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The qualitative (TLC) and quantitative (colorimetry and HPLC) composition of saponins (ginsenosides) in Panax quinquefolium originating from the field cultivation in Poland was determined. The saponin content in roots of P. quinquefolium increased with the age of the plant. The largest content of ginsenosides (33-67 mg in 1 g of dry mass) was determined in roots. Especially rich in ginsenosides was a fraction of the finest tertiary roots. Also leaves contained quite high levels of these compounds (over 30 mg/g). Seeds and stems had the lowest content of studied metabolites - about 2 and about 5 mg/g, respectively, of a sum of the six saponins determined. In almost all the raw materials investigated the Re ginsenoside dominated. However, two ginsenosides, namely Re and Rb,, dominated in roots.
The aspartase overproducing mutant B-715 was used as a donor of the aspartase gene for further construction of the aspartase-hyperproducing strains by molecular cloning. In preliminary experiments activity of transformants and their efficiency in L-aspartic acid biosynthesis were compared. The conditions for recombinant strain multiplication, biomass activation and L-aspartic acid biosynthesis were optimized. The optimum temperature for cells multiplication, their activation and for product biosynthesis was 37°C. Two-stage process of the multiplication of bacteria (first in LB medium, and then in FF medium) eliminates the appearing of the inclusion bodies of aspartase in the cells. The shaking during cell activation improved cells productivity. The change of pH in the course of the biosynthesis process was insignificant but did not influence the process.
The strain of Escherichia coli K-12 with high aspartase activity was irradiated with UV. After mutagenesis and selection, the mutant B-715 was isolated which was 4-times more active in L-aspartic acid biosynthesis than parental K-12 strain. The highest productivity was achieved while the strain was cultivated in the ammonium fumarate medium in 37°C for 18-30 hours. It was found that better results were obtained when before the main production step of biosynthesis of L-aspartic acid, the cells of E. coli B-715 were incubated in the activation medium with ammonium fumarate. Activation at 37°C was the most advisable for high efficiency of L-aspartic acid biosynthesis. The productivity of E. coli B-715 during 1 hour biosynthesis process was at the range 0.19-0.35 g of L-aspartic acid per 1 gram of dry mass (biomass) per minute.
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