Satoru Takamatsu

Production of l-alanine from ammonium fumarate using two immobilized microorganisms

SummaryFor the efficient production of l-alanine from ammonium fumarate using the aspartase activity of immobilized Escherichia coli cells and l-aspartate β-decarboxylase activity of immobilized Pseudomonas dacunhae cells, alanine racemase and fumarase activities should be eliminated. We investigated various procedures to eliminate these side reactions, and found that both activities of intact E. coli cells could be eliminated by treating the culture broth at pH 5.0 and 45° C for 1 h, and those of intact P. dacunhae cells could be eliminated by treating the culture broth at pH 4.75 and 30° C for 1 h. Further, it was confirmed that l-alanine was efficiently produced using these two immobilized pH-treated microorganisms.

Improvement of production of L-aspartic acid using immobilized microbial cells

SummaryIn our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in χ-carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in χ-carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation.

Improvement of nitrogen supply for L-threonine production by a recombinant strain of Serratia marcescens

Serratia marcescens T-2000 was previously reported to be an l-threonine-producing strain that harbors the recombinant plasmid carrying the mutant-type threonine operon. This strain produced 55 g of l-threonine/L of the medium containing urea as a nitrogen source after 72 h of cultivation. In the urea-containing medium, transitory stop of the growth was observed during the early period of cultivation when the entire amount of ammonium ion formed from urea via heat decomposition disappeared in the medium. This indicated that the shortage of ammonium supply in cells might delay both the cell growth and the l-threonine production. The use of ammonia water as a nitrogen source for l-threonine production was therefore studied, because microbial cells generally assimilate this source more readily than urea. When ammonia water was automatically fed to the medium so as to maintain the pH of the medium at around 7, the growth was accelerated, and the l-threonine production reached a maximum of 65 g/L at 48 h. Under these conditions, sucrose, a carbon source, was continuously fed to the medium, resulting in the production of 100 g of l-threonine/L at 96 h. Thus, the l-threonine productivity of the recombinant l-threonine-producing strain could be increased by devising the method for supply of a nitrogen source.

[43] Continuous l-alanine production using two different immobilized microbial cell preparations on an industrial scale

Publisher Summary This chapter describes the method of preparation of immobilized microbial cells suitable for industrial application and the bioreactors used for sequential enzyme reactions. L-Alanine is used as a component of amino acid infusion and as a food additive because of its good taste. It has traditionally been produced from solid L-aspartic acid by a batch enzymatic method using the activity of L-aspartate β-decarboxylase of intact Pseudomonas dacunhae cells. In turn, L-aspartic acid has been efficiently produced from ammonium fumarate using the aspartase activity of immobilized Escherichia coli cells. Tanabe Seiyaku Co. Ltd. successfully industrialized a continuous production system of L-alanine from ammonium fumarate, using a column reactor containing immobilized pH-treated E. coli cells and a closed column reactor containing immobilized pH and glutaraldehydetreated P. dacunhae cells. By this system, L-alanine has been produced at low cost. This is considered to be the first industrial application of sequential enzyme reactions using two immobilized microbial cells.