Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 73, No. 6, 425-430. 1992

Purification and Characterization of the Nitrilase from Alcaligenes faecalis ATCC 8750 Responsible for Enantioselective Hydrolysis of Mandelonitrile KEIZOU YAMAMOTO,* ISAO FUJIMATSU, AND KEN-ICHI KOMATSU

Pharmaceutical Research and Development Department, Asahi Chemical Industry Co. Ltd., 6-2700 Asahi-machi, Nobeoka, Miyazaki 882, Japan Received 13 December 1991/Accepted 24 March 1992 A nitrilase that converts racemic mandelonitrile to R-(-)-mandelic acid was purified to apparent homogeneity from a cell extract of Alcaligenesfaecalis ATCC 8750. The molecular weight of this enzyme was estimated to be 32,000 + 2,000 from SDS-PAGE and that of the native enzyme 460,000-+30,000 from HPLC gel filtration. The enzyme preferentially hydrolyzed substituted aliphatic nitriles, in particular benzyl cyanide and its p-substituted compounds, but hydrolyzed aromatic nitriles only with difficulty. The amino-terminal amino acids were sequenced and their sequences compared with those of other nitrilases. The purified enzyme had a pH optimum of 7.5 and an optimum temperature range of 40 to 45°C. The enzyme was inhibited by various thiol reagents. It hydrolyzed racemic mandelonitrile, producing optically pure R-(-)-mandelic acid and ammonia without the concomitant production of mandelamide, evidence that this nitrilase is highly enantioselective for R-mandelonitrile.

R-(--)-Mandelic acid, an optical resolving reagent and a source of pharmaceuticals, is now produced by optical resolution of the racemate with chiral amines (1); enzymatical producing methods (2-4) have not been used for industrial production because of expensive substrates, coenzymes and enzymes. Therefore, we have been investigating a new production process that uses the action of microorganisms on racemic mandelonitrile to produce the optically active acid. There have been reports on the production of optically active compounds such as amino acids (5) or 2-hydroxyacids (6) from the corresponding nitriles by various microorganisms. The ability of certain microorganisms to produce S-(+)-2-phenylpropionic acid and S-(+)-ibuprofen from nitrile compounds has recently been examined, with the result that S-(+)-ibuprofen has been produced from the corresponding racemic nitrile by an isolated bacterial strain, Acinetobacter sp. AK226 (7). We have purified and characterized the enantioselective nitrilase from Acinetobacter sp. AK226 (8). Elsewhere (9), we reported the screening of microorganisms that produce R-(--)-mandelic acid from racemic mandelonitrile, one of which, Alcaligenesfaecalis ATCC 8750, converted mandelonitrile or benzaldehyde and HCN to R(-)-mandelic acid. This microorganism has an R-enantioselective nitrilase for mandelonitrile and an amidase for mandelamide, but not a nitrile hydratase for mandelonitrile. We here report the purification and properties of the nitrilase from A. faecalis ATCC 8750 that hydrolyzes racemic mandelonitrile to produce R-(--)-mandelic acid.

Co., Inc. Bovine serum albumin was obtained from Sigma Chemical Co. Peptone, yeast extract D-3 and agar were purchased from Wako Pure Chemical Ind., Ltd. (Osaka). DEAE-cellulose (DE-52) was a product of Whatman BioSystems Ltd. Phenyl-Sepharose CL-4B and the reference proteins used to determine molecular weight were purchased from Pharmacia Fine Chemicals Co. Ltd. Membrane filters were obtained from Amicon Co. The other chemicals used were obtained from commercial sources. Microorganism and culture conditions

