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.     

The Acceptor Specificity of Amylomaltase from Escherichia coli IFO 3806

The acceptor specificity of amylomaltase from Escherichia coli IFO 3806 was investigated using various sugars and sugar alcohols. d-Mannose, d-glucosamine, N-acetyl- d-glucosamine, d-xylose, d- allose, isomaltose, and cellobiose were efficient acceptors in the transglycosylation reaction of this enzyme. It was shown by chemical and enzymic methods that this enzyme could transfer glycosyl residues only to the C4-hydroxyl groups of d-mannose, iY-acetyl- d-glucosamine, d-allose, and d-xylose, producing oligosaccharides terminated by 4–0-α-d-glucopyranosyl-d-mannose, 4–0-α-d-glucopyranosyl-yV-acetyl-d-glucosamine, 4-O-α-d-glucopyranosyl-d-allose, and 4–0-α-d-gluco- pyranosyl-d-xylose at the reducing ends, respectively.     

Cloning of the d-Xylose Uptake Gene Linked to the xyl A Gene in Escherichia coli

An Escherichia coli mutant (MX-5) deficient in d-xylose utilization was isolated. The d-xylose uptake and d-xylose isomerase activities of the mutant were much lower than those of the parental strain (C600). The genes responsible for the d-xylose uptake by E. coli were cloned onto vector plasmid pBR322, and the resultant hybrid plasmid was designated as pXP5. Hybrid plasmid pXP5 improved the growth rate of the mutant (MX-5) on d-xylose, and also both the d-xylose uptake and d-xylose isomerase activities of the mutant were recovered when pXP5 was introduced into the mutant cells. Based on these results, it was suggested that one (xyl T) of the d-xylose transport genes could be closely linked to the d-xylose isomerase gene (xylA) known to be present at 80 min on E. coli chromosomal DNA.     

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.     

Molecular analysis of NAD+-dependent xylitol dehydrogenase from the zygomycetous fungus Rhizomucor pusillus and reversal of the coenzyme preference

The zygomycetous fungus Rhizomucor pusillus NBRC 4578 is able to ferment not only d-glucose but also d-xylose into ethanol. Xylitol dehydrogenase from R. pusillus NBRC 4578 (RpXDH), which catalyzes the second step of d-xylose metabolism, was purified, and its enzymatic properties were characterized. The purified RpXDH preferred NAD⁺ as its coenzyme and showed substrate specificity for xylitol, d-sorbitol, and ribitol. cDNA cloning of xyl2 gene encoding RpXDH revealed that the gene included a coding sequence of 1,092?bp with a molecular mass of 39,185?kDa. Expression of the xyl2 in R. pusillus NBRC 4578 was induced by d-xylose, and the expression levels were increased with accumulation of xylitol. The xyl2 gene was expressed in Escherichia coli, and coenzyme preference of the recombinant RpXDH was reversed from NAD⁺ to NADP⁺ in the double mutant D205A/I206R by site-directed mutagenesis.     

Studies on d-Glucose Isomerizing Activity of d-Xylose Grown Cells from Bacillus coagulans,Strain HN-68

A thermophilic spore-forming strain HN-68, only d-xylose grown cells of which have an activity of d-glucose isomerization, was isolated from soil, and identified to be similar to Bacillus coagulans Hammer. The conditions necessary for maximal production of the glucose isomerizing activity by the cells from shaken cultures in d-xylose media were studied. Much higher activities were observed with the cells grown from 14 ~ 16 hours at 40°C on d-xylose medium containing yeast extract, ammonium chloride, manganese sulfate and calcium carbonate. d-Glucose isomerizing activity was also developed inductively by exposing the washed cells grown on d-glucose to d-xylose within one hour. With the use of living cells as an enzyme source, the addition of both cobaltous ion and toluene in reaction system remarkably enhanced the reaction rate of d-glucose isomerization.     

