Three forms (1, II, and III) of R 5-P ketol icomerase (EC 5.3.1.6) were separated and purified from Candida utilis. The Kms for R 5-P by I, II, and III were 0.77mM, 0.67mM and 0.16mM, respectively. The isoelectric points of the forms, I, II, and III were 3.79, 3.87 and 3.99, respectively.
The activation energies of the forms, I, II, and III were 10, 800, 12, 000 and 6360 cal/mole, respectively. The forms, I, II, and III were all inhibited with chelating reagent, EDTA (1mM), but not affected with thiol reagents such as p-CMB, ICH₂COOH, NEM, and DTNB. These properties are quite different from the other known R 5-P ketol isomerases, all of those are not affected with EDTA, but inhibited with the thiol reagents as described above.

View full abstract

Cystathionase decreased in activity during sporulation of Bacillus subtilis. The experimental data obtained from the extract of vegetative cells and sporulating cells suggest that the decrease in cystathionase activity was caused by a heat-labile protease in cells. The extract from sporulating cells was applied to a DEAE-cellulose column and a Sephadex G-75 column. In both chromatograms Fraction I having azocasein-hydrolyzing activity (protease B) showed cystathionase-inactivating activity. Cystathionase was inactivated by protease B in the absence of pyridoxal 5'-phosphate (PLP) but its activity was maintained in the presence of PLP in vitro condition. During sporulation cystathionase level in cells decreased after t_1.5 stage, and the intracellular level of phosphorylated vitamin B₆ decreased after t₂ stage. On the other hand, the production of intracellular protease B commenced at t₀ stage. From these data it is suggested that cystathionase activity in vegetative cells of B. subtilis is maintained by PLP from to t₀ to t_1.5 stage, in spite of the presence of protease B in cells. The inactivation of cystathionase by protease B may begin after intracellular phosphorylated vitamin B₆ decreased rapidly at t₂ stage.

View full abstract

In the screening of actinomycetes' culture filtrate for inhibitor of subtilisin and various microbial alkaline proteinases, a novel inhibitor was found in a cultured broth of strain WT-27. This inhibitor was named as MA-PI, abbreviation of microbial alkaline proteinase inhibitor.
Judging from the morphological and physiological properties of the actinomycetes which produced MA-PI, this strain was identified as Streptomyces nigrescens.
For the production of MAPI, this strain was aerobically cultured at 25_??_27°C in a jar fermentor which contained an optimum medium consisting of polypepton 3%, meat extract 1%, glucose 1%, NaCl 0.1%, K₂HPO₄ 0.1% and MnSO₄•nH₂O 0.0001%, pH 7.0. The production of MAPI reached its maximum after 21_??_24hr cultivation.
MAPI had an inhibitory activity against various microbial alkaline proteinases, α-chymotrypsin and papain but not against trypsin, kallikrein, thermolysin, or pepsin.

View full abstract

It has been shown by the preliminary experiment that the insolubilized DNA prepared by the direct addition of multivalent metal ion to DNA solution differed greatly from that by electrolysis in the contents of water absorbed and protamine combined to insolubilized DNA, etc. For the elucidation of their molecular differences the condition of insolubilization of DNA by electrolysis must be established, which has been investigated in this paper.
Results obtained are summarized as follows:
The yield of insolubilized DNA was positively corelated with the increase in both concentration and pH of DNA solution, while it decreased gradually with increasing temperature. The amount of insolubilized DNA increased with the increment of current passed. Copper or nickel was preferred as the electrode plates. The current efficiency was dependent on the concentration of DNA solution. The current density was not related to the formation of insolubilized DNA.

