The phase change for an amylose solution in the binary solvent system of dimethylsulfoxide (DMSO) with water was investigated under various conditions from sol to gel. The phase change was determined with measurements of the fluorescent depolarization and other methods by varying the solvent constitution at 25°C, and then varying the temperature at 10%ofDMSOconcentration.
The phase diagrams obtained with both variables were substantially similar and were also similar to those for an aqueous agarose solution. This similarity in phase diagram suggests a similar gel formation mechanismof amylose to agarose.
It was found that the phase separation point for the amylose solution agreed with the gel formation point and also with the starting point of retrogradation.

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The phase change for an amylose-butanol complex solution in 10%of dimethylsulfoxide was investigated as a function of temperature. The phase change was determined with measurements of the turbidity, fluorescent depolarization, and viscosity. The phase diagram obtained was qualitatively similar to that for an amylose solution. From the result, the change in solution phase for the amylose-butanol complex is suggested to be similar to that for amylose, i.e., when the solution cools from a higher temperature, amylose molecules in the complex solution change the conformation from a random coil to an interrupted helix, and then separate into two phases. Coacervate particles resulting from the phase separation coalesce with each other to yield precipitates.
An adsorption of uranine on amylose was studied to ascertain its relationship with the fluorescent depolarization method used for detecting phase changes in solution. The result showed that uranine was adsorbed on amylose chains but not on the amylose-butanol complex.

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In order to study the gelling mechanism of gelatin, the phase diagram for a diluted gelatin solution was investigated. Phase boundary curves were obtained by measuring the fluorescent depolarization, optical rotation, reduced viscosity, and light scattering of the gelatin solution. The phase diagram obtained exhibited similar features to those for amylose and agarose gelling systems. Fromthis similarity, the gelling mechanism of gelatin is concluded to be as follows: The randomly coiled gelatin molecule in hot solution partially recovers a collagen fold at the denaturation temperature near 40°C on cooling. This gelatin state is presumed to correspond to the interrupted helical form for amylose. This partial helical solution then separates into two phases, a dilute solution and a coacervate consisting of a concentrated solution, at a phase separation temperature which depends upon the gelatin concentration. When the concentration is sufficiently high, coacervate particles are connected with each other to form a three dimensional macroscopic network, and the solution loses its fluidity to become a gel.

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The intracellular concentration of vitamin B6 (B6) of a wild type strain (WG1) of Escherichia coli B remained almost constant (0.25 to 0.35 nmol per mg cells) when different amounts ofB6 were extracellularly added. However, B6-requiring mutants were strongly affected by extracellular B6.
Activities of tryptophanase in a B6-requiring mutant, strain WG3, grown under various conditions were measured. A clear correlation between intracellular B6concentration and the ratio of holo-tryptophanase activity (i.e., holo-activity/total activity) was observed.
Tryptophanase of strain WG3 grown under B6-deficient conditions and the enzyme of strain WG1 were purified and their properties compared. The purified enzymes of both strains WG3 and WG1 showedsimilar characteristics, but a difference was observed in the antigen activities.

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Biodegradation of 6-methyl-2, 3-dithiolquinoxaline cyclocarbonate or quinomethionate (Q₁) by cucumber seedlings seems to confirm that the thiocarbonate linkage is disrupted during metabolism of the fungicide by the plant.
The sulfur liberated is principally incorporated into sulphates and sulfur amino-acids, while the labeled (2, 3-¹⁴C) quinoxaline nucleus is catabolized to ¹⁴CO₂. This work also suggests that microorganisms can largely share in fungicide biodegradation when they are in the immediate environment of seedlings.

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A terrestrial green unicellular alga of the Ankistrodesmus falcatus genus vigorously degrades the 6-methyl-2, 3-dithiolquinoxaline cyclocarbonate (quinomethionate or Q₁), the active ingredient of Morestan.
A toxicity level is determined for the alga when Q₁, previously dissolved in dimethyl formamide is added to the cells.
When added to the culture medium without anything else, Q₁ is completely degraded. One of the metabolism pathways relates to the halogen metabolism and leads to the transformation of Q₁ into an aminochlorinated compound identified as 6-methyl-2-amino-3-chloroquinoxaline. This compoundis particularly toxic to cucumber plants.

