Explore the vast range of titles available.

Nitrilase (EC 3.5.5.1) is an important biological catalyst with industrial application, which can directly convert nitrile compounds into corresponding carboxylic acids and ammonia under mild conditions. Nitrilase is used to convert molecules containing nitrile groups into carboxylic acid derivatives, and it is also used in many industries such as the textile industry. Studies should be conducted on the production of nitrilase, which is widely used in industry and has the potential to be used in other industrial areas, from different sources. Since reducing the cost in the industry will reflect positively on the consumer, high-activity nitrilases should be obtained. For this purpose, optimization of the production mediums gains importance in the production of nitrilase from microbial sources. The present study deals with the production and optimization of the microbial nitrilase (EC 3.5.5.1). Microbial strains stored in the laboratory were used in this study. All selected strains ( Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), Pseudomonas putida (ATCC 17514), Pseudomonas fluorescens (DSM 6521), Pseudomonas oleovorans (DSM 50188), and Micrococcus luteus (DSM 20030)) from stock were screened on 3 different mediums. The acetonitrile showed to be an inducer for nitrilase production. The most active bacterium produced about 41.11 U/ml of nitrilase activity and was identified as Pseudomonas putida (ATCC 17514). Maximum nitrilase production was obtained using the medium (3) with 0.50 g/L (v/v) acetonitrile as an inducer. The effects of different parameters (chemical and physical) on the nitrilase (EC 3.5.5.1) production were studied. When bacterial growth at pH 8.0 and 28 ºC was cultured for 20 h, and medium containing starch as a carbon source and peptone-yeast extract as a nitrogen source, the nitrilase activity peaked at 172.76 U/ml. Enzyme activity could be increased 4.2 times with optimization studies. By optimization of enzyme production, a significant increase in activity may be achieved. In this study, when compared with similar studies about nitrilase production from bacteria in the literature, high-activity nitrilase production from Pseudomonas putida (ATCC 17514) was achieved. Graphical Abstract

Nitrilases are industrially important biocatalysts due to their ability to degrade nitriles to carboxylic acids and ammonia. In this study, a workflow for simple and fast recovery of nitrilase candidates from metagenomes is presented. For identification of active enzymes, a NADH-coupled high-throughput assay was established. Purification of enzymes could be omitted as the assay is based on crude extract containing the expressed putative nitrilases. In addition, long incubation times were avoided by combining nitrile and NADH conversion in a single reaction. This allowed the direct measurement of nitrile degradation and provided not only insights into substrate spectrum and specificity but also in degradation efficiency. The novel assay was used for investigation of candidate nitrilase-encoding genes. Seventy putative nitrilase-encoding gene and the corresponding deduced protein sequences identified during sequence-based screens of metagenomes derived from nitrile-treated microbial communities were analyzed. Subsequently, the assay was applied to 13 selected candidate genes and proteins. Six of the generated corresponding Escherichia coli clones produced nitrilases that showed activity and one unusual nitrilase was purified and analyzed. The activity of the novel arylacetonitrilase Nit09 exhibited a broad pH range and a high long-term stability. The enzyme showed high activity for arylacetonitriles with a KM of 1.29 mM and a Vmax of 13.85 U/mg protein for phenylacetonitrile. In conclusion, we provided a setup for simple and rapid analysis of putative nitrilase-encoding genes from sequence to function. The suitability was demonstrated by identification, isolation, and characterization of the arylacetonitrilase.Key points• A simple and fast high-throughput nitrilase screening was developed.• A set of putative nitrilases was successfully screened with the assay.• A novel arylacetonitrilase was identified, purified, and characterized in detail.

