RESUMEN
An engineered (S)-selective imine reductase from Streptomyces sp. GF3546 (SIR46) showed twice the activity of the wild type and high thermostability at 50 °C. Using a biocatalyst based on SIR46, we investigated the activity toward high-concentration 2-methyl-1-pyrroline up to 500 mM, conversion rates of other imines, and reductive amination activity.
Asunto(s)
Iminas , Streptomyces , Oxidorreductasas/metabolismo , Aminación , Streptomyces/metabolismo , Aminas , EstereoisomerismoRESUMEN
Rhodococcus sp. 2N was found as a 1,3-propanediols-oxidizing strain from soil samples through enrichment culture using 2,2-diethyl-1,3-propanediol (DEPD) as the sole carbon source. The culture condition of the strain 2N was optimized, and the highest activity was observed when 0.3% (w/v) DEPD was added in the culture medium as an inducer. Chiral HPLC analysis of the hydroxyalkanoic acid converted from 2-ethyl-2-methyl-1,3-propanediol (EMPD) revealed that the strain 2N catalyzed the (R)-selective oxidation of EMPD. The reaction products and intermediates from DEPD and EMPD were identified by nuclear magnetic resonance analyses, and the results suggested that only one hydroxymethyl group of the propanediols was converted to carboxy group via two oxidation steps. Under optimized conditions and after a 72-h reaction time, the strain 2N produced 28 mM (4.1 g/L) of 2-(hydroxymethyl)-2-methylbutanoic acid from EMPD with a molar conversion yield of 47% and 65% ee (R).
Asunto(s)
Butiratos/metabolismo , Glicoles de Propileno/metabolismo , Rhodococcus/metabolismo , Biodegradación Ambiental , Butiratos/química , Medios de Cultivo/química , Medios de Cultivo/farmacología , Cinética , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Glicoles de Propileno/química , Rhodococcus/química , Rhodococcus/efectos de los fármacosRESUMEN
Verticillium sp. GF39, catalyzing the oxidation of 1-methylisoquinoline to 1-methylisoquinoline N-oxide, was found to be the highest N-oxide producer. Under the optimized reaction conditions, the whole cells of Verticillium sp. GF39 formed 5 mM 1-methylisoquinoline N-oxide from 1-methylisoquinoline with a molar conversion yield of 100% after a 10-h incubation at 20°C. The whole cells also acted on pyridine, 2-methylpyridine, quinoline and isoquinoline and formed the corresponding N-oxides.
Asunto(s)
Óxidos N-Cíclicos/metabolismo , Isoquinolinas/metabolismo , Verticillium/metabolismo , Óxidos N-Cíclicos/química , Isoquinolinas/química , Oxidación-Reducción , Picolinas/metabolismo , Piridinas/metabolismo , Verticillium/químicaRESUMEN
The nitrilase gene of Rhodococcus rhodochrous J1 was expressed in Escherichia coli using the expression vector, pKK223-3. The recombinant E. coli JM109 cells hydrolyzed enantioselectively 2-methyl-2-propylmalononitrile to form (S)-2-cyano-2-methylpentanoic acid (CMPA) with 96 % e.e. Under optimized conditions, 80 g (S)-CMPA l(-1) was produced with a molar yield of 97 % at 30 °C after a 24 h without any by-products.
Asunto(s)
Aminohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Ácidos Pentanoicos/metabolismo , Aminohidrolasas/genética , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Nitrilos/química , Nitrilos/metabolismo , Ácidos Pentanoicos/análisis , Ácidos Pentanoicos/química , Rhodococcus/enzimología , Rhodococcus/genética , Estereoisomerismo , TemperaturaRESUMEN
A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5 kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6 nmol min(-1) mg(-1), and their optical purities were 92.7 % ee, 96.4 % ee, and >99 % ee, respectively. Using a NADPH-regenerating system, 10 mM 2-MPN was converted to amine with 100 % conversion and 92 % ee after 24 h. The amino acid sequence analysis revealed that SIR showed about 60 % identity to 6-phosphogluconate dehydrogenase. However, it showed only 37 % identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.
