RESUMO
BACKGROUND: Microbial production of aromatic chemicals is an attractive method for obtaining high-performance materials from biomass resources. A non-proteinogenic amino acid, 4-amino-3-hydroxybenzoic acid (4,3-AHBA), is expected to be a precursor of highly functional polybenzoxazole polymers; however, methods for its microbial production have not been reported. In this study, we attempted to produce 4,3-AHBA from glucose by introducing 3-hydroxylation of 4-aminobenzoic acid (4-ABA) into the metabolic pathway of an industrially relevant bacterium, Corynebacterium glutamicum. RESULTS: Six different 4-hydroxybenzoate 3-hydroxylases (PHBHs) were heterologously expressed in C. glutamicum strains, which were then screened for the production of 4,3-AHBA by culturing with glucose as a carbon source. The highest concentration of 4,3-AHBA was detected in the strain expressing PHBH from Caulobacter vibrioides (CvPHBH). A combination of site-directed mutagenesis in the active site and random mutagenesis via laccase-mediated colorimetric assay allowed us to obtain CvPHBH mutants that enhanced 4,3-AHBA productivity under deep-well plate culture conditions. The recombinant C. glutamicum strain expressing CvPHBHM106A/T294S and having an enhanced 4-ABA biosynthetic pathway produced 13.5 g/L (88 mM) 4,3-AHBA and 0.059 g/L (0.43 mM) precursor 4-ABA in fed-batch culture using a nutrient-rich medium. The culture of this strain in the chemically defined CGXII medium yielded 9.8 C-mol% of 4,3-AHBA from glucose, corresponding to 12.8% of the theoretical maximum yield (76.8 C-mol%) calculated using a genome-scale metabolic model of C. glutamicum. CONCLUSIONS: Identification of PHBH mutants that could efficiently catalyze the 3-hydroxylation of 4-ABA in C. glutamicum allowed us to construct an artificial biosynthetic pathway capable of producing 4,3-AHBA on a gram-scale using glucose as the carbon source. These findings will contribute to a better understanding of enzyme-catalyzed regioselective hydroxylation of aromatic chemicals and to the diversification of biomass-derived precursors for high-performance materials.
Assuntos
Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Glucose , Oxigenases de Função Mista/genética , CarbonoRESUMO
Coordination environment of the Tb(3+) ion in oxygen-sensitive luminescent complexes can be successfully controlled through the size of alkyl substituents on ligands {((RMe)ArOH)4cyclen} (R = tBu or Me; cyclen = 1,4,7,10-tetraazacyclododecane); a newly prepared eight-coordinate complex 1(tBu) shows higher oxygen sensitivity (KSV = 17â¯600) and lower luminescence quantum yield (Φ = 0.67 under N2) than those of the previously reported seven-coordinate analogues 1(Me) and [{((MeMe)ArO)3tacn}Tb(III)(THF)] (KSV = 12â¯600 and 8300, Φ = 0.91 and 0.91 under N2, respectively; tacn = 1,4,7-triazacyclononane; THF = tetrahydrofuran). The oxygen-sensitive mechanism is discussed on the basis of the photophysical properties of the corresponding Gd(III) complexes.
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Many types of superoxide dismutases have been purified and characterized from various bacteria, however, a psychrophilic Mn-superoxide dismutase (MnSOD) has not yet been reported. Here, we describe the purification and the biochemical characterization of the psychrophilic MnSOD from Exiguobacterium sp. strain OS-77 (EgMnSOD). According to 16S rRNA sequence analysis, a newly isolated bacterium strain OS-77 belongs to the genus Exiguobacterium. The optimum growth temperature of the strain OS-77 is 20 °C. The EgMnSOD is a homodimer of 23.5 kDa polypeptides determined by SDS-PAGE and gel filtration analysis. UV-Vis spectrum and ICP-MS analysis clearly indicated that the homogeneously purified enzyme contains only a Mn ion as a metal cofactor. The optimal reaction pH and temperature of the enzyme were pH 9.0 and 5 °C, respectively. Notably, the purified EgMnSOD was thermostable up to 45 °C and retained 50% activity after 21.2 min at 60 °C. The differential scanning calorimetry also indicated that the EgMnSOD is thermostable, exhibiting two protein denaturation peaks at 65 and 84 °C. The statistical analysis of amino acid sequence and composition of the EgMnSOD suggests that the enzyme retains psychrophilic characteristics.
