Riu Shinke

Induction, Purification, and Characterization of Catechol 2, 3-Dioxygenase from Aniline-assimilating Pseudomonas sp. FK-8-2

Catechol 2,3-dioxygenase (EC 1.13.1.2) was induced by aniline in cells of the aniline-assimilating Pseudomonas sp. FK-8-2 isolated from soil. The addition of polypepton besides aniline to the incubation mixture promoted the induction of this enzyme. The enzyme was purified to homogeneity with the high recovery of 95% from a cell-free extract of anilin-grown Pseudomonas sp. FK-8-2. The purified enzyme was stable between pH 6.0 and 7.0 and up to 70°C in the presence of 5% (v/v) acetone, which protected this enzyme from inactivation by air and other oxidizing agents. The molecular weight of the enzyme was 120,000 by gel filtration. It was dissociated into four identical subunits with the molecular weight of 33,000 on a sodium dodecyl sulfate-polyacrylamide gel. This enzyme catalyzed the extradiol cleavage of 3- and 4-chlorocatechol, 3- and 4-methylcatechol, and 3-fluorocatechol.

Tannic acid staining and extraction of enzymes in polyacrylamide gel electrophoresis

Abstract A procedure was developed for a rapid staining of proteins in polyacrylamide gels with tannic acid and the extraction of enzymatic activity from the gels. Lysozyme and Taka-amylase A were stained with tannic acid and localized on pH 4.3, and 8.0 and 9.5 gels, respectively. After the gels were rinsed in buffer solutions, the activities of the enzymes were recovered in good yield from the gels. The use of these techniques is discussed.

Lytic Enzymes toward Aniline-assimilatingRhodococcus erythro-polisAN-13: Purification and Characterization

Three lytic enzymes, C-2, C-4 and C-5, capable of lysing cells of Rhodococcus erythropolis AN13 were purified from the cultural filtrate of Flavobacterium species SH-548 by (NH4)2SO4 fractionation and column chromatographies on CM-Toyopearl and SP-Sephadex. The three purified enzymes gave single protein bands on polyacrylamide gels. C-4 and C-5 were stable between pH 3.0 and 12.5, and C-2 between pH 5.5 and ll.0. The molecular weights ofC-4 and C-5 were 26,000 and that of C-2 was 36,000,>as judged on sodium dodecylsulfate-polyacrylamide gel electrophoresis. C-4 and C-5 also showed proteolytic activity toward casein, but C-2 did not exhibit such activity. C-2 showed higher specific lytic activity toward cells of R. erythropolis AN-1 3 than C4andC-5.