JOURNAL OF BACTERIOLOGY, Sept. 1990, p. 4807-4815 Vol. 172, No. 9 0021-9193/90/094807-09$02.00/0 Copyright X3 1990, American Society for Microbiology Purification and Characterization of a Novel of Rhodococcus rhodochrous K22 That Acts on Aliphatic MICHIHIKO KOBAYASHI,* NORIYUKI YANAKA, TORU NAGASAWA, AND HIDEAKI YAMADA Department ofAgricultural Chemistry, Faculty ofAgriculture, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received 18 April 1990/Accepted 8 June 1990

A novel nitrilase that preferentially catalyzes the hydrolysis of aliphatic nitriles to the corresponding carboxylic acids and ammonia was found in the cells of a facultative crotononitrile-utilizing actinomycete isolated from soil. The strain was taxonomically studied and identified as Rhodococcus rhodochrous. The nitrilase was purified, with 9.08% overall recovery, through five steps from a cell extract of the stain. After the last step, the purified appeared to be homogeneous, as judged by polyacrylamide gel electrophoresis, analytical centrifugation, and double immunodiffusion in agarose. The relative molecular weight values for the native enzyme, estimated from the ultracentrifugal equilibrium and by high-performance liquid chromatog- raphy, were approximately 604,000 + 30,000 and 650,000, respectively, and the enzyme consisted of 15 to 16 subunits identical in molecular weight (41,000). The enzyme acted on aliphatic olefinic nitriles such as crotononitrile and as the most suitable substrates. The apparent Km values for crotononitrile and acrylonitrile were 18.9 and 1.14 mM, respectively. The nitrilase also catalyzed the direct hydrolysis of saturated aliphatic nitriles, such as valeronitrile, 4-chlorobutyronitrile, and glutaronitrile, to the correspond- ing acids without the formation of amide intermediates. Hence, the R. rhodochrous K22 nitrilase is a new type distinct from all other that act on aromatic and related nitriles.

Nitrile compounds are synthesized on a large scale as p-aminobenzoic acid (21) with resting cells. We have purified solvents, plastics, synthetic rubber, pharmaceuticals, herbi- and characterized the nitrilase from R. rhodochrous Jl (20). cides, and starting materials from other industrially impor- Nitrilases that utilize benzonitrile and related aromatic tant chemicals. However, they are notoriously poisonous. nitriles as substrates have been purified from Pseudomonas Crotononitrile and acrylonitrile, which have been proposed sp. (19, 35), Nocardia sp. strains NCIB 11215 (18) and NCIB as specific reagents for the alkylation of protein sulfhydryl 11216 (16), Fusarium solani (17), Arthrobacter sp. (4), R. groups (7, 14), are not only toxic to the central nervous rhodochrous Jl (20), and Escherichia coli transformed with a system of animals but also mutagenic by virtue of their Klebsiella ozaenae plasmid DNA (38). However, aliphatic conjugated bonds (26, 47). waste dispersed through- nitriles are inactive as substrates for all of these nitrilases. It out the biosphere has been recognized as an important form has been shown that aliphatic nitriles are catabolized via of environmental pollution. amides by the combination of nitrile hydratase and amidase In recent years, the most significant development in the (2, 3, 13, 27, 47). To the best of our knowledge, no nitrilases field of synthetic organic chemistry has been the application that act on aliphatic nitriles have been reported. Such a of biological systems to chemical reactions. Reactions cata- nitrilase would be an interesting enzyme from the viewpoints lyzed by cells and enzyme systems display far greater of amelioration of the environmental pollution due to artifi- specificities than more conventional organic reactions. cial nitriles, detoxification, and bioconversion. We have The microbial degradation of nitriles has been found to described the occurrence of a novel nitrilase that acts proceed through two enzymatic pathways. Nitrilase cata- preferentially on aliphatic nitriles in R. rhodochrous K22 lyzes the direct cleavage of nitriles to yield the correspond- isolated from soil. In the work described here, the enzyme, ing acids and ammonia, whereas nitrile hydratase catalyzes tentatively designated as an aliphatic nitrilase, was purified the hydration of nitriles to amides. Both nitrile-converting and characterized. have attracted increasing attention as catalysts for processing organic chemicals, since they can convert nitriles MATERIALS AND METHODS to the corresponding higher-value acids or amides. Recently, Materials. DEAE-Sephacel, phenyl-Sepharose CL-4B, the use of Pseudomonas chlororaphis B23 nitrile hydratase, and a low-molecular-weight standard kit were obtained from which was found in our laboratory, for the industrial pro- Pharmacia (Uppsala, Sweden). Cellulofine GCL-2000 super- duction (6,000 ton per year) of the important chemical fine was purchased from Seikagaku Kogyo Co. (Tokyo, commmodity acrylamide was pioneered in Japan (3, 36). We Japan). Marker proteins for molecular mass determination have purified and characterized the nitrile hydratases of P. by high-performance liquid chromatography (HPLC) were chlororaphis B23 (30), Brevibacterium sp. strain R312 (31), purchased from Oriental Yeast Co. (Tokyo, Japan). All other and Rhodococcus rhodochrous Jl (32). On the other hand, chemicals used were from commercial sources and of re- we recently found an abundance of nitrilase in cells of R. agent grade. rhodochrous Jl (29) and established the optimal conditions Screening and identification. R. rhodochrous K22 was for the production of vitamins, nicotinic acid (24), and isolated from soil samples by means of an enrichment culture technique. Crotononitrile-utilizing bacteria were isolated as follows. A soil sample (5 g) was added to a 500-ml shaking flask containing 80 ml of medium (pH 7.2) consisting of 0.5 g * Corresponding author. of glycerol, 2 g of K2HPO4, 1 g of NaCl, 0.2 g of 4807 4808 KOBAYASHI ET AL. J. BACTERIOL.

