Identification and characterization of a novel nitrilase ... - Springer Link

Appl Microbiol Biotechnol (2009) 83:273–283 DOI 10.1007/s00253-009-1862-6

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Identification and characterization of a novel nitrilase from Pseudomonas fluorescens Pf-5 Jung-Soo Kim & Manish Kumar Tiwari & Hee-Jung Moon & Marimuthu Jeya & Thangadurai Ramu & Deok-Kun Oh & In-Won Kim & Jung-Kul Lee

Received: 6 October 2008 / Revised: 7 December 2008 / Accepted: 6 January 2009 / Published online: 20 January 2009 # Springer-Verlag 2009

Abstract Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0°C and 45°C, respectively, with a specific activity of 1.7 and 1.9 μmol min−1 mg protein−1 for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a Km =17.9 mM and kcat/ J.-S. Kim : M. K. Tiwari : M. Jeya : T. Ramu : I.-W. Kim : J.-K. Lee Department of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 143-701, South Korea H.-J. Moon : D.-K. Oh Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 143-701, South Korea J.-K. Lee (*) Institute of Biomedical Science and Technology, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 143-701, South Korea e-mail: [email protected]

Km =0.5 mM−1 s−1 for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids. Keywords Cloning . Dinitrile . Aliphatic nitrilase . Pseudomonas fluorescens . Substrate specificity

Introduction Nitriles are readily converted to the corresponding carboxylic acids by a variety of chemical processes, but these processes typically require strongly acidic or basic reaction conditions and high reaction temperatures, and usually produce unwanted byproducts. Processes in which enzymecatalyzed hydrolysis converts nitriles to the corresponding carboxylic acids are preferred to chemical methods because these processes are often run at ambient temperature, do not require strongly acidic or basic reaction conditions, and do not produce large amounts of byproducts. Especially advantageous over chemical hydrolysis, the enzymecatalyzed hydrolysis of a variety of dinitriles can be highly regioselective, where only one of the two nitrile groups is hydrolyzed to the corresponding carboxylic acid (Carmela and Arie 1989). The enzymatic hydrolysis of nitriles can either proceed by nitrile hydratases which convert nitriles to the amides, which in turn might be further hydrolyzed by amidases to

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the corresponding carboxylic acids or nitrilases which directly hydrolyze nitriles to the corresponding carboxylic acids without the formation of intermediate such as amide (Nagasawa and Yamada 1989). Since the biological system offers environmentally friendly reaction conditions and potentially also offers the advantages of regio-, stereo-, and/or enantioselective reactions (Bunch 1998; Robertson et al. 2004; Singh et al. 2006), nitrilases have attracted substantial interest as a valuable alternative to chemical hydrolysis in organic chemical process. The occurrence, characteristics, applicability, action mechanism of nitrilases, and the cloning of the nitrilase gene have been reported (Pace and Brenner 2001; Banerjee et al. 2002; Brenner 2002). Nitrilases occur in both prokaryotes and eukaryotes, and their applications include the manufacture of nicotinic acid, ibuprofen, and acrylic acid and the detoxification of cyanide wastes (Banerjee et al. 2002; O’Reilly and Turner 2003). Although some of nitrilases have broad substrate specificity, nitrilase have been roughly classified into three major categories, based on substrate specificity (Kobayashi and Shimizu 1994). Aromatic nitrilases act on aromatic or heterocyclic nitriles, aliphatic nitrilases act on aliphatic nitriles, and arylacetonitrilases act on arylacetonitriles (O’Reilly and Turner 2003). Although there are many reports of enzymes that catalyze the formation of carboxylic acids, i.e., aromatic nitrilase, aliphatic nitrilase, and arylacetonitrilase, respectively, there are a few reports of enzymes that catalyze the formation of cyanocarboxylic acids from dinitriles. However, they are not optimal for enzymatic production of cyanocarboxylic acids because of their poor stability and/or activity (O’Reilly and Turner 2003; Zhu et al. 2008). The dicarboxylic or cyanocarboxylic acids produced are useful as precursors for chemicals of high value in the agricultural, materials, and pharmaceutical industries (Winkler et al. 2007; Zanatta et al. 2007; Zhu et al. 2007). The ability to chemoselectively convert a nitrile functional group to the corresponding carboxylic acid is a powerful tool for the preparation of agrochemicals and pharmaceuticals. Since the elucidation of the Pseudomonas fluorescens Pf-5 genome sequence in 2005 opened the door for genome scale research with this important microbial strain, we attempted to identify the nitrilase in P. fluorescens Pf-5 (Paulsen et al. 2005). Here, we report the cloning, heterologous expression, and characterization of a new nitrilase (NitPf5) from P. fluorescens Pf-5. We provide experimental evidence that NitPf5 is an aliphatic nitrilase with a high preference for dinitriles. This enzyme is one of the most active nitrilases toward dinitriles ever reported. The characterization of the NitPf5 adds a new and interesting member to the family of nitrilase and gives a better understanding for the formation of carboxylic acid compounds in nature.

