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A new nitrilase from Bradyrhizobium japonicum USDA 110
Available online at www.sciencedirect.com
Journal of Biotechnology 133 (2008) 327–333
A new nitrilase from Bradyrhizobium japonicum USDA 110 Gene cloning, biochemical characterization and substrate specificity Dunming Zhu a , Chandrani Mukherjee a , Yan Yang a , Beatriz E. Rios a , D. Travis Gallagher b , N. Natasha Smith b , Edward R. Biehl a , Ling Hua a,∗ b
a Department of Chemistry, Southern Methodist University, Dallas, TX 75275, USA Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA
Received 4 March 2007; received in revised form 31 August 2007; accepted 2 October 2007
Abstract A nitrilase gene blr3397 from Bradyrhizobium japonicum USDA110 was cloned and over-expressed in Escherichia coli, and the encoded protein was purified to give a nitrilase with a single band of about 34.5 kD on SDS-PAGE. The molecular weight of the holoenzyme was about 340 kD as determined by light scattering analysis, suggesting that nitrilase blr3397 self-aggregated to an active form with the native structure being a decamer. The Vmax and Km for phenylacetonitrile were 3.15 U/mg and 4.36 mM, respectively. The catalytic constant kcat and specificity constant kcat /Km were 111 min−1 and 2.6 × 104 min−1 M−1 . This nitrilase is most active toward the hydrolysis of hydrocinnamonitrile among the tested substrates (4.3 times that of phenylacetonitrile). The nitrilase blr3397 shows higher activity towards the hydrolysis of aliphatic nitriles than that for the aromatic counterparts, and can be characterized as an aliphatic nitrilase in terms of activity. This nitrilase also possesses distinct features from the nitrilase bll6402 of the same microbe. © 2007 Elsevier B.V. All rights reserved. Keywords: Nitrilase; Aliphatic nitrile; Hydrolysis; Bradyrhizobium japonicum
1. Introduction Nitriles, in general, are synthetically more accessible than the corresponding carboxylic acids. These compounds thus provide convenient precursors for the synthesis of a wide variety of carboxylic acids, which are important intermediates in the production of fine and commodity chemicals. Chemical hydrolysis of nitriles typically requires strongly basic or acidic conditions and high temperature, and usually produces unwanted byproducts and/or large amounts of inorganic wastes. Nitrilasecatalyzed hydrolysis of organocyanides (nitriles) to carboxylic acids offers a “greener” alternative, because this ecofriendly biotransformation allows clean and mild synthesis combined with high yield and selectivity (Sugai et al., 1997; Effenberger and Osswald, 2001a,b; Martinkova and Kren, 2002; Osswald et al., 2002; Chaplin et al., 2004; Hann et al., 2004; Wang, 2005; Bergeron et al., 2006; Bornscheuer and Kazlauskas, 2006; Singh
∗
Corresponding author. Tel.: +1 214 768 1609; fax: +1 214 768 4089. E-mail address: [email protected] (L. Hua).
0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.10.001
et al., 2006). However, many of the known nitrilases possess considerable disadvantages such as low stability or selectivity. This prevents the industrial application of nitrilases, and there is thus a constant demand for new nitrilases (DeSantis et al., 2002, 2003; Robertson et al., 2004; Kiziak et al., 2005; Kaplan et al., 2006; Xie et al., 2006). The rapidly growing genome sequence data obtained in the course of genome projects offers a tremendous opportunity for rapid discovery of new enzyme catalysts. For example, Stolz and co-workers have characterized a nitrilase from cyanobacterium Synechocystis sp. strain PCC 6803 by genome mining (Heinemann et al., 2003; Mukherjee et al., 2006). Mueller et al. (2006) have cloned a thermoactive nitrilase from hyperthermophilic archaeon Pyrococcus abyssi, which showed highest activity towards malononitrile. Recently, we have cloned and purified a mandelonitrile hydrolase (bll6402) from Bradyrhizobium japonicum USDA110 via rational genome mining and demonstrated that it was an efficient catalyst for the hydrolysis of mandelonitrile and its derivatives (Kamila et al., 2006; Zhu et al., 2007a). Another putative nitrilase gene (blr3397) is also present in the same microorganism (Kaneko et al., 2002). The sequence alignment with known nitrilases
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shows that it has 55 and 52% sequence identity to the nitrilase ZmNIT2 from Zea mays (Park et al., 2003; Mukherjee et al., 2006) and the nitrilase 4 from Arabidopsis thaliana (Bartel and Fink, 1994), respectively. As part of our effort to develop an effective nitrilase catalyst tool-box, this putative nitrilase gene was cloned and expressed in Escherichia coli, the recombinant protein was purified and characterized. The substrate specificity of this nitrilase was evaluated towards a variety of nitriles with diverse structures. 2. Materials and methods 2.1. General methods and materials The GC analysis was performed on a Hewlett Packard 5890 series II plus gas chromatograph equipped with autosampler, EPC, split/splitless injector, FID detector and 25 m × 0.25 mm CP-Chirasil-Dex CB capillary column. The B. japonicum USDA110 was obtained from Dr. Patrick Elia at USDA Soybean Genomics and Improvement Laboratory. E. coli EC100 was used for all cloning experiments. The strain was routinely cultured in Luria–Bertani (LB) medium containing 100 g of ampicillin/ml for selection of plasmid. Rosetta2(DE3) from Novagen was used for expression. The nitrile substrates and the carboxylic acid standards were purchased from Sigma–Aldrich (Milwaukee, USA). 2.2. Amplification of the nitrilase gene by PCR and subsequent cloning of the gene The gene was amplified from the genomic DNA of B. japonicum USDA110 by using the two oligonucleotide primers 5 -TCGCATATGATGGATAGCAACCGACCG-3 and 5 -TTTGGATCCTCAATTGCGCGCGCGAACG-3 , which incorporated NdeI and BamHI restriction sites, respectively. The amplification was performed in a final volume of 100 l, and the reaction mixtures contained 200 ng of genomic DNA, 50 pmol of each primer (Integrated DNA Technologies, Iowa, USA), a 200 M of dNTP, 1× PCR buffer, 1.25 U of Pfx DNA polymerase (Invitrogen Inc., CA) and 1 mM MgSO4 . The following PCR program was used for the amplification of a 966 bp DNA fragment: 2 min of denaturing at 94 ◦ C; 30 cycles of denaturing (45 s at 94 ◦ C), annealing (45 s at 55 ◦ C), and polymerization (1 min at 68 ◦ C) followed. The program was completed by an additional polymerization step (10 min at 68 ◦ C). The amplified DNA fragment was digested with NdeI and BamHI and the insert was cloned into pTXB1 digested with the same restriction enzymes. The resulting plasmid was designated pLH12.1.
tures were incubated at 18 ◦ C on an orbital shaker at 180 rpm for another 11 h. The cells were harvested. 2.4. Preparation of cell-free extracts and partial purification of the enzyme The obtained cell pellet was resuspended in the lysis buffer containing 10 mM potassium phosphate and 1 mM DTT (pH 7.2), and the cells were lysed by a EmulsiFlex®-C5 high pressure homogenizer (Avestin, Inc.). The cell-free extract was mixed with equal volume of PEI (0.25% polyethyleneimine MW 40 K–60 K, 6% NaCl, 100 mM Borax, pH 7.4) to remove lipids (Milburn et al., 1990). The PEI-treated supernatant was then precipitated with 30% ammonium sulfate. The precipitate was separated by centrifugation and dissolved in the potassium phosphate buffer (10 mM, pH 7.2, 1 mM of DTT). The resulting enzyme solution was desalted by diafiltration with the same potassium phosphate buffer. The lysate was lyophilized and used for activity assay. 2.5. Enzyme isolation Further purification of nitrilase was performed in two steps—ion exchange and gel filtration liquid chromatography. The soluble fraction of the cell-free extract was loaded on DEAE Sepharose Fast Flow column (3.4 cm × 27 cm, Amersham Biosciences) and eluted with 0 to 500 mM NaCl gradient (3 l). Fractions showing nitrilase activity were pooled and concentrated on 10K MWCO centrifugal concentrators (Amicon). The resulting sample was loaded on Sephacryl S-300 (Amersham Biosciences) column and eluted with 20 mM Tris/HCl, pH 7.4. Fractions showing nitrilase activity were concentrated on 10K MWCO centrifugal concentrators (Amicon) to 30 mg/ml and appeared by SDS-PAGE (Laemmli, 1970) to be more than 98% pure. 2.6. Determination of oligomer molecular weight The molecular weight of nitrilase blr3397 was measured by light scattering. The measurement was performed at 690 nm using a WYATT DAWN static light scattering instrument, which was attached to an Agilent 1100 series high-performance liquid chromatography station equipped with a SHODEX KW-803 gel filtration column. The protein sample solutions were filtered through an Amicon Ultrafree-MC 0.22 m epitube filter unit and run through the gel filtration column at a flow rate of 0.5 ml/min prior to light scattering measurements. The mass of the native protein was calculated with an ASTRA software from the 90◦ scattering data. Thymoglobulin (Mr 669,000) and ferritin (Mr 440,000) were used as standards.