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A gas-liquid chromatographic method was applied to the determination of anomeric forms of sugar produced by carbohydrases. Anomeric forms of glucose released from maltotriose, phenyl amaltoside and phenyl a-glucoside were determined quantitatively. Thirteen a-glucosidases tested, including a-glucosidase from honey bee and acid α-glucosidase from pig's liver, were found to produce a-glucose exclusively, and two kinds of glucoamylases, only β-glucose. This method proved very useful for the determination of the anomeric forms of sugar produced. It was confirmed that mammalian acid a-glucosidase does not belong to the category of exo-1, 4-α-glucosidase (EC 3.2.1.3).

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Anomeric forms of glucose and maltose produced from phenyl, p-nitrophenyl, p-tert-butylphenyl, p-ethylphenyl and p-chlorophenyl α-maltosides and maltopentaose by α- and β-amylases were determined quantitatively by a gas-liquid chromatographic method. All of the three kinds of α-amylases tested, B. subtilis saccharifying α-amylase, Taka-amylase A, and porcine pancreas α-amylase, were found to produce only α-maltose from the maltosides. Sweet potato and barley β-amylases produced β-maltose from maltopentaose.
Saccharifying α-amylase from B. subtilis also released α-maltose from all the maltosides mentioned above, contrary to the report by Shibaoka et al. that the enzyme released β-maltose from maltosides other than phenyl α-maltoside: FEBS Lett., 16, 33 (1971); J. Biochem., 77, 1215 (1975). It appears unlikely that the a-amylase releases β-maltose, depending on the kind of substrate.

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The aqueous extract of dried bonito (Katsuobushi) was distilled under reduced pressure. The resulting distillate with diethyl ether and the extract was separated into acidic, phenolic, basic and neutral fractions. The neutral fraction was further fractionated into ten sub-fractions by silica gel column chromatography. All these sub-fractions were analyzed by gas chromatography and gas chromatography-massspectrometry.
One hundred and sixty-five compounds were identified and 12 compounds were tentatively identified from the neutral fraction. Amongthem, 111 compounds were newly identified as flavor componentsof Katsuobushi.

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Two active forms (F-1 and F-2) of a maltotetraose-forming amylase [EC 3.2. 1.60 exomaltotetraohydrolase] from Pseudomonas stutzeri were completely separated and highly purified by using Polybuffer exchanger PBE^TM 94, and their enzymatic properties were compared. The purified F-1 and F-2 both showed a single band in polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The optimum pH for the action of F-1 and F-2 on starch was exactly the same at 8.0. The isoelectric points of F-1 and F-2 were estimated to be 5.6 and 5.3 by polyacrylamide gel electrofocusing. Except the properties above, other enzymatic properties were very similar for F-1 and F-2: 1) the optimum temperature (45°C at pH 8.0) and the molecular weight (5.5×10⁴), 2) the stabilization by Ca²⁺, and the inhibition by some metal ions and N-bromosuccinimide, 3) the inhibition by cyclomaltodextrins and the inability to hydrolyze cyclomaltodextrins, 4) the kinetic constants (Km and k⁰) for starch, glycogens and short chain amylose (DP=17), and 5) the type of inhibition and inhibitor constants (Ki) of some microbial a-amylase inhibitors. Both purified F-1 and F-2 attacked starch from the non-reducing end to produce a-anomer oligosaccharides; these results confirmed that both enzymeswere exo-a-amylases as reported previously.

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An extracellular exo-maltohexaohydrolase [EC 3.2.1.98] from a Klebsiella pneumoniae (Aerobacter aerogenes) mutant produced about 40% maltohexaose (G₆) from short-chain amylose (DP=23). Mostly G₆ was produced from maltooligosaccharides larger than G₆ by an exomechanism action. It also hydrolyzed G₆ and shorter maltooligosaccharides to give smaller maltooligosaccharides. Its position specificity of action on G₃ through G₈ was studied with maltodextrins specifically labeled at the reducing-end glucose unit with ¹⁴C. The highest frequency of cleavage was at the second bond from the reducing end in G₃ through G₆. For G₇ and G₈, the sixth bond from the nonreducing end of the substrate was cleaved with absolute specificity by the exo-mechanism action.
Kinetic parameters of the exo-maltohexaohydrolase on various substrates were also studied. The Michaelis constant (Km)for short-chain amylose was the smallest among the various substrates examined.
G₆ was also formed from G₄ by a transfer action of the enzyme, with an action pattern dependent on the substrate concentration.

