Biochemical Studies on Yeast

The growth of Zygosaccharomyces soja was strongly inhibited by the addition of 10^?2 m-monoiodoacetic acid and 10^?3 m-potassium cyanide. The formation of a considerable amount of riboflavin was recognized after 5 days cultivation of this organism in presence of the aforementioned two inhibitors, and its growth was considerably improved after the formation and accumulation of riboflavin. Furthermore, the effect of the aforementioned two inhibitors upon the growth of this organism in young stage was compensated with the addition of riboflavin.     

Biochemical Studies on the Mineral Components in Sake Yeast

Analysing the mineral components in yeasts (sake yeast and some of other brewer’s yeasts), the author found that no remarkable difference was seen on the composition of the major mineral components. The potassium content of yeast cultured in koji extract is lower than that cultured in malt extract or in the synthetic medium. Potassium concentration of koji extract is lower than that of each of other media, and it was possible to increase the potassium content of the cells to the normal level by the enrichment of the koji extract with potassium chloride. From these facts, it is assumed that the potassium concentration of koji extract is insufficient for the saturation in the cells with potassium.

Phosphorus and magnesium contents in the cells are not so much affected by pH of the medium as potassium.     

Biochemical Studies on the Mineral Components in Sake Yeast

The critical concentrations of minerals in a growing medium for maximum fermentation of yeast were as follows: P, 1 mmol/1; Mg, 0.2 mmol/1; and K, 1~2 mmol/1. These values are lower than those for the saturation of the cells with each mineral. The order of the concentration for maximum fermentation (K>P>Mg) is in agreement with that for yeast growth.

Only a small amount of mineral salt was required to increase the fermentative activity. Very small increase of fermentative activity was observed when the starved yeast was enriched with corresponding minerals by incubating cells with the mineral salt and glucose.     

Studies on Yeast Uricase

A method for obtaining a highly purified preparation of yeast uricase was developed. The procedure included extraction of uricase from the uric ase-induced yeast cells, fractionation with ammonium sulfate and chromatography on a DEAE-cellulose column. The purified yeast uricase was shown to be ultracentrifugally homogeneous. The enzyme acted best at pH 8.5 and was stable in a range from pH 7.0 to 11.0 and at temperatures lower than 40°G. The Michaelis constant for urate was calculated to be 5.88 × 10^?6 m at pH 8.5, borate buffer. Activity and stability of the enzyme, however, were found to be significantly affected by the kind of buffer used. The enzyme was sensitive to heavy metal ions such as mercuric and cupric ions, but the sensitivity was influenced by the kind of buffer used.     

Biochemical Studies on Cycasin

Purification of β-glucosidase from the seeds of Japanese cycad, and properties of the purified preparation are described. The enzyme activity was determined by colorimetry using ONPG as substrate. Crude preparation was obtained easily by adsorption on fibrous CMC pulp. It was further purified by chromatography on CMC powder, and a preparation which showed an activity of 135-folds of the original extract was obtained. Influences of pH, temperature, and substrate concentration upon the enzyme activity were examined. Michaelis constant of the enzyme for ONPG was 3.3×10^–3M.     

Studies on Extracellular Ribonuclease from Yeast

An extracellular ribonuclease of Rhodotorula glutinis was purified about 50-fold from the culture broth, by a procedure involving ammonium sulfate fractionation, DEAE-Sephadex column chromatography, acetone fractionation and Sephadex G-100 gel filtration. The purified enzyme was homogeneous when examined by the ultracentrifuge and disc electrophoresis. The molecular weight was calculated to be 58,900 and 56,000 by the sedimentation-diffusion method and the sedimentation equilibrium method, respectively.     

Biochemical Studies on Active Transport

The active transport process, so important in cell function, has been studied in the past with intact cells. Models which have arisen from this work all depend on: first, a specific protein to recognize the substrate; second, translocation of the substrate across the cell membrane; third, release of substrate within the cell and restoration of the system to its initial state. These steps are adequate for facilitated transport, but in active transport an energy input is required to maintain a concentration gradient. Parts of transport systems have been isolated recently. A protein which specifically recognizes β-galactosides has been partially purified. In another case, a protein that appears to be the recognition part of the sulfate transport system of Salmonella typhimurium has been crystallized, and many of its properties have been described. The role of this protein in recognition and in translocation is discussed. Also proteins that phosphorylate a variety of sugars as they enter the cell''s interior provide a mechanism for concentrating sugars as their phosphates, against a gradient.     

Biochemical Studies on Streptomyces sioyaensis

Non-proliferating mycelium of Streptomyces sioyaensis was shown to form siomycin in phosphate buffer without addition of an energy source or precursors. This increase of siomycin in phosphate buffer was suppressed by glucose, acetate, l-cysteine, casamino acid, metals (Fe⁺⁺, Cu⁺⁺), various metabolic inhibitors, and antibiotics (chloramphenicol, erythromycin), whereas it was promoted by yeast extract, beef extract, l-isoleucine, Mg, etc.

The mechanism of the inhibitory effect of glucose on siomycin formation was investigated. Although glucose suppressed siomycin formation, it was the best carbon source for Streptomyces sioyaensis and vigorously metabolized to keto acids and other metabolites. Glucose suppressed siomycin formation by promoting cellular metabolism and mycelial growth. Siomycin formation was not only different from but also competitive to mycelial growth (cellular protein synthesis).     

Biochemical Studies on Fatty Liver

The relation of the fatty liver with some enzyme activities concerning the amino acid metabolism was investigated. Fatty livers were produced by an amino acid imbalanced diet containing 8% of casein supplemented with 0.3% of DL-methionine (threonine deficient), and serine dehydrase ( = threonine dehydrase = cystathionine synthetase), homoserine dehydrase ( = cystine splitting enzyme = cystathionase), and threonine aldolase activities were determined.

