RESUMEN
The leaves of Nandina domestica Thunb. exhibited high hydroxynitrile lyase (HNL) activity in (R)-mandelonitrile synthesis. The specific activity of young leaves was significantly higher than that of mature leaves. We isolated two HNLs with molecular mass of 24.9 kDa (NdHNL-S) and 28.0 kDa (NdHNL-L) from the young leaves. Both NdHNLs were composed of two identical subunits, without FAD and carbohydrates. We purified NdHNL-L and revealed its enzymatic properties. The whole deduced amino acid sequence of NdHNL-L was not homologous to any other HNLs, and the specific activity for mandelonitrile synthesis by NdHNL-L was higher than that by other plant HNLs. The enzyme catalyzed enantioselective synthesis of (R)-cyanohydrins, exhibited high activity at pH 4.0, and high stability in the pH range of 3.5-8.0 and below 55°C. Thus, NdHNL-L is a novel HNL with novel amino acid sequence and has a potential for the efficient production of (R)-cyanohydrins.
Asunto(s)
Aldehído-Liasas/metabolismo , Berberidaceae/enzimología , Aldehído-Liasas/química , Aldehído-Liasas/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Hojas de la Planta/enzimología , Espectrofotometría Ultravioleta , Especificidad por Sustrato , TemperaturaRESUMEN
The mimABCD gene clusters in Mycobacterium smegmatis strain mc(2) 155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B-4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the Rhodococcus-Escherichia coli shuttle vector, pTip-QC2, and then introduced into R. opacus cells. Although whole-cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chaperoninas/metabolismo , Hierro/metabolismo , Oxigenasas de Función Mixta/metabolismo , Mycobacterium/enzimología , Proteínas Bacterianas/genética , Western Blotting , Chaperonina 60/genética , Chaperonina 60/metabolismo , Chaperoninas/genética , Electroforesis en Gel de Poliacrilamida , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Oxigenasas de Función Mixta/genética , Familia de Multigenes , Mycobacterium/genética , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Oxidación-Reducción , Fenol/metabolismo , Proteínas Recombinantes/metabolismo , Rhodococcus/genética , Rhodococcus/metabolismo , Especificidad por SustratoRESUMEN
A bacterial strain, designated PS9(T), was isolated from soil in the Ryukyu Archipelago, Japan. The bacterium grew with racemic phenylsuccinate as the sole carbon and energy source. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain PS9(T) was closely related to Citricoccus muralis, C. alkalitolerans and C. nitrophenolicus with sequence similarities of 97.5, 97.8, and 98.3%, respectively, suggesting that the strain belonged to the genus Citricoccus. Strain PS9(T) was a Gram-positive, non-motile, circular-shaped and aerobic bacterium. The major respiratory quinone was MK-8 (H(2)) and the predominant cellular fatty acid was C(15:0) anteiso, C(17:0) anteiso, and C(15:0) iso. The G+C content was 72.4 mol%. Based on the phylogenetic and phenotypic traits, it was concluded that the organism represents a new species in the genus Citricoccus, with the name Citricoccus yambaruensis. The type strain is PS9(T) (=NBRC102121(T) = DSM18783(T)).
Asunto(s)
Micrococcaceae/clasificación , Microbiología del Suelo , Succinatos/análisis , Ácidos Grasos/análisis , Genes Bacterianos , Micrococcaceae/genética , Micrococcaceae/aislamiento & purificación , Fenotipo , Filogenia , Quinonas/análisis , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Especificidad de la Especie , EstereoisomerismoRESUMEN
The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ(54) factor, a gene encoding the σ(54) factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria.
Asunto(s)
Acetona/metabolismo , Proteínas Bacterianas/genética , Regulación Enzimológica de la Expresión Génica , Genes Reguladores , Oxigenasas de Función Mixta/genética , Familia de Multigenes , Mycobacterium smegmatis/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Mycobacterium/química , Mycobacterium/enzimología , Mycobacterium/genética , Mycobacterium/metabolismo , Mycobacterium smegmatis/química , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/metabolismo , Alineación de SecuenciaRESUMEN
Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.