Enzymatic Assay of d-Xylose Isomerase Activity

The d-xylose isomerase activity was assayed spectrophotometrically as NADH oxidation in a coupled reaction with the d-arabitol dehydrogenase. The assay system is based on the following reactions:

d-Arabitol dehydrogenase was purified from the d-sorbitol-grown cells of Agrobacterium tumefaciens. The standard assay condition is as follows: 5 μmoles of Tris-HCl buffer (pH 7.0), 0.2 μmole of MnCl₂, 2 μl of reduced glutathione (25 mg/ml), 0.05 μmole of NADH, 6 units of d-arabitol dehydrogenase, 5 μmoles of d-xylose and d-xylose isomerase in a total volume of 0.30 ml. The reaction was carried out at 30°C. With the assay system, it was confirmed that d-xylose isomerase did not produce d-xylulose from d-lyxose.     

Purification and Properties of d-Xylose Isomerase from Alkalophilic Bacillus No. KX-6

An alkalophilic Bacillus No. KX-6 isolated from soil produced a d-xylose isomerase in alkaline media. The striking characteristic of this bacterium was its especially good growth in alkaline media. The d-xylose isomerase of this bacterium was purified by ammonium sulfate fractionation, DEAE-Sepharose ion exchange column chromatography and G-200 gel Alteration. The molecular weight and sedimentation constant were approximately 120,000 and 9.35 S, respectively. The enzyme was most active at pH 7~10 and was stable at pH 6.0 to 11.0. Enzyme activity was stimulated by cobalt ion but inhibited by Hg^{2 +}, Ag^{2 +}, and Cu^{2 +}. Substrate specificity studies showed that this enzyme was active on d-xylose, d-glucose, d-ribose, and d-arabinose. The smaller Km value and larger V_max value for d-xylose indicated that this enzyme is essentially d-xylose isomerase.     

Occurrence of Superoxide Dismutase in Bovine Milk

Acidic heteropolysaccharides, d-glucurono-d-xylo-d-mannans were isolated from the water- and alkaline extracts of the fruit body of Tremella fuciformis Berk. Similar polysaccharides were isolated from the growing culture of the haploid cells of two strains (T–19 and T–7) of T. fuciformis, when they were cultured in sucrose or glucose-yeast extract medium. The extracellular polysaccharides contain, d-glucuronic acid, d-xylose and d-mannose [molar ratios, 1.3: 1.0: 3.5 (T–7) and 0.8: 1.0: 2.1 (T–19)], and, in addition, small proportions of l-fucose and O-acetyl groups. Methylation and Smith degradation studies indicated that both fruit body and extracellular polysaccharides are built up of α-(1 → 3)-linked d-mannan backbone chain to which β-linked d-glucuronic acid and single or short chains of β-(1 → 2)-linked d-xylose residues are attached at the C–2 position. l-fucose residues in the extracellular polysaccharides may form the single branches. The structural features of these polysaccharides are discussed in comparison with the similar polysaccharides from other fungi.     

Studies on d-Glucose-isomerizing Enzyme of Bacillus coagulans,Strain HN-68

Cells of Bacillus coagulans, strain HN-68 grown on the medium containing d-glucose, did not show any measurable d-glucose-isomerizing activity. However, when d-glucose-grown cells were shaked for a few hours in an induction medium containing d-xylose, induced formation of d-glucose-isomerizing enzyme occurred in the cells. Cell weight and number of survival cells showed only an increase of 30 and 10%, respectively during 6 hr induction.

The induced formation of d-glucose-isomerizing enzyme required organic nitrogen such as polypeptone in addition to d-xylose. Development of the maximum activity was observed when the concentration of d-xylose and polypeptone were 2 and 3%, respectively. Initial velocity of induced formation of d-glucose-isomerizing enzyme increased in proportion to the decrease of initial pH values of the induction medium, i.e., at 2 hr induction, activity at pH 4.5 was 5-fold increase than that at pH 8.0.

Induced formation of d-glucose-isomerizing enzyme was inhibited strongly by addition of chloramphenicol, tetracycline, streptomycin, cyanide or azide, and was promoted by threonine plus glycine.     

