502
BIOCHEMICAL SOCIETY TRANSACTIONS
Buehring, K. U.,Tamura, T.& Stokstad. E. L.R.(1974) J. Biol. Chem. 249,1081-1089 Houlihan, C. M. & Scott, J. M. (1972) Biochem. Biophys. Res. Commun. 48,1675-1681 Krumdieck, C. L. & Baugh, C. M. (1969) Biochemistry 8,1568-1572 Leslie, G. I. & Baugh, C. M. (1974) Biochemisfry 13,4957-4961 68,1392 fiffner, J. J., Calkins, D. G. Bloom, E. S. &ODell, B. L. (1946)J. Am. Chem. SOC. Rabinowitz, J. C. (1962) Enzymes, 2nd edn., 2,185-252 Scott, J. M., Houlihan, C. M. Bassett, R. & Weir, D. G. (1975). Int. SOC. Haematol. (Absfr.) 3rd Meet. 22,14 Shin, Y.S., Willams, M. A. & Stokstad, E. L. R. (1972) Biochem. Biophys. Res. Commun. 47. 35-43 Shin, Y.S., Buehring, K. U.& Stokstad, E. L. R.(1974) Arch. Biochem. Biophys. 163,211-224
Purification and Properties of an Unusual Nitrilase from Nocardiu N.C.I.B. 11216 DAVID B. HARPER Department of Agricultural and Food Chemistry, Queen's University of Berfast, Newforge Lane, Berfast BT9 SPX, Northern Ireland, U.K. Little is known of the metabolism of the nitrile group by micro-organisms. Robinson & Hook (1964) reported the isolation of a nitrilase enzyme of rather restricted substrate specificity from a Pseudomonas sp. which would grow on the naturally occurring cyanopyridine, ricinine, and Mimura et al. (1969) have described a Corynebacterium sp. which can use aliphatic nitriles such as acetonitrile as sole carbon source. The nitrilase produced by this organism hydrolyses the nitrile to the corresponding amide. In this communication the purification of a nitrilase from Nocardiu N.C.I.B. 11216, which can use benzonitrile as sole carbon and nitrogen source, is described. Respiration studies on intact cells indicate that metabolism of benzonitrile proceeds through benzoic acid and catechol via the meta-cleavage route (Harper, 1974). Cell-free extracts were prepared by sonication of benzonitrile-grown cells suspended in 100mMphosphate buffer, pH 7.5, containing ~ ~ M - E D and T A 2m~-dithioerythritol,and centrifugation at 60000g for 45min. Such extracts show high activity in the conversion of benzonitrile into benzoic acid, the reaction being followed by assay of the NHJ produced by using the phenol/hypochlorite method. Benzamide is not attacked and so does not appear to be a free intermediate in the reaction. The nitrilase was further purified by DEAE-cellulose column chromatography and passage through Sephadex G-200 in the presence and absence of benzonitrile. The resulting purified fraction was shown to be a single protein by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis and isoelectric focusing on polyacrylamide gel. On incubation with substrate the nitrilase shows a time-dependent activation process which is a function of enzyme concentration (Fig. 1). Neither benzoic acid nor ammonia had any effect on the length of the lag period. However, the activation process was highly dependent on both temperature and pH. A possible explanation of such behaviour is that the substrate, benzonitrile, causes association of inactive subunits to form the polymeric active enzyme. The molecular weight of the enzyme was therefore determined on Sephadex G-200 at 0°C in the absence of benzonitrile and also in the presence of 5m~-benzonitrileafter preincubation of the enzyme at 30°C for 4min with 2 0 m ~ benzonitrile. The elution behaviour of the enzyme under these conditions is shown in Fig. 2. In the absence of substrate the nitrilase has an elution volume corresponding to a mol.wt. of 47000, whereas in the presence of substrate the apparent mol.wt. increases to about 560000. The associated enzyme therefore seems to consist of about 12 identical inactive subunits. The molecular weight of the subunits is further confirmed by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis, which indicates a mo1.W. of 45 OOO. 1976
562od MEETING,BANGOR
503
10 h
f*
co
g6
(cl
0
3
--5
4
R2
2
X
0
8
16
24
32
Time of incubation (min) Fig. 1. Time-course of benzonitrile hydrolysis as a function of enzyme concentration Enzyme was preincubated at 30°C for lOmin in 100m-phosphate buffer, pH8.0, containing 1m-EDTA and 0.25mhi-dithioerythritol.Final concentrations in the assay mixture incubated at 30°C were 27 mhi-knzonitrile, 0.1 a - E D T A , 0.025m-dithioerythritol, 100mwphosphate buffer, pH8.0, and enzyme concentrations as shown on the graph.
