Purification and Characterization of a Cyanide-Degrading Nitrilase from Trichoderma Harzianum VSL291

Turkish Journal of Biology Turk J Biol (2015) 39: 248-257 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1406-51

Purification and characterization of a cyanide-degrading nitrilase from Trichoderma harzianum VSL291

Jorge RICAÑO-RODRÍGUEZ, Mario RAMÍREZ-LEPE* Unit for Research and Development in Food, Technological Institute of Veracruz, Veracruz, Mexico

Received: 17.06.2014 Accepted: 02.10.2014 Published Online: 01.04.2015 Printed: 30.04.2015

Abstract: An intracellular nitrilase (Nit1) with cyanide-degrading activity was isolated from Trichoderma harzianum VSL291, cultivated on benzonitrile as the sole carbon source. Nit1 was purified to homogeneity by ion exchange and gel filtration chromatography with a recovery of 7.15% and a fold of 22.5. The molecular weight was estimated to be 47.7 kDa and the purified enzyme was sequenced with a system of liquid chromatography and mass spectrometry (LC-MS). The enzyme consists of 436 amino acids with a predicted molecular weight of 47.088 kDa. The sequence revealed conserved domains for a nitrilase super family such as putative active and binding sites and a Glu-Lys-Cys catalytic triad. Nit1 exhibited maximum activity (19.6 U mg–1) at 40 °C and a pH of 7.5. Nit1 had a strong inhibition in the presence of Al3+, Cu2+, Zn2+, and Ag+ ions and was able to degrade KCN completely at 0.02 mmol/L, 0.05 mmol/L, and 0.1 mmol/L in 15 min, 40 min, and 45 min, respectively. The effect on KCN (0.02 mmol/L) degradation was tested in the presence of Cu2+ and Ag+ ions (0.025 mmol/L to 1.0 mmol/L) and the enzymatic activity was not affected significantly at 0.025 mmol/L, 0.075 mmol/L, and 0.125 mmol/L concentrations. However, when both ions were combined, the activity of the enzyme decreased significantly.

Key words: Cyanide-degrading nitrilase, bioremediation, enzyme characterization, purification, Trichoderma