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A novel serine protease was purified from the crude membranefraction of Bacillus subtilis IFO 3027 by a procedure involving extraction with n-butanol, precipitation in a solution of low ionic strength, washing with organic solvents, Phenyl Sepharose CL-4B column chromatography and Sephacryl S-300 gel filtration. The purified enzyme was homogeneous on SDS-polyacrylamide gel electrophoresis.
The molecular weight of the native enzyme and its subunit were determined to be 540, 000 and 62, 000, respectively. The enzyme was precipitated in the low ionic strength buffer. The enzyme was a serine protease and inhibited by PMSFand certain microbial serine protease inhibitors such as MAPIand chymostatin. The enzyme showed a broad substrate specificity against peptides with arginine and tyrosine bonds. The Km for Z-L-Ala-L-Ala-L-Leu-pNA was calculated to be 1.2 x 10^-4M. Casein hydrolytic activity of the enzyme was maximumat pH ll and at 55°C.

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CWM was isolated from suspension-cultured tobacco cells and was fractionated into EDTAsoluble, 5% potassium hydroxide-soluble, and 24% potassium hydroxide-soluble fractions, and α-cellulose fraction. Sugar composition and methylation analyses of the CWM and each fraction wereperformed and the following polysaccharides were suggested to be present; rhamnogalacturonan, arabinant, galactan AXG, GGM, (4-O-methylglucurono) xylan, cellulose and glycoprotein containing hydroxyproline-arabinosides.

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An arabinoxyloglucan (AXG), isolated from the 24% potassium hydroxide-soluble fraction of the cell wall material of suspension-cultured tobacco cells, was characterized by methylation analysis before and after mild acid hydrolysis and cellulase degradation. The results indicated that AXG was essentially composed of the following repeating units:

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An aminopeptidase was purified from buckwheat seed by using affinity chromatography, ionexchange chromatography and chromatofocusing. The enzyme had a molecular weight of 37, 000 as determined by gel filtration. The aminopeptidase activity, determined with L-leucine-p-nitroanilide (Leu-PNA) as the substrate, exhibited a pH optimum of 7.2. The Km value for Leu-PNAwas 140 μM. The preferred substrates were L-leucine-β-naphtylamide and Leu-pNA, although there was also high activity against L-leucyl-L-alanine and L-leucinamide. Thiol antagonists were found to be potent inhibitors against the enzyme. The enzymeexhibited less or no sensitivity to the endogeneous proteinase inhibitors, benzamidine and TPCK.

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A general, genetic technique for in vivo cloning of bacterial genes is presented. Wepreviously introduced the Muphage into various genera of bacteria including Klebsiella aerogenes with RP4: : Mu.Using these strains carrying RP4: : Mu cts and thermo-inducible Muprophage in the chromosome, we cloned in vivo the arylsulfatase (ats) and tyramine oxidase (tyn) genes by partial thermo-induction. The donor strains carrying the recombinant plasmids were conjugated with K. aerogenes rec strains, which were isolated as UV-sensitive mutants. The resultant recombinant plasmids, pATl and pAT2, were purified and used for the transformation of mutant strains deficient in the ats and tyn genes. The ats-tyn genes seemedto be transposed into the RP4::Mu cts plasmid together with other chromosomal DNA fragments. This in vivo cloning method is applicable to a wide variety of gram-negative bacteria.