2.3. Expression of the nitrilase in E. coli 2.7. Enzyme assay The plasmid pLH12.1 was transformed into Rosetta2(DE3) E. coli strain (Novagen). Overnight precultures were diluted into LB containing 100 g/ml of amplicillin and 34 g/ml of chloramphenicol, the cells were induced with 0.1 mM of IPTG when optical density at 600 nm reached 1.0. The bacterial cul-
The standard assays were carried out by mixing the substrate nitriles (10 mM, final concentration) and the nitrilase enzyme in potassium phosphate buffer (100 mM, pH 7.2). The reaction mixtures were incubated at 30 ◦ C. Aliquots (usually 100 l)
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were taken at different time intervals, and the reactions were quenched by addition of 10% (v/v) 1 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) as follows: to the 100 l aliquot, 100 l of each of the assay reagents (0.02 M NaOCl, 0.33 M PhONa, and 0.01% sodium nitroprusside aqueous solution) were added sequentially. The resulting mixture was heated at 95 ◦ C for 2 min, and then cooled rapidly in cold water bath. A 200 l of the resulting reaction mixture was used to measure the absorbance at 640 nm, and the amount of ammonia content was calculated by comparing with the standard curve. The activity was defined as the number of mol of ammonia produced in 1 min by 1 mg of enzyme (mol min−1 mg−1 ). To determine the kinetic parameters of the purified protein for phenylacetonitrile, the activity assays were performed with different substrate concentrations, and Vmax and Km were calculated by best fitting the experimental data to the v/[A] curve according to Michaelis equation. 2.8. Effects of temperature, pH and CN− anion For the determination of temperature effect, a mixture of nitrilase (0.5 mg) with phenylacetonitrile (50 mM) in 1.0 ml of potassium phosphate buffer (100 mM, pH 7.2) was incubated at 25, 30, 37, 45, 60 or 70 ◦ C, respectively. Aliquots (100 l) were taken after 1, 2, 4, and 16 h intervals, and acidified with 10 l of 1 M HCl. The formed phenylacetic acid was extracted with 200 l of tert-butyl methyl ether, dried over anhydrous sodium sulfate, and quantified by GC analysis after converting to methyl ester with diazomethane. The pH effect was studied using the following buffers: sodium citrate buffer (pH 4.91 and 5.44), potassium phosphate buffer (pH 6.07, 6.52, 7.10, 7.58, 7.98 or 8.49) or sodium bicarbonate buffer (pH 8.98). A mixture of nitrilase (0.5 mg) with phenylacetonitrile (50 mM) in 1.0 ml of the respective buffer (100 mM) was incubated at 30 ◦ C. Aliquots (100 l) were taken after 1, 2, 4 and 16 h, and acidified with 10 l of 1 M HCl. The formed phenylacetic acid was extracted into 200 l of tert-butyl methyl ether, dried over anhydrous sodium sulfate, and quantified by GC analysis after converting to methyl ester with diazomethane. To study the effect of CN− anion, a mixture of nitrilase (0.5 mg) with phenylacetonitrile (50 mM) in 1.0 ml of potassium phosphate buffer (100 mM, pH 7.2) containing 0, 0.1, 0.5, 1.0, 5.0 or 10.0 mM of NaCN was incubated at 30 ◦ C. Aliquots (100 l) were taken after 24 h, and acidified with 10 l of 1 M HCl. The formed phenylacetic acid was extracted with 200 l of tert-butyl methyl ether, dried over anhydrous sodium sulfate, and quantified by GC analysis after converting to methyl ester with diazomethane. 2.9. Thermal stability A solution of 0.5 mg of blr3397 enzyme in 950 l of potassium phosphate buffer (pH 7.2, 100 mM) was heated at 30, 40, 50, 60 or 70 ◦ C for 30 min and 1 h separately. After heat-treatment, substrate phenylacetonitrile (final concentration:
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25 mM) was added to the enzyme solution. The resulting mixture was incubated at 30 ◦ C for 24 h. The product was extracted with tert-butyl methyl ether and methylated with freshly prepared diazomethane, and then analyzed by GC. 2.10. Substrate specificity The specific activities of nitrilase blr3397 toward different nitriles with structural diversity were measured by the quantification of the amount of ammonia released during the hydrolysis. A reaction mixture containing 20 or 40 g of purified blr3397 enzyme, 10 mM substrate in potassium phosphate buffer (1 ml, 100 mM, pH 7.2) was incubated at 30 ◦ C. The conversion was determined by measuring the amount of ammonia produced in the reaction using the Bertholet assay as described above. 2.11. Characterization of hydrolysis products To confirm the carboxylic acid products, a reaction mixture containing purified nitrilase (0.5 mg) and nitrile substrate (25 mM) in 1 ml of potassium phosphate buffer (100 mM, pH 7.2) was incubated at 30 ◦ C overnight. The reaction was then quenched with 100 l of 1 M HCl, and the formed carboxylic acids were extracted into 1 ml of tert-butyl methyl ether and converted to methyl esters with diazomethane. The methyl esters were analyzed by GC and compared with the standard compounds. In some cases, the product acids were extracted directly into chloroform-d for NMR analysis and the data were compared with those of authentic samples. 3. Results and discussion In addition to gene bll6402 which has been found to encode a mandelonitrile hydrolase (Zhu et al., 2007a), the published genome sequence of B. japonicum strain USDA110 (NCBI accession number BA000040) contains another open reading frame (blr3397), which also encodes a putative nitrilase enzyme. The gene blr3397 was amplified from the genomic DNA of B. japonicum USDA110 strain, and over-expressed in E. coli. The encoded protein was purified from the cell-free extract of the recombinant E. coli strain carrying plasmid pLH12.1 as described in Section 2. The purified protein gave a single band on SDS-PAGE with a molecular mass of about 34.5 kD (Fig. 1). The light scattering analysis showed that the molecular weight of the enzyme particle in solution was about 340 kD. This was confirmed by size exclusion high-performance liquid chromatography performed on an Agilent 1100 series highperformance liquid chromatography system with a Superdex 200 10/300 GL column (Amersham Biosciences). The column was calibrated using thyroglobulin (Mr 669,000), ferritin (Mr 440,000) and catalase (Mr 232,000) as references (highmolecular-weight kit from Amersham Biosciences). The eluent was potassium phosphate buffer (50 mM, pH 7.0) with 0.15 M NaCl, and flow rate was 0.4 ml/min at room temperature. This suggested that the active form of nitrilase blr3397 is a decamer. A nitrilase from Rhodococcus rhodochrous J1 has also been found to self-associate to decamer (Nagasawa et al., 2000).
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Fig. 2. Temperature dependence of the nitrilase activity toward phenylacetonitrile. The conversion was measured at pH 7.2 with different reaction time intervals of 1 h (diamond); 2 h (square); 4 h (triangle); 16 h (cross).
Fig. 1. SDS-PAGE of nitrilase blr3397. Left lane: protein marker; right lane: purified enzyme.
