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Enzymatic degradation of nitriles by a Candida guilliermondii UFMG-Y65 João C.T. Dias, Rachel P. Rezende, Carlos A. Rosa, Marc-André Lachance, and Valter R. Linardi

Abstract: Candida guilliermondii UFMG-Y65, isolated from a gold mine, was able to utilize different nitriles and the corresponding amides as sole source of nitrogen, at concentrations up to 2 M. Resting cells cultivated on YCBacetonitrile medium showed nitrile hydrolyzing enzyme activities against acrylonitrile and benzonitrile. These enzymes were inducible and intracellular; the optimum pH was 7.0–8.0, and the optimum temperature 25°C–30°C. Liquid chromatographic analysis indicated that C. guilliermondii UFMG-Y65 metabolized 12 mM benzonitrile to 11 mM benzoic acid and 10 mM acrylonitrile to 7.9 mM acrylic acid. The results suggest that C. guilliermondii UFMG-Y65 may be useful for the bioproduction of amides and acids, and for the bioremediation of environments contaminated with nitriles. Key words: Candida guilliermondii, nitrile hydrolyzing enzyme, amidase, nitriles, amides. Résumé : Candida guilliermondii UFMG-Y65, isolée à l’origine d’une mine d’or, est capable d’utiliser les nitriles et leurs amides respectifs comme source unique d’azote à des concentrations allant jusqu’à 2 M. Des cellules cultivées dans un milieu acétonitrile-YCB ont présenté une activité enzymatique causant l’hydrolyse des nitriles de l’acrylonitrile et du benzonitrile. Les enzymes en cause sont intracellulaires et inductibles et leur optimum de pH est à 7.0–8.0 et de température à 25°C–30°C. Une analyse en chromatographie liquide a révélé que le C. guilliermondii UFMG-Y65 pouvait métaboliser 12 mM de benzonitrile en 11 mM d’acide benzoïque et 10 mM d’acrylonitrile en 7.9 mM d’acide acrylique. Les résultats obtenus suggèrent que le C. guilliermondii UFMG-Y65 pourrait être utilisé dans la production biologique d’amines et d’acides et dans la biorestauration d’environnements contaminés par les nitriles. Mots clés : Candida guilliermondii, hydrolyse enzymatique des nitriles, amidase, nitriles, amides. [Traduit par la Rédaction]

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Introduction Nitriles possess the general structure R–CN, which may occur naturally or synthetically. Naturally occurring nitriles are found in higher plants, bone oils, insects, and microorganisms (Jallageas et al. 1980). They are very versatile substances that can be used for the synthesis of a wide variety of compounds including plastics, synthetic rubber, pharmaceuticals, herbicides, and other chemicals (Henahan and Idon 1971). Unfortunately, there is an increasing dissemination of these chemicals in the environment via industrial waste waters, and toxic activity can occur due to the release of cyanide. Accumulation of nitriles in ecosystems may cause deleterious effects, since most of them are highly

Received October 14, 1999. Revision received March 1, 2000. Accepted March 3, 2000. Published on NRC Research Press web site May 11, 2000. J.C.T. Dias, R.P. Rezende, C.A. Rosa, and V.R. Linardi.1 Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Cx. P. 486, 31270–901 Belo Horizonte, Brazil. M.-A. Lachance. Department of Plant Sciences, University of Western Ontario, London, ON, Canada. 1

Author to whom all correspondence should be addressed (e-mail: [email protected]).

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toxic, mutagenic, and carcinogenic (Tanii and Hashimoto 1984). Biotreatment systems have been developed following two major strategies. The first involves the adaptation of classical activated sludge systems to biodegrade the wastewater. The second and more recent involves the use of specialized cultures of microorganisms with unique metabolic capabilities (Wyatt and Knowles 1995). Generally, microbial metabolism of nitriles occurs via a hydrolytic route, yielding the corresponding carboxylate and ammonia. This may occur in two distinct ways: (i) a onestep mechanism catalyzed by a nitrilase (E.C. 3.5.5.1) (Robison and Hook 1964), or (ii) a two-step mechanism where the nitrile is converted to an amide by nitrile hydratase (E.C. 4.2.1.1.84), and the amide is subsequently converted to a carboxylate with the release of ammonia by an amidase (E.C.3.5.1.4) (Asano et al. 1982). Many rhodococci contain both nitrile hydratase and nitrilase activities in a single isolate, or show evidence for multiple forms of each type of enzyme (Bunch 1998). The use of microbial processes in industrial production of chemicals has increased rapidly, due to the high purity of the products, environmental acceptability, and energy savings (Nagasawa and Yamada 1993). For example, the Nitto Chemical Industry (Yokohama, Japan) research group uses a bacterial nitrile hydratase for industrial production of acrylamide. © 2000 NRC Canada