View full abstract

The activities of polyphosphate glucokinase which synthesizes glucose-6-phosphate from glucose and metaphosphate were found in some microorganisms. This enzyme occurred most abundantly in Micrococcus and Achromobacter species and less in Brevibacterium, Aerobacter and Alcaligenes species. The distribution pattern of this enzyme was closely similar to that of polyphosphate NAD kinase. Polyphosphate glucokinase in A. butyri was different from ATP-dependent glucokinase in some enzymatic properties. The potent phosphoryl donor for this enzyme was metaphosphate, and other chain or ring phosphate polymers were not utilized by this enzyme.

View full abstract

A rapid and large scale isolation of chymosin was achieved by an affinity column including pepstatin-aminohexylagarose with a column of DEAE-cellulose. The affinity column with 3ml wet gel made it possible to isolate 90mg pure chymosins (I, II and III) from 20g crude rennet tablet. Isoelectric points of chymosin-I, -II and -III were 4.6, 4.6 and 4.5, respectively. Each of chymosins was homogeneous electrophoretically and chromatographically.

View full abstract

Egg white solids freeze-dried with or without the addition of glucose were stored at 50°C under 65% relative humidity to study the effect of the Maillard reaction on the solubility and heat stability and the formation of aggregates. A stimulative effect on the former properties was observed on the sample with glucose in the initial stage of the Maillard reaction. By comparing the SDS-polyacrylamide gel electrophoretic patterns, solubilities in SDS and SDS/2-mercaptoethanol and the s_{20, W} values, it was found that glucose, in addition to the effect due to the changes of charged groups in glucose-protein complex occurring in the course of the Maillard reaction, might have a protective effect against aggregate formation through a stable cross-linking, which was not dependent on the SS bond, and through a stable non-covalent bond.

View full abstract

A precursor of a known long life free radical species which gives a triplet ESR signal was detected among the reaction products of dehydroascorbic acid and amino acids. Experimentally, this compound was transformed to the radical species by dilution with 1:1 pyridine-water or some buffer solutions of pH 5_??_7. The yield of the precursor was significantly improved by the addition of ascorbic acid to the reaction system.

View full abstract

Transketolase mutants derived from Bacillus pumilus IFO 12089 produced two unknown compounds. One of them was isolated from the culture broth and was determined to be a new monosaccharide, 1-deoxy-D-altro-heptulose (1-deoxy-sedoheptulose) (1). Compound 1 was easily converted into its non-reducing anhydride in acidic solution at room temperature, which was identified as 2, 7-anhydro-l-deoxy-β-D-altro-heptulopyranose (2). Compounds 1 and 2 were also chemically synthesized from 2, 7-anhydro-β-D-altro-heptulopyranose (3) to confirm the chemical structures.

View full abstract

The structure of a galactan, obtained from the pectic polysaccharides of the midrib of the leaves of Nicotiana tabacum, was investigated by methylation analysis and partial acid hydrolysis. The galactan contained only n-galactose and was composed of a straight chain of β-(1→4)-linked D-galactopyranosyl residues.

View full abstract

Endosperm cell walls were isolated from rice grains and their chemical composition was analyzed. The cell walls were composed of cellulose microfibrils and matrix phase which consisted of hemicellulose and pectic substances. Hemicellulose mainly comprised arabino-xylan, accompanied by a small amount of glucose-containing polysaccharide. Pectic substances contained polygalacturonides, some of which had side chains containing neutral sugars such as galactose and arabinose. Amino acid analysis of these fractions suggested that hydroxyproline-containing glycoproteins were contained in these cell walls and firmly bound to cellulose microfibrils.