Appl Microbiol Biotechnol (2009) 83:273–283

Materials and methods Materials Reagents for PCR, Ex-Taq DNA polymerase, genomic DNA extraction kit, and pGEM-T easy vector were purchased from Promega (Madison, USA). T4 DNA ligase and restriction enzymes were obtained from New England Biolabs (MA, USA). pQE-80L expression vector, plasmid isolation kit, and Ni-NTA super flow column for purification were from Qiagen (Hilden, Germany). Oligonucleotide primers and electrophoresis reagents were obtained from Bioneer (Daejeon, South Korea) and Bio-Rad, respectively. All chemicals for assay were purchased from SigmaAldrich (St. Louis, MO, USA). Bacterial strains and culture condition P. fluorescens Pf5 was obtained from the Korea Research Institute of Bioscience and Biotechnology (Daejeon, South Korea). All cloning experiments and plasmid preparations were carried out in Escherichia coli DH-5α. The E. coli cells for protein expression were cultivated in Luria– Bertani medium containing 100 μg mL−1 ampicillin at 37°C on a rotary shaker at 250 rpm. Cloning and expression of nitrilase gene from P. fluorescens Pf5 The PCR amplification was conducted using PfuTurbo DNA polymerase (Stratagene, La Jolla, CA, USA) to minimize potential point mutations introduced by PCR reactions under standard conditions. The gene was amplified from the genomic DNA of P. fluorescens Pf5 by using the two oligonucleotide primers 5′-TTGGATCCATGCC CAAGTCCGTTG-3’ (BamHI restriction site is underlined) and 5′-TATAAGCTTGGTAAAGCGCACCCCGGGAC-3’ (HindIII restriction site is underlined). The sequence of the oligonucleotide primers was based on the DNA sequence of the putative nitrilase from P. fluorescens Pf5 (GenBank accession number YP260015). The following PCR program was used for the amplification of a 921 bp DNA fragment: 5 min 94°C followed by 30 cycles of 45 s 94°C, 45 s 60°C, 1 min 72°C, and a final elongation of 10 min 72°C. The amplified fragment was cloned into the pGEM-T Easy (Promega, Madison, WI, USA), and DNA sequencing was performed at Macrogen Co. (Seoul, South Korea). The BamHI–HindIII fragment from T-vector containing nitrilase was subcloned into the same site of pQE-80L, and the resulting plasmid was designated pQE80L-nitPf5. The pQE80L-nitPf5 is under the control of the T5 promoter and expresses NitPf5 as a fusion protein to the N terminus of His6-tag. The recombinant plasmid was then transformed


