RESUMEN
Biocatalysis using molecular oxygen as the electron acceptor has significant potential for selective oxidations at low cost. However, oxygen is poorly soluble in water, and its slow rate of mass transfer in the aqueous phase is a major obstacle, even for laboratory-scale syntheses. Oxygen transfer can be accelerated by vigorous mechanical methods, but these are often incompatible with biological catalysts. Gentler conditions can be achieved with shallow, high surface area bag reactors that are designed for single use and generally for specialized cell culture applications. As a less-expensive alternative to these high-end bioreactors, we describe repurposing inflatable shipping pillows with resealable valves to provide high surface area mixing under oxygen for preparative synthesis of glucosone (D-arabino-hexos-2-ulose) from D-glucose using non-growing Escherichia coli whole cells containing recombinant pyranose 2-oxidase (POX) as catalyst. Parallel reactions permitted systematic study of the effects of headspace composition (i.e., air vs 100% oxygen), cell density, exogenous catalase, and reaction volume in the oxidation of 10% glucose. Importantly, only a single charge of 100% oxygen is required for stoichiometric conversion on a multi-gram scale in 18 h with resting cells, and the conversion was successfully repeated with recycled cells.
Asunto(s)
Reactores Biológicos , Escherichia coli/metabolismo , Cetosas/biosíntesis , Oxígeno/metabolismo , Embalaje de Productos , CatálisisRESUMEN
Dihydroxyacetone phosphate (DHAP)-dependent aldolases demonstrate important values in the production of rare ketoses due to their unique stereoselectivities. As a specific example, we developed an efficient Escherichia coli whole-cell biocatalytic cascade system in which rare ketoses were produced from abundant glycerol and catalyzed by four enzymes based on L-rhamnulose-1-phosphate aldolase (RhaD). For the semicontinuous bioconversion in which D-glyceraldehyde was continuously added, once D-glyceraldehyde was consumed, the final yields of D-sorbose and D-psicose were 15.30 g/L and 6.35 g/L, respectively. Moreover, the maximum conversion rate and productivity of D-sorbose and D-psicose were 99% and 1.11 g/L/h at 8 h, respectively. When L-glyceraldehyde was used instead of the D-isomer, the final yield of L-fructose was 16.80 g/L. Furthermore, the maximum conversion rate and productivity of L-fructose were 95% and 1.08 g/L/h at 8 h, respectively. This synthetic platform was also compatible with other various aldehydes, which allowed the production of many other high-value chemicals from glycerol.
Asunto(s)
Aldehído-Liasas/metabolismo , Escherichia coli/metabolismo , Cetosas/biosíntesis , Biocatálisis , Biotransformación , Fructosa/metabolismo , Gliceraldehído/metabolismo , Glicerol/metabolismo , Microbiología Industrial , Sorbosa/metabolismo , Especificidad por SustratoRESUMEN
The high-value ketocarotenoid astaxanthin, a natural red colorant with powerful antioxidant activity, is synthesised from ß-carotene by a hydroxylase and an oxygenase enzyme, which perform the addition of two hydroxyl and keto moieties, respectively. Several routes of intermediates, depending on the sequence of action of these enzymes, lead to the formation of astaxanthin. In the present study, the enzyme activities of 3, 3' ß-carotene hydroxylase (CRTZ) and 4, 4' ß-carotene oxygenase (CRTW) have been combined through the creation of "new to nature" enzyme fusions in order to overcome leakage of non-endogenous intermediates and pleotropic effects associated with their high levels in plants. The utility of flexible linker sequences of varying size has been assessed in the construction of pZ-W enzyme fusions. Frist, in vivo color complementation assays in Escherichia coli have been used to evaluate the potential of the fusion enzymes. Analysis of the carotenoid pigments present in strains generated indicated that the enzyme fusions only possess both catalytic activities when CRTZ is attached as the N-terminal module. Astaxanthin levels in E. coli cells were increased by 1.4-fold when the CRTZ and CRTW enzymes were fused compared to the individual enzymes. Transient expression in Nicotiana benthamiana was then performed in order to assess the potential of the fusions in a plant system. The production of valuable ketocarotenoids was achieved using this plant-based transient expression system. This revealed that CRTZ and CRTW, transiently expressed as a fusion, accumulated similar levels of astaxanthin compared to the expression of the individual enzymes whilst being associated with reduced ketocarotenoid intermediate levels (e.g. phoenicoxanthin, canthaxanthin and 3-OH-echinenone) and a reduced rate of leaf senescence after transformation. Therefore, the quality of the plant material producing the ketocarotenoids was enhanced due to a reduction in the stress induced by the accumulation of high levels of heterologous ketocarotenoid intermediates. The size of the linkers appeared to have no effect upon activity. The potential of the approach to production of valuable plant derived products is discussed.
