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Dec 7, 2000 - Abstract The nitrile hydratase from Rhodococcus equi. A4 consisted of two kinds of subunits which slightly dif- fered in molecular weight (both ...
Appl Microbiol Biotechnol (2001) 55:150–156 DOI 10.1007/s002530000507

O R I G I N A L PA P E R

Irena Prˇepechalová · Ludmila Martínková Andreas Stolz · Mária Ovesná · Karel Bezousˇ ka Jan Kopecky´ · Vladimír Krˇen

Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus equi A4 Received: 22 May 2000 / Received revision: 1 September 2000 / Accepted: 1 September 2000 / Published online: 7 December 2000 © Springer-Verlag 2000

Abstract The nitrile hydratase from Rhodococcus equi A4 consisted of two kinds of subunits which slightly differed in molecular weight (both approximately 25 kDa) and showed a significant similarity in the N-terminal amino acid sequences to those of the nitrile hydratase from Rhodococcus sp. N-774. The enzyme preferentially hydrated the S-isomers of racemic 2-(2-, 4-methoxyphenyl)propionitrile, 2-(4-chlorophenyl)propionitrile and 2(6-methoxynaphthyl)propionitrile (naproxennitrile) with E-values of 5–15. The enzyme functioned in the presence of 5–98% (v/v) of different hydrocarbons, alcohols or diisopropyl ether. The addition of 5% (v/v) of nhexane, n-heptane, isooctane, n-hexadecane, pristane and methanol increased the E-value for the enzymatic hydration of 2-(6-methoxynaphthyl)propionitrile.

Introduction Nitrile hydratases and nitrilases were first investigated as biocatalysts for the manufacture of commodity chemicals (e.g. acrylamide, acrylic acid, nicotinamide; Nagasawa and Yamada 1990). Later on, chemo-, regioand stereoselectivity of these enzymes were examined (see Sugai et al. 1997 for review). A number of nitrilases (EC 3.5.5.1) showed different degrees of enantioselectivity towards α-substituted nitriles (Yamamoto et al. 1990, 1991; Layh et al. 1992; Gradley and Knowles 1994). I. Prˇepechalová · L. Martínková (✉) · M. Ovesná · J. Kopecky´ V. Krˇen Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenˇská 1083, 14220 Prague, Czech Republic e-mail: [email protected] Tel.: +420-2-4752569, Fax: +420-2-4752509 A. Stolz Institute of Microbiology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany K. Bezousˇka Department of Biochemistry, Faculty of Science, Charles University Prague, Hlavova 8, 12840 Prague, Czech Republic

Also many amidases (EC 3.5.1.4) were shown to be highly enantioselective towards α-substituted amides (Hirrlinger et al. 1996; Yamamoto et al. 1996). Previously, nitrile hydratases (NHases; EC 4.2.1.84) were assumed to be relatively non-stereospecific (Faber 1992). However, later on enantioselective hydration of α-arylpropionitriles by whole cells was reported (Kakeya et al. 1991; Cohen et al. 1992; Beard et al. 1993; Bauer et al. 1994; Layh et al. 1994; Blakey et al. 1995; Masumoto et al. 1995; Martínková et al. 1996; Fallon et al. 1997). Enantioselective NHases were purified from the gram-negative microorganisms Pseudomonas putida NRRL-18668 (Payne et al. 1997) and Agrobacterium tumefaciens d3 (Bauer et al. 1998). In contrast, no report has been published on the enantioselective NHases from gram-positive bacteria. Our recent findings on the enantioselectivity of Rhodococcus equi A4 cells for substituted α-arylpropionitriles (Martínková et al. 1996) led us to a detailed study about the properties of the NHase from this strain.