A. faecalis

ATCC 8750 was the source of the enzyme. The culture medium contained (per liter) 10 g of ammonium acetate, 5 g of yeast extract D-3, 5 g of peptone, 1.2g of KH2PO4, 0.8 g of KzHPO4, 0.2 g of MgSO4.7H20, 30 mg of FeSO4. 7H20, 1 g of NaCI and 3 g of n-butyronitrile (pH 7.2). A culture that had first been grown in 3 ml of culture medium was inoculated to 100 ml of the same medium in a 500-ml flask and cultured at 32°C with reciprocal shaking (250 rpm). After 18 h of culture, cells were harvested by centrifugation at 10,000xg at 5°C, then washed with 0.05 M potassium phosphate buffer, pH 7.0, containing 1 mM dithiothreitol (DTT) and 0.1 mM EDTA. The yield of wet ceils was approximately 18 g/l medium. Enzyme assay Nitrilase was assayed in a reaction mixture (1 ml) containing 10/~mol of mandelonitrile, 100/zmol of potassium phosphate buffer, pH 7.5, 1 pmol of DTT, 1 mg of bovine serum albumin and an appropriate amount of enzyme. The reaction was run at 30"C for 30 min then stopped by the addition of 0.2 ml of 80% acetic acid. The amounts of mandelic acid, mandelonitrile, mandelamide and benzaldehyde in the reaction mixture were measured as described elsewhere (9). Alternatively, the ammonia formed was measured by the method of Fawcett and Scott (10). One unit of the enzyme activity was defined as the amount of enzyme liberating 1/zmol of mandelic acid or ammonia per m in in the reaction mixture. Analysis Protein was determined by the method of

MATERIALS AND METHODS

Mandelic acid, mandelonitrile and the other nitrile compounds were purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo) and Aldrich Chemical Chemicals

* Corresponding author. 425

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YAMAMOTO ET AL.

J. FErtMENT.BIOEN6.,

Lowry et al. (11) with bovine serum albumin. The optical purity of the mandelic acid was measured as described previously (9). Total cyanide was assayed by the method of Asmus and Garshagen (12). Purification of nitrilase All purification procedures were done at 0 to 5°C. The buffer solution used throughout was 0.05 M potassium phosphate buffer, p H 7.0, containing 1 mM DTT and 0.1 mM E D T A . Step 1. Preparation o f a cell-free extract Washed cells (28 g) from 1.5 l of culture broth were suspended in 150ml o f 0.05 M buffer then disrupted by sonication at maximum amplitude for 32 rain in a Sonic 300 Dismembrator (Artex Systems Co.). Cell debris was removed by centrifugation for 20 min at 15,000 × g. Step 2. A m m o n i u m sulfate fractionation Saturated a m m o n i u m sulfate solution in 0.05 M buffer was added to the enzyme solution to obtain a concentration o f 10%. After the mixture had been stirred for 1 h, the precipitate formed was removed by centrifugation for 2 0 m i n at 15,000 × g and discarded. The a m m o n i u m sulfate concentration was increased to 35% saturation, and the mixture again stirred for 1 h. The resulting precipitate was collected by centrifugation for 2 0 m i n at 15,000×g, then dissolved in 20 ml of 0.05 M buffer and dialyzed overnight against the same buffer. Step 3. Ultracentrifugation The enzyme solution was centrifuged at 100,000×g for 2 h at 3°C using a RP50-2 rotor with a 55p-72 Himac Centrifuge (Hitachi Co., Tokyo). The supernatant solution was collected.

Step 4. Phenyl-Sepharose CL-4B column chromatography The enzyme solution was applied to a PhenylSepharose CL-4B column (1.7 × 17 cm) equilibrated with 0.05 M buffer. After the column had been washed with buffer containing 30% ethylene glycol (100ml), it was flushed with a linear gradient o f ethylene glycol in the same buffer (30 to 70%, 400 ml). The active fractions were combined, concentrated by filtration on a YM10 membrane in a Diaflo Cell 8050 (Amicon Co., Danvers, Mass., USA), then dialyzed overnight against 0.05 M buffer containing 30% ethylene glycol.

Step

5.

DEAE-cellulose

column

chromatography

The dialyzed solution was applied to a DEAE-cellulose column (1.7 × 17 cm) equilibrated with 0.05 M buffer containing 30% ethylene glycol, and was then flushed with a linear gradient o f NaC1 in the same buffer (0 to 0.5 M, 400 ml). The active fractions were combined, then concentrated by membrane filtration and dialyzed overnight against the buffer containing 30% ethylene glycol. Analytical methods for the nitrilase Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was done on 4 to 12% polyacrylamide slab gels using a Tris-glycine buffer system (13). The molecular weight of the subunit o f the enzyme was calculated from the relative mobilities o f standard proteins. The molecular weight o f the enzyme was determined using analytical high-pressure liquid chromatography (HPLC) at a flow rate o f 1.0 m l / m i n in a Tosoh C C P M system equipped with A s a h i p a k GS-620 (Asahi Chemical Ind. Co. Ltd., Tokyo). A solvent system o f 0.1 M potassium phosphate buffer (pH 6.5) containing 0.3 M NaCI was used. The A2s4 was measured. The molecular weight o f the enzyme was calculated by comparing its retention time with the retention times o f the standard proteins; thyroglobulin (669,000), ferritin (440,000), catalase (232,000), aidolase (158,000), bovine serum albumin (47,000), ovalbumin (43,000), chymotrypsinogen A

