NAD-dependent n-glucose dehydrogenase (NAD-GIcDH) was purified from the cell free extract of alkalophilic Corynebacterium sp. No. 93-1. The preparation obtained was homogeneous on disc gel and SDS-disc gel. The molecular weight of NAD-GIcDH was 51, 000 by SDS-disc electrophoresis and 55, 000 by gel filtration. The isoelectric point of the enzyme was pH 4.05. The optimal pH was 9.8, and it was stable at pH 6 to 8. NAD-GIcDH oxidized D-glucose, D-xylose, cellobiose, cellotriose, gentiobiose and laminaribiose. The Km values for D-glucose, o-xylose, cellobiose and gentiobiose were 51mM, 125mM, 11mM and 3.2mM, respectively. The Km value for NAD was 0.44mM. The enzyme was inhibited completely by PCMB, Ag⁺ and Hg²⁺. NADH had an inhibitory effect on NAD-G1cDH.

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NADP-dependent maltose dehydrogenase (NADP-MalDH) was completely purified from the cell free extract of alkalophilic Corynebacterium sp. No. 93-1. The molecular weight of the enzyme was estimated as 45, 000_??_48, 000. The enzyme did not have a subunit structure. The isoelectric point of the enzyme was estimated as pH 4.48. The pH optimum of the enzyme activity was pH 10.2, and it was stable at pH 6 to 8. The temperature optimum was 40°C, and the enzyme was slightly protected from heat inactivation by 1mM NADP. The enzyme oxidized D-xylose, maltose and maltotriose, and the Km values for these substrates were 150mM, 250mM and 270mM, respectively. Maltotetraose and maltopentaose were suitable substrates. The Km value for NADP was 1.5mM with 100mM maltose as substrate. The primary product of this reaction from maltose was estimated as maltono-b-lactone, and it was hydrolyzed non-enzymatically to maltobionic acid. The enzyme was inhibited completely by PCMB, Ag⁺ and Hg²⁺.

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The enzymes of methanol oxidation were investigated in a new yeast strain, Pichia pastoris IFP 206, with high yield (0.42g cell per g of methanol). The following enzymes were detected in cell free extracts of P. pastoris: alcohol oxidase, catalase, formaldehyde and formate dehydrogenases. The alcohol oxidase was purified from cell free extracts of P. pastoris containing high amount of the enzyme (33%) with a good yield (55%). The preparation was homogenous by immunochemical methods. The enzyme had a molecular weight of 675, 000 and was composed of eight identical subunits of M.W. 80, 000. Each subunit contained one FAD. The N-terminal sequence was found to be: Ala-Ile-Pro-Glu-Glu-Phe-Asp-Ile-Leu-Val-Leu-Gly-
The protein had 65 free -SH groups per molecule. The optimum temperature for the enzyme activity was 37°C and the activation energy was 11.1 kcal/mol. Optimum pH was 7.5 and the enzyme activity was unstable at acidic pH. The apparent Km for methanol were 1.4 and 3.1mM at oxygen concentrations of 0.19 and 0.93 mat. Similarly, the apparent Kms for oxygen were 0.40 and 1.0mM at methanol concentrations of 1, 10 and 100ma. The enzyme oxidized primary alcohols with short carbon chains like ethanol and propanol. Inhibition of enzyme activity by hydrogen peroxide was a consequence of the oxidation of essential -SH groups. The inhibition was reversed by reducing agents.

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A simple chemically defined medium was devised for exoprotein production by Bacillus brevis No. 47. About 2mg/ml of proteins was produced in the synthetic medium containing 4% glucose and 1% ammonium sulfate. An essential component of fermentation medium was Ca salt which is required by this organism for assimilating glucose.
Studies on the effects of various medium components on protein production revealed that the conditions appropriate for growth are also suitable for protein accumulation. Some compounds, especially inhibitors of cell wall synthesis and certain detergents, were found to enhance protein production.

