(Vol.61 No.1 1997)
Functions of Phosphorus Moiety
in Agrochemical Molecules
Morifusa Eto 1
Biosynthesis of Phytotoxins from
Alternaria solani
Akitami Ichihara and Hideaki Oikawa 12
Micro-assay Method for Evaluating
the Allergenicity of the Major Soybean Allergen, Gly m Bd 30K, with Mouse
Antiserum and RBL-2H3 Cells
Rintaro Yamanishi, Hideaki Tsuji, Noriko Bando, Izumi Yoshimoto, and
Tadashi Ogawa 19
Immune Bioactivity in Shellfish
toward Serum-free Cultured Human Cell Lines
Zwe-Ling Kong, Li-Ching Chiang, Francis Fang, Kazuki Shinohara, and
Peter Pan 24
Protein, and Dietary Fiber-rich
New Foodstuff from Brewer's Spent Grain Increased Excretion of Feces and
Jejunum Mucosal Protein Content in Rats
Osamu Kanauchi and Kazue Agata 29
Analysis of Low Temperature Inducible
Mechanism of ม-Glutamyltranspeptidase of Escherichia coli K-12
Wataru Hashimoto, Hideyuki Suzuki, Kenji Yamamoto, and Hidehiko Kumagai
34
Purification and Characterization
of a Thermostable Pyruvate Kinase from the Actinomycete Microbispora thermodiastatica
Ayumi Arai, Seiji Murakami, and Moto-o Nakajima 40
Production of 3-Vinylcatechol
and Physiological Properties of Pseudomonas LF-3, Which Can Assimilate
Styrene in a Two-phase (Solvent-Aqueous) System
Yuriko Yoshida, Yoko Ikura, and Toshiaki Kudo 46
Physiological Characteristics
of a Film-forming Strain of Zygosaccharomyces rouxii, and Its Cellular
Fatty Acid Synthesis
Minoru Tomita, Sumito Yamamoto, Kanoko Yamaguchi, Hajime Ohigashi,
and Koichi Koshimizu 51
Predicted and Unsuspected
Alterations of the Genomes Structure of Genetically Defined Bacillus subtilis
168 Strains
Mitsuhiro Itaya and Teruo Tanaka 56
Physico-chemical
Properties of Purified Isoforms of the 12S Seed Globulin from Mustard Seed
(Brassica alba)
Massimo F. Marcone, Rickey Y. Yada, Wichai Aroonkamonsri, and Yukio
Kakuda 65
Novel Exopolygalacturonases
Produced by Alternaria mali
Kouichi Nozaki, Kazuo Miyairi, Seiji Hozumi, Yohko Fukui, and Toshikatsu
Okuno 75
Identification and Characterization
of a Karyotypic Mutation in Magnaporthe grisea
Teruo Sone, Takumi Abe, Manabu Suto, and Fusao Tomita 81
Molecular Cloning of an Inulin
Fructotransferase (Depolymerizing) Gene from Arthrobacter sp. H65-7 and
Its Expression in Escherichia coli
Hiroaki Sakurai, Atsushi Yokota, and Fusao Tomita 87
Evidence for a Single Active
Site in ภ-D-Glucosidase/ภ-D-Fucosidase from Dalbergia cochinchinensis
Seeds
Rudee Surarit, Hirokazu Matsui, Seiya Chiba, Jisnuson Svasti, and Chantragan
Srisomsap 93
Novel Extracellular Alkaline
Metalloendopeptidases from Vibrio sp. NUF-BPP1: Purification and Characterization
Kazumasa Fukuda, Katsumi Hasuda, Tatsuya Oda, Hiroshi Yoshimura, and
Tsuyoshi Muramatsu 96
Hesperidin as an Inhibitor of
Lipases from Porcine Pancreas and Pseudomonas
Kiyomi Kawaguchi, Takashi Mizuno, Kazuhiko Aida, and Keijiro Uchino 102
Effects of an Escherichia
coli ilvA Mutant Gene Encoding Feedback-resistant Threonine Deaminase on
L-Isoleucine Production by Brevibacterium flavum
Ken-ichi Hashiguchi, Hiroyuki Kojima, Katsuaki Sato, and Konosuke Sano
105
The Role of Microsomal
ภ-Glucuronidase in Ascorbic Acid Biosynthesis Stimulated by Xenobiotics
in Rats
Fumihiko Horio and Toru Horie 109
Expression and Characterization
