1 Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
2 Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
Correspondence
Joachim Klein
joachim.klein{at}lonza.com
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Lonza AG, Biotechnology Research and Development, CH-3930 Visp, Switzerland.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previously, Layh et al. (1992) isolated several bacterial strains with nitrilase activities from the environment. They were isolated from enrichment cultures using different arylacetonitriles such as 2-methyl- or 2-ethylbenzylcyanide as sole sources of nitrogen. One of these strains, Pseudomonas fluorescens EBC191, was able to use different arylacetonitriles (e.g. 2-phenylpropionitrile) as nitrogen sources and converted the nitriles to the corresponding
-substituted carboxylic acids. It was demonstrated that strain EBC191 synthesized a nitrilase, which converted O-acetoxymandelonitrile preferentially to R-acetoxymandelic acid (Layh et al., 1992
). The enzyme was subsequently partially purified, biochemically characterized, and the N-terminal and some internal amino acid sequences were determined (Moser, 1996
; Layh et al., 1998
). This enzyme seems to possess some potential for the enantioselective production of carboxylic acids from racemic nitriles. In the present work, the nitrilase gene was identified in a genomic library of P. fluorescens EBC191, expressed in Escherichia coli and the recombinant protein biochemically characterized.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA preparation, DNA manipulation and cell transformation.
Small-scale plasmid preparation was performed by the method of Kieser (1984). The isolation of genomic DNA and all DNA manipulations were carried out as described by Sambrook et al. (1989)
. All enzymes for DNA manipulation were purchased from Roche Diagnostics and used according to the manufacturer's suggestions. Cells of E. coli were transformed with plasmid DNA by the method of Chung et al. (1989)
.
Genomic library construction and screening for the nitrilase gene.
The amino acid sequence HKKQYKV of the N-terminal part of the nitrilase (Moser, 1996) was used to design oligonucleotide S1061 (5'-CAC/T-AAG/A-AAG/A-CAG/A-TAC/T-AAG/A-GT-3'). The oligonucleotide was radiolabelled at its 5' end by use of T4 polynucleotide kinase and [
-32P]ATP (Sambrook et al., 1989
). Genomic DNA from P. fluorescens EBC191 was digested with SalI, loaded onto an agarose gel and electrophoresed at 10 V cm1 for 1 h. Fragments of 35 kb were isolated and ligated to SalI-digested plasmid pJOE890. Plasmid pJOE890 is a positive selection vector based on palindromic DNA sequences (Altenbuchner et al., 1992
). The plasmid genomic library was screened by colony lift with QIABRANE nylon membranes (Qiagen). The immobilized DNA was hybridized to the
-32P-labelled oligonucleotide S1061. Plasmid DNA from minilysates of positive colonies was analysed by Southern hybridization (Sambrook et al., 1989
) to confirm the presence of the oligonucleotide sequence in the plasmid inserts.
Nucleotide sequence analysis.
DNA sequences were determined by the dideoxy chain-termination method using an automated DNA sequencer (ALF-Sequencer, Pharmacia Biotech). Database searches were performed online with the programs BLASTX, BLASTP and BLASTN provided by the BLAST e-mail server (Altschul et al., 1990; Gish & States, 1993
). CLUSTALW (Thomson et al., 1994
) was used to align the amino acid sequences. All parameters were set at their default values. The sequence alignments were edited and analysed by using the multiple sequence alignment editor and shading utility GeneDoc (version 1.1.004) (Nicholas & Nicholas, 1996
).
Amplification of the nitrilase gene by PCR.
The forward primer P1 (5'-GAA-ATT-CCA-TAT-GAC-GGT-GCA-TAA-AAA-ACA-G-3') and the reverse primer PfluHisB (5'-CGG-GAT-CCC-TTG-TCG-CCT-TGC-TCT-TCT-3') were used to amplify a 1052 bp fragment containing the nitrilase gene. To facilitate cloning of the PCR product, NdeI and BamHI restriction sites were added to the primers. The PCR product was cut with NdeI and BamHI and inserted into the NdeI/BamHI-cleaved plasmid vector pJOE2775 (Volff et al., 1996) to give pIK9. The native stop codon was replaced by (CAT)6TGA, which encodes a C-terminal His6 tag. All PCR reaction mixtures contained 10100 ng genomic DNA in a volume of 40 µl, 0·5 µM of each forward and reverse primer (MWG-Biotech), 10 mM Tris/HCl (pH 9·0), 50 mM KCl, 1·5 mM MgCl2, 0·2 mM dNTP (Pharmacia Biotech) and 2·5 U Taq DNA polymerase (Pharmacia Biotech). The PCR reactions were performed in a PTC-200 thermal cycler (MJ Research). The PCR protocol comprised an initial denaturation for 1 min at 94 °C, followed by 30 cycles of denaturation for 1 min at 92 °C, annealing of primers for 1 min at 50 °C and extension for 2 min at 72 °C, with extension for 5 min during the last cycle. The PCR products were separated by electrophoresis using 1 % (w/v) agarose gels at 10 V cm1 and DNA bands were stained with ethidium bromide.