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Mutants of Bacillus brevis No. 47 that grew in synthetic media containing a high concentration of ammonium sulfate were stable and had high protein production. Among various antibiotics tested, inhibitors of cell wall synthesis, such as bacitracin or β-lactam antibiotics, were effective in greatly increasing the accumulation of exoproteins.
When 60μg/ml of bacitracin was added to the culture at the early logarithmic growth phase, about 9mg/ml of proteins was produced. Such a protein yield was estimated to be nearly maximum from a given amount of glucose. Alterations in cell wall components were found in cells grown in the presence of bacitracin. Possible relationships between cell wall structure and protein production were discussed.

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Cadmium ions at 5 to 100μg/ml induced morphological alterations in Euglena gracilis grown in zinc-sufficient media. The average cell volume was used as a parameter of morphological abnormality. Many types of abnormal-shaped cells were observed when the cadmium-ion concentration was high enough to increase the average cell volume. Most abnormal-shaped cells were starfish-shaped. Scanning electron microscopic observations clearly showed that the structure of the pellicle of starfish-shaped cells was not different from that of normal dividing cells and that these starfish-shaped cells, which were polynucleated and in the process of cytokinesis, resulted from abnormal cytokinesis, but not from cell fusion.

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An Hg²⁺-sensitive mutant strain was isolated from an Hg²⁺-tolerant bacterium Pseudomonas oleovorans G-1 strain by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The Hg²⁺-sensitive mutant strain was about 10-times as sensitive to Hg²⁺ as the parent strain. Moreover, the mutant strain was considerably more sensitive to Cr⁶⁺ than the parent strain, but it did not show an appreciable change in sensitivity to Cd²⁺ and Cu²⁺. The mutant strain was considerably more sensitive to antibiotics achromycin, chloramphenicol and streptomycin than the parent strain. A more rigid structure was observed in the cell envelope of the mutant strain than the parent strain under transmission electron microscope. Higher amounts of DNA but less protein and RNA were found in the mutant strain compared to the parent strain. Disc electrophoretic patterns showed some differences in protein bands between the parent and mutant strain.

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The molecular sizes of hydrolysates of acetylated and succinylated caseins by pepsin-pancreatin were examined by gel filtration on Sephadex G-15. There were more large peptides (average residual number >15) in the hydrolysates of the acylated caseins than there were in the hydrolysate of unmodified casein. In these large peptides from the acylated caseins the contents of N^ε-acyl-lysines were high. The digestibility of N^ε-acetyl and N^ε-succinyl lysine bonds in peptides by aminopeptidases [(EC 3.4.11.1) and (EC 3.4.11.2)] and watermelon carboxypeptidase model enzyme of cathepsin A [(EC 3.4.16.1)] was examined using digest of acylated caseins by pepsin, trypsin and α-chymotrypsin and some synthetic peptides. All peptidases released either N^ε-acetyl or N^ε-succinyl-lysine from peptides.
The hydrolytic processes of acetylated and succinylated proteins before and after intestinal absorption are discussed.

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Pyridoxamine (pyridoxine) 5'-phosphate oxidase purified from baker's yeast was found to have a molecular weight of ca. 55, 000 daltons based on polyacrylamide gel electrophoresis. The size of the enzyme subunit was analyzed by gel electrophoresis in the presence of sodium dodecylsulfate. This showed that the enzyme was composed of two nonidentical subunits with a molecular weight of 27, 000 and 25, 000 daltons. Fluorescence titration of the apoenzyme with FMN suggested that the holoenzyme contained one mol of FMN per mot of the enzyme. The K_m value of FMN for apoenzyme was calculated to be ca. 16nM on both activities of pyridoxamine 5'-phosphate oxidase and pyridoxine 5'-phosphate oxidase.