MgSO4. 7H20, 1 ml of a vitamin mixture (2 mg of nicotinic nitrile, 1 ,umol of dithiothreitol, and an appropriate amount acid, 0.4 g of thiamine hydrochloride, 0.2 g of p-aminoben- of cells or enzyme. The reaction was carried out at 25°C for zoic acid, 0.2 g of riboflavin, 10 mg of folic acid, and 0.4 g of 10 min and stopped by adding 0.2 ml of 1 M HCl to the pyridoxime hydrochloride in 1 liter of distilled water) and reaction mixture. The amounts of crotonic acid and croton- crotononitrile at a final concentration of 0.01% (vol/vol)/liter amide formed in the reaction mixture were determined by of tap water. Cultivation was performed with shaking at 28°C HPLC with a Shimadzu LC-6A system equipped with an for 1 week. Once a week, 0.5 volume (40 ml) of the isolation M&S pack C18 column (reverse-phase column; 4.6 by 150 medium was replaced with 40 ml of fresh medium, and mm; M&S Instruments, Osaka, Japan) at a flow rate of 1.0 crotononitrile was fed to the medium at the concentration of ml/min, using the following solvent system: 5 mM KH2PO4- 0.05% (vol/vol). After 1 month of cultivation, the microor- H3P04 buffer (pH 2.9)-acetonitrile, 3:1 (vol/vol). One unit of ganisms were spread on agar plates and isolated. enzyme is defined as the amount needed to catalyze the Isolation was carried out according to Goodfellow (15). formation of 1 ,umol of crotonic acid per min from crotono- The chemical compositions of cells were analyzed by the nitrile under the conditions described above. methods of Becker et al. (5), Lechevalier and Lechevalier Protein was determined by the Coomassie brilliant blue (23), Suzuki and Komagata (42), Alshamaony et al. (1), and G-250 dye-binding method of Bradford (6), using dye reagent Uchida and Aida (44). supplied by Bio-Rad Laboratories (Richmond, Calif.). For Culture conditions. R. rhodochrous K22 was collected the nitrilase from R. rhodochrous K22, protein was deter- from an agar slant and inoculated into the subculture me- mined from the A280. The absorption coefficient was calcu- dium. The subculture was carried out at 28°C for 30 h with lated to be 1.09 ml/mg per cm by absorbance and dry-mass reciprocal shaking in a test tube containing 4 ml of a medium determinations. Spectrophotometric measurements were consisting of 5 g of Polypepton (Daigo, Osaka, Japan), 5 g of performed with a Shimadzu UV-240 spectrophotometer. meat extract (Mikuni, Tokyo, Japan), 0.5 g of yeast extract Specific activity is expressed as units per milligram of (Oriental Yeast Co.), and 2 g of NaCl per liter of tap water protein. (pH 7.0). Then 8 ml of the subculture was inoculated into a specificity. The standard reaction mixture (2 ml) 2-liter shaking flask containing 500 ml of a medium consisting was composed of 0.2 mmol of potassium phosphate buffer of 50 g of sorbitol, 3 g of yeast extract, 7 g of NZ amine (pH 7.5), 0.2 mmol of nitrile, 1 ,umol of dithiothreitol, 0.2 ml (Humko Sheffield Chemical), 7 g of urea, and 1 ml of of methanol, and an appropriate amount of the enzyme. isovaleronitrile per liter of tap water (pH 7.2). Incubation Methanol was added to enhance the solubility of the sub- was carried out at 28°C with reciprocal shaking. At 76 and strate, the enzyme being stable even in the presence of 10% 100 h during the cultivation, 0.1 and 0.2% (vol/vol) isovale- (vol/vol) methanol (data not shown). However, in the cases ronitrile were fed, respectively, followed by further incuba- of aromatic nitriles, heterocyclic nitriles, alicyclic nitriles, tion for 20 h. At 120 h from the start, the cells were harvested and aralkyl nitriles, the reaction was carried out with a final by continuous-flow centrifugation at 10,000 x g at 4°C and concentration of 10 mM because of their low solubility in then washed twice with 0.1 M potassium phosphate buffer water. Each reaction was carried out at 25°C for 10 to 50 min (pH 7.5) containing 1 mM dithiothreitol. The yield of wet and stopped by adding 0.2 ml of 1 M HC1 to the reaction cells was approximately 3.5 g/liter of medium. mixture. The amount of NH3 produced in the reaction Eight strains were used for studies of the immunological mixture was colorimetrically estimated by the phenol-hy- properties of the nitrilase. The microorganisms came from pochlorite method (12), using a Conway microdiffusion stock cultures of the Institute of Fermentation, Osaka (IFO apparatus (10). strains), the Japan Collection of Microorganisms (JCM The following nitrile compounds were tested for substrate strains), and the American Type Culture Collection (ATCC specificity: the aliphatic olefinic nitriles crotononitrile, strains). The following Rhodococcus strains were examined: acrylonitrile, methacrylonitrile, 3-ethoxyacrylonitrile, 3- R. erythropolis (IFO 12539 and IFO 12682), R. rhodochrous aminocrotononitrile, 2-chloroacrylonitrile, fumaronitrile, (IFO 3338, JCM 2157, JCM 3202, and ATCC 21198), R. diaminomaleonitrile, 2-methyl-2-butenenitrile, 2-methyl-3- rhoseus (JCM 2156), and R. rubropertinctus (JCM 3204). butenenitrile, 2-pentenenitrile, 3-pentenenitrile, 1,4-dicyano- These Rhodococcus strains were cultivated for 48 h in 2-butene, 2,4-dicyano-1-butene, and N-cyano-N'-S-dimeth- medium consisting of 5 g of Polypepton, 3 g of malt extract ylisothiourea; the saturated aliphatic nitriles acetonitrile, (Difco Laboratories, Detroit, Mich.), 3 g of yeast extract, 10 , butyronitrile, isobutyronitrile, valeronitrile, iso- g of glycerol, and 1 ml of isovaleronitrile per liter of tap valeronitrile, capronitrile, isocapronitrile, trimethylacetoni- water (pH 7.2). Cells harvested by centrifugation at 9,000 x trile, methoxyacetonitrile, (S)-(+)-2-methylbutyronitrile, g at 4°C for 20 min were washed with 0.1 M potassium chloroacetonitrile, (±)-2-chloropropionitrile, 3-chloropropi- phosphate buffer (pH 7.5) containing 1 mM dithiothreitol and onitrile, 4-chlorobutyronitrile, cyanoacetic acid, cyanoacetic then disrupted for 50 min by ultrasonic oscillation (19 kHz; acid ethyl ester, 2-cyanoacetamide, 3-cyano-L-alanine, N- Insonator model 201M; Kubota, Tokyo, Japan). The cell methyl-,-alaninenitrile, lactonitrile, maleonitrile, succinoni- debris was removed by centrifugation at 10,000 x g for 30 trile, glutaronitrile, adiponitrile, pimelonitrile, suberonitrile, min. The supernatant solution was designated as the cell sebaconitrile, 3,3'-oxydipropionitrile, iminodiacetonitrile, extract. and 3,3'-iminodipropionitrile; the aromatic nitriles benzoni- Preparation of resting cells. Cells were harvested from trile, monosubstituted (position 2, 3, or 4) benzonitrile culture both by centrifugation at 10,000 x g for 20 min. The derivatives with a functional group (e.g., a hydroxy, amino, harvested cells were washed with 10 mM potassium phos- methyl, nitro, cyano, methoxy, ethoxy, or carboxyl group), phate buffer (pH 7.5) containing 1 mM dithiothreitol and then 4-cyanothiophenol, 1-or 2-cyanonaphthalene, 9-cyanoan- suspended in the same buffer. This cell suspension was used thracene, 4-biphenylcarbonitrile, 4,4'-biphenyldicarbonitrile, for the resting-cell reaction. 2,6-dichlorobenzonitrile, and 3,5-diiodo-4-hydroxybenzoni- Enzyme assay and definition of units. Nitrilase activity was trile; the heteroaromatic nitriles 2-, 3-, or 4-cyanopyridine, assayed in a reaction mixture (2 ml) consisting of 20 jimol of cyanopyrazine, 2,3-dicyanopyrazine, 2-furonitrile, 2-thiophene- potassium phosphate buffer (pH 8.0), 200 ,umol of crotono- carbonitrile, 5-cyanoindole, piperonylonitrile, 3-quinoline- VOL. 172, 1990 NOVEL ALIPHATIC NITRILASE OF R. RHODOCHROUS K22 4809 carbonitrile, and 6-cyanopurine; the alicyclic nitriles final concentration of 80%. The precipitate was centrifuged cyclopropanecarbonitrile, 1,2-dicyanocyclobutane, cyclo- and then dissolved in 0.1 M buffer. The enzyme solution was pentanecarbonitrile, cycloheptanecarbonitrile, 4-cyano-1- dialyzed against 10 mM buffer and then preserved in 10 mM cyclohexene, 5-norbornene-2-carbonitrile, 1-adamantane- buffer containing 50% (vol/vol) glycerol at -20°C. carbonitrile, 1-cyclopenteneacetonitrile, and 1-cyclohexene- Analytical methods for the nitrilase. Sodium dodecyl sul- acetonitrile; the aralkyl nitriles cinnamonitrile, phenylaceto- fate (SDS)-polyacrylamide gel electrophoresis was per- nitrile, (+)-a-aminophenylacetonitrile, 3-xylylene dicyanide, formed in 12.5% polyacrylamide slab gels, using the Tris- 3-indoleacetonitrile, 2-pyridineacetonitrile, 2-or 3-thiophe- glycine buffer system (22). Proteins were stained with neacetonitrile, 2-furanacrylonitrile, a-methylphenylaceto- Coomassie brilliant blue R-250 and destained in ethanol- nitrile, and mandelonitrile; and the N-carbonitriles 1,4-pip- acetic acid-H20 (3:1:6, vol/vol/vol). The relative molecular erazinedicarbonitrile, 4-morpholinecarbonitrile, 1-pyrrolidine weight of the subunit ofthe enzyme was determined from the lcarbonitrile, and 1-piperidinecarbonitrile. relative mobilities of standard proteins. Purification of R. rhodochrous K22 nitrilase. All purifica- To estimate the molecular weight of the enzyme, the tion steps were performed at 0 to 4°C. Throughout purifica- enzyme sample (20 ,ug) was subjected to HPLC (Toyo Soda tion steps 1 to 5, potassium phosphate buffer (pH 7.5) CO-8000 system) on a TSK G-4000SW column (0.75 by 60 containing 1 mM dithiothreitol was used unless otherwise cm; Toyo Soda) at a flow rate of 0.5 ml/min, using 0.1 M N- specified. Centrifugation was carried out for 30 min at 12,000 2-hydroxyethyl-N'-2-ethanesulfonic acid (HEPES)-KOH x g. buffer (pH 7.0) containing 0.2 M NaCl at room temperature. (i) Step 1. Preparation of a cell extract. Washed cells (35 g) The A280 of the effluent was recorded. The relative molecular from 20 liters of culture broth were suspended in 1.0 liter of weight of the enzyme was then calculated from the relative 0.1 M buffer and then disrupted with a Dyno-Mill (W. A. mobility compared with those of the standard proteins, Bachofen, Basel, Switzerland), followed by sonication at 19 glutamate dehydrogenase (Mr = 290,000), lactate dehydro- kHz for 20 min with an ultrasonic oscillator (model 4280; genase (140,000), enolase (67,000), adenylate kinase Kaijo Denki, Tokyo, Japan). The cell debris was removed by (32,000), cytochrome c (12,400) (products of Oriental Yeast centrifugation. The resulting supernatant solution was used Co.), thyroglobulin (bovine; Mr = 669,000; Sigma Chemical as the cell extract and followed by dialysis against 10 mM Co., St. Louis, Mo.), alcohol oxidase (Candida boidinii; Mr buffer. = 673,000; Boehringer Mannheim Biochemicals, Indianapo- (ii) Step 2. DEAE-Sephacel column chromatography. The lis, Ind.), and ,-galactosidase (type II; E. coli; Mr = 540,000; dialyzed solution was applied to a DEAE-Sephacel column Toyobo Co., Osaka, Japan). (5.2 by 50.5 cm) equilibrated with 10 mM buffer. After the Analytical ultracentrifugation was carried out with a column was washed thoroughly with 10 mM buffer followed Spinco model E ultracentrifuge at 20°C. The purity of the by the same buffer containing 0.1 M KCl and 0.2 M KCI, the enzyme and its sedimentation coefficient were determined enzyme was eluted with 2.5 liters of 10 mM buffer containing with a phase plate as a Schlieren diagram and operated at 0.3 M KCI. The active fractions were pooled, and ammo- 12,590 rpm, with the boundary at the meniscus in the sector- nium sulfate was added to give 80% saturation. After cen- shaped centrifuge cell (43). The relative molecular weight trifugation of the suspension, the precipitate was dissolved was determined by the procedure of Van Holde and Baldwin in 0.1 M buffer, followed by dialysis against 10 mM buffer. (46), using Rayleigh interference optics. Multicell operations (iii) Step 3. Ammonium sulfate fractionation. Solid ammo- were carried out for three samples of different initial con- nium sulfate was added to the resulting supernatant solution centrations, ranging from 1.40 to 5.62 mg/ml, using an An-G to give 40% saturation. The pH was maintained at 7.5 with rotor and double cells with different side-wedge angles. The an ammonia solution. After being stirred for 4 h or more, the rotor was centrifuged at 3,848 rpm at 15°C for 16 h, and the precipitate was removed by centrifugation, and ammonium interference pattern was photographed at 30-min intervals. sulfate was added to the supernatant solution to give 50% The relationship between the concentration of the enzyme saturation. The suspension was then centrifuged, and the and the fringe shift was determined by using a synthetic- pellet was dissolved in 0.1 M buffer, followed by dialysis for boundary cell. 36 h against four changes of 10 liters of 10 mM buffer. Amino acid composition. For analysis of the amino acid (iv) Step 4. Phenyl-Sepharose CL-4B column chromatogra- composition of the nitrilase, duplicate samples of the en- phy. After the enzyme solution from step 3 was cooled, zyme were hydrolyzed in 6 M HCI in evacuated and sealed sodium chloride was added in small portions with stirring to Pyrex tubes at 110°C for 20, 42, and 70 h. The hydrolysates bring the solution to 1.6 M saturation. The enzyme solution were analyzed with an amino acid analyzer (KlOl-AS; was placed on a phenyl-Sepharose CL-4B column (2.5 by Kyowa Seimitsu, Mitaka, Japan) by the procedure of Spack- 27.5 cm) equilibrated with 20 mM buffer containing 1.6 M man et al. (37). Cysteine and cystine were determined as saturated sodium chloride. After the column was washed cysteic acid after performic acid oxidation of the sample by thoroughly with 20 mM buffer containing 1.6 M sodium the method of Moore (28). Tryptophan was determined chloride, the enzyme was eluted by lowering the ionic spectrophotometrically at 280 and 288 nm in the presence of strength of sodium chloride (1.6 to 1.0 M) in the same buffer. 6 M guanidine hydrochloride by the method of Edelhoch The active fractions were combined and precipitated with (11). ammonium sulfate at 80% saturation. Metal analysis. All glassware was boiled briefly in 2 M HCI (v) Step 5. Cellulofine GCL-2000 superfine column chroma- and then exhaustively rinsed with distilled water before use. tography. The precipitate from step 4 was centrifuged and Before analysis, the enzyme was dialyzed against 10 mM then dissolved in 0.1 M buffer. This enzyme solution was potassium phosphate buffer (pH 7.5). Enzyme samples con- placed on a Cellulofine GCL-2000 superfine column (1.8 by taining 1.0 to 3.0 mg of protein per ml were analyzed with an 101 cm) equilibrated with 10 mM buffer. The rate of sample inductively coupled radiofrequency plasma spectrophotom- loading and column elution was maintained at S ml/h with a eter (Shimadzu ICPV-1000; 27,120 MHz). The following peristaltic pump (LKB 2121). The active fractions were assay conditions were used for qualitative analysis: cooling combined and then precipitated with ammonium sulfate at a gas, 15 liters/min; plasma gas, 1.2 liters/min; and carrier gas, 4810 KOBAYASHI ET AL. J. BACTERIOL.