Asunto(s)
Aminas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Clonación Molecular , Iminas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Streptomyces/enzimología , Aminas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Expresión Génica , Iminas/química , Datos de Secuencia Molecular , NADP/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificación , Homología de Secuencia de Aminoácido , Microbiología del Suelo , Streptomyces/química , Streptomyces/genética , Streptomyces/aislamiento & purificación , Especificidad por SustratoRESUMEN
Washed cells (62 mg) of Kitasatospora sp. GF12 in 4 ml buffer (pH 7) catalyzed the regioselective hydroxylation of 60 mM 1,3-adamantanediol [1,3-ad(OH)(2)] to 30.9 mM 1,3,5-adamantanetriol [1,3,5-ad(OH)(3)] over 120 h at 24 °C. Glycerol at 400 mM was added to the reaction mixture to recycle the intracellular NADH/NADPH. Whole cells of GF12, also catalyzed the hydroxylation of 10 mM 1-adamantanol (1-adOH), to 3.6 mM 1,3,5-ad(OH)(3).
Asunto(s)
Adamantano/análogos & derivados , Adamantano/metabolismo , Biotecnología/métodos , Streptomycetaceae/metabolismo , Adamantano/química , Glicerol/metabolismo , Hidroxilación , NAD/metabolismo , NADP/metabolismo , Estereoisomerismo , Temperatura , Factores de TiempoRESUMEN
The (R)-imine reductase (RIR) of Streptomyces sp. GF3587 was purified and characterized. It was found to be a NADPH-dependent enzyme, and was found to be a homodimer consisting of 32 kDa subunits. Enzymatic reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) resulted in the formation of 9.8 mM (R)-2-methylpyrrolidine ((R)-2-MP) with 99% e.e. The enzyme showed not only reduction activity for 2-MPN at neutral pH (6.5-8.0), but also oxidation activity for (R)-2-MP under alkaline pH (10-11.5) conditions. It appeared to be a sulfhydryl enzyme based on the sensitivity to sulfhydryl specific inhibitors. It was very specific to 2-MPN as substrate.
Asunto(s)
Iminas/metabolismo , Oxidorreductasas/metabolismo , Subunidades de Proteína/química , Pirroles/metabolismo , Pirrolidinas/metabolismo , Streptomyces/enzimología , Cromatografía en Gel , Dimerización , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Cinética , Metilación , Peso Molecular , NADP/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , Streptomyces/química , Especificidad por Sustrato , Reactivos de Sulfhidrilo/farmacología , Ácido p-Cloromercuribenzoico/farmacologíaRESUMEN
Streptomyces sp. GF3587 and 3546 were found to be imine-reducing strains with high R- and S-selectivity by screening using 2-methyl-1-pyrroline (2-MPN). Their whole-cell catalysts produced 91 mM R-2-methylpyrrolidine (R-2-MP) with 99.2%e.e. and 27.5 mM S-2-MP (92.3%e.e.) from 2-MPN at 91-92% conversion in the presence of glucose, respectively.
Asunto(s)
Aminas/síntesis química , Iminas/química , Pirrolidinas/metabolismo , Catálisis , Glucosa/metabolismo , Oxidorreductasas/química , Pirrolidinas/química , Estereoisomerismo , Streptomyces/metabolismoRESUMEN
To efficiently produce 1,3-adamantanediol (1,3-ad(OH)(2)) from 1-adamantanol (1-adOH), our stocks of culture strains and soil microorganisms were surveyed for hydroxylation activity towards 1-adOH. Among them, the soil actinomycete SA8 showing the highest hydroxylation activity was identified as Streptomyces sp. based on 16S ribosomal DNA sequence analysis. The reaction products were purified by silica gel column chromatography, and from NMR and MS analyses, they were identified as 1,3-ad(OH)(2) and 1,4-ad(OH)(2). Streptomyces sp. SA8 produced 5.9 g l(-1) 1,3-ad(OH)(2)from 6.2 g l(-1) 1-adOH in culture broth after 120 h at 25 degrees C. Using resting cells, 2.3 g l(-1) 1,3-ad(OH)(2) was produced after 96 h of incubation at a 69% conversion rate. In both cases, 1,4-ad(OH)(2) was formed as a byproduct at a rate of about 15%. Strain SA8 also hydroxylated 2-adamantanol and 2-methyl-2-adamantanol.
