Dimethylformamide is a novel nitrilase inducer in Rhodococcus rhodochrous.

Nitrilases are of commercial interest in the selective synthesis of carboxylic acids from nitriles. Nitrilase induction was achieved here in three bacterial strains through the incorporation of a previously unrecognised and inexpensive nitrilase inducer, dimethylformamide (DMF), during cultivation of two Rhodococcus rhodochrous strains (ATCC BAA-870 and PPPPB BD-1780), as well as a closely related organism (Pimelobacter simplex PPPPB BD-1781). Benzonitrile, a known nitrilase inducer, was ineffective in these strains. Biocatalytic product profiling, enzyme inhibition studies and protein sequencing were performed to distinguish the nitrilase activity from that of sequential nitrile hydratase-amidase activity. The expressed enzyme, a 40-kDa protein with high sequence similarity to nitrilase protein Uniprot Q-03217, hydrolyzed 3-cyanopyridine to produce nicotinic acid exclusively in strains BD-1780 and BD-1781. These strains were capable of synthesising both the vitamin nicotinic acid as well as ß-amino acids, a compound class of pharmaceutical interest. The induced nitrilase demonstrated high enantioselectivity (> 99%) in the hydrolysis of 3-amino-3-phenylpropanenitrile to the corresponding carboxylic acid.

Screening of potential bioremediation enzymes from hot spring bacteria using conventional plate assays and liquid chromatography - Tandem mass spectrometry (Lc-Ms/Ms).

The search for an eco-friendly, non-toxic, economical and efficient means of cleaning water through bioremediation is not only more favourable but critical to maintaining water quality globally especially in water-scarce countries. Thermophilic bacteria including Bacillus species are an important source of novel enzymes for biotechnology applications. In this study, 56 bacterial isolates which were cultured from five hot springs in South Africa were identified predominantly as Bacillus sp. or Bacillus-related spp by 16S rDNA gene sequencing. These isolates were screened for potentially useful enzymes for water bioremediation. Using conventional agar plate assays, 56% (n = 43), 68% (n = 38) and 16% (n = 31) were positive for amylase, protease and bromothymol blue decolorisation respectively. In liquid starch culture, three amylase-positive isolates differentially degraded starch by 34% (isolate 20S) to 98% (isolate 9T). Phenol degradation revealed that five out of thirty reduced phenol up to 42% by colorimetric assay. A thermophilic strain of Anoxybacillus rupiensis 19S (optimal growth temperature of 50 °C), which degraded starch, protein and phenol, was selected for further analysis by tandem LC-MS/MS. This newer technique identified potential enzymes for water bioremediation relating to pollutants from the food industry (amylase, proteases), polyaromatic hydrocarbons and dye pollutants (catalase peroxidase, superoxide dismutase, azoreductase, quinone oxidoreductase), antibiotic residues (ribonucleases), solubilisation of phosphates (inorganic pyrophosphatase) and reduction of chromate and lead. In addition, potential enzymes for biomonitoring of environmental pollutants were also identified. Specifically, dehydrogenases were found to decrease as the level of inorganic heavy metals and petroleum increased in soil samples. This study concludes that bacteria found in South African hot springs are a potential source of novel enzymes with tandem LC-MS/MS revealing substantially more information compared with conventional assays, which can be used for various applications of water bioremediation.

Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197.

AIMS: To investigate the production of soluble cross-reacting material 197 (CRM197 ) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines. METHODS AND RESULTS: The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulphide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM197 , at a production rate ~10% of the production of insoluble CRM197 , in equivalent small-scale cultures. Structural analysis of the purified CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favourably on all structural levels. CONCLUSIONS: SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197 in E. coli, which was previously unachievable. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous literature reports have shown that CRM197 can be expressed in E. coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM197 .

A DFT study of ruthenium pincer carboxylate complexes as potential catalysts for the direct carboxylation of arenes with CO2- meridional versus facial coordination.

A recent DFT study of the ruthenium pincer benzoate complex [Ru(PNP)(PhCOO)2] I (PNP = 2,6-bis(diphenylphosphanyl)lutidine) in its meridional form has revealed mer-I to be a promising catalyst lead structure for the direct insertion of CO2 into the C-H bonds of arenes, such as benzene. After the successful synthesis of I, its solid state structure interestingly and unexpectedly showed the pincer ligand to adopt the facial rather than the meridional coordination mode. Recalculation of the catalytic cycle with fac-I including all relevant local minima and transition states revealed (a) fac-I to be significantly more stable (6.1 kcal mol(-1)) than mer-I, (b) that the energetic span (ES; i.e. the effective activation barrier) for the cycle with fac-I amounts to 38.8 kcal mol(-1), while the cycle with mer-I has an ES of 25.5 kcal mol(-1) only. These results are a hint that fac-I is catalytically inactive. Experimental testing of fac-I showed indeed no product formation, which is in full accordance with the computations. To reduce the spatial flexibility of the pincer ligand, its CH2 groups were replaced by O atoms. The resulting complex [Ru(PONOP)(PhCOO)2] II (PONOP = 2,6-bis(diphenylphosphinito)pyridine) was used for the calculation of the catalytic cycle in benzene as the solvent. Gratifyingly, the starting complex mer-II is more stable than fac-II by 1.9 kcal mol(-1) in benzene as the solvent. Consequently, mer-II should be available experimentally. As with fac-I, also fac-II generates a catalytic cycle with a high ES (37.1 kcal mol(-1)), while mer-II generates a cycle with a significantly lower ES (27.2 kcal mol(-1)) indicating mer-II to be a potentially active catalyst. A possible explanation of the much lower ES in the case of the meridionally coordinated species is found in the stronger interaction of the substrate with the metal center in the arene-σ-bond complex. As a result the issue that is created by the mer/fac isomerism can be resolved by creating spatially less flexible structures.

Three-electron interatomic Coulombic decay from the inner-valence double-vacancy states in NeAr.

We have unambiguously identified interatomic Coulombic decay in NeAr from the inner-valence double-vacancy state Ne-Ar(2+)(3s(-2)) to outer-valence triple-vacancy states Ne(+)(2p(-1))-Ar(2+)(3p(-2)) by momentum-resolved electron-ion multicoincidence. This is the first observation of interatomic Coulombic decay where three electrons (3e) participate. The results suggest that this 3e interatomic Coulombic decay is significantly faster than other competing processes like fluorescence decay and charge transfer via curve crossing.

Electron-transfer-mediated decay and interatomic Coulombic decay from the triply ionized states in argon dimers.

We report the first observation of electron-transfer-mediated decay (ETMD) and interatomic Coulombic decay (ICD) from the triply charged states with an inner-valence vacancy, using the Ar dimer as an example. These ETMD and ICD processes, which lead to fragmentation of Ar(3+)-Ar into Ar(2+)-Ar(2+) and Ar(3+)-Ar+, respectively, are unambiguously identified by electron-ion-ion coincidence spectroscopy in which the kinetic energy of the ETMD or ICD electron and the kinetic energy release between the two fragment ions are measured in coincidence.