In a previous paper, a consecutive transition mechanism in Fig.1 has been proposed for the phase transition of tetramethylsilane. In order to confirm whether the mechanism is accepted in general, the phase transition of pentane is investigated by the frequency response (FR) and the pressure-jump methods. The FR data in Fig.12 reveal that the equilibrium system involves at least four different relaxation processes, of which relaxation times are <6×10^-2s, 4.3s, 5min, and >5min. In the pressure-jump method, small amounts of pentane containing tritiated pentane (10^-5 in molar fraction) were added abruptly; the radioactive pentane in the vapor phase decreased exponentially but the relaxation curve of the total vapor pressure was nonexponential (Fig.6). These results may be explained by the consecutive transtion mechanism that a molecule in the vapor phase condenses to bulk liquid via several growing aggregates at the interface. Further, origins of the energy gaps in Figs.1 and 13 are discussed (Figs.14 and 15).
The effects of the addition of methanol as an impurity on the FR data in Figs.9 and 10seem to increase with increasing co, which would support the aggregation model because aggregates of pentane at the interface should be more affected by the impurity.

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A Cupper (II) Hexanoate (C6-Cu soap) was prepared by double-decomposition of sodium hexanoate and copper (II) chloride. A yellowish-brown organosol was formed by heating the copper soap suspended in 1-octanol, 1-decanol and 1-tetradecanol, respectively, at 100-170°C. The organosol particles obtained by heating the suspension in 1-decanol in concentraion of 10mg/5 ml at 170°C were found to be spherical by an electron microscope, whereas the particles obtained at 100°C in concentration of 500 mg/5 ml were cubic. The both spherical and cubic organosol particles were identified as copper(I) oxide by X-ray analysis. The influence of hydroquinone on the organosol formation was also examined.
The mass spectrum of the organosol showed many fine peaks around m/e=100-200, which were not found for copper soap itself, and a peak at m/e=99 of CH₃(CH₂)₄CO⁺. This result corresponds to the fact that an infrared absorption peak in 1590-1600 cm^-1 of COO- disappears and a new peak in 1710 cm^-1 of >C=-0 appears on heating.
These experimental results suggest that the C₈-Cu soap was reduced on heating in long-carbon chain alcohol to copper(I) oxide particles which are stabilized by oxidized product of the alcohol and hexanoyl derivative in the medium.

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Rheological properties of hydrogels of poly (vinyl alcohol) (PVA) (frozen at -10°C and -20°C, and melted at 15°C every 24 h) were examined by the measurement of longitudinal vibration. The freezing time was chosen as 24, 48, 72 and 96 h. The degree of polymerization (DP) and the degree of saponification (DS) of the PVA were 500, 1000, 1700, 2000, 2400 and 98.5±0.5, 99.6±0.3 mol%, respectively. Measurements were done for the PVA solutions of 5, 7, 10, 15, and 20 wt%. The dynamic Young's modulus, E′, and the mechanical loss tangent, tan 8, were obtained at 2.5 Hz by longitudinal vibrations of a cylindrical gel. The E′ increased both increasing freezing time and with decreasing freezing temperature. The E′ increased steeply with increasing DP up to about 1000 and became almost constant in the range of 1700 to 2400. The effect of DS on the E′ was remarkable: the E′ decreased drastically with the decrease of DS of only about 1 mol%. The effects of the concentration and melting temperature on the E′ of PVA gels were enhanced by the increase in the number of steps in the temperature cycle. This phenomenon was explained through the formation of microcrystalline structure within the gels.

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The sulfur species in some blast furnace slags were analyzed by means of the newly developed methods, and their mutual relationships were considered under various conditions (storage environment, standing time, particle size). Sulfide, thiosulfate and sulfite sulfur were determined by iodometric titrimetry, sulfate and total sulfur by chelatometric titrimetry, and elemental sulfur by photometry. It was confirmed that the total sulfur content is approximately equal to the sum of sulfide sulfur, elemental sulfur, thiosulfate sulfur, sulfite sulfur and sulfate sulfur. Each content of elemental sulfur, thiosulfate and sulfate had a close correlation with the sulfide content in the total sulfur con-tent (correlation coefficient 0.96). Therefore, the contents of these sulfur species could be estimated easily from the analytical values of sulfide and total sulfur. It was found that the smaller the particle size of slag, the more oxidation of sulfur species occurred on standing, and that finally the content of sulfide in slag decreased rapidly to produce sulfate when it was exposed to sunlight, wind and rain water in the atmosphere.