View full abstract

A hemicellulosic polysaccharide, which was homogeneous on sedimentation analysis and also on electrophoresis, was isolated from the rice endosperm cell walls by the combination of alkaline extraction, ion exchange chromatography and iodine complex formation. It is composed of arabinose, xylose and glucose (molar ratio, 1.0:2.0:5.7) together with a small amount of galactose and rhamnose. Methylation analysis, Smith degradation and frag-mentation with cellulase showed that this polysaccharide is composed of three distinct polysaccharide moieties i.e., xyloglucan, β-glucan and arabinoxylan. The xyloglucan consists of β-(1→4)-linked glucan back bone and short side chains of single xylose units or galac-tosylxylose both attached to C-6 of the glucose residues. The β-glucan contains both (1→3)-and (1→4)-linkages similarly to the other cereal β-glucans, but differ from them in containing the blocks of (1→3)-linked glucose residues in the chain. The arabinoxylan has a highly branched structure, in which 78% of (1→4)-linked xylose residues have short side chains of arabinose at C-3 position.
On the basis of these findings, the interconnection of these polysaccharide moieties is discussed.

View full abstract

A methanol-utilizing bacterium, strain SK-200, which was isolated as an L-serine producer, was identified as Arthrobacter globiformis. The strain excreted a considerable amount of L-serine when glycine and glucose were added in a methanol medium. Cultural conditions for L-serine production were studied. Derivation of mutants increased the productivity of L-serine. A methionine auxotroph produced 5.2mg per ml of L-serine.

View full abstract

Aromatic hydrocarbons such as anisole, benzene, and toluene, are hydroxylated by treating with the reaction system containing reductone, hydrogen peroxide, and molecular oxygen. Main products were the corresponding phenols. In the hydroxylation of toluene, •OH scavengers and superoxide dismutase were added to this reaction system. The hydroxylation was inhibited by either the scavenger or the enzyme. These results suggest that •OH and O₂ are present in the process of this reaction and the active species in the final step of this reaction is •OH. The mechanism of the hydroxylation is discussed by comparing with the Udenfriend system, Fenton's reagent and photolysis of hydrogen peroxide.

View full abstract

The equilibrium moisture contents of sun-cured (Kroumougrad), flue-cured (Bright Yellow-4) and air-cured (Burley-21 and Matsukawa) tobaccos were measured over a relative humidity range from 5 to 80% at 20°C. The moisture sorption isotherms of tobaccos were of sigmoid type, and classified into two groups. In a lower humidity range below ca. 40% RH, the A group (Kroumougrad and BY-4) had a smaller moisture sorption capacity than B group (Burley-21 and Matsukawa), while in a higher humidity range above ca. 50 % RH the former had a larger moisture sorption capacity than the latter. By extracting with water, the moisture content of BY-4 was increased in the lower humidity range, while it decreased in the higher humidity range. However, the moisture content of Matsukawa was scarecely changed by extracting it with water. These results suggest that the differences in equilibrium moisture content with the type of curing were due to the differences in contents of water soluble components. To control the hygroscopic properties of a tobacco, therefore, the influences of the addition of sucrose and glycerol on the equilibrium moisture content were quantitatively analysed. The moisture sorption capacity of tobacco was greatly different from its nitrogen sorption capacity. The specific surface area of tobacco calculated from moisture sorption isotherm was ca. 110 times larger than the specific surface area calculated from the nitrogen sorption isotherm. Both the nitrogen and moisture sorption data should be necessary for better understanding of the complicated sorption-desorption phenomena in tobaccos.

View full abstract

Crystallized α-globulin of sesame seed sedimented as 13S component in 1M NaCl at pH 8.0. Basic and acidic subunits of the globulin were separated by ion exchange chromatography. The molecular weights of the subunits were estimated to be 21, 000 and 28, 600, respectively. The basic subunit contained glycine and the acidic one leucine (or isoleucine), as N-terminus. The amino acid analysis indicated that the basic subunit was rich in methionine, 4.5%. The 13S globulin showed a temperature-dependent dissociation phenomenon. Only when the globulin had been frozen, it dissociated into 7S component in 1M NaCl solution at 4°C, but not at 20°C.