1.0 liter/min. The spectra were scanned, from 400 nm to 190 TABLE 1. Purification of the aliphatic nitrilase from nm, at a speed of 25 nm/min. R. rhodochrous K22 Antiserum preparation. Antibodies were elicited by injec- Total Total act Yield a Sp tion of 8.0 mg of R. rhodochrous K22 nitrilase homogenized Step proteina activity (U/mg) (%) with an equal volume of complete Freund adjuvant (Difco) (mg) (U) into young white male rabbits. The rabbits received a 1. Cell-free extract 6,110 544 0.0890 100 booster injection after 3 weeks. The booster injection, ad- 2. DEAE-Sephacel 1,980 201 0.102 36.9 ministered subcutaneously in the neck, was of 3.5 mg of 3. (NH4)2SO4 (0.4-0.5) 731 102 0.140 18.8 antigen homogenized in an equal volume of incomplete 4. Phenyl-Sepharose CL-4B 160 53.4 0.334 9.81 Freund adjuvant (Difco). On the day 7 after the booster 5. Cellulofine GCL-2000 67.0 49.4 0.737 9.08 injection, blood was collected and serum was prepared. superfine Ouchterlony plates were made using 1% (wt/vol) special a The concentration of the purified enzyme after step 5 was determined Noble agar (Difco) in 10 mM Tris-H2SO4 (pH 8.0) containing from the A280, using the extinction coefficient (A1% = 10.9). 0.01% sodium azide (34).