Inverse PCR.
Inverse PCR was performed as described by Ochman et al. (1993). To isolate the 5' flanking sequence of the chromosomal DNA region of plasmid pDC5, chromosomal DNA of strain EBC191 was cut with PvuI and hybridized against a 946 bp fragment of plasmid pDC5. A 2 kb fragment was identified corresponding to the 5' flanking sequence of the chromosomal fragment of pDC5. Chromosomal DNA of strain EBC191 was cut with PvuI and ligated. The primers S2299 (5'-GGC-ACC-TCG-CCA-AAT-ACG-3') and S2302 (5'-ATC-AAG-GAC-AAT-ATC-CAG-G-3') were used for PCR with the ligated DNA as template. The 5' flanking fragment of the chromosomal DNA region was cloned into EcoRV-cut pUC18 and sequenced.
Purification of the nitrilase.
Cells of E. coli JM109(pIK9) were grown overnight in 2x YT medium with 100 µg ampicillin ml1. The cells were subsequently diluted (1 : 100) in 600 ml fresh medium. Rhamnose (0·2 %, w/v) was added to induce the nitrilase gene when the OD600 of the cell cultures reached about 0·25. The cells were harvested after 412 h by centrifugation, washed twice with 30 ml Tris/HCl buffer (20 mM, pH 7·5) and resuspended in 30 ml of the same buffer. The cells were disrupted using a French press (16 00020 000 p.s.i., 110138 MPa) and the homogenate was centrifuged at 15 000 g for 30 min. The supernatant (soluble protein fraction) was used for further experiments. Since the recombinant nitrilase carried a C-terminal affinity tag of six consecutive histidine residues, it was purified using the QIAexpress nickel-nitrilotriacetic acid (Ni-NTA) protein purification system (Qiagen). The method is based on the interaction of positively charged metal ions such as Ni2+ to histidine residues. Spin columns were packed with 1 ml Ni-NTA agarose and the protein purification was done with about 3045 mg soluble cell protein (about 1015 mg nitrilase) per ml Ni-NTA agarose. The columns were subsequently washed with Tris/HCl buffer (20 mM, pH 7·5) plus 100 mM NaCl and 1 mM DTT and with Tris/HCl buffer (20 mM, pH 7·5) plus 150 mM NaCl, 1 mM DTT and 50 mM imidazole. Finally, the nitrilase was eluted with Tris/HCl buffer (20 mM, pH 7·5) plus DTT (1 mM), NaCl (100 mM) and 200 mM imidazole.
Enzyme assays.
The nitrilase activity of resting cells was determined in reaction mixtures (1 ml each) containing 50 µmol Tris/HCl buffer (pH 7·5), 10 µmol nitrile and an appropriate amount of cells. The assays with the purified nitrilase contained in addition 1 µmol DTT and 1 µg bovine serum albumin (BSA). The assays with naphthalene-2-acetonitrile, naproxennitrile or ketoprofennitrile contained in addition 20 % (v/v) methanol in order to increase the solubility of the substrates. All nitrile stock solutions (200 mM each) were prepared in methanol. The reaction mixtures were incubated at 30 °C. After different time intervals, samples (200 µl each) were taken and the reactions were stopped by the addition of 1 M HCl (20 µl). The samples were centrifuged at 15 000 g for 10 min and the supernatants were analysed using HPLC. The phenylglycinonitrile decomposes slowly to benzaldehyde, HCN and ammonia. At the same time the cyanohydrine mandelonitrile arises from the spontaneous reaction of benzaldehyde and HCN. In order to suppress the mandelonitrile side reaction by the nitrilase, the sampling intervals were shortened as much as possible by using a high concentration of nitrilase in the phenylglicinonitrile assay. The (R-)-O-acetoxymandelonitrile and the conversion products decomposed to mandelonitrile and the corresponding conversion products even at neutral pH. The arising mandelonitrile served as a competing substrate for the nitrilase reaction. Furthermore, the products of this side reaction were indistinguishable from the decomposition products of (R-)-O-acetoxymandelic acid and amide. This problem was minimized by injecting the assay samples directly into the HPLC system without further treatment. Protein concentrations were determined using the Bio-Rad protein assay.