View full abstract

Solubilization of milk fat globule membrane (MFGM) and its apoprotein was attempted by using the nonionic detergents such as Triton X-100, Tween 20, Nonidet P-40, Liponox NCK and Emulgen 109-P. About 40 to 60% of MFGM and 5% of its apoprotein was solubilized with the nonionic detergents alone. In these cases, selective solubilization was observed on SDS-polyacrylamide gel electrophoresis. The supplementary factors such as the presence of urea and an increase in pH were found to enhance the solubilizing ability of Triton X-100. In particular, a combination of 8 m urea with 2_??_5% nonionic detergents in the presence of 0.2 2-mercaptoethanol accomplished complete solubilization of MFGM and its apoprotein. After complete solubilization the removal of urea by dialysis in the presence of the detergent did not cause the reaggregation of protein-detergent complexes. The biologically active components such as marker enzymes of MFGM in the completely solubilized protein fraction was denatured with urea.

View full abstract

Guanidine hydrochloride (GuHCl) solubilized 35% and 60% of the constituent proteins of isolated milk fat globule membrane (MFGM) at the concentrations of 1.8M and 6.0M, respectively. The hydrophilic amino acid and carbohydrate contents of the soluble fractions were slightly greater than those of the insoluble fractions. Sodium dodecylsulfate-polyacryl-amide gel electrophoresis revealed that the solubilization was highly selective. Polypeptides such as CB-7+8, 12, 13, 14 and 15 were easily solubilized with 1.8M GuHCl, and PAS-1, 2, 3, 4 and CB-1 required 3_??_6M GuHCl to attain their complete solubilization, while CB-5 and 6 were highly insoluble.
Selective release of the proteins from the native MFGM (washed cream) by GuHCI extraction (0_??_2.1M) was also observed; i.e., some minor polypeptides, such as CB-12, 13, 14, 15 and 16 were readily solubilized with 0.6_??_1.2M GuHCl, and a major glycoprotein, CB-7-4 8 (PAS-6-f-7), was solubilized with 1.8_??_2.1M GuHCl. The results suggested that these polypeptides were peripheral proteins and bound with other components of the membrane by relatively weak interaction. On the other hand, the proteins remaining in the cream fraction seem to bind with other components of the membrane by relatively strong interaction.

View full abstract

Cultural conditions of Ps. thiazolinophilum AJ 3854 for the production of the enzyme which could form L-cysteine from DL-2-amino-Δ²-thiazoline-4-carboxylic acid (ATC) and the reaction conditions of this enzyme were investigated. This enzyme was perfectly inducible, intracellular and growth-associated type. A remarkable inactivation of enzyme was observed especially in the growing phase but could be prevented by the addition of 1-10mM Mn²⁺ or by the feeding of ATC as the inducer at the mid-logarithmic phase. Enzymatic degradation of L-cysteine (or L-cystine) formed from ATC could be prevented by the addition of hydroxylamine or semicarbazide. Thus, L-cysteine and L-cystine were quantitatively produced from ATC. Optimal pH and temperature of enzymatic reaction were 8.2 and 42°C (2hr), respectively. A sigmoidal reaction curve was observed when intact cells were used as enzyme source, but sonic treatment of cells made the curve linear.

View full abstract

The kinetics of the tryptic proteolysis of glycinin were studied by the pH-stat method. The results clearly indicated the existence of at least two simultaneous reactions proceeding at different rates. The rate constants suggested that a group of peptide bonds was much more susceptible to proteolytic attack than others.
In addition stable digestion intermediates were expected in the degradation course. In a low ionic strength condition, 3 or 4 electrophoretic bands were formed, whereas only single band was seen in high ionic strength buffer. The major intermediate in a high ionic strength condition (glycinin-T) was isolated by Sepharose 6B chromatography. Glycinin-T showed a single band on polyacrylamide gel electrophoresis. Acidic subunits of glycinin were digested forming 13, 500 and 16, 000 molecular weight fragments, whereas basic subunits were not digested at all.