1. Introduction Moreover, many cyanide complexes are highly Nitrilase enzymes (nitrilases) catalyze the hydrolysis of unstable. Temperature, pH, and light can degrade the nitrile (R-CN) compounds to the corresponding carboxylic components and form free cyanide, which is the most acid and ammonia (Howden and Preston, 2009). They toxic form of cyanide (Rao et al., 2010). There are several belong to the nitrilase super family and have been widely chemical methods used to treat effluents containing acknowledged as valuable alternatives to chemical catalysts cyanide before discharging them into the environment. because of their ability to transform an immense variety The most common are alkaline chlorination, sulfur oxide/ air and hydrogen peroxide processes. However, these of organic nitriles under mild conditions and often in a methods have significant drawbacks: an inability to treat stereoselective or regioselective manner (Martínková and metal-complexed cyanide, costly reagents, equipment, Kren, 2010). maintenance, and royalty payments, and the generation of Cyanide compounds are widely distributed on earth unfavorable by-products such as chlorinated compounds and are produced naturally by many organisms. They are (Maniyam et al., 2011). The biological alternatives for synthesized by plants as a defense mechanism (Moller, treatment of cyanide containing waste do not require 2010), secreted by fungi and bacteria as an antimicrobial addition of toxic or hazardous chemicals. Several attempts compound (Frapolli et al., 2012), and synthesized by to develop biological processes for cyanide detoxification insects as a control over mating behavior (Baxter and have been concentrated on cyanide-degrading enzymes Cummings, 2006). However, natural quantities of obtained from animals, plants, fungi, and prokaryotes cyanogenic compounds do not compare to those produced (Hong et al., 2008). by industrial processes. In addition, these compounds can As mentioned above, most of the enzymes found in be used in mining, electroplating, steel manufacturing, nitrogen-fixing prokaryotes may have disadvantages (e.g. polymer synthesis, pharmaceutical production, and other nitrogenases) and require strictly anaerobic conditions specialized applications including dyes and agricultural due to being subject to the inhibitory effect of oxygen (Goldberg et al., 1987). One example is rhodanese, which products (Dash et al., 2009). * Correspondence: [email protected] 248 RICAÑO-RODRÍGUEZ and RAMÍREZ-LEPE / Turk J Biol requires thiosulfate to function (Westley, 1981). Nitrilases mmol/L sodium phosphate buffer (pH 7.0), and dialyzed from fungi, on the other hand, have many properties that against the same buffer. The resultant dialyzed extract (12 make them promising candidates for remediation. They mL) was loaded onto a Sephadex G100 column (1.5 cm are stable over long periods of time, require no co-factors, by 100 cm; Sigma Chemical Co., St Louis, MO, USA), are readily expressed at high levels, and can function as equilibrated with Tris-HCl (pH 8.0). Fractions (2 mL purified enzymes or crude extracts (Martínková et al., each) were collected, and the nitrilase-positive fractions (3 2009). In the current study an intracellular nitrilase fractions) were lyophilized. that hydrolyzes cyanide was purified from Trichoderma During purification, protein concentration was harzianum VSL291 and the properties of the purified monitored by measuring absorbance at 280 nm, with enzyme were characterized. bovine serum albumin as the standard. The 3 nitrilase- positive fractions (6 mL in total) were loaded onto a DEAE- 2. Materials and methods Sepharose (GE Healthcare, Uppsala, Sweden) column (1.5 cm by 25 cm), pre-equilibrated with 50 mmol/L of 2.1. Microorganisms and their cultivation conditions sodium phosphate buffer (pH 7.0). The nonadsorbed The antagonistic fungiT. harzianum strain VSL291 was materials were washed from the column with the same isolated from soil