Similar self-aggregation was observed for the other nitrilase (bll6402) from the same microorganism, which exists as an oligomer of 12 subunits (Zhu et al., 2007a). The present results are consistent with the previous observations that nitrilases are propitious to self-associate to form large aggregates of 6–26 subunits (Banerjee et al., 2002; O’Reilly and Turner, 2003). The kinetic parameters of the recombinant protein for the hydrolysis of phenylacetonitrile were determined with Vmax and Km being 3.15 U/mg and 4.36 mM, respectively. The catalytic constant kcat and specificity constant kcat /Km were 111 min−1 and 2.6 × 104 min−1 M−1 . The kinetic parameter Vmax of nitrilase blr3397 is lower than that of nitrilase bll6402 (6.2 U/mg) from the same microorganism for the hydrolysis of phenylacetonitrile, and Km is higher (0.1 mM for nitrilase bll6402). Thus nitrilase blr3397 showed lower kcat and kcat /Km than bll6402 (230.0 min−1 and 2.30 × 106 min−1 M−1 , respectively, for bll6402). Phenylacetonitrile was used as the substrate to examine the temperature, pH and CN− anion effects on the activity of nitrilase blr3397. The activities of nitrilase blr3397 toward phenylacetonitrile at different temperatures were studied by measuring the conversion of the substrate to phenylacetic acid at four time intervals, and the data are presented in Fig. 2. It can be seen that the reaction rate first increased as temperature became higher, reached the maximum at 45 ◦ C and then decreased sharply. Since the optimal reaction temperature of nitrilase blr3397 was about 45 ◦ C, a question was raised as how thermostable this enzyme would be. Therefore, the thermostability of nitrilase blr3397 was studied using phenylacetonitrile as the substrate. The enzyme solutions were heated at various temperatures for
Fig. 3. Thermal stability of nitrilase blr3397. Phenylacetonitrile was used as the substrate. The enzyme solutions were heated at various temperatures for 30 or 60 min and then allowed to react with the substrate at 30 ◦ C for 24 h.
30 or 60 min and then allowed to react with the substrate at 30 ◦ C for 24 h. The product was analyzed by GC. The results are presented in Fig. 3. From the results it can be seen that the conversion of phenylacetonitrile to phenylacetic acid decreased slightly after the enzyme was heat-treated at 50 ◦ C for 30 min, indicating that nitrilase blr3397 possesses good thermal stability. The enzyme completely lost its activity after heat-treatment at 70 ◦ C for 30 min. The activity of nitrilase blr3397 was measured in buffers with pH ranging from 5 to 9 using phenylacetonitrile as substrate (Fig. 4). The enzyme was found active in this pH range and the activity was optimal from pH 7.0–8.0. This suggests that nitrilase blr3397 possesses a relatively broad working pH range. The behavior of nitrilase blr3397 against cyanide anion was also investigated using phenylacetonitrile as substrate. The
Fig. 4. pH dependence of the nitrilase activity toward phenylacetonitrile. The conversion was measured with different reaction time intervals of 1 h (diamond); 2 h (square); 4 h (triangle); 16 h (cross).
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results showed that cyanide anion did not exert significant effect on the activity up to 10 mM of CN− anion. The specific activities of the recombinant nitrilase blr3397 toward a variety of nitriles were measured by the quantification of the amount of ammonia released during the hydrolysis. For the convenience of the comparison, the relative activity of phenylacetonitrile was defined as 100, and the results for these nitriles are presented in Table 1. It can be seen from Table 1 that the enzyme showed highest activity for the hydrolysis of hydrocinnamonitrile (entry 8). The activity decreased as the number of methylene group between the phenyl and cyano groups became larger or smaller (entries 9 and 7). For ␣-substitued phenylacetonitriles (entries 1–6), the activity was much lower than that of the parent counterpart (entry 7) regardless of the electronic properties of the substituents. This might be due to crowdness at the ␣-position, which prevents the substrate from entering the active site. Therefore, steric factors may play a dominant role in determining the activity of nitrilase blr3397. This nitrilase showed a clear preference for aliphatic nitriles (entries 13–18) over aromatic and vinyl nitriles (entries 10–12). While nitrilase blr3397 was very active toward methylthioacetonitrile, it had lower activity for the hydrolysis of 3-amino- and 3-hydroxypropionitriles (entries 20 and 21). Aliphatic dinitriles were also effectively hydrolyzed by this enzyme (entries 22 and 23). Table 1 also lists the relative activity of nitrilase bll6402 from the same strain, which shows highest activity towards mandelonitrile. Although
Table 1 The relative activity of nitrilase blr3397 and bll6402 from Bradyrhizobium japonicum strain USDA110 toward various nitrile substrates Entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Substrate Mandelonitrile 2-Trimethylsilyloxy-2phenylacetonitrile 2-Phenylglycinonitrile 2-(N,N-Dimethylamino)2-phenylacetonitrile 2-Phenylpropionitrile 2-Phenylbutyronitrile Phenylacetonitrile Hydrocinnamonitrile 4-Phenylbutyronitrile 3-Indolylacetonitrile Benzonitrile Crotononitrile n-Butyronitrile 4-Chlorobutyronitrile Valeronitrile Hexanenitrile Heptanenitrile Allyl cyanide Methylthioacetonitrile 3-Aminopropionitrile 3-Hydroxypropionitrile 1,4-Dicyanobutane 2-Methylglutaronitrile
blr3397a
331
these two enzymes are highly active towards phenylacetonitrile, they show distinct substrate preference (Zhu et al., 2007a). The activities of nitrilase blr3397 to catalyze the hydrolysis of nitriles to the corresponding carboxylic acids were further confirmed by characterization of the hydrolysis products. The nitriles were treated with the nitrilase at 30 ◦ C overnight. The products were analyzed by gas chromatography or 1 H NMR, and identified by comparing the data with those of the standard samples. No amide was detected for all the substrates. The results are summarized in Table 2. From Table 2 it can be seen that nitrilase blr3397 efficiently catalyzed the hydrolysis of aliphatic nitriles including those with phenyl group at the end. The hydrolysis of benzonitrile and crotonitrile, in which CN was connected with sp2 carbons, was less effective than their counterparts with CN being bonded to sp3 carbons. This enzyme also effectively hydrolyzed aliphatic dinitriles to di-acids. Selective hydrolysis of one of the cyano groups had not been achieved in the present case. This result is different from that of other nitrilase (bll6402) in the same microorganism, in which -cyanocarboxylic acids were obtained exclusively in the hydrolysis of aliphatic dinitriles (Zhu et al., 2007b). The nitrilase blr3397 hydrolyzed mandelonitrile to mandelic acid with moderate enantioselectivity. In conclusion, B. japonicum strain USDA110 harbors a nitrilase gene (blr3397), which possesses 33% sequence identity to the nitrilase bll6402 from the same microbe. These two nitrilases show distinct features. First, although both nitrilases are active for the hydrolysis of phenylacetonitrile, nitrilase blr3397 shows highest activity for hydrocinnamonitrile, while bll6402 is most active toward mandelonitrile (Table 1). Second, the nitrilase blr3397 has compatible activity toward the hydroly-
bll6402b
4 4
460 165
3 <1
3 1
<1 <1 100 431 86 2 1 4 15 59 57 60 107 11 72 7 1 47 63
10 8 100 13 4 3 1 3 3 6 6 16 22 2 1 1 1 5 3
a The specific activity of phenylacetonitrile was 2.32 U/mg and its relative activity was defined as 100. b The specific activity of phenylacetonitrile was 5.3 U/mg and its relative activity was defined as 100. The data are cited from reference (Zhu et al., 2007a).
Table 2 Nitrilase catalyzed hydrolysis of various nitriles Entry
Substrate
Product
Conversion (%)a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Mandelonitrile Phenylacetonitrile Hydrocinnamonitrile 4-Phenylbutyronitrile Benzonitrile Crotononitrile Butyronitrile Valeronitrile Hexanenitrile Heptanenitrile Allyl cyanide Methylthioacetonitrile 1,2-Dicyanoethane 1,4-Dicyanobutane 1,5-Dicyanopentane 1,6-Dicyanohexane 1,8-Dicyanooctane Fumaronitrile
Mandelic acid Phenylacetic acid Hydrocinnamic acid 4-Phenylbutyric acid Benzoic acid Crotonic acid Butyric acid Valeronic acid Hexanoic acid Heptanoic acid 3-Butenoic acid Methylthioacetic acid Succinic acid Adipic acid Pimelic acid Suberic acid Sebacic acid Fumaric acid
44b 100 100 98 10 15 100 62 63 67 66 100 100 100 100 100 100 35
a The conversion was determined by GC analysis for entries 1–10 and by 1 H NMR analysis for entries 11–18. For GC analysis, the acid was converted to methyl ester with freshly prepared diazomethane, and the conversion was calculated for the areas of unreacted nitrile and product methyl ester. For 1 H NMR analysis, the conversion was calculated from the integration of distinguishable protons. b (S)-Mandelic acid was obtained in 36% ee.
