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Christopher J Knowles - One of the best experts on this subject based on the ideXlab platform.
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Biotransformation of benzonitrile to benzohydroxamic acid by Rhodococcus rhodochrous in the presence of hydroxylamine
Biotechnology Letters, 2001Co-Authors: Michael R. Dadd, Alan John Pettman, Timothy D. W. Claridge, Christopher J KnowlesAbstract:Whole cells of the bacterium Rhodococcus rhodochrous LL100-21, which had been grown on benzonitrile to induce the nitrilase enzyme, converted benzonitrile to benzohydroxamic acid in the presence of hydroxylamine.
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The nitrilases of Rhodococcus rhodochrous NCIMB 11216
Enzyme and Microbial Technology, 1998Co-Authors: Alison J Hoyle, Alan W Bunch, Christopher J KnowlesAbstract:Rhodococcus rhodochrous NCIMB 11216 grows on propionitrile or benzonitrile as the sole source of carbon and nitrogen. The possibility that different nitrile-hydrolyzing enzymes were produced under these two growth conditions was investigated. Nitrilase activity in whole cell suspensions from either bacteria grown on propionitrile or benzonitrile were capable of biotransforming a wide range of nitriles. The propionitrile-induced nitrile degrading activity hydrolyzed 3-cyanobenzoate and both the nitrile groups in 1,3-dicyanobenzoate. In contrast, the benzonitrile-induced activity hydrolyzed only one of the nitrile groups in 1,3-dicyanobenzoate, but did not affect 3-cyanobenzoate. Both nitrilases biotransformed α-cyano-o-tolunitrile to produce 2-cyanophenylacetic acid. The nitrilases were purified by fast protein liquid chromatography and the n-terminus of each enzyme sequenced. SDS-PAGE analysis identified a subunit molecular weight of 45.8 kDa for each nitrilase. The n-terminal sequences showed significant similarity with other sequenced nitrilases and with the exception of a single amino acid were identical with each other. Both nitrilases had temperature and pH optima of 30°C and 8.0, respectively. The propionitrile-induced nitrilase had a Km for benzonitrile of 20.7 mm and a Vmax of 12.4 μmol min−1 mg−1 protein whereas the benzonitrile-induced nitrilase had a Km for benzonitrile of 8.83 mm and a Vmax of 0.57 μmol min−1 mg−1 protein.
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The nitrilases of Rhodococcus rhodochrous NCIMB 11216
Enzyme and Microbial Technology, 1998Co-Authors: Alison J Hoyle, Alan W Bunch, Christopher J KnowlesAbstract:Rhodococcus rhodochrous NCIMB 11216 grows on propionitrile or benzonitrile as the sole source of carbon and nitrogen. The possibility that different nitrile-hydrolyzing enzymes were produced under these two growth conditions was investigated. Nitrilase activity in whole cell suspensions from either bacteria grown on propionitrile or benzonitrile were capable of biotransforming a wide range of nitriles. The propionitrile-induced nitrile degrading activity hydrolyzed 3-cyanobenzoate and both the nitrile groups in 1,3-dicyanobenzoate. In contrast, the benzonitrile-induced activity hydrolyzed only one of the nitrile groups in 1,3-dicyanobenzoate, but did not affect 3-cyanobenzoate. Both nitrilases biotransformed alpha-cyano-o-tolunitrile to produce 2-cyanophenylacetic acid. The nitrilases were purified by fast protein liquid chromatography and the iv-terminus of each enzyme sequenced. SDS-PAGE analysis identified a subunit molecular weight of 45.8 kDa for each nitrilase. The N-terminal sequences showed significant similarity with other sequenced nitrilases and with the exception of a single amino acid were identical with each other. Both nitrilases had temperature and pH optima of 30 degrees C and 8.0, respectively. The propionitrile-induced nitrilase had a K-m for benzonitrile of 20.7 mM and a V-max of 12.4 mu mol min(-1) mg(-1) protein whereas the benzonitrile-induced nitrilase had a K-m for benzonitrile of 8.83 mM and a V-max of 0.57 mu mol min(-1) mg(-1) protein
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Asmmetric Hydrolysis of Chiral Nitriles by Rhodococcus-rhodochrous NCI-11216 Nitrlase
1994Co-Authors: Michelle L. Gradley, Christopher J KnowlesAbstract:The enantioselective potential of Rhodococcus rhodochrous NCIMB 11216 nitrilase has been measured, using a range of chiral nitriles with various C-2 group substitutions. The highest selectivity was achieved during the biotransformation of (+/-) 2-methylhexanitrile where the reaction appears enantiospecific for the (+) enantiomer.