into E. coli BL21(DE3), and the expression of recombinant enzyme was performed using 0.5 mM of isopropyl-β-Dthiogalactopyranoside (IPTG). The induced cells were harvested by centrifugation at 4°C for 15 min at 10,000×g, rinsed with phosphate-buffered saline, and stored at −20°C. Purification of the recombinant NitPf5 To purify the recombinant His6-tagged NitPf5, cell pellets were resuspended in 50 mM potassium phosphate buffer (pH 7.5) supplemented with 25 μg mL−1 DNase I. The cell suspension was incubated on ice for 30 min in the presence of 1 mg mL−1 lysozyme. Cell disruption was carried out by sonication at 4°C for 5 min, and the lysate was centrifuged at 14,000×g for 20 min at 4°C to remove the cell debris. The resulting crude extract was retained for purification. The cell-free extract was applied onto a Ni-NTA Super flow column (3.4×13.5 cm, QIAGEN) previously equilibrated with a binding buffer (50 mM Na-phosphate buffer, 300 mM NaCl, pH 8.0). Unbounded proteins were washed out from the column with a washing buffer (50 mM Na-phosphate buffer, 300 mM NaCl, 20 mM imidazole, pH 8.0). Then, the NitPf5 protein was eluted from the column with an elution buffer (50 mM Na-phosphate buffer, 300 mM NaCl, 250 mM imidazole, pH 8.0). Enzyme fractions were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by staining with Coomassie blue R250. All purification steps were carried out at 4°C. Protein quantification and determination of molecular mass Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard protein (Bradford 1976). The molecular mass of the native enzyme was determined by gel filtration chromatography. One milliliter of purified enzyme was applied to a Sephacryl S-300 high resolution column (16/60; Amersham, UK) and eluted with 50 mM potassium phosphate buffer (pH 7.2) containing 0.15 M NaCl at a flow rate of 1 mL min−1. To determine the molecular mass of NitPf5, the column was calibrated with aldolase (MW 168,000), albumin (MW 67,000), ovalbumin (MW 43,000), chymotrypsinogen (MW 25,000), and ribonuclease A (MW 13,700) as reference proteins (Amersham, UK). The subunit molecular weight was examined by SDS-PAGE under denaturing conditions, using the prestained ladder marker (Bio-Rad) as reference proteins. Enzyme assay The standard assays were carried out by mixing the substrate nitrile (10 mM) and the NitPf5 nitrilase in potassium

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phosphate buffer (50 mM, pH 7.0). The reaction mixtures (1 mL final volume) were incubated at 30°C. Reactions were started by addition of enzyme to the reaction mixture. Aliquots were taken at 30 min, and the reactions were quenched by addition of 10% (v/v) 2 M HCl. The conversion was determined by the quantitation of the amount of ammonia formed in the reaction. The amount of ammonia was measured by the Bertholet assay (Weatherburn 1967). One unit (U) of the enzyme activity was defined as the number of micromoles of ammonia produced in 1 min by 1 mg of enzyme (μmol min−1 mg−1). Effect of pH and temperature on purified nitrilase Effect of pH and temperature on NitPf5 was determined by incubating the enzyme with succinonitrile in buffers of different pHs (4–9) or at different temperatures (20–55°C). The pH optimum of the nitrilase was determined by using the sodium acetate (pH 5–6), phosphate (6–8), and Tris– HCl (8–10) buffers. The purified nitrilase was incubated in 1 mL of the respective buffer with succinonitrile (10 mM) in a reaction vial at 30°C. For the determination of the temperature optimum, the NitPf5 was incubated in 1 mL of potassium phosphate buffer (50 mM, pH 7.0) with succinonitrile (10 mM) in a reaction vial at 20–55°C. The reactions were stopped after 30 min by addition of 10 μL of 2 M HCl. Effect of metal ions and other chemicals Before studying the effect of metal ions on NitPf5 activity, the purified enzyme was dialyzed against 20 mM phosphate buffer (pH 7.0) containing 10 mM EDTA for 24h at 4°C. Subsequently, the enzyme was dialyzed against 20 mM EDTA-free phosphate buffer (pH 7.0). Then, the enzyme was assessed under standard conditions in the presence of several metal ions (MgCl2, MnCl2, CoCl2, ZnCl2, CaCl2, FeSO4, CuSO4, and HgCl2) and chemicals with a final concentration of 0.1 and 1 mM. Succinonitrile (10 mM) was used as a substrate, and the NitPf5 activity was measured as described above. The measured activities were compared with the activity of the enzyme without added ions under the same conditions. Determination of kinetic parameters The specific activities of NitPf5 toward different nitriles with structural diversity were measured by the quantification of the amount of ammonia released during the hydrolysis. The standard reaction mixture (1 mL) was composed of 50 mM potassium phosphate buffer (pH 7.0), 10 mM of nitrile, 1 mM of dithiothreitol, 0.1 mL of methanol, and an appropriate amount of the enzyme.

