enzyme loss without introducing important limitations to the diffusion of products. This is the first report concerning the characterization and

Transcription

1 JOURNAL OF BACTERIOLOGY, Dec. 1990, p /90/ $02.00/0 Copyright 1990, American Society for Microbiology Vol. 172, No. 12 Purification, Cloning, and Primary Structure of an Enantiomer- Selective Amidase from Brevibacterium sp. Strain R312: Structural Evidence for Genetic Coupling with Nitrile Hydratase JEAN-FRANCOIS MAYAUX,1* EDITH CERBELAUD,2 FABIENNE SOUBRIER,1 DIDIER FAUCHER,' AND DOMINIQUE PETRE2 Institut de Biotechnologie, Rhone-Poulenc Sante, Centre de Recherches de Vitry, 13 quai Jules Guesde, B.P. 14, Vitry Sur Seine Cedex,' and Laboratoire de Catalyse Enzymatique, Rhone-Poulenc Recherches, Centre de Recherches des Carrieres, Saint-Fons Cedex, France Received 2 March 1990/Accepted 13 August 1990 An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amda. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amda is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 imol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30TC under the control of an E. coli promoter and ribosome-binding site. Nitriles are extensively used in organic synthesis by the chemical industry as precursors to produce compounds such as amides and organic acids. However, the chemical conversion of nitrites presents several disadvantages, such as the need for strongly acidic or basic conditions, a high energy consumption, or the formation of unwanted by-products. More recently developed biochemical procedures in which microorganisms are used as catalysts look promising because temperature and ph conditions are less severe and because very pure products can be formed (23). In addition, bioconversions can be stereospecific and can lead to the production of a single enantiomer, which might be a crucial aspect in the manufacturing of active new drugs (5). However, the use of very efficient microbial strains is necessary to develop a biochemical process at the industrial scale. For instance, an efficient process for the production of acrylamide from acrylonitrile using resting cells of Pseudomonas chlororaphis B23 has recently been developed (21). Many microorganisms can use nitrites as a sole carbon and/or nitrogen source. Among them, the potential use of Brevibacterium sp. strain R312 was recognized several years ago by Arnaud and colleagues (1). This gram-positive coryneform strain was shown to contain two types of enzyme activities for the hydrolysis of nitrites: a nitrile hydratase that hydrates nitrites into amides (6) and probably several amidases which transform amides into the corresponding organic acids (13). The bioconversion of water-soluble amides in a continuous immobilized cell reactor was also demonstrated (3). Such experiments led to the view that this type of corynebacteria may be viewed as "bags," preventing * Corresponding author enzyme loss without introducing important limitations to the diffusion of products. This is the first report concerning the characterization and cloning of an amidase from Brevibacterium sp. strain R312 active on several 2-aryl or 2-aryloxy propionamides. This stereospecific enzyme is significantly homologous to several amidases from different microorganisms. In addition, we showed that the structural gene encoding this enzyme is located immediately upstream of the gene coding for nitrile hydratase, suggesting that both enzymes are coordinately expressed in the cell. Finally, our results, compared with other data (11), strongly suggest that two studied microorganisms in the field, Brevibacterium sp. strain R312 (1) and Rhodococcus (Corynebacterium) sp. strain N-774 (28), are probably identical, or very closely related. MATERIALS AND METHODS Abbreviations. HPLC, High-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; X-gal, 5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside; HPPAmide, 2-(4-hydroxyphenoxy) propionamide; HPPAcid, 2-(4-hydroxyphenoxy)propionic acid; RBS, ribosome-binding site. Bacterial strains, plasmids, and media. Bacterial strains and plasmids used in this study are described in Table 1. Brevibacterium and Escherichia coli strains were routinely grown on LB plates or in liquid LB medium at 30 and 370C, respectively. Ampicillin was used at 100 [ug/ml to maintain E. coli plasmids. Conditions used for the expression of heterologous proteins in E. coli have been described previously (8, 14). Materials. T4 DNA ligase and restriction enzymes were purchased from New England BioLabs; T4 polynucleotide kinase and DNA polymerase I (Klenow fragment) were

2 VOL. 172, 1990 ENANTIOMER-SELECTIVE AMIDASE FROM BREVIBACTERIUM SP TABLE 1. Bacterial strains and plasmids used Strain or plasmid Relevant characteristics Reference or source Strains Brevibacterium sp. strain R312 Natural isolate CBS (1) Escherichia coli DH5a F- endal hasr17 (rk- mk+) supe44 thi-j X- recal gyra96 relal Clontech Laboratory, Palo 4)80 dlaczam15 Alto, Calif. Escherichia coli E103S Lon- Met- D. L. Simon, Waksman Institute of Microbiology, Piscataway, N.J. Escherichia coli B Wild-type strain 20 Plasmids and bacteriophage puc18, -19 Multicloning site 30 M13mpl9 Multicloning site 30 pxl534 Apr Ptrp promoter vector expressing human serum albumin 16a pxl820 Apr PR,cIts promoter vector expressing human serum albumin 14 pxl1650 Apr puc derivative containing 5.4-kbp amda insert This study pxl1651 Apr, same as pxl1650; reverse orientation of insert This study pxl1724 Apr amda with its own RBS under control of Ptrp This study pxl1751 Apr amda under control of cii RBS and Ptrp This study pxl1752 Apr amda under control of c1i RBS and PR cits This study obtained from Boehringer Mannheim Biochemicals and used as recommended by the manufacturer. General methods. Standard procedures for DNA isolation, manipulation, analysis, and amplification were used (18). Brevibacterium genomic DNA was prepared as described previously (9). The Sequenase Version 2-0 (United States Biochemical Corp.) was used for DNA sequencing with [kx-355]datp (Amersham Corp.). 