Uptake of monosaccharides by guinea-pig cerebral-cortex slices

By the use of 1mm-iodoacetate to inhibit glycolysis in guinea-pig cerebral tissue slices, the kinetics of the uptake of monosaccharides on transfer of tissue from 0° to 37° were studied. d-Ribose, d-galactose, d-mannose, l-sorbose, and d-fructose showed diffusion kinetics, whereas 2-deoxy-d-glucose, d-glucose, d-arabinose and d-xylose showed saturation kinetics.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Substrate Specificity of a Mannose-6-Phosphate Isomerase from Bacillus subtilis and Its Application in the Production of l-Ribose

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co²⁺. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter⁻¹ h⁻¹.l-Ribose is a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). l-Ribose has been produced mainly by chemical synthesis from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, or d-mannono-1,4-lactone (2, 17, 23). Biological l-ribose manufacture has been investigated using ribitol or l-ribulose. Recently, l-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of l-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from l-arabinose (8). l-Ribulose has been biochemically converted from l-ribose using an l-ribose isomerase from an Acinetobacter sp. (9), an l-arabinose isomerase mutant from Escherichia coli (4), a d-xylose isomerase mutant from Actinoplanes missouriensis (14), and a d-lyxose isomerase from Cohnella laeviribosi (3), indicating that l-ribose can be produced from l-ribulose by these enzymes. However, the enzymatic production of l-ribulose is slow, and the enzymatic production of l-ribose from l-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11, 12, 21, 22). l-Ribose isomerase from an Acinetobacter sp. (9) and d-lyxose isomerase from C. laeviribosi (3) had activity with l-ribose, d-lyxose, and d-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of l-ribose, because there are no sugar phosphate isomerases relating to l-ribose and d-lyxose. The production of the expensive sugar l-ribose (bulk price, $1,000/kg) from the rare sugar l-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced l-ribulose from the cheap sugar l-arabinose (bulk price, $50/kg) using the l-arabinose isomerase from Geobacillus thermodenitrificans (20) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic representation for the production of l-ribulose from l-arabinose by G. thermodenitrificans l-arabinose isomerase and the production of l-ribose from l-ribulose by B. subtilis mannose-6-phosphate isomerase.In this study, the gene encoding mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in E. coli. The substrate specificity of the recombinant enzyme for various aldoses and ketoses was investigated, and l-ribulose exhibited the highest activity among all pentoses and hexoses. Therefore, mannose-6-phosphate isomerase was applied to the production of l-ribose from l-ribulose.     

The biosynthesis of polysaccharides. Incorporation of d-[1-14C]glucose and d-[6-14C]glucose into plum-leaf polysaccharides

1. The utilization of specifically labelled d-glucose in the biosynthesis of plum-leaf polysaccharides has been studied. After these precursors had been metabolized in plum leaves, the polysaccharides were isolated from the leaves, and their monosaccharide constituents isolated and purified. 2. Both the specific activities and the distribution of ¹⁴C along the carbon chains of the monosaccharides were determined. Significant ¹⁴C activity was found in units of d-galactose, d-glucose, d-xylose and l-arabinose, but their specific activities varied widely. The labelling patterns suggest that in the leaves the other monosaccharides all arise directly from d-glucose without any skeletal change in the carbon chain, other than the loss of a terminal carbon atom in the synthesis of pentoses. 3. The results indicated that within the leaf there are various precursor pools for polysaccharide synthesis and that these pools are not in equilibrium with one another.     

Structural Features of Sugars That Trigger or Support Conidial Germination in the Filamentous Fungus Aspergillus niger

The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia.     

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K⁺-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a K_m of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H⁺-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H⁺-sucrose symporter.     

Substrate specificity of the Bacillus licheniformis lyxose isomerase YdaE and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on d-lyxose, suggesting that the enzyme is d-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²⁺. The apparent K_m values for d-lyxose and d-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k_cat/K_m) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for d-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of d-lyxose and d-glucose from d-xylose and d-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM d-lyxose and d-mannose, 3.7 mM and 3.8 mM d-lyxose and d-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.     