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Several studies of nitrile biotransformation by filamentous fungi and bacteria have been reported in the literature (Arnaud et al. 1977; Harper 1977; Babu et al. 1995). However, yeast studies are limited to few publications (Fukuda et al. 1973; van der Walt et al. 1993; Brewis et al. 1995; Linardi et al. 1996; Rezende et al. 1999). Economically and quantitatively, yeasts are the most important group of microorganisms used by man. Yeasts have considerable potential for biotechnological applications such as large-scale production of enzymes (Vaughan-Martini and Martin 1995). The present study describes (i) the ability of a Candida guilliermindii UFMG-Y65 to utilize a wide range of nitriles and their respective amides as sole source of nitrogen and (ii) the enzymatic degradation of acrylonitrile and benzonitrile.

Materials and methods Yeast isolation The yeast strain was isolated from a gold extraction circuit liquid (Mineração Morro Velho, Nova Lima, Brazil) by an enrichment technique. Briefly, 25 mL samples were added to 1% glucose, 0.1% yeast extract, 0.1% K2HPO4, 0.02% MgSO4 7H2O, 0.01% NaCl and 0.2% acetonitrile and incubated at 25°C for 120 h. The samples were streaked out on plates containing yeast carbon base (YCB-Difco, Detroit, Mich.) and 2% agar to which 0.2% acetonitrile was added as the nitrogen source and the cultures were grown for 120 h at 25°C. Morphologically distinct colonies were isolated.

Yeast identification The strain was characterized by standard methods (van der Walt and Yarrow 1984), and identified by the keys of Kreger-van Rij (1984) and Barnett et al. (1990). Final identification of strain UFMG-Y65 was done by molecular analysis. The DNA extraction and sequencing of the D1 and D2 domains of the large ribosomal DNA subunit were performed as described by Lachance et al. (1998). The primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG) and NL4 (5′-GGTCCGTGTTTCAAGACGG) (O’Donnell 1993) were used to amplify the D1 and D2 domains of the yeast rDNA. The polymerase chain reaction was conducted following the instructions provided by the supplier of Taq polymerase (Boeringher Mannheim) in the presence of 1.5 mM MgCl2 in a Perkin-Elmer System 2400 cycler. The amplified DNA was concentrated and purified by ultrafiltration through a MicroCon 100 concentrator (Amicon) and sequenced in an ABI sequencer at the John P. Robarts Research Institute, London, Ont. The sequence was edited with the DNAMAN program, v. 3.2 (Lynnon BioSoft, Vaudreuil, Que.). Known sequences for the other yeasts were retrieved from GenBank, where they had been deposited by Kurtzman and Robnett (1997). The Clustal (Thompson et al. 1994) algorithm provided in the DNAMAN package was used to align the sequences. The strain was maintained on a glucose yeast extract maltose medium (GYMP agar) slant medium (2% glucose, 0.5% yeast extract, 1% malt extract, 0.2% NaH2PO4, and 2% agar) under a mineral oil layer and stored at 4°C, or in liquid nitrogen.

Can. J. Microbiol. Vol. 46, 2000 amides utilized were: acetonitrile, benzonitrile, isobutyronitrile, methacrylonitrile, propionitrile, and succinamide (Merck); acrylonitrile, cyclopentanecarbonitrile, isobutyramide, glutaramide, and glutaronitrile (Aldrich); acetamide, acrylamide, adiponitrile, 4cyanopyridine, 3-cyanopyridine, 2-cyanopyridine, benzamide (Sigma); succinonitrile, succinamide, and butyronitrile from Pfaltz and Bauer. The MIC of nitriles and amides was determined by monitoring the growth of C. guilliermondii UFMG-Y65 estimated by optical density (480 nm) after 120 h of incubation at 25°C. The MIC was defined as the lowest concentration of the inhibitor at which no growth was observed (Silva-Avalos et al. 1990).