Asunto(s)
Carotenoides/biosíntesis , Cetosas/biosíntesis , Plantas/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Fusión Génica , Ingeniería Metabólica/métodos , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Hojas de la Planta/metabolismo , Plantas/genética , Plantas Modificadas Genéticamente , Plásmidos/genética , Nicotiana/genética , Nicotiana/metabolismo , Xantófilas/biosíntesisRESUMEN
Rare sugars are valuable natural products widely used in pharmaceutical and food industries. In this study, we expected to synthesize rare ketoses from abundant glycerol using dihydroxyacetone phosphate (DHAP)-dependent aldolases. First, a new glycerol assimilation pathway was constructed to synthesize DHAP. The enzymes which convert glycerol to 3-hydroxypropionaldehyde and l-glyceraldehyde were selected, and their corresponding aldehyde synthesis pathways were constructed in vivo. Four aldol pathways based on different aldolases and phosphorylase were gathered. Next, three pathways were assembled and the resulting strains synthesized 5-deoxypsicose, 5-deoxysorbose, and 5-deoxyfructose from glucose and glycerol and produce l-fructose, l-tagatose, l-sorbose, and l-psicose with glycerol as the only carbon source. To achieve higher product titer and yield, the recombinant strains were further engineered and fermentation conditions were optimized. Fed-batch culture of engineered strains obtained 38.1 g/L 5-deoxypsicose with a yield of 0.91 ± 0.04 mol product per mol of glycerol and synthesized 20.8 g/L l-fructose, 10.3 g/L l-tagatose, 1.2 g/L l-sorbose, and 0.95 g/L l-psicose.
Asunto(s)
Corynebacterium glutamicum/metabolismo , Glicerol/metabolismo , Cetosas/biosíntesis , Ingeniería Metabólica , Aldehído-Liasas/metabolismo , Técnicas de Cultivo Celular por Lotes , Biomasa , Vías Biosintéticas , Cromatografía Líquida de Alta Presión , Corynebacterium glutamicum/genética , Escherichia coli/genética , Fermentación , Fructosa/biosíntesis , Glucosa/metabolismo , Gliceraldehído/análogos & derivados , Gliceraldehído/metabolismo , Hexosas/biosíntesis , Hidroliasas/metabolismo , Espectroscopía de Resonancia Magnética , Propano/metabolismo , Sorbosa/biosíntesisRESUMEN
Glycerol phosphate oxidase from Streptococcus pneumoniae (GPOS.pne) was purified and characterized. By the actions of GPOS.pne and dihydroxyacetone phosphate (DHAP)-dependent aldolases, various ketoses including rare sugars were synthesized with glyceraldehydes as acceptors in a one-pot four-enzyme system.
Asunto(s)
Glicerolfosfato Deshidrogenasa/metabolismo , Cetosas/biosíntesis , Streptococcus pneumoniae/enzimología , Fosfatasa Ácida/metabolismo , Aldehído-Liasas/metabolismo , Catalasa/metabolismo , Escherichia coli/metabolismo , Gliceraldehído/química , Gliceraldehído/metabolismo , Glicerolfosfato Deshidrogenasa/genética , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Especificidad por SustratoRESUMEN
Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars.