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following ¹³C-labeling patterns of proteinogenic amino acids after growth on [¹³C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus.d-Xylose, a constituent of the polymer xylan, is the major component of the hemicellulose plant cell wall material and thus one of the most abundant carbohydrates in nature. The utilization of d-xylose by microorganisms has been described in detail in bacteria and fungi, for which two different catabolic pathways have been reported. In many bacteria, such as Escherichia coli, Bacillus, and Lactobacillus species, xylose is converted by the activities of xylose isomerase and xylulose kinase to xylulose 5-phosphate as an intermediate, which is further degraded mainly by the pentose phosphate cycle or phosphoketolase pathway. Most fungi convert xylose to xylulose 5-phosphate via xylose reductase, xylitol dehydrogenase, and xylulose kinase. Xylulose 5-phosphate is also an intermediate of the most common l-arabinose degradation pathway in bacteria, e.g. of E. coli, via activities of isomerase, kinase, and epimerase (1).Recently, by genetic evidence, a third pathway of xylose degradation was proposed for the bacterium Caulobacter crescentus, in analogy to an alternative catabolic pathway of l-arabinose, reported for some bacteria, including species of Azospirillum, Pseudomonas, Rhizobium, Burkholderia, and Herbasprillum (2, 3). In these organisms l-arabinose is oxidatively degraded to α-ketoglutarate, an intermediate of the tricarboxylic acid cycle, via the activities of l-arabinose dehydrogenase, l-arabinolactonase, and two successive dehydration reactions forming 2-keto-3-deoxy-l-arabinoate and α-ketoglutarate semialdehyde; the latter compound is further oxidized to α-ketoglutarate via NADP⁺-specific α-ketoglutarate semialdehyde dehydrogenase (KGSADH).² In a few Pseudomonas and Rhizobium species, a variant of this l-arabinose pathway was described involving aldolase cleavage of the intermediate 2-keto-3-deoxy-l-arabinoate to pyruvate and glycolaldehyde, rather than its dehydration and oxidation to α-ketoglutarate (4). Because of the presence of some analogous enzyme activities in xylose-grown cells of Azosprillum and Rhizobium, the oxidative pathway and its variant was also proposed as a catabolic pathway for d-xylose. Recent genetic analysis of Caulobacter crecentus indicates the presence of an oxidative pathway for d-xylose degradation to α-ketoglutarate. All genes encoding xylose dehydrogenase and putative lactonase, xylonate dehydratase, 2-keto-3-deoxylonate dehydratase, and KGSADH were found to be located on a xylose-inducible operon (5). With exception of xylose dehydrogenase, which has been partially characterized, the other postulated enzymes of the pathway have not been biochemically analyzed.The pathway of d-xylose degradation in the domain of archaea has not been studied so far. First analyses with the halophilic archaeon Haloarcula marismortui indicate that the initial step of d-xylose degradation involves a xylose-inducible xylose dehydrogenase (6) suggesting an oxidative pathway of xylose degradation to α-ketoglutarate, or to pyruvate and glycolaldehyde, in analogy to the proposed oxidative bacterial pentose degradation pathways. Recently, a detailed study of d-arabinose catabolism in the thermoacidophilic crenarchaeon Sulfolobus solfataricus was reported. d-Arabinose was found to be oxidized to α-ketoglutarate involving d-arabinose dehydrogenase, d-arabinoate dehydratase, 2-keto-3-deoxy-d-arabinoate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase (3).In this study, we present a comprehensive analysis of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. This halophilic archaeon was chosen because it exerts several suitable properties for the analyses. For example, it can be cultivated on synthetic media with sugars, e.g. xylose, an advantage for in vivo labeling studies in growing cultures. Furthermore, a shotgun DNA microarray of H. volcanii is available (7) allowing the identification of xylose-inducible genes, and H. volcanii is one of the few archaea for which an efficient protocol was recently described to generate in-frame deletion mutants.Accordingly, the d-xylose degradation pathway was elucidated following in vivo labeling experiments with [¹³C]xylose, DNA microarray analyses, and the characterization of enzymes involved and their encoding genes. The functional involvement of genes and enzymes was proven by constructing corresponding in-frame deletion mutants and their analysis by selective growth experiments on xylose versus glucose. The data show that d-xylose was exclusively degraded to α-ketoglutarate involving xylose dehydrogenase, a novel xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase.     

The Production of Xylitol,l-Arabinitol and Ribitol by Yeasts

The polyalcohol production from the pentoses such as d-xylose, l-arabinose and d-ribose by various genera and species of yeasts was examined. Candida polymorpha dissimilated aerobically these three pentoses and produced xylitol from d-xylose, l-arabinitol from l-arabinose and ribitol from d-ribose at good yield of 30~40% of sugar consumed. The result suggests that these polyalcohols would be major products from pentoses by yeasts, but some unidentified minor polyalcohols were also produced.     

