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.     

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.     

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.     

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.     

A Sulfated Galactan from the Red Seaweed Hypnea charoides

The contents of acetoin and ethyl lactate in Japanese and foreign wines were determined by gas chromatography with direct injection method. Table wines (225 samples) contained from zero to 140 mg/liter(: 15~23 mg/liter) of acetoin and from traces to 534 mg/liter(: 116~196 mg/liter) of ethyl lactate. Sparkling wines (17 samples) showed almost the same ranges of the volatile contents as table wines. Acetoin was formed significantly by aerobic fermentation and also microbial contaminations. Small quantities of ethyl lactate were formed during alcoholic fermentation, while, the formation was promoted by addition of lactic acid to must or to wine, and also by malo-lactic fermentation: the facts revealed that the concentration of lactic acid is related to ethyl lactate formation.

As to the contribution of the volatile compounds on wine flavor, it was found that acetoin has little influence in most wines, however, ethyl lactate has appreciable influence in many wines.     

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 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.     

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.     

Main-hydrolysis of White Birch Chips Wetted with Sulfuric Acid Solution of Medium Strength Using a Through Drying Operation

The purpose of this study is to find the relation between the saccharification ratio and the mixing ratio in a new wood saccharification process with strong sulfuric acid by employing a through drying operation. The saccharification yield in the main-hydrolysis of white birch chips with a sulfuric acid solution of a medium strength is expressed by the following empirical equations according to respective kind of samples of white birch chips used. With mixing ratio R ranging from 0.4 to 1.0.

when raw white birch chips (0.1°10°10 mm) are used and

when pre-hydrolyzed white birch chips (1.5×3.5~4.5×10~15 mm) are used.     

Purification,Crystallization and Some Properties of β-Galactosidase from Saccharomyces fragilis

Crystalline β-galactosidase was prepared from the cell extract of Saccharomyces fragilis KY5463, by procedures including protamine sulfate treatment and DEAE-cellulose, hydroxylapatite and DEAE-Sephadex column chromatographies. Crystals were formed when solid ammonium sulfate was added to solutions of the purified enzyme. This procedure resulted in a 55-fold purification with an over-all yield of l5.4%. The crystalline enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis.

The sedimentation coefficient, , was determined to be 10.0 S. The molecular weight was estimated to be approximately 203,000 by the sedimentation equilibrium method of Yphantis. Electrolysis with carrier ampholytes revealed that this enzyme has an isoelectric point at around pH 4.4.

The enzyme was activated by K⁺ in addition to bivalent cations, such as Mn²⁺, Mg^2? and Co²⁺. The Km values for o-NPG and lactose were 4.0×10^?3m and 21.0×10^?3m, respectively. The enzyme is sulfhydryl dependent and was completely inactivated by mercuric ions or p-chloromercuribenzoate.     

Cofactor Requirements of Tryptophanase from Proteus rettgeri

Crystalline tryptophanase prepared from the cells of Proteus rettgeri is inactive in the absence of added pyridoxal phosphate. Half-maximal enzyme activity is obtained at a concentration of 1.81 µm. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 420 nm. Holotryptophanase requires K⁺ or for its maximal activity, but Na⁺ is inactive. No appreciable spectral change was observed on changing the ionic environments.

The amount of pyridoxal phosphate bound to the enzyme was determined by equilibrium dialysis and spectrophotometric titration to be 4 moles per mole of enzyme. Reduction of holoenzyme with sodium borohydride results in a shift of the absorption peak at 420 to 336 nm. ?-Pyridoxyllysine was isolated from the acid hydrolyzate of the reduced holoenzyme by paper chromatography and electrophoresis.

Addition of the substrate, l-tryptophan, or the competitive inhibitor, l-alanine, to the holoenzyme causes appearance of a new peak near 500 nm which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor. The similar spectral change was observed by the addition of pyruvate, ammonia and indole to the holoenzyme.     

Digestion of Myosin by a Bacterial Protease

A neutral protease, which was prepared from Bacillus polymixa, was used on the digestion of myosin. Myosin was split to HMM and LMM, The HMM fraction was further digested with the protease and a subfragment-1 was prepared.

The sedimentation coefficient, , and the intrinsic viscosity of this subfragment-1 were determined as 5.6S and 0.08dl/g, respectively. The molecular weight was estimated to be about 120,000 by the sedimentation equilibrium method. This subfragment showed the characteristic myosin-type ATPase; the ATPase was activated by Ca²⁺ ion or EDTA and inhibited by Mg²⁺ ion, the maximum activation of ATPase was obtained when 3.5 SH groups per 10⁵ g of subfragment were titrated with PCMB.

The subfragment-1 possessed the ability to interact with F-actin and to accelerate G-actin polymerization.     

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.     

Microbial Transformation of Sterols

The ability of accumulating androsta-1, 4-diene-3, 17-dione (ADD) in the digestion of cholesterol by Arthrobacter simplex IAM 1660 was examined with 167 compounds and (i) chelating agents, (ii) Ni²⁺, Co²⁺, Hg²⁺, As³⁺, Sb³⁺, Bi³⁺, Cd²⁺, , and ions, and (iii) redox dyes were found effective for ADD accumulation. Ionic state of the chelating agents was unfavorable for ADD accumulation but inactive ethylaenediamine tetraacetic acid could be turned effective with aid of surface active agents and penicilline. Lipophilic structure of the chelating agents was required probably for its penetration through the cell membrane. The target process of the ADD accumulating agents was supposed as 9α-hydroxylation and their possible mechanism of inhibiting 9α-hydroxylation is discussed.     

Purification and Characterization of Formaldehyde Dehydrogenase in a Methanol-utilizing Yeast,Kloeckera sp. No. 2201

An NAD-linked formaldehyde dehydrogenating enzyme was found in the cell-extract of Kloeckera sp. No. 2201, which utilized methanol as a sole source of carbon. The enzyme was inducibly formed in methanol-grown cells. This fact suggests that the enzyme may play a significant role in the methanol metabolism of this yeast. The enzyme was purified from a cell-extract by ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and on hydroxylapatite, and Sephadex G-200 gel filtration. From an experiment with the purified enzyme, it was found that the enzyme specifically required reduced glutathione for activity, and was reactive toward methylglyoxal as well as formaldehyde. The enzyme catalyzed the following reaction:

the enzyme was concluded to be a kind of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1). Other properties of the enzyme were also investigated.     

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      

Studies on the Behaviors of Impurities on the Crystallization of l-Glutamic Acid

The behaviors of impurities on the crystallizations of the free acid and the hydrochloride, monosodium, zinc, calcium and barium salts of l-glutamic acid were examined, and a tendency was recognized that coexisting impurities were apt to be taken into the crystals when the crystallization proceeded from the zwitterion, i.e., Glu.^± or The adsorption of l-tyro-sine was compared when l, d and dl-glutamic acid were crystallized with coexistence of l-tyrosine, and an effect of more or less extent of steric configuration on its behavior could be recognized.     

Red Algae (Eucheuma muricatum) Mucilage- Complex Formation and Mechanism

Eucheuma muricatum mucilage which was extracted and purified after irradiation of the seaweed with γ-ray of ⁶⁰Co formed a complex with , and exhibited a new absorption band at 555 nm. The absorbancy observed at that time depended on the concentration of urea and on the temperature. The curves representing relations between absorbancy at 555 nm and the above factors have two inflection points. The fact that their inflection points shift toward the lower temperature side with the increase in urea concentration suggests that the coloring phenomenon may relate closely to the transition of the mucilage. It was also found that the absorbancy at 555 nm depended on the content of pyruvic acid residue in the same mucilages, the absorbancy decreased with the increase pyruvic acid residues, and that the steric hindrance caused by a sugar residue of large demension affected the stable from containing viscous polysaccharide.