KAGAKU KOGAKU RONBUNSHU
Online ISSN : 1349-9203
Print ISSN : 0386-216X
ISSN-L : 0386-216X
Volume 17 , Issue 3
Showing 1-40 articles out of 40 articles from the selected issue
  • Tadashi Hano, Michiaki Matsumoto, Takaaki Ohtake, Fumiaki Hori
    1991 Volume 17 Issue 3 Pages 449-454
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Hydrolysis of olive oil in various organic diluents was carried out by using lipase in a stirred transfer cell. Based on measurement of the interfacial tension between aqueous and organic solutions, it was found that they are adsorbed at the oil-water interface.
    Among diluents investigated, isooctane gave the highest reaction rate as reported previously. From adsorption and kinetic experiments, a new model of interfacial reaction between adsorbed lipase and olive oil was proposed. The effect of diluent on the reaction rate was explained not by the difference in adsorption characteristics of lipase or olive oil but by the difference in interfacial reaction rate constants.
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  • Norio Takayama, Hideaki Nakamura, Kazuhisa Ohtaguchi, Kozo Koide
    1991 Volume 17 Issue 3 Pages 455-461
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Fatty acid desaturation responses of mesophilic yeast Candida lipolytica to upward or downward shifts of temperature were investigated by following the time courses in cell growth and content of C 16 and C 18 fatty acids. The main fatty acids produced by the yeast were linoleic acid (18 : 2), oleic acid (18 : 1), palmitic acid (16 : 0) and palmitoleic acid. (16 : 1). The tendency of cells at low temperature to form more unsaturated fatty acids to facilitate the fluidity of membranes was especially evident among the yeast cells in late logarithmic growth and stationary phases. During logarithmic growth at 20 and 25°C, constant levels of content of linoleic acid and palmitoleic acids were observed. A special response in the formation of unsaturated fatty acids was observed after the upward shift of temperature to 30°C. Such seemingly inconsistent observations are interpreted in terms of two biosynthetic pathways to linoleic acid that have different temperature dependencies.
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  • Teruyuki Nagamune, Takashi Nakamura, Isao Endo, Ichiro Inoue
    1991 Volume 17 Issue 3 Pages 462-469
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    The kinetics of the enzymatic hydrolysis of sucrose by cell-bound invertase of yeast (Saccharomyces cerevisiae) and the change in its maximal reaction rate during cultivation were examined in both batch culture and discontinuous-feeding fed-batch culture systems using a synthetic medium whose composition of sugars is almost the same as that in cane sugar molasses (sucrose : glucose : fructose= 2 : 1 : 1). The experimental results showed that the reaction of sucrose inversion by cell-bound invertase of yeast was inhibited not only by substrate sucrose but also by products, competitively by fructose and partially noncompetitively by glucose. It was also observed that the maximal reaction rate of cell-bound invertase was limited at high total sugar concentration and increased as its concentration was lowered. This response of maximal reaction rate against total sugar concentration was expressed in terms of a dimensionless characteristic curve. These results were incorporated into a kinetic model that has proven satisfactory in simulating hydrolysis of sucrose by yeast in batch culture and discontinuous-feeding fed-batch culture systems.
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  • Masaru Hakoda, Tsunenori Chiba, Kozo Nakamura
    1991 Volume 17 Issue 3 Pages 470-476
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    When protein solutions are separated and concentrated by ultrafiltration, protein molecules are accumulated on the membrane due to concentration polarization and form a gel layer with larger filtration resistance. Electro-ultrafiltration is a method that mitigates filtration resistance where an electric field is used to move electrically charged solutes or colloids away from the membrane. In this work electro-ultrafiltration was applied to enzymatic starch hydrolysis and the effect of the electric field on the activity of glucoamylase and ultrafiltration flux was examined experimentally.
    An unfavorable effect of the electric field on the enzyme was produced with foam separation and electric denaturation. The loss of activity consisted of two terms, each of which was proportional to the amount of electricity or electric energy. When buffer solution was not used in the electro-ultrafiltration bioreactor (EUFBR) to decrease electric current, filtration flux was much improved with small loss of enzyme activity. The experimental results showed that EUFBR could be effective if the experimental conditions were chosen properly.
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  • Muneharu Goto, Masahiro Goto, Fumiyuki Nakashio, Kazuharu Yoshizuka, K ...
    1991 Volume 17 Issue 3 Pages 477-483
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Triolein, a major component of olive oil, was hydrolyzed with lipase in a membrane bioreactor using hollow fiber. The hydrolysis mechanism of triolein was investigated, taking account of the mass transfer of triolein and oleic acid through the membrane.
    It was made clear that oleic acid, one of the products, strongly inhibited the hydrolysis of triolein at high concentration. Glycerin, another product, did not inhibit the reaction.
    This hydrolysis reaction with lipase could be quantitatively explained by the Michaelis-Menten mechanism at the interface between the aqueous and organic phases, taking account of the adsorption of lipase and the permeation rate of triolein and oleic acid.