of Endopeptidase in Suspension-cultured Cells of French Bean
Jae Whune Kim and Takao Minamikawa 113
Hydroxyl Radical-scavenging
Effects of Spices and Scavengers from Brown Mustard (Brassica nigra)
Shin-Kyo Chung, Toshihiko Osawa, and Shunro Kawakishi 118
A New Staining Method for Lyases
Catalyzing Cleavage of a C-S Bond in Sulfur-containing Compounds after
Polyacrylamide Gel Electrophoresis
Koji Ukai and Jiro Sekiya 124
Concise Synthesis of a Racemic
and Diastereomeric Mixture of the Sex Pheromones of Matsucoccus Pine Scales
Hidenori Watanabe, Takeru Watanabe, Takeshi Kitahara, and Kenji Mori
127
Purification and Properties
of 3ฟ-Hydroxysteroid Dehydrogenase from Pseudomonas putida
Osao Adachi, Hirohide Toyama, and Kazunobu Matsushita 131
Development of Thermotolerant
Acetic Acid Bacteria Useful for Vinegar Fermentation at Higher Temperatures
Akihiko Saeki, Gunjana Theeragool, Kazunobu Matsushita, Hirohide Toyama,
Napha Lotong, and Osao Adachi 138
Purification and Characterization
of Dipeptidyl Aminopeptidase from Aureobacterium sp. WO26
Wataru Ogasawara, Noriyuki Inanobe, Keiko Ochiai, Katsuhiko Ando, Hirofumi
Okada, and Yasushi Morikawa 146
A Novel NADP+-dependent Serine
Dehydrogenase from Agrobacterium tumefaciens
Emran Kabir Chowdhury, Kazuhiko Higuchi, Shinji Nagata, and Haruo Misono
152
Note
Bone Resorption Inhibitors
from Hop Extract
Hiroyasu Tobe, Yoshifumi Muraki, Kazuyuki Kitamura, Osamu Komiyama, Yusuke
Sato, Tatsuo Sugioka, Hiromi B. Maruyama, Eriko Matsuda, and Masahiro Nagai
158
Protection by Trehalose of DNA
from Radiation Damage
Koichi Yoshinaga, Hiroe Yoshioka, Hiromu Kurosaki, Miyuki Hirasawa, Masahiro
Uritani, and Kunihiko Hasegawa 160
Neoagarobiose as a Novel
Moisturizer with Whitening Effect
Reijiro Kobayashi, Mikimasa Takisada, Tadashi Suzuki, Kohtaro Kirimura,
and Shoji Usami 162
Syntheses of Alkyl
ภ-D-Mannopyranosides and ภ-1,4-Linked Oligosaccharides Using ภ-Mannosidase
from Rhizopus niveus
Hiroshi Fujimoto, Megumi Isomura, and Katsumi Ajisaka 164
Positional Specificity
and Stereoselectivity of a Lipase Preparation from Oat Seeds Acting on
1,2,3-Trihexanoylglycerol
Yasuhide Ota, Toshio Minesaki, and Aki Oshima 166
Isolation and Molecular
Characterization of Four Arginine/Glutamate Rich Polypeptides from the
Seeds of Sponge Gourd (Luffa cylindrica)
Hisashi Ishihara, Takahiro Sasagawa, Ritsu Sakai, Masateru Nishikawa, Makoto
Kimura, and Gunki Funatsu 168
Quantitative Analysis
of Alkylpyrazines in Regular- and Low-fat Commercial Peanut Butter Preparations
Kwangjee Joo and Chi-Tang Ho 171
Isolation and Characterization
of Chitinase from a Flake-chitin Degrading Marine Bacterium, Aeromonas
hydrophila H-2330
Kazumi Hiraga, Lee Shou, Mitsunori Kitazawa, Saori Takahashi, Masahiko
Shimada, Ryoichi Sato, and Kohei Oda 174
Isolation and Identification
of ฟ-Glucosidase Inhibitors from Tochu-cha (Eucommia ulmoides )
Jun Watanabe, Jun Kawabata, Hideyuki Kurihara, and Ryoya Niki 177
Rapid Detection Method for Bacteriocin
and Distribution of Bacteriocin-producing Strains in Lactobacillus acidophilus
Group Lactic Acid Bacteria Isolated from Human Feces
Yasushi Kawai, Tadao Saito, Junko Uemura, and Takatoshi Itoh 179
Isolation of Isoeugenitin as
a Fruiting Body Inducer for Stigmatella aurantiaca from a Soil Fungus Papulaspora
sp.