Nitrilase-mediated biocatalysis reactions have been continuously arousing wide interests by scholars and entrepreneurs in organic synthesis over the past six decades. Since regioselective nitrilases could hydrolyze only one cyano group of dinitriles into corresponding cyanocarboxylic acids, which are virtually impossible by chemical hydrolysis and of interest for a variety of applications, it becomes particularly appealing to synthetic chemists. The aim of the current review is to summarize the recent advancements on regioselective nitrilases concerning their fundamental researches and applications in synthesis of a series of high-value fine chemicals and pharmaceuticals. Carbon chain lengths and substituent group positions of substrates are found to be two crucial factors in affecting regioselectivity of nitrilase. Practical applications of regioselective nitrilases in synthesis of 1,5-dimethyl-2-piperidone (1,5-DMPD), atorvastatin, gabapentin, ( R )-baclofen, and ( S ) - pregabalin were systematically reviewed. Future perspectives clearly elucidating the mechanism of regioselectivity and further molecular modifications of regioselective nitrilases integrating within silico technology for industrial applications were discussed.

Nitrilases consist of a group of enzymes that catalyze the hydrolysis of organic cyanides. They are found ubiquitously distributed in the plant kingdom. Plant nitrilases are mainly involved in the detoxification of ß-cyanoalanine, a side-product of ethylene biosynthesis. In the model plant a second group of -specific nitrilases (NIT1-3) has been found. This so-called NIT1-subfamily has been associated with the conversion of indole-3-acetonitrile (IAN) into the major plant growth hormone, indole-3-acetic acid (IAA). However, apart of reported functions in defense responses to pathogens and in responses to sulfur depletion, conclusive insight into the general physiological function of the NIT-subfamily nitrilases remains elusive. In this report, we test both the contribution of the indole-3-acetaldoxime (IAOx) pathway to general auxin biosynthesis and the influence of altered nitrilase expression on plant development. Apart of a comprehensive transcriptomics approach to explore the role of the IAOx route in auxin formation, we took a genetic approach to disclose the function of NITRILASE 1 (NIT1) of . We show that NIT1 over-expression (NIT1ox) results in seedlings with shorter primary roots, and an increased number of lateral roots. In addition, NIT1ox plants exhibit drastic changes of both free IAA and IAN levels, which are suggested to be the reason for the observed phenotype. On the other hand, RNAi knockdown lines, capable of suppressing the expression of all members of the NIT1-subfamily, were generated and characterized to substantiate the above-mentioned findings. Our results demonstrate for the first time that Arabidopsis NIT1 has profound effects on root morphogenesis in early seedling development.

Cyanide is a nitrile which is used extensively in many industries like jewelry, mining, electroplating, plastics, dyes, paints, pharmaceuticals, food processing, and coal coking. Cyanides pose a serious health hazard due to their high affinity towards metals and cause malfunction of cellular respiration by inhibition of cytochrome c oxidase. This inhibition ultimately leads to histotoxic hypoxia, increased acidosis, reduced the functioning of the central nervous system and myocardial activity. Different physicochemical processes including oxidation by hydrogen peroxide, alkaline chlorination, and ozonization have been used to reduce cyanide waste from the environment. Microbial cyanide degradation which is considered as one the most successful techniques is used to take place through different biochemical/metabolic pathways involving reductive, oxidative, hydrolytic or substitution/transfer reactions. Groups of enzymes involved in microbial degradation are cyanidase, cyanide hydratase, formamidase, nitrilase, nitrile hydratase, cyanide dioxygenase, cyanide monooxygenase, cyanase and nitrogenase. In the future, more advancement of omics technologies and protein engineering will help us to recoup the environment from cyanide effluent. In this review, we have discussed the origin and environmental distribution of cyanide waste along with different bioremediation pathways and enzymes involved therein.

The use of halogen bond is widespread in drug discovery, design, and clinical trials, but is overlooked in drug biosynthesis. Here, the role of halogen bond in the nitrilase-catalyzed synthesis of ortho-, meta-, and para-chlorophenylacetic acid was investigated. Different distributions of halogen bond induced changes of substrate binding conformation and affected substrate selectivity. By engineering the halogen interaction, the substrate selectivity of the enzyme changed, with the implication that halogen bond plays an important role in biosynthesis and should be used as an efficient and reliable tool in enzymatic drug synthesis.