cultivated with Agave tequilana, cv. ‘Azul’, eluting buffer, and the enzymes were fractionated with a in the State of Jalisco, Mexico (Sánchez and Rebolledo, linear gradient from 0 mmol/L NaCl to 200 mmol/L NaCl 2010). The culture was maintained on potato dextrose agar in 50 mmol/L phosphate buffer at a flow rate of 46 mL/h. (BD Bioxon). Twenty fractions (100 µL each) were collected and 6 of 2.2. Enzyme purification them showed nitrilase activity. A T. harzianum VSL291 spore suspension of 1 mL (spore 2.3. Measurement of enzyme activity and protein 7 density 1 × 10 spores/mL) from agar slant was inoculated concentration aseptically into 2000-mL culture flasks containing 400 mL Nitrilase activity was measured with 4-cyanopyridine of sterilized liquid of potato-dextrose broth (Fluka Sigma as a substrate. Enzyme solution (0.1 mL) was added to St. Louis, MO, USA). Given the fact that T. harzianum is 0.7 mL of substrate solution, which contained 40 µL of a mesophilic microorganism, the flask was incubated at 4-cyanopyridine (50 mmol/L) in a sodium acetate buffer 25 °C and 130 rpm for 5 days, which is an adequate time (100 mmol/L, pH 7.5). The mixture was incubated at 37 period for obtaining the required biomass for subsequent °C for 20 min and then 100 µL of HCl 1M were added to experiments. stop the reaction. A 50-µL volume of reaction mixture The culture was filtered using Whatman filter paper was incubated for 5 min with 500 µL of Nessler’s reagent no. 41. The mycelium was recovered, washed with sterile (Sigma-Aldrich, St. Louis, MO, USA). After centrifugation distilled water, and inoculated aseptically into 2000-mL (12,000 × g, for 3 min) samples were taken from culture flasks containing 400 mL of a sterilized minimal supernatant and absorbance was measured at 435 nm in a medium containing 0.1% (v/v) benzonitrile. The medium Bio-Rad SmartSpec 3000 spectrophotometer. composition (expressed as g/L in distilled water) was A standard curve of absorbance at 435 nm versus ammonium chloride (10–1000 mM) was prepared and as follows: NaNO3, 3; K2HPO4×3H2O, 1.3; KCl, 0.5 residual ammonium was determined (Benedict et al., MgSO4×7H2O, 0.5; FeSO4×7H2O, 0.5; ZnSO4×7H2O, 1983). One unit of nitrilase activity was defined as the 0.001; CaCl, 0.5; FeCl3, 0.001; MnSO4×H2O, 0.001 and pH was adjusted to 5.0 to minimize the risk of bacterial amount of enzyme that formed 1 µmol of ammonium per contamination (Donzelli and Harman, 2001). The flask was 1 min under the above conditions. The specific activity incubated at 25 °C and 130 rpm for 7 days. The mycelium of the enzyme was expressed as the nitrilase activity per was filtered, washed with phosphate buffer (50 mM, pH milligram of protein. Protein was determined according to 8.0), ground with a pestle in a mortar, suspended in the Bradford (1976) using bovine serum albumin as standard. same buffer (approximately 3 mL per 1 g of mycelium), 2.4. Molecular weight estimation, LC-MS and stirred in an ice bath for 20 min, and centrifuged (12,000 × bioinformatic analysis g, at 3 °C, for 30 min). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis The supernatant was filtered using a 0.22-µm (Millipore, (SDS-PAGE) and native PAGE were used to determine Billerica, MA, USA) membrane and the obtained protein purity. The molecular mass of the enzyme was enzymatic extract was filtered using a 500 kDa (Millipore) determined under denaturing conditions using a 7.5% molecular size weight cutoff membrane. All purification acrylamide gel as described by Laemmli (1970). The steps were carried out at 4 °C. The protein extract (50 mL) proteins were Coomassie blue stained. A mixture of was brought to 80% saturation of ammonium sulfate, and high-molecular-weight proteins (HMW electrophoresis the precipitate was recovered by centrifugation (18,000 × standard, Sigma) were used as molecular mass markers. g, at 4 °C, for 40 min), dissolved in a small amount of 50 Electrophoresis was performed at 100 V for 30–40 min.