View full abstract

Particulate alcohol dehydrogenase of acetic acid bacteria was purified to homogeneous state from Acetobacter aceti IFO 3284. The enzyme was purified about 70-fold with an overall yield of 55% from the cell homogenate by solubilization and extraction of the enzyme with Triton X-100 and subsequent fractionations on column chromatography. The purified enzyme was revealed of its properties as flavo-cytochrome complex and was composed of four different subunits having a molecular weight of 63, 000, 44, 000, 29, 000 and 13, 500. A tightly bound cytochrome component was not alcohol dehydrogenase itself and had a function as an electron acceptor in vivo. The first subunit which reacts with ethanol was shown to be a flavoprotein of the particulate alcohol dehydrogenase complex. Catalytic properties of the enzyme were also examined and the data that the enzyme is a representative as the vinegar fermenter were ob-tained.

View full abstract

Molecular weight of α-aminoisobutyrate (AIB) decomposing enzyme was estimated to be about 180, 000 by the Hedrick-Smith's method and dodecylsulfate-polyacrylamide gel electrophoresis of this enzyme showed a single band having molecular weight of 45, 000. Spectrophotometric titration of the enzyme with pyridoxal 5'-phosphate at pH 7.5 gave four equivalent binding sites per mole. Therefore, AIB decomposing enzyme seemed to be a tetrameric form of a single polypeptide chain.
The enzyme was inactivated by phenylglyoxal. In partial inactivation of the enzyme with phenylglyoxal, both AIB decomposing activity and L-alanine: α-ketobutyrate transaminase activity were proportionally lost. This finding was consistent with the concept of a single active site for both activities.
The holoenzyme was converted into simple aminotransferase by modification with diethylpyrocarbonate, indicating that only AIB decomposing activity was inactivated. The com-plete inactivation of AIB decomposing activity was achieved by the modification of 1 to 1.5 histidyl residues per monomer of the enzyme. Amino acid specificity and a-keto acid specificity in transamination of the converted simple aminotransferase were not different from those of native enzyme.

View full abstract

New enzymatic microdetermination of D-gluconate was developed. D-Gluconate was assayed by a direct enzymatic oxidation to 2-keto-D-gluconate by particulate D-gluconate dehydrogenase which was prepared from acetic acid bacteria or other oxidative bacteria. This method was highly specific to D-gluconate and stoichiometry was achieved satisfactorily. Since the particulate D-gluconate dehydrogenase showed its optimum pH in D-gluconate oxida-tion at fairly acidic pH, this enzyme was favorable for the practical purposes in determining D-gluconate content in brewing products or other foodstuffs, many of which are fairly acidic or highly bufferized at acidic pH, without any preadjustment of sample's pH before the assay. It gave a pretty determination of D-gluconate by measuring the initial reaction rate as well as end point determination.
Alternative enzymes for D-gluconate determination, 2-keto-D-gluconate reductase or 5-keto-n-gluconate reductase which were prepared from acetic acid bacteria, were also available for the purpose. D-Gluconate was assayed quantitatively from the initial reaction rate by reading the formation of reduced NADP.

View full abstract

Membrane-bound D-gluconate dehydrogenase, which catalyzes oxidation of D-gluconate to 2-keto-n-gluconate in the presence of an electron transport system, was purified to homo-geneous state from Serratia marcescens IFO 3054. Purification was achieved at about 450-fold with an overall yield of 18% from the particulate fraction by solubilization with Triton X-100, subsequent fractionations on DEAE-cellulose and DEAE-Sephadex column chromato-graphies, and precipitation with polyethylene glycol 6000. The purified enzyme preparation was found to be an enzyme-cytochrome complex and the dehydrogenase protein tightly bound cytochrome c₁ component which had the absorption maxima at 418, 523 and 554nm. The substrate specificity of the enzyme seemed to be restricted to only D-gluconate and the apparent Km value of the purified enzyme for D-gluconate was 0.8×10^-3M. Enzyme activity was inhibited by pyruvate competitively and by oxamate noncompetitively.

