Appl Microbiol Biotechnol (1990) 34:42-46
Applied Microbiology Biotechnology © Springer-Verlag 1990
Aryl acylamidase from Rhodococcus erythropolis N CIB 12273 Peter A. Vaughan 1., Geoffrey F. Hall 2, and David J. Best 2.* 1 MediSense (UK) Inc., Abingdon, Oxfordshire, OX14 1DY, UK 2 Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedford, MK43 0AL, UK Received 22 February 1990/Accepted 17 May 1990
Summary. A Rhodococcus erythropolis strain was isolated from soil on the basis of its ability to use acetaminophen as the sole source of both carbon and energy for growth. When grown in a complex medium containing an anilide inducer compound, the bacterium exhibited aryl acylamidase (EC 3.5.1.13) activity. This activity was not subject to carbon or nitrogen repression by the growth medium constituents as the enzyme was present throughout the exponential growth phase. The anilide was converted to the corresponding aniline, which was not further degraded. The enzyme was partially purified by a variety of methods including a batch ion exchange procedure, column ion exchange chromatography and hydrophobic interaction chromatography. The enzyme had a maximum activity at around p H 8.0 and had a Km for acetaminophen of 0.11 raM. Electrochemical assays of aryl acylamidase activity are described. The enzyme is suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen.
Introduction Aryl acylamidases catalyse the hydrolysis of a N-acyl primary aromatic amine (anilide) to form an aniline and a carboxylic acid anion. The aryl acylamidase of Pseudomonas fluorescens ATCC 39004 has been the subject of studies concerning the large-scale purification of the enzyme and its subsequent use in clinical analysis ( H a m m o n d et al. 1983, 1984). The enzyme has also been found in several other strains of Pseudomonas (Kearney 1965; Alt et al. 1975; Hsuing et al. 1975).
* Present address: Biotechnology Unit, Laboratory of the Government Chemist, Queens Road, Teddington, Middlesex, TW11 0LY, UK ** Present address: Research and Development, Sterling Organics Ltd., Edgefield Avenue, Fawdon, Newcastle-Upon-Tyne, NE3 3TT, UK Offprint requests to: P. A. Vaughan
Aryl acylamidases occurring in Gram-positive bacteria have been generally studied in less detail. A Rhodococcus sp. that could use the anilide N-phenyl-propionamide as the sole source of carbon and energy has been described (Lechner and Straube 1984). The organism possessed an aryl acylamidase activity which was present constitutively at low levels when the organism was grown in a minimal medium and which was induced to higher levels by various anilides. A Corynebaeterium pseudodiphtheriticum, which was capable of growth on the anilides acetanilide, phenacetin and acetaminophen as sole sources of carbon and energy, was described by Grant and Wilson (1973). The organism possessed hydrolytic activity against both phenacetin and acetaminophen. An aryl acylamidase from Bacillus sphaericus ATCC 12123 was purified to homogeneity from bacteria grown on the anilide herbicide Linuron (Engelhardt et al. 1973). This paper reports the isolation and characterization of a Rhodoeoccus bacterium suitable for the production of an aryl acylamidase which could be used for clinical diagnostic purposes, and the partial purification and characterization of the enzyme. The interest in the use of aryl acylamidase as a clinical diagnostic reagent is because of the ability of the enzyme to hydrolyse the drug acetaminophen (paracetamol) to 4-aminophenol, which can be detected subsequently by colorimetric ( H a m m o n d et al. 1984) or electrochemical means.
Materials and methods Isolation o f the microorganism A minimal salts medium (50 ml, pH 7.0), containing acetaminophen (0.1% w/v) as the sole source of carbon and energy, was inoculated with a small quantity of garden soil from a flower bed. The minimal salts medium consisted of (per litre): NaNO3, 0.85 g; KH2PO4, 0.56 g; NaEHPO4, 0.86 g; KaSO4, 0.17 g; MgSO4.7H:O, 0.037 g; CaC12.2H20, 0.007 g; Fe(III) ethylenediaminetetraacetate (EDTA), 0.004g; 2.5 ml trace element solution (ZnSO4.TH20, 0.232g/l; MnSO4.H~O, 0.178g/l; H3BO4, 0.056g/1; CuSO4-SH20, 0.1g/l; Na:MoO4,2H:O, 0.039g/1:
43 COC12 •6H20, 0.042 g / l ; KI, 0.66 g/1; EDTA, 0.1 g/1; FeSO4.7 H20, 0.04 g / l ; NiC12.6H20, 0.0004 g/l; H2SO4 (1 M), 8 ml/l). The initial cultures were incubated for 5 days on an orbital shaker (200 rpm) at 30 ° C. Samples of these enrichment cultures were plated onto minimal salts agar (2% w/v) containing acetaminophen at 0.1%. The organisms which arose on these plates were isolated to purity.
voltammagram at 100 mV was indicative of aryl acylamidase activity and the peak height was proportional to the concentration of 4-aminophenol produced.
Enzyme purification Enzyme and protein assays. A rapid method of monitoring aryl
Growth of the organism for culture profile studies The organism was grown on tryptone soya broth (2%). Colonies from nutrient agar slants were inoculated into flasks (250 ml) containing medium (100 ml) and incubated for approximately 18 h at 30 ° C. The cultures were used to inoculate a fermentor (Bioengineering, Wald, Switzerland; working volume 12 1) containing medium (10 1) to which polyethylene glycol P200 (0.33% v/v) and acetanilide (0.15% w/v) had been added. The vessel was aerated at 2 l / m i n at 30 ° C and a stirring speed of 500 rpm. Samples of the culture were removed periodically and the optical density (600 nm) and pH were measured. Portions of each sample were centrifuged in a microfuge and the resulting supernatants were stored at - 2 0 ° C for later metabolite analysis. The non-centrifuged remainder of each sample was stored at - 20 ° C for analysis of the aryl acylamidase activity in the cells.