Ryosuke Fudo, Toshihiko Ando, Seiichi Sato, Toshiyuki Kameyama, and Shigeru
Yamanaka 183
Mechanism of Stereospecific
Conversion of DL-5-Substituted Hydantoins to the Corresponding L-Amino
Acids by Pseudomonas sp. Strain NS671
Takahiro Ishikawa, Ken Watabe, Yukuo Mukohara, and Hiroaki Nakamura 185
Depolymerization of Hyaluronic
Acid by Low-molecular-weight Amadori-rearrangement Products and Glycated
Polylysine
Jian Zu, Shiro Nishikawa, and Naoki Kashimura 188
Coulometric Electrochemical
Detection of Phospholipid Hydroperoxides by High-performance Liquid Chromatography
Hirofumi Arai, Satoshi Mohri, Tetsuya Suzuki, Kozo Takama, and Junji Terao
191
Enzymatic Resolution of 2,2,2-Trifluoro-1-(1-pyrenyl)ethanol
with Lipases
Katsuya Kato, Masato Katayama, Shozo Fujii, and Hiroshi Kimoto 194
Increased Stability of PEG-PPG
Conjugated Human Urokinase against Autolysis
Jun-ichi Kajihara, Kozue Shibata, and Kazuo Kato 197
Changes of Lipoxygenase and Fatty
Acid Hydroperoxide Lyase Activities in Bell Pepper Fruits during Maturation
Kenji Matsui, Yasuhi Shibata, Hideki Tateba, Akikazu Hatanaka, and Tadahiko
Kajiwara 199
High Efficiency Transformation
of Bacillus brevis by Electroporation
Akiko Okamoto, Akihiko Kosugi, Yukimichi Koizumi, Fujiharu Yanagida, and
Shigezo Udaka 202
Inhibitory Effect of Green
Tea on Injury to a Cultured Renal Epithelial Cell Line, LLC-PK1
Takako Yokozawa, Erbo Dong, Hae Young Chung, Hikokichi Oura, and Hitomi
Nakagawa 204 Rapid Paper
Increase in Lysophosphatidylethanolamine
in the Cell Membrane upon the Regulated Exocytosis of Pancreatic Acinar
AR42J Cells
Yoshiki Ikeda, Shin-Ichi Fukuoka, and Makoto Kito 207
-1-
Review
Functions of Phosphorus Moiety
in Agrochemical Molecules
Morifusa Eto*
Miyakonojo National College of Technology, Miyakonojo 885, Japan
@Organophosphorus (OP) compounds have a great variety of biological activities. The functions of the phosphorus moiety in OP agrochemicals may be classified as follows; 1) the principal of phosphorylation; 2) the leaving group in alkylation; 3) a building block to maintain the shape or physical properties of an active molecule; 4) the analog of a carboxyl group or its tetrahedral transition state in enzyme reactions; 5) a moiety of anti-metabolites mimicking physiological phosphates; 6) a carrier or protective group making a prodrug that produces an active principle after biotransformation; and 7) other unknown functions including as stressors. To have a definite, selective biological activity, the OP molecule should have a specified structure suitable in biodynamic, biokinetic, and environmental aspects.
Key words: alkylation; phosphorylation; stereochemical aspect; stressor; transition-state analog inhibition
-2-
Review
Biosynthesis of Phytotoxins from
Alternaria solani
Akitami Ichihara and Hideaki Oikawa
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060, Japan
@Alternaria solani is a causal fungus of early blight disease of potato and tomato. Two phytotoxins, alternaric acid and solanapyrones, were isolated from the different strains of the fungus. Biosynthetic studies on these two phytotoxins have been done, and in the biosynthesis of solanapyrones, it was proved that a biological Diels-Alder reaction is involved through the polyketide pathway and the participation of the enzyme Diels-Alderase has been proved for the first time.
Key words: alternaric acid; solanapyrones; Alternaria solani; biological Diels-Alder reaction; Diels-Alderase
-3-
Micro-assay Method for Evaluating
the Allergenicity of the Major Soybean Allergen, Gly m Bd 30K, with Mouse
Antiserum and RBL-2H3 Cells
Rintaro Yamanishi,๕ Hideaki Tsuji, Noriko Bando, Izumi Yoshimoto, and Tadashi Ogawa
Department of Nutrition, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770, Japan
Received April 17, 1996
@A micro-assay method for evaluating the allergenicity of soybean allergen was developed by using the mouse antiserum against Gly m Bd 30K, a major soybean allergen, and RBL-2H3, a rat mucosal mast cell line. The antiserum against Gly m Bd 30K was prepared by subcutaneously immunizing BALB/c mice with the allergen. The behavior by affinity-chromatography and the properties against heat treatment show that the reaginic activity of the antiserum resided in the IgE antibody specific for Gly m Bd 30K. The developed assay method is shown to be useful for simulating IgE mediated type-I allergy and to be highly sensitive for detecting the allergen.
Key words: allergy; reagin; soybean; Gly m Bd 30K; RBL-2H3
-4-
Immune Bioactivity in Shellfish
toward Serum-free Cultured Human Cell Lines
Zwe-Ling Kong, Li-Ching Chiang,* Francis Fang, Kazuki Shinohara,** and Peter Pan
Cellular Immunology Laboratory, Department of Marine Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, Republic of China
* King Car Food Industrial Co., Ltd., 230 Roosevelt Road, Sec. 3, Taipei, Taiwan, Republic of China
** National Research Institute of Fisheries Science, Ministry of Agriculture, Forestry, and Fisheries, 2-12-4 Fukuura, Kanazawa-ku, Yokoyama 236, Japan
Received May 7, 1996
@The biologically functional effect of eight kinds of hot-water extracts of shellfish on cultured human cell lines was examined in a serum-free medium model. Meretrix lusoria and Sinonovacula constricta extracts enhanced IgM secretion of both hybridoma HB4C5 and SI102 cells when cultured with the respective extracts. The purified principle exhibited remarked activity in the adsorbed fraction in hydroxyapatite and Concanavalin A columns. The extracts of Corbicula fluminea, Crassostreas gigas, Meretrix lusoria, Anadara granosa, and Sinonovacula constricta enhanced in nitroblue tetrazolium (NBT)-reducing ability of macrophage U-M cells. Meretrix lusoria, Anadara granosa, and Sinonovacula constricta were specifically cytotoxic to both cultures of MCF-7 breast cancer cells and HuH-6KK hepatoblastoma. These findings imply that the extracts of shellfish that were examined exhibited a differential effect on immune cells and tumor cells.