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  • Yoshitaka Sudo, Hajime Okawara, Eiji O'Shima
    1991 Volume 17 Issue 3 Pages 484-490
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Phaeodactylum tricornutum is a microscopic marine diatom belonging to the Bacillariophyceae class of algae and contains 735% of lipids in the form of myristic acid, eicosapentaenic acid and other acids. Due to the high rate of growth of Phaeodactylum tricornutum, which breeds 1000-fold in 10 days in terms of number of cells, it attracts interest as a substitute for the fossil hydrocarbons of petroleum as an evergy source.
    In the present work, Phaeodactylum tricorutum was cultivated in batch operation in artificial sea water with bubbling of air as the carbon source, where the light was turned on and off with a 24-hour cyclic period. The optimum operating conditions were found to be such that the light source is placed at the bottom of the vessel, illuminating upward with an intensity brighter than 10, 000 lux, while the temperature is 20°C, and the aeration rate is 2 (liters/min/liter of culture solution). It was found from the experimental investigations that under such conditions the rate of growth of Phaeodactylum tricoruntum can be represented by the model of integrated intensity of illumination.
    The dependencies of size distribution, dry weight of biomass, and lipid content on illumination intensity were also investigated.
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  • Tetsuo Kobayashi, Sadao Ozawa, Kazumi Sato, Teruyuki Nagamune, Isao En ...
    1991 Volume 17 Issue 3 Pages 491-496
    Published: May 10, 1991
    Released: February 19, 2010
    JOURNALS FREE ACCESS
    By using urethane foam carrier (UFC) as a semi-solid medium, and impregnating it with a liquid medium, we have studied an effective solid-state system for cultivation of Aspergillus oryzae. This system can control the initial substrate concentration and medium composition as well as the feed rate of medium, and enzyme can be separated simply from the culture system.
    It was elucidated that maximum production activity of glucoamylase was obtained under batch culture conditions where the initial amount of sugar was around 35, 000 mg/l -bulk volume and the initial moisture content was from 78 to 80%. By feeding the soluble starch medium intermittently, it was found that glucoamylase could be produced semicontinuously with high production activity when moisture content was maintained around 85% during the repeated -batch culture period.
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  • Chiaki Hatanaka, Toshihide Haraguchi, Shunsuke Ide, Kouji Nishimiya, T ...
    1991 Volume 17 Issue 3 Pages 497-503
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Glucoamylase was immobilized in highly crosslinked polyacrylamide gel by plasma-initiated polymerization, exposing the frozen aqueous monomer to a glow discharge for 90 seconds followed by postpolymerization at room temperature for several days. The polymer gel obtained was refrozen by liquid nitrogen and thawed. By this means, immobilization efficiency was increased drastically. Immobilization efficiency and half-life reached 65% and 520h respectively at a molar ratio of 0.1 and a monomer concentration of 0.20. The productivity of immobilized enzyme was 0.635 mol/g using 30% dextrin as substrate.
    Glucose purity in the saccharificates exceeded 97% by a combination of continuous saccharification through the bioreactor using this enzyme with membrane separation by UF-membrane having a molecular cutoff of 1000.
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  • Yoshitoshi Nakamura, Moniruzzaman Mohammed, Mamoru Nagao, Tatsuro Sawa ...
    1991 Volume 17 Issue 3 Pages 504-510
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Steam explosion is one of the effective pretreatment methods for enzymatic saccharification of plant biomass. Rice straw was treated with high-pressure steam (2.55, 3.04, 3.53 and 4.02MPa) for 0.510 min. The exploded rice straw was separated into water-soluble hemicellulose, holocellulose, methanol-soluble lignin and Klason lignin. Effects of this preteatment on the characteristics of exploded rice straw were studied from experimental data of pH and pore size distibution of exploded material, amounts of extractive components and enzymatic saccharification. The enzymatic saccharification increased with increasing amount of methanol-soluble lignin. The saccharification of the exploded rice straw was expressed as a function of steam pressure and steaming time. It was estimated from the equation that maximum saccharification was obtained at a steam pressure from 3.3 to 3.8MPa and a steaming time of 1.0 to 3.5min.
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  • Akira Hirata, Makoto Hirata
    1991 Volume 17 Issue 3 Pages 511-517
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Three kinds of acetic esters -ethyl acetate, butyl acetate and amyl acetate -were used as the organic component in an aqueous-organic biphasic mixture for aspartame precursor synthesis, accompanied by solvent extraction.
    Firstly, the distribution ratios of the substrates and product between organic and aqueous media were measured at various pH, and it was confirmed that these three acetic esters are suitable as the solvent in this reaction system.
    Secondly, the effect of acetic esters on the native enzyme activity was examined by using three solvent systems : water free from any ester, water saturated with the respective ester and aqueous -ester biphasic mixture. Comparison of the latter two with the former showed that the reaction rate was lowered in the acidic range in the case of ethyl acetate, but no remarkable effect was observed in the cases of butyl acetate and amyl acetate.
