was 60kDa for the putative post-translational modifications; optimal pH shifted to 5.5. The specific activities of alpha-amylase produced by E. coli and P. pastoris were 8.1U/mg and 102U/mg respectively. This result indicated that the alpha-amylase were secreted into the culture medium with high efficiency in the recombinant P. pastoris High activity in high temperature and lower pH properties impart the recombinant amylase potential applications in industry.

[Purification and properties of recombinant extremely thermostable and acid-stable amylase].

Extremely thermostable and acid-stable a-amylase produced by Pichia pastoris GS115/pPIC9K-Amy-228 was purified to electrophoretic homogeneity by the steps of ultrafiltration and PAGE. Purification of about 11.7 fold was achieved with an overall yield of 29.8%. Its molecular weight was estimated to be about 55kD by SDS-PAGE. The isoelectric point was 5.0 (room temperature). Michaelis constant of the enzyme for soluble starch was 1.12g/L. The carbohydrate content was 15.4% by the phenol-sulfuric acid method. The optimum temperature and pH of the enzyme activity were 95 degrees C and 4.5 respectively. The enzyme activity was stable under room temperature in the pH rang of 4.0 - 7.0 for 48 hours. About 60% of the initial enzyme activity was measured after 1h of incubation at 110 degrees C. The activity was strongly inhibited by Fe2+, Cr2+ and Cu2+, While Ca2+ had no effect on it. DTT and EDTA had no effect on the activity.

[Chinese little-fat-tail sheep prion protein gene belongs to PrPARH genotype].

The genomic DNA was extracted from lymphocytes of peripheral blood of Chinese little-fat-tail sheep. The prion protein gene(PrPc) was amplified by polymerase chain reaction. The PCR products were cloned into pUC19 plasmid and was sequenced. The results indicated that 771 bp amplified fragment contains the entire sheep PrPc coding sequence. There were 7 base pairs different with the reported sheep PrPc sequence, leading to three amino acids changes in the protein. It is show that its genotype is PrPARH, and these three polymorphisms may be linked to susceptibility or resistance to scrapie infection. Mature sheep prion protein gene was inserted into pET30a and expressed in E. coli, and 23 kD PrP25-231 protein was obtained. Recombinant PrP25-231 protein was purified by inclusion body washing, ion-exchange chromatography, reverse phase chromatography. Results of SDS-PAGE and Western blot showed that the purified PrP25-231 was obtained.

[Molecular cloning and expression of extremely thermostable and acid-stable amylase gene in Pichia pastoris].

The gene encoding a extremely thermostable and acid-stable alpha-Amylase was amplified by PCR using hyperthermophilic archaebacterium pyrococcus furiosus genomic DNA as template. Then the gene was cloned into the vector of pPIC9K. The recombinant vector pPIC9K-amy was then transformed into E. coli DH5alpha strain. Sequencing test showed that the a-amylase gene cloned consisted of 1305 base pairs and the mature protein encoded by the gene consisted of 435 amino acids. The recombinant vector was transformed into chromosome of methylotrophic yeast Pichia pastoris GS115 strain. Regulated by the alpha-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant a-Amylase was expressed and excreted out of the cells. The expression of the recombinant alpha-amylase was strictly induced by methanol. As induction time increased, the activity of amylase per milliliter medium went up accordingly. After 7 days induction, the activity of the amylase reached the max. The recombinant alpha-amylase exhibited maximal activity at 90 to approximately 100 degrees C and at pHranging from 4.5 to 5.0. The enzyme is so thermostable that after disposed at 100 degrees C for 5 hours over 60% of activity was retained.