Colistin, a fatty acyl peptide antibiotic, was attacked by proteolytic enzymes such as papain, ficin and bromelain, and as degradation product, a peptide portion retaining the ring structure of colistin was liberated. In contrast, an analogous antibiotic polymyxin B showed a characteristic resistance to the catalytic activity of papain.
Colistin nonapeptide and α-N-fatty acyl α, γ-diaminobutyric acid were obtained as products from the above enzymatic hydrolyzates of colistin and their chemical and physicochemical properties were investigated.
Contrary to colistin, this colistin nonapeptide was inactive to Escherichia coli. NIHJ and to many other strains even at a concentration of 800mcg/ml by the agar dilution method. As α-N-fatty acyl α, γ-diaminobutyric acid which is rest part of colistin was added to colistin nonapeptide, antimicrobial activity of colistin nonapeptide did not increase.

View full abstract

Ovalbumin, L-ascorbic acid and cupric sulfate were allowed to react at pH 3.0, 6.0, 6.8 and 7.5. Non-proteinous nitrogen compounds were formed from ovalbumin coupled with autoxidation of ascorbic acid, and a pronounced increase in their formation was observed in the reactions of neutral pH ranges. Non-proteinous nitrogen compounds contained peptides, free amino acids and ammonia. In the reactions of ovalbumin with triose reductone similar results to those with ascorbic acid were obtained. In the ovalbumin degraded with ascorbic acid at pH 6.8 was found an increase of N-terminal amino acid, which could react with carbonyl compounds resulting in browning.

View full abstract

APS-I, an acidic polysaccharide isolated from soy sauce, was subjected to Smith degradation and methylation, and each product obtained was examined by paper chromatography, gas-liquid chromatography and mass spectrometry. Some compounds, such as ethylene glycol, propylene glycol, glycerol, n-threitol, D-galactose, L-rhamnose, D-galacturonic acid and D-threonic acid, were identified as Smith degradation products, and their molar ratios were estimated. Several kinds of methylated sugars were detected in the hydrolysate of methylated APS-I and identified by gas chromatograph-mass spectrometry. On the basis of those findings and previous discussion, the constitution of APS-I was discussed.

View full abstract

Some proteolytic digests of chicken egg white ovomucin were fractionated and characterized. It was shown that there are at least three types of carbohydrate side chains in ovomucin; a chain composed of galactose, galactosamine, sialic acid and sulfate in a molar ratio of about 1:1:1:1, a chain composed of galactose and glucosamine in a molar ratio of about 1:1, and a chain composed of mannose and glucosamine in a molar ratio of about 1:1. It was also shown that the carbohydrate side chain composed of galactose, galactosamine, sialic acid and sulfate is linked O-glycosidically to serine or threonine in the protein core of ovomucin.

View full abstract

The hydrolysis of olive oil by the Humicola lipase was inhibited by the addition of n-alcohols, fatty acids and surface active agents. The inhibition of n-alcohols was overcomed by the addition of more substrate but not by the addition of more enzyme. The inhibition of fatty acids and bile salts was eliminated by adding calcium ion. It was concluded that the inhibition of the Humicola lipase by n-alcohols, fatty acids and bile salts was not due to inactivation of the enzyme directly but due to the displacing of the substrate from the oil/water interface, thus blocking the enzyme from the substrate.

View full abstract

Some physical and chemical properties of the extracellular lipase from the thermophilic fungus, Humicola lanuginosa S-38, were investigated. The results were as follows: Sedimentation coefficient was 2.4×10^-13 (cm•g/sec•dyne); diffusion coefficient was 8.8×10^-7(cm²/sec); and frictional coefficient was 1.22. Molecular weight was 27, 500±500 and α-helix content was 18.9%. The number of amino acid residues contained in 1 mole of protein of Humicola lipase was 224. Sugar and lipid were not detected. The effect of calcium ion and denaturing reagents, such as urea, sodium dodecyl sulfate and dithiothreitol, on the thermostability of Humicola lipase was examined. It was concluded that the thermostability of Humicola lipase was not influenced by protective cofactors but was attributable to the enzyme itself. Some properties of enzyme structure which were concerned with the thermostability of Humicola lipase are also discussed.