Materials and methods Chemicals 2-(2-, 3-, 4-Methoxyphenyl)propionitrile, 2-(4-chlorophenyl)propionitrile, 2-(3-benzoylphenyl)propionitrile, 2-(6-methoxynaphthyl)propionitrile, 2-phenylbutyronitrile and the corresponding amides were prepared according to Bauer et al. (1994, 1998). Standards of cyano benzamides were prepared as described by Martínková et al. (1995). All other chemicals were of analyticalgrade purity and supplied from standard commercial sources. Bacterial strain and culture conditions Rhodococcus equi A4 (Martínková et al. 1995; deposited in the Czech Collection of Microorganisms, Brno) was cultured in 1.2 l of medium (in gl–1, beef extract 3, peptone 10, NaCl 5) at 30°C under shaking (160 rpm, amplitude 23 mm, 0.4 l medium/2-l Erlenmeyer flask). The biomass was used to inoculate a 75-l fermenter (Bioengineering AG, Switzerland) containing 50 l of medium (DiGeronimo and Antoine 1976) with 10 mM methacrylamide and 109 mM glycerol. After 24 h cultivation (30°C,

151 min–1),

300 rpm, aeration 20 l the cells were removed by centrifugation, washed with Na2HPO4/KH2PO4 buffer (54 mM, pH 7.0) and stored at –70°C. Enzyme purification Frozen cells (85 g) were resuspended in Tris/HCl [50 mM, pH 7.5 (further buffer A)] with 1 mM EDTA and 40 mM sodium butyrate to 30% (w/v) and disintegrated with glass beads (0.1–0.3 mm) in a Dyno Mill (Type KDL, Helmut Claus, WAB, Germany) at the maximal power output for 10 min at cooling by a kryostat set to –20°C. Cell debris were removed by centrifugation (12,000 rpm, 30 min, 4°C). An FPLC system (LCC-501 Plus controller, P-500 pump, UV-1 monitor, REC-102 recorder and FRAC-100 autosampler) from Pharmacia Biotech was used at room temperature. The NHase eluted from a Q-Sepharose column (HK 50, Pharmacia) with a 0–1 M NaCl linear gradient in buffer A at 430–470 mM NaCl. Ammonium sulphate was added to the pooled fractions to a concentration of 1 M. The solution was stirred on ice for 20 min, filtered (Millex GV-Filter, 0.22 µm pore size, Millipore) and loaded onto a Phenyl-Sepharose-FF column (HR 16/10, Pharmacia). The NHase eluted with a 1–0 M ammonium sulphate linear gradient in buffer A at 580–450 mM ammonium sulphate.

996 and an HPLC solvent delivery system 600; Waters Associates, Milford, Mass., USA). Reversed-phase columns were Grom-Sil 100 Octyl14 FE (5 µm particles, 125×4 mm, Grom, Herrenberg, Germany) for 3-cyanopyridine and nicotinamide, Nova-Pak C18 (5 µm, 3.9×150 mm, Waters) for 2-phenylpropionitrile, 2-phenylpropionamide, 2-(6-methoxynaphthyl)propionitrile and 2-(6methoxynaphthyl)propionamide and Separon SGX G18 column (7 µm, 3.3×150 mm; Tessek Prague, Czech Republic) for other compounds. 25% (v/v) Acetonitrile (for 2-, 3-, 4-cyanopyridine, 1,2-, 1,3-, 1,4-dicyanobenzene, 2,6-dicyanotoluene, 2-aminobenzonitrile, 3-, 4-hydroxybenzonitrile, benzyl cyanide and the respective amides), 40% (v/v) acetonitrile (for 2-phenylpropionitrile and 2-(2-, 3-, 4-methoxyphenyl)propionitrile and the respective amides) or 50% (v/v) acetonitrile (for other compounds) in water plus 0.1% (v/v) H3PO4 were employed at a flow rate of 1.0 ml/min. For certain experiments with cosolvents, 2-phenylpropionitrile and 2-phenylpropionamide were analysed with 20–30% (v/v) acetonitrile. The absorbance was measured at local spectral maxima in the range from 210 nm to 240 nm or at 210 nm when no local maxima of the compounds were detected in this wavelength range. One unit of NHase activity was defined as the amount of enzyme that catalysed the hydration of 1 µmol of substrate per minute. Protein assay

Molecular weight and isoelectric point The relative molecular mass of the native enzyme was determined by gel filtration on a Superose 12 HR 10/30 column calibrated with protein standards (β-amylase, ovalbumin, myoglobin; Serva). The liquid chromatography system consisted of an LDC Analytical-ConstaMetric 3500 Bio solvent delivery system (LDC Analytical) and Dual path monitor UV-2 (Pharmacia). Proteins were eluted with buffer A plus 150 mM NaCl. The subunit size was determined by SDS gel electrophoresis in 10% polyacrylamide slab gels using a Tris/glycerol buffer system (Laemmli 1970). The gels were stained with Coomassie Brilliant Blue R-250. As marker proteins a Benchmark Prestained Protein Ladder was used (LifeTechnologies; GibcoBRL, USA). Isolelectric points of enzyme subunits were determined by 2D electrophoresis using a combination of isoelectric focusing in the first dimension (Pharmalyte 3–10) and SDS–PAGE in the second dimension (10% polyacrylamide gels as above).