SDS-PAGE.
The proteins were separated on a 9 % (w/v) denaturing SDS-polyacrylamide gel using the discontinuous buffer system of Laemmli (1970) and stained with Coomassie blue R250.
Analytical methods.
The different aromatic nitriles and their corresponding amides and acids were analysed by HPLC. A S1121 solvent delivery system equipped with a S3205 UV-VIS detector from Sykam was used. For the achiral analysis, a reversed-phase column [250 mmx4 mm (internal diameter); GROM] filled with 5 µm diameter particles of Lichrospher RP18 (E. Merck) was used to identify individual compounds which were detected spectrophotometrically at 210 nm. Methanol (50 % v/v) and H3PO4 (0·1 %, v/v) in H2O was used as the mobile phase to analyse the conversion of the different arylacetonitriles. Separation of the enantiomers of mandelic acid, 2-phenylpropionic acid and the corresponding amides was achieved on a Chiral-HSA column (ChromTech). The mobile phases consisted of sodium phosphate buffer (100 mM, pH 7·0) containing 4·5 % (v/v) acetonitrile, or sodium phosphate buffer (10 mM, pH 6·0) plus 0·5 % (v/v) 2-propanol, respectively. The separation of the enantiomers of 2-phenylglycine and 2-phenylglycineamide was achieved on a Crownpak CR+ column (Daicel Chemical Industries) at 38 °C with 10·3 g HClO4 l1 (pH 1) plus 10 % (v/v) methanol as mobile phase. The enantiomers of naproxen were separated on a Chiral-AGP column (ChromTech) with 10 mM sodium phosphate buffer (pH 7·0) as mobile phase. Aliphatic nitriles such as propionitrile, butyronitrile, isobutyronitrile, 2-methylbutyronitrile and valeronitrile and their corresponding acids were analysed by gas chromatography. The reaction mixtures (1 ml each) were acidified with 0·1 ml 1 M HCl and the substrates and products extracted into dichloromethane (0·2 ml). Aliquots of the organic phase (3 µl each) were injected into a gas chromatograph from Packard Instrument (model 437A), equipped with a flame-ionization detector with a FS-Hydrotex -PM column (0·5 mx0·25 mm, Macherey-Nagel) and a Merck chromatointegrator (model 2000, E. Merck). The column temperature was set between 80 and 130 °C; the injector and detector temperature were set at 220 and 250 °C, respectively.
Chemicals.
All chemicals were obtained from Sigma-Aldrich Chemie or E. Merck. The biochemicals were supplied by Roche Diagnostics. The sources of -acetoxymandelonitrile, naproxennitrile and ketoprofennitrile have been previously described (Bauer et al., 1994
; Layh et al., 1992
, 1994
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Upon storage, the activity of the purified enzyme was reduced to approximately 25 % of the initial activity after 14 days at 8 °C in the elution buffer (pH 7·5) or Tris/HCl buffer (pH 7·5±1 mM DTT). Less than 10 % of the initial activity was recovered after storage of the purified enzyme in the same buffer systems at 20 °C. Therefore, the effects of different additives which are known for their protein-stabilizing properties were tested. The addition of glycerol (30 %, v/v), Tween (0·3 %, v/v), sucrose (1 mg ml1), NaCl (100 mM) or ammonium sulfate (1 M) decreased the rate of inactivation of the nitrilase during storage at 20 °C (Table 2). The addition of ammonium sulfate resulted in an almost complete stabilization of the nitrilase. In this way, the purified enzyme could be stored at 20 °C for several months without any significant loss of activity.