7-Deaza-dGTP was substituted for dgtp to reduce compression caused by the high G+C percentage. Polymerase chain reaction experiments were performed using the Taq polymerase from Cetus and the Perkin-Elmer Cetus DNA thermal cycler as recommended by the manufacturer. Proteins were fractionated by PAGE under denaturing conditions (18). Enzyme assay. The enzyme activity on HPPAmide (20 mm) was monitored during stirring at 25 C in 4 ml of 50 mm sodium phosphate buffer (ph 7.0). The reaction was stopped by the addition of a 90:10 (%) (vol/vol) mixture of acetonitrile and 1 N HC1, respectively. After homogenization and centrifugation, the supernatant was applied to an HPLC reverse-phase column (Hibar-Merck RP-18; 5 plm). The elution at 1 ml/min by an 85:15 (vol/vol) mixture of phosphoric acid (0.05 M) and acetonitrile, respectively, was monitored by the optical density at 280 nm, and the respective concentrations of HPPAmide and HPPAcid in the analyzed sample were measured from the peak positions and areas of standards. Enantiomer selectivity assays. The enantiomer excess (ee), defined as the ratio [(R - S)I(R + S)] x 100 (%), where R and S are, respectively, the concentrations of the R(+) and S(-) HPPAcid enantiomers, was deduced from HPLC on a chiral column (Resolvosil BSA 7; Macherey-Nagel) with the following elution buffer: sodium phosphate (ph 6.8)-10 mm isopropanol (95/5, vol/vol). Under these conditions, the retention times of the R(+) and S(-) forms were 7.2 and 13 min, respectively. Purification of enantiomer-selective amidase activity. All procedures were done at 4 C. (i) Step 1. Preparation of crude enzyme solution. Frozen cell paste (40 g [wet weight] of Brevibacterium sp. strain R312) was thawed and suspended in 300 ml of buffer A (50 mm sodium phosphate [ph 7.0], 5 mm P-mercaptoethanol). Cells were broken by ultrasonic treatment, and cell debris was separated by centrifugation at 20,000 x g for 30 min. A 25-ml portion of a 10% solution of streptomycin sulfate was then slowly added under gentle agitation to 310 ml of the supernatant. After 45 min, the solution was clarified as described above and the supernatant was submitted to the following steps. (ii) Step 2. Ammonium sulfate fractionation. The protein fraction precipitating between 30.8 and 56.6% saturation with ammonium sulfate was collected by centrifugation and dissolved in 35 ml of buffer A. This solution was further desalted by extensive dialysis against the same buffer. (iii) Step 3. Phenyl-Sepharose CL-4B chromatography. The protein fraction was brought to 20% saturation with ammonium sulfate, clarified by centrifugation, and applied onto a column of phenyl-sepharose CL-4B (Pharmacia) equilibrated with buffer A at 20% saturation with ammonium sulfate. Protein fractions containing the enzyme activity were eluted with the same buffer, pooled, and concentrated by ultrafiltration with an Amicon Diaflo PM10 cell to a volume of 18 ml. (iv) Steps 4 and 5. Gel filtration. Glycerol (10%) was added to the concentrated protein fraction, and the solution was loaded on an Ultrogel AcA 44 (IBF-biotechnics, Villeneuvela-Gareivne, France) column which had been equilibrated in 50 mm Tris hydrochloride (ph mm NaCl. Protein fractions containing the highest specific activity (about 32% of the total activity loaded on the column) were collected, concentrated, and submitted to an additional filtration step on the same gel. Once again, fractions (about 30% of applied proteins) exhibiting the highest specific activity were analyzed by SDS-PAGE and pooled. The enantiomer selectivity of the purified protein obtained by this procedure was also checked (ee > 95%). Protein sequencing. About 3 nmol of the purified amidase preparation was reduced and carboxymethylated. The major protein component was then desalted and purified to homogeneity by RP C4 reverse-phase HPLC. The N-terminal amino acid sequence was determined by the sequential automated Edman degradation method with an Applied Biosystems model 470 A apparatus. The sequence NH2- ATIRPDDKAIDAAARHYGITLDKTARL... was determined. To obtain an additional internal sequence, we submitted the same quantity of the protein to complete trypsin digestion. The reduced and carboxymethylated fragments were then separated by RP C8 reverse-phase HPLC (2.1 by 10 mm; flow rate, 0.2 ml/min), eluting with a gradient of 0 to