Enzymatic assay The crude enzyme extract was obtained from the growth of C. guilliermondii UFMG-Y65 cells for 120 h in the presence of 2 M acetonitrile. Cells (300 mg wet wt) were disrupted in 2.0 mL sodium phosphate buffer in a vortex mixer with glass beads (0.25– 0.30 mm diameter) for 3 cycles of agitation (1 min/cycle) interspersed with cycles of cooling on ice. The pooled suspension from this treatment was centrifuged, and 0.1 mL of clear cell-free extract was added to 0.9 mL of each nitrile or amide at a final concentration of 120 mM in 25 mM sodium phosphate buffer, pH 7.0, and incubated for 30 min at 30°C (Linardi et al. 1996). The reaction was stopped by the addition of 0.2 mL of 1 M HCl. The enzyme activities as nitrile hydrolyzing enzyme and amidase were determined by ammonia production (Langdanhl et al. 1996). Ammonia was quantified by a colorimetric method (Fawcett and Scott 1960). One unit of enzyme activity was defined as the amount of enzyme which catalyzed the formation of 1 µmol ammonia/min at 30°C, pH 7.0. Production of ammonia by the cell-free extract from yeast cells growing on YCB with 0.1% ammonium sulfate was also investigated. Protein concentration was determined according to Lowry et al. (1951). Controls containing no cell-free extract were included in all experiments.

Determination of substrate degradation Benzonitrile or acrylonitrile degradation was determined using 1.2 mg dry wt · mL–1 of resting cells previously grown on YCBacetonitrile 2 M, and harvested after 120 h of growth. The reaction mixture consisted of 10 mL 25 mM phosphate buffer pH 7.0, to which was added benzonitrile or acrylonitrile to final concentration of 10 mM. An aliquot was removed and filtered at 2-h intervals. The controls were prepared in uninoculated flasks containing 10 mL 25 mM phosphate buffer, pH 7.0, with 12 mM benzonitrile or 10 mM acrylonitrile added. Benzonitrile and acrylonitrile and its degradation products were detected by high-performance liquid chromatography (HPLC) using a Shimadzu, model-10 AD apparatus equipped with 20 µL loop injector and a UV detector (Shimadzu, SPD-M6A). The integration-calculation of the peak areas was performed with a Model C-R7A cromatopac (Shimadzu) processor. The column resin utilized was 5 µm LiChrosorb RP 18 (125 × 4.0 mm I.D., Merck, Darmstadt, Germany). The analytical determinations were done by varying the polarity of the mobile phase, using an isocratic system consisting of 20 mM H3PO4– MeOH–acetonitrile, pH 2.0 (65:15:20, v/v) for benzontrile, and 20 mM H3PO4–MeOH, pH 2.0 (99:1, v/v) for acrylonitrile. The volume of the samples was 10 µL, and the absorbance was measured at 210 or 240 nm. The control samples were prepared without cell suspension.

Determination of minimal inhibitory concentration (MIC)

Effect of pH and temperature on enzymatic activity

The MIC of nitriles and amides was determined from inoculation of the yeast in 125-mL Erlenmeyer flasks containing 20 mL of yeast carbon base (YCB) medium with the addition of different inhibitory compounds at different concentrations. The nitriles and

The activity as nitrile hydratase and amidase was estimated in the 3-to-10 pH range obtained with 100 mM solutions of sodium acetate (pH 3 to 5), sodium phosphate (pH 6 to 8), sodium borate (pH 8 to 9) and bicarbonate buffer (pH 9 to 10) in the presence of © 2000 NRC Canada

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2 M acetonitrile or acetamide. The assays for the determination of optimal temperature from enzymatic activity were carried out at 15, 25, 35, 45, 55, and 65°C.

Table 1. Minimal inhibitory concentration values (MIC) of Candida guilliermondii UFMG-YLF65 on nitriles and amides.

Inducible or constitutive nature of the enzymes

Substrate

The inducible or constitutive nature of the enzymatic system was determined by growth on acetonitrile and by serial transfer of 10 consecutive cultures in solid YCB with 0.1% ammonia sulfate as nitrogen source. The cells obtained from growth on ammonium sulfate and acetonitrile were harvested in the mid-exponential phase by centrifugation at 10 000 × g at 5°C for 15 min. The cellfree extract and resting cell suspensions were used for enzymatic studies. Data were subjected to an analysis of variance. When the main effects were significant (P < 0.005), differences between means were evaluated for significance using Duncan’s multiple range test.