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Amino acids are adsorbed on iron(III) hydroxide from aqueous solution. The adsorption behavior was found to depend on the nature of amino acids. Acidic amino acids are adsorbed strongly in the pH range of 5 to 7. Neutral and basic amino acids are adsorbed, but weakly, in the pH range of 8 to 10. In the optimum pH range as described above, acidic amino acids are present as anions, and neutral and basic amino acids as zwitter ions. The zero point of charge of iron(III) hydroxide was determined to be 7.36. From these results the adsorption of amino acids was explained in terms of the electrostatic interaction between adsorbate and adsorbent. Hydroxyl ion or proton was released during the adsorption reaction of amino acids. The adsorption process was exothermic for all amino acids. The Langmuir equation was applicable to the adsorption of acidic and basic amino acids. The area occupied by glutamic acid on iron(lla) hydroxide was calculated to be 195 m2/g from its saturation adsorption.

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A rapid and highly selective method for the simultaneous determination of 13 organochlorine pesticides and related compounds (Table 1) in soil, bottom deposit and river water was developed by gas chromatography mass spectrometry (GC/MS) utilizing negative ion chemical ionization. The procedure is as follows. [Pretreatment] the modifications were done along the proposed method. Main points are as follows; 1) Elimination of a reflux treatment for removal of free sulfur by copper powder in the case of soil and bottom deposit was done, 2)Developing solvents in Florisil column chromatography were changed to 200 ml portions of diethyl ether/hexane (15/85) and acetone/hexane (5/95) and 2.5 g of Florisil was used, 3) Elimination of silica-gel column chromatography for removal of PCB's was done. [Measurement]the concentrated solution of the effluent from Florisil column is introduced into a GC/MS instrument equipped with a SE-54 fused silica capillary column. Methane is used as a reagent gas. Benzil is used as an internal standard. The mass fragmentograms are recorded by monitoring specific peaks of each compound (Table 2, Fig.2). Because of high selectivity of the proposed method, 13 organochlorine pesticides could be determined simultaneously without preseparation of PCB's and phthalic acid esters (Table 4). The determination values of individual constituents were in the range 0.1-2300 ppb in the case of soil and bottom deposit and 1.9-45 ppt in the case of river water (Table 5, Fig.3). Minimum detectable amounts of pesticides for this method were 0.2-50 pg and time required for GC/MS measurements was within 15 minutes.

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The acetylation of silicic acid with acetyl chloride was investigated. The reaction was monitored by NMR spectroscopy and the products were characterized by IR spectra and by analysis of the acetoxyl group. The degree of acetylation was evaluated by the NMR spectroscopic analysis of the silylate of acetylated silicic acid revealing that acetyl chloride reacted readily with silicic acid. Acetylated silicic acid and its silylate had a low molecular weight. They were unstable and became insoluble after isolation. This indicates that acetyl chloride like chlorotrimethylsilane reacts with silicic acid without an appreciable change of the degree of polymerization of silicic acid.

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Popcorn polymerizations of styrene and methyl methacrylate in the presence of various solvents were carried out by using styrene and butyl methacrylate popcorn polymers as the seed. There were two different linear relationships between the activity of the seed toward vinyl monomer and the chain transfer constant to the solvent. These solvent effects were interpreted in terms of the chain-transfer reaction of the growing radical to the solvent added and the dependence of the solvent quality, e. g., a goodi solvent or a poor solvent, on the polymer graft polymerized in the seed.