Asunto(s)
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Cetosas/biosíntesis , Ingeniería Metabólica/métodos , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Redes y Vías Metabólicas/genética , Mutación , Recombinación GenéticaRESUMEN
The tri-enzyme system pyranose 2-oxidase (P2O), laccase, and catalase was used to study major parameters in the homogeneous and heterogeneous application of a multi-component enzymatic machinery. P2O oxidizes aldoses to 2-ketosugars, which are interesting intermediates in carbohydrate chemistry, and concomitantly reduces oxygen or alternative electron acceptors. The enzyme was immobilized on eleven agarose or acrylic resins using various coupling methods. The binding capacity was determined and an acrylic carrier with the most suitable properties selected for detailed studies. As P2O shows higher turnover numbers with the electron acceptor 1,4-benzoquinone than with oxygen, the use of this alternative electron acceptor was enabled by employing laccase for the continuous reoxidation of hydroquinone. The laccase regeneration system was found to increase the specific productivity up to 3-fold. Catalase was used to disproportionate the formed hydrogen peroxide in close proximity to the oxygen consuming enzymes and applied in different amounts to adjust the hydrogen peroxide concentration, which was found to be the main reason for enzyme deactivation under turnover conditions. In contrast to homogeneous catalysis, the specific productivity of heterogeneous catalysts under the applied experimental conditions was limited primarily by oxygen transfer, an effect significantly reduced by the laccase regeneration system.
Asunto(s)
Deshidrogenasas de Carbohidratos/metabolismo , Cetosas/biosíntesis , Animales , Catálisis , Bovinos , Enzimas Inmovilizadas/metabolismo , Óxido de Etileno/metabolismo , Cinética , Lacasa/metabolismo , Microesferas , Solubilidad , Factores de TiempoRESUMEN
3-Keto-l-gulonate 6-phosphate decarboxylase (KGPDC) and d-arabino-hex-3-ulose 6-phosphate synthase (HPS) are members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemistry 41, 3861-3869], a group of homologous enzymes that share the (beta/alpha)(8)-barrel fold. KGPDC catalyzes a Mg(2+)-dependent decarboxylation reaction in the catabolic pathway of l-ascorbate utilization by Escherichia coli K-12 [Yew, W. S., and Gerlt, J. A. (2002) J.Bacteriol. 184, 302-306]; HPS catalyzes a Mg(2+)-dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate in formaldehyde-fixing methylotrophic bacteria [Kato, N., Ohashi, H., Hori, T., Tani, Y., and Ogata, K. (1977) Agric. Biol. Chem. 41, 1133-1140]. Our previous studies of the KGPDC from E. coli established the occurrence of a stabilized cis-enediolate intermediate [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 43, 6427-6437; Wise, E., Yew, W. S., Gerlt, J. A., and Rayment, I. (2004) Biochemistry 43, 6438-6446]. Although the mechanism of the HPS-catalyzed reaction has not yet been investigated, it also is expected to involve a Mg(2+)-stabilized cis-enediolate intermediate. We now have discovered that the KGPDC from E. coli and the HPS from Methylomonas aminofaciens are both naturally promiscuous for the reaction catalyzed by the homologue. On the basis of the alignment of the sequences of orthologous KGPDC's and HPS's, four conserved active site residues in the KGPDC from E. coli were mutated to those conserved in HPS's (E112D/R139V/T169A/R192A): the value of the k(cat) for the promiscuous HPS activity was increased as much as 170-fold (for the E112D/R139V/T169A/R192A mutant), and the value of k(cat)/K(m) was increased as much as 260-fold (for the E112D/R139V/T169A mutant); in both cases, the values of the kinetic constants for the natural KGPDC activity were decreased. Together with the structures of mutants reported in the accompanying manuscript [Wise, E. L., Yew, W. S., Akana, J., Gerlt, J. A., and Rayment, I., accompanying manuscript], these studies illustrate that large changes in catalytic efficiency can be accomplished with only modest changes in active site structure. Thus, the (beta/alpha)(8)-barrel fold shared by members of the OMPDC suprafamily appears well-suited for the evolution of new functions.