Studies on Glucose Isomerase of Bacteria

A bacterial strain, HN-56, having an activity of d-glucose isomerization was isolated from soil, and was identified to be similar to Aerobacter aerogenes (Kruse) Beijerink. d-Glucose-isomerizing activity was induced when HN-56 was precultured in the media containing d-xylose, d-mannose, lactate, especially d-mannitol. Paper chromatography showed that the ketose formed in reaction system containing d-glucose was d-fructose alone. The optimum pH for the reaction was 6.5~7.0. Sulfhydryl reagents inhibit the reaction, but metal inhibitors affect little if any. With the washed living cells as enzyme source, only arsenate could accumulate d-fructose. In addition, the cells grown with d-mannitol and d-mannose showed no activity of d-xylose isomerase.     

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.     

Properties of Asterosaponin B Isolated from a Starfish,Asterias amurensis

Succeeding to asterosaponin A, the second saponin component has been isolated from a starfish (Asterias amurensis) and designated asterosaponin B. It contains a conjugated ketone and one molecule of sulfuric acid as the sodium salt. The sugar moiety consists of two molecules of d-quinovose and one molecule each of D-fucose, d-xylose, and d-galactose, differing from that of asterosaponin A consisting of two molecules each of d-quinovose and d-fucose. On acid hydrolysis both asterosaponins A and B yielded the similar mixture of aglycon components. The two main components isolated were designated asterogenins I and II, respectively.     

Production of Polyalcohol by a Corynebacterium sp.

A gluconate-utilizing strain of Corynebacterium was found to be capable of utilizing aldopentoses and producing corresponding pentitols when pentoses were added to the medium containing gluconate as a carbon source during the cultivation of the organism.

Pentitols produced from d-xylose, l-arabinose, and d-ribose were isolated from the cultured medium and identified as xylitol, l-arabitol, and ribitol, respectively.

The pentitol production was significantly influenced by the concentration of gluconate in the initial medium and that of pentose added to the medium during the cultivation.

The amount of xylitol, l-arabitol, and ribitol reached 69 mg/ml, 60 mg/ml, and 32 mg/ml, respectively, after 14 days of incubation when pentoses were added to the medium containing 9.6% potassium gluconate to give a final concentration of 150 mg/ml.     

Pectin Isolated from the Midrib of Leaves of Nicotiana tabacum

A pectin isolated from tobacco midrib contained residues of d-galacturonic acid (83.7%), L-rhamnose (2.2%), l-arabinose (2.4%) and d-galactose (11.2%) and small amounts of d-xylose and d-glucose. Methylation analysis of the pectin gave 2, 3, 5-tri- and 2, 3-di-O-methyl-l-arabinose, 3, 4-di- and 3-O-methyl-l-rhamnose and 2, 3, 6-tri-O-methyl-d-galactose. Reduction with lithium aluminum hydride of the permethylated pectin gave mainly 2, 3-di-O-methyl-d-galactose and the above methylated sugars. Partial acid hydrolysis gave homologous series of β-(1 → 4)-linked oligosaccharides up to pentaose of d-galactopyranosyl residues, and 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, and di- and tri-saccharides of α-(1 → 4)-linked d-galactopyranosyluronic acid residues.

These results suggest that the tobacco pectin has a backbone consisting of α-(1 → 4)-linked d-galactopyranosyluronic acid residues which is interspersed with 2-linked l-rhamnopyranosyl residues. Some of the l-rhamnopyranosyl residues carry substituents on C-4. The pectin has long chain moieties of β-(1 → 4)-linked d-galactopyranosy] residues.     

Xylitol Production by an Enterobacter Species

A xylose-utilizing bacterial strain was isolated from soil.

The strain, No. 553, was identified as Enterobacter liquefaciens from the result of the taxonomical studies. This bacterium grew well on D-xylose as a sole carbon source and accumulated pentitol extracellularly in shaking culture.

Pentitol produced was isolated from the culture broth and identified as xylitol.

The xylitol production reached the maximum after the cessation of the cell growth with a yield of 33.3 mg per ml in a medium containing 10% D-xylose as a sole carbon source and no significant decline of the amount of xylitol was observed through the period of the cultivation.     