Yuda Yürüm - One of the best experts on this subject based on the ideXlab platform.
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Biodesulfurization of Turkish lignites: 2. Microbial desulfurization of Mengen lignite by the mesophilic microorganism Rhodococcus rhodochrous
Fuel, 1997Co-Authors: Tülay Durusoy, Tijen Özbaşbozdemir, Evrim Erincin, Yuda YürümAbstract:Abstract The effects of substrate type in the growth medium, mixing time of lignite into the growth medium and the biodesulfurization time on sulfur removal were studied. Biodesulfurization experiments were carried out with Mengen lignite under optimum growth conditions of Rhodococcus rhodochrous . The highest reduction of organic sulfur forms was 27.1% when sodium acetate was the substrate. Sulfate sulfur could be totally reduced when lignite was added to the culture medium 24 h after incubation. Compared with sodium acetate, glycerol yielded higher sulfate sulfur reduction rates when lignite was added at the time of incubation. The highest organic sulfur removal rates were found when sodium acetate was the substrate.
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Biodesulfurization of Turkish lignites: 1. Optimization of the growth parameters of Rhodococcus rhodochrous, a sulfur-removing bacterium
Fuel, 1996Co-Authors: Tijen Ozbas Bozdemir, Tülay Durusoy, Evrim Erincin, Yuda YürümAbstract:Abstract The effects of inoculum percentage, initial pH, growth temperature, shaking rate, substrate type and initial substrate concentration on the growth kinetics of Rhodococcus rhodochrous , a sulfur-removing bacterium, were investigated. The optimum value for each parameter that favoured the maximum specific growth rate was calculated from models established through linear of non-linear regression of the relevant data. Around these optima, biodesulfurization experiments were performed with sterile lignite. An increase in sulfate, pyritic and organic sulfur reduction with increasing reaction time were observed. The highest decreases in the total (30.2%) and organic sulfur (27.1%) contents were obtained with Mengen lignite at 75 h of biodesulfurization.
Tek Chand Bhalla - One of the best experts on this subject based on the ideXlab platform.
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Expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in Pichia pastoris
Biocatalysis and Biotransformation, 2016Co-Authors: Amit Pratush, Amit Seth, Tek Chand BhallaAbstract:The nitrile hydratase (NHase) gene of Rhodococcus rhodochrous PA-34 mutant 4D has been amplified by PCR, cloned and expressed in Pichia pastoris KM-71 using pHIL-D2 expression vector. The recombina...
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Purification and characterization of nitrile hydratase of mutant 4D of Rhodococcus rhodochrous PA-34
3 Biotech, 2012Co-Authors: Amit Pratush, Amit Seth, Tek Chand BhallaAbstract:Nitrile hydratase (NHase; E.C. 4.2.1.84) has been purified and characterized using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography from the mutant 4D of Rhodococcus rhodochrous PA-34. The SDS-PAGE and MALDI-TOF analysis of the purified enzyme revealed that it is dimmer consisting of α- and β-subunits with a molecular mass of 25 and 30 kDa, respectively. The Km and Vmax values were 102 mM and 350.8 μmol/min/mg using 3-cyanopyridine as substrate. The purified NHase was stable in higher concentration of potassium ions and in acidic pH 5.5 as compared to NHase of the wild R. rhodochrous PA-34. The analysis of the N-terminal amino acid sequence of this enzyme revealed that this enzyme has 90 % homology with the high molecular weight nitrile hydratase of R. rhodochrous J1.