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The marker proteins provided protein molecular min at 37 °C. The effects of co-solvents (butanol, toluene, weight standards in the range of 14.4–97 kDa (GE acetonitrile, 2-propanol, acetone, ethanol, methanol, and Healthcare). Proteins were stained with Coomassie hexane) at 5%, 30%, and 50% v/v on nitrilase activity blue. The nitrilase-protein was recovered from the using 4-cyanopyridine 50 mM as substrate were also polyacrylamide gel, reduced with dithiothreitol (DTT), determined. All experiments were performed 3 times and alkylated with iodoacetamide, and digested with trypsin. relative enzymatic activity was obtained by comparing the The enzyme was sequenced with a liquid chromatography enzyme activity with and without the effector. system and mass spectrometry (LC-MS) by the Instituto 2.7. Assays for cyanide degradation de Biotecnología, UNAM Cuernavaca, Mor. Mexico. A Nitrilase was assayed for cyanide degradation. KCN was neighbor-joining (NJ) tree of the amino acid sequence of prepared in 100 mM MOPS, at pH of 7.6. Total volume for T. harzianum VSL291 and 19 nitrilase/cyanide hydratases each reaction was 1000 µL (60 mU in 50 µL of enzymatic from fungi was performed using Clustal X program. extract, 100 µL of KCN and 750 µL of MOPS buffer, at Bioinformatic analyses of homology (i.e. identical amino pH 7.6). Cyanide degradation by nitrilase was tested in 5 acids search) were conducted according to BLAST (http:// different KCN concentrations (0.02 mmol/L, 0.05 mmol/L, www.ncbi.nml.nih.gov/BLAST/Blast.cgi.). The analysis for 0.1 mmol/L, 0.5 mmol/L, and 1 mmol/L) and the effects of hypothetical conserved domains was carried out through copper, silver, and copper and silver were tested at 0.025 MASCOT database search. Conserved residues as well as mmol/L, 0.075 mmol/L, 0.125 mmol/L, 0.25 mmol/L, 0.5 catalytic triad identification, including binding sites, were mmol/L, and 1 mmol/L in 20 µM KCN concentration. For made according to CDD (http://www.ncbinml.nih.gov/ use as a measurement of cyanide concentration at time Structure/cdd/wrpsb.cgi.). point zero, all 3 diluted cyanide samples (KCN, KCN + CuSO , KCN + AgI, and KCN + CuSO + AgI) were set 2.5. Effects of pH and temperature on nitrilase activity 4 4 up at 100%. Samples were incubated at 38 °C for 5 min, and enzyme stability 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, Enzyme activity was measured in the pH range from 4 to 9 45 min, 50 min, and 55 min. The percent of degraded and temperature range from 30 to 50 °C, using 200 µL of 50 cyanide was determined by the picric acid endpoint mmol/L Britton–Robinson buffer (0.04 M acetic acid/0.04 method (Fisher and Brown, 1952). M boric acid/0.04 M phosphoric acid) and 200 µL of the sample with a time reaction of 1 h. Thermal and pH 3. Results stability of the enzyme was tested by incubating a sample- 3.1. Enzyme purification buffer aliquot in duplicate at a particular temperature The purification steps of nitrilase produced by T. harzianum (30 °C to 50 °C) or pH (4 to 9) for 24 h, and assaying its VSL291 are shown in Table 1. Our results showed a yield residual activity and plotting the percent residual activity of 7.15 (per 100), a purification fold of 22.5, and a specific against temperature or pH. activity of 19.6. Trichoderma harzianum VSL291 Km and 2.6. Effects of effectors on enzyme activity Vmax values obtained for 4-cyanopyridine were 19.7 mg/mL The effects of (NH4)2SO4, NaN3 (1 mmol/L), AgNO3 (1 and 4.76 µmol/mg protein/min, respectively. The profile of mmol/L), Al2(SO4)3 (1 mmol/L), CaCl2 (1 mmol/L), CuSO4 the enzyme from the gel filtration Sephadex G-100 column

