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Nitrile hydratase activity in Rhodococcus rhodochrous DAP 96253 can be induced with multiple inducers that include urea, cobalt (Co), iron (Fe) and nickel (Ni). When induced with Co/urea, cells of R. rhodochrous DAP 96253 expressed the highest level of nitrile hydratase activity (~200 units/min·mg-cdw) when compared with the other inducers tested. Cells induced with Co had the second highest nitrile hydratase activity (~7 units/min·mg-cdw), whereas in the uninduced cells, nitrile hydratase activity was lower than 1 unit/min·mg-cdw. Similarly in R. rhodochrous DAP 96622, when induced with Co/urea, the nitrile hydratase activity of R. rhodochrous DAP 96622 cells was around 50 units/min·mg-cdw which was the highest of all inducers tested. When induced with Co only, the nitrile hydratase activity of R. rhodochrous DAP 96622 was around 20 units/min·mg-cdw, and the nitrile hydratase activity of R. rhodochrous DAP 96622 uninduced was the same as the nitrile hydratase activity of uninduced R. rhodochrous DAP 96253. When Co/urea induced R. rhodochrous DAP 96253 cell lysate was examined on gradient SDS-PAGE and analyzed by Image Quant TL, the nitrile hydratase bands (both α and β subunits) accounted for more than 55% of the total cytosolic proteins. Whereas in Co/urea induced R. rhodochrous DAP 96622, the nitrile hydratase bands accounted for around 25% of the total cytosolic proteins. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry results, amidase in R. rhodochrous DAP 96253 was approximately 38 kDa from the nitrilase/cyanide hydratase family and amidase in R. rhodochrous DAP 96622 was 55 kDa from the amidase signature family. In addition, the nitrile hydratase regulation system in both R. rhodochrous DAP 96253 and DAP 96622 strains are different. Moreover, the nitrile hydratase regulation system in R. rhodochrous DAP 96253 is different from R. rhodochrous J1. Purified nitrile hydratase from R. rhodochrous DAP 96253 may form a protein complex with glutamine synthetase, resulting in a nitrile hydratase activity of approximately 1500 units/mg-proteins, and nitrile hydratase from R. rhodochrous DAP 96622 is not a protein complex and results in a nitrile hydratase activity of 950 units/mg-proteins.

ABSTRACT Recent studies have shown that R. rhodochrous DAP 96253 has the ability to delay the ripening of many climacteric fruit, by potentially degrading volatile compounds released by plant cells during the ripening process. Rhodococcus rhodochrous DAP 96253 cells were cultured on YEMEA medium supplemented with inducers, (16mM cobalt and 125mM urea), that over-expressed nitrile hydratase (NHase) and amidase (AMDase) enzymes. Cells were cultured on propylene/ ethylene as sole carbon source to induce alkene monooxygenase (AMO) like activity. Induced R. rhodochrous DAP 96253 cells displayed an 83% increase in final total dry weight compared to cells previously cultured on non-induced medium. Induced R. rhodochrous DAP 96253 cells displayed a 53-85% increase in NHase activity after exposure to propylene/ethylene, and cells displayed a 24-53% increase in NHase activity after exposure to fruit. Non-induced R. rhodochrous DAP 96253 cells displayed a 1-5% increase in NHase activity after propylene/ethylene, and cells displayed an 18-38% increase in NHase activity after exposure to fruit. Propylene/ethylene induced nitrilase activity in non-induced R. rhodochrous DAP 96253cells. Experimental results suggest that R. rhodochrous DAP 96253 may use NHase, amidase, nitrilase, and AMO like activity to delay ripening of climacteric fruit. Rhodococcus rhodochrous 96253 cells cultured on propylene/ethylene and cofactors (16mM cobalt and 125mM urea) displayed improved ability to delay ripening of fruit.

ABSTRACT Recent studies have shown that R. rhodochrous DAP 96253 has the ability to delay the ripening of many climacteric fruit, by potentially degrading volatile compounds released by plant cells during the ripening process. Rhodococcus rhodochrous DAP 96253 cells were cultured on YEMEA medium supplemented with inducers, (16mM cobalt and 125mM urea), that over-expressed nitrile hydratase (NHase) and amidase (AMDase) enzymes. Cells were cultured on propylene/ ethylene as sole carbon source to induce alkene monooxygenase (AMO) like activity. Induced R. rhodochrous DAP 96253 cells displayed an 83% increase in final total dry weight compared to cells previously cultured on non-induced medium. Induced R. rhodochrous DAP 96253 cells displayed a 53-85% increase in NHase activity after exposure to propylene/ethylene, and cells displayed a 24-53% increase in NHase activity after exposure to fruit. Non-induced R. rhodochrous DAP 96253 cells displayed a 1-5% increase in NHase activity after propylene/ethylene, and cells displayed an 18-38% increase in NHase activity after exposure to fruit. Propylene/ethylene induced nitrilase activity in non-induced R. rhodochrous DAP 96253cells. Experimental results suggest that R. rhodochrous DAP 96253 may use NHase, amidase, nitrilase, and AMO like activity to delay ripening of climacteric fruit. Rhodococcus rhodochrous 96253 cells cultured on propylene/ethylene and cofactors (16mM cobalt and 125mM urea) displayed improved ability to delay ripening of fruit.