Key words: bioactivity; shellfish; immunity; functional food; antitumor
-5-
Protein, and Dietary Fiber-rich
New Foodstuff from Brewer's Spent Grain Increased Excretion of Feces and
Jejunum Mucosal Protein Content in Rats
Osamu Kanauchi and Kazue Agata
Applied Bioresearch Center Corporate Research and Development Division, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki-shi, Gunma 370-12, Japan
Received May 9, 1996
@We made a new protein-rich and fibrous foodstuff by milling and sieving brewer's spent grain. This product contained glutamine-rich protein and the dietary fibers cellulose, hemicellulose, and lignin. We called this product germinated barley foodstuff (GBF). GBF had the effect of increasing fecal dry weight and number of feces and of significantly increasing jejunum mucosal protein content in rats over the cellulose group. In GBF, Gln-rich protein is thought to have strong chemical bonds with dietary fiber, an arrangement which would be important in the way these physiological effects arise. As dietary supplements of Gln or dietary fibers (i.e., cellulose, hemicellulose, lignin, and a mixture of these) did not improve defecation and jejunum mucosal protein simultaneously, the effects of GBF are thought to be caused not by the individual ingredients, but by the combination of protein with dietary fiber.
Key words: germination; barley; defecation; intestine; jejunum mucosa
-6-
Analysis of Low Temperature Inducible
Mechanism of ม-Glutamyltranspeptidase of Escherichia coli K-12
Wataru Hashimoto,๕ Hideyuki Suzuki, Kenji Yamamoto, and Hidehiko Kumagai ๕๕
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan
Received May 13, 1996
@Escherichia coli K-12 cultured at 20C has more ม-glutamyltranspeptidase (GGT: EC 2.3.2.2) activity than that cultured at 37 or 42C. On Western blot analysis, E. coli K-12 cells cultured at 20C produced more GGT protein than those cultured at 37C. mRNA of the GGT gene (ggt) in the cells was also measured and it was found that the level of ggt mRNA at 20C was 10-fold higher than that at 37C. When the ggt promoter was replaced by a tac promoter, GGT activity at 37C from the tac promoter was 5-fold higher than that at 37C from the ggt promoter, though there was less difference in GGT activity between both grown at 20C. The ggt mRNA at 20C was found to be more stable than that at 37C. These results suggested that the higher GGT activity in E. coli K-12 cells grown at 20C was due to a higher level of GGT protein at 20C caused by higher level of ggt mRNA at 20C because of a low-temperature dependent ggt promoter as well as the stability of ggt mRNA at 20C.
Key words: Escherichia coli ; gene expression; ม-glutamyltranspeptidase; promoter analysis
-7-
Purification and Characterization
of a Thermostable Pyruvate Kinase from the Actinomycete Microbispora thermodiastatica
Ayumi Arai, Seiji Murakami, and Moto-o Nakajima
Research and Development Division, Kikkoman Corporation, 399 Noda, Noda-shi, Chiba 278, Japan
Received May 14, 1996
@The pyruvate kinase of Microbispora thermodiastatica was purified to homogeneity and some properties of the enzyme were characterized. The molecular weight of the enzyme by gel filtration is 277,000. The subunit molecular weight is 55,000 by SDS-polyacrylamide gel electrophoresis, and only one N-terminal amino acid sequence was obtained. It had a pH optimum around pH 4.5 to 7.0 and was stable over the range of pH 4.0-8.0. The enzyme is thermostable and no activity was lost after heat treatment at 55C for 60 min. AMP activated this enzyme and the saturation curve of the enzyme for PEP changed from sigmoidal type to hyperbolic type in the presence of AMP.