Protein was determined according to Bradford (1976) using bovine serum albumin as a standard. Enzyme enantioselectivity The enantiomers of 2-phenylpropionitrile, 2-(2-chlorophenyl)propionitrile, 2-(2-, 4-methoxyphenyl)propionitrile, 2-phenylbutyronitrile and the respective amides and 2-(6-methoxynaphthyl)propionamide were determined by chiral HPLC (Bauer et al. 1994; Bauer 1997). The enantiomeric ratio (E) was calculated according to Chen et al. (1982). The enantiomeric excess of the biotransformation product of 2-(4-methoxyphenyl)butyronitrile was not determined as no suitable chiral chromatographic method was available.

Results Amino acid sequence The enzyme subunits were separated by SDS–PAGE in 10% polyacrylamide gels and transferred onto a polyvinylidene difluoride membrane. Coomassie Brilliant Blue R-250 stained spots corresponding to enzyme subunits were excised and subjected to automated Edman degradation (Protein Sequencer LF3600D, Beckman) according to the manufacturer’s instructions. Homologous proteins were searched in the NCBI database using the BLAST program (http:\\www.ncbi.nlm.nih.gov/cgi-bin/BLAST/ nph-newblast).

Purification of the NHase A typical purification pattern is shown in Table 1. The specific activity increased nearly ninefold by purification at 12% yield. More than 90% of the enzyme activity was lost when further purification of the NHase by gel filtration on Superdex 200 was attempted. No major peaks but the NHase were eluted from the column. The following experiments were therefore performed with the enzyme obtained after the Phenyl-Sepharose chromatography.

Enzyme activity The NHase activity was assayed with 10–50 µg/ml of protein and different substrates. If not stated otherwise, the reaction proceeded in Na2HPO4/KH2PO4 buffer (54 mM, pH 7.5) at 32°C. The reaction was stopped by adding 1/10 aliquots of volume of 1 M HCl and the precipitated protein was centrifuged (13,000 g, 15 min, 4°C). From biphasic organic-aqueous mixtures the amide was extracted with a tenfold volume of water. Nitriles and amides were determined by HPLC (HPLC Millenium Chromatography Manager 2.0, equipped with a multiwavelength detector model 486 or a photo diode array detector

Molecular weight and isoelectric point The apparent molecular mass of the native NHase was estimated to be approximately 74 kDa by gel filtration. SDS–PAGE showed that the protein consisted of two types of subunits with slightly different molecular weights (both approximately 25 kDa). The enzyme probably occurred both as a dimer and a tetramer like the

152 Table 1 Purification of the NHase from Rhodococcus equi A4. The enzyme was assayed using 5 mM 3-cyanopyridine as substrate. Experimental details are given in the text Step

Volume (ml)

Protein (mg)

Specific activity (U/mg)

Total activity (U)

Recovery (%)

Purification (-fold)

Crude extract Q-sepharose Phenyl-superose

282 34 4

274 20 4

9.3 64.6 82

2630 1300 328

100 49 12

1 6.9 8.8

NHase from Rhodococcus sp. N-771 (Nakasako et al. 1999) which showed similar behaviour during gel chromatography (apparent molecular weight from 51 to 66 kDa due to an equilibrium between the dimeric and the tetrameric structures). According to 2D electrophoresis, the pI of both subunits was 6.5.

inhibition was observed with iodoacetamide and a weak inhibition with EDTA (residual activities 3% and 88%, respectively). According to its sensitivity to oxidizing and reducing agents as well as its temperature sensitivity this enzyme resembled the Fe(III) containing enzyme from Rhodococcus sp. R 312 (Nagasawa et al. 1986).