Nitriles Acetonitrile Acrylonitrile Adiponitrile Benzonitrile Butyronitrile 2-Cyanopyridine 3-Cyanopyridine 4-Cyanopyridine Cyclopentanecarbonitrile Glutaronitrile Isobutyronitrile Metacrylonitrile Propionitrile Succinonitrile Amides Acetamide Adipamide Acrylamide Benzamide Isobutyramide Glutaramide Succinamide

Results Yeast identification The yeast strain was identified as Candida guilliermondii by physiological methods and molecular analysis. Using only physiological tests, the yeast could be identified as C. guilliermondii or C. famata by the keys of Kreger-van Rij (1984). The sequence of the D1 and D2 domains of the rDNA of the gold mine yeast strain was identical to the Pichia guilliermondii (teleomorph of C. guilliermondii) (NRRL Y-2075) rDNA sequence deposited by Kurtzman and Robnett (1997) in GenBank (accession No. U45709). These results confirmed that the strain isolated from gold-mining effluents belongs to the species C. guilliermondii, anamorph of P. guilliermondii. Determination of MIC of nitriles and amides These were estimated as 2.2 M acetonitrile, 1 M acetamide, 900 mM propionitrile, 800 mM succinonitrile, 650 mM isubutyronitrile, 500 mM isubutyramide, and 500 mM 3-cyanopyridine. For the other nitriles and amides the MIC values were less than 300 mM (Table 1). Effect of acetonitrile concentration on Candida guilliermondii UFMG-Y65 growth Increasing acetonitrile concentration up to 2 M had little effect on growth of the yeast. However, a longer lag phase was observed at 2 M acetonitrile (data not shown). A concentration of 2.2 M acetonitrile totally suppressed C. guiliermondii UFMG-Y65 growth (Table 1). Specific activities The cell extract of C. guilliermondii UFMG-Y65 was tested for a number of aliphatic, heterocyclic and aromatic substrates. Table 2 shows the specific activities as nitrile hydrolyzing enzyme and amidase. The activities of both enzymes increased with incubation time and reached a maximum after 72 h of growth, with a decline thereafter (data not shown). The most suitable substrates for nitrile hydrolyzing enzyme were found to be succinonitrile, acetonitrile, acrylonitrile, methacrylonitrile, isobutyronitrile, butyronitrile, 3-cyanopyridine, benzonitrile, cyclopentanecarbonitrile, propionitrile, adiponitrile, glutaronitrile, 4cyanipyridine, and 2-cyanopyridine, whereas the amidase exhibited maximum activity in the presence of iso-

Chemical formula

MIC values* (mM)

CH3CN H2C = CHCN NC(CH2)2CN C7H5N CH3CH2CH2CN C6H4N2 C6H4N2 C6H4N2 C7H5N NC(CH2)3CN (CH3)2CHCN H2C = C(CH3)CN C2H5CN NC(CH2)2CN

2200 300 280 N.G. 300 150 500 150 N.G. 200 650 280 900 800

CH3CONH2 H2NCO(CH2)4CONH2 H2C = CHCNH2 C7H5NH2 (CH3)2CHCONH2 NH2(CH2)6 NH2 C4H5NH2

1000 300 250 N.G. 500 300 150

* Determined after 120 h at 25°C. N.G. = no growth.

butyramide followed by acrylamide, succinamide, acetamide, benzamide, glutaramide, and adipamide. Notably, cultivation of C. guilliermondii UFMG-Y65 in medium containing acetonitrile resulted in the induction of enzyme activities to compounds such as benzonitrile, benzamide, and cyclopentanecarbonitrile which totally suppressed yeast growth (Table 1). Influence of pH and temperature The optimum pH and temperature for enzymatic activity was between 7.0 and 8.0 (Fig. 1) at 25°C–30°C (Fig. 2). These results were similar to the maximum observed with the nitrile hydratase and amidase from Pseudomonas marginalis (Babu et al. 1995) and Rhodococcus erythropolis A10 (Achary and Desai 1997). Above 55°C, the rate of activity decreased, indicating inactivation of the enzymes at higher temperatures. Inducible or constitutive nature of the enzymes The enzymes were found to be inducible because the washed (intact) cells and cell-free extracts obtained from cells transferred after ten subcultures in YCB with 0.1% ammonia failed to hydrolyze nitriles and amides, unlike the cultures that had always been exposed to acetonitrile. In addition, the enzymes responsible for nitrile hydrolysis were found predominantly in the cells and not in the culture filtrate when the cell-free culture broth was tested for the enzymes. © 2000 NRC Canada