Key words: pyruvate kinase; thermostable enzyme; Microbispora thermodiastatica
-8-
Production of 3-Vinylcatechol
and Physiological Properties of Pseudomonas LF-3, Which Can Assimilate
Styrene in a Two-phase (Solvent-Aqueous) System
Yuriko Yoshida,* Yoko Ikura,** and Toshiaki Kudo*,**
* Research Development Corporation of Japan, and ** The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan
Received May 24, 1996
@A microorganism able to grow in mineral salt medium with styrene (0.1-10%) as a sole carbon source was isolated. The bacterium belongs to the genus Pseudomonas. Pseudomonas LF-3 used not only styrene but also toluene as a carbon source. Cell viability, total cell count, and turbidity with 0.1% showed good results. Cell viability reached 7~108 cells/ml with 1.0% styrene and remained near this after reaching its maximum. Maximum cell viability was 1.4~106 cells/ml with 1.0% styrene and 1.1~106 cells/ml with 10% styrene, respectively. Pseudomonas LF-3 grew on 1-phenylethanol, 2-phenylethanol, benzylalcohol, acetophenone, phenyl-1,2-ethanediol, benzoic acid, or protocatechuic acid. The degradation product from styrene was separated and identified as 3-vinylcatechol. The yellow color observed in the medium suggests a meta ring cleavage pathway.
Key words: Pseudomonas sp.; styrene-assimilation; 3-vinylcatechol; styrene-aqueous two-phase medium
-9-
Physiological Characteristics
of a Film-forming Strain of Zygosaccharomyces rouxii, and Its Cellular
Fatty Acid Synthesis
Minoru Tomita, Sumito Yamamoto, Kanoko Yamaguchi,*Hajime Ohigashi,* and Koichi Koshimizu**
Tokushima Prefecture Technology Center, 11-2 Nishibari, Saika-cho, Tokushima 770, Japan
* Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan
** Department of Biotechnological Science, Faculty of Biology-oriented Science and Technology, Kinki University, Iwade-Uchita, Wakayama 649-64, Japan
Received May 27, 1996
@Some physiological characteristics of Zygosaccharomyces rouxii strain No. F51, forming a film only on saline media, were investigated. When the strain was statically cultivated in a NaCl-hypertonic medium, it produced ethanol accompanied with the consumption of glucose in the early stage of the cultivation. Subsequently, the strain assimilated the resulting ethanol to form a film on the surface of the medium after the consumption of glucose. Isobutyl and isoamyl alcohols were transiently accumulated in the medium at an unusually high concentration during the film-forming growth of the strain. The total amount of lipids readily extracted from the film-forming cells (F-cells) grown in a hypertonic medium was about 4-fold as much as that extracted from the sedimentary cells (S-cells) grown in a salt-free hypotonic medium. The total amount of higher fatty acids (C(/)14-C(/)18) from F-cells was over twice as much as that from S-cells. Oleic and linoleic acids were the major fatty acid in F-cells. The quantity ratio of linoleic acid to oleic acid of F-cells was significantly high (0.5). It was suggested that the hydrophobic property of F-cells might be associated with the ability to assimilate ethanol only aerobically and to increase the total fatty acid content of cells.
Key words: Zygosaccharomyces rouxii; film-forming yeast; alcohol metabolism; cellular fatty acids
-10-
Predicted and Unsuspected
Alterations of the Genomes Structure of Genetically Defined Bacillus subtilis
168 Strains
Mitsuhiro Itaya๕ and Teruo Tanaka*,๕๕
Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan
Received June 11, 1996
@We scrutinized the genome structures of Bacillus subtilis 168 derivative strains the isolation procedure of which was well described. NotI and SfiI restriction fragment analyses indicated that strains constructed by transformation using non-168 Bacillus strains as donors had replacement of DNA segments in the predicted region. Interesting examples were found in a lineage including strains 168trpC2-YS11-RM125-MI112, the last two of which have a restriction-deficiency mutation (hsdM ) introduced from a non-168 strain DNA. As expected, the loss of the hsdM gene was a result of replacement of the hsdM region by non-168 DNA fragments. Moreover, RM125 acquired a previously unnoticed NotI site that gave no obvious phenotypes. Among 168 derivative strains investigated, the most extreme case had changes at six different regions of the genome, probably introduced by repeated transformations. Unsuspected DNA alterations were found even in isogenic strains constructed by phage PBS1-mediated transduction. These acquired structural variations of the B. subtilis genome were distinct from the spontaneous DNA deletions previously characterized [M. Itaya, J. Bacteriol., 175, 741-749 (1993)]. Caution should therefore be taken for nominally isogenic strains that are thought to have identical genome structure except for the gene(s) of interest. We propose the term isogenomic strains for strains that shows the same macrorestriction fragment pattern.
