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.     

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.     

Enzymatic Assay of d-Xylose Isomerase Activity

The d-xylose isomerase activity was assayed spectrophotometrically as NADH oxidation in a coupled reaction with the d-arabitol dehydrogenase. The assay system is based on the following reactions:

d-Arabitol dehydrogenase was purified from the d-sorbitol-grown cells of Agrobacterium tumefaciens. The standard assay condition is as follows: 5 μmoles of Tris-HCl buffer (pH 7.0), 0.2 μmole of MnCl₂, 2 μl of reduced glutathione (25 mg/ml), 0.05 μmole of NADH, 6 units of d-arabitol dehydrogenase, 5 μmoles of d-xylose and d-xylose isomerase in a total volume of 0.30 ml. The reaction was carried out at 30°C. With the assay system, it was confirmed that d-xylose isomerase did not produce d-xylulose from d-lyxose.     

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      

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.     

Lysine-derived cross-links in the egg shell membrane

Egg shell membrane protein contains significant quantities of the lysine-derived aldehyde, allysine, and its aldol condensation product. NaB³H₄ reduction followed by alkaline hydrolysis of purified protein revealed that there were six residues/1000 of both allysine and the reduced aldol while only traces of desmosine and isodesmosine were detected. The amino acid composition of the membrane protein did not resemble that of mammalian elastin.     

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.     

The Presence of Intracellular Proteolytic Enzyme Activities in Azotobacter vinelandii Strain O Cultured in Iron-deficient Medium

The effects of alkalic salts on the apparent viscosity of acid precipitated protein (APP) of soybean-suspending systems and heated gels were investigated using a modified coaxial cylinder viscometer. A hybrid program was established. (i) a cyclic temperature test (20 → 90 → 20°C) under a constant shear rate and (ii) a cyclic shearing test (48.7 → 243.7 → 48.7 sec^–1) under isothermal conditions. The apparent viscosity of protein suspending systems (12%, wt/vol) gradually decreased with increasing temperature to about 70°C. The apparent viscosity increased with a rise in temperature in the range of 70 to 90°C, in ascending order of Hofmeister’s series.

With a fall in temperature, the apparent viscosity increased considerably in this order. The effect of anions to apparent viscosity was larger than that of cations at all measured temperatures in the gel-forming period.     

β-Galactosidase in Alkalophilic Bacillus

The changes in ¹³C-NMR and ³¹P-NMR spectra and ¹H-NMR images in soybean cotyledons during germination were investigated. Using ¹³C-NMR, fatty acid signals in the form of triglycerides were observed in dry seeds, and those were observed approximately 18 days after the start of imbibition. Sucrose signals appeared at 16 hr and disappeared at 5 days. A signal was observed after 5 days, suggesting the activation of membrane metabolism in the cotyledons.

³¹P-NMR signals appeared 2 hr after imbibition before any apparent change in the ¹³C-NMR spectrum. The peaks identified as sugar phosphate, inorganic phosphate in the cytoplasm and in the vacuole, and an unassigned compound, were distinguishable after 5 days. The vacuole-associated inorganic phosphate peak became prominent 18 days after imbibition in ³¹P-NMR.

Distribution maps of free water indicated that the stored macro-molecular materials which bound water were consumed heterogeneously within the cotyledon. The relaxation time (T₁) increased suddenly between 18 days and 23 days after imbibition, which indicates the consumption of stored materials.

These findings suggest that cotyledons are the source of such compounds and the energy required for plant growth for approximately 18 days from germination until tri-foliolate leaves begin developing.     

New Strains of Botryodiplodin-producing Fungi

The light-emitting species of chemiluminescence produced in rat liver homogenate on adding autoxidized linseed oil (AOLO) were investigated. The chemiluminescent intensity of liver homogenate was strongly enhanced by the addition of AOLO and showed a proportional relationship to the amount of AOLO. The chemiluminescence was reduced with singlet oxygen (¹O₂) quenchers and free radical scavengers. Among them, β-carotene showed the most effective quenching. The emission spectrum had broad bands in the visible region with eminent chemiluminescent lines at 520, 575 and 640 nm due to the simultaneous transition, . An additional weak line was found at 480 nm corresponding to . In the presence of β-carotene, lines corresponding to the simultaneous transition of ¹O₂ disappeared. These results indicate that the liver homogenate with AOLO generated singlet molecular oxygen as one of the major light-emitters of the chemiluminescence. A possible mechanism for the generation of ¹O₂ is by decomposition of peroxy radicals derived from AOLO in the liver homogenate.     

Studies on Alginase

Chemical investigations were made on a new unsaturated crystalline diuronide isolated from alginase hydrolysate of alginic acid. This uronide has (in water), and m.p. 135.5~136.5°C (decomp.). The presence of an α/β-unsaturated carboxylic acid formulation is supported by the following evidences: (a) an ultraviolet absorption band at 232 m/μ, (b) infrared absorption bands at 1648 cm^-1 due to double bond and at 1720 cm^-1 due to conjugated carboxylic group, (c) the consumption of about 1 mole of bromine per mole of the compound, (d) the production of oxalic acid on oxidation with ozone, (e) the formation of a substance that shows absorption maximum at 550 mμ, caused by the addition of thiobarbituric test. After hydrolysis, crystalline mannuronic lactone was obtained from the unsaturated diuronide. Occurrence of mannuronic moiety in the reducing unit was observed by paper chromatography of the hydrolysate of borohydride-reduced unsaturated compound. From these results it can be seen that the possible structure of this unsaturated diuronide is 4-O- (β-d-Δ4,5 mannoseenpyranosyluronic acid) -d-mannuronic acid.     

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.     

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 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.     

Production and Isolation of Dopastin,an Inhibitor of Dopamine β-Hydroxylase

A new inhibitor of dopamine β-hydroxylase, dopastin, has been isolated. The dopastin-producing strain was found in a mushroom culture, and after being separated, it was confirmed to be a member of Pseudomonas. Dopastin was obtained as colorless needles, mp 116~119°C, (c=0.5, С₂Н₅ОН), C₉H₁₇N₃O₃. The catalytic hydrogenation afforded dihydro-dopastin which also inhibits dopamine β-hydroxylase.     

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.     

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.     

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.     

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.