|
|
Substrate specificity
The conversion of benzonitrile, different substituted phenylacetonitriles and aliphatic nitriles was examined (Table 3). The enzyme showed high relative activities with (substituted) phenylacetonitriles, such as 2-phenylvaleronitrile (230 U mg1), phenylacetonitrile (68 U mg1), mandelonitrile (2-hydroxyphenylacetonitrile) (33 U mg1) and 2-phenylpropionitrile (4·1 U mg1) as substrates. The
-aminonitrile 2-phenylglycinonitrile was also an effective substrate. The corresponding amino acid phenylglycine was produced with high specific activities (26 U mg1). In contrast, (R,S)-O-acetylmandelonitrile and the optically pure (R)-O-acetylmandelonitrile were only converted to the corresponding acid with specific activities of
2 U mg1. The nitrilase also converted heterocyclic arylacetonitriles, such as 2-thiopheneacetonitrile and 3-thiopheneacetonitrile. The differences in the turnover rates of these two substrates suggested a pronounced influence of the position of the heteroatom on the enzyme activity (Table 3
). Indole-3-acetonitrile was hydrolysed to the plant growth hormone indole-3-acetic acid (7·2 U mg1). More hydrophobic substrates such as 1- and 2-naphthaleneacetonitrile were hydrolysed with lower but clearly detectable activities (0·45 and 0·55 U mg1, respectively). In contrast, structurally similar substrates with an additional
-methyl group, such as naproxennitrile [2-(6-methoxy-2-naphthyl)propionitrile] and ketoprofennitril [2-(3-benzoylphenyl)propionitrile], were very poor substrates. The enzyme also converted benzonitrile and short-chain aliphatic nitriles with low specific activities (Table 3
).
|
Amide production
The conversion of most nitriles by the nitrilase resulted not only in the formation of the corresponding carboxylic acids but also in the release of the corresponding amides (Table 3). The highest proportions of amides were obtained with racemic O-acetylmandelonitrile and the optically pure (R)-O-acetylmandelonitrile as substrates (43 % and 28 % of the substrates converted to the amides, respectively). The non-acetylated substrate mandelonitrile was converted to mandelic acid and mandelic acid amide with a product ratio of 80 : 20. The conversion of 2-phenylpropionitrile, phenylacetonitrile, 2-phenylglycinonitrile, thiophene- and naphthylacetonitriles resulted in the formation of considerably lower relative amounts of the corresponding amides (Table 3
). The chiral HPLC analysis of the reactions demonstrated that the formation of the amides was to a certain degree enantioselective. Surprisingly, it was found that the acids and amides that were formed from chiral nitriles demonstrated in most cases opposite enantiomeric excesses. Thus, mandelonitrile was converted by the nitrilase preferentially to R-mandelic acid and S-mandelic acid amide (Table 3
).
Genomic region of the nitrilase gene
Several additional putative ORFs were identified on the 3·8 kb SalI fragment surrounding nitA. Upstream of nitA, ORF3 was identified which encoded a protein of 415 amino acid residues (43 773 Da) that showed weak homologies to indole-3-acetamide hydrolases such as IaaH from Agrobacterium tumefaciens (Barker et al., 1983), which participates in the synthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile. Further upstream of ORF3, the 3' end of an ORF was detected (corresponding to 113 amino acids at the C-terminus of the encoded protein) which showed homologies to transcriptional activators of the LuxR type. In order to obtain the complete sequence of this putative regulator, the region upstream of nitA was isolated via inverted PCR. Chromosomal DNA was cut with PvuI, self-ligated and the ligation mixture used for inverted PCR using the primers S2299 und S2302. Thus, a 2 kb PCR fragment was amplified and cloned into pUC18. One complete and one incomplete ORF were identified via sequence analysis and homology searches. At the 5' end of the isolated PCR fragment, a gene encoding a putative aromatic permease was identified. The 41 amino acids encoded by this gene fragment (ORF1, Fig. 3
, Table 4
) shared 37 % (30 %) identical residues with PcaK (BenK) from Pseudomonas putida (Acinetobacter sp. ADP1), which are involved in the transport of aromatic compounds such as 4-hydroxybenzoate (Harwood et al., 1994
) and benzoate (Collier et al., 1997
). The amino acid sequence deduced from the DNA sequence of ORF2 indicated that the gene product consisted of 234 amino acids and had a molecular mass of 26 236 Da. The putative protein showed only a low degree of similarity to transcriptional activators of the LuxR type such as MoaR from Klebsiella pneumoniae (23 % identical residues, Azakami et al., 1993
) and CarR from Erwinia carotovora (18 % identical residues, McGowan et al., 1995
). A higher degree of sequence similarity was found in the C-terminal helixturnhelix motif (Pabo & Sauer, 1984
) which is responsible for the DNA binding of LuxR-like regulators.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the nitrilase from strain EBC191 converted various sterically hindered phenylacetonitriles and also some bicyclic and heterocyclic arylacetonitriles, some severe steric restriction was also evident which hindered the performance of the catalysis. While the activities observed with 1- and 2-naphthaleneacetonitrile were about 10 % of the activity with 2-phenylpropionitrile, naproxennitrile was only converted with less than 1 % of the activity found with the naphthaleneacetonitriles. The decrease in activity is probably not caused by the additional 6-methoxy group attached to the naphthalene ring system but by the combined effects of the extended ring system and the methyl group at the 2-position of the naproxennitrile molecule.