(1 mmol/L), FeSO4 (1 mmol/L), MgSO4 (1 mmol/L), showed 2 fractions (20 to 21) with nitrilase activity (data

ZnSO4 (1 mmol/L), ions and protein inhibitors like not shown). Fractions were mixed and loaded onto a

DTT (1 mmol/L), cysteine (1%), H2O2 (1 mmol/L), and DEAE column. The elution profile of the enzyme from the EDTA (10 mmol/L) were determined. Each compound anion-exchange chromatography DEAE column is shown was added to the reaction mixture and incubated for 60 in Figure 1. In the final purification step, most of the

Table 1. Summary of purification steps of Nit1 from T. harzianum VSL291.

Volume Total enzymatic Protein Specific activity Purification Yield Purification step (mL) activity (U) (mg) (U/mg) fold (%) Crude enzyme 50 35.77 41 0.87 1 100

(NH4)2 SO4 precipitation 12 17.52 8 2.17 2.49 48.97 Sephadex G100 6 8.91 1.5 5.77 6.63 24.90 DEAE-Sepharose 2 2.56 0.13 19.6 22.52 7.15

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0.06 Proten 1.2 Ntrlase actvty 1 0.05 1 2 0.04 0.8

0.03 0.6

0.02 0.4

0.01 0.2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fracton number Figure 1. Anion-exchange chromatography profile on DEAE-Sepharose of the pool of fractions for nitrilase activity obtained from gel filtration chromatography through a Sephadex G100 column (data not shown). A linear elution of 0–200 mmol/L NaCl was applied. Samples of fractions 1 and 2 were electrophoresed on 12.5% SDS polyacrylamide gels. The gels were stained with Coomassie blue and the purified enzyme is shown (lanes 1 and 2; 47.7 kDa). Line M indicates molecular weight standards (GE Healthcare). ▲ Nitrilase activity, ● protein measured at 280 nm. activity was present in fractions 6 and 7, which were well aeruginosa (NP_253032) amidase sequence was used to separated from other proteins, indicating that the enzyme root the tree (Figure 3). preparation was relatively pure. 3.3. Effects of temperature and pH on enzyme activity The homogeneity of the enzyme preparation was and stability confirmed by performing a 12.5% SDS-PAGE where a The effect of temperature on nitrilase activity was studied in single band was obtained. The molecular weight of the the range of 35–45 °C using 4-cyanopyridine as substrate. purified enzyme, as determined by plotting the log of The nitrilase was active in the range of 30–40 °C showing molecular weights of the markers versus their relative optimal activity at that interval, and its activity decreased mobilities, was 47.7 kDa (Figure 1). abruptly at temperatures above 40 °C (Figure 4). Thermal 3.2. Sequencing and analysis of the purified nitrilase and stability studies indicated that the nitrilase retained more phylogeny than 90% of its activity when exposed to 30–40 °C for The protein band was excised from the gel and digested 24 h. To evaluate the effect of pH, enzyme activity was with trypsin, and the generated peptides were analyzed measured over a range of 4.0 to 9.0. The optimum pH was by LC-MS. The results showed that the enzyme consists found to be around 6.0–8.0 and retained more than 80% of of 426 amino acids with a predicted molecular weight of its activity between 6.0 and 8.0 pH values (Figure 5). The 47.088 kDa (assuming an average molecular mass for an highest stability was detected at 24 h at pH 7.5. amino acid of 108 mass units). This value is similar to the 3.4. Effects of inhibitors and co-solvents on enzyme molecular weight calculated by SDS-PAGE (47.7 kDa). activity The results from the NCBI conserved domains database The effects of metal ions and protein inhibitors on (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) nitrilase activity were studied by measuring the influence showed that the nitrilase had several conserved regions of different ions, protein inhibitors such as EDTA, DTT 42 for the nitrilase super family such as putative active (E , (Figure 6), and co-solvents (Figure 7). No enzyme activity 131 163 165 166 128 129 130 133 T , W , H , I ) and binding (M , K , P , M , was observed in the presence of Al3+, although a strong D140, A141, S142, Q167, P168, Y169, A177, H388, Y389, S390, R391, P392) + 2+ 2+ inhibition by Ag , Zn , Cu , and H2O2 ions was detected. sites, as well as a Glu-Lys-Cys catalytic triad (amino acid On the other hand, nitrilase activity in the presence of residues: E127, K134 C162) (Figure 2). Phylogenetic analysis of ++ ++ ++ + cysteine, EDTA, sodium azide, Ca , Mg , Fe , NH4 , and T. harzianum VSL291 nitrilase and other 19 representative DTT ions decreased due to the decrease of the residual fungi nitrilases generated a tree by the neighbor-joining ++ + activity of Fe , NH4 , and DTT (i.e. 98%, 97%, 91%, 90%, method and formed 2 majors groups, where Pseudomonas 82%, 74%, 60%, and 48%, respectively).

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Figure 2. Alignment of T. harzianum VSL291 nitrilase sequence with nitrilase/cyanide hydratases obtained from 6 other fungi. (*) represents identical amino acids; (:) high conserved residues; (.) low conserved residues. The conserved domains related to nitrilase/ cyanide hydratase enzymes in all fungi (including T. harzianum VSL291) are underlined. The catalytic triad residues (K, E, C) are shaded. ▲ Active site, ◆ Dimer interface. Homology comparison according to BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ Blast.cgi.). Conserved domain search according to CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi.).

The effects of some of these ions on nitrilase activity concentrations and the highest enzyme activities for all are not similar to those reported in other fungal studies. co-solvents were at 5% (v/v) concentrations (Figure 7). We tested 8 co-solvents at 5%, 30%, and 50% (v/v) When the co-solvent concentrations increased to 30% and

