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For L-aspartic acid biosynthesis, high production cells of Escherichia coli mutant B-715 and P1 were immobilized in chitosan gel using a technique developed in our laboratory. The immobilization process reduced initial activity of the intact cells, however, the biocatalyst produced was very stabile for long-term use in multi-repeated batch or continuous processes. Temperature influence on the conversion of ammonium fumarate to L-aspartic acid was investigated. In long-term experiments, over 603 hours, the temperature 40°C was found to be the best for both biocatalyst stability and high conversion rate. The optimum substrate concentration was 1.0 M. Continuous production of L-aspartic acid was investigated in three types of column bioreactors characterized by different volumes as well as different high to biocatalyst bed volume rations (Hz/Vz). The highest conversion rate, 99.8%, and the productivity 6 g/g/h (mass of L-aspartic acid per dry mass of cells in biocatalyst per time unit) was achieved in the bioreactor with the highest value Hz/Vz = 3.1, and liquid hour space velocity value of 5.2, defined as the volume of feeding substrate passed per volume of catalyst in bioreactor per one hour.
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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