Functional Analysis of Two l-Arabinose Transporters from Filamentous Fungi Reveals Promising Characteristics for Improved Pentose Utilization in Saccharomyces cerevisiae

Limited uptake is one of the bottlenecks for l-arabinose fermentation from lignocellulosic hydrolysates in engineered Saccharomyces cerevisiae. This study characterized two novel l-arabinose transporters, LAT-1 from Neurospora crassa and MtLAT-1 from Myceliophthora thermophila. Although the two proteins share high identity (about 83%), they display different substrate specificities. Sugar transport assays using the S. cerevisiae strain EBY.VW4000 indicated that LAT-1 accepts a broad substrate spectrum. In contrast, MtLAT-1 appeared much more specific for l-arabinose. Determination of the kinetic properties of both transporters revealed that the K_m values of LAT-1 and MtLAT-1 for l-arabinose were 58.12 ± 4.06 mM and 29.39 ± 3.60 mM, respectively, with corresponding V_max values of 116.7 ± 3.0 mmol/h/g dry cell weight (DCW) and 10.29 ± 0.35 mmol/h/g DCW, respectively. In addition, both transporters were found to use a proton-coupled symport mechanism and showed only partial inhibition by d-glucose during l-arabinose uptake. Moreover, LAT-1 and MtLAT-1 were expressed in the S. cerevisiae strain BSW2AP containing an l-arabinose metabolic pathway. Both recombinant strains exhibited much faster l-arabinose utilization, greater biomass accumulation, and higher ethanol production than the control strain. In conclusion, because of higher maximum velocities and reduced inhibition by d-glucose, the genes for the two characterized transporters are promising targets for improved l-arabinose utilization and fermentation in S. cerevisiae.     

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb

A Mg²⁺-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and β-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d-myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6-phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3-Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg²⁺. At 4 millimolar, Co²⁺, Fe²⁺ or Mn²⁺ are less effective. Substantial inhibition is obtained with 0.25 molar Li⁺. With β-glycerophosphate as substrate the K_m is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, β-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.     

The metabolism of galactarate, d-glucarate and various pentoses by species of Pseudomonas

1. When NAD⁺ was present, cell extracts of Pseudomonas (A) grown with d-glucarate or galactarate converted 1mol. of either substrate into 1mol. each of 2-oxoglutarate and carbon dioxide; 70–80% of the gas originated from C-1 of the hexarate. 2. The enzyme system that liberated carbon dioxide from galactarate was inactive in air and was stabilized by galactarate or Fe²⁺ ions; the system that acted on d-glucarate was more stable and was stimulated by Mg²⁺ ions. 3. When NAD⁺ was not added, 2-oxoglutarate semialdehyde accumulated from either substrate. This compound was isolated as its bis-2,4-dinitrophenylhydrazone, and several properties of the derivative were compared with those of the chemically synthesized material. Methods were developed for the determination of 2-oxoglutarate semialdehyde. 4. Synthetic 2-oxoglutarate semialdehyde was converted into 2-oxoglutarate by an enzyme that required NAD⁺; the reaction rate with NADP⁺ was about one-sixth of that with NAD⁺. 5. For extracts of Pseudomonas (A) grown with d-glucarate or galactarate, or for those of Pseudomonas fragi grown with l-arabinose or d-xylose, specific activities of 2-oxoglutarate semialdehyde–NAD oxidoreductase were much higher than for extracts of the organisms grown with (+)-tartrate and d-glucose respectively. 6. Extracts of Pseudomonas fragi grown with l-arabinose or d-xylose converted l-arabonate or d-xylonate into 2-oxoglutarate when NAD⁺ was added to reaction mixtures and into 2-oxoglutarate semialdehyde when NAD⁺ was omitted.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

l-Ribose Production from l-Arabinose by Using Purified l-Arabinose Isomerase and Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