    Thirdly, using butyl acetate as the solvent for substrate feeding and product extraction the batchwise synthesis was performed repeatedly. It was found that free enzyme can be used repeatedly without its deactivation, merely by renewing the organic phase.
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  • Ryuichi Ueoka, Yoko Matsumoto, Yasuo Kato
    1991 Volume 17 Issue 3 Pages 518-523
    Published: May 10, 1991
    Released: November 12, 2009
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    The enantioselective hydrolysis of long-chain substrates (p-nitrophenyl N-dodecanoyl-D (L) -phenylalaninate : C12-D (L) -Phe-PNP) and p-nitrophenyl N-dodecanoyl-D (L) -leucinate : C12-D (L) -Leu-PNP) were carried out in cationic micellar (hexadecyltrimethylammonium chloride : CTAC ; hexadecylbenzyldimethylammonium chloride : CBzAC) systems. The rate constants for the hydrolysis of C12-L-Phe-PNP are markedly enhanced by N- (benzyloxycarbonyl) -L-phenylalanyl-L-histidine (Z-L-Phe L-His) through the efficient hydrophobic interaction between the L-Phe residues (in the Z-L-Phe-L-His catalyst and the L-form substrate). The enantioselectivity decreased from a value of kLa, obsd/kDa, obsd = 18 for the N- (benzyloxycarbonyl) -L-phenylalanyl-L-histidyl-L-leucine (Z-L-Phe-L-His-L-Leu) catalyst to that of kLa, obsd/kDa, obsd= 5.8 for the N-dodecanoyl-L-phenylalanyl-L-histidyl-L-leucine (C 12-L-Phe-L-His-L-Leu) catalyst having a long acyl chain. High enantioselectivity (kLa, obsd/kDa, obsd = 38) was attained for the hydrolysis of C12-D (L) -Phe-PNP as catalyzed by Z-L-Phe-L-His-L-Leu with CBzAC micelles above the critical micelle concentration through the efficient hydrophobic (recognizant) interaction between active tripeptide (Z-L-Phe-L-His-L-Leu), L-substrate (C12-L-Phe-PNP), and surfactant (CBzAC).
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  • Xin-hui Xing, Hiroyuki Honda, Naohiro Shiragami, Hajime Unno
    1991 Volume 17 Issue 3 Pages 524-530
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Characteristics of microbial communities retained in polyurethane cubes of different sizes (5, 7 and 10 mm) were investigated. It was found that microbial communities retained by the support particles started forming at the center of the cubes and gradually extended to the periphery. Although the development of the microbial communities depended on the support sizes, their steady-state densities were independent of support size.
    The results also showed that besides organic oxidation and nitrification, which occurred in all support particles, denitrification was also observed in 7-and 10-mm support particles in which an anaerobic region was presumably present. However, denitrification was scarcely observed in 5-mm support particles in which, presumably, only aerobic bacteria existed. Therefore, support particles of appropriate size make it possible to develop a microbial community capable of simultaneous removal of nitrogenous and organic substances.
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  • Masao Sudoh, Yasuyuki Uda, Takayuki Mukouyama, Masayuki Katsumata
    1991 Volume 17 Issue 3 Pages 531-538
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    The relationship between output current and glucose concentration was studied experimentally and theoretically for a glucose sensor having a hydrogen peroxide electrode as a detector. A theoretical analysis was made, considering diffusions of glucose, oxygen, hydrogen peroxide and hydrogen ion, and also the rate of the two-substrate reaction catalyzed by glucose oxidase (GOD) within the GOD-immobilized membrane. Two Michaelis constants of enzyme kinetics were obtained experimentally as functions of pH for soluble enzyme. The activity of immobilized enzyme was estimated from the linear relationship between output current and glucose concentration under the condition of homogeneous reaction across the membrane, and was evaluated to be close to the activity of native enzyme used for membrane preparation. The reaction rate of GOD in the vicinity of the electrode was enhanced by oxygen generated by electrode reaction, but was depressed by hydrogen ion simultaneously generated by electrode reaction. The effect of electrode reaction on output current became dominant at more than 300 mA·m-2 and output current showed the tendency of asymptotic saturation as glucose concentration increased.
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  • Satoshi Matsuoka, Hisao Tanaka, Yutaka Ado
    1991 Volume 17 Issue 3 Pages 539-546
    Published: May 10, 1991
    Released: November 12, 2009
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    A sensor which detects NAD (P) H-dependent fluorescence is commercially available. Some studies on its application to fermentation have been reported. However, many studies are still necessary before such a sensor can be used for monitoring fermentation. For instance, quantitative studies of the interpretation of culture fluorescence have not been reported yet.
    To get more quantitative information from the detected fluorescence, we derived the basic equations between the fluorescence itself and the fluorophore concentrations from the Beer-Lambert Law. And we applied the equations to the estimation of the intracellular fluorophore concentration in culture. There was good similarity between the change of the estimated intracellular fluorophore concentrations and the change of the intracellular NAD (P) H levels judged by the feature of cultural changes in the batch fermentation of Saccharomyces cerevisiae.