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sis of aliphatic nitriles with phenylacetonitrile, but the activity of the nitrilase bll6402 towards the hydrolysis of aliphatic nitriles is much lower than that for phenylacetonitrile (Table 1). The most significant difference between these two nitrilases is that -cyanocarboxylic acids are obtained exclusively in the hydrolysis of aliphatic dinitriles catalyzed by nitrilase bll6402 (Zhu et al., 2007b), while no regioselectivity is observed in blr3397-catalyzed hydrolysis of aliphatic dinitriles. As predicted from the functional analysis of genetic organization of nitrilase gene bll6402 within the chromosome of B. japonicum strain USDA110, nitrilase bll6402 might play an important role in the mandelonitrile metabolic pathway in B. japonicum and can be characterized as a mandelonitrile hydrolase (Zhu et al., 2007a). Similar analysis for the nitrilase gene blr3397 shows that it is flanked by a putative vanillate O-demethylase oxidoreductase (blr3399) (Priefert et al., 1997), a toluate 1,2-dioxygenase ␣subunit (blr3400) and -subunit (blr3401) (Neidle et al., 1992), a crotonobetaine/carnitine-CoA ligase (blr3402) (Jin et al., 2002) and some hypothetical proteins or regulator/transporter proteins. It is difficult to predict the natural function of nitrilase blr3397 and further studies are needed to address this question. However, nitrilase blr3397 clearly shows higher activity toward the hydrolysis of the nitriles with CN being bonded to sp3 carbons than that for the sp2 counterparts. Therefore, this enzyme can be classified as an aliphatic nitrilase in terms of its activity and will be a valuable biocatalyst for the regioselective hydrolysis of aliphatic nitrile groups in the presence of aromatic ones. References Banerjee, A., Sharma, R., Banerjee, U.C., 2002. The nitrile-degrading enzymes: current status and future prospects. Appl. Microbiol. Biotechnol. 60, 33–44. Bartel, B., Fink, G.R., 1994. Differential regulation of an auxin-producing nitrilase gene family in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 91, 6649–6653. Bergeron, S., Chaplin, D.A., Edwards, J.H., Ellis, B.S.W., Hill, C.L., Holt-Tiffin, K., Knight, J.R., Mahoney, T., Osborne, A.P., Ruecroft, G., 2006. Nitrilasecatalyzed desymmetrization of 3-hydroxyglutaronitrile: preparation of a statin side-chain intermediate. Org. Process Res. Dev. 10, 661–665. Bornscheuer, U.T., Kazlauskas, R.J., 2006. Hydrolases in Organic Synthesis: Regio- and Stereoselective Biotransformations. Wiley-VCH, Weinheim. Chaplin, J.A., Levin, M.D., Morgan, B., Farid, N., Li, J., Zhu, Z., McQuaid, J., Nicholson, L.W., Rand, C.A., Burk, M.J., 2004. Chemoenzymatic approaches to the dynamic kinetic asymmetric synthesis of aromatic amino acids. Tetrahedron: Asymmetry 15, 2793–2796. DeSantis, G., Wong, K., Farwell, B., Chatman, K., Zhu, Z., Tomlinson, G., Huang, H., Tan, X., Bibbs, L., Chen, P., Kretz, K., Burk, M.J., 2003. Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). J. Am. Chem. Soc. 125, 11476–11477. DeSantis, G., Zhu, Z., Greenberg, W.A., Wong, K., Chaplin, J., Hanson, S.R., Farwell, B., Nicholson, L.W., Rand, C.L., Weiner, D.P., Robertson, D.E., Burk, M.J., 2002. An enzyme library approach to biocatalysis: development of nitrilases for enantioselective production of carboxylic acid derivatives. J. Am. Chem. Soc. 124, 9024–9025. Effenberger, F., Osswald, S., 2001a. (E)-Selective hydrolysis of (E,Z)-␣,unsaturated nitriles by the recombinant nitrilase AtNIT1 from Arabidopsis thaliana. Tetrahedron: Asymmetry 12, 2581–2587. Effenberger, F., Osswald, S., 2001b. Enantioselective hydrolysis of (RS)-2fluoroarylacetonitriles using nitrilase from Arabidopsis thaliana. Tetrahedron: Asymmetry 12, 279–285. Hann, E.C., Sigmund, A.E., Fager, S.K., Cooling, F.B., Gavagan, J.E., Bramucci, M.G., Chauhan, S., Payne, M.S., DiCosimo, R., 2004. Regioselective
biocatalytic hydrolysis of (E,Z)-2-methyl-2-butenenitrile for production of (E)-2-methyl-2-butenoic acid. Tetrahedron 60, 577–581. Heinemann, U., Engels, D., Buerger, S., Kiziak, C., Mattes, R., Stolz, A., 2003. Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp. strain PCC6803 and heterologous expression and characterization of the encoded protein. Appl. Environ. Microbiol. 69, 4359–4366. Jin, Q., Yuan, Z., Xu, J., Wang, Y., Shen, Y., Lu, W., Wang, J., Liu, H., Yang, J., Yang, F., Zhang, X., Zhang, J., Yang, G., Wu, H., Qu, D., Dong, J., Sun, L., Xue, Y., Zhao, A., Gao, Y., Zhu, J., Kan, B., Ding, K., Chen, S., Cheng, H., Yao, Z., He, B., Chen, R., Ma, D., Qiang, B., Wen, Y., Hou, Y., Yu, J., 2002. Genome sequence of Shigella flexneri 2a provides insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157. Nucleic Acids Res. 30, 4432–4441. Kamila, S., Zhu, D., Biehl, E.R., Hua, L., 2006. Unexpected stereorecognition in nitrilase-catalyzed hydrolysis of b-hydroxy nitriles. Org. Lett. 8, 4429–4431. Kaneko, T., Nakamura, Y., Sato, S., Minamisawa, K., Uchiumi, T., Sasamoto, S., Watanabe, A., Idesawa, K., Iriguchi, M., Kawashima, K., Kohara, M., Matsumoto, M., Shimpo, S., Tsuruoka, H., Wada, T., Yamada, M., Tabata, S., 2002. Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Res. 9, 189–197. Kaplan, O., Vejvoda, V., Plihal, O., Pompach, P., Kavan, D., Bojarova, P., Bezouska, K., Mackova, M., Cantarella, M., Jirku, V., Kren, V., Martinkova, L., 2006. Purification and characterization of a nitrilase from Aspergillus niger K10. Appl. Microbiol. Biotechnol. 73, 567–575. Kiziak, C., Conradt, D., Stolz, A., Mattes, R., Klein, J., 2005. Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme. Microbiology (Reading, England) 151, 3639–3648. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (5259), 680–685. Martinkova, L., Kren, V., 2002. Nitrile- and amide-converting microbial enzymes: stereo-, regio- and chemoselectivity. Biocatal. Biotransfor. 20, 73–93. Milburn, P., Bonnerjea, J., Hoare, M., Dunnill, P., 1990. Selective flocculation of nucleic acids, lipids, and colloidal particles from a yeast cell homogenate by polyethyleneimine, and its scale-up. Enzyme Microb. Technol. 12, 527–532. Mueller, P., Egorova, K., Vorgias, C.E., Boutou, E., Trauthwein, H., Verseck, S., Antranikian, G., 2006. Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi. Protein Expression Purif. 47, 672–681. Mukherjee, C., Zhu, D., Biehl, E.R., Hua, L., 2006a. Exploring the synthetic applicability of a cyanobacterium nitrilase as catalyst for nitrile hydrolysis. Eur. J. Org. Chem., 5238–5242. Mukherjee, C., Zhu, D., Biehl, E.R., Parmar, R.R., Hua, L., 2006b. Enzymatic nitrile hydrolysis catalyzed by nitrilase ZmNIT2 from maize. An unprecedented -hydroxy functionality enhanced amide formation. Tetrahedron 62, 6150–6154. Nagasawa, T., Wieser, M., Nakamura, T., Iwahara, H., Yoshida, T., Gekko, K., 2000. Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association. Eur. J. Biochem. 267, 138–144. Neidle, E., Hartnett, C., Ornston, L.N., Bairoch, A., Rekik, M., Harayama, S., 1992. cis-Diol dehydrogenases encoded by the TOL pWW0 plasmid xylL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short-chain alcohol dehydrogenase superfamily. Eur. J. Biochem. 204, 113–120, FIELD Full Journal Title:European journal of biochemistry/FEBS. O’Reilly, C., Turner, P.D., 2003. The nitrilase family of CN hydrolyzing enzymes—a comparative study. J. Appl. Microbiol. 95, 1161–1174. Osswald, S., Wajant, H., Effenberger, F., 2002. Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana. Eur. J. Biochem. 269, 680–687. Park, W.J., Kriechbaumer, V., Mueller, A., Piotrowski, M., Meeley, R.B., Gierl, A., Glawischnig, E., 2003. The nitrilase ZmNIT2 converts indole3-acetonitrile to indole-3-acetic acid. Plant Physiol. 133, 794–802. Priefert, H., Rabenhorst, J., Steinbuchel, A., 1997. Molecular characterization of genes of Pseudomonas sp. strain HR199 involved in bioconversion of vanillin to protocatechuate. J. Bacteriol. 179, 2595–2607.