Asunto(s)
Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Carboxiliasas/química , Carboxiliasas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Orotidina-5'-Fosfato Descarboxilasa/química , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Alanina/genética , Aldehído-Liasas/genética , Aldehídos/química , Aldehídos/metabolismo , Sustitución de Aminoácidos/genética , Arginina/genética , Ácido Aspártico/genética , Catálisis , Descarboxilación , Estabilidad de Enzimas , Evolución Molecular , Formaldehído/química , Ácido Glutámico/genética , Histidina/química , Cetosas/biosíntesis , Methylomonas/enzimología , Ribulosafosfatos/química , Ribulosafosfatos/metabolismo , Estereoisomerismo , Treonina/genética , Valina/genéticaRESUMEN
The anhydrofructose pathway describes the degradation of glycogen and starch to metabolites via 1,5-anhydro-D-fructose (1,5AnFru). Enzymes that form 1,5AnFru, ascopyrone P (APP), and ascopyrone M (APM) have been reported from our laboratory earlier. In the present study, APM formed from 1,5AnFru was found to be the intermediate to the antimicrobial microthecin. The microthecin forming enzyme from the fungus Phanerochaete chrysosporium proved to be aldos-2-ulose dehydratase (AUDH, EC 4.2.1.-), which was purified and characterized for its enzymatic and catalytic properties. The purified AUDH showing a molecular mass of 97.4 kDa on SDS-PAGE was partially sequenced. Total 332 amino acid residues in length were obtained, representing some 37% of the AUDH protein. The obtained amino acid sequences showed no homology to known proteins but to an unannotated DNA sequence in Scaffold 62 of the published genome of the fungus. The alignment revealed three introns of the identified AUDH gene (Audh; ph.chr), thus the first gene coding for a neutral sugar dehydratase is identified. AUDH was found to be a bi-functional enzyme, being able to dehydrate 1,5AnFru to APM and further isomerizing the APM formed to microthecin. The optimal pH for the formation of APM and microthecin was pH 5.8 and 6.8, respectively. AUDH showed 5 fold higher activity toward 1,5AnFru than toward its analogue glucosone, when tested at concentrations from 0.6 mM to 0.2 M. Based on the characteristic UV absorbance of microthecin (230 nm) and APM (262 nm) assay methods were developed for the microthecin forming enzymes.
Asunto(s)
Fructosa/análogos & derivados , Fructosa/metabolismo , Glucógeno/metabolismo , Hidroliasas/fisiología , Cetosas/biosíntesis , Secuencia de Aminoácidos , Catálisis , Hidroliasas/química , Hidroliasas/genética , Hidroliasas/aislamiento & purificación , Datos de Secuencia Molecular , Especificidad por Sustrato , Terminología como AsuntoRESUMEN
The term "advanced glycation end products" (AGEs) stands for a heterogeneous group of amino acid derivatives that are formed via glycation processes between peptide-bound lysine or arginine derivatives and carbonyl compounds, processes originally known from food systems as "Maillard reactions." AGEs accumulate in plasma and tissues with advancing age, diabetes, and particular renal failure. In vivo and in vitro studies indicate that AGEs represent an important class of uremic toxins. This review focuses on the chemistry behind the formation of AGEs, possible mechanisms underlying the accumulation of AGEs in uremia, clinical and therapeutic implications, and possible nutritional consequences.