(25,000) and ribonuclease (13,700). Amino acid sequencing The purified enzyme was used for amino-terminal amino acid sequence analysis by automated Edman degradation in an Applied Biosystems 470A gas-phase protein sequencer. The phenylthiohydantoin amino acid derivatives were separated and identified with an on-line phenylthiohydantoin analyzer model 120A (Applied Biosystems). RESULTS Nitrilase purification The nitrilase in A. faecalis A T C C 8750 was purified 29.0-fold using the procedures described in Materials and Methods (summarized in Table 1). The purified enzyme showed only one protein band on SDS-polyacrylamide gel electrophoresis (Fig. 1) and a single symmetrical protein peak on gel filtration by H P L C . These results suggest that the enzyme was purified to homogeneity. Molecular weight and subunit structure The molecular weight o f the subunit was estimated to be 32,000_+2,000 by SDS-polyacrylamide gel electrophoresis as described in Materials and Method. The molecular weight o f the enzyme was 36,000--+2,000 on H P L C with a solvent system of 0.1 M potassium phosphate buffer (pH 6.5) containing 0.3 M NaCI. But, when 0.1 M potassium phosphate buffer (pH 7.5) containing 0.3 M NaC1 and 1 mM DTT was used as the solvent in the H P L C , the enzyme showed two protein peaks with molecular weights of 36,000_+2,000 and 460,000_+30,000 (ratio o f protein peaks: about 2 : 1). In the reaction mixture without 1 mM DTT, the time course of the nitrilase reaction as a function o f enzyme concentration showed a lag period, not linearity, in the early phase o f the reaction as did the enzymes from Nocardia sp. (14, 15) and Arthrobacter sp..1"-I (16). This apparently was caused by the activation process, in which the protein subunits aggregated to produce the high molecular weight polymer (the active native protein), as reported by Harper (14). Therefore, the protein of high molecular weight in the buffer (pH 7.5) containing 1 mM DTT (which indicates maximum activity) may represent the native nitrilase (molecular weight: 460,000_+30,000). The enzyme appears to consist of a great number o f identical subunits, as do the enzymes from Nocardia species (15, 17), Fusarium solani (18), Fusarium oxysporum f. sp. melonis (19), Rhodococcus rhodochrous K22 (20) and A. faecalis JM3 (21). The enzyme contained no substance that showed absorbance in the visible region o f 370-850 nm. Nor did any peak other than that at 279 nm appear in the ultraviolet region o f 250-370 nm. Amino-terminal amino acid sequence Results of the amino-terminal amino acids sequencing o f the purified enzyme are shown in Fig. 2. The sequence is partly homologous to the sequences of the nitrilases from Klebsiella TABLE 1. Purification of the nitrilase from A. faecalis ATCC 8750 Total activity (units) Cell-free extract 311 (NH4)2SO 4 fractionation 271 Ultracentrifugation 221 Phenyl-Sepharose CL-4B 81.5 DEAE-cellulose 55.8 Step

Total protein (mg) 2,900 530 166 30.0 18.0

Spec. act. (units/mg)

Yield (%)

0.107 0.511 1.33 2.72 3.10

100 87.1 71.0 26.2 17.9

VOL. 73, 1992

NITRILASE FROM A. FAECALIS

(A)

(B)

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FIG. I. SDS-polyacrylamide gel electrophoresis of the nitrilase from A. faecalis ATCC 8750. The purified enzyme was electrophoresed as described in Materials and Methods. The gel was stained with Coomassie Brilliant Blue R-250. (A) The enzyme, 2#g. (B) Marker proteins: 1, phosphorylase b (94,000); 2, bovine serum albumin (67,000); 3, ovalbumin (43,000); 4, carbonic anhydrase (30,000); 5, soybean trypsin inhibitor (20,100). The direction is from the cathode (top) to the anode.

Acinetobacter sp.