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Methanol was added to enhance the solubility of the substrate, the enzyme being stable even in the presence of 10% (v/v) methanol. Initial velocities at pH 7.0 were determined for nitriles by calculating the initial rate of substrate hydrolysis in the range of 1–60 mM. Km and Vmax values were determined from Lineweaver–Burk plots using standard linear regression techniques. Homology modeling The three-dimensional homology model of NitPf5 was generated using Build Homology Models (MODELER) module in Discovery Studio 2.1 (Accelrys Software, San Diego, CA, USA). Crystal structure of hypothetical protein PH0642 (PDB accession code 1J31, resolution 1.6 Å; Sakai et al. 2004) from Pyrococcus horikoshii was used as template. A BLAST search of the Protein Databank demonstrated strongest similarity between NitPf5 and the protein PH0642 (PDB1j31) belonging to the nitrilase superfamily. The comparative modeling was used to generate the most probable structure of the query protein by the alignment with template sequences, simultaneously satisfying spatial restraints and local molecular geometry. Sequence identity between target and template was found to be 28% (62 of 219) according to BLAST parameters. Fitness of the models sequence in their current 3D environment was evaluated by Profiles-3D Score/Verify Protein (MODELER) as implemented in DS 2.1. Discrete optimized protein energy score in MODELER was also calculated to determine the quality of protein structures. Profile-3D score was 105.27 against 138.34 maximum expected score. The root mean square deviation (RMSD) between the models and template was calculated by superimposing on template crystal structure for the reliability of the models, and RMSD was 0.65 Å based on C-alpha atoms. The generated structure was improved by subsequent refinement of the loop conformations by assessing the compatibility of an amino acid sequence to known PDB structures using the Protein Health module in DS 2.1. The geometry of loop regions was corrected using Refine Loop/MODELER. Finally, the best quality model (Fig. 5) was chosen for further calculations, molecular modeling, and docking studies. The conserved catalytic residues Cys-165, Glu-44, and Lys-131 from P. horikoshii protein have similar orientations and locations in the NitPf5 model. The accuracy of the model is limited as it is based on homology modeling and not on a crystal structure. To confirm our molecular modeling and structural analyses, the crystal structure of NitPf5 should be determined. The resulting model was used for further docking study. Hydrogen atoms were added to protein model. The added hydrogen atoms were minimized to have stable energy


conformation and to also relax the conformation from close contacts. The active site of nitrilase was defined, and sphere of 5 Å was generated around the catalytic triad pocket (CEK). Succinonitrile and n-butyronitrile were docked into CEK binding pocket of nitrilase model using C-DOCKER, a molecular dynamics (MD) simulated-annealing-based algorithm module from DS 2.1 (Wu et al. 2003). Random substrate conformations are generated using high-temperature MD. Candidate poses are then created using random rigidbody rotations followed by simulated annealing. The structure of protein, substrate, and their complexes were subjected to energy minimization using CHARMm (Books et al. 1983) forcefield as implemented in DS 2.1. A fullpotential final minimization was then used to refine the substrate poses. Based on C-DOCKER, energy docked conformation of the substrate was retrieved for post-docking analysis. As a result, the substrate orientation, which gave the lowest interaction energy, was chosen for other round of docking.

