About 2700 strains of various kinds of microorganisms isolated from soil were tested for phospholipase D activity. All of the 200 selected strains were actinomycetes which were divided into two groups on the basis of the isomeric form of diaminopimelic acid (A₂pm) present in the cell wall. Among 18 strains containing meso-A₂pm, two strains were selected on the basis of taxonomical characteristics and productivity of phospholipase D. These two strains were examined for microbiological characteristics and found to belong to the genera Actinomadura and Nocardiopsis.

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Four mannanases (Mannanases I, II, III, and IV) were isolated from the culture filtrate of a Streptomyces sp. by ion exchange chromatography. Mannanase IV was the main component and accounted for 64.4% of the total activity of the four mannanases. Mannanase IV was further purified by gel filtration, and the purified Mannanase IV was homogeneous on disc-gel electrophoretic analysis.
Optimum pH and temperature for the activity of Mannanase IV were 6.8 and 57°C, respectively. It was stable at temperatures up to 45°C when examined at pH 6.8 for 30 min, and lost only 15% of its activity at 70°C for 30min at pH 6.8. The isoelectric point and molecular weight were pH 3.65 and 42, 900, respectively. The enzyme was strongly inactivated by Al³⁺, Hg²⁺, Fe²⁺, Fe³⁺, Cd²⁺, Ag⁺, Sn²⁺, and Cu²⁺, and completely inhibited by iodoacetic acid and N-bromosuccinimide. The enzyme hydrolyzed mannotriose to mannose and mannobiose, but did not hydrolyze mannobiose.

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Changes of the estradiol and lipid concentrations in hen serum, and those in the fatty acids of hen and cockerel livers were studied during the maturing period. The concentrations of estradiol and lipid in serum reached a maximum level a few days before the onset of laying, and then fell. A marked increase in oleate, and decreases in stearate and arachidonate were observed in both the liver and serum lipids of the hen when reaching laying. The fatty acid compositions of the serum and liver of the hen at 145 and 167 days of age were close to those of yolk lipid. In the cockerel, high contents of stearate and arachidonate and a low content of oleate were observed, and the fatty acid composition did not change appreciably with age. On estrogenization of the male chick, the fatty acid cmposition of the liver lipid was similar to that of the laying hen liver. The ratio of the unsaturated fatty acid/saturated fatty acid in the liver of hen increased when reaching laying, the value at 167 days old being close to that of yolk lipid. This ratio in the liver lipid of cockerel did not change significantly throughout the experimental period.

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In the presence of citrate, citrate-fermenting Streptococcus sp. KSM-1106 and KSM-11 12 grew in long chains with innumerable cells. Divalent cations, such as Mn²⁺ and Ca²⁺, arrested the chain elongation. The results with citrate-negative and citrate-resistant variants suggested the involvement of citrate in the cell separation system. A dechaining activity, which was inhibited by citrate, could be demonstrated in the cell extract of strain KSM-1106.Such a special effect of citrate was not observed on the growth of strains of other lactic streptococci examined.

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The combination of flow injection analysis and the electrochemical method was investigated for measuring the organic acid content of citrus fruits. The controlled-potential four-electrode method was used for conductance measurements. The instrumentation of the measuring apparatus used in this study is defined. The optimum conditions of the flow system were as follows : injection volume 35μl, mixing coil length 100cm, flow rate 20ml/min and dilution ratio of samples 1 : 130. A linear correlation between the peak conductivity and the organic acid content was obtained with high correlation coefficient (r=0.992). It is possible to analyze about 50 samples within an hour using the method, and the coefficient of variation with any given juice for 20 assays was less than 0.5%.

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Physicochemical and functional properties of myosin from red (red-myosin) and white (white-myosin) muscles of broilers, grown at different planes of nutrition, were studied. The red- and white-myosin from well-nourished broilers exhibited characteristic difference in certain physicochemical properties. The specific viscosity of red-myosin was lower and sedimentation coefficient (s_{20, w}) was higher than that of white-myosin. The transition of monomer⇔filament phase of white-myosin seemed to occur in a narrower range of ionic strength than that of red-myosin. Under identical conditions (ionic strength, pH, protein concentration, temperature) of the model system, white-myosin always exhibited greater gel strength than that of red-myosin. Characteristic differences were also observed in the three-dimensional structure of red- and white-myosin gel.
Heat-induced gel strength of red-myosin from under-fed broilers was about 50% less than that of control, whereas the gelling property of white-myosin was not influenced by the nutritional states. Myosin from under-fed chicken had low Ca²⁺-activated ATPase activity, although EDTA-activated ATPase, specific viscosity, and helicity of myosin were not different between the nutritional states.