(r
0
Relative elution volume (V./ V,) Fig. 2. Elution behaviour of nitrilasefrom Sephadex G-200at 0°C (a) In the absence of benzonitrile, ----;(b) in the presence of Srn~-benzonitrileafter preincubation of enzyme with 20m-benzonitrile at 30°C for 4min, -. In each case lOOpg of protein was applied to a 2.5cm x 70cm column. In (a) the eluting medium was 100m-phosphate buffer, pH8.0, containing 1m-EDTA and 0.25~-dithioerythritol, and in (b)the medium contained in addition Sm-benzonitrile.
VoL 4
504
BIOCHEMICAL SOCIETY TRANSACTIONS
The associated enzyme had a pH optimum between 7.95 and 8.05, activity falling sharply on either side of this pH range, though the pH optimum for association of the enzyme, i.e. that pH which produced a minimum lag period, was between 7.3 and 7.4. The K, of the nitrilase with benzonitrile as substrate was 4 x 1 0 - 3 and ~ the activation energy of the reaction between 20°C and 40°C as deduced from an Arrhenius plot was 51.8kJ.mol-' (12400cal*mol-'). At temperatures below 20°C the slope of the plot was discontinuous, suggesting that association of the enzyme was not complete even at equilibrium with substrate at such temperatures. The purified enzyme was highly unstable, having a half-life of 10h at 0°C and undergoing almost complete loss of activity on freezing. It showed great sensitivity to thiol reagents such as p-chloromercuribenzoate and N-ethylmaleimide, indicating the presence of active thiol groups in the enzyme. Studies with the different substrates demonstrated that the enzyme was specific for nitrile groups directly attached to the benzene ring. In contrast with the nitrilase isolated from barley leaves by Mahadevan & Thimann (1964), substrate-specificitystudies with the microbial enzyme revealed little correlation between electron-withdrawing capacity of substituentsin the aromatic ring and the rate of attack by the enzyme. ortho-Substitution, except by fluorine, was incompatible with attack presumably because of steric factors. However, nitriles meta- or para-substituted with methyl or halogen groups acted as substratesfor the enzyme, though Michaelis-Menten kinetics were not observed in all cases. The significance of the slow substrate-activated association of this enzyme in the regulation of metabolism in this organism remains obscure. Harper, D. B. (1974)Abstr. Commun. FEBS Meet. 9th,p. 403 Mahadevan, S. & Thimann, K. V. (1964)Arch. Biochem. Biophys. 107,6248 Mimura, A., Kawano, T.& Yamaga, K. (1969)J. Ferment. Technol. 47,631-638 Robinson, W.G.& Hook, R. H. (1964)J. Biol. Chem. 239,4263-4267
The Effect of Growth Conditions on the Wax Content of Various Strains of Acinetobacter LESLIE M. FIXTER and JAMES G. McCORMACK Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
There have been scattered reports of the occurrence of waxes in various strains of Acinetobacter (Gallagher, 1971; Thorne et al., 1973, Fixter & Fewson, 1974) and presumed Acinetobacter (Russell, 1974). The present work was undertaken to extend in a more systematic way knowledge of the distribution of waxes in the Acinetobacter group and to determine whether, as in Acinetobacter calcoaceticus N.C.I.B. 8250, the wax content of the bacteria varies with culture conditions (Fixter & Fewson, 1974). The organisms used are all classified as Acinetobacter (Baumann et al., 1968) and because of the plethora of synonyms will only be referred to by culture-collection numbers. The organisms used were from the National Collection of Type Cultures, N.C.T.C. 7364 (Al), N.C.T.C. 10307 (Bl), N.C.T.C. 10308 (Bl), N.C.T.C. 10304 (B3), N.C.T.C. 10309 (B3), N.C.T.C. 10305 (B4), N.C.T.C. 10306 (B4);from the National Collection of Industrial Bacteria, N.C.I.B. 9205 (Al), N.C.I.B. 8250 (A2), N.C.I.B. 8154 (B2), N.C.I.B. 9689 (B2), N.C.I.B. 9115 (B4); and from the American Type Culture Collection, A.T.C.C. 14290 (Al), A.T.C.C. 17976 (A3) and A.T.C.C. 23055 (A unassigned). The designation in parentheses are the groups (letters) and sub-groups (numbers) of the classification proposed by Baumann et al. (1968) and therefore these strains are representative of the broad spectnun of organisms classified in this genus. Bacteria were grown into stationary phase of batch culture and harvested as described by Livingstone et al. (1972). Lipids were extracted from the bacteria by the method of 1976