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The chemical shifts and profile changes in the TiK_a, Kp and its satellite lines of 17 chemical compounds were measured with a two-crystal X-ray spectrometer. The data obtained were discussed qualitatively and related to the chemical bonding states of titanium. With increasing valence of the titanium, the K_a1 and K_a2 lines shifted to the lower energies, while the K_β5 band shifted significantly to the higher energies. No such a simple dependence on the Ti valence could be found in the K_{β1, 3} shifts and the K_{β1, 3} widths. All compounds were characterized by the K_β5 profiles with complex structures and in certain cases by the appearance of the K_β″ band.

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Themal reaction of sodium crotonate in solid state was found to afford exclusively 1hexene-3, 4-dicarboxylic acid[1] in high yield. Sodium crotonate was heated at 300°C in solid state under vacuum for 4 h. The salt was dissolved in water, and the solution was acidified with HCl to recover crude crystals. Recrystallization of the product from water gave [1] (mp 185-486°C) in 84% yield. Hydrogenation of [1] in methanol solution in the presence of Pt-black yielded a product, of which, the melting point agreed with that reported for the meso-hexane-3, 4-dicarboxylic acid [3] (mp 192°C). The structure of [ 1 ] was elucidated by ¹H-NMR and mass spectra of its methyl ester [2]. Refluxing of (2) at 240°C for 20 h, resulted in double bond migration to give dimethyl 2-hexene-3, 4-dicarboxylate consisting of (E)- [5 a] and (Z)- [5 b] forms in yields of 52% and 43%, respectively.

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Various dye staffs have been used as electron carriers to evolve hydrogen with hydrogenase. Viologen dyes such as ethyl- and propylviologens were found to be suitable electron carriers. Among viologen dyes propylviologen was most effective for hydrogen evolution. Kinetic studies have been carried out by using Safranine T and Neutral Red as electron carriers. In the case of Safranine T system, the hydrogen evolution rate was expressed by equation;
ν=D[H₂]/dt=k₁²[s]²/(1+K₁[s])²
where [S] is the concentration of Safranine T, and k and K₁ are rate constant and equilibrium constant, respectively. On the basis of the above equation, the reaction mechanism is proposed. The hydrogen evolution rate decreased with reaction time. It may be caused by the deactivation of Safranine T itself. When Neutral Red was used as an electron carrier, the turnover number of hydrogen evolution against Neutral Red was 15.2 after 3 h reaction. In this case the hydrogen evolution rate also decreased with reaction time. The inhibition of hydrogenase activity by dimerization of reduced form of Neutral Red or by decrease of the concentration of the leuco form may be responsible for the rate decrease.

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Negamycin, [2-[(3 R, 5R)-3, 6-diamino-5-hydroxyhexanoy1]-1-methylhydrazino]acetic acid, which possesses a strong inhibitory activity against Gram-negative bacteria, has been synthesized in a highly stereocontrolled manner. A combination of enzymatic and chemical procedures was taken as our synthetic strategy.
The 3 R-absolute configuration was created by the enzyme-mediated asymmetric hydrolysis of the prochiral dimethyl 3-(benzyloxycarbonylamino)glutarate. The remaining 5 R-absolute configuration was introduced by the newly developed iodocyclocarbamation with high 1, 3asymmetric induction. Thus, the enzymatically produced chiral half ester, methyl hydrogen (3 S)-3-(benzyloxycarbonylamino)glutarate, was chemically converted to t-buty1(3 R)-3-benzyloxycarbonylamino5-hexenoate in 4 steps, and treatment of the N-(t-butyldimethylsily1)derivative with iodine afforded preferentially trans-cyclic carbamate, effecting high 1, 3-asymmetric induction. Further transformation of the. functional groups efficiently afforded the desired key intermediate, (3 R, 5 R)-6-azido-3-benzyloxycarbonylamino-5-(tetrahydropyran -2yloxy)hexanoic acid, which was then converted to natural negamycin in good overall yields.

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Some analogues of 3-methyl-2-butenylpyrophosphate (DMAPP) having a hetero atom or a cyclopropane ring were found to be a substrate for hog liver farnesylpyrophosphate synthetase. The enzymatic reaction products were analyzed by GLC, TLC and GC-MS. The structures of the products were elucidated to be a farnesyl-PP type. Among the artificial substrates the allylic-PP [3c] having a cyclopropane ring was the best suited. Although its K_m value was larger than that of DMAPP, the V_max values were almost the same between them. These results suggest that the "ionization-condensation-elimination" mechanism proposed by Poulter would be applicable to this enzymatic reaction and that this enzyme can have such a wide specificity that it may be widely applied to bioorganic synthesis.