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Generation of mutant of Rhodococcus rhodochrous PA-34 through chemical mutagenesis for hyperproduction of nitrile hydratase.
Acta microbiologica et immunologica Hungarica, 2010Co-Authors: Amit Pratush, Amit Seth, Tek Chand BhallaAbstract:Rhodococcus rhodochrous PA-34 has been reported to produce nitrile hydratase enzyme that converts 3-cyanopyridine to nicotinamide. A mutant of R. rhodochrous PA-34 was generated through chemical mutagenesis using N-methyl-N-nitro-N-nitrosoguanidine (MNNG) that exhibited 2 times higher nitrile hydratase activity as compared to wild strain. The reaction conditions using resting cells of this mutant strain for the conversion of nicotinamide were optimized. Under the optimized reaction conditions the mutant strain exhibited maximum nitrile hydratase activity [7.8 U/mgdcm (milligram dry cell mass)] at 55 °C in 0.3 M potassium phosphate buffer (pH 5.5).
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Bench scale conversion of 3-cyanopyidine to nicotinamide using resting cells of Rhodococcus rhodochrous PA-34.
Indian Journal of Microbiology, 2007Co-Authors: Shreenath Prasad, Tek Chand BhallaAbstract:The nitrile hydratase (NHase, EC 3.5.5.1) activity of Rhodococcus rhodochrous PA-34 was explored for the conversion of 3-cyanopyridine to nicotinamide. The NHase activity (∼18 U/mg dry cell weight, dcw) was observed in 0.1 M phosphate buffer, pH 8.0 containing 1M 3-cyanopyridine as substrate, and 0.75 mg of resting cells (dry cell weight) per ml reaction mixture at 40°C. However, 25°C was more suitable for prolonged batch reaction at high substrate (3-cyanopyridine) concentration. In a batch reaction (1 liter), 7M 3-cyanopyridine (729 g) was completely converted to nicotinamide (855 g) in 12h at 25°C using 9.0 g resting cells (dry cell weight) of R. rhodochrous PA-34.
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The molecular cloning and sequencing of the nitrilase gene of Rhodococcus rhodochrous PA‐34
Acta Biotechnologica, 1995Co-Authors: Tek Chand Bhalla, M. Aoshima, S. Misawa, R. Muramatsu, K. FuruhashiAbstract:The nitrilase of Rhodococcus rhodochrous PA-34 catalyzes the production of optically active amino acids from aminonitriles. The amino acid sequence of the NH 2 terminus of the purified nitrilase was determined for the preparation of a synthetic oligonucleotide as a southern hybridization probe. A 9.5-kbp Pst I-fragment, which hybridized with the oligonucleotide probe, was isolated from R. rhodochrous PA-34 genomic libraries constructed in pUC 19. Nucleotide sequence analysis revealed that the nitrilase gene codes for a putative polypeptide of 380 amino acids which correspond to a relative molecular weight of 41,723.
Alison J Hoyle - One of the best experts on this subject based on the ideXlab platform.
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The nitrilases of Rhodococcus rhodochrous NCIMB 11216
Enzyme and Microbial Technology, 1998Co-Authors: Alison J Hoyle, Alan W Bunch, Christopher J KnowlesAbstract:Rhodococcus rhodochrous NCIMB 11216 grows on propionitrile or benzonitrile as the sole source of carbon and nitrogen. The possibility that different nitrile-hydrolyzing enzymes were produced under these two growth conditions was investigated. Nitrilase activity in whole cell suspensions from either bacteria grown on propionitrile or benzonitrile were capable of biotransforming a wide range of nitriles. The propionitrile-induced nitrile degrading activity hydrolyzed 3-cyanobenzoate and both the nitrile groups in 1,3-dicyanobenzoate. In contrast, the benzonitrile-induced activity hydrolyzed only one of the nitrile groups in 1,3-dicyanobenzoate, but did not affect 3-cyanobenzoate. Both nitrilases biotransformed α-cyano-o-tolunitrile to produce 2-cyanophenylacetic acid. The nitrilases were purified by fast protein liquid chromatography and the n-terminus of each enzyme sequenced. SDS-PAGE analysis identified a subunit molecular weight of 45.8 kDa for each nitrilase. The n-terminal sequences showed significant similarity with other sequenced nitrilases and with the exception of a single amino acid were identical with each other. Both nitrilases had temperature and pH optima of 30°C and 8.0, respectively. The propionitrile-induced nitrilase had a Km for benzonitrile of 20.7 mm and a Vmax of 12.4 μmol min−1 mg−1 protein whereas the benzonitrile-induced nitrilase had a Km for benzonitrile of 8.83 mm and a Vmax of 0.57 μmol min−1 mg−1 protein.