BIOCHEMICAL SOCIETY TRANSACTIONS
Buehring, K. U.,Tamura, T.& Stokstad. E. L.R.(1974) J. Biol. Chem. 249,1081-1089 Houlihan, C. M. & Scott, J. M. (1972) Biochem. Biophys. Res. Commun. 48,1675-1681 Krumdieck, C. L. & Baugh, C. M. (1969) Biochemistry 8,1568-1572 Leslie, G. I. & Baugh, C. M. (1974) Biochemisfry 13,4957-4961 68,1392 fiffner, J. J., Calkins, D. G. Bloom, E. S. &ODell, B. L. (1946)J. Am. Chem. SOC. Rabinowitz, J. C. (1962) Enzymes, 2nd edn., 2,185-252 Scott, J. M., Houlihan, C. M. Bassett, R. & Weir, D. G. (1975). Int. SOC. Haematol. (Absfr.) 3rd Meet. 22,14 Shin, Y.S., Willams, M. A. & Stokstad, E. L. R. (1972) Biochem. Biophys. Res. Commun. 47. 35-43 Shin, Y.S., Buehring, K. U.& Stokstad, E. L. R.(1974) Arch. Biochem. Biophys. 163,211-224
Purification and Properties of an Unusual Nitrilase from Nocardiu N.C.I.B. 11216 DAVID B. HARPER Department of Agricultural and Food Chemistry, Queen's University of Berfast, Newforge Lane, Berfast BT9 SPX, Northern Ireland, U.K. Little is known of the metabolism of the nitrile group by micro-organisms. Robinson & Hook (1964) reported the isolation of a nitrilase enzyme of rather restricted substrate specificity from a Pseudomonas sp. which would grow on the naturally occurring cyanopyridine, ricinine, and Mimura et al. (1969) have described a Corynebacterium sp. which can use aliphatic nitriles such as acetonitrile as sole carbon source. The nitrilase produced by this organism hydrolyses the nitrile to the corresponding amide. In this communication the purification of a nitrilase from Nocardiu N.C.I.B. 11216, which can use benzonitrile as sole carbon and nitrogen source, is described. Respiration studies on intact cells indicate that metabolism of benzonitrile proceeds through benzoic acid and catechol via the meta-cleavage route (Harper, 1974). Cell-free extracts were prepared by sonication of benzonitrile-grown cells suspended in 100mMphosphate buffer, pH 7.5, containing ~ ~ M - E D and T A 2m~-dithioerythritol,and centrifugation at 60000g for 45min. Such extracts show high activity in the conversion of benzonitrile into benzoic acid, the reaction being followed by assay of the NHJ produced by using the phenol/hypochlorite method. Benzamide is not attacked and so does not appear to be a free intermediate in the reaction. The nitrilase was further purified by DEAE-cellulose column chromatography and passage through Sephadex G-200 in the presence and absence of benzonitrile. The resulting purified fraction was shown to be a single protein by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis and isoelectric focusing on polyacrylamide gel. On incubation with substrate the nitrilase shows a time-dependent activation process which is a function of enzyme concentration (Fig. 1). Neither benzoic acid nor ammonia had any effect on the length of the lag period. However, the activation process was highly dependent on both temperature and pH. A possible explanation of such behaviour is that the substrate, benzonitrile, causes association of inactive subunits to form the polymeric active enzyme. The molecular weight of the enzyme was therefore determined on Sephadex G-200 at 0°C in the absence of benzonitrile and also in the presence of 5m~-benzonitrileafter preincubation of the enzyme at 30°C for 4min with 2 0 m ~ benzonitrile. The elution behaviour of the enzyme under these conditions is shown in Fig. 2. In the absence of substrate the nitrilase has an elution volume corresponding to a mol.wt. of 47000, whereas in the presence of substrate the apparent mol.wt. increases to about 560000. The associated enzyme therefore seems to consist of about 12 identical inactive subunits. The molecular weight of the subunits is further confirmed by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis, which indicates a mo1.W. of 45 OOO. 1976
562od MEETING,BANGOR
503
10 h
f*
co
g6
(cl
0
3
--5
4
R2
2
X
0
8
16
24
32
Time of incubation (min) Fig. 1. Time-course of benzonitrile hydrolysis as a function of enzyme concentration Enzyme was preincubated at 30°C for lOmin in 100m-phosphate buffer, pH8.0, containing 1m-EDTA and 0.25mhi-dithioerythritol.Final concentrations in the assay mixture incubated at 30°C were 27 mhi-knzonitrile, 0.1 a - E D T A , 0.025m-dithioerythritol, 100mwphosphate buffer, pH8.0, and enzyme concentrations as shown on the graph.
