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.     

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.     

Isolation and Constitution of a Xylan from Bamboo

A xylan from bamboo culm was isolated by extraction with aikali of chlorite holocellulose and fractional precipitation as a copper complex. The structure was investigated by means of examination of acid components by controlled hydrolysis, methylation analysis, and periodate oxidation. As a result, 4-O-methyl-α-D-glucuronic acid and 2-O-(4-O-methyl-α-D-glucopyranosyluronic acid) D-xylose were isolated and identified as acid components of the bamboo xylan. Hydrolysis of the fully methylated products afforded 2,3,5-tri-O- methyl-L-arabinose (1.6 moles), 2,3,4-tri-O-methyl-D-xylose (1.2 moles), 2,3,4,6-tetra-O-methyl-D-glucose(0.4 moles), 2,3-di-O-methyl-D-xylose (35.8 moles) and mono-O-methyl-D-xylose (2.6 moles). In addition to the above methylated sugars, 2,3,4-tri-O-methyl-D-glucuronic acid and partially methylated aldobiouronic acid were separated by cellulose column chromatography and identified. These results suggest that the bamboo xylan consists mainly of a linear backbone of 1,4-linked β-D-xylopyranose unit, to which L-arabinofuranose and 4-O-methyl-D-glucuronic acid were attached as a single side chain unit at C₂ or C₃.

Additional evidence for a linear chain structure has been given by periodate oxidation. On oxidation by periodate, the bamboo xylan consumed 1.09 moles of periodate and produced 0.05 mole of formic acid per anhydroxylose unit.     

The Production of Xylitol and d-Xylonic Acid by Yeast

Pichia quercibus Phaff et Knapp produced xylitol and d-xylonic acid by aerobic dissimilation of d-xylose at good yield of 40% of sugar consumed. The products were isolated from the fermented broth and identified. It would be interesting that both of xylitol, a reduction product of d-xylose, and d-xylonic acid, an oxidation product, are accumulated in the fermented broth.     

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.     

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.     

Microbial Production of d-Arabitol by n-Alkane-grown Candida tropicalis

In the course of study on citric acid fermentation by Candida tropicalis KY6224, in which n-alkane mixture (C–12 to C–15) was used as the sole source of carbon, we found that a arabitollike substance was accumulated when the medium-pH was controlled at low level (3.0 to 4.0). This substance was isolated in crystalline forms and identified as d-arabitol.

d-Arabitol production was also observed with ethanol, acetic acid and glucose as the sole source of carbon. Important factors for efficient production of d-arabitol were keeping the medium-pH at low-level (3.0 to 4.0) and the concentration of NaCl or KCl at high level (1 to 5%). This strain produced 75 mg/ml of d-arabitol in 120 hr incubation under optimal culture conditions; this corresponds to 50 % of n-alkane consumed.     

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.     

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.     

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.     

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.     

Carbohydrate Metabolism Mutants of a Bacillus Species

Mutants which could not utilize d-gluconate, l-arabinose, sorbitol, pyruvate or l-glutamate as a sole carbon source and which required shikimic acid for their growth were isolated. Characterization of these mutants by the patterns of carbohydrate utilization revealed that various kinds of carbohydrate metabolism mutants including those of the non-oxidative limb of the pentose phosphate pathway were isolated.

Ability of inosine formation of these mutants and transformants from them was investigated. Consequently, slightly improved strains were found among transformants in comparison with the parent strain.     

Metabolism of l-Theanine,d-Theanine and the Related Compounds in Bacteria

Some strains of Pseudomonas was found capable of utilizing l-theanine or d-theanine as a sole nitrogen and carbon source. The cell-free extract catalyzes the hydrolysis of the amide group of the compounds and the hydrolase activity was influenced remarkably by the nitrogen source in the medium. l-Theanine and d-theanine were hydrolyzed to yield stoichiometrically l-glutamic acid and d-glutamic acid, respectively, and ethylamine, which were isolated from the reaction mixture and identified.