D. Zhu et al. / Journal of Biotechnology 133 (2008) 327–333 Robertson, D.E., Chaplin, J.A., DeSantis, G., Podar, M., Madden, M., Chi, E., Richardson, T., Milan, A., Miller, M., Weiner, D.P., Wong, K., McQuaid, J., Farwell, B., Preston, L.A., Tan, X., Snead, M.A., Keller, M., Mathur, E., Kretz, P.L., Burk, M.J., Short, J.M., 2004. Exploring nitrilase sequence space for enantioselective catalysis. Appl. Environ. Microbiol. 70, 2429–2436. Singh, R., Sharma, R., Tewari, N., Geetanjali Rawat, D.S., 2006. Nitrilase and its application as a ‘green’ catalyst. Chem. Biodiversity 3, 1279–1287. Sugai, T., Yamazaki, T., Yokoyama, M., Ohta, H., 1997. Biocatalysis in organic synthesis: the use of nitrile- and amide-hydrolyzing microorganisms. Biosci. Biotechnol. Biochem. 61, 1419–1427. Wang, M.-X., 2005. Enantioselective biotransformations of nitriles in organic synthesis. Top. Catal. 35, 117–130.
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Weatherburn, M.W., 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39, 971–974. Xie, Z., Feng, J., Garcia, E., Bernett, M., Yazbeck, D., Tao, J., 2006. Cloning and optimization of a nitrilase for the synthesis of (3S)-3-cyano-5-methyl hexanoic acid. J. Mol. Catal. B: Enzymatic 41, 75–80. Zhu, D., Mukherjee, C., Biehl, E.R., Hua, L., 2007a. Discovery of a mandelonitrile hydrolase from Bradyhizobium japonicum USDA110 by rational genome mining. J. Biotechnol. 129, 645–650. Zhu, D., Mukherjee, C., Biehl, E.R., Hua, L., 2007b. Nitrilase catalyzed selective hydrolysis of dinitriles and green access to the cyanocarboxylic acids of pharmaceutical importance. Adv. Synth. Catal. 349, 1667–1670.
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A new nitrilase from Bradyrhizobium japonicum USDA 110 Gene cloning, biochemical characterization and substrate specificity Dunming Zhu a , Chandrani Mukherjee a , Yan Yang a , Beatriz E. Rios a , D. Travis Gallagher b , N. Natasha Smith b , Edward R. Biehl a , Ling Hua a,∗ b
a Department of Chemistry, Southern Methodist University, Dallas, TX 75275, USA Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA
Received 4 March 2007; received in revised form 31 August 2007; accepted 2 October 2007
Abstract A nitrilase gene blr3397 from Bradyrhizobium japonicum USDA110 was cloned and over-expressed in Escherichia coli, and the encoded protein was purified to give a nitrilase with a single band of about 34.5 kD on SDS-PAGE. The molecular weight of the holoenzyme was about 340 kD as determined by light scattering analysis, suggesting that nitrilase blr3397 self-aggregated to an active form with the native structure being a decamer. The Vmax and Km for phenylacetonitrile were 3.15 U/mg and 4.36 mM, respectively. The catalytic constant kcat and specificity constant kcat /Km were 111 min−1 and 2.6 × 104 min−1 M−1 . This nitrilase is most active toward the hydrolysis of hydrocinnamonitrile among the tested substrates (4.3 times that of phenylacetonitrile). The nitrilase blr3397 shows higher activity towards the hydrolysis of aliphatic nitriles than that for the aromatic counterparts, and can be characterized as an aliphatic nitrilase in terms of activity. This nitrilase also possesses distinct features from the nitrilase bll6402 of the same microbe. © 2007 Elsevier B.V. All rights reserved. Keywords: Nitrilase; Aliphatic nitrile; Hydrolysis; Bradyrhizobium japonicum
1. Introduction Nitriles, in general, are synthetically more accessible than the corresponding carboxylic acids. These compounds thus provide convenient precursors for the synthesis of a wide variety of carboxylic acids, which are important intermediates in the production of fine and commodity chemicals. Chemical hydrolysis of nitriles typically requires strongly basic or acidic conditions and high temperature, and usually produces unwanted byproducts and/or large amounts of inorganic wastes. Nitrilasecatalyzed hydrolysis of organocyanides (nitriles) to carboxylic acids offers a “greener” alternative, because this ecofriendly biotransformation allows clean and mild synthesis combined with high yield and selectivity (Sugai et al., 1997; Effenberger and Osswald, 2001a,b; Martinkova and Kren, 2002; Osswald et al., 2002; Chaplin et al., 2004; Hann et al., 2004; Wang, 2005; Bergeron et al., 2006; Bornscheuer and Kazlauskas, 2006; Singh
∗
Corresponding author. Tel.: +1 214 768 1609; fax: +1 214 768 4089. E-mail address: [email protected] (L. Hua).
0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.10.001
et al., 2006). However, many of the known nitrilases possess considerable disadvantages such as low stability or selectivity. This prevents the industrial application of nitrilases, and there is thus a constant demand for new nitrilases (DeSantis et al., 2002, 2003; Robertson et al., 2004; Kiziak et al., 2005; Kaplan et al., 2006; Xie et al., 2006). The rapidly growing genome sequence data obtained in the course of genome projects offers a tremendous opportunity for rapid discovery of new enzyme catalysts. For example, Stolz and co-workers have characterized a nitrilase from cyanobacterium Synechocystis sp. strain PCC 6803 by genome mining (Heinemann et al., 2003; Mukherjee et al., 2006). Mueller et al. (2006) have cloned a thermoactive nitrilase from hyperthermophilic archaeon Pyrococcus abyssi, which showed highest activity towards malononitrile. Recently, we have cloned and purified a mandelonitrile hydrolase (bll6402) from Bradyrhizobium japonicum USDA110 via rational genome mining and demonstrated that it was an efficient catalyst for the hydrolysis of mandelonitrile and its derivatives (Kamila et al., 2006; Zhu et al., 2007a). Another putative nitrilase gene (blr3397) is also present in the same microorganism (Kaneko et al., 2002). The sequence alignment with known nitrilases
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shows that it has 55 and 52% sequence identity to the nitrilase ZmNIT2 from Zea mays (Park et al., 2003; Mukherjee et al., 2006) and the nitrilase 4 from Arabidopsis thaliana (Bartel and Fink, 1994), respectively. As part of our effort to develop an effective nitrilase catalyst tool-box, this putative nitrilase gene was cloned and expressed in Escherichia coli, the recombinant protein was purified and characterized. The substrate specificity of this nitrilase was evaluated towards a variety of nitriles with diverse structures. 2. Materials and methods 2.1. General methods and materials The GC analysis was performed on a Hewlett Packard 5890 series II plus gas chromatograph equipped with autosampler, EPC, split/splitless injector, FID detector and 25 m × 0.25 mm CP-Chirasil-Dex CB capillary column. The B. japonicum USDA110 was obtained from Dr. Patrick Elia at USDA Soybean Genomics and Improvement Laboratory. E. coli EC100 was used for all cloning experiments. The strain was routinely cultured in Luria–Bertani (LB) medium containing 100 g of ampicillin/ml for selection of plasmid. Rosetta2(DE3) from Novagen was used for expression. The nitrile substrates and the carboxylic acid standards were purchased from Sigma–Aldrich (Milwaukee, USA). 2.2. Amplification of the nitrilase gene by PCR and subsequent cloning of the gene The gene was amplified from the genomic DNA of B. japonicum USDA110 by using the two oligonucleotide primers 5 -TCGCATATGATGGATAGCAACCGACCG-3 and 5 -TTTGGATCCTCAATTGCGCGCGCGAACG-3 , which incorporated NdeI and BamHI restriction sites, respectively. The amplification was performed in a final volume of 100 l, and the reaction mixtures contained 200 ng of genomic DNA, 50 pmol of each primer (Integrated DNA Technologies, Iowa, USA), a 200 M of dNTP, 1× PCR buffer, 1.25 U of Pfx DNA polymerase (Invitrogen Inc., CA) and 1 mM MgSO4 . The following PCR program was used for the amplification of a 966 bp DNA fragment: 2 min of denaturing at 94 ◦ C; 30 cycles of denaturing (45 s at 94 ◦ C), annealing (45 s at 55 ◦ C), and polymerization (1 min at 68 ◦ C) followed. The program was completed by an additional polymerization step (10 min at 68 ◦ C). The amplified DNA fragment was digested with NdeI and BamHI and the insert was cloned into pTXB1 digested with the same restriction enzymes. The resulting plasmid was designated pLH12.1.