Asunto(s)
Adamantano/análogos & derivados , Streptomyces/metabolismo , Adamantano/metabolismo , Hidroxilación , Oxidación-Reducción , Análisis de Secuencia de ADN , Streptomyces/genética , Especificidad por SustratoRESUMEN
3,4-Dihydroxybenzoate decarboxylase in Enterobacter cloacae P241 was induced by adding 3,4-dihydroxybenzoic acid, 3-hydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid or 4-acetamidobenzoic acid to the culture medium. After stabilizing the enzyme activity by adding 5 mM dithiothreitol and 20 mM Na(2)S(2)O(3) to a cell-free extract, catechol at 50 mM was carboxylated in the presence of 3 M KHCO(3) to 3,4-dihydroxybenzoic acid with a molar conversion ratio of 28% after 14 h at 30 degrees C.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carboxiliasas/metabolismo , Catecoles/metabolismo , Enterobacter cloacae/enzimología , Enterobacter cloacae/metabolismo , Ácido 4-Aminobenzoico/metabolismo , Bicarbonatos/farmacología , Coenzimas/farmacología , Medios de Cultivo/química , Ácido Gálico/metabolismo , Hidroxibenzoatos/metabolismo , Compuestos de Potasio/farmacología , Estereoisomerismo , para-AminobenzoatosRESUMEN
Alkane-assimilating microorganisms were isolated from enrichment cultures using n-octane, n-dodecane, n-hexadecane, or pristane (2,6,10,14-tetramethylpentadecane) as a sole carbon source to find microbial catalysts oxidizing methyl groups of 2,6-dimethylpyridine. The cells of Exophiala dermatitidis (Kano) de Hoog DA5501, an n-dodecane-assimilating fungus, oxidized a single methyl group of 2,6-dimethylpyridine to produce 6-methylpicolinic acid (6-methylpyridine-2-carboxylic acid) without the formation of dipicolinic acid (pyridine-2,6-dicarboxylic acid); 67 mM 6-methylpicolinic acid (9.2 g/l) accumulated with a molar conversion yield of 89% by 54-h incubation. The fungus cells also oxidized the methyl group of 2,6-dimethylpyrazine and 2,4,6-trimethylpyridine regioselectively.
Asunto(s)
Alcanos/metabolismo , Exophiala/crecimiento & desarrollo , Exophiala/metabolismo , Ácidos Picolínicos/metabolismo , Piridinas/metabolismo , Biotransformación , Cinética , Oxidación-Reducción , Factores de TiempoRESUMEN
5-Hydroxyisophthalic acid-producing microorganisms were isolated from enrichment cultures using 5-sulfoisophthalic acid as a sulfur source. One bacterium, Ochrobactrum anthropi S9, had the highest 5-sulfoisophthalic acid-degrading activity, and stoichiometrically formed 5-hydroxyisophthalic acid, a raw material for polymer synthesis. Under optimum culture conditions, 1.3 mM 5-hydroxyisophthalic acid accumulated in the medium by 60 h. The addition of Na(2)SO(4), L: -methionine or L: -cysteine at 2 mM inhibited the conversion of 5-sulfoisophthalic acid. O. anthropi S9 cells converted 5-sulfoisophthalic acid, benzenesulfonic acid, 3-sulfobenzoic acid, 4-aminobenzenesulfonic acid, naphthalene-1-sulfonic acid and naphthalene-2-sulfonic acid into the corresponding hydroxylated compounds.
