Substrate Specificity of α-Glucosidase II in Rice Seed

The substrate specificity of rice α-glucosidase II was studied. The enzyme was active especially on nigerose, phenyl-α-maltoside and maltooligosaccharides. The actions on isomaltose and phenyl-α-glucoside were weak, and on sucrose and methyl-α-glucoside, negligible. The α-glucans, such as soluble starch, amylopectin, β-limit dextrin, glycogen and amylose, were also hydrolyzed.

The ratio of the maximum velocities for hydrolyses of maltose (G₂), nigerose (N), kojibiose (K), isomaltose (I), phenyl-α-maltoside (?M) and soluble starch (SS) was estimated to be 100: 94.4: 14.2: 7.1: 89.5: 103.1 in this order, and that for hydrolyses of malto-triose (G₃), -tetraose (G₄), -pentaose (G₅), -hexaose (G₆), -heptaose (G₇), -octaose (G₈), and amyloses ( and ), 113: 113: 113: 106: 113: 100: 106: 106. The Km values for N, K, I, ?M and SS were 2.4 mm, 0.58 mm, 20 mm, 1.6 mm and 5.0 mg/ml, respectively; those for G₂, G₃, G₄, G₅, G₆, G₇, G₈, and , 2.4 mm, 2.2 mm, 2.1 mm, 1.5 mm, 1.0 mm, 1.1 mm, 0.95 mm, 1.5 mm and 1.1 mm.

Rice α-glucosidase II is considered an enzyme with a preferential activity on maltooligosaccharides.     

Determination of CGTase from Bacillus ohbensis and Its Optimum pH Using HPLC

Glucose is widely known to be required during superoxide generation in phagocytic cells. However, when an specific chemiluminescence probe with the Cypridina luciferin analog 2-methyl-6-(p-methoxyphenyl)-3, 7 -dihydroimidazo[ 1,2-a]pyrazin-3-one (MCLA) was used, about 60% of the chemiluminescence remained in stimulated macrophages in the presence of the glycolytic inhibitor 2-deoxyglucose. -nonspecific luminol-dependent chemiluminescence disappeared when the same drug was added. These results clearly demonstrate that the generation of by macrophages is not completely glucose-dependent, and strongly suggest that macrophages have both glucoseindependent NADPH-supplying pathway(s) and glucose dependent pathway(s) which generate reactive oxygen species other than .     

Isolation and Physicochemical Properties of a Soluble Glycoprotein Fraction of Milk Fat Globule Membrane

A soluble apoprotein fraction was prepared from milk fat globule membrane lipoproteins by delipidation with a chloroform-methanol mixture and was fractionated into three fractions by gel filtration on Bio-Gel A–5m.

The major fraction, Fraction II, contained about 30% of carbohydrate, i.e. 13.9% of hexoses, 8.1% of hexosamines, 8.0% of sialic acid and 0.8% of fucose, and was therefore designated a soluble glycoprotein fraction. The fraction was apparently homogeneous on sedimentation velocity analysis and DEAE-Sephadex chromatography, and had 6.1, 3.79, 0.719, f/f⁰ 2.16 and molecular weight 139,000 daltons. However, the diffused pattern on disc electrophoresis and the occurrence of plural N-terminal amino acid residues suggest that the protein of this fraction is likely to be formed by intermolecular association of heterogeneous polypeptide chains.     

Cell-wall Polysaccharides of Immature Barley Plants. II. Characterization of a Xyloglucan

A xyloglucan occurring in the hemicellulose II fraction of cell walls of immature barley plants was characterized by methylation and fragmentation analyses. The results indicated that the xyloglucan was mainly composed of the following repeating units:      

Two Forms of Glucoamylase from Mucor rouxianus 1. Purification and Crystallization

Mucor rouxianus produced two forms (isoenzymes) of glucoamylase which could be separated from each other by polyacrylamide gel electrophoresis or by chromatography on SP-Sephadex C-50, and they were designated glucoamylase I and glucoamylase II. Glucoamylases I and II were isolated in crystalline form, and were homogeneous in poly acrylamide gel electrophoresis and in ultracentrifugation, respectively. The sedimentation coefficient () molecular weight of glucoamylase I were 4.39 S and 59,000, and those of glucoamylase II were 4.29 S and 49,000, respectively.     