RESULTS AND DISCUSSION belongs to the genus Rhodococcus. Then, in accordance with references 15 and 39, strain K22 was classified as Identification of the microorganism. We isolated from a soil Rhodococcus rhodochrous. sample a strain, K22, that utilizes crotononitrile as a sole During the screening program for nitrilases, among a nitrogen source. Taxonomical studies on strain K22 were number of nitrile-degrading microorganisms, only a very few carried out. The microbiological characteristics of strain K22 nitrilase-containing microorganisms were found in the envi- were as follows. Morphologically, the cells were gram pos- ronment, whereas nitrile hydratase-containing organisms itive, aerobic, non-endospore forming, non-acid fast, and were found to be widespread (data not shown). Thus, the nonmotile. Cocci germinated and gave rise to branched occurrence of an aliphatic nitrilase is rare even among filaments, which underwent fragmentation into rods (0.5 to nitrilase-containing microorganisms. 1.1 ,um by 1.5 to 5.0 ,um). Elongated cells and snapping Purification of the aliphatic nitrilase. By using the purifi- division were frequently observed. Physiological character- cation procedures described in Materials and Methods, the istics were as follows: reduction of nitrate, positive; denitri- enzyme was purified 8.3-fold with a yield of 9.08% from the fication, methyl red test, indole production, hydrogen sulfide cell extract, using crotononitrile as the substrate (Table 1). production, and Voges-Proskauer test, negative; oxidase, The purified enzyme showed only one band on SDS-poly- negative; catalase and phosphatase, positive; growth range, acrylamide slab gel electrophoresis (Fig. 1, lane B). It pH 5 to 10; growth temperature, 10 to 41°C; growth in 7% sedimented as a single symmetrical peak in 10 mM potas- NaCl, positive; oxygen requirement, aerobic; Hugh-Leifson sium phosphate buffer (pH 7.5) on analytical ultracentrifu- test, weakly oxidative; acid production from glucose, posi- gation. Further evidence for the purity of the enzyme tive; gas production from glucose, negative; decomposition preparation was provided by the results of HPLC on a TSK of tyrosine, positive; decomposition of adenine and urea, negative; growth on sole carbon sources, positive with 1% (wt/vol) maltose, mannitol, sorbitol, and trehalose and with A B C 0.1% m-and p-hydroxybenzoic acid, sodium adipate, sodium benzoate, sodium citrate, sodium lactate, testosterone, ace- tamide, and sebacic acid and negative with 1% inositol and 1- -. rhamnose; hydrolysis of starch, negative; Tween 80 hydrol- ysis, positive; o-nitrophenyl-p-D-galactopyranoside hydrol- ysis, negative. The chemical composition of the cells was as 3 _ _ follows: meso-diaminopimelic acid, arabinose, and galac- tose, present; major fatty acid components, hexadecanoic acid (31.4%), hexadecenoic acid (15.0%), octadecenoic acid, (15.4%), and methyloctadecanoic acid (tuberculostearic 4_* acid) (19.2%); C32 to C46 mycolic acids of the mycolic acid type, present. The strain was positive for the glycolate test, and its G+C content of DNA was 67 to 70% mol (Tm). 5 u Thus, strain K22 is pleomorphic and has some typical characteristics of a coryneform bacterium. Recently, chem- ical examination of the cell wall provided data useful for 6 distinguishing coryneform bacteria at the generic level. FIG. 1. SDS-polyacrylamide gel electrophoresis of the aliphatic From its morphology and physiology and from the presence nitrilase. Conditions are given in Materials and Methods. Lane A of meso-diaminopimelic acid, arabinose, and galactose (23), was loaded with the following molecular weight standards: 1, fatty acids of the straight-chain saturated and monounsat- phosphorylase b (94,000); 2, bovine serum albumin (67,000); 3, urated types, and 10-methyl branched-chain acids (42), ovalbumin (43,000); 4, carbonic anhydrase (30,000); 5, soybean strain K22 was thought to belong to the genus Rhodococcus, trypsin inhibitor (20,100); and 6, a-lactalbumin (14,400). Lane B was Corynebacterium, or Nocardia. To clearly differentiate be- loaded with the purified aliphatic nitrilase (27 jig). Lane C was tween loaded with 82 ,ug of protein of a cell extract of cells cultivated for Corynebacterium and related taxa, the types of my- 60 h in medium consisting of 50 g of sorbitol, 3 g of yeast extract, 7 colic acids and the acyl type in the cell wall were determined g of NZ amine, 7 g of urea, and 1 ml of isovaleronitrile per liter of tap (8, 45). Strain K22 was found to possess mycolic acids with water (pH 7.2), with feeding of 0.2% (vol/vol) isovaleronitrile at 48 32 to 46 carbon atoms and glycolyl residues of the glycan h from the start. The direction is from top (cathode) to bottom moiety. From these results, it was assumed that strain K22 (anode). VOL. 172, 1990 NOVEL ALIPHATIC NITRILASE OF R. RHODOCHROUS K22 4811