View full abstract

Relations among sensory properties, physical characteristics and the kinds of force supporting gel structure were studied by using thiolated gelatin.
Breaking stress of the gelatin gel was greatly influenced by contents of disulfide bonds in the gel. Young's modulus of the gel was affected by its temperature. As to sensory properties of the gelatin gel, “hardness” highly correlated with Young's modulus and “brittleness” correlated with breaking stress.
Therefore, it could be concluded that in the case of the gelatin gel, “hardness” was mainly attributed to hydrogen bonds and “brittleness” was to chemical bonds like disulfide linkages.

View full abstract

By use of pepsin a plastein was synthesized from a peptic hydrolysate of soybean protein and characterized as a protein-like substance, based on its behavior against trichloroacetic acid and its affinity to dyes. The contribution of the S-S bridge to plastein formation was little if any. The fractionation of the protein-like substance by solubility showed that the whole plastein-reaction product was constituted of 81.5 parts of the 50% ethanol-insoluble fraction; 63.8% of this fraction was also insoluble in 0.035M phosphate buffer (pH 7.6). The phosphate buffer-insoluble fraction was mostly solubilized in 0.3M sodium dodecyl sulfate (SDS). Although it was a minor component, there was a 50% ethanol-soluble and water-insoluble (prolamine-like) fraction which showed a reversible aggregation-dispersion change by a repeated heating-cooling treatment. A characteristic point of the SDS-soluble and prolamine-like fractions was found in their amino acid compositions. As compared with the substrate (peptic hydrolysate of soybean protein), they contained larger amounts of hydrophobic amino acids such as leucine than hydrophilic ones typified by glutamic acid.

View full abstract

This paper presents a study on the enzyme reduction of the disulfide bond and the following results have been found.
In enzyme preparation, antioxidants showed a stability effect and EDTA appeared to have both enzyme stabilization and solubilization. On the distribution of the enzyme activity in subcellular fractions, the water soluble fraction appeared to contain the major released enzyme activity. The enzyme was inhibited with several metals. Hg²⁺ and transition metals were the most toxic. The substrate specificity of this enzyme was wide for the low molecular substrates, but the protein disulfide reducing activity was not detected in this preparation. It was assumed that the thiol-disulfide transhydrogenase was coupled with glutathione reductase and the disulfide substrates were reduced by the system involving the two enzymes. A new method for the direct recording of an enzyme-catalyzed thiol-disulfide interchange using diphenyl disulfide and p, p'-dinitro diphenyl disulfide was devised.

View full abstract

A bacterium R-4 which produces a novel type of lytic enzyme which lyses fungal and yeast cell walls was isolated from the air and was identified to belong to the genus Bacillus.
Production of the enzyme appeared to require a high concentration of nitrogen source in medium. No inducing substance was needed for the enzyme production.
A crude preparation of the enzyme was used to characterize the lytic activity. From the lytic spectrum, the enzyme seemed to have the highest activity toward the cell walls of species in the genus Rhizopus among various fungi and yeasts tested. A proteolytic activity was shown to be parallel with the lytic activity. The lytic activity was also accompanied with the liberation of reducing sugars from Rhizopus cell wall, but no activity on some known carbohydrates tested was detected in the preparation.

View full abstract

About 500 strains of dextranase producing microorganisms were examined in detail for pH-activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibaeterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1% dextran, 2% ethanol, 1% polypeptone and 0.05% yeast extract together with trace amounts of inorganic salts.
Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10min.

View full abstract

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.
The crude enzymes, prepared by ammonium sulfate fractionation (0.2_??_0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A-50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5M NaCl. The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.
Fractions I and II were then separated by Sephadex G-100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.
The cell wall fraction prepared from C-3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C-3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.