N-Terminal amino acid sequences of the αand β-subunits

Substrate specificity

The N-terminal amino acid sequences of the smaller and the larger subunit (comprising 26 and 25 amino acid residues, respectively) of the purified enzyme exhibited marked similarities to those of the α- and β-subunits of the apparently identical NHases from Rhodococcus sp. N-774 (Ikehata et al. 1989), Rhodococcus sp. N-771 (Nakasako et al. 1999) and Brevibacterium sp. R 312 (reclassified as Rhodococcus sp. R 312; Huang et al. 1997). Amino acid residues 2 and 9 in the N-terminal region of the smaller subunit and residues 15, 18 and 19 in that of the larger subunit of strain A4 were not definitely identified. The other amino acid residues were identical to those of Rhodococcus sp. N-774. Therefore, it remains unclear whether these sequences were identical or only very similar.

The enzyme hydrated a broad range of aromatic, heterocyclic and arylaliphatic nitriles (Table 2). The lower reactivity of hydroxy- and amino-substituted benzonitriles in comparison with benzonitrile may be explained by electron-donating effects of the substituents. For hydroxy- and methylbenzonitriles (tolunitriles) a pronounced effect of the position of the substituents was observed. In general, the meta derivatives were better substrates than the ortho derivatives, probably due to steric effects. Similarly, 2-cyanopyridine was transformed with a lower rate than 3- and 4-cyanopyridine. Substrates with a methyl- or an ethyl-substituent attached to the α-position relative to the nitrile group (2-phenylpropionitrile, 2-phenylbutyronitrile) were good substrates but the enzyme activity decreased for other 2-phenylpropionitriles substituted on the benzene ring with a methoxy or a chloro group or containing an additional benzene ring, probably due to an increased substrate size.

Temperature and pH optimum The enzyme was incubated at 20–60°C for 5 min and assayed with 15 mM 3-cyanopyridine for 3 min. The temperature optimum of the NHase was 32–35°C. The enzyme activity decreased at 50°C and 60°C to 16% and 4%, respectively, of the maximal activity. The pH optimum of the enzyme was determined with 15 mM cyanopyridine at 32°C within a pH range of 4–9 using Na phosphate/citrate (pH 4–6), Na/K phosphate (pH 6–8) and Tris/HCl (pH 8–9) buffers (100 mM each). The enzyme showed a broad maximum at pH 7.5. At pH 6 and 9, the activity was reduced to 18 and 25% of the maximal activity, respectively. The enzyme was not active at pH 5.

Enantioselectivity of the enzyme for 2-arylpropionitriles

Effect of inhibitors on the enzyme activity

Activity of the enzyme in the presence of cosolvents

The enzyme was completely inhibited by Ag+, H2O2, dithiothreitol and phenylhydrazine (1 mM each). Strong

The NHase from R. equi A4 converted its substrates in biphasic reaction mixtures composed of water and several

The enzyme transformed preferentially the S-isomers of different substituted 2-arylpropionitriles. The highest degree of enantioselectivity was observed with 2-(6-methoxynaphthyl)propionitrile and 2-(4-methoxyphenyl)propionitrile (Table 2). 2-(2-Methoxyphenyl)propionitrile was transformed with a considerably lower enantioselectivity than 2-(4-methoxyphenyl)propionitrile. Also the exchange of the methoxy group in 2-(4-methoxyphenyl)propionitrile by a chlorine atom reduced the enantioselectivity of the enzyme. No enantioselectivity was observed with 2-phenylpropionitrile or 2-phenylbutyronitrile.

153 Table 2 Substrate specificity of the NHase assayed in a reaction mixture containing 54 mM NaH2PO4/K2HPO4 buffer (pH 7.5) and 2.5 mM substrate at 32°C. Enantiomeric ratios were determined from transformations of 0.6 mM of (R, S)2-(4-methoxyphenyl)propionitrile, (R, S)-2-(2-methoxyphenyl)propionitrile or (R, S)-2(6-methoxynaphthyl)propionitrile and 0.2 mM of (R, S)-2(4-chlorophenyl)propionitrile in the same buffer at 30°C. Specific enzyme activity with benzonitrile (55.4 U/mg of protein) was taken as 100%

a Not totally soluble at 2.5 mM b Products cyano benzamides

Substrate

Activity (%)