TABLE 2. Amino acid composition of the aliphatic nitrilasea No. of residues/subunit Amino acid 2.4 Found Integral Aspartic acidb 41.0 41 Threoninec 16.9 17 Serinec 18.5 19 0 0 Glutamic acidb 38.0 38 c 1.6 .8 Proline 20.0 20 .0 Glycine 26.3 26 0 Alanine 43.2 .0 43 49 Valine 19.7 20 Methionine 5.80 6 0.8 . .4 Isoleucine 18.3 18 Leucine 33.6 34 Tyrosine 15.5 16 Phenylalanine 11.4 11 Lysine 19.2 19 Histidine 10.1 10 260 300 400 500 600 Arginine 21.1 21 Wavelength (nm) Tryptophan 5.82 6 Half-cystine 0.75 1 FIG. 2. Absorption spectrum of the native aliphatic nitrilase. The concentration of enzyme protein was 2.57 mg/ml in 0.01 M a Except for those amino acids determined independently and those esti- potassium phosphate buffer (pH 7.5). The same buffer was examined mated by extrapolation, the results are within ±5%. There was no significant as a control. difference in amino acid residues between the 20-h hydrolysate and the 42-or 70-h hydrolysate; each value is the average of the values for these hydroly- sates. b Both free and amide residues. G-4000SW column, which showed a symmetrical single Determined by extrapolation to time zero, assuming first-order decay. protein peak. The purity of the purified enzyme was also examined by double immunodiffusion in agarose. A single precipitin line was observed between antibodies and the Further experiments are in progress to identify the sub- crude cell extract of R. rhodochrous K22. The purified stances showing absorbance at 320 and 380 nm to elucidate enzyme catalyzed the hydrolysis of crotononitrile to cro- the nitrilase reaction mechanism. Qualitative analysis of the tonic acid at 0.737 ,umol/min per mg of protein under the following metals in the concentrated enzyme solution was standard reaction conditions. performed with an inductively coupled radiofrequency Molecular weight and subunit structure. The sedimentation plasma spectrophotometer: Be, B, Mg, Al, Si, P, S, Ca, Ti, coefficient (S20,) and diffusion coefficient (D20,,) of the V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Sr, Zr, Mo, Pd, Ag, Cd, enzyme were calculated to be 10.9S (20°C; 10 mM potassium Sn, Sb, Ba, Ta, W, Pt, Au, Hg, Pb, La, and Ce. However, phosphate buffer, pH 7.5) and 6.27 x 10-6 cm2/s, respec- none of these 35 metals were detected within the limits of the tively. A relative molecular weight of 604,000 ± 30,000 was assay (10 nglml). On the other hand, the nitrile hydratases of determined by the sedimentation equilibrium method (46), Pseudomonas (30) and Brevibacterium (31) species contain assuming a partial specific volume of 0.74. The molecular tightly bound iron (40, 41), and the R. rhodochrous Ji nitrile weight of the enzyme was determined to be 650,000 by hydratase contains cobalt (32). In this respect, strain K22 analytical HPLC. When the enzyme was treated with 1% nitrilase is quite different from these nitrile hydratases. SDS and 50 mM 2-mercaptoethanol and then electro- Amino acid composition. The enzyme contained all of the phoresed on a gel containing 0.1% SDS, a single band was common amino acids (Table 2). The amino acid composition observed upon protein dyeing (Fig. 1, lane B). The molecular of our enzyme resembles those of the R. rhodochrous Jl weight corresponding to the band was estimated to be nitrilase (20) and the Klebsiella enzyme (38) (obtained from 41,000, as judged from its mobility relative to those of the nucleotide sequence) except for valine, threonine, and reference proteins. Thus, the enzyme probably consists of 15 lysine and for aspartic acid, leucine, and lysine, respec- to 16 subunits identical in molecular weight; this molecular tively. The minimum value with the nearest integral numbers weight is closer to those of the enzymes found in Nocardia of all amino acids found was calculated to be 40,671, which (Mr = 560,000; 16, 18) and Fusarium (Mr = 620,000; 17) coincides well with the subunit molecular weight (41,000) species. determined by SDS-polyacrylamide gel electrophoresis, The purified enzyme showed maximum absorption at 278 Stability. The highly purified enzyme could be stored for nm and weak absorption at 320 and 380 nm (Fig. 2); such more than 2 months at 4°C and for 11 months or more at phenomena have not been reported for any other nitrilases. -20°C without loss of activity in a 50% glycerol solution Even when purification of the enzyme was carried out three containing 10 mM potassium phosphate buffer (pH 7.5) and times in the same manner, the enzyme showed this absorp- 1 mM dithiothreitol. tion pattern, which was not eliminated upon thorough dial- The stability of the enzyme was examined at various ysis against 10 mM potassium phosphate buffer (pH 7.5). temperatures. After the enzyme had been preincubated for 1 These findings suggest that a may bind to the h in 10 mM potassium phosphate buffer (pH 7.5) containing enzyme. However, when crotononitrile, p-chloromer- 1 mM dithiothreitol, a sample of the enzyme solution was curibenzoate, HgCl2, or H202 was added at a final concen- taken and the nitrilase activity was assayed under the tration of 0.1 M, 10 mM, 5 mM, or 5 mM, respectively, to 7 standard conditions. It exhibited the following activity: nmol of the enzyme in 1.0 ml of 10 mM potassium phosphate 55°C, 0%; 50°C, 6.7%; 45°C, 28%; 40°C, 87%; 35°C, 92%; buffer (pH 7.5), no appreciable spectral shift occurred. 30°C, 94%; and 25°C, 100%. 4812 KOBAYASHI ET AL. J. BACTERIOL.

