Continuous Production of L-Alanine Using Pseudomonas dacunhae Immobilized with k-Carrageenan

Abstract

To obtain immobilized Pseudomonas dacunhae cells having higher L-aspartate 8-decarboxylase activity and better operational stability on industrial scale, we investigated the glutaraldehydetreatment of the cells in culture broth before immobilization with k-carrageenan.
When the cells were treated at pH 4.75 and 30°C for 1 h and further treated with 5 mmol?dm-3 glutaraldehyde at pH 6.0 and 10°C for 10 min, the immobilized P. dacunhae cells showed highest enzyme activity and operational stability (Fig.3). The low pH-treatment devised previously for elimination of alanine racemase activity was found to be useful for stabilization of L-aspartate 8-decarboxylase activity of immobilized P. dacunhae cells. The reason is considered to be due to removal of unstabilizing factors such as intracellular neutral and/or alkali proteases from P. dacunhae cells by the low pH-treatment (Fig.4). Furthermore, the glutaraldehydetreatment of cells was also found to stabilize the enzyme activity. This treatment of cells was superior to that of immobilized cells reported previously in both enzyme activity and operational stability. That is, in the f ormer case remaining enzyme activity and halflife in continuous operation at 37°C were about 90% and 260 days, respectively, and in the latter case those were about 70% and about 100 days, respectively. The reason of advantages in the former case might be depend on homogeneous contact between cells and glutaraldehyde at optimum conditions. On the other hand, in the latter case cells located at near surface of gel were contacted with a higher concentration of glutaraldehyde than optimum one and cells at near center were contacted with a less concentration of glutaraldehyde than that due to consumption of glutaraldehyde during duffusion in gel.
Continuous production of L-alanine using the immobilized P. dacunhae cells was industrialized in 1982 in Tanabe Seiyaku Co. Ltd.