| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
When Rhodococcus erythropolis AN-13 grew on aniline, a fluorescent substance accumulated in the cultural fluid. It was obtained as crystals and identified as anthranilic acid (AnA). AnA was also produced from aniline following incubation with resting cells of the bacterium grown on aniline. Heated cells lost the activity to produce it, and aniline was essential for its production. The production of AnA was promoted by sodium bicapbonate ; when ^<14>C-sodium bicarbonate was added to the incubation mixture, ^<14>C-AnA formed. the optimal pH for AnA production by the resting cells was 7.0 to 7.5. These results suggest that microbial activities of R. erythropolis AN-13 catalyzed the formation of AnA from aniline.
The production was investigated for 40 bacterial strains in the presence and absence of aniline. Resting cells of all aniline-assimilating R. erythropolis strains produced more AnA than those of other anilineassimilating bacteria. Resting cells of several non-aniline-assimilating s
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trains produced AnA in the absence of aniline. However, its production by these strains was much lower than that by the Rhodococcus strains. The production of AnA by cells of aniline-assimilating R. erythropolis AN-13 was promoted by aliphatic monocarboxylates, ATP, biotin and coenzyme A, and repressed by catechol analogues, N-ethylmaleimide and iodoacetate. on the other hand, its production by non-aniline-assimilating PseudoNonas sp. AN21 was repressed by glucose, mannose and some amino acids.
Cell-free extracts prepared from R. erythropolis AN-13 cells retained the activity capable of producing AnA from aniline. The catalytic factor(s) in these extracts was partially purified by fractionation with streptomycin sulfate and anunonium sulfate and gel filtration on a column of Tyopearl HW55s. Further purification procedures of this catalytic factor(s) are in progress.
A bacterial strain, FK-8-2, that metabolized aniline through a metacleavage pathway was isolated and identified as Pseudomonas. Pseudomonas sp. FK-8-2 produced much catechol 2, 3-dioxygenase in its cells. This enzyme was purified to homogeneity. The purified enzyme differed in chemical and catalytic properties from that obtained from Pseudomonas putida mt-2 growing on benzoate. Another aniline-assimilating bacteria producing isozymes of catechol 1, 2dioxygense in their cells were also isolated. These isolates will be useful materials for investigating on the bacterial production of AnA from aniline. Less
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