Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi

We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl ß-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines.

Process optimization for pectinase production by locally isolated fungal strain using submerged fermentation / Otimização de processos para produção de pectinase por cepa fúngica localmente isolada usando fermentação submersa

The present study deals with the isolation screening and optimization of fungal strain for pectinase production. The fungal strains were isolated from different sources, including soil, fruits etc. Qualitative screening was performed on the basis of the pectin hydrolysis zone. While, quantitative screening was carried out employing submerged fermentation. Among all the strains the strains showing highest pectinolytic potential were selected identified and assigned the code Aspergillus niger ABT-5.The influence of different fermentation media on pectinase production was evaluated. The M5 medium containing 10g wheat bran, nutrient medium containing (g/l) of (NH4)2SO4 6.0, K2HPO4 6.0, KH2PO4 6.0, MgSO4.7H2O 0.1 gave the highest pectinase production. The other important physico chemical parameters including incubation period, temperature, and volume of media, size of inoculum, carbon and nitrogen sources were also optimized for pectinase production. The highest pectinase production (15.5U/ml) was obtained at 72h of incubation, pH 6, temperature 30°C, volume of media 50ml. Fructose and urea were designated as best carbon and nitrogen sources subsequently.

O presente estudo trata da triagem de isolamento e otimização da cepa fúngica para produção de pectinase. As cepas fúngicas foram isoladas de diferentes fontes, incluindo solo, frutas, etc. A triagem qualitativa foi realizada com base na zona de hidrólise da pectina. Enquanto, a triagem quantitativa foi realizada utilizando fermentação submersa. Entre todas as cepas, as cepas que apresentaram maior potencial pectinolítico foram selecionadas e atribuídas ao código Aspergillus niger ABT-5. Avaliou-se a influência de diferentes meios de fermentação na produção de pectinase. O meio M5 contendo 10g de farelo de trigo, meio nutriente contendo (g / l) de (NH4)2SO4 6.0, K2HPO4 6.0, KH2PO4 6.0, MgSO4.7H2O 0.1, proporcionou a maior produção de pectinase. Os outros parâmetros físico-químicos importantes, incluindo período de incubação, temperatura e volume dos meios, tamanho do inóculo, fontes de carbono e nitrogênio também foram otimizados para a produção de pectinase. A maior produção de pectinase (15,5U / ml) foi obtida às 72h de incubação, pH 6, temperatura 30 ºC, volume dos meios 50ml. A frutose e a ureia foram designadas como melhores fontes de carbono e nitrogênio posteriormente.

Evaluation of microbial potential and ripening behavoiur of preclimacteric bananas following gamma irradiation / Avaliação do potencial microbiana e amadurecem comportamento de bananas preclimacteric seguinte irradiação gama

The current study was carried out to evaluate the effects of gamma irradiation on the epiphytic microflora and ripening process of the green Dwarf Cavendish bananas harvested at the three-quarter stage of the maturity. The mature green bananas were irradiated using Cobalt-60 as the source of irradiation at different dosages of 0.5, 0.75 and 1.0 kGy. The mean life of both the experimental and control group of fruits was analyzed under ambient conditions. For all the treatments the microbial potential, the decay percent and the ripening behavior of the fruits were recorded. Results revealed that the applied radiation doses reduced the decay incidence, delayed ripening process and greatly inhibit the microbial growth (total bacterial and fungal count) thereby enhancing the shelf life of bananas. Irradiation dose of 1.0 kGy was found to be the most effective dose to positively maintain the stored bananas under ambient conditions. The mean life of bananas was extended by 14 days. The identification of the enteric bacteriaeaceae through API 20 E strips revealed the presence of Shigella sonnie on the fruit surface along with Escherichia coli and a nonfermentor spp. The dominant spoilage causing fungi identified were Aspergillus niger, Aspergillus flavus, Collotrichum musae, Fusarium oxysporum,Mucor spp, Lasiodiplodia theobromea and Rhizopus stolonifer.

O Presente estudo foi realizado para investigar os efeitos da radiação gama sobre a microflora epífita e amadurecimento das bananas Cavendish Anão verde colhidas no estádio de três quartos da maturidade. As bananas verdes maduros foram irradiadas usando Cobalto-60 como fonte de irradiação a diferentes dosagens de 0,5, 0,75 e 1,0 kGy. A vida média de ambos os grupos experimental e de controlo de frutas foi analisada sob as condições ambientes. Para todos os tratamentos a potenciais microbiana, o percentual decadência e do comportamento do amadurecimento dos frutos foram recorded.Results revelou que as doses de radiação aplicadas reduziu a incidência de podridões, atrasou processo de amadurecimento e inibir significativamente o crescimento microbiano (contagem de bactérias e fungos total), assim aumentar a vida de prateleira das bananas. dose de irradiação de 1,0 kGy foi encontrada como sendo a dose mais eficaz para manter positivamente as bananas armazenada sob condições ambientes. A vida média de bananas foi prorrogado por 14 dias. A identificação do bacteriaeaceae entérico através de API 20 E tiras revelou a presença de Shigella sonnie sobre a superfície do fruto, juntamente com Escherichia coli e um nonfermentor spp. A deterioração dominante causando fungos identificados foram Aspergillus niger, Aspergillus flavus, Collotrichum musae, Fusarium oxysporum, Mucor spp, Lasiodiplodia theobromea e Rhizopus stolonifer.