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100 Botryotinia fuckeliana (hypothetical protein) XP_001547043 36 Sclerotinia sclerotiorum (hypothetical protein) XP_001597458 67 100 Penicillium digitatum (putative cyanide hydratase/nitrilase) EKV07546 Magnaporthe oryzae (nitrilase 2) XP_003716682 94 Beauveria bassiana (cyanide hydratase) EJP62307 Aspergillus fumigatus (cyanide hydratase/nitrilase) XP_756085 100 67 Neosartorya fischeri (hypothetical protein) XP_001261812 Neofusicoccum parvum (putative cyanide hydratase) EOD44220 Metarhizium anisopliae (cyanide hydratase) ABD49722 Gloecercospora sorghi (cyanide hydratase) P32946 100 Fusarium solani (cyanide hydratase) CAM82815 95 100 31 Stereum hirsutum (cyanide hydratase) EIM82695 27 Pyrenophora tritici-repentis (cyanide hydratase) XP_001930992 39 Leptosphaeria maculans (putative cyanide hydratase) AAF66098 99 50 Neurospora crassa (cyanide hydratase) XP_960160 99 Trichoderma virens (hypothetical protein) EHK17305 Trichoderma atroviride (nitrilase) EHK47735 92 Talaromyces stipitatus (nitrilase) XP_002478076 74 Trichoderma harzianum VSL291 (cyanide hydratase/nitrilase) Trichoderma reesei (predicted protein) EGR46746 Pseudomonas aeruginosa (amidase) NP_253032 OUT GROUP

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Figure 3. Neighbor-joining (NJ) tree of the amino acid sequence of T. harzianum VSL291 nitrilase and 19 nitrilase/ cyanide hydratases from fungi obtained from NIG (http://www.ncbi.nlm.nig.gov). Pseudomonas aeruginosa amidase sequence was used as the out group. Nitrilase/cyanide hydratase were aligned using Clustal X program. Numbers of branches indicate bootstrap values from an analysis of 1000 replicates.

100 100 80 80 60 60 Optmum temperature Optmum pH 40 40 Temperature eect pH eect over enzyme stablty Resdual actvty (%)

over enzyme stablty Resdual actvty (% ) 20 20

35 37.5 40 42.5 45 44.555.566.57 7.588.59 Temperature (ºC) pH Figure 4. Effects of temperature on nitrilase activity. ■ Optimum Figure 5. Effects of pH on nitrilase activity.■ Optimum pH temperature (1 h). ● Temperature effects over enzyme stability (1h). ● pH effects over enzyme stability (24 h). The assays were (24 h). The assays were carried out in duplicate. Bars represent carried out in duplicate. Bars represent standard deviation. standard deviation.

50% (v/v), enzyme activity decreased significantly. The was observed (Figure 8A). At 0.02 mmol/L and 5 min most suitable co-solvents tested were the following (in of incubation the KCN concentration in the reaction descending order): hexane, methanol, ethanol, acetone, mixture decreased sharply to less than 10% and at 15 2-propane, acetonitrile, toluene, and butanol. Interestingly, min was not detectable. At 0.05 mmol/L and 1 mmol/L the enzyme showed 28% activity in hexane and 18% in KCN concentrations, the degradation rate decreased toluene at high concentrations (50%, v/v). and KCN concentration was not detectable after 40 min. 3.5. Cyanide-degrading assays Concentrations of 0.5 mmol/L and 1 mmol/L showed ca. The effects of enzyme activity on potassium cyanide 40% and 5% KCN degradation respectively. The effects 2+ 2+ degradation are shown in Figures 8A–8D. For all KCN of Cu and Ag on 0.02 mmol/L KCN degradation concentrations tested (0.02 mmol/L, 0.05 mmol/L, 0.1 by the enzyme are shown in Figure 8B and Figure 8C, mmol/L, 0.5 mmol/L, and 1 mmol/L) KCN degradation respectively.

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100 100 5% V/V 30% V/V 50% V/V 80 80

60 60

40 40

20 20

0 0

Ions Co-Solvents Figure 6. Effects of ions on nitrilase activity. Figure 7. Effects of co-solvents on nitrilase activity.

AB 100 KCN (mmol/L) 100 CuSO4 (mmol/L) 80 0.02 0.025 0.05 80 0.075 60 0.1 0.125 0.5 60 0.25 40 1 0.5 40 1 20

Remanng KCN (%) 20 Remanng KCN (%) 0 0 510152025330 5 40 45 50 55 510152025330 5 40 45 50 55 Tme (mn) CDTme (mn) AgNO3 (mmol/L) 100 AgNO3 (mmol/L) 0.025 + 80 0.075 80 CuSO4 (mmol/L) 0.125 0.025 60 0.25 60 0.075 0.5 0.125 40 1 40 0.25 0.5 20 20 Remanng KCN (%) Remanng KCN (%) 1 0 0 510152025330 5 40 45 50 55 510152025330 5 40 45 50 55 Tme (mn) Tme (mn) Figure 8. Effects of Nit1 activity on KCN degradation at 0.02 mmol/L, 0.05 mmol/L, 0.1 mmol/L, 0.5 mmol/L, and 1 mmol/L (A).