Two enzymes, l-arabinose isomerase and mannose-6-phosphate isomerase, from Geobacillus thermodenitrificans produced 118 g/liter l-ribose from 500 g/liter l-arabinose at pH 7.0, 70°C, and 1 mM Co²⁺ for 3 h, with a conversion yield of 23.6% and a volumetric productivity of 39.3 g liter⁻¹ h⁻¹.l-Ribose, a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, is not abundant in nature (4, 15, 20). l-Ribose has been synthesized primarily from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, and d-mannono-1,4-lactone (1, 13, 20). Recombinant cells containing a NAD-dependent mannitol-1-dehydrogenase produced 52 g/liter l-ribose from 100 g/liter ribitol after fermentation for 72 h (14). However, the volumetric productivity of l-ribose was 26-fold lower than that of the chemical synthetic method starting from l-arabinose (6). l-Ribose isomerase from an Acinetobacter sp., which is most active with l-ribose, showed poor efficiency in the conversion of l-ribulose to l-ribose (9). Recently, l-ribulose was produced with a conversion yield of 19% from the inexpensive sugar l-arabinose using l-arabinose isomerase (AI) from Geobacillus thermodenitrificans (18). l-Ribose has been produced from l-ribulose using mannose-6-phosphate isomerase (MPI) from Bacillus subtilis with a conversion yield of 70% (17). In this study, the production of l-ribose from l-arabinose was demonstrated via a two-enzyme system from G. thermodenitrificans, in which l-ribulose was first produced from l-arabinose by AI and subsequently converted to l-ribose by MPI.The analysis of monosaccharides and the purification and thermostability of AI and MPI from G. thermodenitrificans (2) isolated from compost were performed as described previously (7, 18, 19). The cross-linked enzymes were obtained from the treatment of 0.5% glutaraldehyde (10, 16). The reaction was performed by replacing the reaction solution with 100 g/liter l-arabinose and 1 mM Co²⁺ every 6 h at 70°C and pH 7.0. The reaction volume of 10 ml contained 5 g of the cross-linked enzymes with 8 U/ml AI and 20 U/ml MPI. One unit of AI or MPI activity, which corresponded to 0.0625 or 2.5 mg protein, respectively, was defined as the amount of enzyme required to produce 1 μmol of l-ribulose or l-ribose, respectively, per min at 70°C, pH 7.0, and 1 mM Co²⁺. Unless otherwise stated, the reaction was carried out in 50 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 7.0) in the presence of 1 mM Co²⁺ at 70°C for 4 h. All experiments were performed in triplicate.The recombinant Escherichia coli ER2566 (New England Biolabs, Ipswich, MA) containing pTrc99A plasmid (Pharmacia Biotech, Piscataway, NJ) and the AI or MPI gene was cultivated in a 7-liter fermentor containing 3 liters of chemically defined medium (11). When the cell mass reached 2 g/liter, 10 g/liter lactose was added for enzyme induction. After 14 h, 40 g/liter cells with 13,400 U/liter of AI or 34 g/liter cells with 630 U/liter of MPI was obtained. The enzyme was purified by heat treatment and Hi-Trap anion-exchange chromatography. The purification yields of AI and MPI were 21 and 78%, respectively, and the levels of purity for the concentrated AI and MPI by gene scanning were 48 and 92%, respectively. Maximum l-ribose production from l-arabinose by AI and by MPI in 10 ml of total volume was observed at pH 7.0, 70°C, and 1 mM Co²⁺ (data not shown). Half-lives for the two-enzyme system containing 10 mM l-arabinose, 0.2 U/ml AI, and 0.5 U/ml MPI at 60, 65, 70, 75, and 80°C were 1,216, 235, 48, 26, and 12 h, respectively. The use of Co²⁺ may be disadvantageous, as it is fairly toxic. This problem can be solved by using Mn²⁺ instead of Co²⁺. When Mn²⁺ was used in the reaction with the same amounts of enzymes, the conversion yield was the same as that obtained with Co²⁺, even though the volumetric productivity was lower than that with Co²⁺ (data not shown).The effect of the ratio of AI to MPI in the two-step enzymatic production of l-ribose from l-arabinose was investigated by mixing the enzyme solutions (8 U/ml AI and 20 U/ml MPI) to obtain AI/MPI ratios ranging from 10:90 to 90:10 (vol/vol) (Fig. ​(Fig.1).1). The reactions were run with 300 g/liter l-arabinose. Maximum l-ribose production was observed at a volume ratio of 50:50 of the enzyme solutions. The effects of enzyme concentration on l-ribose production were investigated at the optimal unit ratio (AI/MPI ratio, 1:2.5) with 500 g/liter l-arabinose and AI and MPI concentrations from 0.4 