Method for Enrichment of Rhodopseudomonas sphaeroides from Fresh Water

An inulinase was highly purified from the culture broth of Penicillium purpurogenum by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-65, and Bio-Gel P-100. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was 6.4 × 10⁴ by SDS-disc electrophoresis and gel filtration on Bio-Gel P-150. The isoelectric point was pH 3.6 by isoelectric focusing. The enzyme hydrolyzed inulin rapidly, but did not affect sucrose. By paper chromatography analysis, the major products from inulin were tri-, tetra-, penta-, and hexa-saccharides. The substrate specificity of the enzyme on hydrolyses of fructo-oligosaccharides[1^F(1-β-d-fructofuranosyl)n sucrose (n = 1 to 6 and n (average of polymerization degree) = 8)] were examined. The Km values and relative maximum velocities for the hydrolyses of inulin and fructo-oligosaccharides (GF_n, n = 2 to 7 and n = 9) were as follows: inulin, (DP = 35) 0.21 mM and 100; GF₉, 0.24 mM and 86.5; GF₇, 0.33 mM and 132; GF₆, 0.85 mM and 71.2; GF₅, 3.8 mM and 25.4; GF₄, 2.8 mM and 28.8; GF₃, (nystose) 16 mM and 0.8; GF₂ (1-kestose), 8.4 mM and 0.2. The molecular activities for the hydrolyses of fructo-oligosaccharides (GF_n, n = 2 to 6) were increased depending on the degree of polymerization of fructosyl residues, and were nearly constant if the polymerization degree was over seven. These results strongly suggested that the endo-type inulinase from Penicillium purpurogenum had a subsite structure consisting of at least seven subsites.     

Consolidation of Soybean Protein Coagulate

The process of dehydration of soybean protein coagulate in expression involves two mechanisms, filtration and consolidation. The filtration process was explained by Ruth’s filtration model. The consolidation process, except for secondary consolidation, was analyzed by the modified consolidation model of Shirato et al., taking into account the compressibility of cake. The modified coefficient of consolidation was not affected significantly by the pressure applied. Evaluation of the modified coefficient of consolidation is of importance for an attempt to improve the current production of soybean protein.     

Metabolism of Glycine and Threonine in Growing Rats at Various Dietary Protein Levels

The metabolic fates of the carbon skeletons of [U-¹⁴C]glycine and l-[U-¹⁴C]threonine were investigated in growing rats fed with diets containing different percentages of protein calories (0, 5, 10, 15, and 30PC%) at 4100 kcal of metabolizable energy per kg of diet.

The incorporation of ¹⁴C into the body protein at 12 hr after the injection of ¹⁴C-glycine was about 58% of the dose in rats fed with the 10 or 15 PC% diet, and the values were reduced in both the lower and higher PC% groups. A considerable amount of ¹⁴C was recovered in the soluble fraction, and it was attributed to labeled glycine and serine in the free amino acid pools of the tissues.

The incorporation of ¹⁴C into the body protein from ¹⁴C-threonine was extremely high in the dietary groups of 0 to 10 PC%, and it decreased in the 30PC% group. Conversely, the expired ¹⁴C0₂ production was much less until the dietary protein level reached at 10PC%, and it increased with higher PC% in the diets. The change in the activity of hepatic threonine dehydratase in rats fed diets with increasing protein levels was similar to that of the expired ¹⁴C0₂ production from ¹⁴C- threonine.

These results indicate that, though the metabolic patterns for glycine and threonine differ from each other, their responses to dietary protein levels change at 10 to 15 PC%, where the growth rate reached its approximate maximum.     

Subunits and Sulfhydryl Groups of Beef Liver d-Glycerate Dehydrogenase

The amino acid composition of beef liver d-glycerate dehydrogenase (EC 1.1.1.29) was determined. Results of sodium dodecyl sulfate gel electrophoresis and measurements of the number of NADH bound by the enzyme and the number of the essential sulfhydryl groups suggested that the enzyme was composed of two identical subunits with the molecular weight of 36,000. Close relation between the essential sulfhydryl groups and the coenzyme binding site was also suggested. Effect of an alkylating agent (bromopyruvate) with the structure similar to the substrate was studied. Effects of iodoacetate and iodoacetamide were also studied. It was suggested that these reagents behaved as active-site-directed irreversible inhibitors of the enzyme. Bromopyruvate exhibited a high affinity to the enzyme. Iodoacetate (anionic reagent) had a higher affinity than iodoacetamide (neutral reagent).     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Adaptation of Hydrogenobacter thermophilus toward oxidative stress triggered by high expression of alkyl hydroperoxide reductase

Ferriperoxin is a novel peroxidase essential for aerobiosis of Hydrogenobacter thermophilus. Although the ferriperoxin-deficient mutant (Δfpx) was unable to grow aerobically, a suppressor mutant capable of aerobic growth was obtained after long aerobic cultivation. The alkyl hydroperoxide reductase gene was significantly upregulated in the suppressor mutant, indicating that the enzyme counteracts oxidative stress in the absence of ferriperoxin.     

Isolation and Characterization of a Temperature-sensitive Sporulation Mutant of Bacillus subtilis

Temperature-sensitive sporulation mutants were isolated spontaneously from Bacillus subtilis 168 TT by a sequential transfer method. A representative mutant strain, ts32, was characterized in detail. The mutant grew normally at 30°C and 42°C, but did not sporulate at 42°C. Electron microscopic observation and physiological analysis showed that the mutant was blocked at stage 0-1 of sporulation. Genetic analysis suggested that the mutation was located at the spo0B locus on the B. subtilis chromosome. Temperature-shift experiments clearly showed that the spo0B gene product functions only at the beginning of sporulation.     

Ceramide in Rice Grain

A rice lamina inclination test that is simple and specific for brassinosteroids was used as a micro-quantitative bioassay for brassinolide 1 and its 6-keto congener, castasterone 2, in the concentration range of 5 x 10^–5 /ig/ml to 5 x 10^–3μg/ml, when uniform seedlings of the rice cultivars Arborio J-l and Nihonbare were selected. A phytohormone, indole-3-acetic acid (IAA), showed similar activity in this bioassay. Its lowest effective concentration, however, was 50 /ig/μl, about five orders of magnitude greater than that of brassinolide. Other phytohormones, abscisic acid (ABA) and the cytokinins kinetin and A⁶-benzyladenine, inhibited the lamina inclination of rice seedlings. The addition of a cytokinin reduced the promoting effect of brassinolide. Thus, the rice lamina inclination test can be used both as a micro-quantitative bioassay for brassinosteroids and as a method for detecting antibrassinolide compouds.