The Isolation and Characterization of the Acid-Soluble Nucleotides from the Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

The acid-soluble nucleotides were extracted from the tubers of Jerusalem artichoke with percbloric acid, and separated and purified by means of adsorption on and elution from active charcoal, repeated chromatography on columns of Dowex I (Cl^-), followed by paper chromatography. The following nucleotides have been characterized and/or identified: 5′-AMP, 3′-AMP, ADP, ATP, 5′-GMP, 2′-GMP, 3′-GMP, 2′,3′-cyclic GMP, GDP, GTP, 5′-UMP, UDP, UTP, NADP, UDP-glucose, UDP-galactose, UDP-fructose, UDP-N-acetylhexosamine and GDP-mannose.^** Neither cytosine ribonucleotides nor deoxyribonucleotides have been detected. The significance of these observations is discussed.     

L-Tyrosine production by Polyauxotrophic Mutants of Corynebacterium glutamicum

Polyauxotrophic mutants of Corynebacterium glutamicum which have additional requirements to L-phenylalanine were derived from L-tyrosine producing strains of phenylalanine auxotrophs, C. glutamicum KY 9189 and C. glutamicum KY 10233, and screened for L-tyrosine production. The increase of L-tyrosine production was noted in many auxotrophic mutants derived from both strains. Especially some double auxotrophs which require phenylalanine and purine, phenylalanine and histidine, or phenylalanine and cysteine produced significantly higher amounts of L-tyrosine compared to the parents, A phenylalanine and purine double auxotrophic strain LM–96 produced L-tyrosine at a concentration of 15.1 mg per ml in the medium containing 20% sucrose. L-Tyrosine production by the strain decreased at high concentrations of L-phenylalanine.     

DNA as a Flocculation Factor in Pseudomonas sp.

A component responsible for flocculation was extracted from Pseudomonas strain C-120 by treating the cells with 3 M guanidine hydrochloride. The guanidine hydrochloride-extracted cells were reflocculated, not only with the guanidine hydrochloride extract but with DNA prepared from various bacteria. The reconstituted flocs were deflocculated by deoxyribonuclease or guanidine hydrochloride which indicated that the reconstituted flocs closely resembled natural flocs. In reconstitution experiments using Escherichia coli DNA at different molecular weights, it was found that DNA with a molecular weight higher than about 6 × 10⁶ was required to flocculate the guanidine hydrochloride-extracted cells. Heat-denatured DNA did not flocculate the guanidine hydrochloride-extracted cells. DNA with a high molecular weight was detected in the guanidine hydrochloride extract. It was concluded that the component involved in flocculation of this organism was highly polymerized double stranded DNA.     

Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na₂SiO₃, 600 mM Na₂MoO₄, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.     

Purification and Properties of a Pectate Lyase in Erwinia aroideae

A strain of Erwinia aroideae produced an extracellular pectolytic enzyme under growth conditions with pectin or pectic acid as the inducer. This strain also produced a pectin lyase when nalidixic acid is added to a culture medium. The pectolytic enzyme produced under the growth conditions was purified approximately 40-fold from the culture fluid by carboxy- methyl cellulose and Sephadex G-75 gel column chromatographies. The purified enzyme was almost homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, having a molecular weight of about 36,000 to 38,000. This enzyme, with optimal activity at pH 9.0 to 9.2, produced reaction products which had a strong absorption at 230 nm indicating a lyase type of the reaction. The enzyme activity was markedly stimulated by calcium ion and completely inhibited by cobalt and mercuric ions and by ethylenediaminetetraacetate. Pectic acid or pectin with lower methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectin with higher methoxyl content was used as a substrate. It was concluded that the enzyme produced under the normal growth conditions is an endo-pectate lyase and differs from the pectin lyase induced by nalidixic acid.     

A Transport System for C4-Dicarboxylic Acids in Isolated Membrane Preparations from Escherichia coli

To investigate the mechanism of succinate transport system in Escherichia coli, the isolated membranes were prepared from E. coli W2252 and T5, a mutant defective in succinate uptake derived from W2252. Uptakes of ¹⁴C-substrates by W2252 and T5 membranes and the dilution of accumulated radioactivity by unlabeled C₄-dicarboxylic acids, indicated that C₄-dicarboxylic acids in the tricarboxylic acid cycle are transported by the same system in E. coli which requires a suitable energy source such as NADH, D-lactate or reduced phenazine methosulfate. The uptakes of succinate by W2252 membranes were inhibited by an anaerobic incubation or some of the inhibitors of electron transport chain. Difference spectra of reduced versus oxidized membranes from W2252 and T5 indicated the reduction of flavoproteins and cytochromes by dithionite, NADH or D-lactate. From these results it was concluded that the uptake of the C₄-dicarboxylic acids in isolated membranes is coupled to an electron transport chain involving a specific dehydrogenase system.     

Activities of Cysteine Synthetase and Cystathionase in Bacilhis subtilis during Sporulation

Cysteine synthetase (O-acetylserine sulfhydrylase) was partially purified from cells of Bacillus subtilis by the use of ammonium sulfate fractionation technique and DEAE-Sephadex A–50 chromatography. The cysteine synthetase preparation was compared with cystathionase (cystathionine β-cleavage enzyme) of the same organism in regard to biochemical properties and to changes in activity during sporulation.

The optimal pH and temperature for the cysteine synthetase were 8.5 and 25°C respectively. The enzyme was relatively stable at temperatures below 50°C and fairly resistant to proteases, in contrast to cystathionase. Production by B. subtilis of cysteine synthetase in sulfur-deficient synthetic medium was repressed by the addition of cysteine and derepressed by djenkolic acid. Activity of the enzyme was inhibited by methionine and increased by acetate. The cysteine synthetase activity was almost constant until the late sporulation stage commenced, but the specific activity of cystathionase (Fraction I) decreased rapidly in the course of sporulation and it could not be detected in the free spores.     

Neutral Aroma Constituents in Burley Tobacco

The constituents of the neutral volatiles from air-cured Burley tobacco were studied using distillation, silicic acid column chromatography, preparative gas chromatography and GC–MS. The isolation and identification of 84 compounds are reported of which 27 are newly identified as tobacco constituents and 4 are new natural products.     

Purification and Properties of a Pectin trans-Eliminase in Erwin aroideae Formed in the Presence of Nalidixic Acid

A strain of Erwinia aroideae produces a remarkable amount of pectolytic enzyme when the organism was induced by nalidixic acid for the bacteriocin production. This pectolytic enzyme was purified approximately 60-fold from the induced medium by carboxymethyl-cellulose and Sephadex G–75 gel column chromatographies after batchwise treatment with carboxymethyl- and diethylaminoethyl-celluloses. The purified enzyme was almost homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a molecular weight of about 28,000 to 32,000 was determined for this enzyme. The optimum pH of the enzyme activity was about 8.0 to 8.2. The purified enzyme produced reaction products from pectin and methoxylated pectic acid which had a strong absorption at 235 nm indicating a trans-eliminase reaction. Pectin or pectic acid with higher methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectic acid was a substrate. The limit of degradation of pectin and pectic acid with higher methoxyl content (90% esterified) by the enzyme were 6.5% and 43%, respectively. It was concluded that the enzyme is a new endo-pectin trans-eliminase from bacterial origin.