(A) 100 (B). 100

- .2a

0 30 50 50 'o r a

" - 4 5 6 7 8 9 -20 30 40 50 60 pH Temperature (OC) FIG. 3. Effects of pH (A) and temperature (B) on activity of the aliphatic nitrilase. (A) The reactions were carried out for 20 min at 25°C in the following buffers (final concentration, 0.1 M): CH3COONa-CH3COOH (A), potassium phosphate (0), MES-NaOH (0), and Tris hydrochloride (L). (B) The reactions were carried out for 20 min at various temperatures. Relative activity is expressed as the percentage of the maximum activity attained under the experimental conditions.

The stability of the enzyme was examined at various pHs. agents and H202 were examined. After the enzyme, which The enzyme was incubated at 25°C for 45 min in the was fully dialyzed against 10 mM potassium phosphate following buffers (final concentration, 0.1 M): 2-(N-mor- buffer (pH 7.5), had been incubated with each inhibitor (final pholino)ethanesulfonic acid (MES)-NaOH (pH 5.5 to 7.0), concentration, 1 mM) for 30 min, the reaction was carried potassium phosphate (pH 6.0 to 8.0), Tris hydrochloride (pH out in the standard reaction mixture without the addition of 7.5 to 9.0), and NH4Cl-NH40H (pH 9.0 to 10.0). Then a dithiothreitol. Each reductant was added to the reaction sample of the enzyme solution was taken, and the nitrilase mixture described above, after the aforementioned proce- activity was assayed under the standard conditions. The dure, and then the enzyme was assayed. The inhibition by enzyme was most stable in the pH range of 6.0 to 8.0, 40% of HgCl2 could be partially reversed by the addition of dithio- its initial activity being retained even at pH 10.0. threitol, 2-mercaptoethanol, and reduced glutathione (46, 48, Effects of pH and temperature. The effect of pH on the and 14%, recovery, respectively, compared with the activity activity of the enzyme was examined with crotononitrile as without treatment with inhibitor [100%]). The inhibition by the substrate (Fig. 3A). This enzyme showed maximum H202 could be partially reversed by the addition of dithio- activity in the more acidic region of pH 5.5, whereas the threitol, 2-mercaptoethanol, and reduced glutathione (34, 19, optimum pHs of all other nitrilases previously reported are and 29% recovery, respectively). All other nitrilases charac- in the range of 7.5 to 9 (4, 16-20, 35, 38). However, this terized so far are inactivated by thiol reagents and are nitrilase was similar to those of Nocardia sp. strain NCIB therefore classified as sulfhydryl enzymes. The R. rhodo- 11215 and Fusarium sp. with respect to the broad pH range. chrous K22 enzyme was not inhibited by several thiol The optimal temperature was found to be 50°C. Above 55°C, reagents that were inhibitory for the other nitrilases (4, enzyme activity was rapidly lost (Fig. 3B). 16-20, 35, 38). With respect to the active sulfhydryl group, Inhibitors. Various compounds were investigated for their this nitrilase seems to differ from other nitrilases. inhibitory effects on enzyme activity. Incubation was carried Substrate specificity. The ability of the enzyme to catalyze out at 25°C for 10 min in the standard reaction mixture the hydrolysis of various nitriles was examined (Table 3). containing a tested compound at 1 mM. The enzyme was The R. rhodochrous K22 enzyme is characteristic in its strongly inhibited by HgCl2 and H202 (0 and 3.7%, respec- broad substrate specificity for aliphatic nitriles, which can- tively, of the original activity). However, the enzyme not be hydrolyzed by any nitrilases (4, 16-20, 35, 38). showed relatively high resistance to the thiol reagents Aliphatic olefinic nitriles, especially low-molecular-mass 5,5'-dithio-bis(2-nitrobenzoate), iodoacetate, and N-ethyl- ones with two to five carbon atoms, were remarkably active maleimide. At a concentration of 5 or 10 mM, p-chloro- as substrates for the enzyme. Acrylonitrile is known to be an mercuribenzoate caused appreciable inhibition. Carbonyl effective nucleophilic agent for the modification of protein reagents such as hydroxylamine, phenylhydrazine, semicar- sulfhydryl groups (7, 14). Acrylonitrile, despite its alkylating bazide, cysteamine, amoinouanidine, L-or D-penicillamine, ability, was 3.5 times more suitable as a substrate than and D-cycloserine were- iWt inhibitory toward the enzyme. crotononitrile. When the Michaelis constants for acryloni- This nitrilase seems toer in this respect from the nitrile trile and crotononitrile were estimated from the Lineweaver- hydratase from P. chFroraphis B23, which contains pyr- Burk plot, the former (Km = 1.14 mM) was found to exhibit roloquinoline quinone as a prosthetic group (33). Chelat- higher affinity for the enzyme than did the latter (Km = 18.9 ing reagents, e.g., o-phenanthroline, 8-hydroxyquinoline, mM). Saturated aliphatic nitriles, e.g., acetonitrile, 4-chlo- EDTA, o,a'-dipyridyl, and diethyldithiocarbamate, also had robutyronitrile, and glutaronitrile, were also attacked by the no significant effect on the enzyme. These results are in good enzyme. Branched-chain saturated aliphatic nitriles with two agreement with the finding that this enzyme contains no to five carbon atoms, e.g., trimethylacetonitrile, lactonitrile, metals. (S)-(+)-2-methylbutyronitrile, isovaleronitrile, and 3-cyano- The effects of reductants on the inhibition by thiol re- L-alanine, were less effective substrates than the corre- VOL. 172, 1990 NOVEL ALIPHATIC NITRILASE OF R. RHODOCHROUS K22 4813