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  • Hiroshi Ueda, Masako Kikuchi, Hajime Nishimura, Akira Hasegawa, Shinta ...
    1991 Volume 17 Issue 3 Pages 547-552
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    A hybrid receptor gene that combines the murine immunoglobulin IgM gene and a cDNA encoding the cytoplasmic domain of human epidermal growth factor receptor (EGFR) was constructed to cultivate the possibility of a new biosensor. The construct was transfected into myeloma cell which expresses Ig light chain. After selection, transfectant cells which express IgM-EGFR chimeric receptor protein were obtained.
    The chimeric protein could assemble to H2L2 heterotetramer conformation and had the same antigen 4-Hydroxy-3-Nitrophenacetyl binding activity as its parent murine IgM. While wild-type membrane IgM is not transported to the plasmamembrane of this cell line, the chimeric receptor was transported from endoplasmic reticulum to the plasma membrane through Golgi apparatus. Moreover, this antigen receptor had in vitro protein tyrosine kinase activity, which was necessary for intracellular signal transduction. The results indicate that myeloma cells can be used as an expression system of such biosensor proteins.
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  • Ryoichi Nagata, Mitsugu Tanaka, Shinichiro Gondo
    1991 Volume 17 Issue 3 Pages 553-558
    Published: May 10, 1991
    Released: November 12, 2009
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    Uricase was immobilized on oxirane acrylic beads to attain stabilized activity of uricase. The immobilized uricase particles were packed in a micro bed (5.5 mm in dia. and 1.55.0 mm in depth) and attached to the top of a dissolved oxygen sensor of galvanic type. The concentration response characteristics and the stability of the sensor were studied for uric acid concentrations of 010 mg/dl in 50 mM tris borate buffer solution of pH 8.5 at a temperature of 1535°C. The results are summarized as follows.
    1) Dynamic response of the sensor to stepwise change of uric acid concentration was approximately expressed by an equation of first-order response. The dead time was 0.10.68 min and the constant was 0.180.6 min. 2) Steady-state response of the sensor was proportional to the uric acid concentration up to 2.53 mg/dl. 3) When it was stored in the buffer solution at room temperature while not used, the sensor showed stable response within ±5% fluctuation for 250 days and more. 4) The slope of the linear correlation of sensor response with uric acid concentration was shown theoretically and experimentally to be inversely proportional to the dissolved oxygen concentration.
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  • Hideaki Endo, Masayasu Suzuki, Koji Sode, Eiichi Tamiya, Isao Karube
    1991 Volume 17 Issue 3 Pages 559-564
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    An on-line monitoring system was developed for the measurement of various parameters such as cell concentration, viscosity of broth and sucrose concentration in dextran fermentation. This system was constructed from a piezoelectric gum sensor, a quartz crystal viscous sensor, a sucrose sensor, a bioreactor and a personal computer.
    A piezoelectric gum sensor for the determination of biomass was prepared with two piezoelectric gums, a pulse generator and an amplifier. The calibration curve for L. mesenteroides was linear in the range of 1.0-4.8 g dry cell mass.l-1. A quartz crystal viscous sensor was prepared with AT-cut quartz crystal, an oscillating circuit and a peak level meter. A linear relationship was obtained between resonant resistance (R1) of the quartz crystal and (ρη) 1/2 of dextran solution, where ρ and η were density and viscosity respectively. A sucrose sensor was prepared with three immobilized enzyme layers (glucose oxidase, mutarotase and invertase) and a gold working electrode. The calibration curve for sucrose was linear up to 1000 mg·l-1.
    An on-line monitoring system was applied to dextran fermentation, and these paramenters were measured. Good correlation was obtained between the sensor signals and the values determined by conventional methods.
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  • Hideki Kidoushi, Shigeki Murayama, Shigefumi Shiomi, Katsuji Haneda, Y ...
    1991 Volume 17 Issue 3 Pages 565-571
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Maximization of fermentation productivity by the dynamic programing method was studied using a fed-batch culture of Rhodobacter sp., an aerobic bacteria that produces coenzyme Q10. Our aim was to determine the optimal control pattern of the air flow rate with cell concentration as a state variable, under conditions where glucose concentration is not a limiting factor.
    Based on the experimental data, the relation of both the specific growth rate and the specific production rate to the specific respiration rate were expressed as functional tables, which made possible a simulation study. The study produced the optimal pattern of oxygen supply, which had never been obtained by operators' experience. The relationship data between cell concentration and optimal supply rate of oxygen were mapped and the fermentation was controlled by this map. Fermentation experiments using a dynamic programing method were carried out in a 0.03 m3 jar fermentor, which confirmed higher productivity. In addition a method for decreasing the number of map data was investigated by considering map permission area where productivity decrease was kept within an acceptable range.