Asunto(s)
Productos Finales de Glicación Avanzada/metabolismo , Uremia/metabolismo , Aminoácidos/metabolismo , Animales , Productos Finales de Glicación Avanzada/biosíntesis , Productos Finales de Glicación Avanzada/sangre , Humanos , Cetosas/biosíntesis , Reacción de Maillard , Estado NutricionalRESUMEN
D-Glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) was prepared from D-glucosone (D-arabino-hexos-2-ulose) by enzymatic conversion with hexokinase. The isomeric composition of D-glucosone 6-phosphate in aqueous solution was quantitatively determined by NMR spectroscopy and compared to D-glucosone. The main isomers are the alpha-anomer (58%) and the beta-anomer (28%) of the hydrated pyranose form, and the beta-D-fructofuranose form (14%).
Asunto(s)
Hexoquinasa/metabolismo , Cetosas/biosíntesis , Cetosas/química , Fosfatos/química , Fosfatos/metabolismo , Isomerismo , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO(-)) on the formation of N( epsilon )-(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO(-), CML formation was detected by both enzyme-linked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO(-) concentrations. CML was also formed when glucose, preincubated with ONOO(-), was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for alpha-oxoaldehydes. For identifying the aldehydes that contributed to ONOO(-)-induced CML formation, glucose was incubated with ONOO(-) in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO(-) can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive alpha-oxoaldehydes from glucose.
Asunto(s)
2-Naftilamina/análogos & derivados , Glucosa/metabolismo , Glioxal/metabolismo , Cetosas/biosíntesis , Lisina/biosíntesis , Ácido Peroxinitroso/farmacología , Albúmina Sérica/química , Tirosina/análogos & derivados , 2-Naftilamina/farmacología , Aldehídos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Glucosa/farmacología , Guanidinas/farmacología , Lisina/análogos & derivados , Ratones , Ratones Endogámicos BALB C , Ácido Peroxinitroso/administración & dosificación , Tirosina/metabolismoRESUMEN
5-Acylisoxazolines 3a-d were obtained by 1,3-dipolar cycloaddition from acetoxymethyl vinyl ketone and nitro precursors. Compounds 3a-d were biotransformed by Aspergillus niger into a 1:1 mixture of stereomers of 5-dihydroxyethyl isoxazolines (+)-4a-d (anti) and (-)-5a-d (syn). Both stereomers were obtained in good yields and with high optical purities. Carbonyl reduction by Aspergillus niger produces alcohols of R-configuration thus giving an access to D-sugar analogues: Compound (+)-4d was converted to 3-deoxy-D-erythro-hexulose and several protected derivatives. Total synthesis of 3-deoxy-D-fructose-6-phosphate was also achieved in two steps and 64% overall yield from (+)-4d.
Asunto(s)
Aspergillus niger/metabolismo , Desoxiazúcares/biosíntesis , Isoxazoles/metabolismo , Cetosas/biosíntesis , Acetilación , Biotransformación , Oxidación-Reducción , EstereoisomerismoRESUMEN
Incubation of dTDP-glucose with the enzyme dTDP-glucose-4,6-dehydratase [EC 4.2.1.46] from wild type E. coli B yielded a mixture of 3- and 4-keto-6-deoxy sugars after work-up. Model experiments with chemically synthesized methyl 6-deoxy-4-keto-glucoside (9) revealed that dTDP-6-deoxy-alpha-D-ribo-hexopyran-3-ulose (3) is formed by keto-enol tautomerization during the isolation procedure from initially formed dTDP-6-deoxy-alpha-D-xylo-hexopyran-4-ulose (2). dTDP-3-deoxyglucose (4) and dTDP-3-azido-3-deoxyglucose (6) were substrates and showed Michaelis-Menten kinetics (4: KM = 200 microM and V(max) = 130 mumol/h mg; 6: KM = 300 microM and V(max) = 90 mumol/h mg). In 100-mg-scale experiments, both non-natural substrates gave the respective 6-deoxy-4-keto compounds, dTDP-3,6-dideoxy-alpha-D-erythro-hexopyran-4-ulose (5) and dTDP-3-azido-3,6-dideoxy-alpha-D-xylo-hexopyran-4-ulose++ + (7), in yields ranging from 24 to 40%.