TABLE 3. Substrate specificity of the aliphatic nitrilase Substrate Relative activity (%)Substrate Relative activity Crotononitrile ...... 100a Glutaronitrile .345 Acrylonitrile ...... 348 Adiponitrile .110 Methacrylonitrile ...... 143 Pimelonitrile .27.3 3-Ethoxyacrylonitrile ...... 19.1 Suberonitrile.21.4 3-Aminocrotononitrile ...... 30.3 Sebaconitrile .15.7 2-Chloroacrylonitrile ...... 47.1 3,3'-Oxydipropionitrile .29.0 Fumaronitrile ...... 27.4 Iminodiacetonitrile.261 Diaminomaleonitrile ...... 18.6 3,3'-Iminodipropionitrile .19.6 2-Methyl-2-butenenitrile ...... 14.9 Benzonitrileb .27.1 2-Methyl-3-butenenitrile ...... 46.8 3-Tolunitrileb .67.9 2-Pentenenitrile ...... 6.33 3-Nitrobenzonitrileb .74.5 3-Pentenenitrile ...... 132 Isophthalonitrileb .66.1 1,4-Dicyano-2-butene ...... 42.6 3-Methoxybenzonitrileb 24.8 2,4-Dicyano-1-butene ...... 127 3-Ethoxybenzonitrile .21.1 Acetonitrile ...... 28.3 3-Cyanopyridineb .7.27 Propionitrile ...... 12.7 4-Cyanopyridineb.9.71 Butyronitrile ...... 18.0 Cyanopyrazineb.17.3 Isobutyronitrile ...... 6.74 2-Furonitrileb .52.0 Valeronitrile ...... 40.8 2-Thiophenecarbonitrileb 61.1 Capronitrile ...... 38.7 Piperonylonitrileb .6.80 Isocapronitrile ...... 41.5 Cyclopropanecarbonitrileb .22.9 Methoxyacetonitrile ...... 37.4 Cyclopentanecarbonitrileb .11.7 Chloroacetonitrile ...... 61.7 4-Cyano-1-cyclohexeneb .7.03 (+)-2-Chloropropionitrile ...... 6.80 1-Cyclopenteneacetonitrileb .24.9 3-Chloropropionitrile ...... 113 1-Cyclohexeneacetonitrileb .18.2 4-Chlorobutyronitrile ...... 116 Cinnamonitrileb .31.2 Cyanoacetic acid ethyl ester...... 117 Phenylacetonitrileb .27.3 N-Methyl-p-alaninenitrile...... 11.4 3-Xylylene dicyanide .14.6 Malononitrile.45.1 2-Thiopheneacetonitrile .73.5 ...... 271 3-Thiopheneacetonitrileb ...... 66.3 a The synthesis of crotonic acid, corresponding to 0.737 ,umol/min per mg of protein, was taken as 100%. b Incubated at a final concentration of 10 mM. sponding straight-chain ones such as propionitrile and butyr- acids and ammonia by nitrile hydratase and amidase, respec- onitrile. Steric hindrance of the enzyme by the side chain of tively (2, 13, 27, 30, 31). In the former respect, the R. the branched-chain nitriles clearly influences the rate of rhodochrous K22 enzyme seems to exhibit similarity to hydrolysis. As for aliphatic dinitriles, nitriles with three known benzonitrile-specific nitrilases. Surprisingly, how- atoms between the two cyano groups, such as glutaronitrile ever, the R. rhodochrous K22 nitrilase showed significant and iminodiacetonitrile, were better substrates than those activity toward saturated aliphatic nitriles. These findings with four atoms, such as adiponitrile, 3,3'-oxydipropioni- are not in agreement with the well-known hypothesis men- trile, and 3,3'-iminodipropionitrile. Furthermore, the hydrol- tioned above and lead to the conclusion that this enzyme is ysis rate became lower with an increase in the number of a novel type of nitrilase. carbon atoms of the dinitriles. Nitriles with an aromatic or Specific and molecular activities. The specific activity of heterocyclic ring were hydrolyzed at much lower rates than the aliphatic nitrilase was 0.737 ,umol/min per mg of protein the aliphatic nitriles mentioned above (compared with the at 25°C under the standard assay conditions. Assuming the activity toward acrylonitrile or glutaronitrile, the activity molecular weight of this enzyme to be 650,000, its molecular toward benzonitrile was only 7.8%). Compared with the activity was calculated to be 0.479 kmollmin per mol of activity toward crotononitrile (100%), there was slight for- nitrilase at 25°C under the standard assay conditions. On the mation of ammonia (1 to 5%) from the following nitriles: other hand, the molecular activities of the nitrile hydratases isovaleronitrile, (S)-(+)-2-methylbutyronitrile, cyanoacetic of Pseudomonas (30) and Brevibacterium (31) species were acid, 3-cyano-L-alanine, lactonitrile, 3-aminobenzonitrile, 184 and 161 kmol/min per mol of nitrile hydratase, respec- 4-tolunitrile, 4-nitrobenzonitrile, 4-methoxybenzonitrile, 4- tively, at 20°C with propionitrile as the substrate. The ethoxybenzonitrile, 4,4'-biphenyldicarbonitrile, 3,5-diiodo- marked difference in molecular activity between the nitrilase 4-hydroxybenzonitrile, 2-cyanopyridine, 1,2-dicyanocyclo- and nitrile hydratases might reflect differences with respect butane, 5-norbornene-2-carbonitrile, 3-indoleacetonitrile, to reaction mechanisms and cofactors (33, 40, 41). 2-pyridineacetonitrile, cx-methylphenylacetonitrile, 2-furan- Stoichiometry. The stoichiometry of nitrile consumption acrylonitrile, mandelonitrile, and 1,4-piperazinedicarboni- and acid formation during the hydrolysis of nitriles was trile. Other nitriles (see Materials and Methods) were nearly examined in a reaction mixture consisting of 20 ,umol of inactive as substrates for our enzyme. potassium phosphate buffer (pH 8.0), 1 p.mol of dithiothrei- It has been previously shown that nitriles of which the tol, 0.2 mmol of crotononitrile, and 1.7 nmol of the enzyme cyano group is conjugated with a double bond, such as in a final volume of 2.0 ml. The reaction was carried out at benzonitrile, can be directly hydrolyzed to the correspond- 25°C in an airtight tube. Throughout the experiment (Fig. 4), ing acids and ammonia by nitrilase (4, 16-20, 35, 38), crotononitrile was stoichiometrically hydrolyzed, with the whereas aliphatic nitriles are catabolized in two stages, via concomitant formation of crotonic acid and ammonia. No conversion to the corresponding amides and then to the formation of crotonamide was noted. The results indicated 4814 KOBAYASHI ET AL. J. BACTERIOL.