The effects of Nit1 on KCN (0.02 mmol/L) degradation with 0.025~1 mmol/L of CuSO4 (B), AgNO3 (C), and CuSO4 + AgNO3 (D) are shown.

The results show that as the concentrations of both 4. Discussion ions increase from 0.125 mmol/L to 1 mmol/L, the In the current study we examined the properties of a enzyme activity decreases. The combined effect of Cu2+ cyanide-degrading nitrilase from T. harzianum VSL291. and Ag2+ ions significantly affects enzyme activity (i.e. The specific activities of some aromatic nitrilases of at 0.02 mmol/L KCN concentration the complete time fungi such as Fusarium solani IMI196840, F. solani O1, for degradation increases from 20 min to 35 min). Our Aspergillus niger K10, and Rhodochrous rhodochrous J1 results show that the nitrilase obtained from T. harzianum using 3-cyanopyridine as a substrate have been estimated VSL291 is capable of degrading KCN even in the presence at 29.7 U/mg, 3.5 U/mg, 43.7 U/mg, and 59 U/mg, of Cu2+ and Ag2+ ions. respectively (Kobayashi et al., 1989; Kaplan et al., 2006;

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Vejvoda et al., 2008, 2010). Purified nitrilases obtained activity (a.c. 50%) and other purified nitrilases (Vejvoda from Fusarium lateritium, A. niger CBS 513.88, Penicillium et al., 2008, 2010), suggest that its use in the preparation marneffei ATCC 18224, Gibberella moniliformis, and of nitrilase buffers should be more limited or should Neurospora crassa OR74A have shown molecular weights be replaced by stabilizers that are less aggressive to the from 30 to 45 kDa (Cluness et al., 1993; Kaplan et al., 2011; enzyme. In addition, this compound could break disulfide Petrícková et al., 2012; Vejvoda et al., 2008, 2010). These bonds and alter their tertiary structure, which could have a results are consistent with those observed for T. harzianum destabilizing effect, as reported inPseudomonas fluorescens VSL291 nitrilase since Nit1 specific activity was 19.6 U/mg (Kiziak et al., 2005) and A. niger (Kaplan et al., 2011). and it had a molecular mass of 47.7 kDa. The chelating agents of metallic ions such EDTA showed In contrast, the molecular weight of a hypothetical no important enzymatic inhibition, indicating that Nit1 cyanide hydratase from F. solani CAC69666 that has at could lack metal cofactors. Most nitriles are poorly soluble least 2 subunits has been estimated to be more than 300 in aqueous media and, therefore, the enzyme’s ability to kDa (Barclay et al., 1998). It has been described that degrade nitriles in organic solvents is very important. the nitrilase super family hydrolyzes and condenses a Binding of some inhibitors in high concentrations (e.g., variety of nonpeptide carbon-nitrogen bonds, utilizing a 30%–50% v/v), such as butanol, toluene, acetonitrile, and characteristic catalytic triad of Glu-Lys-Cys (Basile et al., 2-propanol, may prevent the entry of the substrate into the 2008). This triad has been identified in the T. harzianum active site of the enzyme and hinder the corresponding VSL291 nitrilase sequence as along with several conserved reaction. Most of the time enzyme inhibitors react with regions (domains) for the nitrilase super family, such the enzyme covalently and modify the essential amino as putative active sites and putative binding sites. acid residues’ structure required for enzyme activity. Similar sequence architecture is present in some fungal In contrast, reversible inhibitors bind to the enzyme hypothetical nitrilases obtained from the NCBI database noncovalently, resulting in different types of inhibition (e.g., Trichoderma reesei (EGR46746), Trichoderma virens depending on whether the inhibitor binds to the enzyme, (EHK18648), Trichoderma atroviride (EHK18468)), the enzyme-substrate complex, or both (Kaplan et al., cyanide hydratases (F. solani (CAM82815), Aspergillus 