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  • Suteaki Shioya, Hiroshi Shimizu, Patoomporn Chim-anage, Ken-ichi Suga
    1991 Volume 17 Issue 3 Pages 572-578
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Process optimization can be accomplished via the three steps of modelling, optimization and realization, and the grade of errors and difficulties included in each step should be harmonized with one another from a practical viewpoint. In this paper a harmonized method to optimize fed-batch processes of microorganism cultivation is explained. We propose a way to build a mathematical model of specific production rate in fed-batch culture using specific growth rate μ.
    An optimal profile of μ for maximum production could be easily calculated based on the Maximum Principle model and was realized by a combination of exponential fed-batch cultures or by the programmed / feedback-control (PF) system with extended Kalman filter. Maximum production of histidine excreted in the medium and maximum intracellular production of glutathione were accomplished as applied examples of the method proposed here. The applicability and usefulness of the method were also studied and the further investigations required were described.
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  • Hajime Asama, Teruyuki Nagamune, Isao Endo, Makoto Hirata, Akira Hirat ...
    1991 Volume 17 Issue 3 Pages 579-585
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Aiming at supporting research and development of bioproducts, an expert system for diagnosing malfunctions of bioprocesses was prototyped. At first, malfunctions in fermentation processes were analyzed, and the functions required for the diagnosing system are clarified. Next, requirements for the expert system were determined, and an expert system for diagnosing a control system of fermentation processes was designed, focusing on process variables. Concerning knowledge representation, hueristic knowledge for diagnosis was represented by production rules, and the model of a cultivating system and causality of malfunctions were represented by frames. In the inference process, while root causes of the malfunction are searched with activating rule sources, the effects introduced by the root cause were verified by tracing the frame network representing the causality. This inference made reliable diagnosis possible. Finally, an expert system was implemented which diagnoses temperature control system, pH control system, and dissolved-oxygen control system interactively. Diagnosis of typical malfunctions was tested by the expert system. The results proved reasonable.
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  • Akira Hirata, Makoto Hirata, Hiroshi Furuzawa, Naoshi Honda
    1991 Volume 17 Issue 3 Pages 586-588
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    A semi-continuous pulsed extraction column bioreactor retaining free enzyme in an aqueous phase was devised for the enzymatic synthesis of the precursor of the artificial sweetener aspartame, using butyl acetate as an organic solvent for substrates feeding and product extraction. Only organic solvent was fed and flowed out, and free enzyme was retained in the reactor without immobilization. This reactor made it possible to use free enzyme continuously and to carry out stable production and extraction by pulsation.
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  • Seiji Ishida, Jun Saeki, Toshiyuki Kawashima, Eitaro Kumazawa, Shigeo ...
    1991 Volume 17 Issue 3 Pages 589-594
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Endotoxin was separated from a macromolecular antibiotic SN-07 by ion exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC). For IEC and HIC, endotoxin-free columns were prepared by washing the adsorbent gels with alkaline-ethanol followed by repeated wash with buffers of high and low concentrations of salt by turns.
    Using an endotoxin-free column (Sepabeads FP-DA 13) for IEC, 95.5% of charged endotoxin was removed from the SN-07 fractions. For HIC, two types of adsorbent gel, Sepabeads FP-PH 12 and FP-PH 13, were used. FP-PH 13 has a higher ligand concentration than that of FP-PH 12. For both gels the differences in adsorption capacity for endotoxin at these salt concentrations of NaCl and (NH4) 2SO4 were slight. In HIC purification of SN-07, the removal of endotoxin involved in the SN-07 fractions after purification by IEC was as high as 92.8% for FP-PH 12 and 95.7% for FP-PH 13. This result corresponded to the adsorption capacity of both gels for endotoxin.
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  • Hideharu Yagi, Masaaki Nakao, Kiyotaka Bitoh, Kazuhiro Uenishi
    1991 Volume 17 Issue 3 Pages 595-600
    Published: May 10, 1991
    Released: November 12, 2009
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    The salting-in and salting-out of protein result from the variation of state of protein in the solution with the concentration of coexisting salt. These phenomena are encountered in the ultrafiltration of protein dissolved in an aqueous salt solution. The ultrafiltration of an aqueous papain solution containing ammonium sulfate of various concentrations was carried out in a stirred vessel. Although the papain concentrations in the bulk liquid were much lower than its solubilities and the variation in viscosity of solutions was small, the characteristics of ultrafiltration varied with ammonium sulfate concentration. For a given papain concentration the resistance of the gel layer increased with increasing ammonium sulfate concentration in the salting-out region. In the salting-in region the mass transfer coefficients were large and the concentrations of papain at the interface of the gel layer were near the solubilities. In the salting-out region the former were small and the latter were higher than the solubility. The results suggest that papain behaves as molecules in the salting-in region but as aggregates in the salting-out regions.
