10.7603/s40-01-00- Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25

Revisión

Understanding nitrile-degrading : classification, biocatalytic nature and current applications

Jorge Ricaño Rodríguez

Laboratorio de Fitopatología, Departamento de Ciencias del Mar y Biología Aplicada. Instituto Multidisciplinar para Estudios del Medio (IMEM) Ramón Margalef, Universidad de Alicante, Ap. 99, 03080, Alicante, España. Tel. 00 (34) 96 590 3400 Ext. 3280.

Corresponding author: [email protected]

Abstract

Nitrile-degrading enzymes commonly known as nitrilase enzymes are able to metabolize nitrile-substituent compounds and they have several industrial applications, for example: in drugs synthesis. It is also common to observe their exploitation for obtaining chemical compounds with commercial interests related to cosmetics production, paints and additives. In addition, these are frequently used in the active metabolites synthesis of pesticides. Due to the catalytic nature of such proteins, it is possible to take advantage of their biotechnological potential to be applied in various scientific fields including synthetic biocatalysis and environmental remediation, since they have been successfully used for soils nitrile-wastes decontamination such as cyanide, bromoxynil and benzonitrile. On the other hand, these enzymes are considered very important intermediaries of metabolic pathways related to indolic compounds that are produced by bacteria, plants and superior fungi, acting in most cases as vegetal growth hormones. Given the fact that indole-derivative molecules play an important role in physiological responses in superior organisms, nitrilase enzymes may be considered as important part of unknown multi-enzymatic secondary metabolites pathways. In light of the above considerations, this review attempts to summarize the current status of nitrilase research and describing in detail the main characteristics of nitrile-converting enzymes with emphasis on fungal proteins, including their function and catalytic selectivity. Likewise, their relationship with plant metabolism and biotechnological importance in bioremediation processes is discussed.

Keywords: nitrile-degrading enzymes, indolic compounds, indole-3-acetic acid, cyanide, environmental remediation.

Resumen

Las enzimas degradadoras de compuestos nitrilo conocidas comúnmente como enzimas nitrilasas, son capaces de metabolizar compuestos nitrilo-sustituyentes, y tienen diversas aplicaciones industriales como por ejemplo: en la síntesis de fármacos. Asimismo, es común observar su explotación para la obtención de compuestos químicos de interés comercial relacionados con la elaboración de cosméticos, pinturas y aditivos. Además, éstas suelen emplearse también de manera frecuente en la síntesis de metabolitos activos de plaguicidas. Debido a la naturaleza catalítica de dichas proteínas, actualmente es posible explotar su potencial biotecnológico para ser aplicado en diversos campos, incluyendo la biocatálisis sintética y remediación ambiental, ya que han sido utilizadas con éxito para la descontaminación de suelos impactados con cianuro, bromoxinil y benzonitrilo. Por otra parte, dichas enzimas son importantes intermediarias de rutas metabólicas de compuestos indólicos producidos por bacterias, plantas y hongos superiores, actuando en la mayoría de los ©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 8 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 casos como hormonas de crecimiento vegetal. Debido al hecho de que las moléculas derivadas de compuestos indólicos juegan un papel importante en las respuestas fisiológicas de organismos superiores, las enzimas nitrilasas podrían ser consideradas parte importante de rutas metabólicas multienzimáticas desconocidas en la síntesis de metabolitos secundarios. La presente revisión crítica tiene la finalidad de describir a detalle las principales características de las enzimas nitrilasas haciendo énfasis en aquellas de origen fúngico, incluyendo su clasificación, función y selectividad catalítica. Igualmente, se discute su relación con el metabolismo de las plantas y la importancia biotecnológica que tienen en procesos de biorremediación.

Palabras clave: enzimas nitrilasas, compuestos indólicos, ácido indol-3-acético, cianuro, remediación ambiental.

1. Introduction: historical outline organisms was not studied any further at of nitrilase enzymes the time. After these previous studies, Strobel (1966; 1967) examined with no The nitrilase superfamily groups a wide detail earlier the enzymes hydrolyzing 2- range of thiol enzymes which are aminopropionitrile and 4-amino-4- commonly involved in several metabolic cyanobutyric acid in an unidentified pathways, such as biosynthesis and basidiomycete. Until today, it is known post-translational modification in that the first nitrilase purification and eukaryotic and certain prokaryotic species partially characterized was an (Pace and Brenner, 2001). This group of from Pseudomonas sp. (Robinson and enzymes can also refer to CN- Hook, 1964). In the late 1970s, research due to their capacity to catalyse the into nitrilases was intensified in the context hydrolysis of non-peptide carbon-nitrogen of increasing interest of their bonds, being the specificity a biotechnological exploitation. From this common way to classify their nature. The moment, new nitrilases were purified and main biotechnological importance of this characterized from both prokaryotic and type of peptides lies in their high potential eukaryotic organisms (for a review see to degrade several nitrile compounds under Martínková et al. 2009). Since nitrilases different environmental conditions, which from genus Rhodococcus were identified in some cases enantioselectivity may be as promising nitrile-hydrolytic enzymes observed (Martínková and Mylerová, due to their substrate specificity toward 2003). Because of these properties, aromatic and aliphatic nitriles (Kobayashi nitrilase enzymes are considered potential and Shimizu, 1994), many other enzymes candidates for conducting bioremediation belonging to different bacterial genera (i.e. processes. The first studies regarding Alcaligenes, Bacillus, Pseudomonas) have nitrilases were conducted at the end of also been purified and characterized (for a 1950s when Thimann and Mahadevan review see O'Reilly and Turner, 2003). (1958) indentified nitrilase activity in While knowledge of nature and structure plants for the first time. Later, through of prokaryotic nitrilases have substantially several researches nitrilase activity was improved in the last twenty years (for also observed in bacteria (Hook and reviews see Banerjee et al. 2002; Zhou et Robinson, 1964) and fungi from genera al. 2005), almost no further work has been Aspergillus, Penicillium, Gibberella and developed to a complete characterization to Fusarium, which showed the ability to nitrilases from filamentous fungi until convert 3-indolacetonitrile (IAN) into recently. In recent years, a great number of indole-3-acetic acid (IAA) (Thimann and putative fungal nitrilases are available from Mahadevan, 1964). Notwithstanding, the protein databases, although just a few nitrile-hydrolyzing ability of these enzymes have been purified and ©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 9 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 characterized. Nowadays, the purified (Brenner, 2002). Nitrilase enzymes also nitrilases from Fusarium solani O1, catalyse metabolic reactions involving Fusarium oxysporum f. sp melonis, degradation and biotransformation of Aspergillus niger K10, Gibberella complex nitrogenous compounds such as, intermedia CA3-1 are some of the most bromoxynil (3,5-dibromo-4- important complete purified enzymes due hydroxybenzonitrile) and acetonitrile to the fact that have shown reliable which are considered highly hazardous experimental enzymatic properties related substances for human health. Such to nitrilase activity (Vejvoda et al. 2008; metabolization processes take place Kaplan et al., 2006a; Goldlust and Bohak, through reactions that involve the 1989; Wu et al. 2013). Despite the fact of hydrolysis of nitriles into their the high potential for nitrile-hydrolyzing corresponding carboxylic acid and that this group of proteins possesses, the ammonia as subproducts (Howden and unsatisfactory level of knowledge for Preston, 2009). Many organisms belonging nitrilases prompts to screen for these to eukaryotic and prokaryotic domains enzymes, optimize their production and such as plants, fungus, animals and describe their biochemical and biocatalytic bacteria, perform several biochemical properties. In fact, there is little recent reactions related to nonpeptide carbon- information related to nitrilases genome nitrogen hydrolysis through nitrilase sequencing (Luque-Almagro et al. 2013; enzymes biocatalysis. Nitrilase and Kaplan et al. 2013). Due to the above, this amidase reactions produce indolic review aims to summarize current compounds as organic subproducts (i.e. knowledge of nitrilase enzymes including IAA and IAN) as well as biotin and β- those that accept organic cyanides and alanine among others, resulting in a substrates related to specificity for metal deamination of protein and aminoacid cyanides (cyanide hydratases and substrates trough a nucleophilic dihydratases). Its focus will be mainly in substitution of a conserved cysteine that the practical aspects of this topic which attacks a cyano or carbonyl carbon include structure classification and (Harper, 1985; Ambler et al. 1987; Novo et molecular function, their role in metabolic al. 1995; Stevenson et al. 1990; Bork and pathways of nitrogen transformation in Koonin, 1994). The nitrilase superfamily plants as well as biocatalytic applications contains a closely group of related cyanide and biodegradation of environmental hydratase and cyanide dihydratase nitrile-contaminants. enzymes which preferentially hydrolyse cyanide to formamide, while cyanide 2. Nitrilases in nature dihydratase enzymes hydrolyse this compound to formic acid and ammonia 2.1 Classification, function and substrate (Singh et al. 2006). Most branches of the specificity of nitrile-degrading enzymes nitrilase superfamily do not contain There are three categories of nitrilases nitrilase enzymes precisely, due to the fact according to substrate specificity: aliphatic that several amide-hydrolyzing and amide- nitrilases, which act on aliphatic nitriles condensing enzymes among this group can such as acrylonitrile, glutaronitrile and β- be found. Hitherto, nitrilase enzymes can cyano-L-alanine; aromatic or heterocyclic be classified in 13 branches and grouping nitrilases that are able to hydrolyze of each depends on its enzyme activity and benzonitrile and cyanopyridine; and substrate specificity (Pace and Brener, arylacetonitrilases that prefer substrates 2001). like arylacetonitriles such as phenylacetonitrile, phenylpropionitrile and Branches 1-3 IAN, an important intermediary indolic- The first group of proteins related to compound involved in auxins biosynthesis nitrilase enzymes can be found in both ©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 10 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 eukaryotic and prokaryotic organisms. The Granjeaud et al. (1999) developed an EST most representative evidence of nitrilase (Expressed Tag Sequence) description of activity in plants can be seen in the first vanin gene family conserved from Arabidopsis thaliana, which in vivo effect fly to human. Branch 5 is related to β- evidences the conversion of IAN into IAA. ureidopropionase enzymes which are This reaction was confirmed through an involved in the catabolism of pyrimidine experiment related to a recessive mutation bases and the production of β-alanine in a nitrilase gene resulting in reduced (Kvalnes-Krick and Traut, 1993). The sensitivity to the auxin-like effects of IAN reaction type of these proteins is the (Normanly et al. 1997). Aliphatic amidases hydrolysis of linear amides whose and amino-terminal amidases can be found chemical pathway is usually related to in two groups which are branches 2 and 3. thymine degradation, resulting in the These comprise a small compilation of release of (R)-3-amino-2- nearly identical proteins commonly found methylpropanoate. On the other hand, a in Pseudomonas, Bacillus, Brevibacteria, reductive reaction that involves uracil Helicobacteria and Saccharomyces. The degradation may also be seen when enzymes hydrolyze substrates like pyrimidines are reduced to β-aminoacids, glutamine and asparagine, specifically the CO2 and ammonia (Matsuda et al. 1996). amino carbonyl sidechains through the Some nitrilase substitutes like pyrimidines utilization of a conserved cysteine which can be catabolized through different forms part of a on the amino pathways, but the best characterized one is acid sequence of the protein. It is known a reductive pathway in which like uracil that Nta1 protein from Saccharomyces degradation, pyrimidines are reduced to cerevisiae is capable to perform an amino- CO2 and ammonia. Examples like an terminal deamination of asparagine and oxidative pathway are only found in a few glutamine residues, releasing aspartate and bacterial species and have not been glutamate as subproducts (Baker and characterized nearly as well (Kao and Hsu, Varshavsky, 1995). The release of 2003; Walsh et al. 2001). subproducts such as biotin-lysine, biotin peptide-conjugates and biotin methylester Branch 6 is also common in this kind of reactions Carbamylase enzymes comprise another (Cole et al. 1994). Previous studies have type of nitrilases grouped in branch 6. A shown that biotinidases are able to recycle lot of bacteria are able to express the vitamin biotin. Commonly, some enzymes for the decarbamylation of D- persons show biotinidase deficiency as part aminoacids. These enzymes have been of an autosomal disease with a recessively exploited in the production of β-lactam inherited neurocutaneous disorder (Wolf, antibiotics and some other secondary 2012). Due to the fact that biotinidases metabolites with pharmacologic interest posses a conserved carboxy-terminal (Louwrier and Knowles, 1997). domain, vanins and GPI-80 protein are Deinococcus radiophilus, an extremophilic grouped into the biotinidase branch bacterium is capable to use the aspartate (branch 4) which contain a similar amino-N as nitrogen source for glutamine carboxy-terminal domain and a GPI anchor synthesis through a substrate-channelled (Aurrand-Lions et al. 1996; Suzuki et al. pathway delivering glutamine to 1999). carbamoyl-phosphate synthetase (McPhail et al. 2009). In addition, Wang et al. Branches 4 and 5 (2008) found that the binding of carbamoyl Branch 4 of nitrilase superfamily is the phosphate to the enzymes aspartate and only group of proteins with amidase ornithine transcarbamoylase from reaction that prefers secondary amine Escherichia coli reduces the rate of substrates as opposed to simple amides. thermal decomposition of carbamoyl ©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 11 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 phosphate by a factor greater than 50000. This group acts on a wide range of Likewise, a preceding antecedent has aromatic nitriles like acetonitrile and also shown that both of these in some aliphatic nitriles including the transcarbamoylases use a binding corresponding acid amines (Brady et al. mechanism were carbamoyl phosphate 2004). In species like A. thaliana, NIT binds allowing the formation of enzyme- enzymes perform their catalytic activity carbamoyl phosphate complex. through an essential Cys179 and Cys186 residues (Vorwerk et al. 2001). Branches 7-9 Agrobacterium sp. and Alcaligenes faecalis The finding of nitrilase-related domains in nitrilases perform a reaction type that proteins allows correlating the ability of involves a hydrolysis of amide bond bacterial NAD Synthetase to convert (Wieser et al. 1997; Petersen and Kiener, glutamine into a lees complex nitrogen 1999). Likewise, it has been demonstrated source such as ammonia. It has been that Nocardia globerula NHB-2 nitrilase is observed that a nicotinamide capable to catalyse the biotransformation mononucleotide synthetase from of 4-cyanopyridine (a complex nitrogen Francisella tularensis is related to an source) to isonicotinic acid (Sharma et al. alternative rout of NAD synthesis were the 2012). It is worth mentioning that their amidation of NaMN (glycohydrolase) industrial potential lies in the mild and occurs before the adenynylation reaction often stereoselective hydrolysis of nitriles, that converts the intermediate to the NAD due to the fact that nitrilases are factor (Sorci et al. 2009). Spencer and heterogeneous in terms of their substrate Preiss (1967) discovered that NAD specificity. Nowadays, the gene and synthetase represents 20 % of the activity protein databases contain a large number relative to ATP in E. coli, which may be of sequences that encode putative and involved in two possible substrates, characterized nitrilases which enzymatic ammonia or glutamine to perform the final activities have not been studied at all. step in the Press-Handler pathway for When searching for enzymes sequences NAD + biosynthesis (Ozment et al. 1999). with defined substrate specificity, a Braun’s lipoprotein as well as known as comparison of their homology and specific BLP or Murein lipoprotein, is one of the regions with those of known enzymes may most abundant membrane-like peptides be useful (Seffernick et al. 2009). found in some gram-negative cell walls. The protein is bound at its C-terminal end Branches 11-13 to a lysine by a covalent bold to the Branches 11 and 12 are grouped in a peptideglycan layer embedded outside the similar branch due to their distinctive membrane (Seltmann and Holst, 2002). similarity with no characterized members. Braun’s lipoprotein is a major component Branch 12 may content Rosetta Stone of the outer membrane of E. coli and has proteins that are interactive peptides such been studied for decades (Tokunaga et al. as acetate CoA in E. coli that 1982). fuse into a single chain in other organisms. In this case, a branch 12 protein may Branch 10 (NIT proteins) contain a Rosetta Stone in that a particular NIT proteins are perhaps the most nitrilase-related domain is found to be important and studied nitrilase enzymes. fused into a terminal domain (amino) of an NIT was originally identified as an approximately 210 aminoacids (Marcotte approximately 300 aminoacid amino- et al. 1999). The branch 12 is also terminal extension on fly and worm associated with a domain that groups the homologs (Pekarsky et al. 1998), of human RimI superfamily of amino-terminal (Ohta et al. 1996) and murine (Fong et al. acetyltransferases (Yoshikawa et al. 1987). 2000), a Fhit tumor suppressor protein. The enzyme reaction type is an acyl group ©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 12 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 transfer that typically involves acetyl-CoA RimI-bisubstrate complex suggests that plus a ribosomal protein and L-alanine. In several residues change conformation upon E. coli is very common to find a group of interacting with the N terminus of S18, enzymes that acetylate the N-terminal including Glu103, the proposed alanine residues of specific ribosomal base, therefore facilitating proton exchange proteins (EC 2.3.1.88). Crystal structure of and catalysis (Hirosama et al. 2008). RimI has been determined in complex with Figure 1 shows a strict consensus tree CoA, AcCoA and CoA-S-acetyl-ARYFRR grouping some of the most important bisubstrate inhibitor. The structures are characterized nitrilases to date, including consistent with a direct nuclophilic some nitriles accepted as substrates by addition-elimination mechanism with each. Table 1 shows some of the nitriles Glu103 and Tyr115 acting as the catalytic accepted as substrates by nitrilases from base and acid, respectively. This is way the fungus like Aspergillus and Fusarium.

Figure 1. Neighbor-joining (NJ) tree of ten nitrilase/cyanide hydratase and sixteen putative nitrilase/cyanide hydratase proteins from fungi, bacteria and plants. Proteins, indicated as accession numbers (database available in http://ncbi.nlm.nih.gov) were aligned using Clustal X program (Thompson et al. 1997). The NJ tree was generated with PAUP program using the neighbour-joining method. Numbers on branches indicate bootstrap values from an analysis of 1,000 replicates. Amidase protein from Pseudomonas aeruginosa was used as the outgroup. The tree is annotated with the most active substrate for each protein; ARO, aromatic; ALIPH, aliphatic; ARYL, arylacetonitriles; Ala (CN); CN, cyanide).

3. Nitrile degrading enzymes and can be hydrolyzed by nitrilase activity into their role in plant metabolism auxins phenyl acetic acid and IAA. The hydrolysis of IAN into IAA by nitrilase Nitrilases are also present in plants in activity is an extremely well characterized cyanide metabolism, a nitrogenous reaction in plants and bacteria (Bartel, compound synthesized by these during 1997; Spaepen et al. 2007). However, the defense response and that originated as a IAN pathway is one of the several that co-product of ethylene biosynthesis. IAN have been identified for IAA biosynthesis ©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 13 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 in plants (Howden et al. 2009a; Lehmann the database. However, only recently the et al. 2010). The importance of the nitrilase gene of A. niger K10 has been production of auxins in plant development functionally analyzed (Kaplan et al. 2011), and physiology is recognized, as well as in which was found to be highly homologous the biosynthesis of IAA in plant- with putative nitrilases from Aspergillus microorganism interactions (Kobayashi et and Penicillium. IAN is an intermediate for al. 1993). While the biological role of plant IAA production and biosynthesis of this nitrilases is well understood, very little is phytohormone is not limited to higher known about the importance of these plants (Lehmann et al. 2010). Various enzymes in fungi. Some filamentous fungi pathways operate in IAA biosynthesis like Trichoderma species are able to among bacteria that inhabit the rhizosphere colonize the entire root of plants and their of plants: the indole-3-pyruvic pathway effects can be seen as positive promoting (IPyA), the indole-3-acetamide (IAM) lateral root development, leading to pathway, the tryptamine (TAM) pathway enhanced plant growth (Harman et al. and the IAN pathway (Spaepen et al. 2004; Shoresh et al. 2010; Yedidia et al. 2007). According to Zhao (2012), auxin 2001). Recently, it has been observed that biosynthesis regulated through using L-tryptophan (Trp) as an IAA transcriptional and protein level may precursor increased the production of IAA remain unknown. IAA, tryptophol, IAM, in Trichoderma virens cultured, and was IPyA and indole-lactic-acid were linked to the promotion of lateral root indentified from Colletotrichum acutatum development in A. thaliana-T. virens cultures supplemented with Trp, showing fungal interactions (Contreras-Cornejo et that this fungal pathogen may also al. 2009). Several studies have related synthesize IAA using various pathways nitrilase genes and enzymes as mentioned, (Chung et al. 2003). It has been also with the conversion of IAN into the plant documented that biosynthesis of IAA from growth factor IAA (Bartling et al. 1992; Trp proceeds through IPyA and IAAld in 1994; Howden et al. 2009a). Since the Ustilago esculenta (Chung and Tzeng, 1980s, bacteria have been mined as a 2004). The ability of some bacteria to source of nitrilases which have been produce auxins may be associated with exploited for biochemical synthesis and for pathogenicity, symbiosis or plant growth environmental remediation (Kobayashi et promotion. It is known that the nitrilase of al. 1993; Thuku et al. 2007). Fungal Pseudomonas syringae pv. syringae nitrilases have been less exploited than B7286, an arylacetonitrilase, is capable of bacterial nitrilases and although several hydrolyzing IAN into IAA, and allows it to reports suggest that the occurrence of use IAN as nitrogen source. This enzyme aromatic nitrilases in filamentous fungi is may represent an additional mechanism for common (Kaplan et al. 2006b; Martínková IAA biosynthesis by P. syringae, or may et al. 2009), only some fungal aromatic be used to degrade and assimilate nitrilases have been purified and aldoximes and nitriles produced by the characterized to date, these include those plant secondary metabolism (Howden et from F. solani IMI196840, F. oxysporum f. al. 2009b). Little information is available sp. melonis, F. solani O1, A. niger K10, about the role of IAN into IAA conversion Nectria haematococca and Arthroderma in fungal-plant interactions. This pathway benhamiae (Goldlust and Bohak, 1989; has been also found in plant pathogenic Harper, 1977b; Kaplan et al. 2006b; fungal species such as Taphrina wiesneri, Vejvoda et al. 2008; Veselá, et al. 2013). Taphrina deformans and Taphrina pruni, According to GenBank research, a large which cause hyperplastic diseases in plants number of putative nitrilase genes from like cherry, peach, and plum, respectively filamentous fungi have been deposited in (Yamada et al. 1990). Understanding

©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 14 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 indolic-pathways in plants, may lead to a a result new hypotheses in fundamental bioprospecting when coupling the power of biology (Morath et al. 2012). “omics” with biotechnology generating as

Table 1. Nitriles accepted as substrates by nitrilases from: Aspergillus niger K10; Fusarium solani O1; Fusarium solani IMI 196840; Fusarium oxysporum f. sp. melonis. According to Martínková et al. (2009) all nitriles shown have not been examined as substrates of all the four nitrilases. Substrate Aspergillus Fusarium Fusarium Fusarium niger K10 solani O1 solani IMI oxysporum f. 196840 sp. melonis Benzonitrile + + + + 3-hydroxybenzonitrile + + + + 3-chlorobenzonitrile + + + 4-chlorobenzonitrile + + + 1,4-Benzodinitrile + 3-cyanopyridine + + + + 4-cyanopyridine + + + Propionitrile + + + + Butyronitrile + + + + Valeronitrile + + + Acrylonitrile + + represents enzymatic activity observed.

4. Biotechnological importance of pesticides, colorants, fuel, solvents, nitrile-degrading enzymes in polycyclic aromatic hydrocarbons (PAHs), environmental remediation explosive, dyes and nitrile-substituents. Although these chemicals have contributed Nitrilase enzymes are becoming very to develop new technologies and a better important in the production and synthesis lifestyle for the humanity, several of them of different pharmaceutical and industrial may accumulate in soil, water and air chemicals. The importance and versatility (Iwamoto and Nasu, 2001). Because of as biocatalysts of these peptides lies in their nature, these are highly persistent and their potential applications in different clear examples are nitriles. The general scientific fields including synthetic toxicity of nitriles in humans are expressed biocatalysis and bioremediation (Banerjee as gastric system destabilization including et al. 2002). A bioremediation strategy vomiting and nausea, bronchial irritation, refers to an application of a biological respiration problems, convulsions, non process for the cleaning of hazardous induced coma which leads to irreversible pollutants that are present in the brain damage, lameness and skeletal environment, which is continuously deformities. The most severe symptoms of contaminated by a large array of chemicals nitrile intoxication are due to their capacity with different structures and toxicity levels to inactivate the respiratory system by that may be released from several tightly to citocrome-c-oxidase (inhibition anthropogenic activities (Gianfreda and of the electron transport chain) Rao, 2004). The main sources of pollution (Solomonson and Spehar, 1981). Some can be classified in three different orders other nitrile compounds like dihalogenated which comprise: industrial activities, benzonitrile analogues are active in a munitions waste and agricultural practices. number of herbicides and their use, The recent development of chemical chemical and physical properties including industries has produced a large variety of environmental toxicity have been reviewed chemical compounds that include recently (Holtze et al. 2008). As reviewed

©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 15 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 by Banerjee et al. (2002), some fungi and demonstrated that fungal metabolism of bacteria have enzymatic capabilities to cyanide is carried out by a mechanism metabolize natural and synthetic nitrile- consisting of a two-step hydrolytic complex. These enzymes may be of both pathway: conversion of cyanide into constitutive and inducible nature and show formamide by cyanide hydratase, followed similar features like optimum activity at by the conversion of formamide into alkaline pH with good stability in a large formate by an amidase (Dumestre et al. range of temperatures. In the mid 1990s, it 1997). Cyanide is a highly persistent was reported the containing of different chemical compound in the environment nitrile hydrolyzing enzymes in mixed when released indiscriminately, in most cultures of bacteria which were capable to cases, residues are found from industrial biodegrade effluents from acrylonitrile processes or as a by-product from mining manufacturing industries, concluding that exploitation, and today, the biota health 99 % of detectable toxic components repercussion from its contact is well measured decreased (Wyatt and Knowles, known. Likewise, some bacteria like 1995). Likewise, previous studies have Rhodococcus UKMP-5M have been demonstrated that acrylonitrile-containing recently applied to degrade cyanide polymer emulsions direct decontamination (Nallapan et al. 2013). A general metabolic may be treated by pathway proposed by Prasad and Bhalla enzymes, as well as the engineering of (2010) for nitrile synthesis and degradation transgenic plants resistant to the herbicide in microorganisms can be seen in figure 2. bromoxynil resulting from the introduction Cyanide hydratases are primarily fungal of microbial bromoxynil-specific nitrilase enzymes which are considered the first genes, confirming that this group of responsible from cyanide conversion. The proteins are potential prospects for the result of this is the bioremediation of polluted places (Battistel formation of formamide that subsequently et al. 1997; Freyssinet et al. 1996). It is decomposes to carbon dioxide and known that nitrile-degrading enzymes also ammonia by another formamide hydratase act over nitrile herbicides like dichlobenil enzyme (FHL). This kind of peptides (2,6-dichlorobenzonitrle) and bromoxynil. belongs to the leases family in which The peptides degrade these cyano group- hydrolyases can be found. The systematic containing herbicides and prevent them name of this type of enzyme is usually from entering the food chain. Some formamide hydro- (cyanide-forming) microorganisms like Agrobacterium (Gupta et al. 2010). Cyanide hydratases of radiobacter are commonly used for the several microorganisms may have degradation of both herbicides when they similarities among them and represent a are highly persistent in soils. Species like much more closely related group of Trichoderma spp. and Fusarium spp. have enzymes. The first cyanide hydratase was previously been demonstrated to degrade purified by Stemphylium loti, a pathogenic metallocyanides through the release of fungus of a cyanogenic plant. According to extracellular cyanide hydratase and its kinetic study, it was observed that the dihydratase enzymes (Muffadal and Lynch, highest activity occurred in the pH range of 2005). Another clear example of this is the 7.0-9.0. This means that one of the first degradation of simple cyanide by fungi, for reports of this peptide showed the neutral- example; purified cyanide hydratase alkaline nature of the family (Kunz et al. enzymes from Fusarium were found to be 1994). The common cause of nitrile entry capable to catalyze the hydration of into the environment may be effluent from cyanide to formamide (Cluness et al. industrial processes either engaged in 1993). This metabolic pathway was nitrile production or processing (Wyatt and clarified further in F. solani and it was Knowles, 1995). On the other hand,

©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 16 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 accidental spillage of nitrile from storing of nitrile contaminated soil with this strain tank (Deshkar et al. 2003), use of nitrile was successful as the nitrile degrading compounds as chemical herbicides organism became rapidly established (Vosahlova et al. 1997) and processing of within the microbial community of soil and oil shale deposit for extraction of oil lead it was also noted that the addition of to significant contamination of air, soil and acetonitrile significantly affected groundwater (Hawthorne et al. 1985; composition of bacterial community in the Aislabie and Atlas, 1988). Due to this fact, soil. In some cases, the biodegradation of removal of nitrile in nature from industrial nitriles by NHase also leads to hazardous effluents and contaminated places should metabolites, for example: 2,6- be mandatory. NHase in combination with dichlorobenzamide from dichlobenil as some other kind of nitrilase enzymes such dead-end product which is not hydrolyzed as amidases or nitrilases, have an important by amidase, and is more soluble and application in bioremediation processes of mobile than the parent compound in soil hazardous nitriles from the contaminated and groundwater (Holtze et al. 2008). air, soil and water systems. Nitrile- However, nitriles of shale oil can be degrading enzymes originated from selectively degraded by mixed cultures of microorganisms are widely distributed in nitrile hydrolyzing organisms such as soils that degrade a wide range of nitrile as Pseudomonas aeruginosa and carbon and nitrogen source. A consortium Pseudomonas fluorescens (Aislabie and of microorganisms from the adapted sludge Atlas, 1988). The soil microorganisms like and acetonitrile-degrading organisms has A. radiobacter 8/4 (Vosahlova et al. 1997), been proved quite effective for F. solani (Harper, 1977a), Klebsiella biodegradation of organonitriles (i.e. ozaenae (McBride et al. 1986) and saturated, unsaturated aliphatic and Nocardia sp. NCIMB 11215 (Harper, aromatic nitriles) in pharmaceutical waste- 1985) harbouring nitrile metabolizing water treatment plants (Li et al. 2007). enzymes efficiently degrade nitrile Baxter et al. (2006) explored the potential herbicides like bromoxynil, ioxynil (3,5- of a known acetonitrile-metabolizing diiodo-4-hydroxybenzonitrile), and organism (Rhodococcus sp. AJ270) for dichlobenil (2,6-dichlorobenzonitrile). degradation of acetonitrile and investigated its effects on resident soil bacterial 5. Conclusions and future community, concluding that the use of perspectives such microorganism could play an important role in the detoxification of this The exploitation of the catalytic properties toxic compound and thus, decreasing the of nitrile-degrading enzymes is constantly risk of environmental contamination. increasing due to the recognition of their Kobayashi and Shimizu (1998) proposed applicability in the production of several that the use of specialized consortia of synthetic compounds with industrial and microorganisms could be a viable pharmaceutical interest. In addition, alternative to activated sludge for the because of their role in plant metabolism as degradation and management of toxic well as plant-microbe interactions, the chemical wastes. Likewise, Kohyama et al. understanding of their structure and (2006) developed a process for the enzymatic properties allow better advances treatment of acetonitrile-containing wastes in genetic manipulation and biosynthetic by employing two nitrile-degrading regulation. Further structural studies of this microorganisms (viz. Rhodococcus type of proteins including application of pyridinovorans S85-2 and Brevundimonas site-directed mutagenesis or by directed diminuta AM10-C) as sources of NHase evolution, genome-mining and molecular and amidase respectively. Bioremediation characterization, may enable more stable engineered structures. A major ©The Author(s) 2014. This article is published with open access by Sociedad Latinoamericana de Biotecnología Ambiental y Algal. 17 Revista Latinoamericana de Biotecnología Ambiental y Algal Vol. 4 No. 1 p. 8-25 understanding of the physical properties of containing industrial wastewater. Through such enzymes including elucidation of better elucidation of structure and reaction reaction mechanisms, may lead to mechanisms of nitrilase enzymes it is improved properties like enhanced enzyme possible to increase the probability of activity and higher thermostability. With better progress in biosynthetic regulation an adequate manipulation of these including higher enzyme activity, characteristics, bioremediation processes stereospecificity and a wide range of would be easiest and successful. Despite applicability over a range of pH and recent discovers made in the last decade, temperature. Including these properties in a further application-oriented studies may multi-enzyme complex could be required to better exploit their considered for future environmental biotechnological applications. In addition, application-oriented studies that require deeper substrate specificity studies may fully exploit their biotechnological help to understand their catalytic potential. Despite the above properties, which are considered one of the considerations, further work is required to most critical steps in a manipulated optimize the technological application of enzymatic reaction. The discovery of new biological systems both in wastewater and nitrilase/cyanide hydratase enzymes that soils, with emphasis on the development of have the ability to hydrolyze nitrile- microorganism processes that face extreme substituent compounds whether fungal, environmental conditions, including high- plant or bacterial nature, increases level toxicity. In addition, new technology prospects to engineer proteins with may be required to ensure an effective directed-catalytic properties that assist in chemical and physical remediation strategy detoxification processes of cyanide- to combat nitrile pollution.

Figure 2. General metabolic pathway for nitrile synthesis and degradation in microorganisms. Prasad and Bhalla (2010).

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