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Fig. 1. Specific activities of nitrile hydrolyzing enzyme (䊉) and amidase (ⵧ) by crude extract obtained from growth of C. guilliermondii UFMG-Y65 cells at different pH values in acetonitrile at 2 M. Error bars indicate standard deviation.

Fig. 2. Specific activities of nitrile hydrolyzing enzyme (䊉) and amidase (ⵧ) by crude extract obtained from growth of C. guilliermondii UFMG-Y65 cells at different temperature values on pH 7.0 in the presence of 2 M acetonitrile. Error bars indicate standard deviation.

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Fig. 3. Time course of substrate disappearance and product formation of acrylonitrile degradation during assay with resting cells of Candida guilliermondii UFMG-Y65 cultivated in YCB-acetonitrile medium, showing acrylonitrile (䉱), acrylamide (䊏), and acrylic acid (䉬). Error bars indicate standard deviation.

Fig. 4. Time course of substrate disappearance and product formation during benzonitrile degradation in assay with resting cells of Candida guilliermondii UFMG-YLF65 cultivated in YCB-acetonitrile medium, showing benzonitrile (䊏), benzamide (䉬), and benzoic acid (䉱). Error bars indicate standard deviation.

Identification of intermediary compounds of acrylonitrile and benzonitrile metabolism During assay the acrylonitrile was completely consumed after 6 h of reaction (Fig. 3). A maximum accumulate (7.9 mM) of acrylic acid was detected at the same time, when the produced acid begin to decay, suggesting its utilization as a energy source by yeast. Even in lower concentrations, the acrylamide was easily detected during the assay, and remained constant after 6 h (270 µM). During the assay with benzonitrile as a substrate, the conversion of benzonitrile to benzoic acid was observed (Fig. 4). The benzamide was also detected in the reaction mixture at very low concentrations, with the maximum recorded after 6 h (12 µM). The nitrile was completely consumed within 6 h in the same assay.

Discussion The C. guilliermondii UFMG-Y65 isolated and characterized in this study is able to hydrolyze a wide range of aliphatic nitriles, aromatic, cyclic and heterocyclic dinitriles, and corresponding amides. This demonstrates that the yeast possesses the necessary enzymatic mechanisms represented by nitrile hydrolyzing enzymes and amidase. The activity as nitrile degrading enzyme was higher than that of the amidase (Table 2). All the cyanopyridines examined were readily utilized, although 3-cyanopyridine was a highly preferred substrate for hydration by the nitrile hydrolyzing enzyme. It was evident that the breakdown of acrylonitrile (Fig. 3) and benzonitrile (Fig. 4) by C. guilliermondii UFMG-Y65 involves a two-step enzymatic mechanism. It is possible that © 2000 NRC Canada

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Table 2. Specific activity of nitrile- and amide- hydrolyzing enzymes by crude extract obtained from growth of C. guilliermondii UFMG-Y65 cells. Substrate Nitriles Acetonitrile Acrylonitrile Adiponitrile Benzonitrile Butyronitrile 2-Cyanopyridine 3-Cyanopyridine 4-Cyanopyridine Cyclopentanecarbonitrile Glutaronitrile Isobutyronitrile Metacrylonitrile Propionitrile Succinonitrile Amides Acetamide Adipamide Acrylamide Benzamide Isobutyramide Glutaramide Succinamide

Specific activities (µmol of NH3·mg–1 protein·min–1) 13.5 10.4 4.6 6.9 7.3 2.5 7.2 2.7 6.8 3.6 9.2 9.7 5.6 15.8

b c i g f j f j g ij e d h a

8.1 3.5 8.9 5.0 9.2 3.9 8.3

c e b d a de c

Note: Means followed by the same letter are not significantly different (P < 0.005) as determined by Duncan’s multiple range test.

nitrile hydratase (E.C 4.2.1.84), the primary enzyme, transforms the acrylonitrile or benzonitrile into acrylamide or benzamide, which is later degraded by amidase (EC 3.5.1.4) to acrylic acid, or benzoic acid and ammonia. A similar sequential formation of the corresponding amide, carboxylic acid, and ammonia by bacteria and yeasts was reported by Nawaz et al. (1991) and Rezende et al. (1999), respectively. Controversy still exists about the nature and type of enzymes involved in the transformation of nitrile compounds. It has been suggested that aromatic and unsaturated nitriles are metabolized by a single enzyme, nitrilase (Bandyopadhyay et al. 1986; Nagasawa et al. 1988). Simple aliphatic nitriles are thought to be metabolized by a two-step process carried out by nitrile hydratase and amidase (Arnaud et al. 1977; Asano et al. 1982). Kobayashi et al. (1990) have reported a nitrilase enzyme that preferentially acts on aliphatic nitriles. In agreement with van der Walt et al. (1993), it was demonstrated here that the utilization of aliphatic nitriles and their corresponding amides by the yeast is consistent with the view that hydrolysis of nitriles may proceed by a two-step reaction mediated by nitrile hydratase and amidase (Table 2). DiGeronimo and Antonoine (1976) isolated a Rhodococcus rhodochrous strain that metabolized acetonitrile to acetamide, acetic acid, and ammonia. The same organism degraded propionitrile to propionic acid and ammonia, but propionamide was not detected in the culture medium. Similarly, Pseudomonas marginalis (Babu et al. 1995) and R. erythropolis BL1 (Langdanhl et al. 1996) were reported to degrade acetonitrile to acetic acid and ammonia via nitrile

hydratase and amidase. No acetamide was detected in the culture medium. Also, acetamide was never detected extracellularly in resting cell suspensions hydrolyzing acetonitrile. The detection limit for acetamide was 100 µM by the GC method used. However, in rare instances, small amounts of amide were excreted by cells of the two strains. Both bacterial isolates extensively utilized propionamide and acetamide as the sole sources of carbon and nitrogen. Based on these observations, these investigators concluded that they failed to detect the respective amide because it was rapidly utilized. Earlier, Mimura et al. (1969) reported the detection of acetamide and ammonia as a result of acetonitrile degradation by a Corynebacterium sp. Although acetic acid was not detected in the growth medium, the investigators assumed it to be an intermediate of acetonitrile metabolism because of the presence of ammonia. In our study, resting cell suspensions of C. guilliermondii UFMG-Y65 growing on acetonitrile rapidly metabolized benzonitrile to benzamide and benzoic acid. Acrylonitrile was metabolized to acrylamide and acrylic acid. The detection of benzamide and benzoic acid in this study suggests that these compounds may be intermediates of benzonitrile metabolism. The low level detected may be attributed to the rapid utilization of these intermediates by the yeast as additional growth substrates. Nitrilases of Fusarium sp. (Harper 1977), Rhodococcus sp. (Harper 1985), and Arthrobacter sp. (Bandyopadhyay et al. 1986) are responsible for the metabolism of benzonitrile. None of these nitrilases catabolize aromatic or aliphatic amides. We have probably identified a nitrile hydrolyzing enzyme with capacity for aromatic as well as aliphatic nitriles in the C. guilliermondii UFMG-Y65. Microbial metabolism of nitrile compounds has become of considerable interest from the point of view of amide production. The application of microorganisms as biocatalysts for the production of amides and organic acids has attracted commercial interest. Biocatalysts are highly specific and efficient in the transformation of nitrile compounds to their corresponding amides or organic acids. Candida guilliermondii UFMG-Y65 has the advantage of metabolizing aliphatic nitriles to their amides. In addition, the organism can convert aromatic nitriles and their amides to aromatic acids, and therefore may be employed commercially for the production of the respective organic acids. The present study suggests that C. guilliermondii UFMG-Y65 may be used for the degradation of toxic nitrile compounds and also for the production of amides or organic acids. Investigations of the synthesis of amides, or aliphatic and aromatic carboxylic acids by purified nitrile-degrading enzymes, are in progress in our laboratory.

Acknowledgements This work was supported by Fundação de Amparo a Pesquisa do Estado de Minas Gerais (Proc.CBS-1001/97) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (Proc.523158/96).

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