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.     

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.     

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.     

A Novel Reaction of Sugars with Anion Radical of Carbon Dioxide Produced from Formate

Radiolysis of some monosaccharides (fructose, glucose and ribose) in air-free condition was markedly enhanced by the addition of formate at concentrations above 20 mm, while it was inhibited at concentrations below 20 mm. The following compounds were detected in the irradiated sugar solutions containing excess formate (100mm): 1-Deoxy-d-arabinohexulose (1, G=4.4) and 1,3- dideoxy-d-erythrohexulose (2, G= 1.3) from fructose; 2-deoxy-d-ribose (3, G=2.3) and 2-deoxyribitol (4, G =0.6) from ribose; and 2-deoxy-d-glucose (5, G=0.5) and 2-deoxy-d-glucitol (6, G=0.4) from glucose. A mechanism for radiolytic formation of the products was proposed, based on interaction of - formed from formate with sugars.     

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.     

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      

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.     

The Stress Relaxation of Concentrated Poly γ-Benzyl-l-Glutamate

The effect of temperature and concentration on the relaxation of poly γ-benzyl-l-glutamate (PBLG-mol. wt. 394,000 to 397,000) was examined as a model of protein behavior under “stress” leading to the definition of intra- and inter-molecular forces involved. The relaxation of PBLG, unlike that of the random coil polymers, takes places in two stages. The lower the temperature and concentration of the polymer, the greater is the shift of the inflexion point towards the lower values.

The distribution spectrum of relaxation has a three zone characteristic. The possible significance of each of them is discussed.     

Studies on the Polyuronide of Oh-gyo-tye (The Achine of Ficus awkeotsang MAKINO)

A polyuronide, main component of the water extract of achine of Ficus awkeotsang MAKINO (on-gyo-tye), was purified by ion-exchange chromatography on DEAE-cellulose. The polyuronide (Fraction IB) is homogeneous electrophoretically and consists mainly of galacturonic acid. Optical rotation of Fraction IB is and content of methoxyl group is trace. In periodate oxidation of Fraction IB, molar ratio of galacturonic acid residue and periodate consumption was 1, and formic acid formation was very small. Periodate oxidation product of Fraction IB was oxidized further with bromine and the resulted substance was hydrolyzed. In the hydrolyzate, presence of large amount of tartaric and glyoxylic acids and small amount of tartronic acid were detected by paper chromatography. Reduced viscosity of aquous solution of Fraction IB increased with decreasing of the concentration of Fraction IB solution. From these results, it was deduced that Fraction IB has a linear structure of 1→4 linkage of d-galacturonic acid, probably α-linkage.     

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.     

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.     

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.     

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.     

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 .     

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.     

Purification,Crystallization and Properties of Primary Alcohol Dehydrogenase from a Methanol-oxidizing Pseudomonas sp. No. 2941

A simple method for purification and crystallization of primary alcohol dehydrogenase (EC 1.1.99.8) is reported. The purification procedures consisted of four steps: protamine sulfate treatment, ammonium sulfate fractionation, passage through a column of DEAE-cellulose at pH 8.0 and Sephadex G-200 gel filtration. Crystallization was performed by the addition of ammonium sulfate at 65 % saturation with an overall yield of 39 %. The crystalline enzyme had an isoelectric point of pH 7.38 and a sedimentation coefficient 8.44s. A molecular weight of 128,000 was estimated, and the enzyme consisted of two subunits each having a molecular weight of 62,000. The enzyme showed an affinity toward the lower primary alcohols, methanol to n-pentanol. Formaldehyde was also oxidized by the crystalline enzyme. The Km values for methanol and formaldehyde were found to be 20 μm and 70 μm, respectively. Ammonium ions were required for enzyme activity.     

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.     

Fukiic Acid Isolated from the Hydrolysate of a Polyphenol in Petasites japonicus

d-Glucose-isomerizing enzyme was purified in a crystalline form with a good yield from the cells of Bacillus coagulans, strain HN-68, and some phsicochemical properties were investigated.

The purified enzyme was homogeneous on both ultracentrifugal and disc-electrophoretical analyses. The molecular weight of the enzyme was determined to be 175,000 and 160,000 from the sedimentation-viscosity method and the gel filtration method, respectively.

The sedimentation coefficient , partial specific volume, at 280 mμ, and the nitrogen content of the enzyme were determined to be 10.2×10^?13 sec, 0.705 cm³g^?1, 10.6 and 16.2%, respectively. The integral numbers of amino acid residues per molecule calculated on the basis of 160,000 were as follows; Lys₁₂₀, His₄₉, Arg₆₁, Asp₁₈₂, Thr₈₇, Ser₇₀, Glu₁₃₆, Pro₄₄, Gly₁₀₆, Ala₁₄₀, Half-Cys₀, Val₅₃, Met₂₇, Ileu₅₁, Leu₁₃₄, Tyr₅₈, Phe₉₆, Try₁₃, and amide-ammonia₈₀.

Purified enzyme preparation obtained from Bacillus coagulans, strain HN-68 requires Co²⁺ for d-glucose- and d-ribose-isomerizing activities and Mn²⁺ for d-xylose-isomerizing activity. The values of Km for d-glucose, d-xylose and d-ribose were 9×10^?2, 1.1×10^?3, 7.7×1O^?m and of the relative V_max were 0.52, 1.1 and 0.25 mg/min at 40°C, respectively. d-Glucose-isomerizing activity was inhibited by d-xylose and d-ribose. However, there was not a difference among three activities of the enzyme with respect to following properties: Activation energy was 14,600 cal per mol. The enzyme was inhibited in a competitive manner by tris(hydroxymethyl)aminomethane, d-xylitol, d-sorbitol and d-mannitol, and the Ki values for these inhibitor were 3×10^?4, 2.5×10^?3, 2.9×10^?2 and 7×10^?2m, respectively. The ratio of three activities did not change by heat- and pH-treatments. Mn²⁺, Co²⁺ and Ni²⁺ protected strongly the enzyme from heat denaturation. The enzyme can isomerize d-glucose, d-xylose and d-ribose to their corresponding ketose, but the kinetic constants and induction studies indicated that ^d-xylose is the natural substrate for the enzyme.     

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.     

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.