Benzonitrile 2-Aminobenzonitrile 2-Hydroxybenzonitrile 3-Hydroxybenzonitrile 4-Hydroxybenzonitrile 2-Tolunitrile 3-Tolunitrile 4-Tolunitrile 2,6-Dichlorobenzonitrilea 1,2-Dicyanobenzene 1,3-Dicyanobenzene 1,4-Dicyanobenzene 2,6-Dicyanotoluene 2-Cyanopyridine 3-Cyanopyridine 4-Cyanopyridine 2-Phenylacetonitrile Thiophenacetonitrile Naphthylcarbonitrilea Phenoxyphenylacetonitrile (R, S)-2-phenylpropionitrile (R, S)-2-(2-methoxyphenyl)propionitrile (R, S)-2-(3-methoxyphenyl)propionitrile (R, S)-2-(4-methoxyphenyl)propionitrile (R, S)-2-(4-chlorophenyl)propionitrile (R, S)-2-(6-methoxynaphthyl)propionitrile (R, S)-2-phenylbutyronitrile (R, S)-2-(3-benzoylphenyl)propionitrilea (R, S)-2-(4-methoxyphenyl)butyronitrile

100 (KM 1.4 mM) 35 45 90 26 38 128 62 0 17b 73b 35b 80 27 67 (KM 5.7 mM) 75 77 128 150 98 48 (KM 3.9 mM) 16 Traces 26 13 23 110 0 23

Table 3 Effect of organic cosolvents (5%, v/v) on the activity and enantioselectivity of the NHase towards (R, S)-2-(6-methoxynaphthyl)propionitrile (0.6 mM, from 100 mM stock solution in methanol) assayed in 54 mM Na2HPO4/KH2PO4 buffer (pH 7.5) at 30°C. Specific enzyme activity in the absence of cosolvent was 1.55 U mg–1 Cosolvent

Activity (%)a

Enantiomeric ratio (E)

None n-Hexanea n-Heptanea n-Hexadecanea Pristanea Isooctanea Methanol

100 76 73 73 91 124 116

14.8 30.1 30.0 26.0 22.5 41.3 34.4

a Biphasic

mixture

different hydrocarbons, but the activities were generally lower than the control (buffer with 0.6% of methanol; Fig. 1A and Table 3). NHase activity for 2-phenylpropionitrile was found in the presence of 5–98% (v/v) of nhexane, n-heptane, 2, 2, 4-trimethylpentane (isooctane), n-hexadecane, 2, 6, 10, 14-tetramethylpentadecane (pristane) and diisopropyl ether. In general, it was found that the NHase activity in the presence of various water-nonmiscible cosolvents increased with increasing solvent hydrophobicity. When different alcohols were used as cosolvents, the NHase activity was similar to that in water in 5–20% (v/v) ethanol and methanol, but decreased significantly in the series ethanol – 2-propanol – 2-butanol.

Enantiomeric ratio (E)

No selectivity 6.9 (S-selectivity) n.d. 14.2 (S-selectivity) 5.2 (S-selectivity) 14.8 (S-selectivity) No selectivity n.d.

Dimethyl sulphoxide and especially N,N-dimethylformamide, 2-methoxyethanol and 1, 2-dimethoxyethanol also suppressed the enzyme activity even at low concentrations (Fig. 1B). Altered enzyme activity probably resulted from both kinetic effects (KM decrease from 3.9 mM to 1.3 mM in the presence of 20% methanol) and enzyme denaturation. The deleterious effect of 50% methanol on the enzyme activity was irreversible (data not shown). 5% (v/v) Isooctane and methanol increased the rates of 2-(6-methoxynaphthyl)propionitrile conversion (compared to the rates in buffer with 0.6% of methanol; Table 3). In contrast, a slight decrease in the rate of 2phenylpropionitrile hydration was caused by these cosolvents. This suggested that the cosolvent effect on the reaction rates was dependent on the substrate structure, probably because of an improved availability of the highly hydrophobic 2-(6-methoxynaphthyl)propionitrile for the enzyme. Influence of organic solvents on the enzyme enantioselectivity The addition of 5% (v/v) of different hydrocarbons or methanol increased the enantioselectivity for the conversion of 2-(6-methoxynaphthyl)propionitrile from moderate to good (E=14.8 to E>30; Table 3). In biphasic reaction mixtures the altered enantioselectivity may reflect the relatively low substrate concentration in the aqueous phase. (Partition coefficients of 2-(6-methoxy-

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from 14 to about 19 by the addition of 5% (v/v) of nhexane, n-heptane, isooctane, pristane or n-hexadecane.