A potential drawback for the utilization of nitrilases in biotransformation reactions is the generally observed low stability of nitrilases in cell-free preparations. Also in this respect, the purified nitrilase from strain EBC191 clearly resembled other nitrilases. Thus, the arylacetonitrilase from Alc. faecalis strain JM3 lost about 60 % of its activity after 1 week storage at 4 °C in potassium phosphate buffer (pH 7·0) in the presence of 1 mM DTT (Nagasawa et al., 1990). The enzyme from P. fluorescens lost about 75 % of its initial activity after 2 weeks storage under similar conditions. Also the nitrilases from Rhodococcus rhodochrous NCIMB 11215, R. rhodochrous NCIMB 11216, Fusarium species and Arthrobacter sp. strain J1 were rapidly inactivated during storage (Bandyopadhyay et al., 1986
; Harper, 1976
, 1977
, 1985
; Goldlust & Bohak 1989
). Several investigators have added reducing agents such as DTT to the enzyme preparations in order to prevent oxidation of the thiol residues which are supposed to be involved in the hydrolysing reaction (Kobayashi et al., 1992
; Nagasawa et al., 1990
). Furthermore, the addition of glycerol or ethylene glycol was used to avoid denaturation of highly purified enzymes from R. rhodochrous J1 and K22, Arthrobacter sp. strain J1, Alc. faecalis JM3 and ATCC 8750 during long-term storage at 20 °C (Kobayashi et al., 1989
, 1990
; Nagasawa et al., 1990
; Bandyopadhyay et al., 1986
; Yamamoto et al., 1992
). Previously, a stabilizing effect of a combination of ammonium sulfate and glycerol was described for the nitrilase from R. rhodochrous J1. This effect was explained by the high salt concentrations which were supposed to prevent the dissociation of the nitrilase subunits (Nagasawa et al., 2000
). The presence of glycerol also significantly increased the stability of the purified enzyme from strain EBC191. An even more pronounced stabilizing effect was obtained in the presence of ammonium sulfate (1 M) (Table 1
). Under these conditions, storage of the enzyme for several months at 20 °C and also repeated freezethawing cycles are possible almost without loss of enzyme activity. This treatment allows an almost unlimited storage of the purified nitrilase of P. fluorescens, which significantly facilitates the utilization of the enzyme.