AK226 JM3 Alcaligenes faecalis

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Hg2CI2, ZnC12, Pb(NO3h, CuSO4 and p-chloromercuribenzoate. Its activity against inhibition by 1 mM p-chloromercuribenzoate was restored to 87% in the presence of 1 mM DTT, These results indicate that thiol groups have an important role in determining the activity of the enzyme. The chelating reagents such as E D T A , azide and cyanide did not cause inhibition, indicating that the enzyme has no metal ion requirement. This is consistent with the result showing that the enzyme has no special absorbance. FeSO4 caused a decrease in enzyme activity, probably because of redox effects on thiol linkage or formation of a complex with the thiol group. Phenylhydrazine, a carbonyl reagent, had no effect on enzyme activity. Substrate specificity The ability of the enzyme to catalyze the hydrolysis of various nitriles was examined (Table 3). It slightly hydrolyzed the nonsubstituted aliphatic nitriles such as n-valeronitrile and acrylonitrile. It hydrolyzed only with difficulty the aromatic nitrile benzonitrile, but readily hydrolyzed the heterocyclic nitrile furonitrile. The enzyme preferentially hydrolyzed aliphatic nitriles with a chloro, bromo, phenyl, thiophene, or pyridine group substitution. Benzyl cyanide and its p substituted compounds such as p-chlorobenzyl cyanide, p fluorobenzyl cyanide, p-nitrobenzyl cyanide and p aminobenzyl cyanide were the most suitable substrates, indicating that this enzyme is arylacetonitrilase.

p n e u m o n i a e subsp, ozaenae (22) and A c i n e t o b a c t e r sp. AK226 (8), and identical to that of the nitrilase from A. faecalis JM3 (21). Effects of pH and temperature The effects of pH on the activity of the nitrilase were studied (Fig. 3A). The enzyme had maximum activity at about pH 7.5. The optimum temperature range of the reaction was 40 to 45°C, activity being markedly reduced above 50°C (Fig. 3B). Stability Thermal inactivation studies of the purified enzyme were done at 40°C for 30 rain with the following 30 mM buffers containing 10 mM DTT: potassium phosphate (pH 5.5 to 8.0), Tris-hydrochloride (pH 7.5 to 9.0), and borate-NaOH (pH 8.5 to 10.0). Results (not shown) indicate that the enzyme is stable at pH 6.5 to 8.0, but unstable at lower and higher pHs. The purified enzyme could be stored at - - 2 0 ° C for at least 18 months without loss of activity in 0.05 M potassium phosphate buffer (pH7.0) containing 1 mM DTT, 0.1 mM E D T A and 30% ethylene glycol. Inhibitors The effects of various compounds on the enzyme's activity were examined (Table 2). The enzyme is very susceptible to inhibition by the thiol reagents such as Klebsiella pneumoniae subsp, ozaenae

i

8 9 10 20 30 40 50 60 70 pH Temperature (*C) FIG. 3. Effects of pH (A) and temperature (B) on the activity of the A. faeealis ATCC 8750 nitrilase. (A) Reactions were run for 30 min at 30°C in the following 0.1 M buffers containing 1 mM DTT and 1 mg/ml bovine serum albumin: potassium phosphate ( I ) , Trishydrochloride (n) and borate-NaOH (O). (B) Reactions were run for 30 rain at various temperatures. The relative activity is expressed as a percentage of the maximum activity under the experimental conditions used.

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IIMQ T R K I V A A ATCC 8750 L FIG. 2, Amino-terminal amino acid sequence of the nitrilase from A. faecalis ATCC 8750 compared with the sequences of the nitrilases from K. pneumoniae subsp, ozaenae (22), Acinetobacter sp. AK226(8) and A. faecalis JM3 (21). The sequenceshave been aligned empiricallyto maximize the number of identities.

428

YAMAMOTO ET AL.

TABLE 2.