Determination of metabolites in the 9rowth medium Culture supernatant acetanilide and aniline concentrations were determined by gas liquid chromatography using a flame-ionization detector and by the use of external standards. Aliquots (200 I~I) of each thawed supernatant sample were extracted into an equal volume of ethyl acetate by vortex mixing for 5 min. Samples (1 .ul) were analysed using a temperature programme under the following conditions: column, DBWAX (JKW Scientific Inc., Folsore, CA, USA) megabore column (30 m × 0.524 mm, 1 p~m film thickness); carrier gas, nitrogen, 20 ml/min: injector temperature, 200 ° C; detector temperature, 250 ° C; initial column temperature, 155°C; initial time 5 rain; ramp rate, 20°C/rain; final column temperature, 235 ° C; final hold time, 10 rain. Extraction and analysis were performed in duplicate on each sample.
Measurement of aryl acylamidase activity in culture cell samples Aliquots (5 ml) of thawed samples were centrifuged (100009, 10 min, 4 ° C) and the pellet of cells was washed in T R I S / H C I buffer (5 ml, 50 raM, pH 8.6) and resuspended in fresh buffer (5 ml). The bacteria were disrupted by sonication (MSE sonicator, Crawley, UK; 150 W, 3 x 45 s, 16 mix amplitude). The disrupted cell suspension was centrifuged (10 000 9, 30 min, 4 ° C) to remove cell debris and the supernatant removed and kept on ice. The protein content of the cell-free extracts was assayed by the bicinchoninic acid method (Smith et al. 1985). An electrochemical assay, Osteryoung square wave voltametry (Osteryoung and Osteryoung 1985), was employed as a sensitive and simple method to measure specific activities for aryl acylamidase in cell-free extracts. The hydrolysis product of the enzyme reaction, 4-aminophenol, was differentiated from the substrate, acetaminophen, by a characteristic peak in the pulse voltammagram at 100 inV. The assay was conducted at 30°C in a two-compartment electrochemical cell with a glassy carbon working electrode, a saturated calomel reference electrode and a platinum wire auxiliary electrode. The incubation mixture (2 ml) contained acetaminophen (50 raM) in T R I S / H C l buffer (100 raM, pH 8.6). The reaction was initiated by the injection of the cell-free extract sample (100 ixl). The appearance of a peak in the pulse
acylamidase activity during the protein purification procedures was provided by a method based on that described by Alt et al. (1975). The reaction mixture in a 3-ml plastic cuvette consisted of the following: T R I S / H C I buffer (100 raM, pH 8.6, 0.6 ml) and the enzyme substrate 4-nitroacetanilide (1 mM). The reaction was initiated by the addition of the enzyme sample (400 IXl, with the enzyme prediluted in buffer if required) and the reaction mixture was incubated at 30 ° C. The formation of the coloured product of the aryl acylamidase reaction, 4-nitroaniline, was monitored by measuring the A405 of the reaction mixture. The molar extinction coefficient of 4-nitroaniline under these conditions was 10000 1/ mol per cm. The protein assay used in the purification studies was the Coomassie Brilliant Blue method (Bradford 1976).
Cellular disruption. A 15-1 culture of the organism was grown in a 20-1 fermentor as for the culture profile study. When the culture attained an optical density at 600 nm (OD6oo) of 6 (i.e. in the late exponential phase of growth) the bacteria were harvested using a Sharples (Pennwalt Ltd., Camberley, UK) continuous centrifuge and 150 g wet weight of bacteria was recovered. The cells were washed by resuspending them in approximately 300 ml T R I S / HCI buffer (50 raM, pH 7.5) and then centrifuging the suspension at 11000 9 for 90 rain at 4 ° C to produce a supernatant, which was discarded, and a cell pellet. The cell pellet was resuspended to 300 ml in buffer (50 mM, pH 7.5). A 60-ml portion of the harvested cell suspension was used to prepare a cell-free extract. A 10-ml volume of buffer (50 raM, pH 7.5) was added to the 60 ml of suspension, and dithiothreitol (DTT) was added to 1 mM. The suspension was split into two batches, each of which were sonicated (10 × 45 s periods, 16 mix amplitude). The pooled, Sonicated material was centrifuged at 31000 9 for 70 min at 4°C to yield cell-free extract (57 ml). The extract was kept at 4 ° C. DTT (1 raM) was included in the TRIS/HC1 buffers used in all of the subsequent enzyme purification steps, and these buffers were all pH 7.5 and 20 raM, unless indicated otherwise. Batch ion exchange procedure. The cell-free extract (57 ml) was mixed with DE52 ion exchange resin (4 g, Whatman, Maidstone, UK), which had been pre-equilibrated with buffer (50 raM). The mixture was stirred at 4°C for 3 h and then the DE52 resin was allowed to settle out. The supernatant was discarded and the DE52 resin was washed by adding buffer (80 ml, 20 raM), and the suspension stirred for 5 min at 4 ° C. The DE52 resin was again allowed to settle out and the suspension was decanted to leave the ion exchange resin suspended in approximately 4 ml buffer. A 21ml volume of buffer containing 0.4 M sodium chloride was added to the DE52 resin suspension to produce a final salt concentration of approximately 0.35 M. The suspension was stirred for 2 h, during which time the aryl acylamidase was eluted from the DE52 resin, and then filtered (through a 0.4 Ixm filter) to remove the ion exchange resin.
Column ion exchange chromatography. The protein solution produced by the batch DE52 resin procedure was further purified by ion exchange chromatography on a column of Pharmacia (Uppsala, Sweden) Q-Sepharose Fast Flow gel (a cross-linked agarose gel containing quaternary amine functional groups). A 5-ml sample of the protein solution was mixed with 1 ml buffer to dilute the NaC1 concentration of the sample to less than 0.3 M. A 5-ml volume of the diluted sample was applied to a 48-ml Q-Sepharose column (9 cm×2.6 cm diameter) which had been previously equilibrated with buffer containing 0.3 M NaC1. The protein was
44 eluted with 240 ml of the same buffer followed by 240 ml of the buffer containing 0.4 M NaC1. The flow rate was 240 ml/h, and 10 ml fractions of the eluent were collected. The aryl acylamidase eluted after between 60 and 150 ml of the 0.4 M NaC1 buffer had been applied to the column.