3 6766 MAYAUX ET AL. 50% (vol/vol) acetonitrile in 0.07% (vol/vol) trifluoroacetic acid. A peptide eluted in a well-separated peak at 40.8% acetonitrile was sequenced. The sequence NH2-LEWPAL IDGALGSYDVVDQLY... was obtained. Oligonucleotide probes. Oligodeoxynucleotides were synthesized with a Biosearch 8600 automatic DNA synthesizer and purified by PAGE under denaturing conditions as previously described (8). Probe A was derived from part of the internal peptide sequence (IDGALGSYDV) on the noncoding strand as the following degenerated 29-mer: 5'-ACGT CGTAGGAGCC(G,C)AG(G,C,T)GCGCCGTCGAT-3'. An exact probe from the N-terminal sequence was designed by polymerase chain reaction as follows. Briefly, two highly degenerated oligonucleotides corresponding to nearly all coding possibilities of the first and last five amino acids of the N-terminal sequence and flanked with a restriction site were synthesized: 5'-CGGAATTCGCNACNAT (T,C,A)GNCC NGA-3' (direct orientation, EcoRI site) and 5'-CCAAGC1[ GCNGT(T,C)TT(A,G)TCNA(G,A) NGT-3' (reverse orientation, HindIII site), where N corresponds to an equimolar mixture of the four bases. These oligonucleotides were used to prime 30 cycles of enzymatic amplification by the Taq polymerase on the total genomic DNA of Brevibacterium sp. strain R312 as described previously (10). The amplified DNA was then digested with EcoRI and HindIII and cloned between the corresponding sites of M13mpl9. Several recombinant clones were sequenced and were found to contain a DNA fragment encoding the N-terminal peptide sequence. In particular, the unique DNA sequence between both primers was used to design the exact 40-mer synthetic probe B (noncoding strand): 5'-GATGCCGTAATGCCTTG CGGCGGCGTCTATTGCTTTGTCG-3'. Southern blots and colony hybridization. Biodyne A nylon transfer membranes (Pall Industrie, St. Germain-en-Laye, France) were used for both Southern and colony hybridizations according to the manufacturer's specifications and protocols. For Southern blotting experiments, S to 10,ug of genomic DNA was digested to completion and fragments were separated on 0.8 to 1% agarose gels. Because oligonucleotide probes were subsequently used, depurination of DNA was avoided by omitting acid treatment of the gel. Oligonucleotides (100 to 500 ng) were 5' labeled with [,y-32p]atp (3,000 Ci/mmol; Amersham), using T4 polynucleotide kinase as described previously (18), and were used without further purification. Standard hybridization buffer was Sx SSC (lx SSC is 0.15 M NaCl plus M sodium citrate)-5 x Denhardt solution-50 mm sodium phosphate (ph 6.5)-0.1% SDS-250,ug of salmon sperm DNA per ml. For probe A, prehybridization and hybridization (4 h and overnight, respectively) were done at 55 C in hybridization buffer. Filters were then washed several times in 3x SSC- 0.5% SDS at 55 C, allowed to air dry, and exposed overnight to Kodak X-Omat XAR-5 films at -70 C between intensifying screens. For probe B, hybridization buffer containing 50% formamide was used at 45 C, and the filter was washed twice for 1 h at room temperature in 2x SSC-0.1% SDS and for 5 min at 45 C in 0.1 x SSC-. 1% SDS. Similar conditions were used for colony hybridization, except that autoradiography was only for 2 to 3 h without intensifying screens. Plasmid constructions. The BamHI-NcoI (538-bp) and NcoI-EcoRI (1,760-bp) fragments from the 5.4-kbp insert of pxl1650 (Fig. 1) were inserted between the BamHI and EcoRI sites of puc18 to give pxl1675. The 2.3-kbp XbaI fragment of pxl1675 containing the amidase gene with its natural RBS was then inserted in the correct orientation, under the control of the E. coli trp promoter, between the unique XbaI sites of plasmid pxl1599 to give pxl1724. Plasmid pxl1599 is derived from pxl534 (16a) by substitution of the HindIII fragment containing the RBS and coding sequence for human serum albumin for the sequence 5'-..A AGCTCTAGAGCTCTAGAGCTT..-3', which introduces the unique overlapping sites XbaI-SacI-XbaI. An NdeI (CATATG) site containing the ATG initiation codon of the amidase structural gene was created, and the corresponding 83-bp NdeI-XhoI fragment containing the first 26 codons of amidase was obtained by polymerase chain reaction enzymatic amplification of the pxl1724 sequence with the two primers 5'-CAGGAGCACACTTCATATGGCGACAATCC- 3' and 5'-CGGCCACTCGAGCCGGGCTGTTTTG-3' and subsequent digestion by NdeI and XhoI. This fragment was then ligated to the 5.25-kbp XhoI-EcoRI vector fragment of pxl1724 containing the 494 last codons of the amidase gene and to an EcoRI-NdeI fragment containing a promoter and an RBS derived from the X cih gene. pxl1751 was derived by using the 121-bp EcoRI-NdeI fragment of pxl534 containing the trp promoter, whereas pxl1752 contains the 1,228-bp EcoRI-NdeI fragment of pxl820 (14) containing both the A PR promoter and clts. Nucleotide sequence accession number. The sequence shown in Fig. 2 has been submitted to GenBank (accession number M32282). RESULTS J. BACTERIOL. Identification and purification from Brevibacterium sp. strain R312 of an enantiomer-selective amidase active on several 2-aryl or 2-aryloxy propionamides. Following the experimental protocols described in Materials and Methods, Brevibacterium sp. strain R312 (CBS ) (1) was found to contain an amidase activity able to catalyze the stereospecific hydrolysis of several racemic 2-aryl propionamides such as 2-phenylpropionamide or 2-aryloxy propionamides such as HPPAmide into the corresponding S or R acid, respectively (E. Cerbelaud and D. Petre, European Patent Application EP A, 1988). The enantiomer excess of the acid product was found to be consistently higher than 93 to 95% on several substrates, demonstrating the enantiomer selectivity of the amidation reaction. Using HPPAmide as a substrate, we found specific activities of 0.25 and 0.40 U/mg of bacterial proteins on whole cells and soluble cell extracts, respectively (1 unit defined is as 1,umol of HPPAcid formed per min under the conditions described in Materials and Methods). HPPAmide was subsequently used to monitor the reaction during the purification of the investigated enzymatic activity. Through the purification steps described in Materials and Methods, the enantiomer-selective enzyme was purified 31-fold up to a maximal purity of about 80% (Table 2, and see Fig. 4, lanes b and d). The global yield and the maximum specific activity were 6.3% and 15.6 U/mg, respectively, and were not optimized, since the objective was essentially to obtain enough protein for N-terminal sequencing. At that stage, a prominent band of apparent Mr 59, ,000, corresponding to about 80% of total proteins, was detected on an SDS-polyacrylamide gel. That this protein was indeed responsible for the measured activity was further confirmed by the SDS-PAGE analysis of the protein fractions characterized by the highest specific activity during the second Ultrogel AcA 44 gel filtration (data not shown). Moreover, we observed in subsequent experiments (data not shown) using HPLC gel filtration that the amidase was eluted at a position corresponding to an apparent Mr of 120, ,000, suggesting that the enzyme behaves as a homodimer.