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Sperm whale myoglobin, horse myoglobin, bovine hemoglobin, and human hemoglobin produced well-defined catalytic hydrogen current (Brdi_??_ka current) in buffer solutions containing cobalt salts at dme and hmde. The Brdi_??_ka current-activity of the myoglobins was shown to be due to the heme group and that of the hemoglobins to both the heme groups and SH groups in the molecule. The Brdicka current produced by human hemoglobin was reduced by the addition of 2, 3-diphosphoglycerate (DPG), indicating that the formation of a hemoglobin-DPG complex resulted in the reduction of the Brdicka current-activity of the protein.

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The metabolic fate of the carbon skeleton of L-(U-¹⁴C)-histidine has been investigated in growing rats fed diets containing different percentages of protein calories (0, 5, 10, 15 and 30 PC%) at 410 kcal of metabolizable energy per 100g diet.
The incorporation of ¹⁴C into body protein at 12 hr after injection of ¹⁴C-histidine was about 70% of the dose in rats fed 0 to 10 PC% diets, though the value was reduced in rats fed higher PC% diets. The expired ¹⁴CO₂ production was depressed in the low protein groups, and it showed an inverse pattern to that of ¹⁴C incorporation into body protein. Urinary excretion of ¹⁴C was about 20% of the dose in all dietary groups. The activities of hepatic histidase, urocanase and histidine-pyruvate aminotransferase were increased in the 30 PC% group.
These results indicate that the metabolic response of histidine to dietary protein changes around 10 PC%, where growth rate and body protein retention reached approximate plateaus.
The nutritional significance of the metabolism of histidine has been discussed and compared with that of leucine, alanine and serine reported previously.

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Arrhenius plot of cytochrome c oxidase activity in mitochondria from either sweet potato roots or leaves showed discontinuity with a transition point at 24_??_25°C, and the activation energy above the transition temperature was extremely low. In contrast, the plot with potato tuber mitochondria showed continuity. Both natural and synthesized phospholipids did not significantly alter the transition temperature (15_??_17.5°C) for activity of cytochrome c oxidase purified from sweet potato root tissue. The enzyme in sweet potato leaf mitochondria was more heat-stable than that in root mitochondria.

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A globulin fraction which was salted out from egg white by ammonium sulfate was constituted of five kinds of proteins. One of them was macroglobulin and two were G2 and G3 of Longsworth et al. [J. Am. Chem. Soc., 62, 2580 (1940)]. Remaining two proteins were assumed to be ovoinhibitors. G₂ and G₃ were separated from each other by CM-cellulose chromatography. The molecular weights of G₂ and G₃ were almost the same, being about 49, 000. An euglobulin-like protein which was precipitated during the dialysis of the globulin fraction consisted mainly of macroglobulin and aggregates of some other egg white proteins.

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Myosin rod was prepared from hen myosin by chymotryptic digestion. The indigested myosin was successfully removed by ultracentrifugation following alcohol treatment. No significant difference in UV absorption and CD spectra was observed between pH 7.0 and pH 10.5 for both myosin rod and myosin. When pH was raised to 11.7, the phenolic groups of the tyrosyl residues were ionized, and the helical configuration of the myosin rod and myosin could not withstand the electrostatic repulsion. When pH was further raised to 13.6, “abnormal” tyrosyl residues were ionized, resulting in decreased helix content. However, the myosin rod was stabler and less flexible against pH change than myosin, because of the lower content of tyrosyl residues in myosin rod.

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Ferredoxin-sulfite reductase (Fd-SiR) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from spinach leaves has been purified to homogeneity by a new procedure. Subunit analysis by sodium dodecyl sulfate gel electrophoresis yielded a single protein band with a molecular weight of 71, 000. Gel electrophoresis in non-denaturing media at different acrylamide concentrations gave a molecular weight of 270, 000, suggesting that the native enzyme was composed of four identical subunits. In the presence of 0, 2M sodium chloride, however, gel filtration produced a value of 136, 000, indicating the presence of dimer in this ionic environment. A plot of substrate (sulfite) concentration versus enzymatic (Fd-SiR) activity yielded a sigmoidal curve, giving a Hill coefficient (ñ) of 2.1. Purified Fd-SiR, in the oxidized form, had absorption maxima at 279, 385, 588 and 714 nm. Thus the enzyme has the property of a siroheme-containing protein.

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Volatile compounds in solid swine manure were separated by cold-trapping of head-space gas, steam distillation and vacuum distillation, and characterization was performed by gas chromatography-mass spectrometry. Fifty-four components except stearic acid were identified, and the relationships between the components and the separation techniques were studied. It was investigated which components were important for malodor of swine manure, using pattern similarity of gas chromatograms. Carboxylic acids, p-cresol, and skatole were essential components for the malodor. Vacuum distillation was the best technique for separation of the volatile components from solid swine manure, although carboxylic acids and phenols were not recovered completely.

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Callus induction was carried out by tissue culture using leaves of Derris elliptica to obtain rotenoids. A small amount of rotenoids (2.9μg/gDW) was detected in the callus tissue thus induced and subcultured for 4 months. Rotenoid biosynthesis, however, decreased with frequent subcultures of callus tissue, and was finally lost. Then, a callus with imperfectly differentiated rootlets was induced from the leaves or stems of derris by regulating plant hormones. This root-like organ was found to contain rotenoids which were identified as rotenone and deguelin by GC-MS. The content was 160μg/gDW as rotenone.

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Mammary gland chromatin from lactating rats was fractionated according to the Mg²⁺-solubility method after mild digestion with DNase II. The chromatin properties were compared between the Mg²⁺-soluble (S₂) and the Mg²⁺-insoluble (P₂) fractions. The weight ratio of RNA to DNA was much greater in the S₂ fraction than in the P₂ fraction (S₂, 0.92; P₂, 0.03). The DNA repeat length of the nucleosome was very similar between the two fractions (S₂, 193±7; P₂, 196±8 nucleotide pairs). A significant difference was seen in electrophoretic pattern of H1 histone between the two fractions in the acid-urea gel system. Nonhistone proteins with molecular weights higher than about 40, 000 dalton were found to be enriched in the S₂ fraction.

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Yeast mitochondria were immobilized easily and efficiently by entrapment with a photo-crosslinkable resin prepolymer or a urethane prepolymer. The immobilized gel-entrapped organelles showed a relatively high activity of adenylate kinase, converting ADP to ATP and AMP. Immobilization greatly enhanced the stability of mitochondrial adenylate kinase during storage at 4°C or incubation at 30°C. The immobilized mitochondria retained almost the original enzyme activity even after 19 batches of the reaction (total operational period, 380min). These results indicate the utility of the immobilized mitochondria as an immobilized preparation of adenylate kinase.

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Asymmetric hydrolysis of (±)-1-pentyl-2-propynyl and 1-pentyl-2-propenyl acetates by selected microorganisms produced chiral 1-octyn-3-ol and 1-octen-3-ol, respectively, with high optical purities and acetates of their antipodes. Enantioselectivity of microbial hydrolysis changed with the microorganisms used. Also, (±)-1-ethylhexyl acetate was asymmetrically hydrolyzed by microorganisms to give (S)-3-octanol and (R)-1-ethylhexyl acetate of relatively low optical purity and hydrolytic ratio, compared with those of (±)-1-pentyl-2-propynyl acetate.

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A component responsible for the aggregation of cells was extracted from Flavobacterium strain B by treatment of cells with 5M guanidine hydrochloride and partially purified by gel filtration. The guanidine hydrochloride-extracted cells were reaggregated with the component after dialysis against 0.3mM of CaCl₂. Various divalent cations were effective in place of Ca²⁺, but Ca²⁺ was most effective for reconstitution. The reconstituted flocs were deflocculated by the treatment of Pronase or ethylenediaminetetraacetic acid indicating that reconstituted flocs closely resemble natural flocs.

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Crude ammonium sulfate fraction of a cell free extract from Bacillus natto contained an enzyme (or enzymes) which catalyzed the transamidation reaction specific for glutamine. Both L- and D-isomers of glutamine were active as substrate. On incubation of L- Or D-glutamine with the enzyme preparation, two peptides consisting of glutamic acid and glutamine were formed. The main component of the peptides was readily isolated by ion-exchange chromatography and identified as γ-glutamylglutamine by paper chromatography and by paper electrophoresis using authentic peptides. The optical configuration of the amino acid residues in the dipeptide was determined by digestion of the acid hydrolyzate with L-glutamic acid decarboxylase, and the result showed that the dipeptide obtained from L-glutamine was a L-L isomer, while the dipeptide from D-glutamine was a D-D isomer.

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High frequency transfection of Streptomyces parvulus with actinophage R4 DNA was performed by modifying the procedure of protoplast transformation of S. coelicolor A3(2) with SCP2 plasmid DNA [Bibb et al., Nature, 274, 398 (1978)]. Optimum conditions for protoplast transfection included the presence of 16_??_24% (w/v) polyethyleneglycol 4000, and the maximum efficiency of transfection was 3×10^-5 per phage DNA molecule. This value was at least 100 times higher than the efficiency of previously reported transfection systems in Streptomyces.

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Mutants of Klebsiella aerogenes that synthesized tyramine oxidase and arylsulfatase constitutively were isolated. The properties of four of seven constitutive mutants isolated were unstable, segregating spontaneously to the parental type at high frequency. Some of these segregants also lost arylsulfatase (AtsA^-) or tyramine oxidase (TynA^-) spontaneously. These unstable constitutive mutations were shown by genetic analysis to involve mutations of tynP and tynR, and transductants receiving the tynP gene were also unstable. These results showed that the instability was due to unstable tynP gene, which may be the promoter region for tyramine oxidase.

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The subunit structure of phenoloxidase from the larvae of housefly, Musca domestica vicina Maquart, was investigated by urea treatment, SDS-polyacrylamide gel electrophoresis and electron microscopy. Phenoloxidase was dissociated into a single type of subunit (MW 6.5×10⁴) by 5M urea treatment. A similar subunit (MW 6.1×10⁴) was also detected by SDS-polyacrylamide gel electrophoresis. Furthermore, four additional protein bands with molecular weights of 4.8×10⁴, 3.5×l0⁴, 2.4×10⁴ and 1.2×10⁴ were detected by SDS-polyacrylamide gel electrophoresis by extensive incubation of phenoloxidase with SDS. A cylindrical molecular structure was deduced from the electron microscopic analysis. The outside and inside diameter and the height of phenoloxidase molecule were evaluated to be 100, 50 and 70Å, respectively.

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α_s1-Casein was dissolved in 50mM cacodylate-HCl-70mM KCl buffer containing 0.02% of sodium azide (pH 7.1), and the size and shape of α_s1-caseins in the absence and presence of calcium ions were observed with the electron microscope. In the absence of calcium ions, most α_s1-caseins existed as spherical particles of which the smallest diameter was 5_??_6nm. The particles were polymerized into bent chains by adding 3mM calcium. It seemed that the smallest particles were the polymerizing units. The mean length of α_s1-casein in the absence of calcium was 8.7nm, and it increased as the calcium concentration increased. From these results, it was speculated that α_s1-casein in the absence of calcium had one binding site and the calcium-induced conformational changes produced a second binding site. The probability distributions were calculated with the above speculation, and compared with the frequency distributions obtained from electron micrographs. It was suggested from the comparisons that the second binding site produced in α_s1-casein might be responsible for the calcium-induced aggregation.

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K-Casein and α_s1-K-casein complex with a weight ratio of unity were dissolved in 50mM cacodylate-HCl-70mM KCl buffer containing 0.02% of sodium azide (pH 7.1), and their size and shape in the absence and/or presence of calcium ions were observed with the electron microscope. In the absence of calcium ions, both K-casein and α_s1-K-casein complex were spherical particles. However, the mean length of α_s1-K-casein complex (12nm) was smaller than that of K-casein (17nm), which suggested that complex formation led to dissociation of the K-casein polymer. The addition of calcium ions to the complex led to the formation of bent chains, though micelle-like aggregates were not observed even at 20mM calcium. Comparison of the frequency distributions of α_s1-K-casein complex at 0, 5, 10, 15 and 20mM of calcium with the calculated probability distributions suggested that most α_s1-K-casein complexes had two binding sites above 10mM of calcium, which seemed to be essential for the stability of casein micelle.

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An acid-sensitive fraction (ASF) was prepared from defatted soybean meals by two procedures. ASF1 was prepared by precipitation at pH 4.5 followed by removal of 1M NaCl-soluble materials from the precipitate. ASF2 was prepared by precipitation in solution containing 1M NaCl at pH 4.5. The protein components of the two fractions were analyzed by gel electrophoresis in a dissociating-buffer system and found to contain β-conglycinin, glycinin and whey proteins. In addition to these, several other bands appeared.
Appreciable amounts of lipid (8.2% in ASF1 and 8.8% in ASF2) were also found in the fractions. They were separated by column chromatography and thin-layer chromatography. Glycolipids were the major components of the lipids. Both glycolipid and phospholipid fractions contained slower-moving materials on thin-layer chromatography.

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The hypocholesterolemic property of experimental agent 4-O-methylascochlorin (MAC) was examined in male mice. MAC exerted hypocholesterolemic activity without hepatomegaly in mice given MAC mixed in a standard laboratory diet for a week. MAC also significantly reduced plasma total cholesterol (p-TC) in mice when it was administered immediately after meals by gastric intubation in controlled feeding. However, the oral administration through gastric tube was completely ineffective on p-TC in mice with empty stomach. These results suggest a close relationship between hypocholesterolemic efficacy and the timing of diet intake. Hypercholesterolemia also induced by a high fat-cholesterol diet was unaffected by MAC when it was given mixed with the diet.
From the isotopic study, three plausible mechanism were proposed for the mode of action: (I) enhanced output of biliary cholesterol, (II) inhibition of intestinal cholesterol absorption followed by an increment of fecal neutral sterols and (III) modulation of cholesterol partition in the plasma.

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Polyamine oxidase was found in mycelia of fungi belonging to the genera of Aspergillus, Mucor, Penicillium, Rhizopus, Cylindrocarpon, Fusarium and Gibberella when they were grown in medium containing spermine or spermidine as the sole source of nitrogen. The maximal formation of the enzymes of Penicillium chrysogenum and Aspergillus terreus was observed in early stationary phase of growth, and thereafter, the enzymes disappeared with consumption of substrate. The oxidation products of spermine and spermidine by the two enzymes were identified as putrescine, 3-aminopropionaldehyde and H₂O₂. Therefore, the enzymes were characterized as a type of polyamine oxidase of rat liver.

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A strong bitter taste was reported previously in highly autoxidized soybean oil, especially in the free fatty acid fraction. In the present study, for the elucidation of bitter substances in autoxidized oils, autoxidized linoleic acid was fractionated by silicic acid column chromatography, and the fraction with maximum peroxide value was treated with cysteine-ferric trichloride, reduced with sodium borohydride, oxidized with chromic acid and esterified with diazomethane. The results indicated that the bitter substances of autoxidized linoleic acid possessed a particular structure between the carboxyl group and other functional groups. To verify this fact, four oxidized fatty acids [ricinoleic(l2-hydroxyoctadecenoic), 12-oxooctadecenoic, 9, 12-dioxooctadecenoic, and di-hydroxyoctadecenoic acids] were synthesized from castor oil, and their correlation to the bitter taste was investigated. The results showed that ricinoleic acid had a strong bitter taste, along with a strong stimulation, but this bitter taste was greatly reduced by methyl esterification. On the other hand, 12-hydroxy- and 9, 10-dihydroxystearic acids were tasteless. From these results we conclude that the presence of an unsaturated bond, a carboxyl, a hydroxyl and/or peroxyl groups is a requisite for the bitterness of ricinoleic and autoxidized fatty acids.

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