D-Alanine and several D, L-amino acids were administered to pea seedlings to investigate formation of D-amino acid conjugates. The N-malonylation was found to occur with all the D-amino acids administered. D-Alanine was specifically converted to additional γ-L-glutamyl conjugate, and this γ-L-glutamylation predominated over the N-malonylation with respect to D-alanine. The other D-amino acids tested were slightly or scarcely utilized for γ-L-glutamylation The overall results indicate that two systems exist for formation of D-amino acid conjugates in pea seedlings: (a) N-malonylation of common D-amino acids and (b) γ-L-glutamylation highly specific to D-alanine.

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Bacillus sp. 11-1S, a thermophilic acidophilic bacterial strain, produced an extracellular amylase with unusual characteristics. The enzyme was purified 40-fold by SE-Sephadex column chromatography. The pH optimum for activity was 2.0, and substantial activity was noted in the pH range of 1.5-3.5. The optimal temperature was 70°C, but the activity decreased markedly in lower reaction temperatures. Arrhenius plots of the reaction showed two straight lines intersecting at about 50°C. The activity or stability of the enzyme was not likely to depend on Ca²⁺. The molecular weight of the enzyme was 54, 000 calculated from the electrophoretic mobility. The enzyme behaved like an a-amylase (1, 4-α-D-glucan glucanohydrolase, E.C. 3.2.1.1). About 34% of glucosidic linkages of soluble starch was hydrolyzed at 65°C and pH 2.0, in 24 hr, and the major products were maltotriose and maltose.

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This paper reports on a modified procedure for determination of lysine in maize seeds. The modified procedure was economical, accurate and fast, and has been used to determine the available lysine in large numbers of maize grains. Protein sufficiently representative of total protein was extracted from samples ground in a water-cooled grinder. It was proposed that for samples with more than 15% protein the volume of NaOH-ETOH extractant be doubled. For maize samples with 15-30% oil content, 20ml of acetone was used for fat extraction, as opposed to 15 ml of acetone used for samples with less than 15% oil content. The sub-sampling variation was not significant for the modified 2, 4, 6-trinitrobenzene-1-sulfonic acid (TNBS) procedure. The correlation coefficient between lysine values determined by the amino acid analyser and the TNBS method for 90 opaque-2 samples was high (r=0.806). The coefficient of variation for TNBS was 6.5% against 12.6% for the microbiological assay (MBA), indicating that more variation was associated with the MBA than with the TNBS method.

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The roles of magnesium and cobalt ions in the reaction of glucose isomerase from Streptomyces griseofuscus S-41 were kinetically investigated. Magnesium was superior to cobalt as an activator, but was inferior as a protector of the enzyme. The enzyme was highly stable against heat and acid inactivation in the presence of cobalt, but labile in the presence of magnesium. The cobalt-activated enzyme was also tolerant to the inhibitory effect of other metal ions.
The mechanism of metal activation of glucose isomerase seemed to be expressed by the random rapid equilibrium system. Dissociation constants of metals from binary, enzyme-metal complexes were estimated to be 1.2 × 10^-3M for magnesium and 1.5 × 10^-5M for cobalt, and those from ternary, enzyme-metal-substrate complexes were 4.8 × 10^-4M for magnesium and 9.0 × 10^-6M for cobalt. The dissociation constant of glucose from enzyme-substrate complex was estimated to be 6.4 × 10^-1M, and dissociation constants of glucose from enzyme-magnesium-substrate complex and enzyme-cobalt-substrate complex were 2.4 × 10^-1M and 4.3 × 10^-1M, respectively. Thus, the affinity of the substrate for enzyme was enhanced by combining a metal with the enzyme and similarly, the affinity of the metal for enzyme was enhanced by combining a substrate with the enzyme.
Magnesium and cobalt were found to behave competitively in the reaction of glucose isomerase, and the competition profile altered depending on the relative concentrations of the two metals. It was, therefore, suggested that both metals combine at the same site or at very close sites of the enzyme.

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The role of cobalt in the stabilization of the molecular structure of glucose isomerase from Streptomyces griseofuscus S-41 was investigated using various denaturants. The enzymatic activity and molar ellipticity at 220nm were significantly reduced in 8M urea solution, but restored to the original values on removing urea. Therefore, the enzyme was thought not to suffer a drastic conformational change with urea. On the other hand, the destruction of ordered structure involving a complete loss of activity was observed from circular dichroism and fluorescence spectra in 6M guanidine hydrochloride solution. The enzyme showed somewhat peculiar behavior in organic solvents; in linear-chained solvents the activity correspondingly decreased with solvent concentrations, whereas it increased slightly in side-chained solvents and acetone. The change in activity observed here were not reflected in the circular dichroism and fluorescence spectra. Three of four cobalt ions originally contained in the enzyme were eliminated by treatment with EDTA or 8M urea without significant loss of activity. However, a cobalt-free enzyme was quite difficult to obtain in stable form. For elimination of all cobalts, drastic treatment was required, such as with 6M guanidine hydrochloride, acid-8M urea or EDTA-8M urea, indicating considerable dissociation into subunits. The cobalt addition showed a protective effect on the enzyme from denaturation in such drastic conditions, whereas it did not in less drastic conditions, with 8M urea or organic solvents. It was, therefore, considered that one of the four cobalts was tightly bound to the enzyme and had an essential role in holding the ordered Conformation, especially the quaternary structure of the enzyme, while the other three were bound loosely and might be less important in stabilizing the structure.

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Glutathione peroxidase (EC 1.11.1.9) was purified about 1500-fold from rat liver cytosol by means of ammonium sulfate fractionation and chromatographies on CM-Sephadex C-50, Sephacryl S-200 and activated thiol-Sepharose 4B. The final enzyme preparation proved to be homogeneous on polyacrylamide gel electrophoresis and to be composed of four identical subunits with a molecular weight of 20, 000. The rabbit antiserum against the purified enzyme gave a single fused precipitation line between the crude and purified enzymes. A quite similar displacement curve was observed for both liver cytosols from rats fed 20% casein diets with and without selenium supplementation, as a result of immunochemical titration with the antiserum. It therefore is apparent that an immunochemically recognizable protein other than glutathione peroxidase does not accumulate in a detectable quantity, if there is any in rat liver; dietary selenium-induced increase of glutathione peroxidase activity in the liver is accompanied with protein synthesis.

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More than 400 methanol-utilizing microorganisms isolated from soil were examined for L-methionine excretion in methanol medium supplemented with L-homoserine as a precusor of L-methionine. The strain OM 33 excreted 70 μg/ml of L-methionine when L-homoserine (5 mg/ml) and Na₂S•9H₂O (2 mg/ml) were added to the medium. This strain utilized only methanol and monomethylamine as sole sources of carbon, and assimilated methanol via the ribulose monophosphate pathway. Ethionine-resistant mutants were induced from strain OM 33 by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. A mutant, OE 120, excreted L-methionine into the medium without the addition of L-homoserine. Cultural conditions for L-methionine accumulation were investigated. After 4- to 5-days of cultivation, this mutant accumulated 420 μg/ml of L-methionine under optimum conditions.

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A cell-free extract of obligate methylotroph strain OM 33 catalyzed the formation of O-succinyl-L-homoserine from L-homoserine and succinyl-CoA, while the corresponding homoserine derivative from acetyl-CoA was scarcely formed. These results indicate that the acylation of L-homoserine, the initial step of L-methionine biosynthesis, was catalyzed by homoserine-O-transsuccinylase. In this bacterium, homoserine-O-transsuccinylase was subject to strict feedback inhibition by S-adenosyl-L-methionine (SAM). On the other hand, the enzyme of an ethionine-resistant mutant OE 120 derived from strain OM 33, was scarcely affected by SAM. These observations suggest the important role of homoserine-O-transsuccinylase in the biosynthesis of L-methionine.

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Bacillus brevis No. 47, a high protein producer, was treated with deoxyribonucleic acid (DNA) from different strains of B. subtilis and B. amyloliquefaciens, the latter being an efficient producer of extracellular enzymes such as α-amylase, protease and ribonuclease (RNase). From the new clones obtained by the transformation procedure, about eighty were purified and used for examining the productivity and nature of trichloroacetic acid precipitable extracellular proteins, and the levels of activities and characteristics of α-amylase, protease and RNase secreted into the extracellular medium. The incorporation of various donor genetic markers into the new clones was also examined.
Despite little homology between the donor and the recipient DNAs, the appearance of the new clones was most probably the result of donor DNA mediated recombination events, because these clones exhibited unpredictably diverse combinations of phenotypes (e.g., extracellular enzyme activities or expression of donor markers). Among the recombinant clones obtained were those which produced greater amounts of extracellular proteins or possessed many-fold higher extracellular enzyme activities than their respective parents. The results presented in this paper show the usefulness of heterologous transformation for obtaining bacteria with new and varied phenotypes.

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A thermophilic fungus, strain YH-78, identified as Humicola grisea var. thermoidea Cooney et Emerson, was newly isolated from compost heaps. This strain exhibited high CMCase activity in cultures on wheat bran medium (50°C, 4 days), but Avicelase activity in the culture filtrate was very low. The culture filtrate significantly disintegrated filter paper and hydrolyzed carboxymethyl cellulose to 45% as glucose at 50°C. The activity of CMCase in the culture filtrate was partially adsorbed onto Avicel. The CMCase component adsorbed onto Avicel was purified to homogeneity by disc electrophoresis. The purified CMCase component was optimally active at 50°C and pH 5.0, and completely adsorbed onto Avicel at pH 5.0 at 4°C for 10 min. The enzyme showed high activity toward CMC and no activity toward Avicel, but showed a high synergistic action together with the cellulase from Humicola insolens YH-8 on hydrolysis of Avicel.

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On incubation with subtilisin, the raw starch-adsorbable and raw starch-digestive glucoamylase I (MW 90, 000) of Aspergillus awamori var. kawachi was converted to raw starch-inadsorbable and raw starch-indigestive glucoamylase I' (MW 83, 000), with the liberation of glycopeptide I (MW 7, 000). This inactive peptide showed significant adsorbability onto raw starch, fungal cell wall and chitin but not onto chitosan. It consisted of 42.1 % carbohydrate residues and amino acid residues, such as Asp₅, Thr₈, Ser₄, Glu₃, Gly₄, Ala₄, Val₃ and His₂, characteristically abundant in hydroxy amino acid. The adsorbability of glucoamylase I and glycopeptide I was supposed to take place through hydrogen bonds between the insoluble raw starch and the hydroxy amino acid situated at the position of glycopeptide I. The specific blocking of the amino acid residue suggested that the seryl residue on glycopeptide I played a role in the raw starch-affinity site on glucoamylase I that is essential for the digestion of raw starch.

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The effect of tryptic digestion on the solubility and emulsifying properties of heat-denatured and native soy protein (crude glycinin) was studied as a function of pH. Fractionation of the tryptic digests of heat-denatured soy protein was achieved by centrifugation and ultra-filtration. The resulting precipitate (PPT), high molecular weight fraction (HMF) and low molecular weight fraction (LMF) were constituted mainly with polypeptide. They had molecular weights of approximately 30, 000, 20, 000 and less than 10, 000, respectively. The isoelectric region of HMF and that of DNG (digest of native glycinin) shifted to the acidic side, and the solubility of PPT and LMF was not influenced by pH. Emulsifying properties, such as emulsifying capacity (EC) and emulsion stability (ES) after 30 min and 24 hr, were measured. It was found that the emulsifying properties were improved by tryptic hydrolysis but the low molecular weight digests showed poorer emulsifying properties. Among these digests, digestion intermediates (HMF and DNG) showed the highest emulsifying properties. The behavior of EC and ES after 30 min of these digests was related to that of their solubility and these values showed the minimum in the isoelectric region. Conversely, the ES value after 24 hr was maximum in this region.

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α, β-Unsaturated ketones were reduced by selected fungi (which are known steroid hydroxylators) incubated under limited aerobic conditions.

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A prototrophic revertant was derived from a citrate synthase-defective glutamate auxotroph of Brevibacterium flavum. It showed low citrate synthase activity and overproduced L-aspartic acid. The maximum production was 10.6g/liter (about a 30% yield) when the strain was cultured in medium containing 36 g/liter of glucose for 48 hr. The amount of aspartic acid produced depended greatly on the concentration of biotin in the medium, as the case of glutamate production by the original wild strain. When the revertant strain was cultured with excess biotin, acetic acid accumulated instead of aspartic acid. A mutant resistant to S-(2-aminoethyl)-L-cysteine plus threonine derived from the revertant strain produced 36 g/liter of L-lysine (as hydrochloride salt) when it was cultured in medium containing 100 g/liter of glucose for 72 hr (36% yield). The amount of lysine production by this mutant was greater than those by the resistant mutants derived directly from the original wild strain. The frequency of the appearance of mutants which produced more than 30 g/liter of lysine was about one per 250 strains of resistant mutants. A high concentration of ammonium sulfate was required for a high yield of lysine production.
A homoserine auxotroph derived from the revertant strain produced 33 g/liter of L-lysine (33% yield), which was almost the same amount as that derived directly from the original wild strain.

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An alkalophilic bacterium isolated from soil and designated as strain GIR-277 was found to efficiently macerate bast fibers of mitsumata (Edgeworthia papyrifera Sieb. et Zucc.). The organism identified as a Bacillus sp. was successfully applied to alkaline retting in papermaking. The optimum concentration of alkali (Na₂CO₃) for this process was 1.0-1.5%, which corresponded to an initial pH of 10.1-10.3. The retting was accomplished within a few days at 30°C. Substantial maceration was ascribed primarily to pectate lyase (EC 4.2.2.2) secreted by the bacterium, and to a lesser extent to the β-eliminative splitting of pectic substances. The overall yield of pulp was about 70%. The strength of the unbeaten pulp resulting from bacterial retting was as higher than that by the conventional soda ash-cooking method. Paper sheets were quite uniform and very soft to touch. It is concluded that bacterial retting under alkaline conditions is a potentially useful process to produce pulp of excellent quality from non-woody pectocellulosic fibers.

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Acetone-dried cells of Escherichia coli with an enhanced level of biodegradative L-threonine deaminase (EC 4.2.1.16) were easily and tightly immobilized to the inner surface of glass tubes by use of an emulsion-type of photo-crosslinkable resin prepolymer. A continuous flow system equipped with the microbial cell tube thus prepared was proved to be applicable for assay of L-threonine or L-serine in fermentation broth with rapidity (each assay, less than 9min), excellent substrate specificity (specific for L-threonine and L-serine) and high stability over repeated assays (at least 60 assays).

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Acetone-dried cells of Citrobacter freundii having cephalosporinase were easily and tightly immobilized to the inner surface of glass tubes by use of an emulsion-type of photo-crosslinkable resin prepolymer. The microbial cell tubes thus prepared were used in a continuous flow system to measure cephalosporin C by potentiometry. A good response was observed in the concentration range from 0.1mM to 4mM of cephalosporin C. This system was active on cephalosporin C, deacetylcephalosporin C and deacetoxycephalosporin C, indicating the possibility for assaying the total amount of cephalosporins in fermentation broth as cephalosporin C values. The enzyme in the microbial cell tubes was stable after repeated use in at least 200 assays.

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D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans var. α IFO 3254 by a procedure involving solubilization of the enzyme with Triton X-100 in the presence of KCl and D-sorbitol. Purification of the enzyme was performed by fractionation with polyethylene glycol 6000, and column chromatographies on DEAE- and CM-cellulose in the presence of Triton X-100. The enzyme was purified about 240-fold from the membrane fraction of the organism. The purified enzyme was tightly bound to a c-type cytochrome existing as a dehydrogenase-cytochrome complex. The dehydrogenase was found to be a flavoprotein, and its flavin moiety was covalently bound to the dehydrogenase protein. The optimum pH of the enzyme activity was 4.5, and the optimum temperature was 25°C. D-Sorbitol was specifically oxidized by the purified enzyme, and D-mannitol was also oxidized to 5% of the reaction rate with D-sorbitol.

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Isocitrate epimerase activity catalyzing the reversible epimerization between threo-D-isocitrate and erythro-L-isocitrate (L-alloisocitrate) was determined in the cell-free extract of Penicillium purpurogenum rubrisclerotium, which accumulated a large amount of the latter acid from glucose.
The presence of 15 mM or a higher concentration of citrate was essential for detection of the activity in vitro. The epimerase was located in mitochondoria and seemed to require structural organization in cells for its full activity. A pathway was proposed in fungal cells for accumulation of erythro-L-isocitrate through threo-D-isocitrate. An enzymological method to determined radioactive erythro-L-isocitrate was developed using a specific bacterial dehydrogenase.

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UV irradiation of α-, β- or δ-isomer of 1, 2, 3, 4, 5, 6-hexachlorocyclohexane (BHC) in 2-propanol resulted in the formation of a new compound. The compound isolated from each irradiation product was found to be an isomer of pentachlorocyclohexane, and the steric orientation for chlorine atoms was determined to be 1a, 2e, 3e, 4e, 5e from X-ray structural analysis. In the case of α-and δ-BHC, the pentachlorocyclohexane may be produced by hydrogen abstraction of the radiation-induced pentachlorocyclohexyl radicals, but migration of an equatorial chlorine atom to the vicinal axial position seems to occur at the intermediate pentachlorocyclohexyl radicals in the case of β-BHC.

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The antimicrobial effect of a variety of essential oil components was examined in the presence of various concentrations of NaCl, using air-borne microorganisms and purely cultured fungi. Even at a NaCl concentration of 15%, various kinds of microorganisms grew in 7 to 10 days of incubation at 27°C. All the essential oil components examined, at a concentration of as high as 1 mM, allowed the growth of various microorganisms within a few days of incubation at 27°C when the NaCl concentration of culture media was less than 3%.
However, in the presence of 7 to 10% NaCl, cinnamaldehyde, perillaldehyde, citral (α, β-unsaturated aliphatic aldehydes), citronellol, perillalcohol and geraniol (primary alcohols) all exhibited a potent antimicrobial effect at a concentration of less than 1 mM. Cuminaldehyde and eugenol were also potent in the respect. L-Menthol at 1 mM was only modest, but at 2mM was potent in this effect. Citronellal, D-carvone, vanillin, and linalool were only modestly effective, and 1, 8-cineole, anethole, and safrole were almost ineffective even at a concentration of 2mM. Hydrocarbons (α-pinene, β-pinene, camphene, β-myrcene, β-caryophyllene, and p-cymene) even at a concentration of 2 mM were all ineffective under the same condition. These results suggest that certain essential oil components are applicable to effectively preserve foods containing more than 7% NaCl.

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In random screening of nematicidal compounds against the pine wood nematode Bursaphelenchus lignicolus using the so-called immersion test, a series of mono- and di-functional alkanes, aromatics and other miscellaneous compounds (total 105) were tested. Among these, the n-alkylamines were identified as the most potentially-active nematicidal compound. Relationship between nematicidal activity and the carbon-chain length of amines, mono-basic acids and alcohols was observed. Among those tested, n-octadecylamine showed the highest activity (LC₅₀; 8.61 × 10^-6 M, 2.08 ppm) on a molar basis.

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A papain-catalyzed reaction involving the covalent attachment of L-leucine n-dodecyl ester [Agric. Biol. Chem., 44, 1979 (1980)] was applied to gelatin and succinylated fish protein concentrate. Proteinaceous surfactants formed were found suitable for emulsification of soybean oil. The emulsions prepared with these surfactants were characterized by having a variety of functional properties in terms of hardness, adhesiveness, viscosity and viscoelasticity. Any particular property could be reproduced by intentionally setting the proper conditions for emulsification; for example, the use of a high surfactant concentration resulted in gel formation. The functions of the proteinaceous surfactants were different in many respects from those of Tween-60 and a type of sucrose fatty acid ester used as controls. Several data were added explaining such differences. The feasibility of preparing a mayonnaise-like concentrated emulsion by use of the proteinaceous surfactants is discussed.

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The accumulation of L-glutamate (L-Glu)by Brevibacterium flavum No. 2247 was investigated from the standpoint of cellular permeability toward L-Glu under electro-energizing (E-E) conditions. In both biotin-poor (4 μg/liter) and biotin-rich (10 μg/liter) conditions, an increase in K⁺ uptake and easy leakage of intracellular L-Glu were induced by E-E. In addition, under the biotin-rich conditions, E-E culture caused changes in cellular fatty acid composition and in the net charge of the cell surface, thus causing the cells to become similar to the biotin-poor cells. These cellular changes facilitated the leakage of intracellular L-Glu.

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A new thermophilic methane-oxidizing bacterium (strain H-2) was isolated from a gas field. The organism grew at 30-55°C, and the optimum temperature for growth was 50°C. Methane was the sole carbon and energy source for growth, and no growth occurred on nutrient broth. When cultivated in a mixed culture with another kind of bacterium (Bacillus sp.), H-2 grew better on methane than in pure culture. Effects of gas composition, nitrogen sources, trace metals and vitamins on growth were studied. Cupric sulfate was found to be essential for growth of a pure culture of strain H-2, and vitamin B₁₂ stimulated its growth. Under the optimum culture conditions, the doubling times of mixed and pure culture were about 2 and 3hr, respectively.

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Pectin lyases of four bacteriocinogenic Erwinia carotovora strains, Er, Ar, Ar13 and 6083, were purified to near homogeneity from cultures treated with mitomycin C. Pectin lyases from these strains had similar molecular weights of around 28, 000, and their optimal pH for reaction was about 8.0. All these enzymes cleaved a highly methylesterified polygalacturonic acid (90% esterified) well, but with pectin (64% esterified) they showed reduced activities. They did not attack polygalacturonic acid which was not esterified. In contrast to these similarities, the isoelectric points of the enzymes were slightly different from each other, ranging between pI 9.55 and 9.62, and suggests that these enzymes were not identical protein molecules. Antiserum against the pectin lyase of strain Er similarly inactivated the pectin lyases of the other strains, as well as the pectin lyase of strain Er. No immunoprecipitate line was formed between the antiserum against pectin lyase of strain Er and polygalacturonic acid (pectate) lyase of this strain or between antiserum against pectate lyase of strain Er and pectin lyases of the four strains. Antiserum against carotovoricin from strain Er neutralized carotovoricins from strains Er, Ar, Arl3 and 6083 to a similar extent. These results imply that these pectin lyases share a common genetic background and that these carotovoricins might be derived from the same source.

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An NAD⁺ analog, N⁶-[(6-aminohexyl)carbamoylmethyl]-NAD⁺, was immobilized on bovine caseins by the action of transglutaminase. Binding of NAD⁺ was not observed. It appears that NAD⁺ analog binds with α_s1- and β-caseins through formation of the γ-glutamylamine bond between the amino groups attached to the hexyl chain in NAD⁺ analog and the glutaminyl residues in caseins. The NAD⁺ analog immobilized on the caseins was enzymatically reducible by alcohol dehydrogenase; the numbers of Coenzymatically active NAD⁺ analog molecules were 0.4, 0.2, 0.9, and 0.3 per mol of α_s1-, dephosphorylated α_s1-, acetylated α_s1-, and β-caseins, respectively. The number of immobilized NAD⁺ analog molecules was always larger than that of the enzymatically reducible one, indicating that some of the immobilized NAD⁺ was inactive. Michaelis constants of immobilized NAD⁺ analog in alcohol dehydrogenase reaction were similar to those of free forms of NAD⁺ and NAD⁺ analog. Maximum velocities were reduced to 15-30% of the free NAD⁺ analog. Immobilized NAD⁺ analog was much more stable at alkaline pH than free NAD⁺ and its analog. The coenzyme • casein conjugates were recovered almost completely in casein precipitated by calcium.

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The effect of indole on the mono-oxygenase systems was studied in liver microsomes from control, indole-, phenobarbital-and 3-methylcholanthrene-treated rats. No induction was observed by indole of either cytochrome P-450 or the mono-oxigenase activities. It was, however, a potent competitive inhibitor of aniline hydroxylase (Ki=0.03mM-0.04mM). A spectroscopic study showed that this competition occurred at the aniline binding site on cytochrome P-450. The inhibitory effect was observed on aminopyrine N-demethylase with less efficiency (Ki=0.1-0.5 mM) and on benzo[a]pyrene hydroxylase. These inhibitions were also observed in the systems of mice, rabbits and monkeys. The physiological effect of indole on liver drug metabolism was also discussed.

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Spore suspensions of 15 strains in 15 species of Micromonospora prepared with ultrasonication-technique were tested for resistance to moist heat, acid, alkali, and organic solvents (5 alcohols, 4 ketones and ether). More than 50% spore-survival was found in most organisms heated at 60°C for 20min, but less than 0.5% survived at 80°C. The spore-viability did not change at pH 6 to 8, but decreased beyond this range, and remarkably at acidic pH. A maximum reduction in viability was found with most organic solvents at a concentration of around 80%, and the spores were more resistant to ketone than alcohols and dioxane. Several Streptomyces species were also studied, and their spores were less resistant to heat and organic solvents than those of Micromonospora.

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Hemolysin in the phytoflagellate Prymnesium parvum was separated into six components by thin layer chromatography. The major component named hemolysin I was isolated and identified as a mixture of 1'-O-octadecatetraenoyl-3'-O-(6-O-β-D-galactopyranosyl-β-D-galactopyranosyl)-glycerol (1) and 1'-O-octadecapentaenoyl-3'-O-(6-O-β-D-galactopyranosyl-β-D-galactopyranosyl)-glycerol (2).

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A simple method has been developed for DNA isolation from purified chloroplasts of Marchantia polymorpha L. (liverwort) cell suspension cultures. Purified chloroplasts exhibited ribulose-bisphosphate carboxylase activity comparable to that of Fraction 1 protein obtained from Nicotiana tabacum. Fraction 1 protein isolated from purified chloroplasts clearly showed large and small subunits when subjected to isoelectric focussing. These results indicate that the purified chloroplasts are intact. DNA isolated from purified chloroplasts showed a covalently closed circular form, and restriction endonuclease digestions of the chloroplast DNA showed clear fragmentation indicating that the DNA was sufficiently free from those of other organelles.

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The growth characteristics of the oxygen-resistant strain of hydrogen bacterium N34 in a 40% O₂-culture were compared with those in a 4% O₂-culture in order to clarify the mechanism of oxygen-resistance. Growth inhibition was observed with decreased CO₂ tension indicating the inhibition of CO₂-fixation. Cell growth in 40% O₂-culture was associated with stimulated oxyhydrogen reaction. There was no significant difference in cell composition. Each of the efficiency of energy conversion and the yield coefficients, Y_H2 and Y_O2, in the 40% O₂-culture was about half of that in the 4% O₂-culture. The yield coefficient, Y_CO2, or the carbon recovery, however, was almost equal between the two kinds of cultures. The mechanism of oxygen-resistance was discussed.

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Three fractions with nucleolytic activities were isolated from rice bran by DEAE-cellulose column chromatography and designated as RB-1, RB-2 and RB-3. RB-1, RB-2 and RB-3 had molecular weights of approximately 6, 200, 35, 000 and 14, 500, respectively, by gel filtration. The main fraction (RB-3) was purified by Sephadex G-75 and CM-cellulose column chromatography. The pH optimum was 5.0. The nucleolytic activity of RB-3 was strongly inhibited by Cu²⁺, while EDTA had no effect on the activity. Seventy-five percent of the original activity of RB-3 still remained after 16 minutes of heating at 100°C. It appeared to be an endonuclease which hydrolyzes yeast RNA to yield purine nucleotides.

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Ten diether-type monoglycosyl and glycobiosyl glycerolipids, including 3-O-(4-O-β-D-galactopyranosyl-β-D-glucopyranosyl)-1, 2, -di-O-n-tetradecyl-sn-glycerol, a synthetic analogue of lactosyl ceramide, were synthesized and their stereochemistry was assigned unambiguously by ¹³C NMR using the values of C-H one bond couplings. Their ¹³C NMR were further analysed to show the diagnostic α-effect of glycosylation in these compounds depending on the anomeric configuration of the glycosyl residue linked to C-3'-O atom.

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Synthetic routes for the following mannooligosylglycerolipids of biological interest were developed by using regioselectively protected monosaccharide synthons and 1, 2-di-O-alkyl-sn-glycerol; 3-O-(2-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-1, 2-di-O-tetradecyl-sn-glycerol; 3-O-[2-O-(2-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-α-D-mannopyranosyl]-1, 2-di-O-tetradecyl-sn-glycerol; 3-O-(6-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-1, 2-di-O-tetradecyl-sn-glycerol; and 3-O-(3, 6-di-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-1, 2-di-O-tetradecyl-sn-glycerol.

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Changes in Ultrastructure of protein bodies in subaleurone cells of rice endosperm during germination were studied by transmission electron microscopy. The subaleurone cells contained two different types of protein bodies: PB-I (spherical) and PB-II (crystalline). Both types of protein bodies were deconstructed during germination. But there was a considerable difference in digestibility between PB-I and PB-II. PB-II which did not have a dense core was easily digested from the central portion when germination began. At 6 days of germination, PB-II was almost deconstructed. On the other hand, PB-I which displayed concentric rings with a dense core was digested from the outside after 3 days of germination. At 9 days of germination, many kernels of the spherical protein bodies remained.
Changes in subunit composition of protein bodies during germination were investigated by SDS-polyacrylamide gel electrophoresis. Protein body fractions were isolated from germinating grains at various stages by enzymatic digestion and two-phase system, then subjected to SDS-polyacrylamide gel. As germination proceeded, 15 (b₁), 20 (d₁), 24 (e), 35 (f₁) and 37 (f₃) k daltons subunits decreased. On the other hand, 16 (b₂), 21 (d₂) and 36 (f₂) k daltons subunits remained at the later stage of germination. We think that PB-I contains b₂, d₂ and f₂ subunits and is attacked only from the outside at middle and later stages of germination by de novo protease. On the contrary, PB-II contains b₁ d₁, e, f₁ and f₃ subunits is utilized at an early stage of germination. PB-II may possibly contain latent protease. The breakdown process of PB-I was by exo-type digestion, on the contrary, that of PB-II was by endo-type digestion.

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Our previous work on the allergenic structure of ovomucoid has shown that the allergenic reactive sites reside within the two fragments (domain I-II and domain II-III) comprising two-thirds of ovomucoid. In the present study, domain II was isolated and its allergenic reactivity was determined with a passive cutaneous anaphylaxis reaction using specific IgE antibody from mouse. The reactivity was estimated to be 40% as compared with the total reactivity in ovomucoid. The allergenic reactive sites still reside within domain II but the combination of domain II with either domain I or domain III is essential for full exhibiting of the allergenic reactivity in ovomucoid.

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