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The nitrilases of Rhodococcus rhodochrous NCIMB 11216
Enzyme and Microbial Technology, 1998Co-Authors: Alison J Hoyle, Alan W Bunch, Christopher J KnowlesAbstract:Rhodococcus rhodochrous NCIMB 11216 grows on propionitrile or benzonitrile as the sole source of carbon and nitrogen. The possibility that different nitrile-hydrolyzing enzymes were produced under these two growth conditions was investigated. Nitrilase activity in whole cell suspensions from either bacteria grown on propionitrile or benzonitrile were capable of biotransforming a wide range of nitriles. The propionitrile-induced nitrile degrading activity hydrolyzed 3-cyanobenzoate and both the nitrile groups in 1,3-dicyanobenzoate. In contrast, the benzonitrile-induced activity hydrolyzed only one of the nitrile groups in 1,3-dicyanobenzoate, but did not affect 3-cyanobenzoate. Both nitrilases biotransformed alpha-cyano-o-tolunitrile to produce 2-cyanophenylacetic acid. The nitrilases were purified by fast protein liquid chromatography and the iv-terminus of each enzyme sequenced. SDS-PAGE analysis identified a subunit molecular weight of 45.8 kDa for each nitrilase. The N-terminal sequences showed significant similarity with other sequenced nitrilases and with the exception of a single amino acid were identical with each other. Both nitrilases had temperature and pH optima of 30 degrees C and 8.0, respectively. The propionitrile-induced nitrilase had a K-m for benzonitrile of 20.7 mM and a V-max of 12.4 mu mol min(-1) mg(-1) protein whereas the benzonitrile-induced nitrilase had a K-m for benzonitrile of 8.83 mM and a V-max of 0.57 mu mol min(-1) mg(-1) protein
A. S. Yanenko - One of the best experts on this subject based on the ideXlab platform.
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Optimization of Expression of Nitrilase from Alcaligenes denitrificans in Rhodococcus rhodochrous to Increase the Efficiency of Biocatalytic Synthesis of Ammonium Acrylate
Biotekhnologiya, 2019Co-Authors: E.g. Grechishnikova, S. P. Voronin, A.o. Shemyakina, A.d Novikov, T.i. Kalinina, A.s. Epremyan, S.a. Glinskii, R.a. Minasyan, A. S. YanenkoAbstract:The Rhodococcus rhodochrous M33-2nit strain containing in its chromosome two copies of a gene for A. denitrificans В-9582 NitC1 nitrilase under the control of a promoter region of genes of R. rhodochrous M8 nitrile hydratase has been constructed. The culturing of the strain was optimized and it was shown that using a two-substrate cultivation scheme on glucose and acetate, it was possible to obtain up to 17 g cdw/L of cells with the specific activity of 7 units/mg cdw. A capacity of synthesizing ammonium acrylate from acrylonitrile by the cells of A. denitrificans В-9582 and A. rhodochrous M33-2nit under the conditions simulating the industrial process was compared. It was shown that the cells of R. rhodochrous M33-2nit were able to produce ammonium acrylate at higher rates of acrylonitrile feeding, than the cells of A. denitrificans В-9582. Using the R. rhodochrous M33-2nit cells, high-concentration solutions of ammonium acrylate (450 g/L) were obtained with the conversion rate of 99.5%. Rhodococcus rhodochrous, nitrilase, nitrile hydratase promoter, ammonium acrylate, acrylonitrile, biocatalysis.