View full abstract

The enzymatic properties of intracellular myrosinase produced by Aspergillus niger AKU 3302 were investigated. Maximum activity occurred at pH 6.2, and the enzyme was stable in a pH range of 7.6 to 8.0 at 5°C for 24 hr. Optimum temperature was about 34°C. Enzyme activity was stimulated by copper (I), (II), manganese (II) and cobalt (II) and was inhibited by mercury (II) and stannous (II) ions. However, metal complexing agents and DFP had little effect, while PCMB was a strong inhibitor. In contrast to plant myrosinase, this enzyme was neither activated nor inhibited by L-ascorbic acid. Glucosides and δ-gluconolactone inhibited enzyme activity but sugars were ineffective. The Km value for sinigrin was 3.3×10^-3M and that for p-nitrophenyl β-glucoside was 1.5×10^-3M. The relation between fungous myrosinases and β-glucosidase is discussed in comparison to plant myrosinase.

View full abstract

The degree of racemization in the several activated ester methods of the peptide synthesis was measured in using the critical racemization test, Pro-Val +Pro, with help of gas chromatography. The results were compared with that in the coupling reaction, Leu-Phe+Val, in which no racemization had been reported in the corresponding reaction conditions by F. Weygand et al., when the activated dipeptide esters had been prepared from Z-Leu+Pheactivated esters. The significantly higher racemization was observed in the methods of N-hydroxypiperidine ester and thiophenyl ester, even when the activated dipeptide esters were prepared from Z-Pro+Val-activated esters. On the other hand, almost no racemization was observed in the N-hydroxysuccinimide ester and p-nitrophenyl ester methods. A great extent of the racemization was detected when the activated dipeptide esters were prepared directly from Z-Pro-Val-OH.

View full abstract

The degree of racemization in the coupling reaction, Pro-Val+Pro, with the several other methods than the activated ester methods was measured and the results were compared with that in the coupling reaction, Leu-Phe+Val, as well as in the previous paper.¹⁾ In the azide and the mixed anhydride methods, no or almost no racemization was detected, whereas in the other tested methods of peptide synthesis (Pachornik-, DCCD- and phosphazo-methods) the significantly large racemization was observed. It can be attributed to the strong nucleophilic N-atom in the penultimate amino acids (Pro) and the steric hindrance of C-terminal amino acid (Val), which are favourable to the formation of the oxazolone ring.
This assumption was further systematically confirmed in the synthesis of the other several tripeptides with the DCCD method. The separation of the diastereoisomers (LLL and LDL) of the resulting tripeptides by gas chromatography with a packed column was also here reported.

View full abstract

Bacillus No. Ku-1 isolated from soil produced and alkaline catalase in alkaline media. The characteristic point of this bacteria was especially good growth in alkaline media. The alkaline catalase in the culture fluid was purified by DEAE-cellulose and Sephadex columns. The enzyme was most active at pH 10.0 and was stable at pH 7.0 to 8.5. The sedimentation constant was about 12.5S. The enzyme was strongly inhibited by NaN₃, KCN, FeSO₄ and Fe₂ (SO₄)₃. Properties of the enzyme are almost same as those of catalases so far reported except optimum pH for enzyme action and Kat.f. value (4.4×10⁴).

View full abstract

Deoxy-fructosazine and its 6-isomer were isolated from the browning mixture prepared by the reaction between glucose and ammonia in weak acidic medium, and were identified, respectively. The two compounds among all pyrazine derivatives formed in the reaction were incomparably abundant in approximately equal ratio. It was supposed that deoxy-fructosazine and its 6-isomer would be formed concurrently by condensation of ammonia with 3-deoxy-glucosone and isoglucosamine.