and 1.0 U/ml, respectively, to 9.2 and 23.0 U/ml, respectively (Fig. ​(Fig.2A).2A). l-Ribose production increased with increasing amounts of enzymes until reaching a plateau at 8 U/ml AI and 20 U/ml MPI. The effect of substrate concentration on l-ribose production was evaluated at l-arabinose concentrations ranging from 15 to 500 g/liter with 8 U/ml AI and 20 U/ml MPI (Fig. ​(Fig.2B).2B). The production of both l-ribose and l-ribulose, an intermediate, increased with increasing substrate level. The results suggest that concentrations of substrate above 500 g/liter l-arabinose might cause the increased production. The conversion yields of l-ribose and l-ribulose from l-arabinose were constant at 32% and 14%, respectively, within an initial concentration of 100 g/liter l-arabinose, indicating that the reactions reached equilibrium at an l-arabinose/l-ribulose/l-ribose ratio of 54:14:32, which was in agreement with the calculated equilibrium (17). However, at l-arabinose concentrations above 100 g/liter, the conversion yields of l-ribose and l-ribulose from l-arabinose decreased with increasing l-arabinose concentration. The l-arabinose/l-ribulose/l-ribose ratio, with an initial l-arabinose concentration of 300 g/liter, was 71:6:23 after 4 h of reaction. To obtain near-equilibrium (54:14:32) at this high concentration of l-arabinose, more effective enzymes are required.Open in a separate windowFIG. 1.Effect of the ratio of AI to MPI on l-ribose production from l-arabinose by the purified AI and MPI from G. thermodenitrificans. Data are the means for three separate experiments, and error bars represent standard deviations. Symbols: •, l-ribose; ▪, l-ribulose.Open in a separate windowFIG. 2.(A) Effect of enzyme concentration on l-ribose production from l-arabinose at the optimal unit ratio (AI/MPI ratio, 1:2.5). Symbols: •, l-ribose; ▪, l-ribulose; ○, l-arabinose. (B) Effect of l-arabinose concentration on l-ribose production. Symbols: •, l-ribose; ▪, l-ribulose. Data are the means for three separate experiments, and error bars represent standard deviations.A time course reaction of l-ribose production from l-arabinose was monitored for 3 h with 8 U/ml AI and 20 U/ml MPI (Fig. ​(Fig.3).3). As a result, 118 g/liter l-ribose was obtained from an initial l-arabinose concentration of 500 g/liter after 3 h, with a conversion yield of 23.6% and a productivity of 39.3 g liter⁻¹ h⁻¹. Recombinant E. coli containing MDH yielded 52 g/liter l-ribose from an initial ribitol concentration of 100 g/liter after 72 h, with a productivity of 0.72 g liter⁻¹ h⁻¹ (14). The production and productivity obtained in the current study using AI and MPI from G. thermodenitrificans were 2.3- and 55-fold higher, respectively, than those obtained from ribitol and 17- and 21-fold higher than those obtained with the production of l-ribose from l-arabinose using resting cells of recombinant Lactobacillus plantarum (5). The chemical synthetic method is capable of producing 56.5 g/liter l-ribose from 250 g/liter l-arabinose after 3 h, corresponding to a productivity of 18.8 g liter⁻¹ h⁻¹ (6). Still, both the production and productivity of l-ribose using the method described herein were 2.1-fold higher. Thus, the method of production of l-ribose in the present study exhibited the highest productivity and production, compared to other fermentation methods and chemical syntheses.Open in a separate windowFIG. 3.Time course of l-ribose production from l-arabinose by purified AI and MPI from G. thermodenitrificans. Data are the means for three separate experiments, and error bars represent standard deviations. Symbols: •, l-ribose; ▪, l-ribulose; ○, l-arabinose.Several rounds of conversion reusing the cross-linked enzymes were performed (Fig. ​(Fig.4).4). The immobilized enzymes showed more than 20% conversion of l-ribose from l-arabinose for the 9th batch, and the concentration of l-ribose was reduced to 43% after the 20th batch. These results suggest that the immobilization of enzyme facilitates separation of product and enzyme, and it enables the enzyme to function continuously, as reported previously (3, 8, 12). Thus, the reuse of enzyme by immobilization improves the economic viability of this enzymatic process.Open in a separate windowFIG. 4.Reuse of immobilized AI and MPI from G. thermodenitrificans for l-ribose production from 100 g/liter l-arabinose. Data are the means for three separate experiments, and error bars represent standard deviations.     