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  • Yumi Yoshikawa, Kazuhisa Nagata, Kanji Matsumoto, Haruhiko Ohya
    1991 Volume 17 Issue 3 Pages 601-606
    Published: May 10, 1991
    Released: November 12, 2009
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    The extraction rate of trehalose from baker's yeast with pure water and ethanolwater solutions was affected mainly by ethanol concentration, extraction temperature and cell disruption. In particular the suppression of enzyme activity of trehalase, which hydrolyzes trehalose to glucose, in the vacuole was important in obtaining a good trehalose extraction rate. One method of suppressing the enzyme activity was to use ethanol-water solution above 343K, and the maximum extraction rate was obtained at an ethanol concentration of 4550 wt%. Another more effective method to inactivate trehalose was to dry the yeast. In this case extraction with the pure water was possible.
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  • Ryoichi Kuboi, Yasushi Yamada, Yoshiaki Mori, Isao Komasawa
    1991 Volume 17 Issue 3 Pages 607-613
    Published: May 10, 1991
    Released: November 12, 2009
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    Forward and backward liquid-liquid extraction of Chromobacterium viscosum lipase were successfully carried out using reverse micellar systems. The reverse micellar system prepared by dissolving both AOT and taurodeoxycholic acid (TDCA) in isooctane was found to form a stable mixed micelle showing distinct advantages in both activity yield and selectivity for lipase over the conventional AOT reverse micellar system.
    The sizes of mixed micelles measured by using a dynamic light-scattering method were not different from those of AOT micelles (50mM AOT) and did not vary with TDCA concentration in the range of 0-4 mM. Nearly 6.2 molecules of AOT were replaced with a TDCA molecule at the micellar interface. The partition coefficient of lipase was selectively improved up to eight-fold in contrast to α-chymotrypsin and papain. Its activity yield was also improved by using a simple phase transfer or liquid membrane extraction method.
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  • Kazuyuki Kishi, Shintaro Furusaki
    1991 Volume 17 Issue 3 Pages 614-619
    Published: May 10, 1991
    Released: November 12, 2009
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    Low-molecular weight peptides were extracted into the oil phase by reversed micelles using AOT. The effect of hydrophobicity and charge density of the peptides on the partition coefficient between the water phase and the oil phase was investigated. The partition coefficient of the peptides increased with the increase of charge density. The peptides with higher hydrophobicity had higher partition coefficient than those with lower hydrophobicity. This result suggests the possibility of a separation procedure using the reversed micelle system, which is based on the combined interaction of the hydrophobicity and the electric charge. The liquid-liquid equilibrium and the extraction behavior were investigated for the specific peptides Lys-Lys-Gly-Glu and the eelcalcitonin-fragment. Enrichment of the peptides in the micellar phase was observed, similaly to the case of amino acid extraction.
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  • Seiji Sugimoto, Yoshiharu Yokoo
    1991 Volume 17 Issue 3 Pages 620-622
    Published: May 10, 1991
    Released: November 12, 2009
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    A purification procedure for highly purified and highly uniform recombinant eel growth hormone (EGH) was established and found to be applicable to large-scale production. Inclusion bodies of recombinant EGH expressed in Escherichia coli were obtained by the disruption of cells and washing. The inclusion bodies were solubilized in urea solution and refolded by dilution and addition of oxidized glutathione. EGH analogues such as oxidized EGH at methionine and formylated EGH at N-terminal, which were produced during the fermentaion and/or purification process, were removed by subsequent hydrophobic interaction chromatography.
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  • Kenji Hashimoto, Yoshihito Shirai, Shuji Adachi, Masaharu Horie
    1991 Volume 17 Issue 3 Pages 623-626
    Published: May 10, 1991
    Released: November 12, 2009
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    Maltose and glucose were separated either in a batchwise chromatographic column or in a simulated moving -bed adsorber packed with ion -exchange resin. Feeding rates of the sample solution per unit bed volume, P, were compared between these operation modes. P was three or four times higher in the simulated moving bed system than in the batchwise one. This tendency was confirmed experimentally.
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  • Atsushi Hashimoto, Masaru Shimizu, Hideo Igarashi
    1991 Volume 17 Issue 3 Pages 627-633
    Published: May 10, 1991
    Released: November 12, 2009
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    The present study aims at determining the influence of far-infrared radiation (FIR) on the pasteurization effect. Escherichia coli 745 or Staphylococcus aureus 9779, suspended in phosphate-buffered saline (pH 7.0), was used as the test bacterium. The bacterial suspension was irradiated by FIR or heated by a water bath. Pasteurization was performed under the condition that the transient behavior of the bulk temperature of the suspension irradiated by FIR was the same as that of the suspension heated by the water bath. The numbers of viable cells and of injured cells were estimated from colony counts. After the heat treatments, the number of viable cells in the suspension irradiated by FIR was less than that heated by the water bath, and the ratio of the number of injured cells to that of viable cells irradiated by FIR was higher than that heated by the water bath. It was suggested that the pasteurization effect of FIR was due to the absorption of infrared radiation energy by suspension in a very thin domain near the surface and to the bulk temperature of the suspension.
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  • Atsushi Hashimoto, Makoto Takahashi, Taijiro Honda, Masaru Shimizu, At ...