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Both enantiomers of ethyl 3-hydroxybutanoate which are useful chiral starting materials for the synthesis of natural products containing CH, CH(OH)CH, R group have been prepared in a 3-18 g scale by microbial methods. Ethyl acetoacetate was enantio-selectively reduced to the S-enantiomer by baker's yeast (in 83, -90% e. e. ) and by high temperature yeast (in 96% e. e., see Table 3). Asymmetric reductions by several brewer's yeasts (in 43--92% e. e. ) and by commercially available HLADH, YADH (in 100% e. e. ) gave also the S-enantiomer. The Renantiomer was prepared from poly (3-hydroxybutanoate) (PHB) produced by Zoogloea ramigera, in 100% e. e. Their optical purities were determined by the HPLC analyses of the corresponding MTPA esters.

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In contrast to the usual lipase reactions which are carried out on liquid oil (below 40°C) as a substrate, unstirred reactions of solid fats have now been investigated. The solid fat index (SFI) was used as a solid fat content in the substrates. The enzymes employed here were Candida cylindracea lipase (CC), Mucor miehei lipase (MM) and Geotrichum candidum lipase (GC). When lipases CC and GC were used, the hydrolysis rates decreased with the increase of SFI of the substrates. Lipase MM showed an opposite behavior. The trends became less clear in the prolonged reactions or in high hydrolysis conversion in these three lipases. The positional and substrate specificities of the lipases were high in the lower conversion. Lipase reaction sequences were found to differ depending on the origin of the enzyme and on SFI of the substrates for a given lipase. The amount of the monoglycerides accumulated in the reaction product became as high as 6-s7% in contrast to 1-2% in stirred reaction. Lipase MM with the specificity at the 1- and 3-position did not show any reactivity towards the ester bond at the 2-position of the glyceride. Since the reaction sequence was controlled by positional migration, it was different from those of the other lipases. This was confirmed by lower hydrolysis rates and higher accumulation of mono- and diglycerides than in other lipase reactions.

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Application of hydrolytic enzyme to organic syntheses is attracting attention, because enzyme would promote selective reaction under moderate conditions. This paper describes an application of a protease to peptide synthesis with use of a photochromic compound and light energy.
A new photo-stable spiropyran, 3', 3'-dimethyl-8-methoxy-6-nitrospiro[2 H-1-benzopyran-2, 2'-indoline]-1'-propionic acid C 3 J was synthesized by the authors. The stability of photoresponsive activity was 3.4 times higher than that of 3', 3'-dimethyl-6-nitrospiro[2 H-1benzopyran-2, 2'-indoline]-1'-propionic acid 2 J (Fig.3). Improvement of the stability was mainly caused from introducing electron-donating groups at the ortho position.
α-Chymotrypsin was immobilized in collagen membrane modified with the spiropyran [3]. The enzyme-spiropyran-collagen membrane showed a reverse photochromism and catalyzed the synthesis of N-acetyl-L-phenylalanyl-L-phenylalanine ethyl ester from N-acetyl-L-phenylalanine and L-phenylalanine ethyl ester. As shown in Fig.4, the yield of the reaction for 55 h was 2% in the dark. In contrast, that for 35 h reached 6% under visible light (30000 lux). Under illumination, the spiropyran became an apolar state and the hydrophobicity of the enzyme bound membrane increased. Therefore, the synthetic reaction by the photosensitive immobilized enzyme was promoted by visible light irradiation.