Asunto(s)
Hidroquinonas/metabolismo , Oxigenasas de Función Mixta/genética , Mycobacterium/enzimología , Mycobacterium/genética , Fenol/metabolismo , Acetona/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Hidroquinonas/química , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mycobacterium/metabolismo , Oxidación-Reducción , Fenol/química , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Hydroxynitrile lyase (MeHNL, EC 4.1.2.39) is a useful enzyme for production of optically active cyanohydrin compounds. Production of MeHNL can be increased by substituting rare codons of the natural sequence of cassava (Manihot esculenta) MeHNL. However, most of the MeHNL produced by this method was in an insoluble form in Escherichia coli expression system. In order to increase the productivity of active form of MeHNL, the effects of cultivation temperature were investigated. When the cultivation temperature was reduced, the cell yield and the ratio of soluble MeHNL increased significantly. The enzyme activity and yield at low-temperature cultures (17 degrees C) were 850 times higher than those obtained at the optimum growth temperature of 37 degrees C. The rate of MeHNL production in the present study was calculated as 3,000 U/h. Low-temperature cultivation is very effective in improving the productivity of the active form of MeHNL and has more potential for large-scale production of MeHNL for optically active cyanohydrin production.
Asunto(s)
Aldehído-Liasas/biosíntesis , Aldehído-Liasas/metabolismo , Escherichia coli/genética , Ingeniería Genética/métodos , Manihot/enzimología , Temperatura , Aldehído-Liasas/química , Aldehído-Liasas/genética , Secuencia de Bases , Biocatálisis , Codón/genética , Escherichia coli/crecimiento & desarrollo , Manihot/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
Racemic phenylsuccinate was stereospecifically degraded by the actinomycetes PS9 and PS17 isolated from soil obtained from Okinawa Island, Japan. Strain PS9, identified as a Citricoccus sp., preferentially degraded the R-form, while strain PS17, identified as a Microbacterium sp., preferentially degraded the S-form of phenylsuccinate. Analysis of the culture broths of these species with phenylsuccinate as the sole carbon source revealed that benzoic acid was produced as a metabolic intermediate. Benzoic acid was further degraded by strain PS9 with m- and/or p-hydroxybenzoic acid but not o-hydroxybenzoic acid as possible intermediates.
Asunto(s)
Actinobacteria/metabolismo , Succinatos/metabolismo , Actinobacteria/clasificación , Biodegradación Ambiental , Filogenia , EstereoisomerismoRESUMEN
Hydroxynitrile lyase from cassava, Manihot esculenta (MeHNL), catalyzes the formation of (S)-cyanohydrins from HCN and aldehydes or ketones. (S)-Mandelonitrile was produced on a bench scale with immobilized MeHNL, after optimizing the enzyme expression system using recombinant technology. MeHNL was cloned from a cDNA library prepared from a leaf of Manihot esculenta, and then expressed in a multi-auxotrophic mutant of Saccharomyces cerevisiae cells. The maximum yield of active MeHNL was obtained by integrating transformation 4 times with a tandemly repeated expression cassette. Silica gel was the most suitable support for immobilization of the prepared enzyme from the recombinant yeast. Using this immobilized enzyme, 22 batches of (S)-mandelonitrile synthesis were performed in a 20 liters bioreactor (1 M benzaldehyde and 1.5 M HCN). During this operation, about 29 kg of (S)-mandelonitrile was produced from 23.3 kg of benzaldehyde, giving 98 mol % yield and a mean enantio excess of 98.9% ee.
Asunto(s)
Acetonitrilos/metabolismo , Aldehído-Liasas/metabolismo , Enzimas Inmovilizadas/metabolismo , Expresión Génica , Manihot/enzimología , Saccharomyces cerevisiae/enzimología , Aldehído-Liasas/genética , Reactores Biológicos , Enzimas Inmovilizadas/genética , Vectores Genéticos/genética , Manihot/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Overexpression and production of the high concentration of hydroxynitrile lyase from cassava (Manihot esculenta (MeHNL, EC 4.1.2.39)) were investigated. Hydroxynitrile lyase is a useful enzyme for the production of optically active cyanohydrin compounds. The production of MeHNL was increased by changing the rare codons of the original sequence of cassava MeHNL. However, most of the produced MeHNL was in the insoluble form. In order to increase the solubility of MeHNL, the effects of the cultivation temperature were investigated. When the cultivation temperature was reduced, the cell yield and the ratio of soluble MeHNL increased significantly. The enzyme activity and yield at low-temperature cultures (17 degrees C) were 850 times higher than those obtained at the optimum growth temperature of 37 degrees C. The rate of MeHNL production in the present study was calculated as 3,000 unit/h. Low-temperature cultivation was very effective in improving the productivity of the active form of MeHNL. Unlike the temperature-shift method, low-temperature cultivation has more potential for the large-scale production of MeHNL for the optically active cyanohydrin production.