Lipolysis by Intracellular Lipase of Streptococcus lactis against Its Neutral Lipids Obtained by Growth at Low Temperature

Lipolytic activities of intracellular lipase obtained from Streptococcus lactis 527 cells grown at 30°C were determined using bacterial neutral lipids extracted from cells grown at 10 and 30°C. The amounts of free fatty acids liberated from lipids by lipase were in the order: 30°C neutral lipid > 10°C neutral lipid > triolein > intracellular membrane fraction. Glycerides hydrolyzed partially by lipase were detected on thin-layer plates and were composed of 1,3- and 1,2-diglycerides, fatty acids and unhydrolyzed triglycerides. Fatty acids liberated from neutral lipids by lipase were determined by gas chromatography. It was found that the major acid was cy-C₁₀ and the minor among the acids liberated from 10°C neutral lipid, whereas the major acid was and the minors and cy-C₁₀ from 30°C lipid.     

Radiation Chemistry of Foods

When 10^?3m cysteine solution was irradiated in the presence of glucose at the concentration of ten-fold of cysteine, the G-values of products produced from cysteine were similar to those from 10^?3m cysteine solution. On the other hand, the yield of carbonyl compound from glucose was suppressed completely by interaction between cysteine and radicals which are secondarily produced from glucose.

Methionine could not suppress the yield of carbonyl compound from glucose, and, G-values of products from methionine varied in comparison with those from solution containing methionine only.

From the results using scavenger, it was concluded that oxidation to methionine sulfoxide and cleavage to α-aminobutyric acid was caused by OH and attack, respectively.     

The Initial Attack Sites of a Streptomyces Alkalophilic Proteinase on Oxidized Insulin B-Chain

The sites on oxidized insulin B-chain substrate initially attacked by an alkalophilic proteinase from a Streptomyces sp., were investigated under incubation conditions employing one part enzyme to one thousand parts of substrate at 0°C.

Analysis of the peptides produced after 10 to 40 seconds of incubation revealed that the enzyme, which has an optimum pH of around 13, first attacks two peptide linkages “-Leu (15)Tyr (16)-Leu (17)-” of the oxidized insulin B-chain with equal efficiency.     

Studies on Oxidation-Reduction Potentials (ORP) of Microbial Cultures

At maximum production of l-glutamic acid, the oxidation-reduction potential of the culture broth in l-glutamic acid fermentation showed a stable value of 9.0 to 9.6 as rH value. When biotin concentration in the medium was high (40γ/liter), the production of l-glutamic acid decreased, and the rH was 8.0 and it was out of accordance with that of the control (biotin-poor; 2γ/liter). Under “less-aerobic” conditions, its rH rose to 10.4.

From these results, it was concluded that the rH during maximum production of l-glutamic acid showed a stable value affected actively by the redox system, l-glutamic acid/α-ketoglutaric acid and      

Physical and Chemical Properties of Yeast Proteinase C

A simple purification method which enables us to obtain homogeneous proteinase C from S. cerevisiae was developed. Physical and chemical properties of the purified enzyme were determined. The extinction coefficient at 280 mμ, , of yeast proteinase C was 14.8, and its isoelectric point was pH 3.60. Partial specific volume, intrinsic viscosity and the sedimentation and diffusion coefficients of homogeneous protein were , 0.71 ml/g, [η], 4.83 × 10^?2ml/g, , 4.23 S and , w, 6.1 × 10^?7 cm²/sec. From these values, molecular weights, M_[·],D, M_S,D and M_[·],S, of 60,000, 59,000 and 58,000, respectively, were obtained. The sedimentation equilibrium experiment gave a molecular weight, M_equil, of 61,000. Yeast proteinase C contained 11.9% nitrogen and was a glycoprotein with 16.7% carbohydrate: The value of β-function, 2.163×l0⁶ or 2.20×l0⁶ indicates that the molecular shape of yeast proteinase C is a plorate with an axial ratio of 4.0, assuming 35% hydration. Furthermore, yeast proteinase C may be a compact, asymmetric ellipsoidal model having semi-axes 30Å × 30Å × 130Å.     

Changes in Amino Acid Composition during Germination of Soybean

Malonogalactan, a malonylated polysaccharide (—74° (c=1.6, H₂O)) produced by Penicillium citrinum, consisted of d-galactose and malonic acid in the approximate molar ratio of 3:1. Molecular weight of the demalonylated galactan (-99° (c=4.6, H₂O)) was about 40,000. From the data regarding optical rotation, nuclear magnetic resonance spectrum, infrared spectrum, glycosidase susceptibility, periodate oxidation, Smith degradation, methylation and acid hydrolysis, the possible structure of the Penicillium malonogalactan is deduced as follows: A galactan, 1,5-β-galactofuranoside polymer esterified with malonic acid at the position of 2 or 3.     