J. FERMENT. BIOENG.,

Effects of various compounds on the activity of the A. faecalis ATCC 8750 nitrilase"

Compound None AgNO3 Hg2Cl2 CaC12 MnSO4 MgSO4 FeSO4 CuSO4 CoSO4 ZnCI2 Pb(NO3)2 N-Ethylmaleimide Iodoacetamide p-Chloromercuribenzoate EDTA Sodium azide Potassium cyanide a,a'-Dipyridyl Phenylhydrazine DTT Glutathione (reduced form)

(mM)

Relative activity (%)

0.1 0.01 0.1 0.1 0.1 0. I 0.1 0.01 0.1 0.1 0.01 0.1 0.01 0.1 0. I 0.1 0.01 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

100 34.7 37.7 5.4 110 104 Il0 82.2 60. I 0.0 3.7 12.8 2.8 26.2 0.0 29.3 22.5 18.2 0.0 124 98.0 101 101 109 124 105

• After the enzyme had been incubated for 10 min with the test compound, enzyme activity was assayed under the standard conditions described in Materials and Methods.

The enzyme appears to have a strict substrate specificity for hydroxynitriles since it readily attacked mandelonitrile, but showed no activity for other hydroxynitriles such as hydroxyacetonitrile, lactonitrile and 2-hydroxy4-phenylbutyronitrile. The aliphatic and aromatic dinitrile compounds such as malononitrile, succinonitrile and telephthalonitrile were somewhat effective substrates, but the corresponding mononitrile-monocarboxylic acids were inert. Similar results have been reported for nitrilases from two R. rhodochrous species (23, 24) and Acinetobacter sp. AK226 (8). Michaelis parameter The enzyme efficiently attacked mandelonitrile with an apparent Km of 5.75 mM and Vma~ of 5.28 units/mg, but there was substrate inhibition when the concentration of mandelonitrile was more than 10 raM. It also yielded R-(--)-mandelic acid from mandelonitrile stimultaneously formed in the reaction mixture containing benzaldehyde and H C N (apparent Kin; 1.18 and 13.0 mM, respectively), but the reaction was inhibited by benzaldehyde with a concentration of more than 1.50 mM. As mandelonitrile in aqueous solution is in a state of equilibrium (mandelonitrile ~ benzaldehyde+ HCN), substrate inhibition for mandelonitrile is therefore considered to be caused by the inhibition of benzaldehyde. Production of R-(-)-mandelic acid by the purified enzyme The time course of R-(--)-mandelic acid production from racemic mandelonitrile was studied using the purified enzyme. Results are shown in Fig. 4. Because mandelonitrile is in equilibrium in aqueous solution (mandelonitrile ~ benzaldehyde + HCN), the substrate was taken to be the total amount of mandelonitrile and benzaldehyde present, or the total cyanide for mandelonitrile and H C N . The presence of identical molar concentrations of R-(--)-mandelic acid and ammonia corresponded to the

TABLE 3.

Substrate specificity of the A. faecalis ATCC 8750 nitrilase"

Substrate KCN n-Butyronitrile n-Valeronitrile Acrylonitrile Cyclopropyl cyanide 3-Direct hylaminopropionitrile Chloroacetonitrile 2-Chloropropionitrile 3-Chloropropionitrile 2-Bromopropionitrile 3-Chloro-2-methylpropionitrile Mandelonitrile Benzonitrile 2-Cyanopyridine 3-Cyanopyridine (Nicotinonitrile) 4-Cyanopyridine 2-Furonitrile 2-Thiophenecarbonitrile Benzyl cyanide (Phenylacetonitrile) o-Chlorobenzyl cyanide m-Chlorobenzyl cyanide p-Chlorobenzyl cyanide p-Fluorobenzyl cyanide p-Nitrobenzyl cyanide p-Aminobenzyl cyanide 2-Phenylpropionitrile 3-Phenylpropionitrile 2-Phenylbutyronitrile 2-Phenylglycinonitrile 2-Thiopheneacetonitrile 3-Thiopheneacetonitrile 2-Pyridineacetonitrile 3-Pyridineacetonitrile 2-Phenoxypropionitrile Malononitrile Succinonitrile Fumaronitrile Glutaronitrile Adiponitrile Terephthalonitrile (1,4-Benzodinitrile)

Relative activityb (%) 6.7 4.5 I 1.6 6. I 6.7 9.4 387 243 45.4 224 15.0 100 1.1 25.0 3.5 14.4 149 17.9 838 112 218 843 1080 1040 1670 43.8 71.3 51.7 686 26.3 340 11.5 287 20.1 6.0 10.8 10.7 3.5 9.5 8.5