View full abstract

ω-Amino acid: pyruvate aminotransferase of Pseudomonas sp. F-126 catalyzes stoichiometrically a transamination between various amines and pyruvate. Most of alkyl and aromatic monoamines served as an amino donor. The enzyme activity was affected by carbon number of straight-chain alkylmonoamines with a maximum activity at 5-carbon unit, n-amylamine. Michaelis constants for n-butylamine and pyruvate were calculated to be 66.6mM and 5.5mM respectively. The enzyme was active in the alkaline range with a maximum at pH 10.5_??_11.0, though not any activity was observed at the pH below 8.0. The optimum temperature for the reaction was at 60°C.

View full abstract

The structural features of the sugars required as an acceptor of cyclodextrin glycosyl-transferases from Bacillus megaterium and B. macerans were investigated using 22 kinds of monosaccharides and their derivatives. It is concluded on the basis of the experimental results that the requirement for an acceptor of the intermolecular transfer reaction catalyzed by this enzyme is the pyranose structure having the same configurations of the free C2-, C3- and C4-hydroxyl groups as D-glucopyranose. The reaction products of the B. megaterium enzyme from starch and 2-deoxy-D-glucose, a poor acceptor, were analyzed by paper chromatography. The main product was isolated and identified by the chemical and enzymatic methods as 4-O-α-D-glucopyranosyl-2-deoxy-D-glucose. The products contained a series of ordinary maltooligo-saccharides besides a series of maltooligosaccharides each terminated by a single 4-O-α-D-glucosyl-2-deoxy-D-glucose residue at the reducing end.

View full abstract

A simple method for simultaneous determination of diphenyl and o-phenylphenol in citrus fruits was established. Fruits were distilled with a distillable oil analyzer. The citrus fruit extract was taken from this apparatus and anthracene was added as an internal standard. Gas chromatography was carried out with a column packed with 10% FFAP and a flame ionization detector. Column temperature was increased from 150 to 210°C. The retention times of diphenyl, o-phenylphenol and anthracene were 4.2, 14.0 and 15.4min, respectively.
This method was completed within 2.5 hr and applied to samples of grapefruits, lemons, oranges, navel oranges, ponkan, iyokan, hassaku, unshu mikan and amanatsu mikan. In the recovery tests with these fruits, diphenyl was recovered in the range from 90.1 to 96.9% and o-phenylphenol was recovered in the range from 86.5 to 99.3%.

View full abstract

Syntheses of the key enzymes of the glyoxylate cycle, in Candida lipolytica, were highly repressed by glucose. Syntheses of the key enzymes of the methylcitric acid cycle were also slightly repressed by glucose but the degrees of repression in the syntheses of these enzymes were nearly equal to those of repression in the syntheses of several enzymes of the citric acid cycle. All enzyme syntheses repressed by glucose were derepressed during incubation with succinate as well as with n-alkanes: enzyme syntheses of the methylcitric acid cycle did not necessitate the addition of propionate or odd-carbon n-alkanes. The enzymes of the methylcitric acid cycle seem to be constitutive, similarly as those of the citric acid cycle.
In the parent strain, the respective enzyme levels of the cells grown on an odd-numbered n-alkane were similar to those of the cells grown on an even-numbered n-alkane. But in the mutant strain lacking 2-methylisocitrate lyase, the cells grown on the odd-numbered alkane contained aconitate hydratase, NADP-linked isocitrate dehydrogenase, isocitrate lyase, 2-methylcitrate synthase and 2-methylaconitate hydratase all at higher levels than the cells grown on the even-numbered alkane. Both the parent cells and the mutant cells grown on the same carbon source contained at individually similar levels of the following six enzymes; citrate synthase, NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase, and malate synthase. The pleiotropic changes of enzyme activities in the mutant cells grown on the odd-numbered alkane seem to be ascribable to direct or indirect stimulation caused by threo-D_s-2-methylisocitric acid accumulation.

View full abstract