View full abstract

S-PI inhibited various acid proteases including pepsin, Rhodotorula glutinis acid protease and Cladosporium acid protease, but the rate of inhibition was different for each acid protease.
S-PI made an equimolar complex with these acid proteases. A part of the enzyme-S-PI complex dissociated in the reaction mixture and showed proteolytic activity. The specific activity of the enzyme-S-PI complex depended on the concentration of the complex in the reaction mixture. Compared with native (S-PI free) enzyme, each of the enzyme-S-PI complex showed 50% activity at the following concentrations, pepsin; 7.5×10^-10M, Rh. glutinis acid protease; 1.8×10^-7M, Cladosporium acid protease; 3.0×10^-6M.
These acid proteases were stabilized from heat or acid denaturation by making the enzyme-S-PI complex. S-PI protected the modification of these acid proteases by diazoacetyl-DL-norleucine methyl ester.
Binding between these acid proteases and S-PI dissociated at around neutral pH. S-PI was separated from enzyme-S-PI complex by dialysis at pH 7.5. In this case, pepsin underwent denaturation, while denaturations of Rh. glutinis acid protease and Cladosporium acid protease were slight. Rh. glutinis acid protease and Cladosporium acid protease were recovered from enzyme-S-PI complex by DEAE cellulose column chromatography as a native form.

View full abstract

An exopolygalacturonase was partially purified from mycelia_??_ extracts of a strain of Acrocylindrium. The enzyme was most active at pH 4.5 and showed a higher affinity for polygalacturonic acid than for oligogalacturonic acids. The enzyme was found to hydrolyze glycosidic linkages at the non-reducing end of polygalacturonic acid molecules, giving only monogalacturonic acid as the reaction product.

View full abstract

ATP-inhibited ribonuclease was first found in Bacillus amyloliquefaciens cells and was isolated by the authors previously. The distribution of the ATP-inhibited ribonuclease was surveyed on various bacteria. ATP-inhibited ribonuclease was found in only several species of Bacillus genus, i.e., B. subtilis (i), B. amyloliquefaciens (ii), B. cereus (iii), B. cereus var. mycoides (iv), and B. thuringiensis (v). ATP-inhibited ribonuclease of Bacillus genus was subdivided into two types. The first type of enzyme found in (i) and (ii) cells had optimum pH at 5.7 and was sensitive to 5mM EDTA. The second type of enzyme found in (iii), (iv), and (v) cells had optimum pH at 6.8 and was partially inhibited by 5mM EDTA.

View full abstract

The stability of RNA preparations which were prepared from soybean cotyledons was examined by incubating the RNA solutions of high salt at 20°C in the presence or absence of PVS. Sedimentation profiles of incubated RNA were given by ultracentrifugal analysis and compared with that of original RNA. RNA retained its original size after incubating for 4 hr in the presence of PVS and 200mM KCl, while RNA was completely degraded into small fragments in the absence of PVS after the same treatment.
The purified rRNA which was prepared from 3 day-old hypocotyls was treated with heating, EDTA or urea in the presence of PVS. L-rRNA component was obviously disappeared by heating at 50°C for 5min. Partial disruption of L-rRNA component occurred by dialyzing against urea solution. L-rRNA separated by zonal ultracentrifugation was decomposed into components by heat treatment or leaving at 4°C for 20 hr in the buffer from which KCl was omitted. No back-conversion of heated RNA to original L-rRNA occurred by gradual cooling at room temperature for 40min. S-rRNA, however, seemed to be stable in these treatments compared with L-rRNA.

View full abstract

The contents of plasma free amino acids, the amounts of urinary excreted amino acids and urea, and the activities of liver serine dehydratase, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase were determined in weanling rats fed ad libitum a 10% casein diet (control), a 10% casein diet containing 7% glycine and 10%. casein diets containing 7% glycine supplemented with 1.4% L-arginine and/or 0.9% L-methionine for 14 days.
The remarkable increase of glycine and the moderate increase of serine in the plasma of animals fed excess glycine diets were observed. The amount of excreted glycine in the urine of animals fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than that of animals given the excess glycine diet. Urinary excreted urea of rats fed the excess glycine diet was a little greater and that of rats fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than the control. Liver serine dehydratase activity of animals given the excess glycine diets with or without L-arginine was higher than the control and the highest activity was observed in the liver of animals fed the excess glycine diet containing L-arginine and L-methionine. The activity of liver glutamicoxalacetic transaminase of rats fed the excess glycine diet containing L-arginine and L-methionine was a little higher than that of rats given the other diets. Liver glutamic-pyruvic transaminase activity was a little higher in animals given the excess glycine diets with or without L-arginine and further higher in animals fed the excess glycine diet containing L-arginine and L-methionine than the control.