• The reaction was carried out under the standard conditions described in Materials and Methods, except that mandelonitrile was replaced by the test nitriles. Activity was determined by measuring the amount of ammonia produced in the reaction mixture. u The activity for mandelonitrile that corresponded to 3.10 units/mg was taken as 100%. The following compounds slightly served as substrates (l to 3%); acetonitrile, propionitrile, isobutyronitrile, methacrylonitrile, m-tolunitrile, m-chlorobenzonitrile, 2-(4"isobutylphenyl) propionitrile, trans-l,4-dicyanocyclohexane and c/s-l,4-dicyanocyclohexane. The following compounds did not serve as substrates; aminoacetonitrile, 3-aminopropionitrile, 2,3-dibromopropionitrile, hydroxyaeetonitrile, laetonitrile, 2-hydroxy-4-phenylbutyronitrile, 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 4phenylbutyronitrile, 2-cyanoacetamide, 2-cyanoacetic acid and peyanobenzoic acid.

decrease in the amount of substrate. No mandelamide was detected at any time, indicating that the reaction was catalyzed by a nitrilase. No detectable amount of S-(+)mandelic acid was produced throughout the reaction. These findings demonstrate that the nitrilase is highly enantioselective for R-mandelonitrile. The reaction yield of R-(--)-mandelic acid-production against the racemic substrate reached 95%. This means that the S-mandelonitrile remaining in the reaction was spontaneously racemized by the chemical equilibrium and supplied as substrate; consequently, almost all the mandelonitrile is con-

NITRILASE FROM A. FAECALIS

VOL. 73, 1992

40

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3

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FIG. 4. Timecourse of the R-(-)-mandelic acid-producingreaction by the nitrilase from A. faecalis ATCC 8750. A reaction mixture (1 ml) containing 10/~mol of mandelonitrile, 100/Jmol potassium phosphate buffer (pFI7.5), 1mg of bovine serum albumin, 1/~mol of DTr and the enzyme(0.06 unit) was incubated at 30°C with shaking for 0, 1, 2, 4, and 6 h, after which the metabolites were analyzed as described in Materials and Methods. Symbols: O, total amount of mandelonitrile and benzaldehyde; ca, total amount of cyanide for mandelonitrile and HCN; o, R-(-)-mandelic acid; A, ammonia. sumed and converted to R-(--)-mandelic acid, as reported previously (9). On the other hand, this nitrilase had hydrolyzing activity for HCN as shown in substrate specificity. Therefore, the consumption of HCN by the enzyme is considered to be the cause that this reaction yield did not reach 100%. DISCUSSION The nitrilase of A. faecalis ATCC 8750 was purified from a cell-free extract 29.0-fold in a yield of 17.9%, evidence that nitrilase corresponding to 3.5% of the soluble protein was produced in the cells grown under experimental conditions in which ammonium acetate was the carbon source and n-butyronitrile the inducer. Nitrilases from two Nocardia species (rhodochrous group) (15, 17) and from F. solani (18), F. oxysporum f. sp. melonis (19), Arthrobacter sp. strain J-1 (16), K. pneumoniae subsp, ozaenae (22) and R. rhodochrous J-1 (23) preferentially hydrolyze aromatic nitriles such as benzonitrile and bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), but not aliphatic nitriles. Nitrilases with broad substrate specificities, which hydrolyze both aromatic and aliphatic nitriles, have been purified from Acinetobacter sp. AK226 (8) and R. rhodochrous K22 (20). Because the nitrilase from A. faecalis ATCC 8750 efficiently hydrolyzed substituted aliphatic nitriles, in particular benzyl cyanide and its p-substituted compound, it appears to be a new type of nitrilase distinct from the above enzymes. As judged by its substrate specificity, this enzyme is an arylacetonitrilase.