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The enzymatic, physicochemical and immunochemical properties of pig liver neutral α-glucosidase were compared with those of pig serum neutral α-glucosidase. The enzyme purified from pig's liver showed a pH-optimum of 6.5 and was stable in the range of pH 5.0 to 9.0. This enzymealso showed a temperature-optimum of 60°C and was stable up to 60°C. These properties of pH and temperature of the pig liver a-glucosidase were very similar to those of the pig serum α-glucosidase reported previously. The isoelectric points and the molecular weights of the two α-glucosidases were same: pi, 4.2 and MW, 2.7-3.0 x 10⁵. The Km values for maltose, maltotriose and phenyl a-maltoside of both α-glucosidases were 8.5 mM, 2.2mM and 2.3 mM, respectively. The essential ionizable groups of the enzymes were identified to be a carboxyl group (-COO^-) in the acidic side and an imidazole group (NH⁺) in the alkaline side. The pig liver a-glucosidase produced a clear precipitin line with the antiserum to the pig serum a-glucosidase, but a difference was found in their mobilities on crossed immunoelectrophoresis.

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Ovomucoid domains I (Ala¹ … Met⁶⁸) and III (Leu¹³¹ …Cys¹⁸⁶) were prepared from chicken egg white ovomucoid by CNBrcleavage and Staphylococcus aureus V8 protease digestion, and antigenic properties of each domain were investigated by immunodiffusion, immunoprecipitation and the rat passive cutaneous anaphylaxis (PCA) test. Both domains I and III formed a precipitin arc or immunoprecipitation against rabbit anti-ovomucoid serum, and inhibited the PCAreaction of mouseanti-ovomucoid serum. These results indicated that ovomucoidantigenic sites existed in both domains I and III, and that the domains retained their antigenicity even after they were separated.

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The apparent molecular weights of soluble ovalbumin aggregates occurring under various heating conditions were determined by using the low angle laser light scattering technique in combination with high performance liquid chromatography. The soluble ovalbumin aggregates were rapidly formed near the melting point (76°C) for thermal denaturation. The apparent molecular weight of the soluble ovalbumin aggregates increased with the heating temperature from 4, 000, 000 at 76°C to 35, 000, 000 at 100°C. It also increased in proportion to the protein concentration, ionic strength and standing time. In the presence of sodium dodecyl sulfate, the soluble ovalbumin aggregates dissociated into lower molecular weight fractions, mainly monomeric and oligomeric. On the other hand, the molecular weight of the soluble ovalbumin aggregates did not change in the presence of mercaptoethanol or N-ethylmaleimide. The soluble ovalbumin aggregates were formed in proportion to an increase in the surface hydrophobicity of heatdenatured ovalbumins. From these results, it was suggested that the soluble ovalbumin aggregates were formed mainly by hydrophobic interaction.

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On addition of soy bean phospholipid fraction to serum-free medium (TES medium), immunoglobulin producing myeloma MPC-11 cells could grow under stirring in the serum-free mediumalmost as well as in serum medium. This is the first success in culturing cells in stirred serum-free conditions. Technologically important parameters such as temperature, stirring velocity and aeration of the mediumwere also optimized in the serum-free stirred culture. These results will provide somefundamental knowledge for developing a large-scale suspension culture system especially for immunoglobulin producing cells.

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Qualitative and quantitative analyses of the volatiles from peel of Citrus iyo was performed by the use of a combined gas-liquid chromatograph and mass spectrometer (GC-MS)and a gas chromatograph (GC-FID) with a computing integrator.
Among 53 oxygenated compounds, 2 aldehydes, 19 esters and 9 alcohols were reported for the first time as componentsof C. iyo.
By comparing the volatile compositions in peel among the fruits at four different stages of storage, i.e. at harvest, pre-treatment, ripening and overripening, it was found that the contributory aroma constituents such as C₁₀-, C₁₁-aldehydes and C₆-, C₈-, neryl-, geranyl-, citronellyl-acetates increased during storage, up to ripening.

