ABSTRACT
Disulfide bond formation protein A, DsbA, is one of the important proteins located in E. coli periplasm, which is a foldase facilitating the folding of nascent secreted proteins, especially for those with many pairs of disulfide bonds. The crystal structure and phylogenetic analysis of DsbA and DsbA-mediated protein folding, alternatively in vivo and in vitro, are summarized. Both the extremely low pKa of Cys30 , about 3.5, and the destabilizing effect of the active site disulfide contribute to its strong oxidizing power. The Cys30 is also considered as the most important residue closely related to its activity using site-directed mutagenesis methodology. DsbA could effectively assist proteins folding, both in vivo coexpressed with the target protein, and in vitro replenished as foldases. Moreover, DsbA also has the chaperone-like activity in the assistant refolding of genetically engineered inclusion bodies.
Subject(s)
Amino Acid Sequence , Disulfides , Chemistry , Metabolism , Escherichia coli , Genetics , Escherichia coli Proteins , Chemistry , Classification , Metabolism , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Disulfide-Isomerases , Chemistry , Classification , Metabolism , Protein Folding , Protein Structure, Tertiary , Sequence Homology, Amino AcidABSTRACT
The 11-hydroxylation of 16alpha,17alpha-epoxy-4-pregenene-3,20-dione as a useful intermediate for the preparation of hormones can be achieved by the mycelium of Absidia coerulea at higher conversion rate than using other strains. In this paper 16alpha,17alpha-epoxy-4-pregenene-3,20-dione mixed with a little water, beta-cyclodextrin, Tween-80 was introduced into the fermentation broth after ultrasonication to increase pseudo-water-solubility of the hydrophobic substrate. This pseudo-crystallo feed could avoid the toxicity of organic solvents and was more available for the microbial transformation. The multi layer feed-forward neural network was used to setup a model which indicated the relationship between medium and feed components and the conversion rate. Particle swarm optimization (PSO), which was a stochastic global optimization algorithm and of which the convergence speed was high, was applied to obtain the optimal concentration of the medium and feed components. At optimum conditions with the pseudo-crystallo feed, the conversion rate of 16alpha,17alpha-epoxy-4-pregenene-3,20-dione at an initial concentration of 10 g/L was 87.5% in shaking flasks. The conversion rate of the substrate was up to 86.6% at higher concentration of 20 g/L feed in a 3.7 L fermentor.
Subject(s)
Absidia , Metabolism , Fermentation , Hydroxylation , Pregnenediones , MetabolismABSTRACT
The membrane of sodium cellulose sulphate ( NaCS)-poly dimethyldiallylammonium chloride (PDMDAAC) microcapsule is compact and has low molecular weight cut-off, which would delay the mass transfer and affect the cell growth immobilized in the capsule. Macroporous NaCS-PDMDAAC microcapsules were prepared using the degradation of the starch by amylase in the membrane of the capsules. The pore size and the permeability in the membrane were improved obviously. As model cells, the Candida krusei CK1 and E. coli EC1 immobilized in the capsules were cultured in the shake flask and bubble column respectively. It was shown that the cell density immobilized in the microcapsules cultured in the bubble column was higher than that cultured in the shaking flask. It implied that the limiting factor of the cell growth in the capsule lied in the diffusion of the oxygen. Since the rate of the oxygen transporting across the membrane was greatly enhanced due to the enlarged pore size, the maximum cell density in the macroporous capsules was 20%-110% over than that in the standard capsules in the bubble column. However, the extent of E. coli cell density increasing was higher than that of the yeast, which may be due to the difference of the oxygen requirement between the two microbes.
Subject(s)
Amylases , Metabolism , Bioreactors , Candida , Capsules , Chemistry , Cell Culture Techniques , Methods , Cells, Immobilized , Cellulose , Chemistry , Escherichia coli , Membranes, Artificial , Polyethylenes , Chemistry , Porosity , Quaternary Ammonium Compounds , Chemistry , Sodium , Surface Properties , Temperature , Time FactorsABSTRACT
A novel multi-components microcapsule--SA/CS-CaCl2/PMCG system was introduced. The effects of PMCG and SA/CS-CaCl2/PMCG microcapsules on the growth of free E. coli and Saccharomyces cerevisiae were studied respectively. In addition, the growth of immobilized E. coli and Saccharomyces cerevisiae were also investigated. The results showed that: Just like other synthetic polycations, PMCG above certain concentration (0.5%) strongly inhibited the growth of free E. coli and Saccharomyces cerevisiae, but SA/CS-CaCl2/PMCG microcapsules almost had no effects on their growth and on the consumption of glucose concentration by Saccharomyces cerevisiae. What's more, immobilized E. coli and Saccharomyces cerevisiae grew almost as normally as free cultivation. As a whole, SA/CS-CaCl2/PMCG microcapsules had good biocompatiability and can be used as a new immobilization system.
Subject(s)
Alginates , Toxicity , Biocompatible Materials , Toxicity , Calcium Chloride , Toxicity , Cellulose , Toxicity , Escherichia coli , Glucuronic Acid , Hexuronic Acids , Polymers , Toxicity , Saccharomyces cerevisiaeABSTRACT
The colorimetric method has been well developed to detect the sensitivity of most antiproliferative drugs with the advantages of objectivity and accuracy. Many parameters including the dosage and the absorbance of crystalline violet, the extraction time of the solvent, plate effect and marginal effect were investigated, then the optimized method was applied further to measure the biological activity during the refolding of recombinant human IFN-? inclusion bodies.