4 VOL. 172, 1990 ENANTIOMER-SELECTIVE AMIDASE FROM BREVIBACTERIUM SP BamHI Not XhoI/ I \/ XhoI HindIII PstI amda NHase 5,4K bp a subunit BamHI Xhol - k - -O NcoI S hi BamHI I Clal, SaIII PstI 4-. 4~~~ A~4 - a.4--. FIG. 1. Upper: Restriction map of the 5.4-kbp PstI fragment, the insert of plasmids pxl1650 and plx1651, is shown with the positions (arrows) of the coding regions for the enantiomer-selective amidase (amda) and the a subunit of nitrile hydratase (NHase). The sequenced BamHI-PstI region is indicated by the dashed arrow. Lower: More precise structure of the BamHI-PstI region. Unique sites are underlined. The sequencing strategy on both strands is shown by the arrows. Arrows with filled squares indicate sequencing from specific oligonucleotide primers. Fragments subcloned in M13 are indicated by shaded boxes. The region common with the Rhodococcus sp. strain N-774 fragment independently sequenced by Ikehata et al. (11) is shown by the black bar. Cloning of the enantiomer-selective amidase. The N-terminal peptide sequence (27 residues) and the sequence of an internal fragment (21 residues) of the major polypeptide species in the purified preparation were determined as described in Materials and Methods. From these peptide sequences, two different oligonucleotide probes were designed. Probe A is a six-fold-degenerated 29-mer derived from the internal fragment, taking into account the codon usage in the tryptophan operon of Brevibacterium lactofermentum (19). For the other probe, the polymerase chain reaction procedure described by Girgis et al. (10) was performed to generate an exact 40-mer nucleotide probe (probe B) from the N-terminal sequence. These synthetic DNAs were then 32p labeled and used as hybridization probes. Southern hybridization with these probes against several digestions of the Brevibacterium sp. chromosomal DNA revealed that, at least for several restriction enzymes, probes A and B hybridized with the same fragment (data not shown). In particular, both probes hybridized with a single 5.4-kb PstI fragment. The PstI-digested chromosomal fragments of 4.8 to 6.3 kb were purified by agarose gel electrophoresis, ligated with PstI-digested puc19 plasmid, and introduced into E. coli DH5a. About 500 ampicillin-resistant white transformants on X-gal medium obtained in this way were individually screened by colony hybridization with probe B. Two colonies showing a strong positive hybridization to both probes A and B were found. Restriction analysis of corresponding plasmid DNAs (pxl1650 and pxl1651) indicated that they had inserted the same 5.4-kb PstI fragment in either of the two possible orientations. A restriction map of this fragment is shown in Fig. 1. Supercoil DNA sequencing with both probes as primers and additional Southern analysis of the plasmids confirmed that the correct chromosomal fragment had been cloned, indicated that the two previously determined peptide sequences were next to each other in the protein, and showed that the studied gene was contained on a 2.3-kb BamHI-PstI fragment (Fig. 1). Nucleotide sequence of enantiomer-selective amidase gene and structural evidence for coupling with the gene coding for nitrile hydratase. The nucleotide sequence of the BamHI- PstI fragment was determined as detailed in Fig. 1. The 2,447-bp-long sequence is shown in Fig. 2. The overall G+C composition of this sequence is 61.5%. An open reading frame coding for a 521-amino-acid protein (Mr 54,671) containing the amino acid sequences determined from the presumed enantiomer-selective amidase is found between nucleotides 245 and In this coding sequence, the mean G+C composition of positions 1, 2, and 3 of the codons is 65.8, 52.5, and 70%, respectively. Such a pattern in codon usage has already been observed for high-g+c-containing organisms (4). An additional open reading frame coding for at least 188 amino acids is found 73 nucleotides downstream

5 6768 MAYAUX ET AL. J. BACTERIOL. cgatccggaaacagtacttcggcagcttgccacgacaccgaaaagctctacgaacaccggtgttccactgcatcggccgattctgatcgctgaatcggcccgtgggcgactgtacccccg 120 BamHI tctctcttgagcgcacgtaacccgaacttaacgagtcaatatgtcgatacctattgacgcaattatggatcoggccctagtctgaaagacaagtgaagccgatcacatcaggagcacactt 241 amidase > ctc ATG GCG ACA ATC CGA CCT GAC GAC AAA GCA ATA GAC GCC GCC GCA AGG CAT TAC GGC ATC ACT CTC GAC AAA ACA GCC CGG CTC GAG Met Ala Thr Ile Arg Pro Asp Asp Lys Ala Ile Asp Ala Ala Ala Arg His Tyr Gly Ile Thr Leu Asp Lys Thr Ala Arg Leu Glu 331 TGG CCG GCA CTG ATC GAC GGA GCA CTG GGC TCC TAC GAC GTC GTC GAC CAG TTG TAC GCC GAC GAG GCG ACC CCG CCG ACC ACG TCA CGC 421 Trp Pro Ala Leu Ile Asp Gly Ala Leu Gly Ser Tyr Asp Val Val Asp Gln Leu Tyr Ala Asp Glu Ala Thr Pro Pro Thr Thr Ser Arg GAG CAC GCG GTG CCA AGT GCG AGC GAA MT CCT TTG AGC GCT TGG TAT GTG ACC ACC