A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp. denitrificans. Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H20-HCOOH + NH3) without forming formamide as a free intermediate. The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media. Cyanate (OCN-) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A. xylosoxidans subsp. denitrificans DF3. Strain DF3 hydrolyzed cyanide with great efficacy. Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h. Enzyme purification established that cyanide hydrolysis by A. xylosoxidans subsp. denitrificans DF3 was due to a single intracellular enzyme. The soluble enzyme was purified approximately 160-fold, and the first 25 NH2-terminal amino acids were determined by automated Edman degradation. The molecular mass of the active enzyme (purity, >97% as determined by amino acid sequencing) was estimated to be >300,000 Da. The cyanide-hydrolyzing enzyme of A. xylosoxidans subsp. denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles.

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A novel class of pullulanase (E.C. 3.2.1.41) has recently been isolated from a new Bacillus species. This novel taxonomic group, Bacillus acidopullulyticus is characterized by production of pullulanases with temperature optima above 6OoC and pH optima around 5. Two of these enzymes have been purified to homogeneity. These two enzymes are compared with the purified pullulanase from Aerobacter aerogenes. The enzyme activity and enzyme kinetics are also compared.The pullulanase A was produced by a Bacillus strain deposited under the number NClB 11647. Pullulanase B was produced by a Bacillus strain deposited under the number NClB 11777. Pullulanase C was from Aerobacter aerogenes. The soluble extracellular enzymes were purified by successive chromatography through CM-Sepharose and DEAE-Sepharose. The purified enzymes were analyzed by sieve chromatography (Sephacryl S200).Enzyme activity was determined by incubation with pullulan followed by determination of reducing power by the method of Somogyi and Nelson. One pullulanase unit NOVO (PUN) is defined as the amount of enzyme that under the given standard conditions hydrolyzes pullulan-liberating reducing carbohydrate with a reducing power equivalent to 1 pmol glucose per minute. The purified enzymes were more than 90% pure and the specific activity could not be increased by further purification.The purified enzymes were analyzed by SDS-PAGE electrophoresis. The molecular weight of A was -100,000, of B -90,000, and of C about 140,000, which is in agreement with Eisele et a/.* The two new purified enzymes were hydrolyzed and the amino-acid composition analyzed on a Beckman analyzer. The results are presented in TABLE 1 and compared with the amino acid composition of C from Eisele et a/.* The isoelectric point of the purified enzyme was 5.0 for pullulanase A, 5.4 for pullulanase B, and 4.0 for pullulanase C. The PI was determined on a LKB Multiphor unit with LKB Anholine PAC plates. The immunological method described by Weeke3 was used for crossed immunoelectrophoresis.The enzymes were characterized by crossed immunoelectrophoresis (CIE). Sera against the two purified proteins did not react against pullulanase C or culture filtrate from pullulanase-producing Bacillus strains meguterium (ATCC 6459) or mycoides (ATCC 31 1027). Duplicates of the CIE plates were incubated overnight with pullulan followed by precipitation of the pullulan with acetone. A clearing zone indicates pullulanase activity. Both enzymes reacted with their own serum and with the other serum, although 3 to 10 times less antigen was necessary for obtaining the same area of immunoprecipitate with the heterologous antisera, indicating partial immunological identity.The purified enzymes were incubated with pullulan at different temperatures and different pH values. The results are presented in FIGURES 1 and 2. It can be seen that 271