tures were incubated at 18 ◦ C on an orbital shaker at 180 rpm for another 11 h. The cells were harvested. 2.4. Preparation of cell-free extracts and partial purification of the enzyme The obtained cell pellet was resuspended in the lysis buffer containing 10 mM potassium phosphate and 1 mM DTT (pH 7.2), and the cells were lysed by a EmulsiFlex®-C5 high pressure homogenizer (Avestin, Inc.). The cell-free extract was mixed with equal volume of PEI (0.25% polyethyleneimine MW 40 K–60 K, 6% NaCl, 100 mM Borax, pH 7.4) to remove lipids (Milburn et al., 1990). The PEI-treated supernatant was then precipitated with 30% ammonium sulfate. The precipitate was separated by centrifugation and dissolved in the potassium phosphate buffer (10 mM, pH 7.2, 1 mM of DTT). The resulting enzyme solution was desalted by diafiltration with the same potassium phosphate buffer. The lysate was lyophilized and used for activity assay. 2.5. Enzyme isolation Further purification of nitrilase was performed in two steps—ion exchange and gel filtration liquid chromatography. The soluble fraction of the cell-free extract was loaded on DEAE Sepharose Fast Flow column (3.4 cm × 27 cm, Amersham Biosciences) and eluted with 0 to 500 mM NaCl gradient (3 l). Fractions showing nitrilase activity were pooled and concentrated on 10K MWCO centrifugal concentrators (Amicon). The resulting sample was loaded on Sephacryl S-300 (Amersham Biosciences) column and eluted with 20 mM Tris/HCl, pH 7.4. Fractions showing nitrilase activity were concentrated on 10K MWCO centrifugal concentrators (Amicon) to 30 mg/ml and appeared by SDS-PAGE (Laemmli, 1970) to be more than 98% pure. 2.6. Determination of oligomer molecular weight The molecular weight of nitrilase blr3397 was measured by light scattering. The measurement was performed at 690 nm using a WYATT DAWN static light scattering instrument, which was attached to an Agilent 1100 series high-performance liquid chromatography station equipped with a SHODEX KW-803 gel filtration column. The protein sample solutions were filtered through an Amicon Ultrafree-MC 0.22 m epitube filter unit and run through the gel filtration column at a flow rate of 0.5 ml/min prior to light scattering measurements. The mass of the native protein was calculated with an ASTRA software from the 90◦ scattering data. Thymoglobulin (Mr 669,000) and ferritin (Mr 440,000) were used as standards.
2.3. Expression of the nitrilase in E. coli 2.7. Enzyme assay The plasmid pLH12.1 was transformed into Rosetta2(DE3) E. coli strain (Novagen). Overnight precultures were diluted into LB containing 100 g/ml of amplicillin and 34 g/ml of chloramphenicol, the cells were induced with 0.1 mM of IPTG when optical density at 600 nm reached 1.0. The bacterial cul-
The standard assays were carried out by mixing the substrate nitriles (10 mM, final concentration) and the nitrilase enzyme in potassium phosphate buffer (100 mM, pH 7.2). The reaction mixtures were incubated at 30 ◦ C. Aliquots (usually 100 l)
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were taken at different time intervals, and the reactions were quenched by addition of 10% (v/v) 1 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) as follows: to the 100 l aliquot, 100 l of each of the assay reagents (0.02 M NaOCl, 0.33 M PhONa, and 0.01% sodium nitroprusside aqueous solution) were added sequentially. The resulting mixture was heated at 95 ◦ C for 2 min, and then cooled rapidly in cold water bath. A 200 l of the resulting reaction mixture was used to measure the absorbance at 640 nm, and the amount of ammonia content was calculated by comparing with the standard curve. The activity was defined as the number of mol of ammonia produced in 1 min by 1 mg of enzyme (mol min−1 mg−1 ). To determine the kinetic parameters of the purified protein for phenylacetonitrile, the activity assays were performed with different substrate concentrations, and Vmax and Km were calculated by best fitting the experimental data to the v/[A] curve according to Michaelis equation. 2.8. Effects of temperature, pH and CN− anion For the determination of temperature effect, a mixture of nitrilase (0.5 mg) with phenylacetonitrile (50 mM) in 1.0 ml of potassium phosphate buffer (100 mM, pH 7.2) was incubated at 25, 30, 37, 45, 60 or 70 ◦ C, respectively. Aliquots (100 l) were taken after 1, 2, 4, and 16 h intervals, and acidified with 10 l of 1 M HCl. The formed phenylacetic acid was extracted with 200 l of tert-butyl methyl ether, dried over anhydrous sodium sulfate, and quantified by GC analysis after converting to methyl ester with diazomethane. The pH effect was studied using the following buffers: sodium citrate buffer (pH 4.91 and 5.44), potassium phosphate buffer (pH 6.07, 6.52, 7.10, 7.58, 7.98 or 8.49) or sodium bicarbonate buffer (pH 8.98). A mixture of nitrilase (0.5 mg) with phenylacetonitrile (50 mM) in 1.0 ml of the respective buffer (100 mM) was incubated at 30 ◦ C. Aliquots (100 l) were taken after 1, 2, 4 and 16 h, and acidified with 10 l of 1 M HCl. The formed phenylacetic acid was extracted into 200 l of tert-butyl methyl ether, dried over anhydrous sodium sulfate, and quantified by GC analysis after converting to methyl ester with diazomethane. To study the effect of CN− anion, a mixture of nitrilase (0.5 mg) with phenylacetonitrile (50 mM) in 1.0 ml of potassium phosphate buffer (100 mM, pH 7.2) containing 0, 0.1, 0.5, 1.0, 5.0 or 10.0 mM of NaCN was incubated at 30 ◦ C. Aliquots (100 l) were taken after 24 h, and acidified with 10 l of 1 M HCl. The formed phenylacetic acid was extracted with 200 l of tert-butyl methyl ether, dried over anhydrous sodium sulfate, and quantified by GC analysis after converting to methyl ester with diazomethane. 2.9. Thermal stability A solution of 0.5 mg of blr3397 enzyme in 950 l of potassium phosphate buffer (pH 7.2, 100 mM) was heated at 30, 40, 50, 60 or 70 ◦ C for 30 min and 1 h separately. After heat-treatment, substrate phenylacetonitrile (final concentration:
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25 mM) was added to the enzyme solution. The resulting mixture was incubated at 30 ◦ C for 24 h. The product was extracted with tert-butyl methyl ether and methylated with freshly prepared diazomethane, and then analyzed by GC. 2.10. Substrate specificity The specific activities of nitrilase blr3397 toward different nitriles with structural diversity were measured by the quantification of the amount of ammonia released during the hydrolysis. A reaction mixture containing 20 or 40 g of purified blr3397 enzyme, 10 mM substrate in potassium phosphate buffer (1 ml, 100 mM, pH 7.2) was incubated at 30 ◦ C. The conversion was determined by measuring the amount of ammonia produced in the reaction using the Bertholet assay as described above. 2.11. Characterization of hydrolysis products To confirm the carboxylic acid products, a reaction mixture containing purified nitrilase (0.5 mg) and nitrile substrate (25 mM) in 1 ml of potassium phosphate buffer (100 mM, pH 7.2) was incubated at 30 ◦ C overnight. The reaction was then quenched with 100 l of 1 M HCl, and the formed carboxylic acids were extracted into 1 ml of tert-butyl methyl ether and converted to methyl esters with diazomethane. The methyl esters were analyzed by GC and compared with the standard compounds. In some cases, the product acids were extracted directly into chloroform-d for NMR analysis and the data were compared with those of authentic samples. 3. Results and discussion In addition to gene bll6402 which has been found to encode a mandelonitrile hydrolase (Zhu et al., 2007a), the published genome sequence of B. japonicum strain USDA110 (NCBI accession number BA000040) contains another open reading frame (blr3397), which also encodes a putative nitrilase enzyme. The gene blr3397 was amplified from the genomic DNA of B. japonicum USDA110 strain, and over-expressed in E. coli. The encoded protein was purified from the cell-free extract of the recombinant E. coli strain carrying plasmid pLH12.1 as described in Section 2. The purified protein gave a single band on SDS-PAGE with a molecular mass of about 34.5 kD (Fig. 1). The light scattering analysis showed that the molecular weight of the enzyme particle in solution was about 340 kD. This was confirmed by size exclusion high-performance liquid chromatography performed on an Agilent 1100 series highperformance liquid chromatography system with a Superdex 200 10/300 GL column (Amersham Biosciences). The column was calibrated using thyroglobulin (Mr 669,000), ferritin (Mr 440,000) and catalase (Mr 232,000) as references (highmolecular-weight kit from Amersham Biosciences). The eluent was potassium phosphate buffer (50 mM, pH 7.0) with 0.15 M NaCl, and flow rate was 0.4 ml/min at room temperature. This suggested that the active form of nitrilase blr3397 is a decamer. A nitrilase from Rhodococcus rhodochrous J1 has also been found to self-associate to decamer (Nagasawa et al., 2000).
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Fig. 2. Temperature dependence of the nitrilase activity toward phenylacetonitrile. The conversion was measured at pH 7.2 with different reaction time intervals of 1 h (diamond); 2 h (square); 4 h (triangle); 16 h (cross).
Fig. 1. SDS-PAGE of nitrilase blr3397. Left lane: protein marker; right lane: purified enzyme.
Similar self-aggregation was observed for the other nitrilase (bll6402) from the same microorganism, which exists as an oligomer of 12 subunits (Zhu et al., 2007a). The present results are consistent with the previous observations that nitrilases are propitious to self-associate to form large aggregates of 6–26 subunits (Banerjee et al., 2002; O’Reilly and Turner, 2003). The kinetic parameters of the recombinant protein for the hydrolysis of phenylacetonitrile were determined with Vmax and Km being 3.15 U/mg and 4.36 mM, respectively. The catalytic constant kcat and specificity constant kcat /Km were 111 min−1 and 2.6 × 104 min−1 M−1 . The kinetic parameter Vmax of nitrilase blr3397 is lower than that of nitrilase bll6402 (6.2 U/mg) from the same microorganism for the hydrolysis of phenylacetonitrile, and Km is higher (0.1 mM for nitrilase bll6402). Thus nitrilase blr3397 showed lower kcat and kcat /Km than bll6402 (230.0 min−1 and 2.30 × 106 min−1 M−1 , respectively, for bll6402). Phenylacetonitrile was used as the substrate to examine the temperature, pH and CN− anion effects on the activity of nitrilase blr3397. The activities of nitrilase blr3397 toward phenylacetonitrile at different temperatures were studied by measuring the conversion of the substrate to phenylacetic acid at four time intervals, and the data are presented in Fig. 2. It can be seen that the reaction rate first increased as temperature became higher, reached the maximum at 45 ◦ C and then decreased sharply. Since the optimal reaction temperature of nitrilase blr3397 was about 45 ◦ C, a question was raised as how thermostable this enzyme would be. Therefore, the thermostability of nitrilase blr3397 was studied using phenylacetonitrile as the substrate. The enzyme solutions were heated at various temperatures for
Fig. 3. Thermal stability of nitrilase blr3397. Phenylacetonitrile was used as the substrate. The enzyme solutions were heated at various temperatures for 30 or 60 min and then allowed to react with the substrate at 30 ◦ C for 24 h.
30 or 60 min and then allowed to react with the substrate at 30 ◦ C for 24 h. The product was analyzed by GC. The results are presented in Fig. 3. From the results it can be seen that the conversion of phenylacetonitrile to phenylacetic acid decreased slightly after the enzyme was heat-treated at 50 ◦ C for 30 min, indicating that nitrilase blr3397 possesses good thermal stability. The enzyme completely lost its activity after heat-treatment at 70 ◦ C for 30 min. The activity of nitrilase blr3397 was measured in buffers with pH ranging from 5 to 9 using phenylacetonitrile as substrate (Fig. 4). The enzyme was found active in this pH range and the activity was optimal from pH 7.0–8.0. This suggests that nitrilase blr3397 possesses a relatively broad working pH range. The behavior of nitrilase blr3397 against cyanide anion was also investigated using phenylacetonitrile as substrate. The
Fig. 4. pH dependence of the nitrilase activity toward phenylacetonitrile. The conversion was measured with different reaction time intervals of 1 h (diamond); 2 h (square); 4 h (triangle); 16 h (cross).
