[Isolation of spheroplasts from Escherichia coli 85 for aspartate-ammonia-lyase localization]. / Poluchenie sferoplastov iz Escherichia coli 85 dlia opredeleniia lokalizatsii spartat-ammiak-liazy.

Conditions were determined for preparation of spheroplasts from E. coli under the action of lysozyme in the presence of EDTA. The preparation took from 10 to 15 min. The degree of conversion to spheroplasts was monitored spectrophotometrically at 660 nm. The spheroplasts formed were unstable in Tris-HCl buffer and immediately lysed, but they were more stable in 1 M sucrose. At lysozyme concentrations above 40 micrograms/ml of the reaction mixture, the cells lysed to a greater extent. The distribution of aspartate ammonia-lyase activity between the precipitate of the spheroplasts and the supernatant allowed the authors to suggest that aspartase should be located in the cytoplasm.

[Aspartase activity of Escherichia coli, strain 85, cells grown on glucose-mineral medium with yeast extract]. / Aspartaznaia aktivnost' kletok Escherichia coli, shtamm 85, vyrashchennykh na gliukozomineral'noi srede s drozhzhevym ékstraktom

The conditions for cultivation of E. coli 85 on a glucose-mineral medium with a yeast extract were optimized. The cells obtained had a high aspartate ammonia-lyase activity. The aspartase activity was determined kinetically both by consumption of the substrate, ammonium fumarate, and by accumulation of the product, aspartic acid.

[Kinetics mechanism of L-aspartate synthesis from ammonium fumarate catalyzed by free and immobilized cells of E. coli]. / Kineticheskii mekhanizm reaktsii sinteza L-asparaginovoi kisloty iz fumarata ammoniia, kataliziruemoi svobodnymi i immobilizovannymi kletkami Escherichia coli.

A detailed study of kinetic peculiarities of the L-aspartate-ammonium-lyase reaction catalyzed by free and immobilized E. coli 85 cells incorporated into polyacrylamide gel, has been carried out. The effects of different types of bacterial cell "activation", substrate concentration and temperature on the reaction rate have been investigated. It was shown that the rate of the reaction is limited by the rate of the substrate transfer through the cell and cytoplasmic membranes and at sufficiently high values of the substrate can be described in terms of zero-order kinetics with respect to substrate and reaction products concentrations A kinetic model based on the diffuse and transfer processes of translocation of the aspartate-ammonium-lyase reaction participants through the cell and cytoplasmic membranes is proposed.

[Dependence of the rate of aspartate ammonia-lyase reaction catalyzed by free and immobilized cells of E. coli on temperature and preliminary thermal treatment]. / Zavisimost' skorosti aspartat-ammiak-lia3noi reaktsii, katali3iruemoi svobdnymi i immobilizovannymi kletkami Escherichia coli ot temperatury i predvaritel'noi teplovoi obrabotki kletok.

The effects of temperature (45--55 degrees) and duration of thermal treatment on the L-aspartase activity of free and immobilized on polyacrylamide gel cells of E. coli, strain 85 were studied. It was found that preliminary thermal treatment of the cells at 50 degrees for 40--60 min is optimal for a high aspartase activity. Within the temperature interval of 20--55 degrees the temperature dependence of effective rate constants of L-aspartate synthesis obeys the Arrhenius equation, whereas the effective energy of activation is decreased from 12,6 to 3,6 kcal/mole, when the "activation" of the cells shows an increase.