Key words: transformation; Bacillus subtilis; isogenic; NotI; SfiI
-11-
Physico-chemical
Properties of Purified Isoforms of the 12S Seed Globulin from Mustard Seed
(Brassica alba)
Massimo F. Marcone,๕ Rickey Y. Yada, Wichai Aroonkamonsri, and Yukio Kakuda Department of Food Science, Ontario Agricultural College, University of Guelph, Guelph, Ontario, Canada, N1G2W1
Received June 13, 1996
@The 12S mustard seed globulin from Brassica alba was isolated from a variety of extraction solutions (i.e., 1.0 M NaCl, pH 7.5 (ส=1.0); 32.6/2.6 mM KH2PO4 /K2HPO4, pH 7.5, containing 0.4 M NaCl (ส= 0.5) and distilled water, pH 7.5, ส=0. Gel filtration chromatography of the crude globulin from the various extracts revealed two heterogeneous forms of the globulin; i.e., a polymerized (490 kDa) and non-polymerized (304 kDa) form. Further purification of the non-polymerized form by anion-exchange chromatography indicated the presence of two isoforms. Characterization of one of these isoforms (i.e., isoform B) from the various extractions indicated the following: pI of 7.10, thermal denaturation temperature in the range (89.73-90.16C), identical amino acid composition that was particularly high in glutamic/glutamine residues, and a similar subunit composition. Isoform B globulins from an extraction with a high ionic strength solution had overall comparable lower negatively and higher positively charged surfaces above and below their pIs, respectively; similar secondary and tertiary structures; and similar solubility and spectroturbidity profiles compared to those globulins extracted with distilled water. The isoform B globulins from all three extractions were found to be cryoprecipitable at low temperatures.
Key words: extraction; purification; globulin; mustard; physico-chemical properties
-12-
Novel Exopolygalacturonases
Produced by Alternaria mali
Kouichi Nozaki, Kazuo Miyairi,๕ Seiji Hozumi, Yohko Fukui, and Toshikatsu Okuno
Department of Bioresources Science, Faculty of Agriculture, Hirosaki University, Hirosaki-shi, Aomori 036, Japan
Received June 20, 1996
@Three exopolygalacturonases (exoPG) were purified from the culture filtrate of Alternaria mali and characterized. Three exoPGs were distinguished by chromatographic properties. They contained a large amount of carbohydrates, and the molecular masses were estimated to be 51-80 kDa (exoPG I) and 51-58 kDa (exoPG II and III) by SDS-PAGE. After treatment with endo-ภ-N-acetylglucosaminidase, their molecular masses decreased equally to 43 kDa. In addition, the amino acid sequences of the N-terminal 20 residues of the three enzymes were identical except for a few amino acid residues. The pH- and thermal stabilities, optimum pHs, and Kms for unsatd. oligoGAs among the three exoPGs were very similar. However their substrate specificities were clearly different. ExoPG I hydrolyzed satd. oligoGAs faster than 4,5-unsatd. oligoGAs. On the contrary, exoPG II and III preferred to hydrolyze 4,5-unsatd. oligoGAs. No enzyme with a substrate specificity like exoPG II and III has so far been reported. It was found that A. mali also produced pectate lyase (PL), pectin lyase (PNL), and pectinesterase (PE) but no endoPG under these growth conditions.
Key words: exopolygalacturonase; pectic enzyme; Alternaria mali
-13-
Identification and Characterization
of a Karyotypic Mutation in Magnaporthe grisea
Teruo Sone, Takumi Abe,๕ Manabu Suto, and Fusao Tomita๕๕
Laboratory of Applied Microbiology, Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060, Japan
Received June 21, 1996
@We have identified karyotypic mutants of Magnaporthe grisea isolate Ina168. The mutants have a karyotype with an extra band of chromosome-v in addition to six bands of the normal karyotype in contour-clamped homogeneous electric field (CHEF) electrophoresis. We named a karyotype of the original isolate as ฟ, and that of mutants as ฟ '. The mutation has not affected the DNA fingerprint pattern, host specific pathogenicity (pathogenic race), or crossing abilities with the isolate Guy11. Karyotypic analyses of isolates from a repetitive single-spore isolation indicated that the frequency of mutation was 12.5% and that the mutation was limited in the manner of ฟ to ฟ ', and never reverted. Hybridization analyses showed that chromosome-v originated from the chromosome in band IIIb of type ฟ and might be produced by deletion.
Key words: Pyricularia; rice blast; chromosomal rearrangement; karyotype
-14-
Molecular Cloning of an Inulin
Fructotransferase (Depolymerizing) Gene from Arthrobacter sp. H65-7 and
Its Expression in Escherichia coli
Hiroaki Sakurai, Atsushi Yokota, and Fusao Tomita*
Laboratory of Applied Microbiology, Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
Received July 2, 1996
@The gene encoding an extracellular inulin fructotransferase (depolymerizing) (inulase II) (EC 2.4.1.93), designated ift gene, was cloned from the genomic DNA of Arthrobacter sp. H65-7, and expressed in Escherichia coli for the first time. Sequence analysis showed a single open reading frame consisting of 1314 base pairs that encoded a signal peptide of 32 amino acids and a mature protein of 405 amino acids. The primary structure showed a homology of 49.8% with that of the inulin fructotransferase (DFA I-producing) (EC 2.4.1.200) from Arthrobacter globiformis S14-3. E. coli cells carrying the ift gene produced the active enzyme under control of the lac promoter. The expression of the ift gene was improved by a plasmid, pIFT-B, in which the ift gene was immediately downstream from the lac promoter. An E. coli transformant carrying pIFT-B expressed twice as much activity of inulase II as that of the original strain, Arthrobacter sp. H65-7. Most of the enzyme activity existed within the cells.