The nitrilase from strain EBC191 produced significant amounts of amides from different substituted phenylacetonitriles. The formation of amides has also been previously reported for other nitrilases from different sources. A nitrilase from Fusarium oxysporum released the amides (45 % of the respective products) largely independent of the structure of the nitriles converted (Goldlust & Bohak, 1989). The nitrilase from Rhodococcus sp. strain ATCC 39484 produced the amide (2 %) only during conversion of phenylacetonitrile (Stevenson et al., 1992
). Furthermore, a ricinine-specific nitrilase from Pseudomonas sp. released the corresponding amide (710 % of the amount of the acid formed) and two nitrilases from the plant Arabidopsis thaliana hydrolysed 2-fluoroacetonitriles (AtNit1) or
-cyano(L-)-alanine (AtNit4) to significant amounts of the corresponding amides (Hook & Robinson, 1964
; Robinson & Hook, 1964
; Effenberger & Osswald, 2001
; Osswald et al., 2002
; Piotrowski et al., 2001
). The nitrilase from strain EBC191 demonstrated a clear correlation between the structure of the substrates and the relative proportion of the respective amides formed. Thus it was evident that large amounts of the amides were formed from O-acetoxymandelonitrile, mandelonitrile, and phenylglycinonitrile (43, 20 and 8 %, respectively). In contrast, significantly lower relative amounts of the amides were formed from the other substrates tested. The negative inductive (I) effect at the 2-position of the substrates tested increased from 2-phenylvaleronitrile (C3H7 group) to 2-phenylbutyronitrile (C2H5 group), 2-phenylpropionitrile (CH3 group), phenylacetonitrile (H atom), 2-phenylglycinonitrile (NH2 group), mandelonitrile (OH group) and O-acetylmandelonitrile (O-acetyl group). This suggested that the synthesis of the amide intermediates increased with an increasing polarity (increasing I effect) of the substituents at the 2-position, although it can not be excluded that other properties of the substituents might influence the degree of amide formation. A similar effect was recently described for the enzymic hydrolysis of 2-fluoroacetonitriles using the nitrilase AtNit1 from Arabidopsis thaliana. This enzyme converted simple aliphatic nitriles mainly to the corresponding acids. In contrast, 2-fluoroarylacetonitriles were preferentially converted to the amides and only minor amounts of the corresponding carboxylic acids were formed. It was suggested that the increased formation of the amides was due to the presence of highly electron-withdrawing substituents such as fluorine or nitro-groups (Effenberger & Osswald, 2001
; Osswald et al., 2002
). The results of the present study substantiate this observation and demonstrate that it may be possible to utilize classical nitrilases with certain substrates as nitrile hydratases forming stoichiometric amounts of the corresponding amides.
The nitrilase gene was physically connected in the genome of P. fluorescens to the genes encoding several enzymes of the mandelate pathway. Previously the genes surrounding a nitrilase gene have only been studied in detail for R. rhodochrous J1 and Bacillus sp. OxB-1. It was found that the R. rhodochrous J1 gene for a transcriptional regulator (nitR) similar to araC from E. coli was located directly downstream of nitA (Komeda et al., 1996). In contrast, the Bacillus sp. OxB-1 nitrilase gene was structurally connected to a gene encoding a phenylacetaldoxime dehydratase (Kato et al., 2000
). Thus on a molecular level the nitrilase organization was completely different in P. fluorescens compared to the Gram-positive strains previously studied: the nitrilase gene was physically connected to the mandelate pathway genes, the nitrilase converted mandelonitrile and the strain grew with mandelic acid. The ability of the P. fluorescens strain to grow with mandelonitrile (which could not be tested directly as growth substrate because of its rapid decomposition at neutral pH to benzaldehyde and cyanide) was supported by the following observations. Mandelonitrile is formed in nature from different cyanogenic glycosides which are synthesized by different plants in order to prevent feeding of insects. Thus it seems reasonable that the natural function of the nitrilase in P. fluorescens might be the utilization of these hydroxynitriles produced by plants in large quantities. A similar combination of the mandelate pathway with a functionally interrelated enzyme has recently been described for P. putida ATCC 12633. In this strain the genes of the mandelate pathway are connected to a mandelic acid amide hydrolase (McLeish et al., 2003
). It appears that in various pseudomonads an expanded mandelate pathway has a function in the metabolism of nitrogen containing derivatives of mandelic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipmann, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403410.[CrossRef][Medline]
Azakami, H., Sugino, H., Yokoro, N., Iwata, N. & Murooka, Y. (1993). moaR, a gene that encodes a positive regulator of the monoamine regulon in Klebsiella aerogenes. J Bacteriol 175, 62876292.[Abstract]
Bandyopadhyay, A. K., Nagasawa, T., Asano, Y., Fujishiro, K., Yoshiki, T. & Yamada, H. (1986). Purification and characterization of benzonitrilase from Arthrobacter sp. strain J-1. Appl Environ Microbiol 51, 302306.
Barker, R. F., Idler, K. B., Thompson, D. V. & Kemp, J. D. (1983). Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2, 335350.[CrossRef]
Bauer, R., Hirrlinger, B., Layh, N., Stolz, A. & Knackmuss, H.-J. (1994). Enantioselective hydrolysis of racemic 2-phenylpropionitrile and other (R,S)-2-arylacetonitriles by a new bacterial isolate, Agrobacterium tumefaciens strain d3. Appl Microbiol Biotechnol 42, 17.[CrossRef]
Bunch, A. W. (1998). Nitriles. In Biotechnology, vol. 8a, Biotransformations I, pp. 277324. Edited by H. J. Rehm & G. Reed. Weinheim: VCH Wiley.