Assuntos
Bacillales/enzimologia , Proteínas de Bactérias/metabolismo , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Bacillales/genética , Bacillales/isolamento & purificação , Proteínas de Bactérias/química , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Superóxido Dismutase/químicaRESUMO
The present study investigated high-yield monoacylglycerol (MAG) synthesis by bacterial lipolytic enzymes in a solvent-free two-phase system. Esterification by monoacylglycerol lipase from Bacillus sp. H-257 (H257) required a high glycerol/fatty acid molar ratio for efficient MAG synthesis. Screening of H257 homologues revealed that carboxylesterase derived from Geobacillus thermodenitrificans, EstGtA2, exhibited a higher esterification rate than H257. Moreover, neutralizing the pH of the acidic reaction solution by adding potassium hydroxide (KOH) solution further increased the esterification rate. The esterification rate by EstGtA2 reached 75% under conditions of equivalent molar amounts of glycerol and fatty acid, and the MAG rate (MAG/total glyceride) was 97%. The neutralized pH of the reaction solution likely affected the thermal stability of EstGtA2 during the esterification reaction. Screening for thermal-tolerant variants revealed that the EstGtA2S26I variant was stable at 75 °C for 30 min, a condition under which wild-type EstGtA2 was completely inactivated. The esterification rate by the EstGtA2S26I variant reached 90%, and the MAG rate was 96%. The addition of alkali and the use of a thermal-tolerant enzyme were important for obtaining high-yield MAG in a solvent-free two-phase system utilizing EstGtA2.
Assuntos
Carboxilesterase , Geobacillus , Carboxilesterase/metabolismo , Enzimas Imobilizadas , Esterificação , Ácidos Graxos , Geobacillus/genética , Glicerol , Monoglicerídeos , SolventesRESUMO
We propose a modified mechanism for the inhibition of [NiFe]hydrogenase ([NiFe]H(2)ase) by CO. We present a model study, using a NiRu H(2)ase mimic, that demonstrates that (i) CO completely inhibits the catalytic cycle of the model compound, (ii) CO prefers to coordinate to the Ru(II) center rather than taking an axial position on the Ni(II) center, and (iii) CO is unable to displace a hydrido ligand from the NiRu center. We combine these studies with a reevaluation of previous studies to propose that, under normal circumstances, CO inhibits [NiFe]H(2)ase by complexing to the Fe(II) center.
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Materiais Biomiméticos/metabolismo , Monóxido de Carbono/metabolismo , Hidrogenase/antagonistas & inibidores , Hidrogenase/metabolismo , Níquel/metabolismo , Compostos de Rutênio/metabolismo , Materiais Biomiméticos/química , Modelos Biológicos , Modelos Moleculares , Níquel/química , Compostos de Rutênio/químicaRESUMO
This paper presents a 1,4,7,10-teraazacyclododecane-based tetrakis-phenol as a protonated ligand precursor and its oxygen-sensitive luminescent terbium(III) complex with an extendable phenol pendant arm (Φ = 0.91 under N2, Φ = 0.031 under air), in which the potentially N4O4-octadentate ligand unprecedentedly coordinates to the Tb(3+) ion in a N4O3-heptadentate fashion.
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This communication presents a new terbium(III) complex that shows the highest luminescence quantum yield among the oxygen-sensitive lanthanide complexes (Φ = 0.91 under N2, Φ = 0.054 under air).
Assuntos
Complexos de Coordenação/química , Compostos Heterocíclicos/química , Oxigênio/química , Térbio/química , Complexos de Coordenação/síntese química , Ligantes , Conformação Molecular , Teoria Quântica , Espectrofotometria UltravioletaRESUMO
[NiFeSe]hydrogenases are promising biocatalysts in H2-based technology due to their high catalytic activity and O2-stability. Here, we report purification and characterization of a new membrane-associated [NiFeSe]hydrogenase from Desulfovibrio vulgaris Miyazaki F ([NiFeSe]DvMF). The [NiFeSe]DvMF was composed of two subunits, corresponding to a large subunit of 58.3 kDa and a small subunit of 29.3 kDa determined by SDS-PAGE. Unlike conventional [NiFeSe]hydrogenases having catalytic bias toward H2-production, the [NiFeSe]DvMF showed 11-fold higher specific activity of H2-oxidation (2444 U/mg) than that of H2-production (217 U/mg). At the optimal reaction temperature of the enzyme (65°C), the specific activity of H2-oxidation could reach up to 21,553 U/mg. Amperometric assays of the [NiFeSe]DvMF clearly indicated that the enzyme had a remarkable O2-stability. According to the amino acid sequence alignment, the conserved cysteine residue at position 281 in medial cluster of other [NiFeSe]hydrogenases was specifically replaced by a serine residue (Ser281) in the [NiFeSe]DvMF. These results indicate that the [NiFeSe]DvMF can play as a new H2-oxidizing and O2-stable biocatalyst, along with providing helpful insights into the structure-function relationship of [NiFeSe]hydrogenases.
Assuntos
Desulfovibrio vulgaris/enzimologia , Hidrogenase/metabolismo , Sequência de Aminoácidos , Biocatálise , Desulfovibrio vulgaris/genética , Hidrogenase/química , Hidrogenase/genética , Dados de Sequência Molecular , Oxirredução , Alinhamento de SequênciaRESUMO
This communication reports the successful merging of the chemical properties of a natural [NiFe]hydrogenase (Desulfovibrio vulgaris Miyazaki F) and our previously reported [NiRu] hydrogenase-mimic. The catalytic activity of both the natural enzyme and the mimic is almost identical, with the exception of working pH ranges, and this allows us to use them simultaneously in the same reaction flask. In such a manner, isotope exchange between D(2) and H(2)O could be conducted over an extended pH range (about 2-10) in one pot under mild conditions at ambient temperature and pressure.