    1991 Volume 17 Issue 3 Pages 634-638
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    The present study aims at determining the influence of the heat transfer mechanism on the amount of maltose produced by heating sweet potato in a packed-bed. The yield of maltose did not depend on whether the heat transfer mechanism in the bed was controlled by heat radiation or by heat conduction. It did depend, however, on the time required to raise the center temperature of the sweet potato from 338K to 353K. When that time was about 8 min and total time for heating the sweet potato was 3036 min, the yield was maximum.
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  • Yoshihito Shirai, Ryuichi Matsuno, Kazuhiro Nakanishi
    1991 Volume 17 Issue 3 Pages 639-641
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    A method of predicting ice crystal size distribution in a continuous crystallizer was extended. Ice crystal growth rate was estimated by a mass transfer resistance-limiting model. Based on the nucleation rate of ice crystals per crystal obtained in a batchwise crystallization experiment and the model, ice crystal size distribution formed in a sugar solution with dextran was predicted with reasonable accuracy.
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  • Masami Shimoda, Masatoshi Matsumura, Hiroshi Kataoka
    1991 Volume 17 Issue 3 Pages 642-648
    Published: May 10, 1991
    Released: November 12, 2009
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    Using a perfusion culture with bleeding, dense cultivation of mouse-mouse hybridoma cells was carried out in serum -free medium under glucose -limiting conditions. The effects of glucose concentration on cell growth and metabolic rates of carbon sources were investigated.
    The specific growth and death rates were not influenced by glucose concentration, but the metabolism of glucose and glutamine was found to be remarkably affected by glucose concentration. High glucose concentrations resulted in both overutilization of glucose and high yields of lactate from glucose. Conversely, high ammonia accumulation was observed when glucose concentration in the broth was kept very low. The above results suggest that there exists an optimum glucose concentration for reducing specific lactate and ammonia production rates.
    Cell growth was found to be well correlated with ATP production calculated from glucose and glutamine metabolisms. This means that the energy required for cell growth was constant in spite of varied metabolic behaviour. Hybridoma cells could adapt to glucose -limiting conditions and metabolize the alternative energy substrate of glutamine.
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  • Taku Matsushita, Kazuhisa Ishibashi, Masako Kizu, Kazumori Funatsu
    1991 Volume 17 Issue 3 Pages 649-654
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    To cultivate plant cells at high density, we tried to make a suitable shape of air -lift column with draft tube so as to obtain good circulation and mixing, along with a high kLα at a lower flow rate. It was found to be important to use a sparger which generated numerous bubbles of homogeneous size, and a draft tube having side holes for efficient separation of medium and cells, the diameter of which fitted the area of bubble flow.
    By using this air -lift column (culture volume of 1 liter), we could cultivate carrot cells at high cell density (1.09 × 107 cells/ml, 15.2 g-dry weight/l) for 713 h of culture time, in which 50% of medium exchange was performed three times. This cell density was superior than that of other culture vessels. These results suggest that this air -lift column is usable for high-density culture of plant cells.
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  • Naoki Nakatsugawa, Koki Horikoshi
    1991 Volume 17 Issue 3 Pages 655-666
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    We have embarked on research attempting to isolate “super-methanogens” which grow in extreme environments, for use in devising an effective methane fermentation system and removing technical restrictions on the methane fermentation process.
    We have developed new and effective screening methods of “super-methanogens” and have isolated a variety of halophilic, alkaliphilic, thermophilic and psychrotolerant methanogenic archaebacteria.
    Strain NY-218, one of our isolates, is a new halophilic, alkaliphilic and psychrotolerant methanogen which was isolated from a hypersaline, alkaline environment in California, USA. The optimum NaCl concentration and pH for growth and methanogenesis were 2.53.0 M and 8.08.5, respectively. Strain NY-218 is the first extremely halophilic and alkaliphilic methanogen to be reported.
    Strain NY-728, another of our isolates, is a novel alkaliphilic and psychrotolerant methanogen belonging to the genus Methanosarcina. It was isolated from a lake sediment in Japan. The optimum pH for growth and methanogenesis was 8.1-8.7. Strain NY-728 is the first reported alkaliphilic Methanosarcina and is a new species.
    Growth characteristics and applicability of strains NY-218 and NY-728 to methane fermentation were examined by using 1- to 3-liter scale cultures under various conditions.
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  • Motoyuki Suzuki, Yasuyuki Sakai
    1991 Volume 17 Issue 3 Pages 667-670
    Published: May 10, 1991
    Released: November 12, 2009
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    Adult rat hepatocytes in primary culture inoculated on polylysine-coated surfaces were found to form spheroidal aggregates, which have recently been reported to express higher hepatic functions than those in monolayer cultures.
    To establish the culture condition for industrial utilization, the effects of serum, epidermal growth factor (EGF) and aprotinin on the formation and maintenance of spheroids were examined. Serum was effective in the maintenance of spheroids, but delayed their formation. EGF improved their formation greatly. Aprotinin inhibited spheroid formation but improved their maintenance after formation was completed.