Key words: inulin; inulin fructotransferase; DFA III; Arthrobacter sp. H65-7; inulin fructotransferase gene
-15-
Evidence for a Single Active
Site in ภ-D-Glucosidase/ภ-D-Fucosidase from Dalbergia cochinchinensis
Seeds
Rudee Surarit,1,5 Hirokazu Matsui,2 Seiya Chiba,3 Jisnuson Svasti,4,5 and Chantragan Srisomsap5
1 Department of Physiology and Biochemistry, Faculty of Dentistry, Mahidol University, Yothi Street, Bangkok 10400, Thailand
2 Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapproro 060, Japan
3 Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
4 Department of Biochemistry, Faculty of Science, Mahidol University, Yothi Street, Bangkok 10400, Thailand
5 Laboratory of Biochemistry, Chulabhorn Research Institute, Vibhavadee Rangsit Road, Bangkok 10210, Thailand
Received June 24, 1996
@Kinetic analyses have been done on the hydrolysis of p-nitrophenyl ภ-D-glucoside (PNPG) and p-nitrophenyl ภ-D-fucoside (PNPF) by the ภ-D-glucosidase/ภ-D-fucosidase enzyme from Thai Rosewood (Dalbergia cochinchinensis Pierre). PNPF showed a competitive inhibition of PNPG hydrolysis with a Ki of 0.42 mM. Hydrolysis of mixtures of PNPG and PNPF at fractional ratios ranging from 0 to 1 showed Lineweaver-Burk plots intermediate between the two extremes. The apparent Km and apparent V(/)max at each fractional ratio showed good correspondence with the theoretical curves predicted for the existence of a single common active site for the hydrolysis of the two substrates.
Key words: active site; ภ-d-glucosidase; ภ-d-fucosidase; Dalbergia cochinchinensis
-16-
Novel Extracellular Alkaline
Metalloendopeptidases from Vibrio sp. NUF-BPP1: Purification and Characterization
Kazumasa Fukuda, Katsumi Hasuda,* Tatsuya Oda, Hiroshi Yoshimura,** and Tsuyoshi Muramatsu๕
Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852, Japan
* POLA Pharmaceutical R & D Laboratory, Yokohama 244, Japan
** Kakuyo-Maru, Faculty of Fisheries, Nagasaki University, Nagasaki 852, Japan
Received June 27, 1996
@We found two types of novel alkaline metalloendopeptidases (AP1 and AP2) from a marine bacterium, isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) and identified as Vibrio sp. (NUF-BPP1). We studied the structure-function relationship of these marine bacterial proteases. The electrophoretically homogeneous proteases had a molecular mass of 48 kDa for AP1 and 36 kDa for AP2 on SDS-PAGE, and showed optimum activity at around pH 9.5-10.0 (casein as substrate). Calcium chloride (5 mM) stabilized the activities over pH 6-11, but o-phenanthroline and EDTA inhibited the activities of both AP1 and AP2. The EDTA-inactivated proteases were partly restored to activity by addition of either zinc or calcium. Sodium chloride (3.5 M) increased the activities toward Z-Gly-Leu-NH2. N-Terminal sites of hydrophobic amino acid residues (Leu, Ile, Phe, Tyr, and Trp) of the peptide substrates were cleaved by AP1 and by AP2. Autolysis of AP1 in the absence of calcium ion probably produced AP2 by releasing a fragment (molecular mass of about 12 kDa) from the C-terminal end of AP1.
Key words: alkaline protease; metalloprotease; microbial protease; Vibrio sp.
-17-
Hesperidin as an Inhibitor of
Lipases from Porcine Pancreas and Pseudomonas
Kiyomi Kawaguchi, Takashi Mizuno, Kazuhiko Aida, and Keijiro Uchino
Central Laboratory, Nippon Flour Mills Co., Ltd., 2114-2 Nurumizu, Atsugi, Kanagawa 243, Japan
Received June 28, 1996
@In the course of our screening work for new types of lipase inhibitors, hesperidin was identified as a simple and small molecular weight inhibitor in the peels of Citrus unshiu. Hesperidin inhibited lipase activity from porcine pancreas and that from Pseudomonas, and their IC50 was 32 and 132 สg/ml, respectively. Hesperidin, neohesperidin, narirutin, and naringin are known as the main flavonoids in Citrus unshiu. Neohesperidin also inhibited the lipase from procine pancreas, but did not have any effect on Pseudomonas. Narirutin and naringin did not show this effect on lipases from porcine pancreas and Pseudomonas. In animal experiments, the concentration of plasma triglyceride in rats fed a diet containing 10% hesperidin were significantly lower than that fed the control diet.
