Saccharomycopsis lipolytica developed mycelial cells in media containing both olive oil and bovine milk casein. Olive oil could be replaced by other lipids including triolein, oleic acid, linoleic acid and oleyl alcohol. On the other hand, bovine milk casein could be replaced by a soybean fraction and meat extract, but not by casamino acids or individual commonamino acids. The mycelial development was inhibited with a deficiency of magnesium sulfate and ferric chloride or with the addition of cysteine and reduced glutathione.
The mycelial development began after 8 hr from the start of cultivation and the mycelial cell ratio was maximumafter 20 hr. Mycelial cells and yeast-form ones were separated from each other on the basis of cellular specific gravity and this method was used to determine the mycelial cell ratio in the present study.

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Galactomyces reessii L, isolated as a protopectin-solubilizing enzyme-producing strain, produced protopectin-solubilizing enzyme in the culture filtrate. The enzyme was purified by repeated CM-Sephadex C-50 column chromatography, and isolated as a crystalline form with a yield of 16% of the initial activity. The enzyme was a glycoprotein containing about 2.6% carbohydrate (as pentose). Its isoelectric point was around pH 8.4, and the sedimentation coefficient (s_{20, w}) was determined to be 3.83 S. The molecular weight was determined to be 30, 000 by gel filtration on Sephadex G-75 and 29, 300 by ultracentrifugal analysis. The enzyme catalyzed the release of highly polymerized pectin from various protopectins. The enzyme also catalyzed the depolymerization of pectic acid or galacturonic acid oligomers, and was confirmed to be an endopolygalacturonase.

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The production of pectolytic enzyme in the genus Kluyveromyces was investigated. The production of the enzymewas dependent on the strain, and some strains belonging to K. fragilis, K. marxianus, and K. wickerhamii produced this enzyme among 11 species (29 strains) of the genus Kluyveromyces. K. fragilis IFO 0288 produced at least four endo-polygalacturonases which have different molecular weights. The dominant endo-polygalacturonase in the culture filtrate of the strain was purified and isolated as crystals. The purified enzyme was homogeneous based on analysis by polyacrylamide gel electrophoresis and ultracentrifugation. The enzyme was a glycoprotein having an isoelectric point around pH 5.6. The sedimentation coefficient (S_{20, w}) was 3.77S, and the molecular weight was around 33, 000. The enzyme contained aspartic acid (asparagine), serine, threonine, and glycine at relatively high levels. The enzyme showed the highest activity around pH 5.0 and was stable at pH 5.0 up to 30°C. With the enzyme, and activity which releases highly polymerized pectin from various protopectins (protopectinase activity) was found.

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(-)-Epicatechin-3-gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG), major tea catechins, formed precipitates with soybean lipoxygenase (LOX) in the pH range of 4-7, although with accompanying 10-30%loss of the LOXactivity. Yeast alcohol dehydrogenase also was precipitated by EGCG. Polyvinylpyrrolidone, Tween 20 and Triton X-100 dissociated the LOX activity from the EGCG-precipitated LOX. However, the MWof the dissociated LOX(1 14, 000) differed from that of the native LOX(100, 000). Enzymeactivities of the EGCG-precipitated LOX and the dissociated LOXfrom the precipitate were less stable than the activity of the native LOX. These findings suggest the altered natures of proteins in the presence of tea catechins, ECG and EGCG.

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Glucose oxidase was immobilized on the surface of a graphite electrode by irreversible adsorption. Anelectrocatalytic steady-state current for the oxidation of D-glucose was observed using this electrode in the presence of p-benzoquinone as an electron transfer mediator. The electrocatalytic current at 0.5 V vs. SCEwas analyzed as a function of the concentrations of dglucose and p-benzoquinone, and the maximum current, I^max_s, and the Michaelis constants (K₁ and K₂ for D-glucose and p-benzoquinone, respectively) of the electrocatalysis were determined. The dependence of the current on the electrode potential, pH, and temperature was also investigated. The results indicate that the kinetics of the immobilized enzyme are essentially the same as those of the enzyme in the solubilized state. The effect of various electron transfer mediators on the electrocatalytic current was also examined and evaluated in terms of I^max_s, K₁, and K₂ values.

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An immobilized chloroplast film, prepared by immobilizing spinach chloroplasts in 2 wt% agar gel, was attached to a SnO₂ optically transparent electrode to obtain the immobilized chloroplast film electrode. The immobilized chloroplast film electrode worked as a photoanode under illumination in the presence of methyl viologen, which was an electron carrier from chloroplasts to the SnO₂ optically transparent electrode. Water photolysis for producing hydrogen by a photoelectrochemical cell using the immobilized chloroplasts film electrode was successfully achieved. A smooth platinum electrode was used as a cathode to produce hydrogen. The pH and temperature of the anolyte were kept at 7.8 and 25°C. Optimizations of the concentrations of methyl viologen and chlorophyll in the immobilized chloroplast film were studied. The optimumthickness for the immobilized chloroplast film was about 0.8 mm.The immobilized chloroplasts had higher storage stability than that of isolated chloroplasts and they retained more than 50% of the initial activities of photosystem I and photosystem II after 10 days when they were stored at 4°C in the dark. It was conceived from the relationship between the photocurrent and the photosystem I and II activities that the main cause for the decrease in the photocurrent was the photochemical inactivation of photosystem II.

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We have constructed phage cloning vectors from an actinophage, R4. A deletion derivative (R4 22B) which had a BamHllinker inserted at the unique PvuII site was used to clone the thiostreptone resistant (tsr) gene derived from plasmid vector pIJ365. The tsr derivative obtained, R422B-tsr1, was shown to have the same level of thiostreptone resistance in lysogenized cells as that of pIJ365-carrying cells. Under the optimal conditions, R422B-tsrl phage was lysogenized at a frequency of 5×10^-2 per infected phage. The usefulness of R4 phage derivatives for gene cloning is discussed.

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L-Methionine y-lyase (EC 4.4. 1. 1 1) catalyzes α, γ-elimination of O-substituted L-homoserines (i.e., ROCH₂CH₂CH(NH₂)COOH; R = acetyl, succinyl, or ethyl) to produce a-ketobutyrate, ammonia, and the corresponding carboxylate or alcohol, and also their γ-replacement reactions with various thiols to produce the corresponding 5-substituted L-homocysteines. The reactivities of Osubstituted L-homoserines in α, γ-elimination relative to that of L-methionine were as follows: Oacetyl, 140%; Osuccinyl, 17%; and O-ethyl-L-homoserine, 99%. However, the enzyme does not catalyze the synthesis of O-substituted L-homoserines from alcohol or carboxylic acids in a γ-replacement reaction. Wehave analyzed the α, γ-elimination of O-acetyl-L-homoserine in deuterium oxide by ¹H-NMR. The [β-²H, γ-²H]-species of α-ketobutyrate was exclusively formed from 0-acetyl-L-homoserine. The enzyme catalyzes deamination of L-vinylglycine to give the identically labeled α-ketobutyrate species. Incubation of the enzymewith O-acetyl-L-homoserine resulted in the appearance of a new absorption band at 480 nm, which was observed also with L-vinylglycine. These results strongly suggest that the α, γ-elimination and γ-replacement reactions of O-acetyl-Lhomoserine proceed through the stabilized α-carbanion of a SchifF base between pyridoxal 5'-phosphate and vinylglycine, which has been suggested as the key intermediate of L-methionine γ-lyase-caralyzed reactions of S-substituted L-homocysteines [N. Esaki, T. Suzuki, H. Tanaka, K. Soda and R. R. Rando, FEBS Lett., 84, 309 (1977).

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To investigate the characters of Agrobacterium sp. KY-8589(an ethionine-resistant mutant) showing excellent potential for coenzyme Q₁₀ (CoQ₁₀) production, two kinds of agar media were used for colony isolation. Marked differences in the form and color of colonies were seen between the agar media of glucose-yeast-bouillon (A) and glucose-yeast (B). Especially, the rough-white (Rw) and smooth-light yellow (Sy) types were isolated on agar mediumB. The effects of the two types purified morphologically on CoQ₁₀ fermentation were further investigated. Although both types have the same ethionine-resistance, significant differences in growth, broth viscosity and CoQ₁₀ production were found between them. The amount of CoQ₁₀ accumulated in the broth by Rwshowed an increment of 25%compared with that by Sy. In this case, the intracellular contents of CoQ₁₀ for Rw and Sy were 4.1 and 2.8mg/g-cell, respectively. In addition, the reduced production with repeated use of KY-8589was shown to be due to the decrease in Rw/Syratio, which was caused by the rapid growth of Sy.

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The amounts of carboxylic acids released from electron beam irradiated polyethylene film, which can be indicators of the intensity of off-odor, were examined. The conditions for trapping acetic acid, propionic acid, n-butyric acid, and n-valeric acid from two grams of film irradiated with a dose of 20 kGy in air were established as follows; carboxylic acids volatilized at 80°C from irradiated film were concentrated on Tenax GCby passing nitrogen gas at a flow rate of 20 ml/min for 30 minutes. The amounts of carboxylic acids varied considerably depending upon the properties of the resin, the presence of additives, etc. Without additives, the total amounts of the acids from the film which gave the strongest off-odor was three times the total amounts of the acids from the film which gave the weakest off-odor. The addition of butylated hydroxytoluene to film reduced remarkably the formation of carboxylic acids.

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The effects of conditions for electron beam irradiation on the amounts of volatiles from irradiated polyethylene film were studied. The concentration of oxygen up to 5%in the atmosphere affected the amounts of carbonyl compounds, and their amounts decreased remarkably with a decrease in the concentration of oxygen. A low irradiation temperature of -75°C, a radiation energy of 1.5 MeVor lower, and a higher beamcurrent were effective in reducing the amounts of volatiles formed by irradiation. The effect of conveyor speed was not so significant. It has also been found that electron beam irradiation is better in restraining the formation of volatiles than γ-irradiation.

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Two forms of formaldehyde dismutase distinguishable on disc-gel electrophoresis were isolated from the cell-free extract of Pseudomonas putida F61. The mobilities on SDS-gel electrophoresis and the NH₂-terminal amino acids (arginine) of the two enzyme species were identical. The COOHterminal amino acid sequence was found to be -Ser-Gly-Lys. The enzyme was inhibited by carbonyl, reducing and sulfhydryl reagents.
The enzyme catalyzed the cross-dismutation reaction between formaldehyde and an aldehyde, such as propionaldehyde, acrolein, butyraldehyde, isobutyraldehyde and crotonaldehyde. The enzyme also catalyzed a coupled oxidoreduction between an alcohol and an aldehyde (RCH₂OH+ R'CHORCHO+R'CH₂OH) without addition of an electron acceptor. Aliphatic alcohols and aldehydes of C₂ to C₄ were utilized in this reaction.

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Soluble and insoluble portions obtained after chymosin treatment of purified humanK>casein were isolated by CM-Sephadexcation exchange chromatography. The soluble portion included all sugars present in intact human κ-casein and the insoluble portion was devoid of sugars. They were designated as human glycomacropeptide and para-κ-casein following the example of bovine κ-casein. The results of amino acid analyses showed that humanglycomacropeptide contained a large amount of acidic amino acids and a small amount of basic amino acids (His and Arg were not contained). Onthe other hand, para-κ-casein contained both acidic and basic amino acids. Human para-κ-casein was positively charged at pH 8.6 and migrated toward the cathode on polyacrylamide gel electrophoresis. Kinetic parameters (Km) for the chymosin action on human and bovine κ-caseinswere 10.6×10^-5M and 6.6×10^-5M, and thoseforthepepsinactionwere 5.2×10^-5M and 4.0×10^-5M, respectively.

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To clarify the mechanism of the cellular DNA-breaking reaction of epinephrine (Ep), we examined the interaction between Ep and chromatin components. The Ep-binding activity of histone increased after the dissociation of histone subunits. The Ep bound to DNAincreased with the increase of Ep concentration and pH. Solubilized chromatin, ν bodies, showed Ep-oxidizing activity in the absence of Cu²⁺. The binding of Ep to ν bodies occurred immediately after mixing and was highly specific. These data suggest the presence of some Ep-specific binding protein(s) in chromatin which oxidizes Ep and induces DNA breakage.

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Various bacteria which can grow on biotin as a sole carbon source were isolated from soil samples. These bacteria were classified into three groups according to the biotin-degrading pattern. Cell-free extracts from all these bacteria grown on biotin possessed high biotinyl-CoA synthetase activities. Cultural conditions for strain No. 166, the highest biotinyl-CoA synthetase producer, were examined. Biotinyl-CoA synthetase was induced in the presence of biotin, but not in the presence of any other carboxylic acids or biotin intermediates. The addition of lactose and yeast extract to the mediumenhanced both the growth and the enzyme activity. The enzyme reaction for biotinyl-CoA synthesis required ATP, CoA and Mg²⁺ as well as biotin. From taxonomical studies, bacterium No. 166 was identified as a strain of Mycoplana.

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Temperature-sensitive sporulation mutants were isolated spontaneously from Bacillus subtilis 168 TT by a sequential transfer method. A representative mutant strain, ts32, was characterized in detail. The mutant grew normally at 30°C and 42°C, but did not sporulate at 42°C. Electron microscopic observation and physiological analysis showed that the mutant was blocked at stage 0-1 ofsporulation. Genetic analysis suggested that the mutation was located at the spo0B locus on the B. subtilis chromosome. Temperature-shift experiments clearly showed that the spo0B gene product functions only at the beginning of sporulation.

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Enzymes and microorganisms were screened for the asymmetric hydrolysis of (R, S)-5-acetoxymethyl-3-tert-butyl-oxazolidin-2-one 1. Lipases from Pseudomonas aeruginosa and Alcaligenes species, and microorganisms which belong to Enterobacter species or Klebsiella species were found to hydrolyze 1 enantioselectively to give (R)-5-hydroxymethyl-3-tert-butyl-oxazolidin-2-one (R)-2 and (S)-1. (S)-2, one of the typical intermediates for preparing optically active β-blocking agents (β-blockers), was obtained with high enantiomeric excess (91- 98% e.e.) from (S)-1.

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(S)-(+)-Gregatin B and racemic gregatin B were synthesized from tetrahydro-2-methyl-5-oxo-2-furancarboxylic acid and the absolute configuration of natural gregatin B was determined as (S).

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Using cold resistant mutants, MET1 and MET2, obtained from Escherichia coli K-12, genetic mapping of the cold resistance gene(s) of E. coli was performed by the conjugation and transduction techniques. The gene(s) was confirmed to be located close to trpB at 28 min (revised chromosome linkage map, 1983) on the E. coli chromosome.

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Sulfaguanidine-resistant mutant S-225, derived from a tryptophan-producing 5-fluorotryptophan-resistant mutant of Brevibacterium flavum, accumulated 19 g/liter of L-tryptophan at maximumwhencultured for 72hr in a mediumcontaining 13%glucose as carbon source, 1.8-fold higher than did the parent. Strain S-225 accumulated 17 g/liter of L-tryptophan in a medium containing 10% sucrose as carbon source (17% yield based on the sugar). It also accumulated 450mg/liter of chorismate, an intermediate commonto the biosyntheses of tryptophan and p-aminobenzoate. The accumulation was 1.7-fold higher than that by the parent, suggesting that the intracellular concentration of chorismate was increased through acquisition of the sulfaguanidine resistance. Sulfaguanidine-resistant mutants were also derived from a tryptophanproducing mutant of Corynebacterium glutamicum. The mutants showed 2.2-fold higher maximumtryptophan production than did the parent.

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Two new antioxidative compounds, named epirosmanol (4a) and isorosmanol (6a), were isolated from the leaves of rosemary (Rosmarinus officinalis L.). The structures have been determined to be 7β, 11, 12-trihydroxy-6, 10-(epoxymethano)abieta-8, 11, 13-trien-20-one and 6α, 11, 12-trihydroxy-7, 10-(epoxymethano)abieta-8, 11, 13-trien-20-one, respectively, on the basis of chemical and spectroscopic evidence. Both are isomers of rosmanol (1a), whose structure was revised to be 7α, 11, 12-trihydroxy-6, 10-(epoxymethano)abieta-8, 11, 13-trien-20-one by the NOE experiment and X-ray analysis.
These two antioxidants showed high activity in both lard and linoleic acid and, particularly in lard, were about four times more active than such synthetic antioxidants, as BHA and BHT.

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Catechol 1, 2-dioxygenase (EC 1.13.11.1) was purified to homogeneity from a cell-free extract of aniline-grown Rhodococcus erythropolis AN-13. The purified enzyme was stable between pH 7.0 and 10.5 and up to 40°C. The optimal pH was 7.5 for catechol as a substrate. The molecular weight of the enzyme was 37, 000 by gel filtration and 36, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 3mol of sulfhydryl groups per mol of protein, but not disulfide bonds of cystine residues. The enzymatic activity was inhibited by AgNO₃, HgCl₂, iodoacetic acid and p-chloromercuribenzoic acid. FeSO₄ and FeCl₃ also inhibited the activity, although the enzyme contained 1.3g atoms of iron per mol of protein. This enzyme catalyzed the intradiol cleavage of 3- and 4-methylcatechol, but not the extradiol cleavage of 3-methylcatechol.

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When Frateuria species ANA-18was grown on aniline, two catechol 1, 2-dioxygenases (CD I and CDII, EC 1.13.11.1) were found in a cell-free extract of the strain. CDI and CDII were separated from each other by DE-52 chromatography and purified to homogeneity by successive column chromatography. The molecular weights of CDI and CD II were 38, 000 and 36, 000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CD I contained 2 mol of sulfhydryl groups per mol of protein, but CDII did not have cysteine or cystine residues. CDI was more stable on the acidic side and against heating than CDII. The activity of CDI was inhibited by AgNO₃, HgCl₂, iodoacetic acid and p-chloromercuribenzoic acid, but that of CDII was not inhibited or was less affected by them because of the lack of sulfhydryl groups. CD I exhibited a high activity for the extradiol cleavage of 3-methylcatechol, and the ratio of intradiol to extradiol activity was 100:120.

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Protocatechuate 3, 4-dioxygenase was isolated from a gram-positive bacterium, Nocardia erythropolis, the enzyme participates in the phthalate ester metabolism in the bacterium. Cultural conditions for production of the enzyme, the purification procedure, and some properties of the enzyme were studied. A bouillon (beef) mediumwas the most effective amongthose tested for cell growth and enzyme formation. The effect was due to the ring closure type of creatine compounds. Protocatechuate 3, 4-dioxygenase was purified from the cell-free extract ca. 1, 400-fold and it gave a single band on polyacrylamide gel electrophoresis. The molecular weight was estimated to be ca. 150, 000. The optimal pH and temperature were ptt 8.0 and 40°C, respectively. The enzyme was stable in a pH range from 7.6 to 8.6 and below 42°C. The enzyme was inhibited by several metals such as Pb²⁺, Cd²⁺ and Hg²⁺. The enzymewas active on a wide range of o-dihydroxyphenyl compounds, in contrast to the high specificity of similar enzymes from gram-negative bacteria (Pseudomonas). The enzyme had a broad absorption band in the visible region with a peak around 450nm, suggesting the presence of non-heme ion(s) bound to the enzyme as a co factor. The spectrum changed markedly upon addition of the substrate, possibly showing the formation of an enzyme-substrate complex.

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A strain of Erwinia carotovora was found to produce extracellularly four kinds of endo-pectate lyase pl-isozyme (PATE-I, -II, -III and -IV) having similar properties. The four pl-isozymes were purified to homogenous states by ion exchange chromatography, gel nitration and isoelectric focusing. The approximate molecular weights were 28, 000 for PATE-I and -III, 32, 000 for PATE-II and 33, 000 for PATE-IV, respectively, and they had isoelectric points of 10-11. The optimum pHs for the reaction catalyzed by these pl-isozymes were 9.3 for PATE-II, 9.5 for PATE-IV and 9.7 for PATE-I and III, respectively. There were no differences in the optimum Ca²⁺ concentration (0.5-0.6mM) or Km and V_max among these four pl-isozymes. The mode of action of all the plisozymes was determined to be the endo-type.

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Human κ-casein was tested as a bifidus growthpromoting factor before and after hydrolysis by chymosin or pepsin. The strain used was Bifidobacterium infantis SI2, originating from the feces of a breast-fed infant. Human κ-casein itself had bifidus growth-promoting activity without any treatment but became muchmore effective after treatment with chymosin or pepsin. Human glycomacropeptide, released by the action of chymosin or pepsin from human κ-casein, was effective as the factor even at low concentrations such as 50 ppm. By further hydrolysis with pronase, the activity was drastically decreased. This suggested that not only the sugar portion but also the polypeptide portion of human glycomacropeptide might play a significant role as a bifidus factor.

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A selective inactivating enzyme for cytosolic aspartate aminotransferase (GOT) was found in the culture filtrate of Streptomyces violaceochromogenes No. 401, newly isolated from soil. The enzyme was purified and crystallized. The enzymespecifically inactivates cytosolic GOT but shows no activity on the mitochondrial isozyme. The enzymeproved to be a serine proteinase. Limited proteolysis of native GOTby the enzyme results in a loss of enzymatic activity.

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