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aliphatic amidase from Rhodococcus rhodochrous m8 is related to the nitrilase cyanide hydratase family
Biochemistry, 2005Co-Authors: S. I. Pertsovich, A. S. Yanenko, D. T. Guranda, D. A. Podchernyaev, Vytas K. ŠvedasAbstract:A comparative study of amino acid sequence and physicochemical properties indicates the affiliation of an amidase from Rhodococcus rhodochrous M8 (EC 3.5.1.4) to the nitrilase/cyanide hydratase family. Cluster analysis and multiple alignments show that Cys166 is an active site nucleophile. The enzyme has been shown to be a typical aliphatic amidase, being the most active toward short-chain linear amides. Small polar molecules such as hydroxylamine and O-methyl hydroxylamine can serve as effective external nucleophiles in acyl transfer reactions. The kinetics of the industrially important amidase-catalyzed acrylamide hydrolysis has been studied over a wide range of substrate concentrations; inhibition during enzymatic hydrolysis by the substrate and product (acrylic acid) has been observed; an adequate kinetic scheme has been evaluated and the corresponding kinetic parameters have been determined.
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Aliphatic amidase from Rhodococcus rhodochrous M8 is related to the nitrilase/cyanide hydratase family.
Biochemistry. Biokhimiia, 2005Co-Authors: S. I. Pertsovich, A. S. Yanenko, D. T. Guranda, D. A. Podchernyaev, Vytas K. ŠvedasAbstract:A comparative study of amino acid sequence and physicochemical properties indicates the affiliation of an amidase from Rhodococcus rhodochrous M8 (EC 3.5.1.4) to the nitrilase/cyanide hydratase family. Cluster analysis and multiple alignments show that Cys166 is an active site nucleophile. The enzyme has been shown to be a typical aliphatic amidase, being the most active toward short-chain linear amides. Small polar molecules such as hydroxylamine and O-methyl hydroxylamine can serve as effective external nucleophiles in acyl transfer reactions. The kinetics of the industrially important amidase-catalyzed acrylamide hydrolysis has been studied over a wide range of substrate concentrations; inhibition during enzymatic hydrolysis by the substrate and product (acrylic acid) has been observed; an adequate kinetic scheme has been evaluated and the corresponding kinetic parameters have been determined.
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Research letterCobalt-dependent transcription of the nitrile hydratase gene in Rhodococcus rhodochrous M8
Fems Microbiology Letters, 1996Co-Authors: Tatyana E. Pogorelova, L. E. Ryabchenko, Nicola I. Sunzov, A. S. YanenkoAbstract:The effects of cobalt ions on the activities of Rhodococcus rhodochrous M8 enzymes for nitrile utilization, nitrile hydratase and amidase, were investigated. In contrast to amidase, synthesis of nitrile hydratase and its activity required cobalt ions in the growth medium. Northern blot analysis showed that in the presence of cobalt ions, the level of mRNA for nitrile hydratase genes was several times higher than that under cobalt-limited conditions. It was assumed that the low nitrile hydratase activity in cells grown in the absence of cobalt ions is connected either with the weak expression of nitrile hydratase genes or with the rapid degradation of nitrile hydratase mRNA.
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Cobalt‐dependent transcription of the nitrile hydratase gene in Rhodococcus rhodochrous M8
FEMS Microbiology Letters, 1996Co-Authors: Tatyana E. Pogorelova, L. E. Ryabchenko, Nicola I. Sunzov, A. S. YanenkoAbstract:The effects of cobalt ions on the activities of Rhodococcus rhodochrous M8 enzymes for nitrile utilization, nitrile hydratase and amidase, were investigated. In contrast to amidase, synthesis of nitrile hydratase and its activity required cobalt ions in the growth medium. Northern blot analysis showed that in the presence of cobalt ions, the level of mRNA for nitrile hydratase genes was several times higher than that under cobalt-limited conditions. It was assumed that the low nitrile hydratase activity in cells grown in the absence of cobalt ions is connected either with the weak expression of nitrile hydratase genes or with the rapid degradation of nitrile hydratase mRNA.