AGC ATC CCG CCG ACG TCG GAC GGC GTC CTG ACC 511 Glu His Ala Val Pro Ser Ala Ser Glu Asn Pro Leu Ser Ala Trp Tyr Val Thr Thr Ser Ile Pro Pro Thr Ser Asp Gly Val Leu Thr GGC CGA CGC GTG GCG ATC AAG GAC MAC GTG ACC GTG GCC GGA GTT CCG ATG ATG AAC GGA TCT CGG ACG GTA GAG GGA TTm ACT CCG TCA 601 Gly Arg Arg Val Ala Ile Lys Asp Asn Val Thr Val Ala Gly Val Pro Met Met Asn Gly Ser Arg Thr Val Glu Gly Phe Thr Pro Ser CGC GAC GCG ACT GTG GTC ACT CGA CTA CTG GCG GCC GGT GCA ACC GTC GCG GGC AAA GCT GTG TGT GAG GAC CTG TGT TTC TCC GGT TCG 691 Arg Asp Ala Thr Val Val Thr Arg Leu Leu Ala Ala Gly Ala Thr Val Ala Gly Lys Ala Vaal Cys Glu Asp Leu Cys Phe Ser Gly Ser AGC TTC ACA CCG GCA AGC GGA CCG GTC CGC AAT CCA TGG GAC CGG CAG CGC GAA GCA GGT GGA TCA TCC GGC GGC AGT GCA GCA CTC GTC 781 Ser Phe Thr Pro Ala Ser Gly Pro Val Arg Asn Pro Trp Asp Arg Gln Arg Glu Ala Gly Gly Ser Ser Gly Gly Ser Ala Ala Leu Val GCA AAC GGT GAC GTC GAT TTT GCC ATC GGC GGG GAT CAA GGC GGA TCG ATC CGG ATC CCG GCG GCA TTC TGC GGC GTC GTC GGG CAC AAG 871 Ala Asn Gly Asp Val Asp Phe Ala Ile Gly Gly Asp Gln Gly Gly Ser Ile Arg Ile Pro Ala Ala Phe Cys Gly Val Val Gly His Lys CCG ACG TTC GGG CTC GTC CCG TAT ACC GGT GCA TTT CCC ATC GAG CGA ACA ATC GAC CAT CTC GGC CCG ATC ACA CGC ACG GTC CAC GAT 961 Pro Thr Phe Gly Leu Val Pro Tyr Thr Gly Ala Phe Pro Ile Glu Arg Thr Ile Asp His Leu Gly Pro Ile Thr Arg Thr Val His Asp GCA GCA CTG ATG CTC TCG GTC ATC GCC GGC CGC GAC GGT AAC GAC CCA CGC CAA GCC GAC AGT GTC GM GCA GGT GAC TAT CTG TCC ACC 1051 Ala Ala Leu Met Leu Ser Val Ile Ala Gly Arg Asp Gly Asn Asp Pro Arg Gln Ala Asp Ser Val Glu Ala Gly Asp Tyr Leu Ser Thr CTC GAC TCC GAT GTG GAC GGC CTG CGA ATC GGA ATC GTT CGA GAG GGA TTC GGG CAC GCG GTC TCA QAG CCC GAG GTC GAC GAC GCA GTC 1141 Leu Asp Ser Asp Val Asp Gly Leu Arg Ile Gly Ile Val Arg Glu Gly Phe Gly His Ala Val Ser Gln Pro Glu Val Asp Asp Ala Val SphI CGC GCA GCG GCA CAC AGT CTG ACC GAA ATC GGT TGC ACG GTA GAG GAA GTA AAC ATC CCG TGG CAT CTG CAT GCT TTC CAC ATC TG6 AAC 1231 Arg Ala Ala Ala His Ser Leu Thr Glu Ile Gly Cys Thr Val Glu Glu Val Asn Ile Pro Trp His Leu His Ala Phe His Ile Trp Asn GTG ATC GCC ACG GAC GGT GGT GCC TAC QAG ATG TTG GAC GGC AAC GGA TAC GGC ATG AAC GCC GAA GGT TTG TAC GAT CCG GAA CTG ATG 1321 Val Ile Ala Thr Asp Gly Gly Ala Tyr Gln Met Leu Asp Gly Asn Gly Tyr Gly Met Asn Ala Glu Gly Leu Tyr Asp Pro Glu Leu Met GCA CAC TTT GCT TCT CGA CGC ATT QAG CAC GCC GAC GCT CTG TCC GAA ACC GTC AAA CTG GTG GCC CTG ACC GGC CAC CAC GGC ATC ACC 1411 Ala His Phe Ala Ser Arg Arg Ile Gln His Ala Asp Ala Leu Ser Glu Thr Val Lys Leu Val Ala Leu Thr Gly His His Gly Ile Thr ACC CTC GGC GGC GCG AGC TAC GGC AAA GCC CGG AAC CTC GTA CCG CTT GCC CGC GCC GCC TAC GAC ACT GCC TTG AGA CAA TTC GAC GTC 1501 Thr Leu Gly Gly Ala Ser Tyr Gly Lys Ala Arg Asn Leu Val Pro Leu Ala Arg Ala Ala Tyr Asp Thr Ala Leu Arg Gln Phe Asp Val CTG GTG ATG CCA ACG CTG CCC TAC GTC GCA TCC GAA TTG CCG GCG AMG GAC GTA GAT CGT GCA ACC TTC ATC ACC AMG GCT CTC GGG ATG 1591 Leu Val Met Pro Thr Leu Pro Tyr Val Ala Ser Glu Leu Pro Ala Lys Asp Val Asp Arg Ala Thr Phe Ile Thr Lys Ala Leu Gly Met ATC GCC AAC ACG GCA CCA TTC GAC GTG ACC GGA CAT CCG TCC CTG TCC GTT CCG GCC GGC CTG GTG AAC GGG CTT CCG GTC GGA ATG ATG 1681 Ile Ala Asn Thr Ala Pro Phe Asp Val Thr Gly His Pro Ser Leu Ser Val Pro Ala Gly Leu Val Asn Gly Leu Pro Val Gly Met Met HindIII ATC ACC GGC AGA CAC TTC GAC GAT GCG ACA GTC CTT CGT GTC GGA CGC GCA TTC GAA AAG CTT CGC GGC GCG TTT CCG ACG CCG GCC GAA 1771 Ile Thr Gly Arg His Phe Asp Asp Ala Thr Val Leu Arg Val Gly Arg Ala Phe Glu Lys Leu Arg Gly Ala Phe Pro Thr Pro Ala Glu CGC GCC TCC MC TCT GCA CCA CAA CTC AGC CCC GCC tagtcctgacgcactgtcagacaacaaattccaccgattcacacatgatcagcccacataagaaaaggtga 1878 Arg Ala Ser Asn Ser Ala Pro Gln Leu Ser Pro Ala *** *** NHase > accagatg TCA GTA ACG ATC GAC CAC ACA ACG GAG AAC GCC GCA CCG GCC CAG GCG CCG GTC TCC GAC CGG GCG TGG GCA CTG TTC CGC GCA 1970 Met Ser Val Thr Ile Asp His Thr Thr Glu Asn Ala Ala Pro Ala Gln Ala Pro Val Ser Asp Arg Ala Trp Ala Leu Phe Arg Ala CTC GAC GGT AAG GGA TTG GTA CCC GAC GGT TAC GTC GAG GGA TGG AAG AAG ACC TTC GAG GAG GAC TTC AGT CCA AGG CGC GGA GCG GAA 2060 Leu Asp Gly Lys Gly Leu Val Pro Asp Gly Tyr Val Glu Gly Trp Lys Lys Thr Phe Glu Glu Asp Phe Ser Pro Arg Arg Cly Ala Glu TTG GTA GCG CGC GCA TGG ACC GAC CCC GAG TTC CGG CAG CTG CTT CTC ACC GAC GCT ACC GCC GCA GTT GCC CAG TAC GGA TAC CTG GGC 2150 Leu Val Ala Arg Ala Trp Thr Asp Pro Glu Phe Arg Gln Leu Leu Leu Thr Asp Gly Thr Ala Ala Val Ala GCn Tyr Gly Tyr Leu Gly CCC CAG GGC GM TAC ATC GTG GCA GTC GAA GAC ACC CCG ACA CTC AAG MC GTG ATC GTG TGC TCG CTG TGT TCA TGC ACC GCG TGG CCC 2240 Pro Gln Gly Glu Tyr Ile Val Ala Val Glu Asp Thr Pro Thr Leu Lys Asn Val Ile Val Cys Ser Leu Cys Ser Cys Thr Ala Trp Pro ATC CTC GGT CTG CCA CCC ACC TGG TAC MG AGC TTC GAA TAC CGT GCG CGC GTG GTC CGC GAA CCA CGG MG GTT CTC TCC GAG ATG GGA 2330 Ile Leu Gly Leu Pro Pro Thr Trp Tyr Lys Ser Phe Glu Tyr Arg Ala Arg Val Val Arg Glu Pro Arg Lys Val Leu Ser Glu Met Gly ACC GAG ATC GCG TCG GAC ATC GAG ATT CGC GTC TAC GAC ACC ACC G(C GAA ACT CGC TAC ATG GTC CTC CCG CAG CGT CCC GCC GGC ACC 2420 Thr Glu Ile Ala Ser Asp Ile Glu Ile Arg Val Tyr Asp Thr Thr Ala Glu Thr Arg Tyr Met Val Leu Pro Gln Arg Pro Ala Gly Thr Pst I GM GGC TGG AGC CAG GAA CAA CTG CAG 2447 Glu Gly Trp Ser Gln Glu Gln Leu Gln FIG. 2. Nucleotide and amino acid sequences of the BamHI-PstI region shown in Fig. 1 (lower panel). S.D., Potential RBS. The sequenced peptides from the purified Brevibacterium sp. strain R312 enantiomer-selective amidase and nitrile hydratase a subunit are underlined. S.D. S.D.