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Some physico-chemical and heat-induced gelling properties of myosin heavy chains (MHCs) from rabbit skeletal muscle were studied. MHCs were found to be almost devoid of ATPase activity, possibly because of the absence of myosin light chains. MHCs formed precipitates below and ionic strength of 0.3, and above this ionic strength MHCs became soluble. On heating to 65°C at low concentrations of the protein, MHCs lost their solubility and formed aggregates even at high ionic conditions. Partially irreversible change in the conformation of MHC (from helical to random coil) also occurred in heated MHCs.
The heat-induced gel strength of MHCs was found to be almost equal to that of intact myosin molecules with ide tical protein concentrations. This suggests that the gelling potential of myosin is solely confined in MHC. Although myosin light chains do not seem to contribute to the gelling power of myosin, possibly they provide some stability to the gel if the pH is varied above the optimum value, i.e., 6.0. The addition of actin to a MHC system weakened the gel strength of the latter proportionally to the quantity of added actin.

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The stability of recombinant strains of Escherichia coli carrying hybrid plasmids comprised of pBR322 and the threonine operon (thrA, thrB and thrC) of E. coli was examined. (1) Recombinant strains were classified into two types according to the direction of the an inserted DNA fragment containing the threonine operon, which was cloned into the HindIII site of pBR322. Type I had a more stable pBR322-thr hybrid plasmid, and Type II had a less stable one and easily lost it. L- Threonine productivity of Type I strains was much higher than that of Type II, presumably depending on the stability of each plasmid. (2) Repeated transfer of the host cells carrying pAJ294, a pBR322-thr hybrid plasmid obtained from a Type I strain, without any selective pressure caused a serious loss of the plasmid. Culturing at high temperature or in a poor medium accelerated the loss, and it was prevented by the addition of ampicillin or tetracycline to the culture medium. (3) pAJ294 was stable under some storage conditions, stocking on an agar medium or after lyophilization. (4) pAJ294 was fairly stable in L-threonine production medium at 30°C during growth of up to approximately 17 generations without any antibiotic.

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Protoplast fusion was carried out between a sake brewer's yeast strain, Saccharomyces cerevisiae Kyokai 7, and a lactose utilizing yeast strain, Kluyveromyces lactis T396. A stable hybrid, PN 13, which was selected from the many resultant fusants, showed physiologically complemented traits with respect to sugar utilization, vitamin requirements and so on. Biochemical investigations also revealed that fusant PN 13 was an intermediate hybrid between the parental strains. In glucose and lactose media, moreover, the fusant grew and produced ethanol at higher rates than K. lactis T396.

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Cigarette flavor ingredients adsorbed on activated carbon were recovered by an organic solvent extraction method and a stem desorption method. Recovery by dichloromethane extraction showed the maximum effect and many volatile components, especially the thermal degradation products from sugar analogues, were recovered in a good yield. Their recovered yield and desorption rate depended on the physical properties of the carbons. Most of the volatile components were also recovered by the steam desorption method, but the recovery of components with high boiling points was very low by steam desorption. The difference in recovered yield from the carbons is explained by the specific surface area of the carbons measured before and after the desorption procedure.

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A microorganism, which produced a potently bacteriolytic endopeptidase, was isolated from soil and classified taxonomically as Cytophaga sp. B-30. This enzyme was purified 740-fold from the culture broth by fractionations with ammonium sulfate and acetone, column chromatographies on CM-cellulose and hydroxyapatite twice, and gel filtration on Sephadex G-75. It was found to be homogeneous on PAGE and SDS-PAGE. The molecular weight and isoelectric point of this enzyme were estimated to be 9, 000 daltons and pH 9.5, respectively, and the optimal pH for its activity was 9.5. The enzyme acivity was completely inhibited by Mn⁺⁺, Zn⁺⁺, Cu⁺⁺, Hg⁺⁺, 2- mercaptoethanol and 2, 3-dimercapto-1-propanol but markedly stimulated by EDTA, potassium oxalete and sodium pyrophosphate at the concentration of 1mM. This enzyme catalyzed both cell wall lysis and proteolysis. A polysaccharide peptide of long chain length was isolated from a digest of Staphylococcus epidermidis peptidoglycan with this enzyme.

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A water-insoluble glucan was synthesized by the β-transglycosylase of Trichoderma longibrachiatum in a reasonable yield from 1% cellopentaose as the substrate after 48hr incubation. The glucan could be completely solubilized by cellulase to give glucose, cellobiose and cellotriose, although the reaction time was about 9 times longer than that required for the complete solubilization of the higher cellodextrin (DP 14) synthesized by the β-transglucosylase of Sclerotinia libertiana. The glucan was separated into two main fractions according to the solubility in NaOH solution, the major soluble in 4N-NaOH and the minor insoluble in it. The structure of the major fraction was investigated by methylation analysis, and it was clearly shown to be a linear β-1, 4- glucan having an average degree of polymerization of 19. These results indicated that the β- transglycosylase of Trichoderma longibrachiatum had a strict specificity of forming β- 1, 4-glucosidic linkages and also had a capacity to elongate the linkage to a higher extent.

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Acidic non-protein amino acids and peptides in five species belonging to the family Liliaceae -Smilax china, Lilium maximowiczii, Tulipa gesneriana, Allium cepa and A. sativum- were surveyed. Two new amino acids -4-hydroxymethyl- and 4-ethyl-4-hydroxyglutamic acid- were isolated from bulbs of T. gesneriana. The first unequivocal identification of 4-ethylglutamic acid was performed from the same plant tissue. A new dipeptide -S-(2-carboxy-n-propyl)cysteinyl glycine- was isolated from A. cepa. Besides the compounds described above, 4-methylene-, 4- methyl- and 4-hydroxy-4-methylglutamic acid were isolated from S. china, L. maximowiczii, and T. gesneriana, but 4-ethylideneglutamic acid was isolated only from T. gesneriana. 4- Methyleneglutamic acid showed no appreciable biological activity, although this amino acid has an α, β-unsaturated carboxyl group and reacts easily with cysteine in vitro. The most abundant free amino acid in the plant tissues examined in this study is arginine ; nevertheless, acidic N^α- acylarginine derivatives which have been found in S. china and L. maximowiczii and published elsewhere could not be detected in bulbs of T. gesneriana, A. cepa, and A. sativum.

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Four neutral sesquiterpenes were isolated from the culture filtrate of of Aspergillus terreus THOM No. 14 which produces two sesquiterpene antibiotics, terrecyclic acid A and terrecyclol. On the basis of spectroscopic data the structures of four neutral sesquiterpenes were elucidated. One of these is quadrone, a known antitumor substance, but isoquadrone, 8-hydroxyquadrone and 6- hydroxyisoquadrone are new sesquiterpenes. Although terrecyclic acid A exhibits antimicrobial and antitumor activities, these four scarcely show these activities.

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The ability to reduce ketopantoyl lactone added to the culture medium to pantoyl lactone was surveyed in a variety of microorganisms. Many of the microorganisms including molds, yeasts, bacteria, actinomycetes and basidiomycetes exhibited this ability. The ratios of D-(-)- and L-(+)- isomers of the yielded pantoyl lactone, however, showed no relation to the genera or sources of strains. Among them, Rhodotorula minuta IFO 0920, Candida parapsilosis IFO 0708 and Aspergillus niger IFO 4415 were found to convert ketopantoyl lactone (45 mg/ml) completely and almost specifically to D-(-)-pantoyl lactone. The main enzyme catalyzing this asymmetric reduction was suggested to be ketopantoyl lactone reductase (EC 1.1.1.168).

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In survey studies on microbial accumulation of S-adenosyl-L-methionine (AdoMet), various yeasts were found to accumulate AdoMet intracellularly to a high concentration when they were grown in medium containing L-methionine. A group of sake yeasts (Saccharomyces sake) exhibited especially high accumulation. Of these yeasts, S. sake Kyokai No. 6 (K-6), which exhibited the highest accumulation, produced 12.6 μmol (5.03 mg) of AdoMet/ml broth. Almost all AdoMet produced was accumulated in cells, extracellular accumulation of AdoMet being very low. The maximum content of AdoMet of cells was 5.31 μmol (205 mg)/g dry cells. This was the highest value that had been reported. Methionine adenosyltransferase (EC 2.5.1.6) activity was significantly higher in this yeast compared to those in other yeasts tested. Ultraviolet photomicrographic studies on S. sake K-6 suggested that AdoMet was gradually accumulated in vacuoles with the passage of cultivation time.

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The volatile components of roasted and unroasted dried coconut shreds, isolated by steam distillation, were analyzed by GC and GC-MS. Seventeen compounds were identified from the unroasted coconut, and nine of them were newly identified in coconut meat aroma. Saturated delta- C₈, C₁₀, and C₁₂ lactones were determined as the main components giving the characteristic mild, sweet, and pleasant coconut flavor. The roasted coconut gave the strong characteristic sweet and nutty aroma, and the GC-MS indicated the saturated delta-lactones as main components, and six pyrazines, two furans, and two pyrroles also found seemed to contribute greatly to the nut-like aroma of roasted coconut. The defatted and roasted meal gave a strong nut-like and burnt odor, but not the characteristic sweet aroma. A large increase in pyrazines and other Maillard reaction products and an absence of lactones and fatty acid esters were observed in the volatiles of the roasted-defatted coconut meal.

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When Rhodococcus erythropolis AN-13 grew on aniline, a fluorescent substance accumulated in the cultural fluid. It was obtained as crystals and identified as anthranilic acid (AnA). AnA was also produced from aniline following incubation with resting cells of the bacterium grown on aniline. Heated cells lost the activity to produce it, and aniline was essential for its production. The production of AnA was promoted by sodium bicarbonate ; when [¹⁴C] sodium bicarbonate was added to the incubation mixture, [¹⁴C] AnA was formed. The optimal pH for AnA production by the resting cells was 7.0 to 7.5. These results suggest that microbial activities of R. erythropolis AN-13 catalyzed the formation of AnA from aniline.

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Optically active trans-cycloheximide isomers such as cycloheximide [(2S, 4S, 6R, αR)-form (1)], naramycin B[(2S, 4S, 6R, αR)-form (4)], and new stereoisomers (2S, 4S, 6S, αS)-form (8) and (2S, 4S, 6R, αS)-form (9) were synthesized by an aldol condensation of trans-2, 4-dimethyl-1-cyclohexanone (5b), with 4-(2-oxoethyl)-2, 6-piperidinedione (6). The antimicrobial activity of trans-cycloheximide isomers (1, 4, 8, and 9) was examined against S. cerevisiae and P. oryzae. The stereoisomers 1 and 4 exhibited marked antimicrobial activity against both microorganisms as compared with their C-α-epimers 8 and 9.

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A stereoisomeric mixture of (±)-8, 16, 26, 34-tetrahydroxy-6, 10, 14, 18, 24, 28, 32, 36-octaoxohentetracontane (C₄₁) was synthesized together with (±)-8, 16-dihydroxy-6, 10, 14, 18-tetraoxotricosane (C₂₃), utilizing bis addition of pentyl-1, 5-dimagnesiumbromide to two moles of a C₁₈-aldehyde.

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The enzymatic hydrolysis of (R, S)-5-acyloxymethyl-3-alkyl-oxazolidin-2-one I and the behavior of (S)-I for extraction with an organic solvent were examined so as to extend the biological resolution to racemates, and to learn about more appropriate combinations of substrates with lipases on the asymmetric hydrolysis. The combination of (R, S)-5-hexanoyloxymethyl-3-tert- butyl-oxazolidin-2-one 4 with lipoprotein lipase Amano 3 (L. P. L. Amano3, origin ; Pseudomonas aeruginosa) and that of (R, S)-5-octanoyloxymethyl-3-isopropyl-oxazolidin-2-one 14 with L. P. L. Amano 3 efficiently gave (S)-5-hydroxymethyl-3-tert-butyl-oxazolidin-2-one (S)-11a (99% e.e.) and (S)-5-hydroxymethyl-3-isopropyl-oxazolidin-2-one (S)-IIb (99% e.e.), respectively. (S)-IIa and (S)-IIb could be considered to be favorable intermediates for preparing optically active β-blockers.

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In this paper, lipoxygenase lacking mutants were characterized in comparison with normal soybeans. The three lipoxygenase isozymes (L-1, L-2, and L-3) in crude seed extracts of normal soybeans were resolved clearly by an improved SDS-polyacrylamide gel electrophoresis. As expected, the three mutant types, L-1-less (P. I. 408251 and 133226), L-2-less (P. I. 86023), and L-3 less (Wasenatsu and Ichigowase) soybeans did not give L-1, L-2, and L-3 protein bands, respectively on a single dimension SDS gel.
An anti L-2 serum obtained from a rabbit reacted not only with the purified L-2 protein, but also partially with the purified L-1 and L-3 proteins. By double immunodiffusion and immuno-disc gel electrofocusing analyses using the anti L-2 serum, L-1, L-2, and L-3 isozymes could not be detected in crude seed extracts from P. I. 408251, P. I. 86023, and Wasenatsu soybeans, respectively.
Three lipoxygenase activity peaks (L-1, L-2, and L-3 enzyme peaks) and a small unknown activity peak eluted right after the L-1 peak were fractionated by DEAE-Sephacel column chromatography of crude seed extracts of Raiden (normal) soybeans. The chromatographic analyses have demonstrated that both the L-1 and the unknown enzyme activities disappear completely in the L-1-less type soybean seeds, and that the L-2 and L-3 enzyme activities disappear completely in P. I. 86023 and the L-3-less type soybean seeds, respectively.

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Transglutaminase catalyzes an acyl-transfer reaction in which the γ-carboxyamide groups of peptide-bound glutaminyl residues are the acyl donors. Primary amino groups in a variety of compounds act as acyl acceptors with the subsequent formation of the monosubstituted γ-amide of the peptide-bound glutamic acid. When one wants to bind a desired amino compound into glutamine residues in a protein using this enzyme, concurrent generation of intra- and intermolecular ε-(γ-glutamyl)lysine crosslinks of the protein lowers the binding efficiency because the reactive glutamine residues are consumed by the crosslinking. Maleylation of amino groups in proteins was attempted with maleic anhydride in order to suppress this unfavorable crosslink formation. Maleylation of caseins, mainly α_s1-casein, increased the rate and the extent of transglutaminase-catalyzed incorporation of amino compounds such as putrescine, methionine ethyl ester, and NAD⁺ analog into the modified proteins. Generation of intermolecular crosslinks during the incorporation reactions was completely repressed by maleylation. Bovine serum albumin and ovalbumin are non-reactive for transglutaminase, i.e. both lysine and glutamine residues in these proteins are unavailable to this enzyme. Maleylation of both proteins induced a conformational change by which glutamine residues were exposed to be available as acceptors in the transglutaminase-catalyzed incorporation of amino compounds. Maleyl residues in proteins can be readily removed by treating the modified proteins at acidic pH. Thus maleylation improves substrate proteins for preparing a specified product with a high yield using transglutaminase.

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Several oxidizing agents were examined for their ability to demethylthiolate adenosine- and cytidine 5'-S-methyl phosphorothiolates.
Iodine dissolved in an aqueous potassium iodide solution or in dimethyl sulfoxide (DMSO) was the most effective demethylthiolating agent of those tested in the present study, rapidly giving the demethylthiolated products in quantitative yields. The iodine-DMSO solution demethylthiolated the ribonucleoside 5'-S-methyl phosphorothiolates to give ribonucleoside 5'-monophosphates even under anhydrous conditions, DMSO acting as an oxygen donor in this reaction.
Hydrogen peroxide has high demethylthiolating ability in spite of its low reaction rate. Isoamyl nitrite, an effective demethylthiolating agent for O-alkyl S-methyl phosphorothiolates, was not effective for the demethylthiolation of ribonucleoside 5'-S-methyl phosphorothiolates, because the unprotected amino groups of the S-methyl nucleotides were attacked by the reagent to give deaminated products. N-Chlorosuccinimide had no effect on the demethylthiolation of S-methyl phosphorothiolates.

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Gibberellins A₄ and A₃₆ were identified from flowering and vegetative apices of ten month-old sugarcane
(Saccharum spp. hybrids) plants. The identifications were based on retention times, relative to authentic standards, on sequential silica gel partition column chromatography→ bioassay→C₁₈ reverse phase high performance liquid chromatography→-bioassay→capillary gas chromatography (GC), and on GC-selected ion monitoring
(SIM), the relative intensities of six characteristic ions being monitored in comparison with authentic standards.

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