Hydrophobic interaction chromatography. The active fractions from the above step were combined, desalted by diafiltration and concentrated, using an Amicon 30000 molecular weight cut-off hollow-fibre cartridge and buffer. The desalted solution was further concentrated to a volume of 5 ml using an Amicon (Danvers, MA, USA) ultrafiltration cell, fitted with a 30000 molecular weight cut-off membrane. A 0.5-ml sample of the concentrated desalted protein solution was applied to a 4-ml (2 cmx 1.6 cm diameter) column of Pharmacia (Uppsala, Sweden) phenyl-Sepharose gel (a pbenyl-substituted cross-linked agarose gel) which had been pre-equilibrated with 0.5 M ammonium sulphate in buffer. The column was washed with 10 ml of the same solution and the enzyme eluted with a 20-ml linear descending gradient of 0.50.0 M ammonium chloride in the same buffer. The flow rate was 0.33 ml/min and 1 ml fractions were collected. Determination of the K,~ of the aryl acylamidase for acetaminophen The assay was carried out at 30° C in a stirred electrochemical cell containing a glassy carbon working electrode, a silver/silver chloride reference electrode and a platinum wire counter electrode. The cell contained TRIS/HC1 buffer (2.9 ml, pH 8.6, 100 mM) which incorporated acetaminophen at concentrations ranging between 17 p.M and 1.67 mM. The working electrode was poised at 300 mV relative to the reference electrode, using a potentiostat, and the electrical current produced was recorded on a chart recorder. When a steady current had been reached, 100 pJ of diluted enzyme solution was injected into the cell to initiate the reaction. The product of the enzyme reaction, 4-aminophenol, was oxidized at the working electrode giving rise to an electrical current proportional to the concentration of the 4-aminophenol. The initial increase in the current after the addition of the enzyme was linear. Each substrate concentration was assayed in triplicate and the mean rate was used for kinetic analysis using Lineweaver-Burk, Hanes and Eadie-Hofstee plots.
Determination of the effect of pH on the aryl acylamidase The effect of pH on aryl acylamidase activity was measured using the 4-nitroacetanilide colorimetric assay and the buffers (50 mM) K2HPO4/KH:PO4 (pH 6.0-8.0), TRIS/HCI (pH 7.5-8.6), and 3[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid/NaOH (pH 8.6-9.5).
Results and discussion
Isolation and identification of the organism Six bacterial isolates were obtained from soil by the isolation procedure described in Materials and methods. One of these bacteria was found to be a Gram-positive organism capable of growth upon minimum agar containing either 0.1% acetaminophen or 0.1% phenacetin. Discolouration (blackening) of the medium occurred in both cases. Medium discolouration was used as an indication of the formation and reaction of substituted anilines, which in turn indicated that the organism was
able to hydrolyse the anilide substrate and therefore possessed aryl acylamidase activity. The bacterium was identified by the National Collection of Industrial and Marine Bacteria, UK, as a strain o f Rhodococcus erythropolis ( N C I B No. 12273) (Best and Vaughan 1987). Rhodococcus erythropolis is a c o m m o n non-pathogenic soil organism. Members of the Rhodococcus genus of bacteria, which includes organisms previously classified as Nocardia rhodochrous species (Goodfellow and Anderson 1976), have been previously found to be capable of the hydrolysis of a wide range of aliphatic, aromatic and heterocyclic amides (Miller and Gray 1982; Linton and Knowles 1986; Vaughan et al. 1988).
Effect of pH, temperature and the presence of an anilide on the growth of the organism Initial studies on the growth of the organism in complex media were performed using shake flasks (250 ml) containing tryptone soya broth (100 ml, 2%). An examination of the effect of p H indicated that the organism will grow over the p H range examined (pH 6.6-8.4) with optimum growth rates seen in cultures with an initial p H between p H 7.0 and 8.0. A comparison of growth rates at 26 °, 30 ° and 35°C indicated that growth was fastest at 30 °C. The presence of the aryl acylamidase inducer acetanilide (0.1%) in the tryptone soya broth appeared to result in a slower initial growth rate but gave an equivalent final culture optical density to that seen in the absence o f acetanilide.
Changes in inducer substrate concentration, enzyme product concentration and aryl acylamidase activity during growth of the organism on a complex medium containing acetanilide The growth of the organism on tryptone soya broth (2%) containing acetanilide (0.15%) and the changes in various culture parameters are shown in Fig. 1. Acetanilide was depleted during the mid-exponential phase of growth with concomitant accumulation of aniline, which did not appear to be further utilized by the bacteria. The non-utilization of the aniline product by the bacteria is consistent with the apparent non-utilization of the substituted aniline products of acetaminophen and phenacetin hydrolysis by the organism, as indicated by the medium discolouration during growth of the organism on either of the two substituted acetanilides. Aryl acylamidase activity, as measured in cell-free extracts of the bacteria, was induced throughout the exponential growth phase with the maximum total and specific activity being expressed at the end of the growth period (approximately 0.68 ~tmol substrate hydrolysed/min per milligram protein, corresponding to 0.66 Ixmol substrate hydrolysed/min per millilitre culture). The specific activity of the aryl acylamidase declined in the stationary phase. No aryl acylamidase activity was detected in a cell-free extract prepared from
45
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Fig. 1. Rhodococcus erythropolis NCIB 12273 was grown on 2% tryptone soya broth containing0.15%v/v acetanilidein a 12-1fermenter and various parameters were measured, as described in Materials and methods: optical density at 600 n m (OD6o0) ( 0 ) ; acetanilide concentration (.6); aniline concentration (V); aryl acylamidase specific activity in cell-free extracts of the bacteria (=)
bacteria grown on trypone soya broth in the absence of acetanilide and harvested in the mid-exponential growth phase. The above results indicate that the aryl acylamidase activity was induced by acetanilide and that this induction was not subject to repression by carbon or nitrogen sources in the complex tryptone soya medium. If such repression had operated it would be expected that the acetanilide would not have been utilized before the end of the growth period. This lack of repression of aryl acylamidase induction by components of a complex growth medium also occurred in a culture of Pseudomonas fluorescens ATCC 39004 during growth of the organism on tryptone soya broth containing acetanilide, in the study reported by Hammond et al. (]983).