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The uptake and metabolism of methotrexate were examined in Crithidia fasciculata ATCC 12857. Whenthe cells grown in a mediumcontaining, biopterin were incubated with methotrexate, the uptake ofmethotrexate by the cells was completed within 1 min, and the amounts ofmethotrexate per 10⁸ cells were maintained between 19 and 37 fmol/min throughout the remaining growth period. In contrast, the uptake offolate was linear for at least 20 min, and the uptake rate offolate by the cells varied from 31 to 247 fmol/min/10⁸ cells during the growth. The folate uptake was inhibited by methotrexate and aminopterin. When C. fasciculata was cultivated in a medium containing methotrexate, the methotrexate taken up by the cells was metabolized to several compounds. Oneof the metabolites was isolated from the cells and the culture supernatant, using DEAE-cellulose and Sephadex G-10 column chromatographies, and identified as 4-amino-4-deoxy-10-methylpteroic acid (AMPte) on the basis of its behavior on thin-layer chromatography, and of its ultraviolet and infrared absorption spectra. An enzyme activity converting methotrexate to AMPtewas found in the cell-free extract of C. fasciculata.

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A new virulent phage, φr5, was isolated from soil on Streptomyces ribosidificus SF733. Electron microscopic observation showed that this phage belongs to group B of Bradley's morphological classification. The molecular weight of φr5 DNAwas about 29×10⁶. φr5 DNA showed resistance to many restriction enzymes. φr5 required Ca^{+ +} for plaque formation and grew at 28°C on S. ribosidificus SF733, S. chartreusis SF1623, S. flavus, S. flavovirens, S. sulfonofaciens SF2103, S. sulfonofaciens SF21 38 and S. sulfonofaciens SF2 1 44. The restriction-modification of φr5 by the strains susceptible to this phage was examined.

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To establish a procedure for high frequency transfection in streptomycetes, the conditions and factors affecting the polyethyleneglycol (PEG) mediated transfection of S. chartreusis SF1623 by actinophage φr5 DNA were studied. Protoplasts of S. chartreusis SF1623 prepared by treatment with lysozyme and achromopeptidase were very stable. Protoplasts from 20 to 22hr culture cells were more competent for transfection. The optimal pHof the mediumfor transfection was pH 7.6. The presence of NaCl, thymidine, ATP, ADP or adenosine in the transfection mediumenhanced the frequency of transfection. The optimal conditions determined for protoplast transfection were 12.5% PEG 4, 000, 300 min NaCl, 1 mMthymidine, final concentration, φr5 DNAand protoplasts in P3 medium(pH 7.6). The frequency oftransfection under the optimal conditions was 5×10⁵ perμg φr5 DNA and was about 3×10^-3 per regenerated protoplasts.
Progenitively mature phages appeared 4 hr after incubation in the regeneration solution and their number continued to increase for about 11 hr. The burst size was estimated to be about 400.

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Aspergillus rager NRRL 337, a significant lipase producer, also produced esterases in a semisolid medium. From the culture extract, four esterases (Esterase-I, -II, -III, -IV) were purified by means of ion-exchange chromatography, gel nitration, gel permeation with high performance liquid chromatography, and preparative disc gel electrophoresis. Homogeneity of each esterase was confirmed using disc gel electrophoresis. The four esterases were found to exhibit low isoelectric points (pH 3.63.9). The molecular weights determined by gel permeation chromatography of Esterase-I, Esterase-II, Esterase-III and Esterase-IV were 23000, 127000, 29500 and 27000, respectively.

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Properties and substrate specificities of four esterases (Esterase-I, -II, -III, -IV) from Aspergillus niger were studied. Esterase-I and Esterase-II were found to be markedly stable to heat. WhenEsterase-I was assayed at 35°C using methylacetylsalicylate as a substrate, even after heating at 100°C for 15 min 60% of its activity remained. However, Esterase-I scarcely hydrolyzed the substrate at 70°C or over, because of a reversible change in conformation by heating as found by CDmeasurement. The maximumactivity of Esterase-I was found at 55°C at 20 min of reaction time. Esterase-II was stable up to 80°C and had an optimum temperature for reaction at 80°C, but was irreversively inactivated by heating for 15 min at 90°C.
The four esterases hydrolyzed aliphatic esters of short chain fatty acids and acetyl esters of phenols, but neither methyl esters of aromatic carboxylic acids nor acetyl esters of aromatic alcohols.