Aromatic Amino Acid Residues in Japanese-radish Peroxidase a and Apoenzyme†

The nature of aromatic amino acid residues in Japanese-radish peroxidase a and the apoprotein was investigated by means of spectrophotometry and fluorospectrophotometry. The tyrosine residues in the holoenzyme were masked in the alkali-titration, giving an abnormally high value of 12.6, while they were exposed in the apoenzyme, exhibiting a value of 10.8. The difference spectra in the ultraviolet region between the holo-and apo-enzyme showed characteristic bands of tryptophan and phenylalanine as well as tyrosine. The perturbation of the aromatic amino acid residues by 50% ethyleneglycol was observed in the apoenzyme but not in the holoenzyme. The fluorophotometric experiments also revealed that the aromatic amino acid residues were in different environments in the holo- and apoenzyme. The difference between the conformation of peroxidase and that of the apoprotein was discussed.     

Studies on Bacterial Protease

A crystalline alkaline protease was prepared from B. amylosacchariticus, which was isolated as a strain of saccharogenic α-amylase-producing Bacillus subtilis. The enzyme was most active at pH values between 10.3 and 10.7 towards casein and was stable at pH values from 6 to 11 on twenty hour incubation at 30°C. Calcium ions were effective to stabilize the enzyme especially at higher temperatures. The enzyme was markedly inactivated by DFP as well as protease inhibitor from potato and slightly by surface active agents, but not affected by sulfhydryl reagents and divalent metal ions except Hg⁺⁺ .Hemoglobin was the best substrate for the enzyme and more than 20% of the peptide bonds were hydrolyzed. Of numerous synthetic peptides tested, only the two compounds, and , were found to be hydrolyzed. A cyclic peptide, gramicidin S, was split by the enzyme only at the peptide bond of -l-valyl-l-ornithyl-. Methyl n-butyrate and tributyrin were also good substrates for the alkaline protease obtained here.     

Degradation of Dextran by Free Radical Systems

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E.C. 1.1.1.1) from Thea sinensis seeds (variety: Zairai) was isolated and purified about 1,500-fold using preparative disc electrophoresis. The specific activity was about 3.4 units/mg protein against ethyl alcohol.

Its value was 6.96 S and its molecular weight was approximately 150,000 using gel filtration on Sephadex G–200. The physical, chemical and catalytic properties of the enzyme are described. The oxidoreduction products formed by the enzyme were identified by gas chromatography, and for the unsaturated compounds the conversion of double bond and geometrical isomerization was observed.

The substrate specificity of tea enzyme is discussed in comparison with the enzymes from Leuconostoc mesenteroides, and horse and human livers. Paticularly a tendency for reactivity in the oxidoreduction of unsaturated alcohols and aldehydes were discribed by comparing the effects of geometry, the position of the double bond and the length of chain in substrates.     

Kinetics of Carbon Dioxide Evolution in Agitation System

Kinetics of carbon dioxide evolution was investigated in agitation system. Reaction steps of carbon dioxide evolution in submerged fermentations may consist of three steps; the first, hydration of carbon dioxide liberated from living cells, the second, dehydration of bicarbonate ions and the third, formation of carbon dioxide bubbles. Taking into account the equilibrium between hydration of carbon dioxide and dehydration of bicarbonate ions at physiological pH value, the fallowings may be rate-limiting steps in mass transfer of carbon dioxide in submerged fermentations, dehydration of bicarbonate ions and the bubble formation. The overall velocity constant of these two reaction steps was determined in the agitation vessel This reaction obeyed good first-order kinetics and the term of was introduced as a velocity constant. This value was influenced by agitation speed, temperature, viscosity of the fluid and carbonic anhydrase. The value of carbon dioxide coefficient (Kd)_CO2 was higher than the oxygen absorption coefficient Kd. The driving force of mass transfer for carbon dioxide, DCO₂—pCO₂, therefore, was lower than that for oxygen, P_B—P_L. The relationship between the overall coefficient of oxygen transfer across gas-liquid interface K_La and the overall velocity constant of carbon dioxide evolution was expressed in the formula      

Ascotoxin (Decumbin), a Metabolite of Ascochyta imperfecta PECK.

From the mycelium of Ascochyta imperfecta decumbin, C₁₆H₂₄O₄, mp 203°C, was obtained in one percent yield.

The absolute structure of decumbin was presented as [II] by the following evidences: The configuration about C₄ was determined as (S) by the benzoate rule on the tetrahydromonoketone (21). The hydroxyl at C₇ is α, because tetrahydrodecumbin (23) showed no intramolecular hydrogen bond, while its C₇ epimer (24) did. Ring juncture was determined by ORD of a five membered ketone (16). Two double bonds were found to be trans from IR data. The stereochemistry of decumbin monoepoxide (7), tetrahydropyrans (12 and 13) was also studied. Plant tests of the twenty derivatives of decumbin on lucerne and rape revealed that the growth inhibition activity has close relation with the presence of double bond in the thirteen membered lactone ring.     