mond et al. (1983) was not improved by the presence of the thiol protective agent 2-mercaptoethanol. The proteinase inhibitor phenylmethyl-suiphonyl fluoride, at 0.1 mM, almost totally inhibited the aryl acylamidase activity in cell-free extracts, indicating that the enzyme might operate by a similar mechanism to the serine proteases (Price and Stevens 1982). In several vertebrate systems aryl acylamidase activity has been found to be associated with the same protein as that responsible for acetylcholine esterase activity. In one study it was found that the enzymic activities appeared to arise from non-identical but partially overlapping active sites on the same protein (Balasubramanian 1984). Several sequential procedures were developed for the partial purification of the R. erythropolis aryl acylamidase as described in Materials and methods. The batch ion exchange procedure gave a 1.8-fold protein purification, i.e. an increase in the aryl acylamidase specific activity from 0.58 to 1.05 ~tmol substrate hydrolyses/min per milligram protein. The recovery of activity was 77%. The column ion exchange chromatography gave a further 4.9-fold purification with a yield for this step of 90%. The diafiltration and concentration procedures gave an approximately two-fold purification of the enzyme. The hydrophobic interaction chromatography step gave 1.9-fold purification, resulting in a protein solution with an aryl acylamidase specific activity of 23.3 ~tmol substrate hydrolysed/min per milligram protein. The partially purified enzyme was used for studies of the effect of pH on the aryl acylamidase activity and to determine the Km of the enzyme for acetaminophen. The enzyme has a broad pH optimum with maximum activity at around pH 8.0. The pH optima of other microbial aryl acylamidases ranges from just above neutrality to pH 10.0 (Hammond et al. 1983). The Km of the enzyme for acetaminophen (determined as described in Materials and methods) was 0.11 mM from the Lineweaver-Burk plot, 0.12 mM from the Hanes plot and 0.11 mM from the Eadie-Hofstee plot.
Suitability of R. erythropolis aryl acylamidase for clinical diagnostic measurement of acetaminophen R. erythropolis NCIB 12273 cultures with aryl acylami-
Enzyme purification and properties Preliminary studies indicated that the R. erythropolis aryl acylamidase activity was unstable in cell-free extracts in the absence of a thiol protecting agent, and therefore ] mM DTT was added to the solutions used throughout the purification procedure and during storage of the enzyme. The enzyme, in cell-free extracts in TRIS/HC1 buffer, pH 7.5, containing 1 mM DTT, was stable when stored at - 20 ° C. When stored at 4 ° C, 66% of the activity was retained after 7 days. The stability of the pseudomonad aryl acylamidase studied by Ham-
dase activity can be produced in a straightforward manner and the enzyme can be partially purified from cell extracts by a variety of methods in acceptable yields. The cell growth and enzyme purification methods described are suitable for further optimization and scaleup. The stability of the enzyme in solution, the pH optimum of the enzyme and its K m for acetaminophen make the enzyme suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen. Acetaminophen (paracetamol) is a commonly used analgesic drug for which the clinically significant concentration range in blood plasma is between 0.0 and approximately 3.0 mM for overdose patients. The R. erythropolis enzyme is currently being used in the develop-
46 m e n t o f a n o v e l b i o s e n s o r to m e a s u r e a c e t a m i n o p h e n ( p a r a c e t a m o l ) in clinical s a m p l e s b y e l e c t r o c h e m i c a l d e t e c t i o n o f the 4 - a m i n o p h e n o l p r o d u c e d b y a c e t a m i n o p h e n h y d r o l y s i s ( S h a n n o n et al. 1989).
References Air J, Krisch K, Hirsch P (1975) Isolation of an inducible amidase from Pseudomonas acidovorans AE1. J Gen Microbiol 87:260272 Balasubramanian AS (1984) Have cholinesterases more than one function? Trends Neurosci 7:467-468 Best DJ, Vaughan PA (1987) Rhodococcus bacterium for the production of aryl acylamidase. European patent application 87306778.9 Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:348-354 Engelhardt G, Wallnofer PR, Plapp R (1973) Purification and properties of an aryl acylamidase from Bacillus sphaericus catalysing the hydrolysis of various phenylamide herbicides and fungicides. Appl Microbiol 26:709-718 Goodfellow M, Alderson G (1976) The actinomycete genus Rhodococcus: a home for the 'rhodochrous' complex. J Gen Microbiol 100:99-122 Grant DJW, Wilson J (1973) Degradation and hydrolysis of amides by Corynebacterium pseudodiphtheriticum NCIB 10803. Microbios 8:15-22 Hammond PM, Price CP, Scawen MD (1983) Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004. Eur J Biochem 132:651-655
Hammond PM, Scawen MD, Atkinson A, Campbell RS, Price CP (1984) Development of an enzyme based assay for acetaminophen. Anal Biochem 143:152-157 Hsuing KP, Kuan SS, Guilbault GG (1975) An inducible amidase from Pseudomonas striata. Biochem Biophys Res Commun 66:1225-1230 Kearney PC (1965) Purification and properties of an enzyme responsible for the hydrolysing of phenylcarbamates. J Agric Food Chem 13:561-564 Lechner U, Straube G (1984) Influence of substrate concentration on the induction of amidases in herbicide degradation. Z Allg Mikrobiol 24:581-584 Linton EA, Knowles CJ (1986) Utilization of aliphatic amides and nitriles by Nocardia rhodochrous LL100-21. J Gen Microbiol 132:1493-1501 Miller JM, Gray DO (1982) The utilization of nitriles and amides by a Rhodococcus species. J Gen Microbiol 128:1803-1809 Osteryoung JG, Osteryoung RA (1985) Square wave voltammetry. Anal Chem 57:101A-110A Price NC, Stevens L (1982) Fundamentals of enzymology. Oxford University Press, Oxford, pp 173-183 Shannon MW, Saladino R, McCarty DL, Parker KM, Scott LDL, Vaughan PA (1989) Field trial of a rapid acetaminophen meter. Vet Hum Toxicol 31:358 Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85 Vaughan PA, Cheetham PSJ, Knowles CJ (1988) The utilization of pyridine carbonitriles and carboxamides by Nocardia rhodochrous LL100-21. J Gen Microbiol 134:1099-1107
Applied Microbiology Biotechnology © Springer-Verlag 1990
Aryl acylamidase from Rhodococcus erythropolis N CIB 12273 Peter A. Vaughan 1., Geoffrey F. Hall 2, and David J. Best 2.* 1 MediSense (UK) Inc., Abingdon, Oxfordshire, OX14 1DY, UK 2 Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedford, MK43 0AL, UK Received 22 February 1990/Accepted 17 May 1990
Summary. A Rhodococcus erythropolis strain was isolated from soil on the basis of its ability to use acetaminophen as the sole source of both carbon and energy for growth. When grown in a complex medium containing an anilide inducer compound, the bacterium exhibited aryl acylamidase (EC 3.5.1.13) activity. This activity was not subject to carbon or nitrogen repression by the growth medium constituents as the enzyme was present throughout the exponential growth phase. The anilide was converted to the corresponding aniline, which was not further degraded. The enzyme was partially purified by a variety of methods including a batch ion exchange procedure, column ion exchange chromatography and hydrophobic interaction chromatography. The enzyme had a maximum activity at around p H 8.0 and had a Km for acetaminophen of 0.11 raM. Electrochemical assays of aryl acylamidase activity are described. The enzyme is suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen.