Studies on Glucose-Isomerizing Enzyme of Bacteria

d-Glucose-isomerizing enzyme from Escherichia intermedia HN-500, which converts d-glucose to d-fructose in the presence of arsenate, was purified by treating with manganous sulfate, rivanol, and DEAE-Sephadex column chromatography. About 180-fold purified enzyme preparation was obtained by the above procedures. The purified preparation was free from the activities of d-glucose-, d-galactose-, glucose-6-phosphate-, mannitol-, and sorbitol-dehydrogenases and was homogeneous on polyacrylamide gel in zone electrophoresis. Optima of pH and temperature for the enzyme were found to be pH 7.0 and 50°C, respectively. The enzyme was completely inactivated by heating at 60°C for ten minutes and stable in the pH range of 7.0~9.0 at 30°C. Activation energy for the isomerizing enzyme was calculated to be 15,300 calories per mole degree from Arrhenius' equation. Either in the absence or presecne of arsenate, d-mannose, d-xylose, d-mannitol and d-sorbitol could not be isomerized by the purified enzyme at all, but the present enzyme isomerized exclusively glucose-6-phosphate and fructose-6-phosphate in the absence of arsenate.     

Structure of Cell Wall Polysaccharide Isolated from Cotyledon of Tora-bean (Phaseolus vulgaris)

The cell wall polysaccharide of cotyledon of Tora-bean (Phaseolus vulgaris), which surrounds starch granules, was isolated from saline-extraction residues of homogenized cotyledon, as alkali-insoluble fibrous substance. Alkali-insoluble residue, which had been treated with α-amylase (Termamyl), had a cellulose-like matrix under the electron microscope. It was composed of l-arabinose, d-xylose, d-galactose and d-glucose (molar ratio, 1.0: 0.2: 0.1: 1.2) together with a trace amount of l-fucose. Methylation followed by hydrolysis of the polysaccharide yielded 2, 3, 5-tri-O-methyl-l-arabinose (3.3 mol), 2, 3, 4-tri-O-methyl-d-xylose (1.0 mol), 2, 3-di-O-methyl-l-arabinose (3.7 mol), 3, 4-di-O-methyl-d-xylose (1.0 mol), 2-O-methyl-l-arabinose and 2, 3, 6-tri-O-methyl-d-glucose (12.7 mol), 2, 6-di-O-methyl-d-glucose (1.2 mol) and 2, 3-di-O-methyl-d-glucose (1.0 mol).

Methylation analysis, Smith degradation and enzymatic fragmentation with cellulase and α-l-arabinofuranosidase showed that the l-arabinose-rich alkali-insoluble polysaccharide possesses a unique structural feature, consisting of β-(1 → 4)-linked glucan backbone, which was attached with side chains of d-xylose residue and β-d-galactoxylose residue at O-6 positions and α-(1 → 5)-linked l-arabinosyl side cains (DP=8) at O-3 positions of β-(1 → 4)-linked d-glucose residues, respectively.     

The Role of a d-Mannosyl-Lipid as an Intermediate in the Synthesis of Polysaccharide in Phaseolus aureus Seedlings

Particulate preparations from Phaseolus aureus produce a d-mannosyl-lipid when treated with GDP-d-mannose. This lipid complex appears to be an active d-mannose donor, and some investigators have proposed that its role might be an obligatory intermediate in mannan synthesis of higher plants. When the partially purified d-mannosyl-lipids, isotopically labeled in the d-mannose moiety, were treated with particulate enzymes under a variety of conditions, a negligible amount of material was produced that behaved as a polysaccharide. Endogenous, particle-bound d-mannosyl-¹⁴C-lipid prepared from P. aureus particles readily transferred d-mannose to GDP to yield GDP-d-mannose and was hydrolyzed to free d-mannose when treated briefly with 0.01 n HCl at 100 C. The d-mannosyl-lipid, therefore, exhibits active d-mannose transfer potential in its endogenous state. When endogenous glycosyl-lipid was incubated in the absence of GDP-d-mannose-¹⁴C, little or no polysaccharide was produced. It was, instead, slowly degraded to d-mannose. Addition of several different unlabeled sugar nucleotides had no effect on the results. Our studies to date, therefore, offer no evidence that the mannosyl-lipid is an obligatory precursor of polysaccharide.