View full abstract

A novel synthesis of a N-acetyllincosamine isomer (8) was accomplished.

View full abstract

A novel synthesis of N-acetyllincosamine derivatives (3) and (14) was accomplished.

View full abstract

A novel synthesis of N-acetyllincosamine derivatives (17) and (14) was accomplished.

View full abstract

Self-association and the state of a tyrosine residue of the active fragment of bovine growth hormone were investigated by gel filtration and difference spectroscopy with various concentrations of acetic acid. In acetic acid solutions more concentrated than 1.5M, the active fragment was found to exist as a monomeric form. Decrease in the concentration of acetic acid promoted self-association of the active fragment. Dimeric and trimeric forms were observed in 0.1M acetic acid. By difference spectroscopy, the tyrosine residue in the active fragment showed a typical blue shift in 2.5M acetic acid compared to 0.5M acetic acid. The transition concentration, 1.25M of acetic acid for the association-dissociation of the fragment was consistent with that for the appearance of the blue shift difference spectrum. It was concluded that a tyrosine residue in the active fragment was exposed on the surface of the fragment molecule in the monomer form and was held between the fragment molecules under conditions in which the active fragment forms dimer and trimer.

View full abstract

In the course of selecting a useful mutant strain for a fermentative production of L-valine, it was found that an arginine-pyrimidine auxotroph of Paracolobactrum coliforme accumulated N^δ-acetyl-L-ornithine (δ-AO) in the culture medium. The accumulation of it reached a level of 16mg/ml with medium containing 12.5% glucose, 2.2% (NH₄)₂SO₄, 0.5% peptone and 300μg/ml of uracil. The wild strain 775 also accumulated 1.4mg/ml of δ-AO in the medium supplemented with a high level (300μg/ml) of uracil when L-ornithine (10mg/ml) was added in the middle phase of fermentation. The mutant cells elongated under the condition with limited supply of uracil.
The mechanism of the accumulation of δ-AO was discussed from the information of relevant biosynthetic regulation in other organisms.

View full abstract

Alterations in the degrees of saturation of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin of Escherichia coli K-12 were determined after raising or lowering the growth temperature during the exponential growth phase. After raising the growth temperature from 17 to 42°C, the cells continued to grow with increasing degrees of saturation of the three phospholipids. cis-9, 10-Methylenehexadecanoic acid increased only in phosphatidylethanolamine. During growth after lowering the growth temperature from 42 to 17°C, no increase was found in cyclopropane fatty acid content of phosphatidylethanolamine, in which cis-vaccenic acid increased. Significance of cyclopropane fatty acid formation in phospholipids was discussed.

View full abstract

Various lipid extraction methods were applied to Lipomyces starkeyi, freeze-dried, heatdried and intact cells. It was found that the freeze-dried cells were usually more extractable than other types of cells. A high lipid recovery was obtained by a lytic enzyme (Corticium centrifugum) treatment, conc. HCl treatment and Pedersen's method using chloroform-methanol (1:1). The first two methods, however, hydrolyzed phospholipids and released free fatty acids during the extraction of lipids. From the results we have obtained, the best method for lipid extraction from L. starkeyi is that in which the freeze-dried cells are extracted by the Pedersen's method. The results obtained from the application of these methods to a hydrocarbon-grown yeast are also described.

View full abstract

The extra-cellular filtrates of Trichoderma viride ITCC-1433 showed considerable cellulolytic activity against native celluloses, cellulose derivatives and raw materials. Newspaperyellow and the rice straw were the prominent waste materials which were preferentially attacked by the enzyme. The alkali treatment of the latter doubled the sugar formation from it. As a result of cellulase action 80.4 per cent of the MN-Cellulose and 60.4 per cent of thealkali treated rice straw lost weight in 96 and 48 hr respectively. The weight loss was more or less equivalent to the reducing sugars formed.

View full abstract