Discussion

Fig. 1 Effect of buffer-saturated water-nonmiscible cosolvents (A) and water-miscible cosolvents (B) on the activity of the NHase towards (R, S)-2-phenylpropionitrile (0.6 mM, from 100 mM stock solution in the respective cosolvent) assayed in 54 mM Na2HPO4/KH2PO4 buffer (pH 7.5) at 30°C. The specific enzyme activity (5.6 U mg–1) in the aqueous reaction mixture was taken as 100%

naphthyl)proprionitrile between water and the waternonmiscible cosolvents used were approx. 10–2.) No further increase in E was observed if the concentration of isooctane was raised to 10–50%. This effect was observed at a lesser extent also with 2-(4-methoxyphenyl)propionitrile. The enantiomeric ratio (E) increased

NHases have been found in various bacterial genera (e.g. Agrobacterium, Bacillus, Pseudomonas and Rhodococcus) (Nagasawa et al. 1987; Bauer et al. 1994; Pereira et al. 1998; Wieser et al. 1998). Most of this work was concerned with the transformation of aliphatic nitriles (especially acrylonitrile) or heterocyclic nitriles (especially 3-cyanopyridine) (Nagasawa and Yamada 1990). Surprisingly, there is only very limited information available about the transformation of aromatic compounds by purified NHases. From the literature it appears that most of the well characterized NHases which were purified and for which the gene sequences were determined (as those from Pseudomonas chlororaphis B23 and Rhodococcus (formerly Brevibacterium) sp. R312) do not convert simple aromatic nitriles such as benzonitrile (Nagasawa et al. 1986, 1987). On the other hand, there are some examples (in most cases whole cell experiments) which describe the conversion of aromatic nitriles (in most cases substituted arylacetonitriles or 2-arylpropionitriles) by bacteria with NHase activity (Beard et al. 1993; Geresh et al. 1993; Bauer et al. 1994; Layh et al. 1994; Meth-Cohn and Wang 1997). The biochemical results and the sequence data which were obtained for the NHase from strain A4 accepting a broad range of benzonitrile derivatives clearly demonstrated that this enzyme strongly resembles the NHases found previously in strains such as Rhodococcus sp. R312 (presumably identical to the enzyme from Rhodococcus sp. N-774 and Rhodococcus sp. N-771), Pseudomonas chlororaphis B23 (Nishiyama et al. 1991) or Rhodococcus rhodochrous J1 (Kobayashi et al. 1991). This may suggest the existence of two different evolutionary highly related NHase groups one of which also hydrates aromatic nitriles. However, data about the conversion of aromatic nitriles by the NHase from Brevibacterium sp. R 312 are contradictory. In contrast to Nagasawa et al. (1986, 1987), Bui et al. (1984) reported that the enzyme from strain R312 showed a low activity for benzonitrile (1% of that for propionitrile) and conversion of benzonitrile was used in a qualitative enzyme assay with strain R 312 (Osprian et al. 1999). The NHase from R. equi A4 also exhibited a much higher activity for propionitrile (about 400 U/mg) than for benzonitrile (about 55 U/mg). This would suggest that this enzyme shows a significantly higher relative activity with benzonitrile compared to propionitrile than the NHase from Brevibacterium sp. R312. The main reason for the present study was the observation that whole cells of strain A4 highly enantioselectively converted (R, S)-2-(4-methoxyphenyl)propionitrile (apparent E about 89; Martínková et al. 1996). In contrast to the purified NHase from A. tumefaciens d3 (Bauer et al. 1998) affording slightly higher enantiomer-

