33 research outputs found
Producción de una fitasa recombinante en Pichia pastoris
En el presente trabajo se construyeron cepas recombinantes
de Pichia pastoris capaces de producir y secretar
una fitasa de origen bacteriano, factible de ser
empleada como aditivo en alimentos para animales.
Las células recombinantes construidas produjeron
en 72 h de cultivo hasta 0.75 U/mL de la fitasa
C de Bacillus subtilis, superando los valores reportados
para las producción de la fitasa C nativa. Pichia
pastoris ofrece una alternativa competitiva para la
producción de esta fitasa bacteriana
Rapid method for Mycobacterium tuberculosis identification using electrospray ionization tandem mass spectrometry analysis of mycolic acids
Mycolic acids (MAs), which play a crucial role in the architecture of mycobacterial cell walls, were analyzed
using electrospray ionization tandem mass spectrometry. A targeted analysis based on the 10 most abundant
and characteristic multiple reaction monitoring pairs was used to profile the crude fatty acid mixtures from
Mtb and several nontuberculous mycobacterial strains. Comparative analysis yielded unique profiles for MAs,
enabling the reliable identification of mycobacterial species. In a case-control study of tuberculosis (TB) and
non-TB Polish patients, we demonstrated the potential diagnostic utility of our approach for the rapid
diagnosis of active TB with sensitivity and specificity surpassing those of existing methods. This robust
method allows the identification of TB-positive patients after 2 h of sample preparation in the case of direct
sputum analysis or 10 days of culturing, both of which are followed by 1 min of liquid chromatography–
tandem mass spectrometry analysis
Diseño de una vacuna in silico contra Naegleria fowleri causante de la Meningitis Amebiana Primaria.
Enhanced heterologous protein production in Pichia pastoris under increased air pressure
Pichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β-galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1-inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β-galactosidase-specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris.The authors acknowledge the financial support provided by FCT (grant SFRH/BD/47371/2008 to Marlene Lopes and grant SFRH/BDP/63831/2009 to Carla Oliveira), by FCT Strategic Project PEst-OE/EQB/LA0023/2013 and by the Project "BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028" Co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER