The capability of Aureobasidium pullulans S-1 to produce pullulan varied considerably with the initial pH; that is, pullulan was hardly elaborated with initial pH 2_??_2.5, and the organism converted ca. 50% (by shaking culture) or ca. 60% (with jar fermenter) of consumed sugar into pullulan with initial pH 6.0. Initial pH, the fall of pH during culture, and the agitation rate greatly influenced the ability of the organism to elaborate pullulan.

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Many microorganisms (17 strains of gram-positive bacteria, 8 strains of gram-negative bacteria, 2 strains of fungi) capable of assimilating di-2-ethyl hexyl phthalate (DEHP) were isolated from soil and other natural sources. When Pseudomonas acidovorans 256-1 among these microorganisms was aerobically cultured in media containing 0.5% of DEHP, DEHP disappeared completely in 72 hr when assayed gaschromatographically. Most of phthalate esters could be assimilated, regardless of their side-chain types. In addition, branched-alkyl phthalate was assimilated better than n-alkyl phthalate. Based on degradation rate of n-alkyl phthalate in relation to its side chain and carbon number, two peaks were observed in n-alkyl phthalate with four and seven carbon number on its side-chain.

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1. The membrane-bound acid protease of Aspergillus oryzae was solubilized by treatments with snail gut extract and phospholipase A, and with several detergents including Triton X-100, Tween 80, and cholic and deoxycholic acids. Other hydrolytic enzymes tested were not effective in the solubilization of enzyme.
2. Membrane-bound acid protease was activated with lipid mixture extracted from membrane fraction of A. oryzae as well as with detergents.

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In order to clarify the interaction of calcium ion with casein, the volume change associated with the interaction was measured by dilatometric procedures. When CaCl₂ was added to the casein solutions at neutral pH, a volume increase occurred and reached a constant saturated value of about 700ml per 10⁵g protein with increasing CaCl₂ concentrations for whole-, α_s- and β-casein solutions, but there was no volume change for κ-casein solution. On the other hand, the binding of calcium ion to the casein fractions was determined by a gel filtration procedure at pH 6.0 to 9.0. The number of Ca²⁺ ions bound to the caseins increased with the CaCl₂ concentration and pH value, and the relative order of binding capacities for the caseins was: as α_s-casein>whole-casein>β-casein>κ-casein.
It was found that the volume changes obtained by the dilatometry were smaller than the calculated volume increases based on the assumption that these are caused by the binding of Ca²⁺ ion to the caseins. Therefore it is necessary to introduce another factor which reduces the volume increase due to the Ca²⁺ ion binding in order to reasonably explain the measured volume changes. At present it is presumed that there occurs the unfolding of peptide chain of casein molecule on Ca²⁺ ion binding, which has been known to decrease the volume of the protein solution.

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Both of the two forms of glucoamylase (glucoamylases I and II) from the wheat bran culture of Mucor rouxianus hydrolyzed amylopectin, amylose, glycogen, soluble starch, maltotriose, and maltose, but did not act on isomaltose and isomaltotriose. Phenyl α-maltoside was hydrolyzed into glucose and phenyl α-glucoside by both glucoamylases. Maltose was hydrolyzed about one-fifth as rapidly as amylopectin. Both enzymes produced glucose from amylopectin, amylose, glycogen, soluble starch in the yields of almost complete hydrolysis. They hydrolyzed amylose with the inversion of configuration, producing the β-anomer of glucose. Glucoamylase II hydrolyzed raw starch at 3-fold higher rate than glucoamylase I. The former hydrolyzed rice starch almost completely into glucose, whereas the latter hydrolyzed it incompletely (nearly 50%).

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Aspergillus awamori produced multiple forms (isoenzymes) of α-glucosidase, three of which could be separated from one another by DEAE-cellulose column chromatography, and a glucoamylase. The α-glucosidases isolated were designated as α-glucosidase I, α-glucosidase II and α-glucosidase III. They were homogeneous on gel electrofocusing and ultracentrifugation, respectively. The glucoamylase preparation was homogeneous on disc gel electrophoresis, gel electrofocusing and ultracentrifugation. The molecular weight of α-glucosidase I, α-glucosidase II, and α-glucosidase III, and glucoamylase was 125, 000, 140, 000, 130, 000 and 88, 000 to 83, 700, respectively. The three α-glucosidases hydrolyzed maltose, maltotriose, phenyl α-maltoside, isomaltose, panose, phenyl α-glucoside, and amylose liberating glucose, but did not act on sucrose. Glucoamylase hydrolyzed maltose, maltotriose, phenyl α-maltoside, soluble starch, amylose, amylopectin, and glycogen, and glucose was the sole product formed in the digests of these substrates. They hydrolyzed phenyl α-maltoside into glucose and phenyl α-glucoside. Glucoamylase hydrolyzed rice starch, amylose, amylopectin, and glycogen, converting them almost completely into glucose. It was found that β-glucose was liberated from amylose by the action of glucoamylase. Isomaltose and panose were the main α-glucosyltransfer products formed from maltose by the three α-glucosidases. Certain properties of these enzymes also were investigated in the present work.

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Effect of heating on the functional properties of egg white acylated with either acetic anhydride or succinic anhydride was studied. The coagulation temperature of egg white greatly increased with acylation in the alkaline pH region. The foaming properties of egg white were improved by acylation, and heat-induced damage on the foaming properties was greatly reduced by acylation. These phenomena were observed in egg white over 30% of whose available amino groups were acylated.

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Abilities of washed suspension of rumen ciliate protozoa to form δ-aminovaleric acid (DAV) and some precursors of DAV were chromatographically and autoradiographically examined. DAV was accumulated linearly in the medium during incubation of the ciliates. When proline or ornithine was added to the medium, the amount of DAV was increased to the level over the control value. The amount of proline in the medium after incubation was increased by addition of ornithine, while the amount of ornithine was increased by addition of arginine. L-Proline-U-¹⁴C was converted to radioactive DAV by the ciliates. But the radioactive DAV was not converted further by them, so it seemed to be the end product in proline metabolism of rumen ciliates. L-Ornithine-U-¹⁴C was converted to proline and DAV, and L-arginine-U-¹⁴C was converted to ornithine, proline, DAV and four other unidentified compounds by the ciliates. All these results led us to presume that the serial pathway such as from arginine to ornithine, from ornithine to proline and from proline to DAV would exist in biochemical systems of rumen ciliate protozoa.

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Ureolytic activity of rumen ciliate protozoa was examined in comparison with that of rumen bacteria. Freshly obtained rumen ciliates which were washed five times with B-9 buffer solution previously, decomposed 1.3 μmole/ml of urea to give ammonia after incubation for 6 hr in the buffer solution containing streptomycin, while rumen bacterial suspension decomposed 84.6_??_97.8 μmole/ml of urea after incubation for 2 hr. The amount of ammonia-N/bacteria-N produced from urea by the suspension of rumen bacteria was usually higher than that by the coexistent suspension consisting of rumen bacteria and ciliates. When the freshly obtained rumen ciliates were previously incubated in the buffer solution containing 100 μg/ml each of streptomycin, penicillin and chloramphenicol (starved ciliates), ureolytic activity of the ciliates decreased with the incubation time and reached zero after 24 hr of incubation. ¹⁴C-Urea remained unchanged in the medium of the starved ciliates even after incubation for 6 hr. All these results led us to the conclusion that rumen ciliate protozoa did not have ureolytic activity and that ureolytic activity of the freshly obtained ciliates might be due to contaminating bacteria.

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The acetonitrilase from Brevibacterium R 312 has an activity between pH 5 and 10, with an optimum pH of 7, the optimum temperature is between 30°C and 35°C. It is a thermolabile enzyme and the temperature denaturation starts at 35°C. The Michaelis constant is near 2, 5.10^-2M. The enzyme is localized in the 180000g supernatant. It is an non adaptative enzyme.

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A pectate lyase was purified from the culture filtrate of Streptomyces nitrosporeus by column chromatography on QAE-Sephadex A-50 and gelfiltration on Sephadex G-100. The purified enzyme was demonstrated to be homogeneous by disc electrophoresis. The optimum pH for the activity was 9.3 to 9.5 and the enzyme required calcium ions for maximum activity. The initial reaction rate was higher on partially esterified pectin than on polygalacturonic acid. The enzyme was more specific for tetra-, penta-, and hexagalacturonic acids than for polygalacturonic acid. But di- and trigalacturonic acids were not good substrates for the enzyme. The Km values of the enzyme for tri-, tetra-, penta-, hexagalacturonic acids and acid soluble polygalacturonic acid at 1.0mM calcium ion concentration were 1.5, 0.19, 0.23, 0.38mM and 0.051%, respectively.

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When polygalacturonic acid was degraded by the purified pectate lyase of Streptomyces nitrosporeus, unsaturated trigalacturonic acid was formed as the main end reaction product. Paper chromatographic analysis of reaction products showed that the enzyme cleaved polygalacturonate by a terminal mechanism. The attack sites of oligogalacturonates by the enzyme were only third bonds from the reducing ends of the molecular. Reduced poly- and pentagalacturonates with sodium borohydride were resistant to the action of the enzyme. From these results it has been shown that the pectate lyase of S. nitrosporeus is a new type exopolygalacturonate lyase removing terminally units of unsaturated trigalacturonic acid from the reducing ends of polygalacturonate chains.

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A useful method of synthesis of pyrethronyl acetate (VII) and pivalate (VIII) from readily available allethrolone (II) is described.

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In the nutrition of the silkworm, Bombyx mori, the free forms of aspartic and glutamic acids possessed low effect, but their salts exerted high effect. Potassium, magnesium, or calcium salt was more effective than sodium salt. Asparagine was utilized well, whereas glutamine was of reduced value. Neutralization of free aspartic acid with KOH or NaOH produced the same effect as brought about by K- or Na-aspartate, and shifted pH of the diet at the same time. It was considered that the neutralization favored the acceleration of larval feeding.

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A methanol-utilizing yeast Kloeckera sp. No. 2201, when grown with methanol as a sole carbon and energy source, accumulated about three times much flavin as those grown with glucose, ethanol, or glycerol. A high proportion of the total flavin was FAD in methanol-grown cells. A remarkable derepression of FAD pyrophosphorylase accompanied by an inducible formation of an FAD-dependent alcohol oxidase which catalyzes oxidation of methanol, the first step in the oxidation sequence, was observed during growth of the yeast on methanol. Significant elevations of riboflavin synthetase and flavokinase were also found. Formate, as well as methanol, effectively induced both FAD pyrophosphorylase and methanol-oxidizing enzymes (alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and catalase). Observations with other methanol-utilizing yeasts also gave essentially same results. These results led to the conclusion that cellular flavin level might be under control with level of flavoprotein physiologically required.

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Amylase inhibitor (S-AI) was purified by about 25 times from culture filtrate of Streptomyces diastaticus subsp. amylostaticus No. 2476 through the methods of adsorption on active carbon, column chromatographies on Dowex 50 W×2 (H-form) and Dowex 50 W×2 (NH₄-form), gel filtration on Sephadex G-25, EI-complex formation with BLA, isolation of complex by gel filtration on Sephadex G-75, dissociation from complex by the method of acid denaturation, rechromatographies on Dowex 50 W×2 (NH₄-form) and Sephadex G-25. Homogeneity of this S-AI was examined by means of TLC, where S-AI gave a single spot in various solvent systems. S-AI specially inhibited α-amylases and glucoamylase, but not β-amylases and other glucoside hydrolases.
S-AI was a very stable substance, as it retained 100% of its original activity after being kept for 30min at 100°C in a pH range between 3.0 and 10.0. The molecular weight of S-AI was estimated to be about 1500 by gel filtration on Sephadex G-15.
S-AI was regarded to be an oligosaccharide which was mainly composed of glucose in an amount of about 85%. S-AI was hydrolyzed by β-amylase from non-reducing terminal and released two moles of maltose succesively.

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As a growth factor, Rhizobium meliloti required cobalt ion, or vitamin B₁₂ which was found to be incorporated into the cells without decomposition to cobalt ion. Trial of replacement for cobalt ion by the addition of various compounds to the cobalt-deficient medium revealed that methionine could substitute for cobalt ion and promote the growth in response to its concentration. Furthermore, B₁₂-dependent methionine synthetase was demonstrated in the cell-free extracts of this microorganism. The morphological change of R. meliloti by the additions to the medium was observed microscopically.

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Proteinase inhibitors I and II from adzuki beans were very stable even at 100°C in acidic pH range. Inhibitor I was a polypeptide chain having aspartic acid as a carboxyterminal residue and inhibitor II was that having asparagine. Inhibitor I may have two different reactive sites against trypsin and one of them will be a particular Lys-X peptide bond, judging from the fact that about 50% of the inhibitory activity against trypsin remained, even if 80% of the amino-groups were modified by trinitrobenzenesulfonic acid. Inhibitor II inhibited trypsin and chymotrypsin separately, but not simultaneously. The inhibitor, however, could be said to have separate and independent reactive sites against the enzymes, because the reaction of inhibitor II with trinitrobenzenesulfonic acid abolished the tryptic inhibitory activity of the inhibitor, but left the chymotryptic inhibitory activity intact.

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The formation of aromatic L-amino acid decarboxylase in bacteria was studied with intact cells in a reaction mixture containing the aromatic L-amino acids, 3, 4-dihydroxy-L-phenyl-alanine, L-tyrosine, L-phenylalanine, L-tryptophan and 5-hydroxy-L-tryptophan. Activity was widely distributed in such genera as Achromobacter, Micrococcus, Staphylococcus and Sarcina. Bacterial strains belonging to the Micrococcaceae showed especially high decarboxylase activity toward L-tryptophan, 5-hydroxy-L-tryptophan and L-phenylalanine. M. percitreus AJ 1065 was selected as a promising source of aromatic L-amino acid decarboxylase. Results of experiments with this bacterium showed that the aromatic amine formed from L-tryptophan by the enzymatic method was identical with tryptamine. M. percitreus constitutively produced an enzyme which exhibited decarboxylase activity toward L-tryptophan. However, when large amounts of the aromatic L-amino acids listed above or the tryptamine formed from L-tryptophan were added, enzyme formation was repressed.
Cells with high enzyme activity were prepared by cultivating this bacterium at 30°C for 24 hr in a medium containing 0.5% glycerol, 0.5% yeast extract, 0.5% Polypepton, 3.0 vol % soybean protein hydrolyzate, 0.1% KH₂PO₄, 0.1% MgSO₄•7H₂O, 0.001% FeSO₄•7H₂O and 0.001% MnSO₄•5H₂O in tap water (pH 8.0).

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Leaf aldehyde, (E)-2-hexenal, in diethyl ether, was converted to an ether adduct, 3-(1-ethoxyethyl)-hexanal (diastereomeric mixture) regioselectively, by irradiation with a 100 W high-pressure lamp.

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Quaternary structure of ribulose-1, 5-bisphosphate (RuP₂) carboxylase from the autotrophically grown cells of blue-green alga, Anabaena cylindrica, was studied. Sedimentation coefficient (s_{20, w}) of the enzyme was determined to be 18.3 S by the sucrose density gradient centrifugation. The molecular weight was estimated to be 5.0×10⁵ by the Sepharose 4 B gel filtration technique. The purification of the enzyme from the algal cells was undertaken by means of sucrose density gradient centrifugation and DEAE-Sephadex A-50 ion-exchange column chromatography, and the structural make-up of the enzyme containing two subunits, A (M. W., 5.2×10⁴) and B (M. W., 1.2×10⁴) was established by the Na-dodecylsulfate polyacrylamide gel electrophoresis experiment. Structural similarity of the algal RuP₂ carboxylase with the spinach enzyme was further demonstrated by the Ouchterlony double immunodiffusion experiment.

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Callus was induced from the veins and young petioles of mulberry leaves on modified Linsmaier and Skoog medium with the addition of 3% sucrose, 10^-5M of auxin and 10^-7M of kinetin. The growth rates of both solid and liquid cultures were nearly the same. The best growth rate was 3_??_4 fold during a 2 week culture period in a jar-fermenter.
To use cultured mulberry cells instead of fresh leaves as the major ingredient in artificial silkworm food, cells needed to be cultured under light to produce a small amount of chlorophyll. IBA was the most effective auxin for obtaining slightly green cells while 2, 4-D was ineffective.
Silkworms fed artificial food which included these cultured mulberry cells, spun good cocoons.

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Taxonomic and some other properties of a yeast strain, Candida sp. 36, which characteristically assimilates n-alkanes, were described. Identification of coenzyme Q, NMR spectroscopy of cell wall polysaccharides, determination of G+C content of DNA and some DNA-DNA hybridization experiments were carried out, in addition to the morphological and physiological observations. All the data were consistent with the suggestion that Candida cloacae Komagata, Nakase and Katsuya and Candida subtropicalis Nakase, Fukazawa and Tsuchiya are the synonyms of Candida maltosa Komagata, Nakase and Katsuya. Candida sp. 36 was identified as C. maltosa, too. The yeast was found to grow most abundantly on n-hexadecane and on n-octadecane in the presence of biotin.

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An attempt was made to estimate the base composition of DNA from UV absorption spectrum of its enzymatic digest. Preheated DNA sample was denatured, dialyzed and hydrolyzed to nucleotides with nuclease P 1. A half of the digest was dialyzed and absorbance of the un-dialyzed digest was read at 16 different wavelengths with the dialyzed one as a reference. The data were input to a computer with standard extinction coefficients. The G+C contents thus obtained were fairly reproducible and reliable. An easier computing with a table calculator was found to be satisfactory for a routine work. It was noted that mathematically correct program does not always give reasonable answers. Base compositions of some DNA estimated by this method were also reported.

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