155

ic excesses than the whole cell biocatalyst, the apparent enantioselectivity of the NHase from R. equi A4 for (R, S)-2-(4-methoxyphenyl)propionitrile (maximum E of 19) was significantly lower than the value found with whole cells. This observation may be explained by the following reasons. Recently it was found that the related NHase from Rhodococcus sp. N-771 existed either as a dimer or a tetramer depending on its concentration (Nakasako et al. 1999). The NHase is presumably present in high concentrations in the cells of R. equi A4 and the enzyme may therefore exist in the cells as a tetramer. On contrary, a dimeric form may be expected in a diluted enzyme solution. It is possible that the dimer and the tetramer may possess different enantioselectivities. The enantioselectivity could be also influenced by the environment of the enzyme. In fact, it was shown in the present work that the enantioselectivity of the enzyme can be changed in the presence of hydrophobic compounds. The enantioselective operation of the NHase from R. equi A4 was strongly dependent on the substrate structure. The enantioselectivity of the NHase was most pronounced with substrates carrying a substituent in the para-position of the benzene ring of 2-phenylpropionitrile (in 2-(4-methoxyphenyl)propionitrile) or an additional benzene ring (in 2-(6-methoxynaphthyl)propionitrile). These substrate structures are probably needed to favour the binding or conversion of the S-enantiomers in comparison to the R-enantiomers. A positive effect of the p-methyl substitution of 2-phenylpropionitrile on the enzyme enantioselectivity was shown with the biocatalyst SP 361 (Beard et al. 1993). In contrast to the NHase from R. equi A4, the selectivity of the NHase from A. tumefaciens d3 was somewhat higher with 2-phenylpropionitrile than with its para-substituted (methoxy-, chloro-) derivatives (Bauer et al. 1998). Thus it appears that currently no generalization about the correlation of substrate structure and enantioselectivity of different NHases is possible. Improvement of an enzyme enantioselectivity by organic cosolvents has been tested with hydrolases (see Faber 1992; Carrea et al. 1995 for reviews). However, to the best of our knowledge, it has not been tested with either nitrilases or NHases although these enzymes were functional in the presence of organic cosolvents (Layh and Willetts 1998). Hydrocarbons, preferably isooctane, were optimal cosolvents at higher concentrations (50–90%, v/v) for the NHase from strain A4. Due to low water solubility, these solvents can hardly reach denaturating concentrations in the water phase despite their high denaturation capacity (Khmelnitsky et al. 1990). Methanol or ethanol were suitable as cosolvents at up to 20% (v/v). Lower convenience of higher alcohols (2-propanol, 2-butanol) is in agreement with their increased denaturation capacities compared with methanol (Khmelnitsky et al. 1990). While the activity of enzymes in organic solvents usually increases with an increasing log P (medium hydrophobicity), no such rule has been formulated for enzyme enantioselectivity. Although correlations of enan-

tioselectivity with either solvent hydrophobicity or dielectric constant can be found, such models are not generally applicable. The lack of general correlation of enzyme selectivity and physicochemical properties of the solvent is explained by an interaction of the solvent with the enzyme inside or near the active site (Carrea et al. 1995). This hypothesis could explain the observation that 1–11% (v/v) of N, N-dimethylformamide and formaldehyde could change the enantiomeric ratio of hydrolysis of ibuprofen methyl ester catalysed by the lipase from Candida cylindracea (Lee et al. 1995). The enantioselectivity of the NHase from R. equi was also significantly influenced by small amounts of cosolvents. Different effects of the cosolvent on the enzyme selectivity are plausible, such as an increased rigidity of the active site or a changed conformation of the solvent–enzyme complex. The cosolvents resulting in biphasic reaction mixtures may also change the substrate concentration in the proximity of the active site. Considering different KM values of the enzyme for individual enantiomers, this may also explain the changed enantioselectivity. In conclusion, a versatile NHase was purified which was efficient in the preparation of amides from different nitriles and enantioselective for some chiral 2-arylpropionitriles. Medium modifications as well as substrate derivatization leading to enantioselectivity improvement were suggested. The N-terminal amino acid homology of the enantioselective enzyme from strain A4 with previously reported ferric NHases suggests that the latter enzymes may also be enantioselective towards particular nitriles. Alternatively, an influence of small differences in the enzyme structure on enantioselectivity is plausible. Observations on the substrate specificity of the NHase from strain A4 suggest that the enzyme may be novel. However, this needs to be proved by determination of its primary structure. Acknowledgements Support of this work by the Grant Agency of the Academy of Sciences of the Czech Republic (Project A4020802/1998), by the Czech and German Ministries of Education (project “Bacterial Nitrile Hydratases”), by the Czech– German Future Fund (Project E/X/027, “Stereoselective Biotransformations of Nitriles”) and by DAAD (fellowship to L. Martínková), is gratefully acknowledged. We also wish to thank Ms M. Balvínová for technical assistance.

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