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  • Masayuki Sato, Takao Shinozawa, Hiroyoshi Ueno, Masayoshi Sadakata
    1991 Volume 17 Issue 3 Pages 671-673
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    The authors proposed a valuable method for the cultivation of adherent cells on an oil-water interface. It was found experimentally that A-375 cell line adhered to a fluorocarbon-medium interface and spread all over the plane interface where there were good or had combinations of oil and water for cell growth. Cultivation on oil droplet surfaces was also studied with a small amount of surface-active agent added, where the percentage of droplets with/without cell adhesion varied with oil droplet diameter and amount of surface-active agent. By the present method, about 90% of cultivated cells was obtained without the usual practice of using trypsin solution to remove cells from the wall surface.
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  • Naofumi Shiomi, Hideki Fukuda, Yasuki Fukuda, Kousaku Murata, Akira Ki ...
    1991 Volume 17 Issue 3 Pages 674-679
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    To apply genetically engineered yeast cells to practical processes for the production of useful compounds, the construction of vector plasmids having high stability and expressive selection markers in polyploid wild yeast cells is required. For this purpose, we selected an ethionine -resistance phenotype of a yeast Saccharomyces cerevisiae as a marker and constructed a vector plasmid pER 9 containing ethionine-resistance gene. The vector plasmid pER9 was 9.8 kb in length, a suitable size for manipulation, and had available cloning sites (Bam H I and SalI) for foreign DNAs. The plasmid was stably maintained in haploid yeast cells and almost all the cells (98%) contained the plasmid, even after 40 generations, when ethionine was in the growth medium as a selection pressure. Since the ethionine-resistance gene on plasmid pER9 could be expressed and maintained in cells of yeast strains such as sake and beer yeasts, pER9 would make it possible to breed industrially useful polyploid strains of yeats at the molecular level.
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  • Kong-Hua Lin, Shinji Iijima, Shih-Yow Huang, Fumio Hishinuma, Takeshi ...
    1991 Volume 17 Issue 3 Pages 680-686
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Saccharomyces cerevisiae harbouring a recombinant plasmid pNW052 was cultured in a shake flask and a jar fermentor. In the recombinant plasmid, the mouse amylase gene was fused to S. cerevisiae PGK (phosphoglycerate kinase) promoter. In shake flask cultivations we found that ammonium sulphate inhibited the expression of mouse amylase from PGK promoter. By replacing ammonium sulphate with yeast extract, gene expression was enhanced and 10 kg/m3 yeast extract gave sufficient gene expression and cell growth. In addition to nitrogen source, we investigated the effect of glucose concentration on mouse amylase production from PGK promoter using an on-line glucose analyzer in a jar fermentor culture. When glucose concentration was controlled at 10 kg/m3, gene expression was repressed. On the other hand, when glucose was controlled at 0.15 kg/m3, mouse amylase was produced and secreted into the medium very efficiently. This amount of the enzyme was almost 10 -fold that obtained at 10 kg/m3 glucose. By the combination of an on-line glucose monitoring and control system and yeast PGK promoter, effective production of gene product could be achieved.
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  • Hideo Nakano, Toshiro Takai, Yasushi Kawakami, Hajime Nishimura, Masah ...
    1991 Volume 17 Issue 3 Pages 687-693
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Several fusion proteins composed of the cytoplasmic protein Escherichia coli dihydrofolate reductase (DHFR) and E. coli α-hemolysin (HlyA) were constructed and their secretion to the medium was examined in order to develop a specific secretion system. Although no chimeric proteins having an intact DHFR region were secreted, those lacking a C-terminal moiety of DHFR were secreted to the culture medium by the hemolysin transport system.
    The C-terminal region of HlyA necessary for its secretion was shown to exist at least within the final 61 amino acids stretch according to the results of secretion of the fused proteins.
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  • Kimi Ojima, Tsutomu Fukumoto, Shuichi Takei, Takao Igarashi, Kazuhisa ...
    1991 Volume 17 Issue 3 Pages 694-700
    Published: May 10, 1991
    Released: November 12, 2009
    JOURNALS FREE ACCESS
    Expression of the gene coding for 3-isopropylmalate dehydrogenase (3-IPM dehydrogenase) from Bacillus caldotenax in Escherichia coli was investigated by following the time courses of cell mass concentration, levels per unit cell mass of the mass of plasmid pTMY2 carrying this gene and the activity of 3-IPM dehydrogenase. The cells were grown on both Davis minimal medium and YT medium at different temperatures. Constant levels in the mass of DNA and the activity of 3-IPM dehydrogenase during logarithmic growth were observed at temperatures higher than 34°C on the chemical defined medium and higher than 31°C on the complex medium. Those levels decreased exponentially with time at lower temperatures, indicating an appearence of plasmid DNA that failed to duplicate during the cell cycle. Equations describing the replication and expression of the gene were also developed and applied to the experimental data. On the grounds that cells are capable of the expression of 3-IPM dehydrogenase genes in both media, we surmise the involvement of a promoter in the DNA fragment from the thermophie.
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