Smith Degradation and Methylation of an Acidic Polysaccharide from Soy Sauce

APS–I, an acidic polysaccharide isolated from soy sauce, was subjected to Smith degradation and methylation, and each product obtained was examined by paper chromatography, gas-liquid chromatography and mass spectrometry. Some compounds, such as ethylene glycol, propylene glycol, glycerol, D-threitol, D-galactose, L-rhamnose, D-galacturonic acid and D-threonic acid, were identified as Smith degradation products, and their molar ratios were estimated. Several kinds of methylated sugars were detected in the hydrolysate of methylated APS–I and identified by gas chromatograph-mass spectrometry. On the basis of those findings and previous discussion, the constitution of APS–I was discussed.     

The PMR Spectra of 1-Alkyl-2-carbo-azetidines: The Effect of the Nitrogen Lone Pair

Screening was carried out to obtain microorganisms which produced the enzyme to reduce the disulfide bond, from our type cultures of yeast. Among many strains of yeast showing activity to reduce the disulfide bond, Candida claussenii, Candida brumptii and Candida rugosa were selected to have the highest activity. The enzyme activity was detected in the cell free extracts, but not in culture broth.

The highest enzyme formation occured during the exponential growth phase, and rapid decrease of activity was observed in the stationary phase. Pantothenate and boron ion contributed to enzyme formation, and biotin and zinc ion to growth. The maximum enzyme activity was obtained in the following synthetic medium: 10% sucrose, 0.3% (NH₄)₂SO₄, 0.5% KH₂PO₄, 0.15% MgCl₂·6HO₂ 0.05% CaCl₂, 0.015% MnCl₂, 0.001% pantothenate, 0.0001% biotin, 0.0001% H₃BO₃, 0.00004% FeCl₃·6H₂O and 0.00008% ZnCl₂. In addition, 30°C of the cultural temperature and vigorous aeration showed good results for enzyme formation.     

Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.     

Applying Proteolytic Enzymes on Soybean

Soy proteins were incubated with a microbial acid protease (Molsin) under the following condition: substrate concentration, 1%; enzyme-substrate ratio (by weight), 1/100; pH, 2.8; and temperature, 40°C—flavor components and related impurities are removable from crude soy-protein concentrates by their incubation for 2 hr under the above condition. The acid-precipitated fraction of soy protein incubated for 2 hr with Molsin (i.e. 2 hr-proteolyzate) showed the following composition: 10% trichloroacetic acid (TCA) insoluble fraction, 47.52%; 10% TCA soluble peptide fraction, 52.02%; and free amino acid fraction, 0.46%. Gel filtration of the 2 hr-proteolyzate gave an elution pattern showing its molecular weight distribution.

In the process of the incubation of the acid-precipitated protein, the 10% TCA insoluble fraction showed increase in amino nitrogen content, its solubility in a phosphate buffer increased to change at 6 hr, and a hydrophobic amino acid share in this fraction increased gradually.

In vitro digestibility of the acid-precipitated fraction were improved and the lipoxygenase activity in this fraction decreased through the Molsin treatment.

Ultracentrifugal analysis showed a decreasing tendency of the cold-insoluble fraction of soy protein during its incubation with Molsin. Optical rotatory dispersion and circular dichroism study elucidated conformational changes in this fraction during its incubation either with or without Molsin.     

Guanosine Diphosphate Mannose in Conformational Relation to Other Suger Nucleotides and Phosphates in Solution

Molecular conformational transition of GDPMan and solution conformation of α-d- mannopyranose moiety in Man-l-P and GDPMan were examined in relation to other sugar nucleotides and phosphates. GDPMan and other sugar nucleotides examined revealed changes in the optical rotation in sigmoidal curve in water by addition of urea. The change was reversible without significant decomposition and is attributable to dissociation of an ordered form into a random form. Optical conformational values in 8m urea solution were+116° for GDPMan, +58°~+79° for UDPGlc, +79° for UDPGal, +135°~+143° for UDPGlcNAc, and +138°~ +155° for UDPGIcA.

NMR analysis and periodate oxidation study revealed the ⁴C₁ conformation of α-d-hexopyranose moieties in Man-1-P, Glc-l-P, GDPMan, UDPGlcNAc and UDPGalNAc.     

Isolation and Identification of Ubiquinone 10 from Cultured Cells of Tobacco

Callus tissues were induced from stem and root segments of Rauwolfia serpentina. Growth and alkaloid production of the callus tissues were examined under various culture conditions. The growth was strikingly promoted in the presence of 2,4-D (0.5~1 ppm), kinetin (0.2~0.5 ppm) and yeast extract (0.1~0.2%). At favourable conditions, the growth value in 4 weeks’ culture was ca. 40 (F.W.), and ca. 25 (D.W.) for stem callus tissues, and ca. 15 (F.W.), and ca. 8 (D.W.) for root callus tissues. Stem and root callus tissues produced ajmaline and some other unidentified Rauwolfia alkaloids. The ajmaline content in root callus tissues was 10~20mg % and in stem callus tissues was 1~10mg %. The ajmaline production was strikingly reduced when 2,4-D concentration increased, or kinetin was omitted in the culture medium. Phytosterols including stigmasterol, β-sitosterol or cholesterol were also produced.     

Growth Profile of Crown Gall Cells of Tobacco in Suspension Culture

In order to obtain a basic information of plant cell suspension culture as a step toward the development of large scale culture, culture conditions of crown gall cells (auxin non-requiring cells) were investigated. Addition of yeast extract to culture medium was significantly effective for the growth and cell dispersion.

In experiments on the ability of the cultured cells to utilize sugars as the carbon source, it was observed that galactose, added to the culture medium, markedly inhibited the cell growth.

Pasteurization of the medium containing fructose as carbon source made it brownish by Maillard reaction and the medium apparently restrained the cell growth. However, the fructose medium sterilized by filtration was excellent for the cell growth as well as sucrose or glucose medium. In a jar fermentor, even the glucose medium became brownish by heat sterilization and the brown colored medium restrained the cell growth. Under optimum conditions, the doubling time was 1.1 day in exponential phase and 2.0 g of cell (dry weight) per 100 ml culture was obtained as the maximum yield.     

A design for the control of apoptosis in genetically modified Saccharomyces cerevisiae

We have engineered a system that holds potential for use as a safety switch in genetically modified yeasts. Human apoptotic factor BAX (no homolog in yeast), under the control of the FBP1 (gluconeogenesis enzyme) promoter, was conditionally expressed to induce yeast cell apoptosis after glucose depletion. Such systems might prove useful for the safe use of genetically modified organisms.