2011). fumigatus (XP_756085), and Penicillium digitatum Another important characteristic of nitrilases is that (EKV07546)). they are able to show activity in the presence of mixtures of As mentioned above, Basile et al. (2008) described the organic compounds such as dimethyl sulfoxide, methanol, catalytic triad on DNA sequences from degrading-cyanide and heptane (Heinemann et al., 2003). The results of nitrilases belonging to 4 species of fungi: Neurospora cyanide degrading assays corroborate previous studies crassa, Gibberella zeae, Gloeocercospora sorghi, and that propose the use of nitrilase enzymes obtained from Aspergillus nidulans. Trichoderma harzianum VSL291 microorganisms as an alternative to detoxification of waste nitrilase is grouped with Talaromyces stipitatus nitrilase waters (e.g., the plastic industry and the extraction of gold (X_002478076) and is close to T. atroviride nitrilase in mines). In bacteria, the genera Rhodococcus, Alcaligenes, (EHK47735) and the hypothetical T. virens nitrilase Pseudomonas, and Bacillus degrade metallo-cyanide (EHK17305). The enzymatic parameters described for complexes and free cyanide because they use them as Nit1 are also consistent with those observed for nitrilases nitrogen sources (Barclay et al., 1998; Dubey and Holmes, of fungal origin. For example, Aspergillus niger K10 and F. 1995; Wang et al., 2012; Maniyam et al., 2013). solani 01 nitrilases have higher activities at 40–45 °C and To date, most of the purified enzymes from fungi and exhibit traces at temperatures close to 50 °C. Likewise, the bacteria belong to the xylanase, glucanase, and lipase range of their optimum pH is between 7.0 and 8.5 (Vejvoda families (Dobrev and Zhekova, 2012; Pham et al., 2012; et al., 2010; Kaplan et al., 2011). Gökbulut and Arslanoğlu, 2013). Several fungal genera It has been suggested that Ag+ ions act on the cysteine that produce cyanide-degrading enzymes have also been of the catalytic triad of nitrilases, destabilizing their activity identified:Neurospora (Dent et al., 2009), Nocardia (Linton (Kiziak et al., 2005). The strong inhibition of the enzymes and Knowles, 1986), and Fusarium (Cluness et al., 1993). by these ions is similar to that observed for nitrilase of F. Demestre et al. (1997) isolated cyanide metabolizing solani (Vejvoda et al., 2008). To date, it is not clear why microorganisms from contaminated alkaline wastes and nitrilases behave differently in the presence of different soil, and they found in batch culture that cyanide (2 mM) ions. One of the possible causes could be the different disappeared in 72 h in an exponential manner in the degrees of purification of the studied enzymes, as well as the presence of F. solani. Recombinant forms of fungi cyanide presence of contaminating proteins on enzymatic extracts hydratases from N. crassa, A. nidulans, G. zeae, and G. (e.g., chaperone proteins). DTT is used commonly as an sorghi reached their maximum rate at 60 mM KCN and N. enzyme stabilizer. However, its effects on Nit1 enzymatic crassa enzyme degraded 90% of the cyanide in less than 5

255 RICAÑO-RODRÍGUEZ and RAMÍREZ-LEPE / Turk J Biol h (Basile et al., 2008). Ezzi and Lynch (2002) showed that Acknowledgments some Trichoderma strains have high capacity to degrade Jorge Ricaño-Rodríguez was a graduate student supported cyanide via both the cyanide hydratase and the rhodanese by the Consejo Nacional de Ciencia y Tecnología pathways, and suggest a constitutive nature of both (CONACYT). This research was supported by the enzymes in this organism. Biodegradation by Trichoderma Dirección General de Educación Superior Tecnólogica spp. using cyanide (2000 ppm) as the sole carbon source and PROMEP (DGEST.4545.12). was achieved in 90 days (Ezzi and Lynch, 2005).

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