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Two problems which are currently urged to be solved in enzymatic synthesis of peptid are described. One is low solubility of substrate which reduces the efficiency of enzymatic reaction, especially in the case of continuous flow system of immobilized enzyme. The other is an enzyme immobilization with high enzyme activity which brings economical profit by repeating use.
The water-soluble polyethylene glycol esters, [1], [4], [5], [6] in Table 1, reacted with leucinamide in the presence of ce-chymotrypsin to give the corresponding amides which were perceived by the precipitation of insoluble products. The ester [7] also gave CET by enzymatic displacement with 7-ACA. The water-soluble esters having suitable poly(oxyethylene)chain length is considered to be applicable to another field of enzymatic synthesis as substrates.
The five kinds of immobilized protease carriers were prepared and the efficiency was tested in the coupling reaction of porcine des-alanyl(B30)-insulin(p-DAI) with Thr-OBu^t which is the key step in converting porcine insulin into human insulin (Fig.2). Carrier-5 (poly (glutamic acid)) was found to be good for immobilizing Achromobacter protease I with high activity. Immobilized Achromobacter protease I on carrier-5 (Poly-Glu-A in Table 4) showed 74 per cent yield in the coupling step. Protease activities immobilized on carrier-1, -2, -3, and -4 were not so high as to be used in the coverting 'processes.

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The biochemical potentiality of plant cultured cells to transform foreign substrates is of considerable interest in connection with the use of the cultured cells as a bioreactor. We have now investigated the bioconversion of the foreign substrates, such as monoterpenoid alcohols and cycloalkanols, with the cultured cells of Nicotiana tabacum for generalizing the bioconversion pattern.
The cultured cells were found to have the ability to transform these foreign substrates as follow: ( i ) the regio- and stereo-selective hydroxylation at the C-C double bond as well as its allylic positions, ( ii ) the hydrolysis of the acetoxyl group, (iii) the interconversion between 5- to 8-membered cycloalkanols and the corresponding ketones; it is at equilibrium and the balance in the equilibrium depends on the number of carbon atoms in the carbocyclic ring of the substrates, and (iv) the enantioselective bioconversion. It was thus elucidated these bioconversion patterns are determined by the functional group existing in the substrates and the structure in the vicinity of the functional group.

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In order to systematically synthesize oligoribonucleotides with defined sequence, trimer unit having different bloking groups at 3'-hydroxyl and 5'-phosphate groups were enzymatically synthesizd. Deblocking at 3'- or 5'-end of the trimer unit gives an acceptor or a donor for the T4RNA ligase, respectively. For the blocking of the 3'-end, o-nitrobenzyl group was attached to the 2'-hydroxyl group, while for the blocking of 5'-phosphate, anilidate was employed. The f ormer was deblocked by photo-irradiation and the latter was by an acid. The hexamer produced by the ligation of the acceptor and the donoralso has the same blocking groups at 3'- and 5'-ends, and is capable to be utilized for the next synthetic reaction as an acceptor or a donor for T4RNA ligase.

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A new process for enzymatic hydrolysis of beef tallow fat was studied for industrialization purposes. An emulsion in solid phase was used for effective contact of fat with an enzyme solution, without using any surfactant and organic solvent. Conditions conductive to effective emulsion and reaction conditions for hydrolysis were studied. One hundred units/g-oil of lipase MY hydrolysed Bleachable Fancy tallow fat in solid phase at 30°C to give fatty acids in 95% yield.

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Reduction of fluorinated ketones and keto esters with baker's yeast was studied. The mono(perfluoroalkyl) ketones and keto esters were asymmetrically reduced to give the corresponding optically active alcohols and hydroxy esters. Bis(perfluoroalkyl) ketones were, however, strongly resistant to the action of the baker's yeast and none of alcohols were obtained (Tables 1 and 2). Furthermore, a variety of highly diastereoselective reductions of a-monofluoro-8keto esters and 2-fluoro-1-phenyl-1-propanone were performed.