6 VOL. 172, 1990 ENANTIOMER-SELECTIVE AMIDASE FROM BREVIBACTERIUM SP TABLE 2. Purification of enantiomer-selective amidase Vol Am f Sp act Recovery Purification cationstep (ml) p(mg) (U/mg) (%) (atonl) Crude extractb 325 1, Ammonium sulfate precipitate Phenyl-Sepharose eluate First gel filtration eluate Second gel filtra tion eluate a One unit (U) is 1 Lmol of HPPAcid formed per min under the conditions described in Materials and Methods. b From 40 g of wet cells after streptomycin sulfate precipitation. from the above sequence. During a systematic search for homologies with published amino acid sequences, we found that the protein sequence deduced from this open reading frame was identical with the recently determined primary structure of the nitrile hydratase a subunit from Rhodococcus sp. strain N-774 (11). Furthermore, a close inspection demonstrated that the nucleotide sequence of the 1,238-bp fragment between SphI and PstI was strictly identical with the published nucleotide sequence of the nitrile hydratase fragment from Rhodococcus sp. strain N-774, with a single nucleotide difference, a C instead of a G at position 1664 (changing Val to Leu in the amidase sequence (Fig. 2), which may reflect either a sequence error or a minor allelic variation. The identity at the DNA level between the sequences of such long fragments from the two strains strongly argues in favor of Rhodococcus sp. strain N-774 (28) and Brevibacterium sp. strain R312 (1) being identical, or at least very closely related, microorganisms. An additional proof was provided by the cloning of the 3' overlapping 3.9-kbp HindIII-SphI genomic fragment from Brevibacterium sp. strain R312 (data not shown). The restriction map of this fragment was found to be in total agreement with the structure of the corresponding Rhodococcus sp. strain N-774 fragment (11). It was also verified that the structural genes for the two subunits of nitrile hydratase were identical for both microorganisms (data not shown). It can thus be concluded that the genes coding for the enantiomer-selective amidase and nitrile hydratase, two enzymes catalyzing successive reactions from nitriles to the corresponding acids, are closely linked on the bacterial genome. Sequence homology with known amidases. A systematic search of the NRBF and GENPRO protein data bases for good local homologies with the amidase sequence (15) gave known amidases as the best matches. Highly significant homologies were found with indoleacetamide hydrolase from Pseudamonas syringae (29) and Agrobacterium tumefaciens (24), also known as the protein encoded by the tms2 locus of plasmid Ti. These amidases are used by their phytopathogenic hosts to produce indoleacetic acid from indoleacetamide, indoleacetic acid being recognized as the principal auxin of higher plants. As shown in Fig. 3A by dot matrix analysis, the homology is clustered in the N-terminal half of the proteins (a40% match with the P. syringae enzyme), in particular between residues 150 and 220 of the strain R312 amidase. Two stretches of 10 and 12 consecutive identical residues between the P. syringae and the R312 enzymes are found between residues 168 and 204 of the R312 amidase. This region is also highly conserved in both the indoleacetamide hydrolase of A. tumefaciens and the acetamidase of Aspergillus nidulans (7) (Fig. 3B), strongly suggesting that it is part of the active site of these amidases. Finally, we noted that the homology between the enantiomer-selective amidase of Brevibacterium sp. strain R312 and the indoleacetamide hydrolase of P. syringae is of the same order as the homology between the indoleacetamide hydrolases of P. syringae and A. tumefaciens. Expression of Brevibacterium sp. strain R312 amidase activity in E. coli. The expression of amidase was first analyzed in the recombinant E. coli DH5ot strain containing either of the original clones pxl1650 and pxl1651. These plasmids carry the same 5.4-kbp chromosomal PstI fragment (Fig. 1) cloned in either orientation in the polylinker of plasmid puc19. Even under conditions known to derepress the lac promoter, we could not detect by Coomassie blue staining any expression of the cloned gene (data not shown). In another experiment, the 2.26-kbp BamHI-PstI fragment containing the complete amidase gene and the 58 bp upstream of the ATG codon was subcloned into aptp expression vector (8, 16a) to give pxl1724. Once again, no detectable synthesis of the presumed amidase protein could be evidenced by Coomassie blue staining, whatever the conditions (data not shown). The intrinsic expression capacity of these vectors could not be questioned since a protein of the expected size was specifically synthesized in an in vitro coupled transcription-translation assay (Amersham) when pxl1650, pxl1724, and, to a much weaker extent, pxl1651 were used as templates (data not shown). All these results strongly suggested that neither the amidase promoter, if present in the 3 kbp 5' to the gene, nor the translation initiation signals of the gene were efficient in E. coli. Two other constructions were then made in which the structural gene for amidase was linked to both a strong E. coli promoter, such as the Ptrp or X PR promoter, and an efficient RBS, such as that derived from the X cii gene (8). Expression of amidase from these vectors, pxl1751 (Ptrp) and pxl1752 (PR), was studied in E. coli B and E103S, respectively, two host strains known to give a high level of expression of heterologous proteins. Results shown in Fig. 4 demonstrate the specific high-level expression of an Mr- 55,000 protein, corresponding to at least 20% of total protein and comigrating with the main.protein species of the purified amidase preparation from Brevibacterium sp. strain R312 (lanes b and c). Subsequent analysis revealed that in fact only a minor proportion (<10%) of this protein was expressed as a soluble enzyme (Fig. 4, lanes g and i or n and p) when the recombinant strain was cultivated at 37 or 42 C, indicating that it was mainly produced as an aggregated, denatured protein as already observed for nitrile hydratase (11). However, when E. coli B(pXL1751) was grown at 30 C, similar amounts of the enzyme were synthesized but mainly (--80 to 90O) as a soluble protein (data not shown), suggesting that the temperature is a major parameter for the correct folding of this protein. That the cloned protein was indeed responsible for the observed enantiomer-selective amidase activity in Brevibacterium sp. strain R312 was demonstrated by activity measurements of the recombinant E. coli strains, compared with strictly isogenic strains expressing recombinant human interleukin-1, (14) as a control (Table 3). Only strains expressing the 55,000-Mr protein could hydrolyze HPPAmide. It was verified that this reaction was highly enantiomer selective, as described above. With or without sonication of the cells, specific amidase activities on HPPAmide were at least 20-fold higher for E. coli recombinants grown at 30 C compared with those grown at 37 C. In the best expression conditions (30 C in minimal medium