Results Identification and characterization of the nitPf5 gene encoding a nitrilase The sequence analysis of the whole genome of P. fluorescens Pf-5 suggested the presence of a nitrilase. The orf was annotated as a putative nitrilase, suggesting that this orf might encode a nitrilase that converts nitrile to carboxylic acid directly. Thus, we considered nitPf5 as a candidate nitrilase in P. fluorescens Pf-5. The nitPf5 gene encodes a polypeptide of 307 amino acids, with a calculated molecular mass of 32,973 Da and an overall GC content of 67%, which is similar to that of the chromosomes of Pseudomonas species (58.4% to 66.6%; Paulsen et al. 2005). The deduced nitPf5 gene product contains the Nit-domain presented in nitrilase super family. In the Cterminal region of NitPf5, a highly conserved catalytic region, which plays a role in the essential catalytic mechanism of nitrilase (Chauhan et al. 2003), was found. A homology search revealed that the deduced nitPf5 gene product showed 46.0%, 43.6%, 25.9%, and 23.7% amino acid identity with Arabidopsis thaliana Nit 2 NP190016 (Bartling et al. 1994), A. thaliana Nit 1 NP851011 (Bartling et al. 1994), Bacillus sp. nitrilase (P82605; Kato et al. 2000), and Rhodococcus rhodochrous K22 nitrilase Q02068 (Kobayashi et al. 1992), respectively (Fig. 1). It also showed the significant amino acid identity with predicted nitrilases including Oryza sativa japonica BAD25100, Schizosaccharomyces pombe CAA19069, Leishmania major CAJ05570, Rhizobium leguminosarum bv. viciae CAK06154, and Clostridium kluyveri EDK34037.


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Fig. 1 Multiple sequence alignments of various nitrilases. 1 NIT1, 2 NIT2, 3 NIT3, 4 NIT4 nitrilase of A. thaliana, 5 NIT4 nitrilase of N. tabacum, 6 NitPf5 nitrilase of P. fluorescens, 7 nitrilase of R. rhodochrous K22, 8 nitrilase of Bacillus sp. The conserved active-site residues (catalytic triad) are highlighted with a box

Heterologous expression of nitPf5 gene and identification of a nitrilase In order to confirm its proposed function, nitPf5 was cloned in the T5 RNA polymerase-based plasmid pQE80L to give pQE80L-nitPf5 and heterologously expressed in E. coli BL21(DE3). Analyses carried out with the extracts of E. coli BL21(DE3) harboring pQE80L-nitPf5 revealed the presence of a high level of nitrilase compared with the control extracts of E. coli BL21(DE3) cells harboring plasmid pQE80L. To ascertain that carboxylic acid production in the presence of a nitrile (succinonitrile) was carried out specifically by the NitPf5 protein and not by another enzyme induced in the host cell as a consequence of the overexpression of the nitPf5 gene, the NitPf5 enzyme was purified. Total protein extracts from E. coli BL21(DE3) transformed with pQE80L-nitPf5 or with pQE80L as control were analyzed by SDS-PAGE. A 33 kDa protein, which was in agreement with the predicted molecular mass for the NitPf5 protein, could be identified in total and soluble extracts only from cells harboring pQE80L-nitPf5 and induced by IPTG. This protein was assigned as NitPf5, and it accounted for ∼90% of total protein based on the

specific activity in the cell-free extracts. The enzyme was purified by Ni-NTA affinity chromatography about 30-fold to near homogeneity (Fig. 2a). The purified nitrilase was colorless, and the UV–visible spectrum showed no evidence of any chromogenic cofactor. Oxidation of nitrile to carboxylic acid was monitored by the production of ammonia referred to the absorbance at 640 nm. In the presence of DTT, NitPf5 oxidized succinonitrile with specific activity of 1.7 U mg protein−1 (Km =17.9 mM). These findings strongly supported the assumption that the nitrilase activity observed in crude extracts of E. coli BL21 (DE3) harboring pQE80L-nitPf5 corresponded to that of the NitPf5 protein. Determination of molecular weight and quaternary structure In gel filtration chromatography on Sephacryl S-300 high resolution column (16/60; Amersham, UK), NitPf5 eluted as a symmetrical peak between aldolase and bovine albumin, corresponding to a Mr of approximately 138 kDa (Fig. 2b). The subunit molecular weight of the enzyme was 33±0.5 kDa (n=4), as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

278


a

Relative activity (%)

a

b

100 80 60 40 20 0

180

5

6

7

8

9

10

pH

150

b

NitPf5

120


Molecular weight (kDa)

