RESUMO
Ginkgo biloba is a medicinal plant used in complementary and alternative medicines. Ginkgo biloba extracts contain many compounds with medical functions, of which the most critical is ginkgolide B (GB). The major role that GB plays is to function as an antagonist to the platelet-activating factor, which is one of the causes of thrombosis and cardiovascular diseases. Currently, GB is obtained mainly through extraction and purification from the leaves of Ginkgo biloba; however, the yield of GB is low. Alternatively, the immobilized cultivation of ginkgo calluses with biomaterial scaffolds and the addition of organic elicitors to activate the cell defense mechanisms were found to stimulate increases in GB production. The aim of this study was to use Ginkgo biloba calluses for immobilized cultures with different elicitors to find a more suitable method of ginkgolide B production via a recycling process.
RESUMO
An heterologous expression of Vitreoscilla hemoglobin (VHb) for improving cell growth and recombinant protein production has been successfully demonstrated in various hosts, including Pichia pastoris. Lower temperature cultures can enhance target protein production in some studies of P. pastoris. In this study, the strategy of combining heterologous VHb expression and lower temperature cultures in P. pastoris showed that final cell density and viability of VHb+ strain at 23 °C were higher than that at 30 °C. In addition, the effects of VHb expression on recombinant β-galactosidase production and oxygen uptake rate were also higher at 23 °C than at 30 °C. Consequently, lower temperature cultures can enlarge VHb effectiveness on cell performance of P. pastoris. This is because VHb activity obtained at 23 °C cultures was twofold higher than that at 30 °C cultures, due to a different heme production. This strategy makes P. pastoris an excellent expression host particularly suitable for increasing the yields of the low-stability and aggregation-prone recombinant proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Hemoglobinas Truncadas/metabolismo , Proteínas de Bactérias/genética , Biomassa , Proliferação de Células/efeitos dos fármacos , Hemina/farmacologia , Oxigênio/metabolismo , Pichia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Temperatura , Hemoglobinas Truncadas/genética , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Pichia pastoris has been used to produce various recombinant proteins under high oxygen demand conditions. To improve the heterologous production of ß-galactosidase, the vgb gene encoding Vitreoscilla hemoglobin (VHb) was co-expressed in the P. pastoris cytoplasm under the control of the methanol-inducible promoter. Co-expression of VHb under different aeration conditions improved cell performance in terms of growth, viability, respiratory rate, and ß-galactosidase production. Under limiting aeration conditions, the VHb(+) strain produced 28.2% more biomass but 31.2% less total ß-galactosidase activity than the VHb(-) strain. Under non-limiting aeration conditions, the VHb(+) strain showed 20.3% higher cell growth and 9.9% more total ß-galactosidase activity than the VHb(-) strain. Moreover, under these conditions, the VHb(+) strain was 7.7% more viable and had a 28.2% higher oxygen uptake rate (OUR) than the VHb(-) strain. Evidently, VHb can enhance the OUR and promote methanol metabolism, thereby improving cell performance and ß-galactosidase production.
Assuntos
Proteínas de Bactérias/genética , Regulação Fúngica da Expressão Gênica , Pichia/enzimologia , Hemoglobinas Truncadas/genética , beta-Galactosidase/biossíntese , Citoplasma/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Oxigênio/metabolismo , Pichia/genética , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vitreoscilla/genéticaRESUMO
Some pyrogallol and catechol derivatives were synthesized, and their urease inhibitory activity was evaluated by using acetohydroxamic acid (AHA), a well known Helicobacter pylori urease inhibitor, as positive control. The assay results indicate that many compounds have showed potential inhibitory activity against H. pylori urease. 4-(4-Hydroxyphenethyl)phen-1,2-diol (2a) was found to be the most potent urease inhibitor with IC(50)s of 1.5±0.2 µM for extracted fraction and 4.2±0.3 µM for intact cell, at least 10 times and 20 times lower than those of AHA (IC(50) of 17.2±0.9 µM, 100.6±13 µM), respectively. This finding indicate that 2a would be a potential urease inhibitor deserves further research. Molecular dockings of 2a into H. pylori urease active site were performed for understanding the good activity observed.