Isolation and Structure of Maingayic Acid,a Piscicidal Constituent of Callicarpa maingayi

A piscicidal constituent (1), C₂₀H₂₈O₃, (chloroform), which was named maingayic acid, was isolated from the leaf of Callicarpa maingayi. On the basis of the chemical spectral studies, the pK_MCS evaluation and the octant rule on the ORD curves, we have and deduced that maingayic acid is a furanoid diterpene acid possessing a rearranged labdane skeleton shown as 1’a.     

Asymmetric Reduction of Iminium Salt with Chiral Dihydropyridines

A gram positive bacterium (strain No. 109) isolated from soil as a producer of cyclodextrinase was identified as Bacillus coagulans. The cyclodextrinase from B. coagulans was purified to a homogeneous state by disc-electrophoresis after Streptomycin treatment, DEAE-Sephadex column chromatography, Ultrogel AcA44 gel filtration and hydroxyapatite column chromatography. The molecular weight of the enzyme was determined to be 6.2}10⁴ by sodium dodecyl-sulfate gel electrophoresis. The isoelectric point of the enzyme was pH 5.0. The enzyme was most active at pH 6.2 and 50°C, and stable up to 45°C at pH 7.0 and in the range of pH 6.0 ~ 7.3 at 40°C on 2 hr incubation. This enzyme hydrolyzed linear maltooligosaccharides (such as maltotetraose (G4), maltopentaose (G5) and maltohexaose (G6)) and α-, β- and α-cyclodextrins (CDs) faster than maltotriose (G3) and short chain amylose ( 18), but did not hydrolyze maltose. The rates of hydrolysis for polysaccharides (such as starch, amylose and amylopectin) were below 1 % as compared to that for β-CD. The Km values for G3, G4, G5, G6, short chain amylose ( 18) and α, β- and γ-CD were 4.5, 4.0,2.3,1.5,1.5,10,2.8 and 0.47 mM, respectively. The products with this enzyme had the α-configulation.     

Hydrolysis Rate of Resistant Pentosan of White Birch Wood in Sulfuric Acid of Medium Strength

The purpose of this study is to find optimal conditions for pre-hydrolysis in the new wood saccharification process with strong sulfuric acid. In the experiment, the hydrolysis rate of resistant fraction of pentosan of white birch (Shirakamba, Betula platyphylla Sukatchev var. japonica Hara) wood and the decomposition rate of xylose are measured in acid concentrations ranging from 30 to 60% at temperatures ranging from 30 to 90°C. The hydrolysis of resistant pentosan of white birch and the decomposition of xylose are the first-order reactions. The first-order reaction constant of hydrolysis of resistant pentosan, k_B min^-1, is expressed by the following empirical equations as the function of percentage concentration of sulfuric acid, C, and reaction temperature described by absolute temperature, T°K, ranging from 40 to 80°C:

where sulfuric acid concentrations range from 30 to 50%;

where sulfuric acid concentration is 60%.

The first-order reaction constant of decomposition of xylose, k₂ min^-1, is expressed by the following empirical equation as the function of sulfuric acid strength described by acidity function, H₀, and reaction temperature described by absolute temperature, T°K, in sulfuric acid concentrations ranging from 30 to 60% at temperatures within the range of 40 to 100°C.

where C is sulfuric acid strength described by acidity function, H₀.     

DNA Strand Breakage In Vitro by Autoxidized Unsaturated Fatty Acids

Nitrate and nitrite were successfully extracted from deproteinized chicken egg with aqueous solution, and analyzed by gasliquid chromatography with an electron capture detector without further cleaning. The distribution of these anions in 50 egg samples was the logarithmic normal distribution in each case, that is, N and p{0.052 ppm ≤ ¼ ≤ 0.076ppm} = 0.95 for nitrate-N, and N and p{0.026ppm ≤ ¼ ≤ 0.034 ppm} = 0.95 for nitrite-N. When the chickens were fed with a commercial diet containing elevated levels (1,000 or 5,000 ppm) of nitrate- or nitrite-N, the concentration of these anions in their eggs markedly increased and proceeded to the steady state within 2 or 3 days, where the level was proportional to that of anions added to the diet. After withdrawing the excess of anions from the diet, the concentrations of anions in the eggs decreased exponentially, where the rate constants for nitrate and nitrite were about 0.6 day^?1 and 1.0 day^?1, respectively. In the series of experiments, it was assumed that the reactions proceed simultaneously in the body of chickens.