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We have constructed restriction maps of actinophage R4 for PstI, PvuII, EcoRI, ScaI, KpnI, BglII and BelI. DNA of R4 phage had no cleavage sites for seven restriction endonucleases, BamHI, ClaI, HindIII, SacII, SlaI (XhoI) or XbaI. The entire genome size of the R4 phage was estimated to be 53.7 kilobases. Three deletions of the R4 phage were delimitated on the R4 genome. All three deletions were located on the right hand side of the R4 restriction map.

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The biological effects of raw winged bean seeds were investigated with feeding experiments on rats, and the effects of lectin (phytohemagglutinin) present in the seeds are discussed. Administration of a 30%raw winged bean diet caused strong growth depression in young rats, and led to death within 10-20 days, inducing severe damage to the small intestine of the rats. Significant morphological changes of the intestinal mucosa were observed with a microscopic investigation. As the lethal effect was eliminated by autoclaving but not removed with supplementation of 0.5% L-methionine to the raw winged bean diet, the lectin was assumed to be closely related to the deleterious effects of raw winged bean. In vitro and in vivo digestion tests of the lectin revealed that the winged bean lectin had resistance to peptic, pancreatic and membrane digestions. The hemagglutinating activity was also detected in the intestinal mucosa and faeces from rats ingesting the raw winged bean or its purified lectin. The binding action of lection to mucosal epitheliums of the gastrointestinal tract is suggested to be the initial step of the deleterious effects induced by the winged bean lectin.

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α-Amino-ε-caprolactam racemase, which occurs in the cytoplasmic fraction of Achromobacter obae, has been purified to homogeneity. It has a monomericstructure with a molecular weight of approximately 50, 000. The absorption spectrum of the enzymeexhibits maximaat 280 and 412 nm at pH 7.3, and is independent of pH from 6.0 to 8.0. One mole ofpyridoxal 5'-phosphate is bound per mol of the enzyme.Incubation of the enzymewith hydroxylamineresulted in the formation of the apoenzyme.d- and L-a-Amino-a-caprolactamsare the only substrates. Themaximumactivity is found at pH 8.8 for both the isomers. Michaelis constants are as follows: 8him for D-α-amino-ε-caprolactam, 6 mMfor L-α-amino-e-caprolactam and 2.1×10^-7 M for pyridoxal 5'-phosphate. The enzyme is inhibited significantly by CuSO₄, HgCl₂, thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate, and carbonyl reagents (e.g., phenylhydrazine and hydroxylamine). a- Amino-e-caprolactam racemase catalyzes the α-proton exchange of the substrate with deuteron during racemization in deuterium oxide.

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(±)-(2Z, 4E)-α-Ionylideneacetic acid (2) was enantioselectively oxidized to (-)-(1'S)-(2Z, 4E)- 4'-hydroxy-α-ionylideneacetic acid (3), (+)-(1'R)-(2Z, 4E)-4'-oxo-α-ionylideneacetic acid (4) and (+)-abscisic acid (ABA) (1) by Cercospora cruenta IFO 6164, which can produce (+)-ABA and (+)-4'-oxo-α-acid 4. This metabolism was confirmed by the incorporation of radioactivity from (±)-(2-¹⁴C)-(2Z, 4E)-α-acid 2 into three metabolites. (-)-4'-Hydroxy-α-acid 3 was a diastereoisomeric mixture consisting of major 1', 4'-trans-4'-hydroxy-α-acid 3a and minor 1', 4'-cis-4'-hydroxy-α-acid 3b. These structures, 3a and 3b, were confirmed by ¹³C-NMR and ¹H-NMR analysis. Also, the enantioselectivity of the microbial oxidation was reexamined by using optically pure aacid (+)-2 and (-)-2, as the substrates.

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Eclosion hormone was purified 5, 000-fold from the extracts of male adult heads of the silkworm, Bombyx mori. The fourteen-step purification procedure consisting of solvent extractions, fractional precipitations and chromatographies afforded a partially purified preparation of eclosion hormone, 1.8 μg of which showed activity in a Bombyx pharate adult. The hormone was inactivated by proteolytic enzymes. The molecular weight of eclosion hormone was estimated to be 8, 400±1, 000 daltons by gel-filtration on Sephadex G-50.

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