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results showed that cyanide anion did not exert significant effect on the activity up to 10 mM of CN− anion. The specific activities of the recombinant nitrilase blr3397 toward a variety of nitriles were measured by the quantification of the amount of ammonia released during the hydrolysis. For the convenience of the comparison, the relative activity of phenylacetonitrile was defined as 100, and the results for these nitriles are presented in Table 1. It can be seen from Table 1 that the enzyme showed highest activity for the hydrolysis of hydrocinnamonitrile (entry 8). The activity decreased as the number of methylene group between the phenyl and cyano groups became larger or smaller (entries 9 and 7). For ␣-substitued phenylacetonitriles (entries 1–6), the activity was much lower than that of the parent counterpart (entry 7) regardless of the electronic properties of the substituents. This might be due to crowdness at the ␣-position, which prevents the substrate from entering the active site. Therefore, steric factors may play a dominant role in determining the activity of nitrilase blr3397. This nitrilase showed a clear preference for aliphatic nitriles (entries 13–18) over aromatic and vinyl nitriles (entries 10–12). While nitrilase blr3397 was very active toward methylthioacetonitrile, it had lower activity for the hydrolysis of 3-amino- and 3-hydroxypropionitriles (entries 20 and 21). Aliphatic dinitriles were also effectively hydrolyzed by this enzyme (entries 22 and 23). Table 1 also lists the relative activity of nitrilase bll6402 from the same strain, which shows highest activity towards mandelonitrile. Although
Table 1 The relative activity of nitrilase blr3397 and bll6402 from Bradyrhizobium japonicum strain USDA110 toward various nitrile substrates Entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Substrate Mandelonitrile 2-Trimethylsilyloxy-2phenylacetonitrile 2-Phenylglycinonitrile 2-(N,N-Dimethylamino)2-phenylacetonitrile 2-Phenylpropionitrile 2-Phenylbutyronitrile Phenylacetonitrile Hydrocinnamonitrile 4-Phenylbutyronitrile 3-Indolylacetonitrile Benzonitrile Crotononitrile n-Butyronitrile 4-Chlorobutyronitrile Valeronitrile Hexanenitrile Heptanenitrile Allyl cyanide Methylthioacetonitrile 3-Aminopropionitrile 3-Hydroxypropionitrile 1,4-Dicyanobutane 2-Methylglutaronitrile
blr3397a
331
these two enzymes are highly active towards phenylacetonitrile, they show distinct substrate preference (Zhu et al., 2007a). The activities of nitrilase blr3397 to catalyze the hydrolysis of nitriles to the corresponding carboxylic acids were further confirmed by characterization of the hydrolysis products. The nitriles were treated with the nitrilase at 30 ◦ C overnight. The products were analyzed by gas chromatography or 1 H NMR, and identified by comparing the data with those of the standard samples. No amide was detected for all the substrates. The results are summarized in Table 2. From Table 2 it can be seen that nitrilase blr3397 efficiently catalyzed the hydrolysis of aliphatic nitriles including those with phenyl group at the end. The hydrolysis of benzonitrile and crotonitrile, in which CN was connected with sp2 carbons, was less effective than their counterparts with CN being bonded to sp3 carbons. This enzyme also effectively hydrolyzed aliphatic dinitriles to di-acids. Selective hydrolysis of one of the cyano groups had not been achieved in the present case. This result is different from that of other nitrilase (bll6402) in the same microorganism, in which -cyanocarboxylic acids were obtained exclusively in the hydrolysis of aliphatic dinitriles (Zhu et al., 2007b). The nitrilase blr3397 hydrolyzed mandelonitrile to mandelic acid with moderate enantioselectivity. In conclusion, B. japonicum strain USDA110 harbors a nitrilase gene (blr3397), which possesses 33% sequence identity to the nitrilase bll6402 from the same microbe. These two nitrilases show distinct features. First, although both nitrilases are active for the hydrolysis of phenylacetonitrile, nitrilase blr3397 shows highest activity for hydrocinnamonitrile, while bll6402 is most active toward mandelonitrile (Table 1). Second, the nitrilase blr3397 has compatible activity toward the hydroly-
bll6402b
4 4
460 165
3 <1
3 1
<1 <1 100 431 86 2 1 4 15 59 57 60 107 11 72 7 1 47 63
10 8 100 13 4 3 1 3 3 6 6 16 22 2 1 1 1 5 3
a The specific activity of phenylacetonitrile was 2.32 U/mg and its relative activity was defined as 100. b The specific activity of phenylacetonitrile was 5.3 U/mg and its relative activity was defined as 100. The data are cited from reference (Zhu et al., 2007a).
Table 2 Nitrilase catalyzed hydrolysis of various nitriles Entry
Substrate
Product
Conversion (%)a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Mandelonitrile Phenylacetonitrile Hydrocinnamonitrile 4-Phenylbutyronitrile Benzonitrile Crotononitrile Butyronitrile Valeronitrile Hexanenitrile Heptanenitrile Allyl cyanide Methylthioacetonitrile 1,2-Dicyanoethane 1,4-Dicyanobutane 1,5-Dicyanopentane 1,6-Dicyanohexane 1,8-Dicyanooctane Fumaronitrile
Mandelic acid Phenylacetic acid Hydrocinnamic acid 4-Phenylbutyric acid Benzoic acid Crotonic acid Butyric acid Valeronic acid Hexanoic acid Heptanoic acid 3-Butenoic acid Methylthioacetic acid Succinic acid Adipic acid Pimelic acid Suberic acid Sebacic acid Fumaric acid
44b 100 100 98 10 15 100 62 63 67 66 100 100 100 100 100 100 35
a The conversion was determined by GC analysis for entries 1–10 and by 1 H NMR analysis for entries 11–18. For GC analysis, the acid was converted to methyl ester with freshly prepared diazomethane, and the conversion was calculated for the areas of unreacted nitrile and product methyl ester. For 1 H NMR analysis, the conversion was calculated from the integration of distinguishable protons. b (S)-Mandelic acid was obtained in 36% ee.
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sis of aliphatic nitriles with phenylacetonitrile, but the activity of the nitrilase bll6402 towards the hydrolysis of aliphatic nitriles is much lower than that for phenylacetonitrile (Table 1). The most significant difference between these two nitrilases is that -cyanocarboxylic acids are obtained exclusively in the hydrolysis of aliphatic dinitriles catalyzed by nitrilase bll6402 (Zhu et al., 2007b), while no regioselectivity is observed in blr3397-catalyzed hydrolysis of aliphatic dinitriles. As predicted from the functional analysis of genetic organization of nitrilase gene bll6402 within the chromosome of B. japonicum strain USDA110, nitrilase bll6402 might play an important role in the mandelonitrile metabolic pathway in B. japonicum and can be characterized as a mandelonitrile hydrolase (Zhu et al., 2007a). Similar analysis for the nitrilase gene blr3397 shows that it is flanked by a putative vanillate O-demethylase oxidoreductase (blr3399) (Priefert et al., 1997), a toluate 1,2-dioxygenase ␣subunit (blr3400) and -subunit (blr3401) (Neidle et al., 1992), a crotonobetaine/carnitine-CoA ligase (blr3402) (Jin et al., 2002) and some hypothetical proteins or regulator/transporter proteins. It is difficult to predict the natural function of nitrilase blr3397 and further studies are needed to address this question. However, nitrilase blr3397 clearly shows higher activity toward the hydrolysis of the nitriles with CN being bonded to sp3 carbons than that for the sp2 counterparts. Therefore, this enzyme can be classified as an aliphatic nitrilase in terms of its activity and will be a valuable biocatalyst for the regioselective hydrolysis of aliphatic nitrile groups in the presence of aromatic ones. References Banerjee, A., Sharma, R., Banerjee, U.C., 2002. The nitrile-degrading enzymes: current status and future prospects. Appl. Microbiol. Biotechnol. 60, 33–44. Bartel, B., Fink, G.R., 1994. Differential regulation of an auxin-producing nitrilase gene family in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 91, 6649–6653. Bergeron, S., Chaplin, D.A., Edwards, J.H., Ellis, B.S.W., Hill, C.L., Holt-Tiffin, K., Knight, J.R., Mahoney, T., Osborne, A.P., Ruecroft, G., 2006. Nitrilasecatalyzed desymmetrization of 3-hydroxyglutaronitrile: preparation of a statin side-chain intermediate. Org. Process Res. Dev. 10, 661–665. Bornscheuer, U.T., Kazlauskas, R.J., 2006. Hydrolases in Organic Synthesis: Regio- and Stereoselective Biotransformations. Wiley-VCH, Weinheim. Chaplin, J.A., Levin, M.D., Morgan, B., Farid, N., Li, J., Zhu, Z., McQuaid, J., Nicholson, L.W., Rand, C.A., Burk, M.J., 2004. Chemoenzymatic approaches to the dynamic kinetic asymmetric synthesis of aromatic amino acids. Tetrahedron: Asymmetry 15, 2793–2796. DeSantis, G., Wong, K., Farwell, B., Chatman, K., Zhu, Z., Tomlinson, G., Huang, H., Tan, X., Bibbs, L., Chen, P., Kretz, K., Burk, M.J., 2003. Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). J. Am. Chem. Soc. 125, 11476–11477. DeSantis, G., Zhu, Z., Greenberg, W.A., Wong, K., Chaplin, J., Hanson, S.R., Farwell, B., Nicholson, L.W., Rand, C.L., Weiner, D.P., Robertson, D.E., Burk, M.J., 2002. An enzyme library approach to biocatalysis: development of nitrilases for enantioselective production of carboxylic acid derivatives. J. Am. Chem. Soc. 124, 9024–9025. Effenberger, F., Osswald, S., 2001a. (E)-Selective hydrolysis of (E,Z)-␣,unsaturated nitriles by the recombinant nitrilase AtNIT1 from Arabidopsis thaliana. Tetrahedron: Asymmetry 12, 2581–2587. Effenberger, F., Osswald, S., 2001b. Enantioselective hydrolysis of (RS)-2fluoroarylacetonitriles using nitrilase from Arabidopsis thaliana. Tetrahedron: Asymmetry 12, 279–285. Hann, E.C., Sigmund, A.E., Fager, S.K., Cooling, F.B., Gavagan, J.E., Bramucci, M.G., Chauhan, S., Payne, M.S., DiCosimo, R., 2004. Regioselective
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