7 * F 6770 MAYAUX ET AL. R312 amidase A 1 r, IL % 521 N. A.% 1% 6. %. 1% '% I. lb I indoleacetamide \ v. \ hydrolase I,.. * * \. w I * s o In F X v. *\ 9 * N.1% \ \ A., fi. N ' \ \ 1% *... 1%. *.. *... \s, ;. \. *.. * * 8 t *..... s h \ "..*......\ lk, v ;:., ^. lk N. l'. 1.,, \ v INK. s. 455 L * In. Ba PASfaPVRSWDRQRE ES.MAALYANGDVDFAI GGD-QÆGEIBI AAFCGW.QHKPTFGLVPYTGAFP YATGAVRNPWNPDLIPGGSSGGVAAAVASRIMLGGIGTDTGASVRLPAALCGWGFRPTLGRYPGDRI IP J. BACTERIOL. b PASGPVRNPWDRQREAGGSSGGSAALVANGDVDFAIGGDQGGSIRIPAAFCGWGHKPTFGLVPYTGAFP PHWGTVGNPVAPGYCAGGSSGGSAAAVASGIVPLSVGTDTGGS IRIPAAFCGITGFRPTTGRWSTAGI IP C PASGPVRNPWDRQREAGGSSGGSAALVA-NGDVDFAIGGDQGGS IRIPAAFCGVVGHKPTFGLVPYTG-A NI IGRTVNPRNKNWSCGGSSGGEGAIVGIRGGV-IGVGTDIGGS IRVPAAFNFLYGLRPSHGRLPYAKMA FIG. 3. Amino acid homologies with known amidases. (A) Dot matrix analysis (25) of the comparison between the amino acid sequences of indoleacetamide hydrolase from P. syringae (29) (residues 1 to 455) and R312 enantiomer-selective amidase (residues 1 to 521) showing the level of homology between the N-terminal parts of the proteins. (B) Amino acid sequence comparisons between region 150 to 220 of R312 amidase (upper sequence) and the corresponding sequences (lower sequences) of indoleacetamide hydrolase (TMS2 protein) from A. tumefaciens (a), indoleacetamide hydrolase from P. syringae (b), and acetamidase from A. nidulans (c). Identical and similar residues are indicated by = and -, respectively. Residues strictly conserved in all four sequences are underlined in row a. without tryptophan), recombinant E. coli strains with up to threefold-higher specific activities than the original Brevibacterium sp. strain R312 were obtained. DISCUSSION This is the first report of the cloning and analysis of an amidase gene from a coryneform species. The R312 strain was isolated by Arnaud and co-workers (1) from a natural earth sample among strains able to utilize acetonitrile as the sole nitrogen source and was shown to hydrolyze a wide variety of nitrile compounds. It was then demonstrated that the metabolism of nitrites did not operate via a direct nitrilase reaction but that an amide intermediate was involved (12). The first enzymatic step, namely, the hydration of nitrites into amides, is performed by an enzyme named nitrile hydratase, as first proposed by Asano et al. (2) for Arthrobacter sp. strain J-1. That this enzyme is unique in Brevibacterium sp. strain R312 is suggested by two types of results. Only one biochemical entity was detected during the purification steps of the enzymatic activity, and a mutant strain, Brevibacterium sp. strain 19, was isolated which had completely lost its ability to hydrate nitrites, while retaining

8 VOL. 172, 1990 ENANTIOMER-SELECTIVE AMIDASE FROM BREVIBACTERIUM SP k 0 a b c d e kd0 f g h j k m n o p q 66, ,2 No 45, 0 f..: 3 l, 0 i5,0 - -._; i, q.:. 21,5-1 4, FIG. 4. Expression of R312 amidase in E. coli. Coomassie blue staining of SDS-polyacrylamide gels. An 8.5% gel and a 12.5% gel were used for lanes a to e and f to q, respectively. The positions of amidase (55 kda) and interleukin-1, (17 kda) are indicated by the arrowheads. Lanes: a and k, molecular size standards; b and d, purified amidase from R312; c and e, total cell extracts of E103S(pXL1752) after promoter induction at 42 C and incubation at 30 C, respectively; f, h, and j, soluble proteins, total cell extract, and insoluble proteins, respectively, of control E103S(pXL1029) expressing recombinant soluble interleukin-11; g and i, soluble and insoluble proteins, respectively, E103S(pXL1752) expressing amidase; m, o, and q, soluble proteins, total cell extract, and insoluble proteins, respectively, of control E. coli B(pXL906) expressing interleukin-13 under the control of the trp promoter; l, n, and p, same extracts as in lanes m, o, and q, respectively, of the isogenic strain E. coli B(pXL1751) expressing amidase. a wild-type amidase activity (6). Nitrile hydratase from Brevibacterium sp. strain R312, characterized by a wide substrate spectrum (6), has been purified to homogeneity and is composed of two types of subunits of apparent Mr near 27,000 (22). The enzyme biosynthesis is not influenced by TABLE 3. Enantiomer-selective amidase activity of different strains on HPPAmide and enantiomeric excess of the produced R(+) HPPAcid Strain' Induction Sonication Sp act ee (%Y' Brevibacterium sp. NMAd strain R312 E. coli(pxl1751)e -Trp (37"C) Trp (30'C) ND' -Trp (30'C) ND E. coli(pxl906) -Trp (37"C) - 0 E. coli(pxl1752) 42"C "C C E. coli(pxl1029) 42"C + 0 ae. coli B and E. coli K-12 E103S were used as hosts for expression from the trp (px1751 and pxl906) and A PR (pxl1752 and pxl1029) promoters, respectively. amda is expressed from pxl1751 and pxl1752, whereas interleukin-ilp is expressed from pxl906 and pxl1029 (14). b Specific activity in micromoles per minute per milligram of protein. ' Enantiomeric excess of the R(+) acid formed. d NMA, N-Methylacetamide. e Expression from trp promoter was determined either at 300C or at 37 C during the whole culture. f ND, Not determined. the nature of the carbon or nitrogen source but can be repressed by amides and amide analogs (27). On the other hand, it has been demonstrated that Brevibacterium sp. strain R312 harbors, as other microorganisms, several different types of amidase activities. For instance, an acylamide amidohydrolase (EC ) with a wide activity spectrum (wide-spectrum amidase) has been characterized (17). The purified enzyme is apparently a tetramer of four identical subunits (Mr 43,000), is able to hydrolyze a large number of amides into their corresponding organic acids, and also possesses an acyl transferase activity (26). In a mutant strain, called A4, in which this enzyme is inactivated, another amidase activity able to hydrolyze a large number of a-amino amides has also been identified. An interesting property of this second candidate enzyme, name a-aminoamidase, is its stereospecificity. Contrary to the wide-spectrum amidase, this enzyme is able to produce only L a-amino acids from a-amino amides (16). We described in the present work the preliminary characterization and the cloning of a stereospecific amidase which was identified by its ability to hydrolyze racemic 2-aryl or 2-aryloxy propionamides (R312 amidase in this discussion). It is clear from the structures of the purified proteins that this enzyme is different from the wide-spectrum amidase. In addition, we recently performed N-terminal sequencing analysis of the purified wide-spectrum amidase (provided by A. Arnaud, Montpellier, France) and confirmed that the two proteins are indeed different. However, we still cannot exclude that the R312 amidase described here may be identical to the previously identified a-aminoamidase, although a preliminary study of the latter seemed to indicate a very different structure (16). More work on the substrate