429

The amino-terminal amino acid sequence of this enzyme showed partial homology to the sequences of the nitrilases from K. pneumoniae subsp, ozaenae (22) and Acinetobacter sp. AK226 (8) although the three enzymes do not have common features in molecular conformation or substrate profile. The disclosed sequences of this enzyme and the A. faecalis JM3 nitrilase (21) were identical. Two nitrilases from A. faecalis ATCC 8750 and A. faecalis JM3 are the same kind of arylacetonitrilase. These arylacetonitrilases differ, however, in many properties such as molecular structure (the respective molecular weights of the enzyme and subunit; 460,000 and 32,000 for ATCC 8750 and 275,000 and 44,000 for JM3), salting out concentration with ammonium sulfate (10 to 35% saturation for ATCC 8750 and 30 to 55% saturation for JM3), substrate specificity and specific activity (the respective specific activities of the nitrilase from ATCC 8750 and JM3; 0.815 U/mg and 144 U/mg for 2-thiopheneacetonitrile, 8.90 U/mg and 124 U/mg for 3-pyridineacetonitrile, 0.189 U/mg and 11.1 U/mg for acrylonitrile, 3.10 U/mg and 12.4U/mg for mandelonitrile). Therefore, granted that experimental conditions differ, in each experiment these enzymes are thought to be distinct nitrilases. This should be more clearly shown by further studies of the subunit association and the amino acid sequence other than the amino-terminal. The strong inhibition of this nitrilase activity by thiol reagents shows that the thiol group of this enzyme has an important role in its activity. Based on the findings about molecular structure, we consider that the nitrilase in the reaction buffer (pH 7.5) containing 1 mM DTT was in a state of equilibrium between the subunits and an associated high-molecular weight polymer, an active protein. Inhibition by thiol reagents therefore appears to be caused both by the reaction at the active site of the nitrilase and by a reaction during the association process of the subunits. As reported elsewhere (9), the high enantioselective activity of the A. faecalis ATCC 8750 nitrilase provides a simple and efficient process for producing R-(--)-mandelic acid from racemic mandelonitrile or benzaldehyde and HCN. The nitrilase has substrate inhibition for benzaldehyde, which can be countered by the use of a substratefeeding system. The establishment of an effective reaction system for the industrial production of R-(--)-mandelic acid from benzaldehyde and HCN is now in progress. There may be many nitrilases besides the A. faecalis enzyme that have diverse enantiselectivities for their substrate. Because many a-hydroxy acids of commercial importance are synthesized chemically from their corresponding nitrile or aldehyde compounds, enzymatic processes that use a nitrilase as presented here should be developed in order to make possible the industrial production of a large variety of optically active a-hydroxy acids. ACKNOWLEDGMENTS

We are indebted to Mr. A. Tanaka, Analytical Research Center, Asahi Chemical Ind. Co. Ltd., for his help in the amino-terminai amino acid sequencing of the enzyme. REFERENCES I. Gattermann, L. and Wieland, H.: Die praxis des organischen

chemikers, p. 203-205, 2nd ed. Waiter de Gruyter & Co., Berlin (1927). 2. Mori, K. and Akao, H.: Synthesis of optically active aikenyl alcohols and a-hydroxyesters by microbial asymmetrichydrolysis by

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YAMAMOTO ET AL. the corresponding acetates. Tetrahedron., 36, 91-96 (1980).

3. Patterson, M. A. K., Szajewski, R.P., and Whitesldes, G.M.:

4.

5.

6. 7.

8.

Enzymatic conversion of a-keto aldehydes to optically active ahydroxy acids using glyoxalase I and II. J. Org. Chem., 46, 46824685 (1981). ¥amazaki, Y. and Maeda, H.: Enzymatic synthesis of optically pure (R)-(--)-mandelic acid and other 2-hydro×ycarboxylic acids: screening for the enzyme, and its purification, characterization and use. Agric. Biol. Chem., 50, 2621-2631 (1986). Choi, S. Y. and Goo, Y. M.: Hydrolysis of the nitrile group in aaminophenylacetonitrile by nitrilase; development of a new biotechnology for stereospecific production of S-a-phenylglycine. Arch. Pharm. Res., 9, 45--47 (1986). Fukuda, Y., Harada, T., and Izumi, Y.: Formation of L-ahydroxyacids from DL-a-hydroxynitriles by Torulopsis candida CN405. J. Ferment. Technol., 51, 393-397 (1973). Yamamoto, K., Ueno, Y., Otsuho, K., Kawakami, K., and Komatsu, K.: Production of S-(+)-ibuprofen from a nitrile compound by Acinetobacter sp. strain AK226. Appl. Environ. Microbiol., 56, 3125-3129 (1990). Yamamoto, K. and Komatsu, K.: Purification and characterization of nitrilase responsible for the enantioselective hydrolysis from Acinetobacter sp. AK226. Agric. Biol. Chem., 55, 14591466 (1991).

9. Yamamoto, K., Oishi, K., Fujimatsu, I., and Komatsu, K.:

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