Introduction Aryl acylamidases catalyse the hydrolysis of a N-acyl primary aromatic amine (anilide) to form an aniline and a carboxylic acid anion. The aryl acylamidase of Pseudomonas fluorescens ATCC 39004 has been the subject of studies concerning the large-scale purification of the enzyme and its subsequent use in clinical analysis ( H a m m o n d et al. 1983, 1984). The enzyme has also been found in several other strains of Pseudomonas (Kearney 1965; Alt et al. 1975; Hsuing et al. 1975).
* Present address: Biotechnology Unit, Laboratory of the Government Chemist, Queens Road, Teddington, Middlesex, TW11 0LY, UK ** Present address: Research and Development, Sterling Organics Ltd., Edgefield Avenue, Fawdon, Newcastle-Upon-Tyne, NE3 3TT, UK Offprint requests to: P. A. Vaughan
Aryl acylamidases occurring in Gram-positive bacteria have been generally studied in less detail. A Rhodococcus sp. that could use the anilide N-phenyl-propionamide as the sole source of carbon and energy has been described (Lechner and Straube 1984). The organism possessed an aryl acylamidase activity which was present constitutively at low levels when the organism was grown in a minimal medium and which was induced to higher levels by various anilides. A Corynebaeterium pseudodiphtheriticum, which was capable of growth on the anilides acetanilide, phenacetin and acetaminophen as sole sources of carbon and energy, was described by Grant and Wilson (1973). The organism possessed hydrolytic activity against both phenacetin and acetaminophen. An aryl acylamidase from Bacillus sphaericus ATCC 12123 was purified to homogeneity from bacteria grown on the anilide herbicide Linuron (Engelhardt et al. 1973). This paper reports the isolation and characterization of a Rhodoeoccus bacterium suitable for the production of an aryl acylamidase which could be used for clinical diagnostic purposes, and the partial purification and characterization of the enzyme. The interest in the use of aryl acylamidase as a clinical diagnostic reagent is because of the ability of the enzyme to hydrolyse the drug acetaminophen (paracetamol) to 4-aminophenol, which can be detected subsequently by colorimetric ( H a m m o n d et al. 1984) or electrochemical means.
Materials and methods Isolation o f the microorganism A minimal salts medium (50 ml, pH 7.0), containing acetaminophen (0.1% w/v) as the sole source of carbon and energy, was inoculated with a small quantity of garden soil from a flower bed. The minimal salts medium consisted of (per litre): NaNO3, 0.85 g; KH2PO4, 0.56 g; NaEHPO4, 0.86 g; KaSO4, 0.17 g; MgSO4.7H:O, 0.037 g; CaC12.2H20, 0.007 g; Fe(III) ethylenediaminetetraacetate (EDTA), 0.004g; 2.5 ml trace element solution (ZnSO4.TH20, 0.232g/l; MnSO4.H~O, 0.178g/l; H3BO4, 0.056g/1; CuSO4-SH20, 0.1g/l; Na:MoO4,2H:O, 0.039g/1:
43 COC12 •6H20, 0.042 g / l ; KI, 0.66 g/1; EDTA, 0.1 g/1; FeSO4.7 H20, 0.04 g / l ; NiC12.6H20, 0.0004 g/l; H2SO4 (1 M), 8 ml/l). The initial cultures were incubated for 5 days on an orbital shaker (200 rpm) at 30 ° C. Samples of these enrichment cultures were plated onto minimal salts agar (2% w/v) containing acetaminophen at 0.1%. The organisms which arose on these plates were isolated to purity.
voltammagram at 100 mV was indicative of aryl acylamidase activity and the peak height was proportional to the concentration of 4-aminophenol produced.
Enzyme purification Enzyme and protein assays. A rapid method of monitoring aryl
Growth of the organism for culture profile studies The organism was grown on tryptone soya broth (2%). Colonies from nutrient agar slants were inoculated into flasks (250 ml) containing medium (100 ml) and incubated for approximately 18 h at 30 ° C. The cultures were used to inoculate a fermentor (Bioengineering, Wald, Switzerland; working volume 12 1) containing medium (10 1) to which polyethylene glycol P200 (0.33% v/v) and acetanilide (0.15% w/v) had been added. The vessel was aerated at 2 l / m i n at 30 ° C and a stirring speed of 500 rpm. Samples of the culture were removed periodically and the optical density (600 nm) and pH were measured. Portions of each sample were centrifuged in a microfuge and the resulting supernatants were stored at - 2 0 ° C for later metabolite analysis. The non-centrifuged remainder of each sample was stored at - 20 ° C for analysis of the aryl acylamidase activity in the cells.