Soil salinization is increasing globally, driving a reduction in crop yields that threatens food security. Salinity stress reduces plant growth by exerting two stresses on plants: rapid shoot ion-independent effects which are largely osmotic and delayed ionic effects that are specific to salinity stress. In this study we set out to delineate the osmotic from the ionic effects of salinity stress. Arabidopsis thaliana plants were germinated and grown for two weeks in media supplemented with 50, 75, 100, or 125 mM NaCl (that imposes both an ionic and osmotic stress) or iso-osmolar concentrations (100, 150, 200, or 250 mM) of sorbitol, that imposes only an osmotic stress. A subsequent transcriptional analysis was performed to identify sets of genes that are differentially expressed in plants grown in (1) NaCl or (2) sorbitol compared to controls. A comparison of the gene sets identified genes that are differentially expressed under both challenge conditions (osmotic genes) and genes that are only differentially expressed in plants grown on NaCl (ionic genes, hereafter referred to as salt-specific genes). A pathway analysis of the osmotic and salt-specific gene lists revealed that distinct biological processes are modulated during growth under the two conditions. The list of salt-specific genes was enriched in the gene ontology (GO) term “response to auxin.” Quantification of the predominant auxin, indole-3-acetic acid (IAA) and IAA biosynthetic intermediates revealed that IAA levels are elevated in a salt-specific manner through increased IAA biosynthesis. Furthermore, the expression of NITRILASE 2 ( NIT2 ), which hydrolyses indole-3-acetonitile (IAN) into IAA, increased in a salt-specific manner. Overexpression of NIT2 resulted in increased IAA levels, improved Na:K ratios and enhanced survival and growth of Arabidopsis under saline conditions. Overall, our data suggest that auxin is involved in maintaining growth during the ionic stress imposed by saline conditions.

Cyanogenic glucosides (CG), the monoglycosides linamarin and lotaustralin, as well as the diglucosides linustatin and neolinustatin, have been identified in flax. The roles of CG and hydrogen cyanide (HCN), specifically the product of their breakdown, differ and are understood only to a certain extent. HCN is toxic to aerobic organisms as a respiratory inhibitor and to enzymes containing heavy metals. On the other hand, CG and HCN are important factors in the plant defense system against herbivores, insects and pathogens. In this study, fluctuations in CG levels during flax growth and development (using UPLC) and the expression of genes encoding key enzymes for their metabolism (valine N-monooxygenase, linamarase, cyanoalanine nitrilase and cyanoalanine synthase) using RT-PCR were analyzed. Linola cultivar and transgenic plants characterized by increased levels of sulfur amino acids were analyzed. This enabled the demonstration of a significant relationship between the cyanide detoxification process and general metabolism. Cyanogenic glucosides are used as nitrogen-containing precursors for the synthesis of amino acids, proteins and amines. Therefore, they not only perform protective functions against herbivores but are general plant growth regulators, especially since changes in their level have been shown to be strongly correlated with significant stages of plant development.

Cyanide is one of the most toxic chemicals for living organisms described so far. Its toxicity is mainly based on the high affinity that cyanide presents toward metals, provoking inhibition of essential metalloenzymes. Cyanide and its cyano-derivatives are produced in a large scale by many industrial activities related to recovering of precious metals in mining and jewelry, coke production, steel hardening, synthesis of organic chemicals, and food processing industries. As consequence, cyanide-containing wastes are accumulated in the environment becoming a risk to human health and ecosystems. Cyanide and related compounds, like nitriles and thiocyanate, are degraded aerobically by numerous bacteria, and therefore, biodegradation has been offered as a clean and cheap strategy to deal with these industrial wastes. Anaerobic biological treatments are often preferred options for wastewater biodegradation. However, at present very little is known about anaerobic degradation of these hazardous compounds. This review is focused on microbial degradation of cyanide and related compounds under anaerobiosis, exploring their potential application in bioremediation of industrial cyanide-containing wastes.