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.

Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622 by GENE KIRK DRAGO Under the Direction of George E. Pierce ABSTRACT Bench- and pilot plant scale fed-batch fermentations were performed in stirred-tank bioreactors (STBR) with Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 in an attempt to elucidate parameters that may affect the optimization of a fermentation process for high biomass production and high inducible expression of cobalt-high-molecular-mass nitrile hydratase (Co-H-NHase. The effects of these factors on amidase (AMDase) activity were also investigated. Biomass and NHase production were inhibited by a total addition of acetonitrile and acrylonitrile (AC / AN) at 500 ppm during a 48 h run. Biomass and enzyme activity were uncoupled when the inoculum mass was increased from 4 g (wet weight) to ¡Ý 19 g. Other factors that allowed for the uncoupling of biomass production from enzyme activity were the reduction of the AC / AN feed rate from a step-addition at 2500 ¦Ìl / min to a continuous addition at 80 ¨C 120 ¦Ìl / min, and the delay to 18 h post-inoculation the time of initial inducer addition. The inhibition of both biomass production and NHase activity was relieved when both the total concentration of AC / AN was reduced to ¡Ü 350 ppm and the AC / AN feedrate was reduced. The factors with the greatest influence were shown to be the inducer, the inducer concentration, inoculum mass and source as well as the major carbohydrate and nitrogen source. In addition, this lab is the first to report high AN-specific NHase induction by asparagine (1300 ppm) in a fed-batch fermentation system. Prior to this program, 250 mg of cells (wet weight) per liter could be provided in 4 ¨C 10 days with an activity of 1 U NHase per mg of cells (dry weight). Current production is > 50 g / L in 48 h with an NHase activity > 150 U / mg of dry cell weight. INDEX WORDS: Amidase, Asparagine, Biodetoxification, Fermentation, Nitrile, Nitrile Hydratase, Rhodococcus

Treatment of industrial wastewaters contaminated with toxic and hazardous organics can be a costly process. In the case of acrylonitrile production, due to highly volatile and toxic nature of the contaminant organics, production wastewaters are currently disposed by deepwell injection without treatment. Under the terms granting deepwell injection of the waste, alternative treatments must be investigated, and an effective treatment identified. Cells of two Gram-positive bacteria, Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 were evaluated for their potential as biocatalysts for detoxification of acrylonitrile production wastewaters. Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 when multiply induced, are capable of utilizing the hazardous nitrile and amide components present in the wastewater as sole carbon and/or nitrogen sources, employing a 2-step enzymatic system involving nitrile hydratase (NHase) and amidase enzymes. There is a significant potential for overproduction of NHase upon multiple induction. However, high-level multiple induction required the presence of highly toxic nitriles and/or amides in the growth medium. Asparagine and glutamine were identified as potent inducers with overexpression at 40% of total soluble cellular protein as NHase. In native form (either cell free enzymes or whole cells) the desired NHase is very labile. In order to develop a practical catalyst to detoxify acrylonitrile production wastewaters, it is necessary to significantly improve and enhance the stability of NHase. Stabilization of desired NHase activity was achieved over a broad range of thermal and pH conditions using simultaneous immobilization and chemical stabilization. Previously where 100% of NHase activity was lost in 24 hours in the non-stabilized cells, retention of 20% of initial activity was retained over 260 days when maintained at 50-55 C, and for over 570 days for selected catalyst formulations maintained at proposed temperature of the biodetoxification process. In addition, NHase and amidase enzymes from Rhodococcus sp. DAP 96253 were purified. Cell free NHase was characterized for its substrate range and effect of common enzyme inhibitors and was compared to available information for NHase from other organisms. As a result of this research a practical alternative to the deepwell injection of acrylonitrile production wastewaters is closer to reality.

Rhodococcus is an important industrial organism that possesses diverse metabolic capabilities, it also has a unique cell envelope, composed of an outer layer of mycolic acids and glycolipids (free or bound lipids generally linked to the sugar trehalose). Rhodococcus is able to transform nitriles to the corresponding amide by the enzyme Nitrile Hydratase (NHase), therefore rhodococcal cells can be utilized as biocatalysts in the detoxification of nitrile waste water or in the production of industrially important amides such as acrylamide. However, the NHase within the native cells must be stable with high activity. This research examined how NHase activity and stability can be increased in native cells by changing growth media composition, the impact on the rhodococcal cell envelope was also studied. Growth media composition was altered by supplementing different sugars such as fructose, maltose or maltodextrin to replace glucose in rich solid media containing cobalt and urea for induction of NHase. The supplementation of maltose or maltodextrin resulted in significantly higher NHase activities and greater NHase stability at 55„aC. The supplementation of these different sugars was shown to alter cellular and lipid bound trehalose levels, a sugar known to stabilize proteins and a component of the rhodococcal cell envelope. Cells that had higher levels of cellular trehalose had significantly greater NHase stability at 55„aC. The effect of the different sugar supplements and inducers of NHase, such as cobalt, on cell envelope components such as mycolic acids and glycolipids were examined by High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC). The results showed that changes in mycolic acids and glycolipids occurred when the cells were grown in the presence of different sugar supplements and when the cells were induced for NHase. Susceptibility of Rhodococcus sp DAP 96253 to different antibiotics was examined to indicate if changes were occurring in the cell envelope. Differences in antibiotic susceptibility were observed when the cells were grown on media with different sugar supplements and when the cells were induced for NHase. In the presence of cobalt Rhodococcus sp DAP 96253 showed a significant increase in sensitivity to antibiotics. Changes in growth media composition influences the cell envelope of Rhodococcus sp DAP 96253 and also affects NHase activity and stability. Therefore, achieving increased enzyme activity and stability is not entirely dependent on the actual enzyme, but is related to other aspects of the cell, such as the cell envelope and metabolites of the cell.

The strain Rhodococcus erythropolis A4 is a source of enzymes nitrilhydratase and amidase, that catalyse conversion of nitriles and amides. These enzymes are used in industrial biotransformation and bioremediation. Since it was difficult to carry out genetic manipulations aimed at increasing the production of these enzymes in the strain A4, the corresponding genes (ami and nha1 + nha2) of a related strain R. erythropolis CCM2595, in which both plasmid and chromosome manipulations can be routinely performed, were identified and analyzed in this diploma theses. The ami and nha1 + nha2 genes from the strain R. erythropolis CCM2595 were isolated and sequenced together with the flanking regions (5.5 kb in total). The organization of these genes and the expected regulatory genes was described in the strain CCM2595 and mechanisms of regulation of expression of these genes were studied. For the analysis of transcription of amidase and nitrilhydratase genes from both strains of R. erythropolis, the promoter-probe vector pEPR1 replicating in Escherichia coli and R. erythropolis was used. Transcriptional fusion of Pami promoters of the strains A4 and CCM2595 and the reporter gfp gene were constructed. The activity of the Pami promoter was measured by means of fluorescence of gfp gene product (green fluorescent...

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