Asunto(s)
Ochrobactrum anthropi/metabolismo , Ácidos Ftálicos/metabolismo , Biodegradación Ambiental/efectos de los fármacos , Ochrobactrum anthropi/citología , Ochrobactrum anthropi/efectos de los fármacos , Ochrobactrum anthropi/crecimiento & desarrollo , Ácidos Ftálicos/química , Ácidos Sulfónicos/metabolismo , Azufre/farmacología , Factores de TiempoRESUMEN
trans-Stilbene degradation was examined by the reaction using resting cells of microorganisms isolated through the enrichment culture using trans-stilbene. The strain SL3, showing the highest trans-stilbene-degrading activity, was identified as Arthrobacter sp. One of the reaction products was identified to be cis,cis-muconic acid. Arthrobacter sp. SL3 cells also transformed benzaldehyde, benzoic acid and catechol into cis,cis-muconic acid, suggesting that one benzene ring of trans-stilbene was converted into cis,cis-muconic acid via benzaldehyde formed by its C(alpha)=C(beta) bond cleavage.
Asunto(s)
Arthrobacter/metabolismo , Estilbenos/metabolismo , Arthrobacter/aislamiento & purificación , Estructura Molecular , Estilbenos/química , Factores de TiempoRESUMEN
The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20 degrees C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.
Asunto(s)
Benzaldehídos/metabolismo , Escherichia coli/metabolismo , Eugenol/análogos & derivados , Oxigenasas de Función Mixta/metabolismo , Pseudomonas putida/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Eugenol/química , Eugenol/metabolismo , Oxigenasas de Función Mixta/genética , Estructura Molecular , Pseudomonas putida/genéticaRESUMEN
An isoeugenol-degrading enzyme was purified to homogeneity from Pseudomonas putida IE27, an isoeugenol-assimilating bacterium. The purified enzyme was a 55 kDa monomer and catalyzed the initial step of isoeugenol degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Another reaction product of isoeugenol degradation besides vanillin was identified to be acetaldehyde. The values of Km and k (cat) for isoeugenol were 175 muM and 5.18 s(-1), respectively. The purified enzyme catalyzed the incorporation of an oxygen atom from either molecular oxygen or water into vanillin, suggesting that the isoeugenol-degrading enzyme is a kind of monooxygenase. The gene encoding the isoeugenol-degrading enzyme and its flanking regions were isolated from P. putida IE27. The amino acid sequence of the enzyme was similar to those of lignostilbene-alpha,beta-dioxygenases, carotenoid monooxygenases and 9-cis-epoxycarotenoid dioxygenases.
Asunto(s)
Eugenol/análogos & derivados , Oxigenasas de Función Mixta , Pseudomonas putida/enzimología , Acetaldehído/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biotecnología/métodos , Clonación Molecular , Eugenol/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/aislamiento & purificación , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Pseudomonas putida/genética , Análisis de Secuencia de ADNRESUMEN
The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 degrees C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.
Asunto(s)
Benzaldehídos/metabolismo , Eugenol/análogos & derivados , Pseudomonas putida/metabolismo , Biotransformación , Eugenol/metabolismo , Fermentación , Temperatura , Factores de Tiempo , Ácido Vanílico/metabolismoRESUMEN
The tandem conversion process involving nitrile hydratase- and amidase-producing microorganisms has potential for use in the treatment of acetonitrile-containing wastes. In that process, the acetamide hydrolysis step catalyzed by amidase is very slow compared with the acetonitrile hydration step catalyzed by nitrile hydratase, and a small amount of acetamide remains in the resulting solution. This study aimed to improve the efficiency of the acetamide hydrolysis step. An amidase-producing microorganism, Rhodococcus sp. S13-4, was newly obtained, whose use enabled rapid acetamide degradation. Though residual acetamide was still detected, it was successfully reduced by the addition of cation/anion mixed ion exchange resin or calcium hydroxide after the acetamide hydrolysis reaction using Rhodococcus sp. S13-4 cells. This result implies that acetamide hydrolysis and acetamide formation are in equilibrium. The incubation of Rhodococcus sp. S13-4 cells with high concentrations of ammonium acetate produced acetamide. The purified amidase from Rhodococcus sp. S13-4 revealed the acetamide formation activity (specific activity of 30.6 U/mg protein). This suggests that the amidase-catalyzed amide formation may cause the remaining of acetamide in the acetonitrile conversion process.