Determination of metabolites in the 9rowth medium Culture supernatant acetanilide and aniline concentrations were determined by gas liquid chromatography using a flame-ionization detector and by the use of external standards. Aliquots (200 I~I) of each thawed supernatant sample were extracted into an equal volume of ethyl acetate by vortex mixing for 5 min. Samples (1 .ul) were analysed using a temperature programme under the following conditions: column, DBWAX (JKW Scientific Inc., Folsore, CA, USA) megabore column (30 m × 0.524 mm, 1 p~m film thickness); carrier gas, nitrogen, 20 ml/min: injector temperature, 200 ° C; detector temperature, 250 ° C; initial column temperature, 155°C; initial time 5 rain; ramp rate, 20°C/rain; final column temperature, 235 ° C; final hold time, 10 rain. Extraction and analysis were performed in duplicate on each sample.
Measurement of aryl acylamidase activity in culture cell samples Aliquots (5 ml) of thawed samples were centrifuged (100009, 10 min, 4 ° C) and the pellet of cells was washed in T R I S / H C I buffer (5 ml, 50 raM, pH 8.6) and resuspended in fresh buffer (5 ml). The bacteria were disrupted by sonication (MSE sonicator, Crawley, UK; 150 W, 3 x 45 s, 16 mix amplitude). The disrupted cell suspension was centrifuged (10 000 9, 30 min, 4 ° C) to remove cell debris and the supernatant removed and kept on ice. The protein content of the cell-free extracts was assayed by the bicinchoninic acid method (Smith et al. 1985). An electrochemical assay, Osteryoung square wave voltametry (Osteryoung and Osteryoung 1985), was employed as a sensitive and simple method to measure specific activities for aryl acylamidase in cell-free extracts. The hydrolysis product of the enzyme reaction, 4-aminophenol, was differentiated from the substrate, acetaminophen, by a characteristic peak in the pulse voltammagram at 100 inV. The assay was conducted at 30°C in a two-compartment electrochemical cell with a glassy carbon working electrode, a saturated calomel reference electrode and a platinum wire auxiliary electrode. The incubation mixture (2 ml) contained acetaminophen (50 raM) in T R I S / H C l buffer (100 raM, pH 8.6). The reaction was initiated by the injection of the cell-free extract sample (100 ixl). The appearance of a peak in the pulse
acylamidase activity during the protein purification procedures was provided by a method based on that described by Alt et al. (1975). The reaction mixture in a 3-ml plastic cuvette consisted of the following: T R I S / H C I buffer (100 raM, pH 8.6, 0.6 ml) and the enzyme substrate 4-nitroacetanilide (1 mM). The reaction was initiated by the addition of the enzyme sample (400 IXl, with the enzyme prediluted in buffer if required) and the reaction mixture was incubated at 30 ° C. The formation of the coloured product of the aryl acylamidase reaction, 4-nitroaniline, was monitored by measuring the A405 of the reaction mixture. The molar extinction coefficient of 4-nitroaniline under these conditions was 10000 1/ mol per cm. The protein assay used in the purification studies was the Coomassie Brilliant Blue method (Bradford 1976).
Cellular disruption. A 15-1 culture of the organism was grown in a 20-1 fermentor as for the culture profile study. When the culture attained an optical density at 600 nm (OD6oo) of 6 (i.e. in the late exponential phase of growth) the bacteria were harvested using a Sharples (Pennwalt Ltd., Camberley, UK) continuous centrifuge and 150 g wet weight of bacteria was recovered. The cells were washed by resuspending them in approximately 300 ml T R I S / HCI buffer (50 raM, pH 7.5) and then centrifuging the suspension at 11000 9 for 90 rain at 4 ° C to produce a supernatant, which was discarded, and a cell pellet. The cell pellet was resuspended to 300 ml in buffer (50 mM, pH 7.5). A 60-ml portion of the harvested cell suspension was used to prepare a cell-free extract. A 10-ml volume of buffer (50 raM, pH 7.5) was added to the 60 ml of suspension, and dithiothreitol (DTT) was added to 1 mM. The suspension was split into two batches, each of which were sonicated (10 × 45 s periods, 16 mix amplitude). The pooled, Sonicated material was centrifuged at 31000 9 for 70 min at 4°C to yield cell-free extract (57 ml). The extract was kept at 4 ° C. DTT (1 raM) was included in the TRIS/HC1 buffers used in all of the subsequent enzyme purification steps, and these buffers were all pH 7.5 and 20 raM, unless indicated otherwise. Batch ion exchange procedure. The cell-free extract (57 ml) was mixed with DE52 ion exchange resin (4 g, Whatman, Maidstone, UK), which had been pre-equilibrated with buffer (50 raM). The mixture was stirred at 4°C for 3 h and then the DE52 resin was allowed to settle out. The supernatant was discarded and the DE52 resin was washed by adding buffer (80 ml, 20 raM), and the suspension stirred for 5 min at 4 ° C. The DE52 resin was again allowed to settle out and the suspension was decanted to leave the ion exchange resin suspended in approximately 4 ml buffer. A 21ml volume of buffer containing 0.4 M sodium chloride was added to the DE52 resin suspension to produce a final salt concentration of approximately 0.35 M. The suspension was stirred for 2 h, during which time the aryl acylamidase was eluted from the DE52 resin, and then filtered (through a 0.4 Ixm filter) to remove the ion exchange resin.
Column ion exchange chromatography. The protein solution produced by the batch DE52 resin procedure was further purified by ion exchange chromatography on a column of Pharmacia (Uppsala, Sweden) Q-Sepharose Fast Flow gel (a cross-linked agarose gel containing quaternary amine functional groups). A 5-ml sample of the protein solution was mixed with 1 ml buffer to dilute the NaC1 concentration of the sample to less than 0.3 M. A 5-ml volume of the diluted sample was applied to a 48-ml Q-Sepharose column (9 cm×2.6 cm diameter) which had been previously equilibrated with buffer containing 0.3 M NaC1. The protein was
44 eluted with 240 ml of the same buffer followed by 240 ml of the buffer containing 0.4 M NaC1. The flow rate was 240 ml/h, and 10 ml fractions of the eluent were collected. The aryl acylamidase eluted after between 60 and 150 ml of the 0.4 M NaC1 buffer had been applied to the column.