The theanine hydrolase of Pseudomonas aeruginosa was purified approximately 200-fold. It was shown that the activities of l-theanine hydrolase, d-theanine hydrolase and the heat-stable l-glutamine hydrolase and d-glutamine hydrolase are ascribed to a single enzyme, which may be regarded as a γ-glutamyltransferase from the point of view of the substrate specificity and the properties. This theanine hydrolase catalyzed the transfer of γ-glutamyl moiety of the substrates and glutathione to hydroxylamine. l-Glutamine and d-glutamine were hydrolyzed by the theanine hydrolase and also by the heat-labile enzyme of the same strain of Pseudomonas aeruginosa, whose properties resembled the common glutaminase.     

Some Physicochemical Properties of Invertase of Candida utilis

This study examined the microbial transformation of carbazole (CZ) by an isolated bacterium that can use CZ as a sole carbon and nitrogen source. The strain identified as Pseudomonas stutzeri produced a large amount of anthranilic acid (AA) from CZ in the medium containing a nonionic surfactant. In dialysis culture using ion-exchange resin, 7.9 g/liter (58mm) of AA was accumulated from 15g/liter (90mm) of CZ and the molar yield of AA reached about 64%.     

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.     

Formation of Threonine Aldolase by Bacteria and Yeasts

Threonine aldolase was found to be formed in various strains of bacteria and yeasts when they were grown in media containing l-threonine as a sole source of carbon. As the other sources of carbon, d, l-allothreonine, l-serine and glycine were effective but glucose and sucrose were inert for the formation of the enzyme.

The maximal formation of the enzyme was observed in the initial of stationary phase of growth and, thereafter, the enzyme disappeared with the consumption of l-threonine. It seems that the enzyme is adaptive in nature and that it is responsible for the growth in threonine as the carbon source.     

SYNTHESIS AND ANTIVIRAL ACTIVITY OF D- AND L-2′-AZIDO-2′,3′-DIDEOXY-4′-THIOPYRIMIDINE AND PURINE NUCLEOSIDES

Novel D- and L-2′-azido-2′,3′-dideoxy-4′-thionucleosides were synthesized starting from L- and D-xylose via D- and L-4-thioarabitol derivative as key intermediates and evaluated for antiviral activity, respectively. When the final nucleosides were tested against HIV-1, HSV-1, HSV-2, and HCMV, they were found to be only active against HCMV without cytotoxicity up to 100 μg/ml.     

Gluconate Metabolism by a Thiamine-requiring Species of Corynebacterium

A thiamine-requiring strain of Corynebacterium was found to accumulate a ketopentose extracellularly from gluconate. The ketopentose was isolated from the culture medium and identified as d-ribulose. The accumulation of d-ribulose was significantly influenced by the concentration of thiamine in the medium. The maximum yield of d-ribulose was obtained at a thiamine concentration of 10 μg per liter, whereas good growth was favored at thiamine concentrations greater than 50 μg per liter. The accumulation of d-ribulose reached the concentration of 9.5 mg per ml after cessation of cell growth in shake culture at 30°C in a medium containing 6% potassium gluconate as a carbon source.     

SYNTHESIS OF NOVEL D- AND L-3′-DEOXY-3′-C-HYDROXYMETHYL NUCLEOSIDE WITH EXOCYCLIC METHYLENE AS POTENTIAL RIBONUCLEOTIDE REDUCTASE INHIBITOR

D- and L-3′-Deoxy-3′-C-hydroxymethyl thymidine substituted with exocyclic methylene at 2′-position were synthesized, starting from D- and L-xylose as potential ribonucleotide reductase inhibitor, respectively, but they were found to be inactive against several tumor cell lines.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

The present investigation is concerned with l-glutamic acid production in the presence of pyrrolidone carboxylic acid and glucose in Bacillus megaterium st. 6126. This strain does not grow on dl-pyrrolidone carboxylic acid (dl-PCA)¹⁾ as the sole source of carbon and nitrogen. The optimal concentration of yeast extract required for the maximal production of l-glutamic acid was 0.005% under the conditions used. As the yeast extract concentration was increased, growth increased proportionally; but the l-glutamic acid production did not exceed the control’s to which glucose and ammonium chloride had been added. l-Glutamic acid produced by both growing cultures and resting cells was derived from glucose and ammonium salt of dl-PCA. Isotope experiments suggested that the l-glutamic acid produced was partially derived from ammonium salt of dl-PCA in the growing culture which had been supplemented with d-glucose-U-¹⁴C or dl-PCA-1-¹⁴C and that ammonium salt of dl-PCA was consumed as the source of nitrogen and carbon for l-glutamic acid.