Chung, C. T., Niemela, S. L. & Miller, R. H. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A 86, 21722175.
Collier, L. S., Nichols, N. N. & Neidle, E. L. (1997). BenK encodes a hydrophobic permease-like protein involved in benzoate degradation by Acinetobacter sp. strain ADP1. J Bacteriol 179, 59435946.
Dorn, E., Hellwig, M., Reineke, W. & Knackmuss, H.-J. (1974). Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad. Arch Microbiol 99, 6170.[CrossRef][Medline]
Effenberger, F. & Osswald, S. (2001). Enantioselective hydrolysis of (RS)-2-fluoroarylacetonitriles using nitrilase from Arabidopsis thaliana. Tetrahedron: Asymmetry 12, 279285.[CrossRef]
Gish, W. & States, D. J. (1993). Identification of protein coding regions by database similarity search. Nat Genet 3, 266272.[CrossRef][Medline]
Goldlust, A. & Bohak, Z. (1989). Induction, purification, and characterization of the nitrilase of Fusarium oxysporum f. sp. melonis. Biotechnol Appl Biochem 11, 581601.
Harper, D. B. (1976). Purification and properties of an unusual nitrilase from Nocardia NCIMB11216. Biochem Soc Trans 4, 502504.[Medline]
Harper, D. B. (1977). Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani. Biochem J 167, 685692.[Medline]
Harper, D. B. (1985). Characterization of a nitrilase from Nocardia sp. (Rhodochrous group) N.C.I.B. 11215, using p-hydroxybenzonitrile as sole carbon source. Int J Biochem 17, 677683.[CrossRef][Medline]
Harwood, C. S., Nichols, N. N., Kim, M. K., Ditty, J. L. & Parales, R. E. (1994). Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate. J Bacteriol 176, 64796488.[Abstract]
Hook, R. H. & Robinson, W. G. (1964). Ricinine nitrilase: II. Purification and properties. J Biol Chem 239, 42634267.
Kato, Y., Nakamura, K., Sakiyama, H., Mayhew, S. G. & Asano, Y. (2000). Novel heme-containing lyase, phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1: purification, characterization, and molecular cloning of the gene. Biochemistry 39, 800809.[CrossRef][Medline]
Kieser, T. (1984). Factors affecting the isolation of ccc DNA from Streptomyces lividans and Escherichia coli. Plasmid 12, 1936.[CrossRef][Medline]
Kiziak, C. (1998). Heterologe Produktion der Nitrilase aus Pseudomonas fluorescens EBC191 und chimärer Enzymvarianten in E. coli. Diplomarbeit, Universität Stuttgart.
Kobayashi, M., Nagasawa, T. & Yamada, H. (1989). Nitrilase from Rhodococcus rhodochrous J1. Purification and characterization. Eur J Biochem 182, 349356.[CrossRef][Medline]
Kobayashi, M., Yanaka, N., Nagasawa, T. & Yamada, H. (1990). Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles. J Bacteriol 172, 48074815.[Medline]
Kobayashi, M., Komeda, H., Yanaka, N., Nagasawa, T. & Yamada, H. (1992). Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue. J Biol Chem 267, 2074620751.
Komeda, H., Hori, Y., Kobayashi, M. & Shimizu, S. (1996). Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase. Proc Natl Acad Sci U S A 93, 1057210577.
Laemmli, U. K. (1970). Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Layh, N., Stolz, A., Förster, S., Effenberger, F. & Knackmuss, H.-J. (1992). Enantioselective hydrolysis of O-acetylmandelonitrile to O-acetylmandelic acid by bacterial nitrilases. Arch Microbiol 158, 405411.
Layh, N., Stolz, A., Böhme, S., Effenberger, F. & Knackmuss, H.-J. (1994). Enantioselective hydrolysis of racemic naproxen nitrile and naproxen amide to S-naproxen by new bacterial isolates. J Biotechnol 33, 175182.[CrossRef][Medline]
Layh, N., Hirrlinger, B., Stolz, A. & Knackmuss, H.-J. (1997). Enrichment strategies for nitrile-hydrolysing bacteria. Appl Microbiol Biotechnol 47, 668674.[CrossRef]
Layh, N., Parratt, J. & Willets, A. (1998). Characterization and partial purification of an enantioselective arylacetonitrilase from Pseudomonas fluorescens DSM 7155. J Mol Catal B Enzym 417424.