Asunto(s)
Desoxiazúcares/biosíntesis , Escherichia coli/enzimología , Glucosa/análogos & derivados , Hidroliasas/metabolismo , Nucleótidos de Timina/metabolismo , Glucosa/metabolismo , Isomerasas/metabolismo , Cetosas/biosíntesis , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Ramnosa/análogos & derivados , Especificidad por SustratoRESUMEN
A pyranose oxidase was isolated from mycelium extracts of the basidiomycete Peniophora gigantea. This enzyme was purified 104-fold to apparent homogeneity with a yield of about 75% by steps involving fractionated ammonium sulphate precipitation, chromatography on DEAE-Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native pyranose oxidase has a relative molecular mass (M(r)) of 322,800 +/- 18,300 as determined on the basis of its Stokes' radius (rs = 6.2 nm) and sedimentation coefficient (S20,w = 10.6), dynamic light-scattering experiments, gradient-gel electrophoresis and cross-linking studies. SDS/PAGE resulted in one single polypeptide band of M(r) 76,000 indicating that the enzyme consists of four subunits of identical size. The pyranose oxidase was shown to be an extremely stable glycoprotein with an isoelectric point of pH 5.3. It contains covalently bound FAD with an estimated stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates D-glucose, D-xylose, L-sorbose, D-galactose, methyl beta-D-glucoside, maltose and D-fucose. Regioselective oxidation of D-glucose, L-sorbose and D-xylose to 2-keto-D-glucose, 5-keto-D-fructose and 2-keto-D-xylose, was demonstrated by identifying the reaction products by mass spectroscopy 13C-NMR spectroscopy and 1H-NMR spectroscopy after purification and derivatization. The pH optimum of the pyranose oxidase was in the range pH 6.0-6.5 in 0.1 M potassium phosphate, and its activation energy (delta H degree) for the conversion of D-glucose was 34.6 kJ/mol. The reactions with the sugars exhibited Michaelis-Menten kinetics, and the Km values determined for D-glucose, L-sorbose, D-xylose and oxygen were 1.1 mM, 50.0 mM, 29.4 mM and 0.65 mM, respectively. The activity of pyranose oxidase was only slightly affected by chelating reagents, thiol reagents, reducing reagents and bivalent cations each at 1 mM.
Asunto(s)
Basidiomycota/enzimología , Deshidrogenasas de Carbohidratos/metabolismo , Glicoproteínas/metabolismo , Cetosas/biosíntesis , Monosacáridos/metabolismo , Basidiomycota/crecimiento & desarrollo , Deshidrogenasas de Carbohidratos/química , Deshidrogenasas de Carbohidratos/aislamiento & purificación , Flavinas/análisis , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Cinética , Peso Molecular , Sorbosa/metabolismo , Espectrofotometría , Especificidad por Sustrato , Xilosa/metabolismoRESUMEN
Pyranose oxidase and pyranosone dehydratase (aldos-2-ulose dehydratase), enzymes which convert in coupled reactions D-glucose to beta-pyrone cortalcerone, peaked coincidently during idiophasic growth of Phanerochaete chrysosporium under agitated conditions. The enzymes were purified from mycelial extracts of the fungus and separated from each other by hydrophobic interaction chromatography on Phenyl-Sepharose and Phenyl-Superose. Two pyranosone dehydratase activity peaks, PD I and PD II, were resolved. The major PD I fraction, consisting about 74% of the total dehydratase activity, was further purified by anion exchange chromatography on Mono Q to yield apparently pure enzyme as judged by SDS-PAGE and gel filtration on Superose 12. Isoelectric focusing indicated microheterogeneity of the protein by the presence of at least five protein bands with pI 5.1-5.3. PD II had a pI of 5.75. Overall PD I purification was 60.7-fold with 50% yield. The enzyme acted on several osones (glycosuloses), with the preferred substrate being D-glucosone. D-Xylosone and 6-deoxy-D-glucosone were dehydrated at C-3-C-4 to give the corresponding 5-hydroxy-2,3-dioxoalcanals (4-deoxy-2,3-glycosdiuloses), new enzymatically produced sugar derivatives. The latter labile compounds were trapped as diphenylhydrazine or o-phenylenediamine derivatives and spectroscopically identified. The analogous D-glucosone dehydration product did not accumulate due to its further transformation. pH optimum of PD I activity was 6.0 and its pH stability was optimal at pH 7-11. The enzyme was sensitive to Me2+ chelating agents and some heavy metal ions (Hg2+, Cu2+).