Student Number : 0009756Y - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / A versatile nitrile-degrading bacterium was isolated through enrichment culturing of soil samples from Johannesburg, South Africa. It was identified as Rhodococcus rhodochrous and submitted to the ATCC culture collection as strain BAA-870. This organism was determined to be a potential biocatalyst in that it contains a two enzyme system with strong nitrile-converting activity comprising nitrile hydratase and amidase. The development of a suitable assay for measuring the activity of the enzymes of interest was explored. A pHsensitive indicator-based assay was found to be suitable only for colorimetrically identifying highly concentrated enzymes with acid-forming activity. An ophthaldialdehyde- based fluorimetric assay was found to be applicable to conversions of select compounds, but the assay could not be used to measure the activity of Rhodoccocus rhodochrous ATCC BAA-870. High performance liquid chromatography was the most suitable method for reliable and quantitative measurement of nitrile hydrolysis, and is applicable to monitoring activities of whole-cell and cell-free extracts. Initial analysis of six compounds, benzonitrile, benzamide, benzoic acid, hydrocinnamonitrile, 3-hydroxy-3- phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid, was performed by HPLC to measure linearly the average retention area, amount and absorbance of the compounds up to 10 mM concentrations. The conversion of the substrates benzonitrile, benzamide and 3- hydroxy-3-phenylpropionitrile were further analysed with respect to time and enzyme concentration. Conversion of benzonitrile to benzamide by the nitrile hydratase was rapid and could be measured in 10 minutes. Conversion of benzamide to benzoic acid by the amidase was considered the rate-limiting step and could be followed for 90 minutes of the reaction at the concentrations tested. Conversion of 3-hydroxy-3-phenylpropionitrile was linearly measured over 20 minutes. Mass spectral analysis was used to confirm, at a structural level, relatively less volatile reactant compounds with a higher thermal stability, including benzamide, 3-hydroxy-3-phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid. Protein concentration studies indicated that activity against benzonitrile was probably due to a nitrile hydratase with potent activity rather than a concentrated enzyme, since formation of benzamide from benzonitrile showed first order reaction kinetics at protein concentrations less than 0.2 mg/ml. Formation of benzoic acid from benzamide was linear up to 1.3 mg total protein and product formation from 3-hydroxy-3-phenylpropionitrile was linear up to 1.4 mg total protein. Overlapping activities against benzonitrile and 3- hydroxy-3-phenylpropionitrile indicate that the nitrile hydratase has differing substrate specificity for the two compounds, with higher activity toward the small aromatic mononitrile, benzonitrile, than the arylaliphatic b-hydroxy nitrile, 3-hydroxy-3- phenylpropionitrile. The nitrile-converting activity of Rhodococcus rhodochrous ATCC BAA-870 would be suitable for biocatalysis as the conversions take place under a wide pH range, require low concentrations of enzyme and reactions are fast. Separation of nitrileconverting activities in Rhodococcus rhodochrous ATCC BAA-870 was undertaken using various chromatography methods to establish a simple, one-step protocol for biocatalytic enzyme preparations. HPLC was not suited to assaying nitrile-converting activity in chromatofocusing fractions, and chromatofocusing Ampholyte buffers were found to interfere with activity measurements. Gel exclusion chromatography of the soluble protein extract from Rhodococcus rhodochrous ATCC BAA-870 indicated the enzyme/s responsible for nitrile hydratase activity are high molecular weight proteins ranging from 40 to 700 kDa in size, while the amidase native enzyme is proposed to be roughly 17 to 25 kDa. SDS-PAGE analysis of gel exclusion and ion exchange chromatography fractions indicated nitrile converting activity in Rhodococcus rhodochrous ATCC BAA-870 is likely due to multimer-forming enzymes made up of 84, 56, 48 and 21 kDa subunits. It is postulated that nitrile hydratase is made up of ab and a2b2 tetramers that may form larger enzyme aggregates. Ion exchange chromatography was used to separate nitrile hydratase with high activity against benzonitrile and 3-hydroxy-3-phenylpropionitrile from amidase activity, and showed that an additional, substrate specific nitrile hydratase may exist in the organism.

Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 are contained in a cluster that includes also a gene specifying an oxygenase. Given the possible connection of Nit1C-endoded nitrilase and oxygenase enzymes to enzymatic cyanide degradation, there is strong reason for thinking that the genes specifying these enzymes contribute to bacterial growth on cyanide in those bacteria containing the Nit1C cluster. Because the biological function of the Hyp1 protein is currently unknown, it was cloned and the protein expressed in E. coli so that its properties could further be explored. Unfortunately, the expression of the protein in an insoluble form complicated these analyses. However, at least two lines of evidence suggest a possible role as a regulator of gene expression. First, over-expression of the protein was accompanied by the parallel elevation of the putative vector-encoded b-lactamase, implying that Hyp1Pf11764 can affect the expression of other genes. Second, a comparison of the amino acid sequence of select peptide fragments of Hyp1Pf11764, by conducting searches for homology with proteins in the existing nonredundant protein database, consistently revealed motifs in common with those present in bacterial response regulators that are part of two-component signal transduction systems widely distributed in bacteria.

Nitrilases are enzymes able to convert toxic nitriles to corresponding carboxylic acids or amides. Thus they might be used in the detoxification of dyes, herbicides and pharmaceutical intermediates and byproducts. They can be used also for enzymatic syntheses of carboxylic acids not available by standard procedures. The aim of this diploma thesis is a recombinant expression of nitrilases from Neurospora crassa and the optimization of their purification. Cells of E. coli (BL 21 Gold) were utilized as an expression system. The purification was performed by ion-exchange chromatography, chelation chromatography and gel filtration - all under reducing conditions. Purified enzymes were studied by sedimentation analysis in an analytical ultracentrifuge. They were also used for searching of optimal conditions for their crystallization. Keywords: nitrilase, Neurospora crassa, recombinant expression

Cyanide is produced throughout a plant's life cycle alongside the hormone ethylene by oxidation of 1-aminocyclopropane-1-carboxylic acid. Production increases during certain developmental stages such as seed germination, seedling elongation, fruit ripening and senescence. Abiotic stresses increase ethylene production giving rise to `stress cyanide'. Cyanide also comes from metabolism of cyanogenic compounds. Cyanide is however, a toxic chemical which readily binds to metallo-enzymes inhibiting primary metabolic processes. Plants have mechanisms to maintain cyanide homeostasis such as the β-cyanoalanine pathway whereby cysteine reacts with cyanide forming β--cyanoalanine, mediated by β-cyanoalanine synthase and cysteine synthase. A dual nitrilase 4 enzyme then converts the β-cyanoalanine into asparagine or aspartate and ammonium. Studies have suggested that the physiological function of the pathway is not restricted to detoxification and assimilation of excess cyanide. The overall research goal was to investigate the role of the pathway in plant tolerance to water deficit and exogenous cyanide exposure in Arabidopsis thaliana. The first objective was to investigate responsiveness of the pathway to duration and intensity of water deficit and cyanide exposure. The second was to investigate the contribution of enzymes associated with the pathway to cyanide metabolism. The questions addressed were whether there is enzymatic redundancy in enzymes associated with the first step of cyanide detoxification and whether there is pathway redundancy between the β-cyanoalanine and an alternative sulfurtransferase pathway. A. thaliana Col-0 and three SALK-line mutants with a T-DNA insertion for the genesAtCysA1, AtCysC1 and AtNIT4 were grown and exposed to water stress. Physiological and biochemical measurements were taken. The results showed a transient increase in cyanide concentration and β-cyanoalanine synthase activity on exposure to stress. The response pattern was similar regardless of intensity or duration of stress. Knocking out AtCysA1 or AtCysC1 did not impair the ability of plants to metabolize cyanide and tolerate stress i.e the enzymes were functionally redundant. The AtNIT4 mutant however, was impaired in cyanide metabolism and exhibited a sensitive phenotype under both stresses, suggesting that the cyanoalanine pathway is the sole pathway in cyanide detoxification. The results show that the pathway may be an important tool in improvement of plant tolerance to abiotic stress.

Nitrilases are enzymes which catalyze the hydrolysis of nitriles to corresponding carboxylic acids. These enzymes have a great potential in biocatalysis, for example in the synthesis of mandelic acid and mandelamide, because of their chemo- and enantioselectivity. As bioremediation agents they are also applicable to sites contaminated with organic nitriles. In this work, activities of recombinant strains of E. coli expressing hypothetical nitrilases from fungi Giberella moniliformis and Nectria haematococca mpVI 77-13-4 were studied, as well as the biodegradation potential of bacteria from Rhodococcus and Nocardia genera towards benzonitrile herbicides dichlobenil (2,6-dichlorobenzonitrile), ioxynil (3,5-diiodo-4- hydroxybenzonitrile) and bromoxynil (3,5-dibromo-4-hydroxybenzonitrile). The hypothetical fungal nitrilases were expressed as functional enzymes. Nitrilase from G. moniliformis showed highest activity towards benzonitrile (30.9 U/mg protein), total activity yield was 2,560 U/l cell culture. The preferred substrate of the nitrilase from N. haematococca was phenylacetonitrile (12.3 U/mg prot.), total activity yield was 28,050 U/l cell culture. Nitrilase from N. haematococca was also able to hydrolyze mandelonitrile (5.9 U/mg prot.). Soil bacteria Rhodococcus rhodochrous PA-34, Nocardia globerula...