Protocatechuate 3,4-dioxygenase (EC 1.13.1.3) was purified to homogeneity from extracts of Azotobacter vinelandii. The molecular weight of the oligomeric protein was estimated to be 510 000 by gel filtration and 480 000 by ultracentrifugation. The oligomer appears to be formed by association of equal amounts of nonidentical subunits which were estimated by sodium dodecyl sulfate gel electrophoresis to have respective molecular weights of 23 300 and 25 250. Ten gram-atoms of iron was associated with each mol of oligomer. Therefore, the enzyme appears to be a decamer with the structure 10(alpha beta Fe). T-HE AMINO ACID COMPOSITION OF Azotobacter protocatechuate oxygenase closely resembles the amino acid compositions of protocatechuate 3,4-dioxygenases from Pseudomonas aeruginosa and Thiobacillus sp. These proteins from P. aeruginosa and P. putida are known to be formed by association of nonidentical subunits of a physical size similar to the subunits of the Azotobacter enzyme. Furthermore, antisera prepared against the Azotobacter oxygenase cross-reacted strongly with the isofunctional enzymes from the two fluorescent Pseudomonas species. A weak immunological cross-reaction was observed when the antisera were tested against protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus. The results favor the conclusion that the bacterial protocatechuate 3,4-dioxygenases were derived from a common ancestral protein.

Experiments were conducted to determine the pathway of melamine metabolism by Klebsiella terragena (strain DRS-1) and the effect of added NH 4 ؉ on the rates and extent of melamine metabolism. In the absence of added NH 4 ؉ , 1 mM melamine was metabolized concomitantly with growth. Ammeline, ammelide, cyanuric acid, and NH 4 ؉ accumulated transiently in the culture medium to maximal concentrations of 0.012 mM, 0.39 mM, trace levels, and 0.61 mM, respectively. In separate incubations, in which cells were grown on either ammeline or ammelide (in the absence of NH 4 ؉), ammeline was metabolized without a lag while ammelide metabolism was observed only after 3 h. In the presence of 6 mM added NH 4 ؉ (enriched with 5% 15 N), ammeline, ammelide, and cyanuric acid accumulated transiently to maximal concentrations of 0.002 mM, 0.47 mM, and trace levels, respectively, indicating that the added NH 4 ؉ had little effect on the relative rates of triazine metabolism. These data suggest that the primary mode of melamine metabolism by K. terragena is hydrolytic, resulting in successive deaminations of the triazine ring. Use of 15 N-enriched NH 4 ؉ allowed estimates of rates of triazine-N mineralization and assimilation of NH 4 ؉-N versus triazine-N into biomass. A decrease in the percent 15 N in the external NH 4 ؉ pool, in conjunction with the accumulation of ammelide and/or triazine-derived NH 4 ؉ in the culture medium, suggests that the initial reactions in the melamine metabolic pathway may occur outside the cytoplasmic membrane.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 5327O), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of -4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. ]Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity it pH 12 over a 24-h incubation period at 25°C; protease HS exhibited similar alkaline stability properties ¢o pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50°C and 25 min at 60°C. At pH above 9.5, protease HS readily lost enzymrtic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60°C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.Alkaline proteases secreted by both neutralophilic and alkalophilic bacilli are of interest because they represent a major source of commercially produced proteolytic enzymes (1; 11, 16). These proteases exhibit optimal activity at pHs of 9 to 11 and are inactivated by serine active-site inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In general, these enzymes have molecular weights ranging from 20,000 to 30,000, are stabilized by Ca2+, and have characteristically high isoelectric points (2,11,12,16,19,23). Alkaline proteases, or subtilisins, secreted by neutralophilic Bacillus spp. are stable from pH 5 to 10 at low temperatures, but are readily inactivated at higher temperatures and alkalinities in the absence of Ca2+ (1, 2). The subtilisins have been divided into two groups based on differences in amino acid composition and immunological and kinetic properties (12). Recent results, however, suggest that these proteases possess identical amino acid residues in 63% of their primary sequences (17). Alkaline proteases from alkalophilic bacteria have been studied in less detail, yet many similarities exist between subtilisins and alkaline proteases from alkalophilic bacilli (11). However, serine proteases from alkalophiles have superior alkaline...

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