Martinková, L. & Kren, V. (2002). Nitrile- and amide-converting microbial enzymes: stereo-, regio- and chemoselectivity. Biocatal Biotrans 20, 7993.
Mauger, J., Nagasawa, T. & Yamada, H. (1990). Occurrence of a novel nitrilase, arylacetonitrilase, in Alcaligenes faecalis JM3. Arch Microbiol 155, 16.[CrossRef]
McGowan, S., Sebaihia, M., Jones, S. & 7 other authors (1995). Carbapenem antibiotic production in Erwinia carotovora is regulated by CarR, a homologue of the LuxR transcriptional activator. Microbiology 141, 541550.[Medline]
McLeish, M. J., Kneen, M. M., Gopalakrishna, K. N., Koo, C. W., Babbitt, P. C., Gerlt, J. A. & Kenyon, G. L. (2003). Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633. J Bacteriol 185, 24512456.
Moser, R. (1996). Charakterisierung, Reinigung und N-terminale Sequenzierung der Nitrilase aus P. fluorescens EBC191 Diplomarbeit, Universität Stuttgart.
Nagasawa, T., Mauger, J. & Yamada, H. (1990). A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3. Purification and characterization. Eur J Biochem 194, 765772.[CrossRef][Medline]
Nagasawa, T., Wieser, M., Nakamura, T., Iwahara, H., Yoshida, T. & Geck, K. (2000). Nitrilase of Rhodococcus rhodochrous J1: conversion into the active form by subunit association. Eur J Biochem 267, 138144.[CrossRef][Medline]
Nicholas, K. B. & Nicholas, H. B., Jr (1996). GENEDOC: a tool for editing and annotating multiple sequence alignments. Distributed by the author per anonymous ftp.
Ochman, H., Ayala, F. J. & Hartl, D. L. (1993). Use of polymerase chain reaction to amplify segments outside boundaries of known sequences. Methods Enzymol 218, 309321.[Medline]
Osswald, S., Wajant, H. & Effenberger, F. (2002). Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana. Eur J Biochem 269, 680687.[CrossRef][Medline]
Pabo, C. O. & Sauer, R. T. (1984). Protein-DNA recognition. Annu Rev Biochem 53, 293321.[CrossRef][Medline]
Piotrowski, M., Schönfelder, S. & Weiler, E. W. (2001). The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode -cyano-L-alanine hydratase/nitrilase. J Biol Chem 276, 26162621.
Robinson, W. G. & Hook, R. H. (1964). Ricinine nitrilase: I. Reaction product and substrate specificity. J Biol Chem 239, 42574262.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schulze, B. (2002). Hydrolysis and formation of C-N bonds. In Enzyme Catalysis in Organic Synthesis, vol. II, pp. 699715. Edited by K. Drauz & H. Waldmann. Weinheim: VCH Wiley.
Stevenson, D. E., Feng, R., Dumas, F., Groleau, D., Mihoc, A. & Storer, A. C. (1992). Mechanistic and structural studies on Rhodococcus ATCC39484. Biotechnol Appl Biochem 15, 283302.[Medline]
Thomson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Tsou, A. Y., Ransom, S. C., Gerlt, J. A., Buechter, D. D., Babbitt, P. C. & Kenyon, G. L. (1990). Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli. Biochemistry 29, 98569862.[CrossRef][Medline]
Vieira, J. & Messing, J. (1982). The pUC plasmids and M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259268.[CrossRef][Medline]
Volff, J.-N., Eichenseer, C., Viell, P., Piendl, W. & Altenbuchner, J. (1996). Nucleotide sequence and role in DNA amplification of the direct repeats composing the amplifiable element AUD1 of Streptomyces lividans 66. Mol Microbiol 21, 10371047.[CrossRef][Medline]
Yamamoto, K., Ueno, Y., Otsubo, K., Kawakami, K. & Komatsu, K.-I. (1990). Production of S-(+)-ibuprofen from a nitrile compound by Acinetobacter sp. strain AK226. Appl Environ Microbiol 56, 31253129.[Medline]
Yamamoto, K., Fujimatsu, I. & Komatsu, K.-I. (1992). Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC8750 responsible for enantioselective hydrolysis of mandelonitrile. J Ferment Bioeng 73, 425430.[CrossRef]
Received 2 June 2005;
revised 8 August 2005;
accepted 9 August 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |