Expression of Aspergillus niger N5-5 in E. coli and purification and identification of products

Due to the feature of high hydrolysis, tannase is widely used in food, beverage, brewing and other fields. However, high cost in producing natural tannase makes it difficult to apply tannase to industry in a large-scale. Microbial expression systems can be used for preparing numerous amount of enzyme at low cost, so in this paper Aspergillus niger N5-5 was expressed using E. coli system. Specific primers were designed based on the Aspergillus niger N5-5 sequence N3 (GenBank, No.: KP677552), and tannase gene tan was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification. Then, tannase gene tan was connected to expression vector by NdeI/HindIII enzyme cutting. In this way, recombinant expression vector tan-pET43.1a was formed. Then, the expression vector pET43.1a by NdeI/HindIII enzyme cutting was transformed into E. coli BL21 (DE3) to induce expression of Aspergillus niger N5-5. When the induced fungi were disrupted by the ultrasonic wave, the crude enzyme was extracted and purified by using the IMAC, and then the activity of the crude enzyme and pure enzyme was determined. According to the results of determination of the tannase activity, the tannase activity of the crude enzyme was greatly improved after the crude enzyme was purified, and the specific activity of the pure enzyme was about 8 times of that of the crude enzyme. The results of SDS-PAGE of the pure enzyme showed that the molecular mass of the pure enzyme was about 65 kDa/64–65 kDa, which was consistent with the expected result (64.2 kDa), It can be concluded that the crude enzyme solution was purified successfully. The results of pure enzyme’s protein identification by Western Blotting showed that clear protein bands pro-3 were observed. Molecular mass of clear protein bands pro-3 was about 65 kDa, which was in line with the expected results (64.2 kDa). It can be seen that the aforementioned expression protein could be specifically combined with His tag. It proved expression protein to be a recombinant fusion protein with 6 His tag.     

Molecular Epidemiology of Nontyphoidal Salmonella in Poultry and Poultry Products in India: Implications for Human Health

Human infections with non-typhoidal Salmonella (NTS) serovars are increasingly becoming a threat to human health globally. While all motile Salmonellae have zoonotic potential, Salmonella Enteritidis and Salmonella Typhimurium are most commonly associated with human disease, for which poultry are a major source. Despite the increasing number of human NTS infections, the epidemiology of NTS in poultry in India has not been fully understood. Hence, as a first step, we carried out epidemiological analysis to establish the incidence of NTS in poultry to evaluate the risk to human health. A total of 1215 samples (including poultry meat, tissues, egg and environmental samples) were collected from 154 commercial layer farms from southern India and screened for NTS. Following identification by cultural and biochemical methods, Salmonella isolates were further characterized by multiplex PCR, allele-specific PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR and pulse field gel electrophoresis (PFGE). In the present study, 21/1215 (1.73 %) samples tested positive for NTS. We found 12/392 (3.06 %) of tissue samples, 7/460 (1.52 %) of poultry products, and 2/363 (0.55 %) of environmental samples tested positive for NTS. All the Salmonella isolates were resistant to oxytetracycline, which is routinely used as poultry feed additive. The multiplex PCR results allowed 16/21 isolates to be classified as S. Typhimurium, and five isolates as S. Enteritidis. Of the five S. Enteritidis isolates, four were identified as group D Salmonella by allele-specific PCR. All of the isolates produced different banding patterns in ERIC PCR. Of the thirteen macro restriction profiles (MRPs) obtained by PFGE, MRP 6 was predominant which included 6 (21 %) isolates. In conclusion, the findings of the study revealed higher incidence of contamination of NTS Salmonella in poultry tissue and animal protein sources used for poultry. The results of the study warrants further investigation on different type of animal feed sources, food market chains, processing plants, live bird markets etc., to evaluate the risk factors, transmission and effective control measures of human Salmonella infection from poultry products.     

An unusual new species of the genus Brachypalpus Macquart (Diptera: Syrphidae) from Eastern Asia

Brachypalpus (Brachypalpus) longifacies sp. nov. is described and illustrated from Eastern Asia (Russian Far East and Japan). It differs from other species of the nominative subgenus by several unusual characters, i.e. face in profile weakly convex dorsally and concave ventrally, antennae very elongated, and abdomen with reddish transversal maculae on 2nd tergum (male) or 2nd and 3rd terga (female). A key is given for known species of the subgenus Brachypalpus.LSID: urn:lsid:zoobank.org:pub:634CB6E2-B2C1-40EC-8811-BF3DD4BDE69A     

Phosphoproteomic Approaches to Discover Novel Substrates of Mycobacterial Ser/Thr Protein Kinases

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Highlights

• •Mapping kinase-substrate relationships is vital in discovering new tuberculosis drug targets.
• •LC-MS/MS-based phosphoproteomics expand mycobacterial STPK substrate catalogues.
• •We review and integrate MS-generated datasets on novel candidate substrates.
• •Validation studies are necessary to confirm true physiological substrates of STPKs.

     

Purification and Characterisation of Keratinase from a Thermotolerant Feather-degrading Bacterium

Summary Isolation and identification of a thermotolerant feather-degrading bacterial strain from Thai soil as well as purification and properties of its keratinase were investigated. The thermotolerant bacterium was identified as Bacillus licheniformis. The keratinase was purified to homogeneity by three-step chromatography. The purified enzyme exhibited a high specific activity (218 U mg⁻¹) with 86-fold purification and 25% yield. The enzyme was monomeric and had a molecular mass of 35 kDa. The optimum pH and temperature for the enzyme were 8.5 and 60 °C, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA and iodoacetamide, but was stimulated by metal ions. It hydrolysed soluble proteins with a relative activity of 4–100% and insoluble proteins, including keratins, with a relative activity of 3–35%. Therefore, the enzyme could improve the nutritional value of meat- and poultry-processing wastes containing keratins, collagen and gelatin.     

注射用重组人干扰素βser17的分离纯化及鉴定

重组人干扰素βser17(rhIFN βser17)工程菌经发酵培养后,采用高压匀浆破菌、离心分离包涵体、有机抽提、酸沉淀、SephacrylS -200、SephadexG -75等进行分离纯化,通过各种方法对终产品进行鉴定。结果显示,纯化的rhIFN- βser17比活性超过 2×107IU/mg,纯度超过 99%,其它各项质量指标均符合质量标准。通过研究建立了大规模制备rhIFN- βser17的纯化工艺,研制出了符合规程要求的注射用rhIFN -βser17纯品,为临床研究奠定了基础。