9 6772 MAYAUX ET AL. specificity of these enzymes is clearly needed to clarify this point. We found important homologies between the R312 amidase and both the A. nidulans acetamidase and the indoleacetamide hydrolases of two different plant pathogens. The significance of these homologies in terms of substrate specificity is not clear. For instance, we found a similar, or even higher, homology between R312 amidase and the P. syringae indoleacetamide hydrolase (32.1%) than between both indoleacetamide hydrolase enzymes from P. syringae and A. tumefaciens (26.4%) (29), although the latter ones are supposed to carry the same enzymatic functions. However, the very high conservation of nearly 40 residues around position 190 in all these amidases (between 57 and 75% identity) strongly suggests that this portion of the protein constitutes the active catalytic site responsible for the hydrolysis of the amide bond. Another important conclusion from this work is the colocalization of the amda gene coding for the stereospecific amidase with the gene for nitrile hydratase. The identification of the latter gene is based on its total identity at the nucleotide level with the corresponding gene of Rhodococcus sp. strain N-774. In fact, it was already known that enzymes from both microorganisms had similar structures (11, 22), and we recently found (data not shown) that the N-terminal peptide sequences of the two subunits of the purified Brevibacterium enzyme (kindly provided by A. Arnaud, Montpellier) were identical to those reported for the enzyme from Rhodococcus sp. (the sequenced N terminus of the a subunit is indicated in Fig. 2). We also take the identity at the nucleotide level of the common sequenced fragment from Brevibacterium sp. strain R312 and Rhodococcus sp. strain N-774 (one nucleotide difference in more than 1.55 kb) as a strong indication that both microorganisms should be at least very closely related, if not identical. It seems plausible from the above observations that nitrile hydratase can be expressed from a polycistronic mrna initiated upstream from amda since no sequence reminiscent of a transcription terminator can be found in the intercistronic region between the two genes; moreover, it has been shown, at least in E. coli (11), that nitrile hydratase can be expressed from a promoter inserted at the SphI site, located in amda. We interpret this gene arrangement as an indication that the bacterial cell needs to coregulate, at least under some conditions, the gene expression of both enzymes. The lack of significant expression of the R312 amidase in E. coli either from the PstI fragment (native promoter and RBS) or from the BamHI-PstI fragment (native RBS and E. coli promoter) is difficult to interpret in the context of previous results suggesting that the transcriptional and translational machineries could recognize similar signals in E. coli and coryneform bacteria (at least Corynebacterium glutamicum). Work is in progress to map the promoter and to check whether R312 amidase can be readily expressed from native signals in prototype coryneform bacteria such as C. glutamicum or B. lactofermentum. It is also interesting to observe that, when expressed from E. coli signals, the solubility of the overexpressed protein is very dependent on the growth temperature between 30 and 37 C, although similar expression levels are obtained. That amidase cannot properly refold and reach its active three-dimensional conformation in vivo at 37 C appears to be the most obvious explanation. Moreover, specific activities measured on whole cells are well correlated to the observed solubilities. These results suggest not only that the production of an active Brevibacterium amidase can be obtained in E. coli but J. BACTERIOL. also that whole recombinant E. coli cells can indeed be used in the bioconversion of amides. ACKNOWLEDGMENTS We thank P. Galzy and A. Arnaud (I.N.S.A., Montpellier, France) for the gift of purified R312 nitrile hydratase and widespectrum amidase and T. Ciora (Vitry) for the synthesis of oligonucleotides. P. Yeh and F. Blanche (Vitry) are gratefully acknowledged for expert advice and help about corynebacteria and enzyme quaternary structure, respectively. The technical help of C. Ortuno during the purification of the enzyme is also acknowledged. Sequence data were compiled and analyzed by using programs from CITI2 (Centre InterUniversitaire d'informatique a Orientation Biom6dicale, Paris, France). LITERATURE CITED 1. Arnaud, A., P. Galzy, and J. C. Jallageas Remarques sur l'activitd nitrilasique de quelques bactdries. C.R. Acad. Sci. 282: Asano, Y., Y. Tani, and H. Yamada A new enzyme "nitrile-hydratase" which degrades acetonitrile in combination with amidase. Agric. Biol. Chem. 44: Bernet, N., A. Thidry, M. Maestracci, A. Arnaud, G. M. Rios, and P. Galzy Continuous immobilized cell reactor for amide hydrolysis. J. Ind. Microbiol. 2: Bibb, M. J., P. R. Findlay, and M. W. Johnson The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene 30: Brown, J. M., and S. G. Davies Chemical asymmetric synthesis. Nature (London) 342: Bui, K., H. Fradet, A. Arnaud, and P. Galzy A nitrile hydratase with a wide substrate spectrum produced by Brevibacterium sp. J. Gen. Microbiol. 130: Corrick, C. M., A. P. Twomey, and M. J. 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10 VOL. 172, 1990 ENANTIOMER-SELECTIVE AMIDASE FROM BREVIBACTERIUM SP plasmids: a comparative analysis of expression potentials in Escherichia coli. DNA Cell Biol. 9: Maestracci, M., A. Thiery, K. Bui, A. Arnaud, and P. Galzy Activity and regulation of an amidase (acylamide amidohydrolase E.C ) with a wide substrate spectrum from a Brevibacterium sp. Arch. Microbiol. 138: Maniatis, T., J. Sambrook, and E. F. Fritsch Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 19. Matsui, K., K. Sano, and E. Ohtsubo Sequence analysis of the Brevibacterium lactofermentum trp operon. Mol. Gen. Genet. 209: Monod, J., and E. Wollman Inhibition de la croissance et de l'adaptation enzymatique chez les bactdries infectdes par le bactdriophage. Ann. Inst. Pasteur (Paris) 73: Nagasawa, T., H. Nanba, K. Ryuno, K. Takeuchi, and H. Yamada Nitrile hydratase of Pseudomonas chlororaphis B23-purification and characterization. Eur. J. Biochem. 162: Nagasawa, T., K. Ryuno, and H. Yamada Nitrile hydratase of Brevibacterium R312-purification and characterization. Biochem. Biophys. Res. Commun. 139: Nagasawa, T., and H. Yamada Microbial transformations of nitriles. Trends Biotechnol. 7: Schroder, G., S. Waffenschmidt, E. W. Weiler, and J. Schroder The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid. Eur. J. Biochem. 138: Staden, R Methods to define and locate patterns of motifs in sequences. CABIOS 4: Thiery, A., M. Maestracci, A. Arnaud, P. Galzy, and M. Nicolas Purification and properties of an acylamide amidohydrolase (E.C ) with a wide activity spectrum from Brevibacterium sp. R312. J. Basic Microbiol. 26: Tourneix, D., A. Thiery, M. Maestracci, A. Arnaud, and P. Galzy Regulation of nitrile hydratase synthesis in a Brevibacterium species. Antonie von Leeuwenhoek J. Microbiol. 52: Watanabe, I., Y. Satoh, and K. Enomoto Screening, isolation and taxonomical properties of microorganisms having acrylonitrile-hydrating activity. Agric. Biol. Chem. 51: Yamada, T., C. J. Palm, B. Brooks, and T. Kosuge Nucleotide sequences of the Pseudomonas savastanoi indoleacetic acid genes show homology with Agrobacterium tumefaciens T-DNA. Proc. Natl. Acad. Sci. USA 82: Yanisch-Perron, C., J. Vieira, and J. Messing Improved M13 phage vectors and host strains: nucleotide sequences of the M13mpl8 and puc19 vectors. Gene 33: