Nitrilase

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Introduction

Nitrilase enzymes (nitrilases) are responsible for catalyzing the hydrolysis of nitrile (R-CN) compounds into carboxylic acids and ammonia. Nitrilases are widely distributed and have been identified and characterized in plants, bacteria and fungi, and homologues have also been found in the genomes of yeasts and animals. Since the enzyme activity was first identified in plants in 1958 and in bacteria in 1964, more than 30 nitrilase enzymes have been characterized.

However, the purpose of identifying most bacterial nitrilases is to elucidate new mechanisms of chemical synthesis or degradation, rather than to study their functions in nature, so many of the biological effects of the enzymes are still unknown. Fortunately, more and more enzymes have now been identified, and their substrates and expression patterns have also been determined, so clues about their biological effects have slowly been revealed. Nitrile compounds are especially abundant in plant environments. A large amount of evidence shows that microbial nitrilase is an important part of the mechanism of promoting plant microbial colonization, which may play an important role in the synthesis of plant hormones, nitrogen utilization, and detoxification of nitriles and cyanides. Therefore, a deeper understanding of nitrilase and its role in plant-microbe interactions can have significant benefits for a range of biotechnology applications, including promoting plant growth, helping bioremediation, and disease control.

The nitrilase superfamily

The nitrilase superfamily (also known as CN-hydrolases) is composed of enzymes that catalyze the hydrolysis of non-peptide carbon-nitrogen bonds. The members of this superfamily are divided into 13 branches according to their catalytic activity and sequence identity, including aliphatic amidase, N‐terminal amidase, carbamylase, biotinidase, and nitrilase, among others. These enzymes are responsible for hydrolyzing the CN group in the nitrile compound, synthesizing the corresponding carboxylic acid and releasing ammonia. The reaction catalyzed by nitrilase is shown in Figure 1.

Figure 1. The nitrilase reaction. Nitrilases catalyse the hydrolysis of nitriles to the corresponding carboxylic acid plus ammonia (Howden, A.J.M.; Preston, G.M. 2009)

The nitrilase branch also contains the closely related cyanide dihydratase and cyanide hydratase enzymes. The nitrilase branching can be distinguished from other members of the superfamily by the conserved cysteine-tryptophan-glutamate motif located at the cysteine residues of the catalytic triad. This cysteine residue is considered to be the active site of the enzyme, and may be the site to which substrate groups attach prior to hydrolysis. Nitrilases can generally be divided into three categories based on substrate specificity: aliphatic nitrilase, aromatic and heterocyclic nitrilases, and arylacetonitrilases. Examples of each type of nitrile compound are shown in Figure 2.

Figure 2. Examples of nitrile compounds (Howden, A.J.M.; Preston, G.M. 2009)

Some nitrilase enzymes have extremely high substrate specificity, such as Klebsiella pneumoniae sp. ozaenaenitrilase. Some other enzymes have a wide range of substrates, such as the nitrilase of Bacillus pallidus Dac521, which can hydrolyze aliphatic, aromatic, and heterocyclic nitriles. Nitrilases with the same substrate specificity usually show amino acid sequence similarity and may belong to the same clade in phylogenetic analysis (Figure 3).

Figure 3. Strict consensus tree of characterized nitrilases (Howden, A.J.M.; Preston, G.M. 2009)

Industrial applications of nitrilases

Nitrilases can be used to synthesize industrially important carboxylic acids, other carboxylic acids are produced by chemical methods under extreme temperature and pH conditions. Nitrilases can also be used to bioremediate land and water resources contaminated with toxic nitrile compounds. Nitrile compounds such as bromoxynil can be used as herbicides, and cyanide is used in plastic manufacturing and precious metal extraction, and is released into the environment during mining, metal finishing and other processes. Due to the excellent cyanide degradation characteristics and the ability to tolerate high concentrations of KCN, the cyanide dihydratase of Pseudomonas stutzeri has been extensively studied. The nitrilase of K. pneumoniae sp. ozaenae is highly specific to the herbicide bromoxynil, which has been expressed in plants and confers herbicide resistance to transgenic lines.

Although nitrilases have great potential, it is difficult to identify and characterize the enzyme. Recently, scientists have developed a high-throughput strategy for the identification of bacterial nitrilase. DNA samples from the environment are transformed into bacterial expression vectors and screened for nitrilase activity. The availability of genome sequence data also helps to discover new nitrilase enzymes. For example, genomic sequence data has been used to identify a functional nitrilase from the cyanobacterium, Synechocystis sp.