Aldolase

90 Bovine albumin

60

Ovalbumin

30

Chymotrypsinogen Ribonuclease A

100

80

60

40

0 30

40

50

60

70

80

90

100

Running time (min) Fig. 2 a Purification of NitPf5 nitrilase by Ni-NTA chromatography. Coomassie-stained 12% SDS-PAGE gel. (Lane 1, marker; lane 2, crude extract; lane 3, flow through; lane 4, wash fraction; lane 5, first elute fraction; lane 6, second eluted fraction; lane 7, third eluted fraction; lane 8, fourth eluted fraction) b Native molecular weight of the enzyme was estimated by gel filtration to be 138 kDa. It can be assumed that the enzyme consists of four identical subunits (tetramer). The column was calibrated with standard molecular weight proteins such as aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa)

20 20

30

40

50

60

Temperature (oC) Fig. 3 Effect of pH (a) and temperature (b) on NitPf5 activity toward succinonitrile. The purified enzyme exhibited pH and temperature optima at 7.0°C and 45°C, respectively. The buffer conditions used for pH optimum were 100 mM sodium acetate pH 4∼6 (filled circles), 100 mM phosphate pH 6∼8 (empty circles), 100 mM Tris–HCl pH 8∼10 (inverted triangles). For the determination of temperature optimum, the NitPf5 was incubated in potassium phosphate buffer (50 mM, pH 7.0) at 20–55°C

Substrate specificity These results indicate that the enzyme migrates as a tetramer in gel filtration and thus may also be present and active as a homotetramer in solution. Optimum pH and temperature The optimum pH for hydrolysis of a nitrile (succinonitrile) by purified NitPf5 was 7.0 (Fig. 3a). The isoelectric point of the NitPf5 was pH 5.4 as determined by isoelectric focusing. This value agrees with the theoretical value (5.5) estimated from the amino acid sequence. The optimum temperature for the hydrolysis reaction was 45°C (Fig. 3b). The stability of NitPf5 was tested at pH 7.0 in standard 50 mM potassium phosphate buffer containing 1 mM DTT. Preparations were incubated at 16°C, 30°C, 45°C, 55°C, and 65°C and retained 50% of their initial activities after 80 h, 30 h, 300 min, 180 min, and 45 min, respectively.

The characterization of NitPf5 as a nitrilase then allowed for the investigation of its substrate specificity for various nitriles. The nitrilase activities for various nitrile substrates are shown in Table 1. The P. fluorescens NitPf5 is active with various nitrile substrates, and its high affinity for aliphatic dinitriles, such as succinonitrile and fumarodinitrile, is atypical of nitrilases. Aliphatic nitriles are followed in catalytic effectiveness by aromatic and arylacetonitriles. While 1-chlorobenzonitrile, mandelonitrile, and 2-aminobenzonitrile showed slight activity, the other aromatic and arylacetonitriles did not serve as substrates for NitPf5. Instead, when dinitriles such as succinonitrile, glutaronitrile, sebaconitrile, and fumarodinitrile (all at 10 mM) were examined as a substrate, NifPf5 showed a high preference for dinitriles. The narrow substrate specificity and high catalytic efficiency of NifPf5 for dinitriles are apparent from Table 1.


279

Table 1 Substrate specificity of P. fluorescens Pf-5 nitrilase Substrate

Specific activity (U/mg protein)

Aliphatic mononitriles Iso-valeronitrile Acrylonitrile Propionitrile n-Butyronitrile Aliphatic dinitriles Succinonitrile Glutaronitrile Sebaconitrile Fumarodinitrile Aromatic nitriles Benzonitrile 1-Chlorobenzonitrile 2-Aminobenzonitrile Arylacetonitriles Mandelonitrile Phenylacetonitrile 2-Thiopheneacetonitrile


0.02 0.10 0.07 0.04

1.2 5.9 4.1 2.4

1.7 1.0 0.8 1.9

100 59 48 112

Km (mM)

kcat (s−1)

kcat/Km (s−1 mM−1)

– 23.3 43.9 35.3

– 0.33 0.17 0.20

– 0.014 0.004 0.006

17.9 24 25.3 23

8.8 5.0 3.3 9.2

0.50 0.25 0.16 0.40

0.08 0.03 0.01

4.7 1.8 0.6

23.8 35.2 –

0.22 0.17 –

0.009 0.005 –