(r
0
Relative elution volume (V./ V,) Fig. 2. Elution behaviour of nitrilasefrom Sephadex G-200at 0°C (a) In the absence of benzonitrile, ----;(b) in the presence of Srn~-benzonitrileafter preincubation of enzyme with 20m-benzonitrile at 30°C for 4min, -. In each case lOOpg of protein was applied to a 2.5cm x 70cm column. In (a) the eluting medium was 100m-phosphate buffer, pH8.0, containing 1m-EDTA and 0.25~-dithioerythritol, and in (b)the medium contained in addition Sm-benzonitrile.
VoL 4
504
BIOCHEMICAL SOCIETY TRANSACTIONS
The associated enzyme had a pH optimum between 7.95 and 8.05, activity falling sharply on either side of this pH range, though the pH optimum for association of the enzyme, i.e. that pH which produced a minimum lag period, was between 7.3 and 7.4. The K, of the nitrilase with benzonitrile as substrate was 4 x 1 0 - 3 and ~ the activation energy of the reaction between 20°C and 40°C as deduced from an Arrhenius plot was 51.8kJ.mol-' (12400cal*mol-'). At temperatures below 20°C the slope of the plot was discontinuous, suggesting that association of the enzyme was not complete even at equilibrium with substrate at such temperatures. The purified enzyme was highly unstable, having a half-life of 10h at 0°C and undergoing almost complete loss of activity on freezing. It showed great sensitivity to thiol reagents such as p-chloromercuribenzoate and N-ethylmaleimide, indicating the presence of active thiol groups in the enzyme. Studies with the different substrates demonstrated that the enzyme was specific for nitrile groups directly attached to the benzene ring. In contrast with the nitrilase isolated from barley leaves by Mahadevan & Thimann (1964), substrate-specificitystudies with the microbial enzyme revealed little correlation between electron-withdrawing capacity of substituentsin the aromatic ring and the rate of attack by the enzyme. ortho-Substitution, except by fluorine, was incompatible with attack presumably because of steric factors. However, nitriles meta- or para-substituted with methyl or halogen groups acted as substratesfor the enzyme, though Michaelis-Menten kinetics were not observed in all cases. The significance of the slow substrate-activated association of this enzyme in the regulation of metabolism in this organism remains obscure. Harper, D. B. (1974)Abstr. Commun. FEBS Meet. 9th,p. 403 Mahadevan, S. & Thimann, K. V. (1964)Arch. Biochem. Biophys. 107,6248 Mimura, A., Kawano, T.& Yamaga, K. (1969)J. Ferment. Technol. 47,631-638 Robinson, W.G.& Hook, R. H. (1964)J. Biol. Chem. 239,4263-4267
The Effect of Growth Conditions on the Wax Content of Various Strains of Acinetobacter LESLIE M. FIXTER and JAMES G. McCORMACK Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
There have been scattered reports of the occurrence of waxes in various strains of Acinetobacter (Gallagher, 1971; Thorne et al., 1973, Fixter & Fewson, 1974) and presumed Acinetobacter (Russell, 1974). The present work was undertaken to extend in a more systematic way knowledge of the distribution of waxes in the Acinetobacter group and to determine whether, as in Acinetobacter calcoaceticus N.C.I.B. 8250, the wax content of the bacteria varies with culture conditions (Fixter & Fewson, 1974). The organisms used are all classified as Acinetobacter (Baumann et al., 1968) and because of the plethora of synonyms will only be referred to by culture-collection numbers. The organisms used were from the National Collection of Type Cultures, N.C.T.C. 7364 (Al), N.C.T.C. 10307 (Bl), N.C.T.C. 10308 (Bl), N.C.T.C. 10304 (B3), N.C.T.C. 10309 (B3), N.C.T.C. 10305 (B4), N.C.T.C. 10306 (B4);from the National Collection of Industrial Bacteria, N.C.I.B. 9205 (Al), N.C.I.B. 8250 (A2), N.C.I.B. 8154 (B2), N.C.I.B. 9689 (B2), N.C.I.B. 9115 (B4); and from the American Type Culture Collection, A.T.C.C. 14290 (Al), A.T.C.C. 17976 (A3) and A.T.C.C. 23055 (A unassigned). The designation in parentheses are the groups (letters) and sub-groups (numbers) of the classification proposed by Baumann et al. (1968) and therefore these strains are representative of the broad spectnun of organisms classified in this genus. Bacteria were grown into stationary phase of batch culture and harvested as described by Livingstone et al. (1972). Lipids were extracted from the bacteria by the method of 1976