Asunto(s)
Acetamidas/metabolismo , Acetonitrilos/metabolismo , Aminohidrolasas/biosíntesis , Hidroliasas/biosíntesis , Rhodococcus/metabolismo , Adsorción , Aminohidrolasas/aislamiento & purificación , Hidróxido de Calcio/metabolismo , Resinas de Intercambio IónicoRESUMEN
We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its Km value for 4-hydroxybenzoate was 596 microM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the Km value was 6.80 mM. In the presence of 3 M KHCO3 and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The Km value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96-97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.
Asunto(s)
Carboxiliasas/genética , Carboxiliasas/aislamiento & purificación , Enterobacter cloacae/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Carboxiliasas/metabolismo , Clonación Molecular , Enterobacter cloacae/genética , Datos de Secuencia MolecularRESUMEN
We found a bacterium, Pandoraea sp. 12B-2, of which whole cells catalyzed not only the decarboxylation of 2,6-dihydroxybenzoate but also the regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate. The whole cells of Pandoraea sp. 12B-2 also catalyzed the regioselective carboxylation of phenol and 1,2-dihydroxybenzene to 4-hydroxybenzoate and 2,3-dihydroxybenzoate, respectively. The molar conversion ratio of the carboxylation reaction depended on the concentration of KHCO(3) in the reaction mixture. Only 5 or 48 % of 1,3-dihydroxybenzene added was converted into 2,6-dihydroxybenzoate in the presence of 0.1 M or 3 M KHCO(3), respectively. The addition of acetone to the reaction mixture increased the initial rate of the carboxylation reaction, but the final molar conversion yield reached almost the same value. When the efficient production of 2,6-dihydroxybenzoate was optimized using the whole cells of Pandoraea sp. 12B-2, the productivity of 2,6-dihydroxybenzoate topped out at 1.43 M, which was the highest value so far reported. No formation of any other products was observed after the carboxylation reaction.
Asunto(s)
Burkholderiaceae/enzimología , Carboxiliasas/metabolismo , Acetona/metabolismo , Bicarbonatos/metabolismo , Catecoles/metabolismo , Hidroxibenzoatos/metabolismo , Parabenos/metabolismo , Fenol/metabolismo , Compuestos de Potasio/metabolismoRESUMEN
This study aimed to construct an acetonitrile-containing waste treatment process by using nitrile-degrading microorganisms. To degrade high concentrations of acetonitrile, the microorganisms were newly acquired from soil and water samples. Although no nitrilase-producing microorganisms were found to be capable of degrading high concentrations of acetonitrile, the resting cells of Rhodococcus pyridinivorans S85-2 containing nitrile hydratase could degrade acetonitrile at concentrations as high as 6 M. In addition, an amidase-producing bacterium, Brevundimonas diminuta AM10-C-1, of which the resting cells degraded 6 M acetamide, was isolated. The combination of R. pyridinivorans S85-2 and B. diminuta AM10-C-1 was tested for the conversion of acetonitrile into acetic acid. The resting cells of B. diminuta AM10-C-1 were added after the first conversion involving R. pyridinivorans S85-2. Through this tandem process, 6 M acetonitrile was converted to acetic acid at a conversion rate of >90% in 10 h. This concise procedure will be suitable for practical use in the treatment of acetonitrile-containing wastes on-site.