Hydrophobic interaction chromatography. The active fractions from the above step were combined, desalted by diafiltration and concentrated, using an Amicon 30000 molecular weight cut-off hollow-fibre cartridge and buffer. The desalted solution was further concentrated to a volume of 5 ml using an Amicon (Danvers, MA, USA) ultrafiltration cell, fitted with a 30000 molecular weight cut-off membrane. A 0.5-ml sample of the concentrated desalted protein solution was applied to a 4-ml (2 cmx 1.6 cm diameter) column of Pharmacia (Uppsala, Sweden) phenyl-Sepharose gel (a pbenyl-substituted cross-linked agarose gel) which had been pre-equilibrated with 0.5 M ammonium sulphate in buffer. The column was washed with 10 ml of the same solution and the enzyme eluted with a 20-ml linear descending gradient of 0.50.0 M ammonium chloride in the same buffer. The flow rate was 0.33 ml/min and 1 ml fractions were collected. Determination of the K,~ of the aryl acylamidase for acetaminophen The assay was carried out at 30° C in a stirred electrochemical cell containing a glassy carbon working electrode, a silver/silver chloride reference electrode and a platinum wire counter electrode. The cell contained TRIS/HC1 buffer (2.9 ml, pH 8.6, 100 mM) which incorporated acetaminophen at concentrations ranging between 17 p.M and 1.67 mM. The working electrode was poised at 300 mV relative to the reference electrode, using a potentiostat, and the electrical current produced was recorded on a chart recorder. When a steady current had been reached, 100 pJ of diluted enzyme solution was injected into the cell to initiate the reaction. The product of the enzyme reaction, 4-aminophenol, was oxidized at the working electrode giving rise to an electrical current proportional to the concentration of the 4-aminophenol. The initial increase in the current after the addition of the enzyme was linear. Each substrate concentration was assayed in triplicate and the mean rate was used for kinetic analysis using Lineweaver-Burk, Hanes and Eadie-Hofstee plots.
Determination of the effect of pH on the aryl acylamidase The effect of pH on aryl acylamidase activity was measured using the 4-nitroacetanilide colorimetric assay and the buffers (50 mM) K2HPO4/KH:PO4 (pH 6.0-8.0), TRIS/HCI (pH 7.5-8.6), and 3[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid/NaOH (pH 8.6-9.5).
Results and discussion
Isolation and identification of the organism Six bacterial isolates were obtained from soil by the isolation procedure described in Materials and methods. One of these bacteria was found to be a Gram-positive organism capable of growth upon minimum agar containing either 0.1% acetaminophen or 0.1% phenacetin. Discolouration (blackening) of the medium occurred in both cases. Medium discolouration was used as an indication of the formation and reaction of substituted anilines, which in turn indicated that the organism was
able to hydrolyse the anilide substrate and therefore possessed aryl acylamidase activity. The bacterium was identified by the National Collection of Industrial and Marine Bacteria, UK, as a strain o f Rhodococcus erythropolis ( N C I B No. 12273) (Best and Vaughan 1987). Rhodococcus erythropolis is a c o m m o n non-pathogenic soil organism. Members of the Rhodococcus genus of bacteria, which includes organisms previously classified as Nocardia rhodochrous species (Goodfellow and Anderson 1976), have been previously found to be capable of the hydrolysis of a wide range of aliphatic, aromatic and heterocyclic amides (Miller and Gray 1982; Linton and Knowles 1986; Vaughan et al. 1988).
Effect of pH, temperature and the presence of an anilide on the growth of the organism Initial studies on the growth of the organism in complex media were performed using shake flasks (250 ml) containing tryptone soya broth (100 ml, 2%). An examination of the effect of p H indicated that the organism will grow over the p H range examined (pH 6.6-8.4) with optimum growth rates seen in cultures with an initial p H between p H 7.0 and 8.0. A comparison of growth rates at 26 °, 30 ° and 35°C indicated that growth was fastest at 30 °C. The presence of the aryl acylamidase inducer acetanilide (0.1%) in the tryptone soya broth appeared to result in a slower initial growth rate but gave an equivalent final culture optical density to that seen in the absence o f acetanilide.
Changes in inducer substrate concentration, enzyme product concentration and aryl acylamidase activity during growth of the organism on a complex medium containing acetanilide The growth of the organism on tryptone soya broth (2%) containing acetanilide (0.15%) and the changes in various culture parameters are shown in Fig. 1. Acetanilide was depleted during the mid-exponential phase of growth with concomitant accumulation of aniline, which did not appear to be further utilized by the bacteria. The non-utilization of the aniline product by the bacteria is consistent with the apparent non-utilization of the substituted aniline products of acetaminophen and phenacetin hydrolysis by the organism, as indicated by the medium discolouration during growth of the organism on either of the two substituted acetanilides. Aryl acylamidase activity, as measured in cell-free extracts of the bacteria, was induced throughout the exponential growth phase with the maximum total and specific activity being expressed at the end of the growth period (approximately 0.68 ~tmol substrate hydrolysed/min per milligram protein, corresponding to 0.66 Ixmol substrate hydrolysed/min per millilitre culture). The specific activity of the aryl acylamidase declined in the stationary phase. No aryl acylamidase activity was detected in a cell-free extract prepared from
45
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Fig. 1. Rhodococcus erythropolis NCIB 12273 was grown on 2% tryptone soya broth containing0.15%v/v acetanilidein a 12-1fermenter and various parameters were measured, as described in Materials and methods: optical density at 600 n m (OD6o0) ( 0 ) ; acetanilide concentration (.6); aniline concentration (V); aryl acylamidase specific activity in cell-free extracts of the bacteria (=)
bacteria grown on trypone soya broth in the absence of acetanilide and harvested in the mid-exponential growth phase. The above results indicate that the aryl acylamidase activity was induced by acetanilide and that this induction was not subject to repression by carbon or nitrogen sources in the complex tryptone soya medium. If such repression had operated it would be expected that the acetanilide would not have been utilized before the end of the growth period. This lack of repression of aryl acylamidase induction by components of a complex growth medium also occurred in a culture of Pseudomonas fluorescens ATCC 39004 during growth of the organism on tryptone soya broth containing acetanilide, in the study reported by Hammond et al. (]983).