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To obtain immobilized Pseudomonas dacunhae cells having higher L-aspartate 8-decarboxylase activity and better operational stability on industrial scale, we investigated the glutaraldehydetreatment of the cells in culture broth before immobilization with k-carrageenan.
When the cells were treated at pH 4.75 and 30°C for 1 h and further treated with 5 mmol?dm-3 glutaraldehyde at pH 6.0 and 10°C for 10 min, the immobilized P. dacunhae cells showed highest enzyme activity and operational stability (Fig.3). The low pH-treatment devised previously for elimination of alanine racemase activity was found to be useful for stabilization of L-aspartate 8-decarboxylase activity of immobilized P. dacunhae cells. The reason is considered to be due to removal of unstabilizing factors such as intracellular neutral and/or alkali proteases from P. dacunhae cells by the low pH-treatment (Fig.4). Furthermore, the glutaraldehydetreatment of cells was also found to stabilize the enzyme activity. This treatment of cells was superior to that of immobilized cells reported previously in both enzyme activity and operational stability. That is, in the f ormer case remaining enzyme activity and halflife in continuous operation at 37°C were about 90% and 260 days, respectively, and in the latter case those were about 70% and about 100 days, respectively. The reason of advantages in the former case might be depend on homogeneous contact between cells and glutaraldehyde at optimum conditions. On the other hand, in the latter case cells located at near surface of gel were contacted with a higher concentration of glutaraldehyde than optimum one and cells at near center were contacted with a less concentration of glutaraldehyde than that due to consumption of glutaraldehyde during duffusion in gel.
Continuous production of L-alanine using the immobilized P. dacunhae cells was industrialized in 1982 in Tanabe Seiyaku Co. Ltd.

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The stereospecific C-C bond forming reaction of farnesyl pyrophosphate synthetase of pig liver was efficiently utilized in the asymmetric synthesis of the stereoisomers of faranal, a trail pheromome of the Pharaoh's ant, Monomorium pharaonis, to establish the absolute configuration of the pheromone to be (3 S, 4R, 6 E, 10 Z)-3, 4, 7, 11-tetramethy1-6, 10-tridecadienal, [14 b]. Similar applications of the prenyltransferase reaction to the asymmetric syntheses of four faranal homologs, (3 S, 4R, 6 E)-3, 4, 7, 11-tetramethyl-6, 10-dodecadienal [24], (3 S, 4 R, 6 E)-7-ethyl-3, 4, 11-trimethyl-6, 10-dodecadienal [25], (3 S, 4R, 6 E, 10 Z)-7-ethyl-3, 4, 11trimethy1-6, 10-tridecadienal [26], and (3 S, 4 R, 6 E, 10 E)-3, 4, 7, 11-tetramethy1-6, 10-tridecadienal [27] were also carried out.

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Prostaglandin [1] methel ester (1) and its analogues [2] and [ 3] were easiliy hydrolyzed with pig liver esterase. In this hydrolysis, saturated esters [1] and [ 2] were hydrolyzed about 15 times faster than a, 8-unsaturated ester [ 3]. In the hydrolysis of three other esters [4], [5] and[ 6], the reaction rate decreased in order: saturated ester [4]trans-α, β-unsaturated ester [5], and cis-α, β-unsaturated ester[6]. In comparison with other enzymatic hydrolysis, this method has the advantages of no side reaction and availability of higher substrate concentration.

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The cells of Streptoinyces roseochromogenes NRRL-B-1233 were immobilized in photo-crosslinked resin, 2, 2-bis[4-[methacroyloxypoly (oxyethylene)]phenyl]propane(BPE-30). The immobilized microorganism catalyzed the transformation of dehydroepiandrosterone (DHEA) to 16 ahydroxyDHEA. The reaction was followed with the aid of thin-layer chromatography and gas chromatography. The original activity of the free cells was found to be retained about 100% after immobilization. The DHEA-16 α-hydroxylase activity of the entrapped cells in a 5 to 10% dimethyl sulfoxide system was approximately 40 to 80% higher than the original activity of the free cells. The entrapped cells showed maximal steroid 16 a-hydroxylase activity at 4.1 × 10^-4mol·dm^-4 DHEA. However, at higher concentrations a weak but signi ficant substrate inhibition appeared. The stability of the steroid 16 a-hydroxylase activity of the cells over repeated reactions was greatly improved by immobilization. These results strongly indicate the usefulness of the BPE-30 hydrophilic resin in iffniobilization of S. roseochromo genes having steroid 16 cr-hydroxylation activity.

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