Asunto(s)
Basidiomycota/enzimología , Desoxiglucosa/análogos & derivados , Hidroliasas/aislamiento & purificación , Cetosas/metabolismo , Deshidrogenasas de Carbohidratos/biosíntesis , Quelantes/farmacología , Cromatografía por Intercambio Iónico , Desoxiglucosa/biosíntesis , Desoxiglucosa/metabolismo , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Calor , Hidroliasas/biosíntesis , Hidroliasas/química , Hidroliasas/metabolismo , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Cetosas/biosíntesis , Especificidad por SustratoRESUMEN
A total of 20 laboratory substrains of Agrobacterium tumefaciens strain B6 were compared with respect to six characteristics, including 3-ketolactose production, lysogeny, octopine catabolism, tumorigenic host range, and plasmid content. Within this group of strains diversity was found for all characteristics except 3-ketolactose production. Six substrains were lysogenized with an omega-type phage, whereas one substrain appeared neither sensitive to nor lysogenized with this bacteriophage. All but two substrains catabolized octopine and induced tumors on carrot disks. These 18 substrains harbor deoxyribonucleic acid sequences homologous to pTiB6-806. The two substrains unable to catabolize octopine were nontumorigenic and lacked detectable Ti plasmid sequences. Of the 20 substrains, 13 also contained sequences homologous to the cryptic plasmid pAtB6-806; 2 of the 18 substrains tumorigenic on carrots failed to induce tumors on Kalanchoe leaves. Their inability to induced tumors on this host, could not be correlated with lysogeny, with the presence or absence of pAtB6-806, or with the very large cryptic plasmid recently described. The Ti plasmids from these two strains were indistinguishable from pTiB6-806 by restriction enzyme analysis and could genetically convert a cured A. tumefaciens strain to tumorigenicity on both plant species. The results with these two strains suggest that parameters of tumorigenicity, such as host range, may be controlled by the bacterial chromosome.
Asunto(s)
Rhizobium/fisiología , Arginina/análogos & derivados , Arginina/metabolismo , Bacteriófagos/crecimiento & desarrollo , Cetosas/biosíntesis , Lactosa/análogos & derivados , Lactosa/biosíntesis , Lisogenia , Tumores de Planta/microbiología , Plásmidos , Rhizobium/genética , Especificidad de la EspecieAsunto(s)
Fructosa-Bifosfato Aldolasa/metabolismo , Cetosas/metabolismo , Hígado/enzimología , Músculos/enzimología , Fosfatos de Azúcar/metabolismo , Animales , Arabinosa/metabolismo , Catálisis , Técnicas In Vitro , Cetosas/biosíntesis , Cinética , Especificidad de Órganos , Conejos , Ribosamonofosfatos/metabolismo , Fosfatos de Azúcar/biosíntesisRESUMEN
The selective paradoxical sleep (PS) deprivation in rats entailed during first 2 days a drop in activity of glucose-6-phosphatehydrogenase by 80 and 60%, and of 6-phosphogluconatdehydrogenase -- by 20--35 and 40--50% in the brain-stem and the cortex, respectively. Activity of the transketolase decreased by 27--29% on the 2nd day of PS deprivation only. On the 4th day, normalizing of activity of all the enzymes under study except transketolase, was observed in both regions of the brain. The consumption of riboso-5-phosphate by the homogenate and level of phosphoketopentoses formation in it reflected, mainly, the dynamics of transketolase activity changes.