Isopullulanase from Arthrobacter globiformis T6

The physico-chemical properties of the purified glucose isomerases [d-xylose ketol isomerase, EC 5.3.1.5] of Streptomyces olivochromogenes and Bacillus stearothennophilus were examined. The molecular size and shape of both enzymes were similar. The molecular weights, sedimentation coefficients, partial specific volumes, diffusion constants and Stokes’ radii of the Streptomyces and Bacillus enzymes were determined to be 120,000 and 130,000, 7.55 S and 9.35 S, 0.725 and 0.736 ml/g, 5.87 × 10^-7 and 6.82 × 10^-7 cm²/sec, and 51 and 53 Å, respectively. The Streptomyces glucose isomerase was found to consist of two subunits, each having a molecular weight of 56,000. Large differences were found in the amino acid compositions of these two enzymes, especially in their serine, proline, tyrosine, lysine and arginine contents. The enzymatic properties of both these purified glucose isomerases were also examined, and it was seen that they both displayed activity on d-xylose, d-xylulose, d-glucose, d-fructose, d-arabinose and d-ribose. The smaller Km values and the larger molecular activities for d-xylose and d-xyluIose indicated that both enzymes are essentially d-xylose isomerases. The optimum temperature was 80°C for both enzymes. The optimum pH was 8 to 10 for the Streptomyces enzymes and 7.5 to 8.0 for the Bacillus enzyme. The Bacillus enzyme was more thermostable than the Streptomyces enzyme, but required cobalt ions in addition to magnesium ions for the full expression of its activity.     

Xylitol Production by a Corynebacterium Species

The washed cells of a gluconate-utilizing Corynebacterium strain grown in a gluconate- xylose medium produced xylitol from D-xylose in the presence of gluconate. The amount of xylitol was progressively increased with increasing gluconate concentration.

An extract of cells grown in the gluconate-xylose medium showed NADPH-dependent D-xylose reductase activity and NADP-dependent 6-phosphogluconate dehydrogenase activity.

These enzymes in the cell-free extract were purified by Sephadex G–100 gel filtration.

The reduction of D-xylose to xylitol was demonstrated by the coupling the D-xylose reductase activity to the 6-phosphogluconate dehydrogenase activity with NADP as a cofactor using the cell-free extract and the fractionated enzymes.     

Studies on Glucose Isomerase of Bacteria

A bacterial strain, HN-500, having an activity of d-glucose isomerization was newly isolated from soil, and was identified to be similar to Escherichia intermedia (Werkman and Gillen) Vaughn and Levine. The strain, grown on wide varieties of carbon sources, shows definitely d-glucose isomerizing activity in the presence of arsenate. d-Fructose formed in reaction mixture was identified by paper chromatography and was isolated in crystalline form from calcium-fructose complex. In order to increase the production of d-glucose isomerase, d-glucose and ammonium nitrogen were effective carbon and nitrogen sources, respectively, but none of the metallic ions tested were effective, furthermore manganese, ferrous and ferric ions present mOre than 10^-5m in growth medium fully repressed the enzyme formation. The cells grown on carbon sources other than d-xylose showed no activity of d-xylose isomerase.     

The Structure of Arabinoxylan and Arabinoglucuronoxylan Isolated from Rice Endosperm Cell Wall

A neutral and an acidic arabinoxylan fraction (H-l and H-2) were obtained from rice endosperm cell wall. The results of methylation analysis and partial hydrolysis of these fractions showed that both of them have highly branched structures in which approximately 6 out of 7 (H-l) and 5 out of 6 (H-2) of the (1→4)-linked d-xylose residues are branched. Most of the side chains in H-l consists of single α-l-arabinofuranose residues, whereas some of them in H-2 were substituted with α-d-glucuronic acid or 4-O-methyl-α-d-glucuronic acid residues, both attached to the O-2 position of d-xylose residues. These highly branched arabinoxylans are not readily hydrolyzed by an endoxylanase of Streptomyces sp.