Under this condition, the threonine aldolase activity was hardly altered, but the serine dehydrase and the homoserine dehydrase activities were fairly variable. However, the variation of these enzyme activities did not seem to have appreciable relation with the fatty liver, but rather had a connexion with the dietary protein level or calory content.     

Biochemical Studies on Fatty Liver

The effects on the fat content in livers of amino acid supplements to various cereal diets at 1.12% dietary nitrogen level, which causes fatty livers in young rats, have been investigated. When rats are fed on diets supplemented with amino acid to retain the requirement pattern of essential amino acids, they grow very well, but the accumulation of liver fat fails to decrease. However, when a part of the requirement pattern diets is replaced with 0.30% of l-threonine, the deposition of liver fat shows a clear decrease. The results obtained suggest that this excess part of threonine has a specific action for the prevention of fat deposition in livers regardless of protein utilization, together with the so-called “supplementary effects” of amino acids.     

锌酵母中酵母甘露多糖组分的特征和结构

本文研究从锌酵母中分离出的酵母甘露多糖ＸＰ的特征和结构。ＸＰ经全水解和＾１３ＣＮＭＲ谱显示除甘露糖基外,还有少量Ｌ－鼠李糖基和甲氧基。甲基化分析、过碘酸盐氧化、Ｓｍｉｔｈ降解、乙酰解和部分酸水解显示ＸＰ的主链是１→６连接的甘露糖,侧链是１→２连接的甘露糖。＾１Ｈ及＾１３Ｃ ＮＭＲ谱表明所有糖苷键均为α型,结合元素分析ＸＰ基本是酵母甘露多糖和蛋白质以及锌的络合物。     

Studies on Crystalline Yeast Phosphoglycenc Acid Mutase

Effects of pH and some chemical agents (1) on the solubilization of insoluble pectin bound to suspended particles, (2) on the viscosity decrease of native soluble pectin, (3) on the flocculation of suspended particles and (4) on the over-all clarification of apple juice have been studied Optimum pH ranges were: (1), 3.2~4.0; (2), 3.6~4.1; (3), lower than 3.0; (4), 3.2~3.8. Gelatin had no effect on (1) and (2) but stimulated (3) and (4). NaCl had no detectable effect on (2) and (3) but slightly stimulated (1) and (4). CaCl₂ strongly inhibited (1), (2) and (3). SnCl₄ stimulated (3) but strongly inhibited (1), (2) and (4). EDTA (ethylenediamineteraacetic acid) accelerated (1) and (4), and had no detectable effect on (2) and (3).     

Studies on Crystalline Yeast Phosphoglyceric Acid Mutase

In order to study the properties of crystalline phosphoglyceric acid mutase, the polarimetric method was employed for the direct measurement of the enzyme activity. As a result, it was found that the enzyme was inhibited by various metallic ions, chelating agents and phosphoryl enolpyruvate, but not influenced by SH-inhibitors. In addition, fluoride was found to inhibit the enzyme activity in a special manner. Some observations on the basic properties are also presented.     

Biochemical Studies on Myo-inositol in Microbes

The chemical analyses showed that inositol deficiency caused especially the increase in content of glucan fraction and the decrease in contents of inositol, phospholipids and free-pool fraction. Other components, however, did not change in contents in inositol deficiency. More mannan fraction and free-pool substances were found to be released from the cells in inositol deficiency than in sufficiency. The respiratory and fermentative activities were lost in inositol deficient cells of 24 hr culture, which was considered to be the consequence of unbalanced growth death. But the respiratory activity did not so much decrease in inositol deficient cells of 8 hr culture as the fermentative activity, especially the aerobic fermentative activity, did. The release of mannan fraction and the decrease in intracellular free-pool fraction were accompanied with the loss of viability.

These results suggest that inositol deficiency caused the abnormality of the cell structure and permeability, and that this abnormality may be the possible cause of loss of viability due to inositol deficiency.     

Biochemical Studies on Rice Bran Lipase

Some chemical properties of the rice bran lipase were studied. The enzyme protein contained 14.98% nitrogen and consisted of 312 amino acid residues. It also contained a certain amount of lipid. The amino-terminal amino acids of the enzyme protein were shown to be glutamic acid and the carboxyl-terminal amino acids to be glycine and serine. The treatment of the enzyme protein with 8 m urea containing 1×10^?3m EDTA (ethyl-enediaminetetraacetic acid) seemed to cause dissociation of the subunits of the enzyme protein. From this observation and the results of the terminal amino acids analysis, it was presumed that the enzyme protein was composed of at least two types of subunits.     

Biochemical Studies on Rice Bran Lipase

Lipase extracted from defatted rice bran with calcium chloride solution was purified by ammonium sulfate precipitation, followed by successive column chromatographies on DEAE-cellulose, Sephadex G-75, CM-Sephadex C-50 in the presence of calcium ion. The specific activity of the purified enzyme was 4.7 units/mg protein and 480 times that of starting crude extract. The homogeneity of the enzyme protein was criticized by polyacrylamide gel disc electrophoresis and ultracentrifugation. The enzyme protein also behaved homogeneously in ampholine electrophoresis, indicating the isoelectric point of 8.56. The sedimentation coefficient of the enzyme was determined to be 2.97 S, and the molecular weight to be 40,000 by Archibald’s method. According to the measurement of optical rotatory dispersion of the enzyme, ORD constant, λ_c, Moffitt-Yang parameters, a₀ and b₀, were evaluated to be 239 mμ, ?164 and ?123, respectively.