mond et al. (1983) was not improved by the presence of the thiol protective agent 2-mercaptoethanol. The proteinase inhibitor phenylmethyl-suiphonyl fluoride, at 0.1 mM, almost totally inhibited the aryl acylamidase activity in cell-free extracts, indicating that the enzyme might operate by a similar mechanism to the serine proteases (Price and Stevens 1982). In several vertebrate systems aryl acylamidase activity has been found to be associated with the same protein as that responsible for acetylcholine esterase activity. In one study it was found that the enzymic activities appeared to arise from non-identical but partially overlapping active sites on the same protein (Balasubramanian 1984). Several sequential procedures were developed for the partial purification of the R. erythropolis aryl acylamidase as described in Materials and methods. The batch ion exchange procedure gave a 1.8-fold protein purification, i.e. an increase in the aryl acylamidase specific activity from 0.58 to 1.05 ~tmol substrate hydrolyses/min per milligram protein. The recovery of activity was 77%. The column ion exchange chromatography gave a further 4.9-fold purification with a yield for this step of 90%. The diafiltration and concentration procedures gave an approximately two-fold purification of the enzyme. The hydrophobic interaction chromatography step gave 1.9-fold purification, resulting in a protein solution with an aryl acylamidase specific activity of 23.3 ~tmol substrate hydrolysed/min per milligram protein. The partially purified enzyme was used for studies of the effect of pH on the aryl acylamidase activity and to determine the Km of the enzyme for acetaminophen. The enzyme has a broad pH optimum with maximum activity at around pH 8.0. The pH optima of other microbial aryl acylamidases ranges from just above neutrality to pH 10.0 (Hammond et al. 1983). The Km of the enzyme for acetaminophen (determined as described in Materials and methods) was 0.11 mM from the Lineweaver-Burk plot, 0.12 mM from the Hanes plot and 0.11 mM from the Eadie-Hofstee plot.
Suitability of R. erythropolis aryl acylamidase for clinical diagnostic measurement of acetaminophen R. erythropolis NCIB 12273 cultures with aryl acylami-
Enzyme purification and properties Preliminary studies indicated that the R. erythropolis aryl acylamidase activity was unstable in cell-free extracts in the absence of a thiol protecting agent, and therefore ] mM DTT was added to the solutions used throughout the purification procedure and during storage of the enzyme. The enzyme, in cell-free extracts in TRIS/HC1 buffer, pH 7.5, containing 1 mM DTT, was stable when stored at - 20 ° C. When stored at 4 ° C, 66% of the activity was retained after 7 days. The stability of the pseudomonad aryl acylamidase studied by Ham-
dase activity can be produced in a straightforward manner and the enzyme can be partially purified from cell extracts by a variety of methods in acceptable yields. The cell growth and enzyme purification methods described are suitable for further optimization and scaleup. The stability of the enzyme in solution, the pH optimum of the enzyme and its K m for acetaminophen make the enzyme suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen. Acetaminophen (paracetamol) is a commonly used analgesic drug for which the clinically significant concentration range in blood plasma is between 0.0 and approximately 3.0 mM for overdose patients. The R. erythropolis enzyme is currently being used in the develop-
46 m e n t o f a n o v e l b i o s e n s o r to m e a s u r e a c e t a m i n o p h e n ( p a r a c e t a m o l ) in clinical s a m p l e s b y e l e c t r o c h e m i c a l d e t e c t i o n o f the 4 - a m i n o p h e n o l p r o d u c e d b y a c e t a m i n o p h e n h y d r o l y s i s ( S h a n n o n et al. 1989).
References Air J, Krisch K, Hirsch P (1975) Isolation of an inducible amidase from Pseudomonas acidovorans AE1. J Gen Microbiol 87:260272 Balasubramanian AS (1984) Have cholinesterases more than one function? Trends Neurosci 7:467-468 Best DJ, Vaughan PA (1987) Rhodococcus bacterium for the production of aryl acylamidase. European patent application 87306778.9 Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:348-354 Engelhardt G, Wallnofer PR, Plapp R (1973) Purification and properties of an aryl acylamidase from Bacillus sphaericus catalysing the hydrolysis of various phenylamide herbicides and fungicides. Appl Microbiol 26:709-718 Goodfellow M, Alderson G (1976) The actinomycete genus Rhodococcus: a home for the 'rhodochrous' complex. J Gen Microbiol 100:99-122 Grant DJW, Wilson J (1973) Degradation and hydrolysis of amides by Corynebacterium pseudodiphtheriticum NCIB 10803. Microbios 8:15-22 Hammond PM, Price CP, Scawen MD (1983) Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004. Eur J Biochem 132:651-655
Hammond PM, Scawen MD, Atkinson A, Campbell RS, Price CP (1984) Development of an enzyme based assay for acetaminophen. Anal Biochem 143:152-157 Hsuing KP, Kuan SS, Guilbault GG (1975) An inducible amidase from Pseudomonas striata. Biochem Biophys Res Commun 66:1225-1230 Kearney PC (1965) Purification and properties of an enzyme responsible for the hydrolysing of phenylcarbamates. J Agric Food Chem 13:561-564 Lechner U, Straube G (1984) Influence of substrate concentration on the induction of amidases in herbicide degradation. Z Allg Mikrobiol 24:581-584 Linton EA, Knowles CJ (1986) Utilization of aliphatic amides and nitriles by Nocardia rhodochrous LL100-21. J Gen Microbiol 132:1493-1501 Miller JM, Gray DO (1982) The utilization of nitriles and amides by a Rhodococcus species. J Gen Microbiol 128:1803-1809 Osteryoung JG, Osteryoung RA (1985) Square wave voltammetry. Anal Chem 57:101A-110A Price NC, Stevens L (1982) Fundamentals of enzymology. Oxford University Press, Oxford, pp 173-183 Shannon MW, Saladino R, McCarty DL, Parker KM, Scott LDL, Vaughan PA (1989) Field trial of a rapid acetaminophen meter. Vet Hum Toxicol 31:358 Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85 Vaughan PA, Cheetham PSJ, Knowles CJ (1988) The utilization of pyridine carbonitriles and carboxamides by Nocardia rhodochrous LL100-21. J Gen Microbiol 134:1099-1107