that these enzymes do not share any antigenic determinants with the R. rhodochrous K22 enzyme. This enzyme is recognized as having considerable com- mercial potential as well as being of general scientific inter- est. Furthermore, the development of stable activated sludge systems involving Rhodococcus nitrilases would represent a major advance in the treatment of the toxic wastes, i.e., E nitriles, from factories, whereas the conventional activated sludge systems are highly susceptible to inactivation by the wastes. Very recently, we determined the nucleotide se- 60 quence of the nitrile hydratase gene of R. rhodochrous Jl 0 (unpublished experiments). To clarify whether the R. rhodochrous K22 and Jl nitrilases, the Klebsiella nitrilase, and the R. rhodochrous Jl nitrile hydratase exhibit any cross-homology at the gene level, studies involving the 20 molecular cloning of both nitrilase genes are in progress.

ACKNOWLEDGMENTS We thank J. Imanishi, Kyoto Prefectural University of Medicine, for the antibody preparation and helpful advice. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. M. Kobayashi is the recipient of a JSPS Fellowship for Japanese Junior Scientists. 0 1 2 3 4 LITERATURE CITED Time 1. Alshamaony, L., M. Goodfellow, and D. E. Minnikin. 1976. Free (h) mycolic acids as criteria in the classification ofNocardia and the FIG. 4. Time course and stoichiometric analysis of the nitrilase rhodochrous complex. J. Gen. Microbiol. 92:188-199. reaction, using crotononitrile as the substrate. Symbols: [1, croto- 2. Asano, Y., K. Fujishiro, Y. Tani, and H. Yamada. 1982. Ali- nonitrile; *, crotonic acid; O, ammonia. The assay conditions and phatic nitrile hydratase from Arthrobacter sp. J-1: purification method for analysis of reactant and are described in Results and characterization. Agric. Biol. Chem. 46:1165-1174. and Discussion. 3. Asano, Y., T. Yasuda, Y. Tani, and H. Yamada. 1982. A new enzymatic method of acrylamide production. Agric. Biol. Chem. 46:1183-1189. 4. Bandyopadhyay, A. K., T. Nagasawa, Y. Asano, K. Fujishiro, Y. that crotonic acid was formed stoichiometrically with the Tani, and H. Yamada. 1986. Purification and characterization of consumption of crotononitrile. When crotonamide was benzonitrilases from Arthrobacter sp. strain J-1. Appl. Environ. added as the substrate, no formation of crotonic acid was Microbiol. 51:302-306. detected. This finding suggests that crotonamide is not an 5. Becker, B., M. P. Lechevalier, R. E. Gordon, and H. A. intermediate in the course of nitrile hydrolysis by the en- Lechevalier. 1964. Rapid differentiation between Nocardia and zyme. Hitherto, enzymes that catalyzed the hydrolys'is of Streptomyces by paper chromatography of whole-cell hydroly- nitriles to acid and ammonia and the of nitriles to sates. Appl. Microbiol. 12:421-423. hydration 6. Bradford, M. 1976. A rapid and sensitive method for the amides were generically called nitrilases (9, 25). Although quantification of microgram quantities of protein utilizing the these enzymes belong to a group of nitrile-degrading en- principle of protein-dye binding. Anal. Biochem. 72:248-254. zymes, from these results it must be concluded that the 7. Cavins, J. F., and M. Friedman. 1968. Specific modification of nitrilase catalyzes only the direct hydrolysis of nitriles to protein sulfhydryl groups with a,p-unsaturated compounds. J. acids and ammonia, without the formation of amides as Biol. Chem. 243:3357-3360. intermediates, and in this respect it is completely different 8. Collins, M. D., M. Goodfellow, and D. E. Minnikin. 1982. A from nitrile hydratases. survey of the structures of mycolic acids in Corynebacterium Serological properties. The following eight Rhodococcus and related taxa. J. Gen. Microbiol. 128:129-149. strains from the IFO, JCM, and ATCC type culture collec- 9. Collins, P. A., and C. J. Knowles. 1983. The utilization of nitriles were and amides by Nocardia rhodochrous. J. Gen. Microbiol. tions examined, using crotononitrile as a substrate, for 129:711-718. the ability to form the nitrilase: R. erythropolis (IFO 12539 10. Conway, E. J., and A. Byrne. 1933. An absorption apparatus for and IFO 12682), R. rhodochrous (IFO 3338, JCM 2157, JCM the micro-determination of certain volatile substances. I. The 3202, and ATCC 21198), R. rhoseus (JCM 2156), and R. micro-detemination of ammonia. Biochem. J. 27:419-429. rubropertinctus (JCM 3204).. Only R. erythropolis (IFO 11. Edelhoch, H. 1967. Spectroscopic determination of tryptophan 12682) showed low activity (4.04 x 10-2 U and 3.32 x 10-4 and tyrosine in proteins. Biochemistry 6:1948-1954. Ulmg). 12. Fawcett, J. K., and J. E. Scott. 1960. A rapid and precise method Immunodiffusion techniques (34) were used to examine' for the determination of urea. J. Clin. Pathol. 13:156-159. the ability of antise'rum prepared against the purified nitrilase 13. Firmin, J. L., and D. 0. Gray. 1976. The biochemical pathway of R. rhodochrous K22 to cross-react with the for the breakdown of methyl cyanide (acetonitrile) in bacteria. nitrilase from Biochem. J. 158:223-229. R. erythropolis (IFO 12682). No precipitin band was formed 14. Friedman, M., J. F. Cavins, and J. S. Wall. 1965. Relative with this enzyme. The nitrilase from R. rhodochrous Jl (20) nucleophilic reactivities of amino groups and mercaptide ions in and the nitrile hydratases from R. rhodochrous Jl (32), P. addition reactions with ax,-unsaturated compounds. J. Am. chlororaphis B23 (30), and Brevibacterium sp. strain R312 Chem. Soc. 87:3672-3682. (31) also did not form pre'cipitin bands. These results indicate 15. Goodfellow, M. 1986. Genus Rhodococcus Zopf 1891, 28AL, p. VOL. 172, 1990 NOVEL ALIPHATIC NITRILASE OF R. RHODOCHROUS K22 4815

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