Key words: hesperidin; lipase inhibitor; Citrus unshiu; triglyceride
-18-
Effects of an Escherichia
coli ilvA Mutant Gene Encoding Feedback-resistant Threonine Deaminase on
L-Isoleucine Production by Brevibacterium flavum
Ken-ichi Hashiguchi, Hiroyuki Kojima, Katsuaki Sato, and Konosuke Sano
Central Research Laboratories, Ajinomoto Co., Inc., Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan
Received July 2, 1996
@A mutated ilvA gene of Escherichia coli encoding a threonine deaminase that is resistant to feedback inhibition by L-isoleucine was obtained. It was functional in Brevibacterium flavum, and a wild strain of B. flavum into which it was introduced became able to convert exogeneous L-homoserine and L-threonine to L-isoleucine. When it was introduced into a L-threonine-producing B. flavum strain, the transformant accumulated 20 g/liter L-isoleucine from 100 g/liter glucose.
Key words: l-isoleucine production; Escherichia coli; ilvA; threonine deaminase; Brevibacterium flavum
-19-
The Role of Microsomal
ภ-Glucuronidase in Ascorbic Acid Biosynthesis Stimulated by Xenobiotics
in Rats
Fumihiko Horio๕ and Toru Horie*
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Nagoya 464-01, Japan
* Drug Metabolism Research Section, Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki 300-26, Japan
Received July 4, 1996
@We (Horio et al., 1993) have reported that the stimulation of the expression of the UDPglucuronosyltransferase (UDPGT) gene played a key role in the ascorbic acid biosynthesis induced by xenobiotics, such as 3-methylcholanthrene (3MC) and phenobarbital (PB). ภ-Glucuronidase catalyzes the hydrolysis of glucuronide formed by UDPGT. The role of microsomal ภ-glucuronidase in ascorbic acid biosynthesis induced by xenobiotics was investigated using homozygous and heterozygous EHBRs (Eisai hyperbilirubinuria rats) which are deficient in microsomal ภ-glucuronidase. Homozygous EHBRs, heterozygous EHBRs, and Sprague Dawley (SD) rats were injected intraperitoneally once with 3MC (20 mg/kg body weight), or once a day for 2 d with PB (100 mg/day kg body weight). In these three strains of rats, the hepatic level of xenobiotics-inducible UDPGT mRNA was elevated similarly by the treatment with xenobiotics. The increases of the hepatic concentration of ascorbic acid and the urinary excretion of ascorbic acid by the treatment with 3MC or PB were markedly suppressed in both EHBRs compared with those in the control SD rats. These results indicate that the microsomal ภ-glucuronidase has an important role in the hepatic ascorbic acid biosynthesis induced by xenobiotics.
Key words: ascorbic acid; xenobiotics; ภ-glucuronidase; UDPglucuronosyltransferase; EHBR (Eisai hyperbilirubinuria rat)
-20-
Expression and Characterization
of Endopeptidase in Suspension-cultured Cells of French Bean
Jae Whune Kim and Takao Minamikawa๕
Department of Biology, Tokyo Metropolitan University, Minami-ohsawa 1-1, Hachioji, Tokyo, 192-03, Japan
Received July 16, 1996
@When suspension cells of French bean (Phaseolus vulgaris L. cv. Goldstar) were cultured in Murashige and Skoog (MS) liquid medium containing 10-6 M 1-naphthaleneacetic acid (NAA) and 10-6 M 6-benzylaminopurine (BAP) (control medium), endopeptidase activity was expressed throughout the cell growth. In the medium containing 10-5 M gibberellic acid (GA3), the endopeptidase activity increased notably during the cell culture, but in the medium containing amino acids the activity decreased. Protein immunoblotting with an antiserum raised against a French bean cysteine endopeptidase (EP-C1) showed that a 34-kDa polypeptide corresponding to EP-C1 in molecular mass occurred in extracts from cultured cells. Five forms of endopeptidase in extracts of cells cultured in the control medium and the four forms in the medium containing GA3 were detected by activity staining after non-denaturing polyacrylamide gel electrophoresis. Experiments with various protease inhibitors indicated that a serine endopeptidase was expressed at high levels in cultured cells in the control medium and the activity of a cysteine endopeptidase was increased by GA3.
Key words: endopeptidase; gibberellic acid; Phaseolus vulgaris ; protease; suspension cells
-21-
Hydroxyl Radical-scavenging
Effects of Spices and Scavengers from Brown Mustard (Brassica nigra)
Shin-Kyo Chung,๕ Toshihiko Osawa,* and Shunro Kawakishi*
Department of Food Science and Technology, Kyung-pook National University, Taegu 702-701, Korea
* Laboratory of Food and Bio-dynamics, Nagoya University, Nagoya 464-01, Japan
Received July 16, 1996
@The effects of methanol extracts of 51 spices on EOH scavenging were studied in detail. 2-Deoxyribose oxidation and sodium benzoic acid hydroxylation methods were used for detecting the scavenging activity of EOH. Mustard varieties, thyme, oregano, clove, and allspice all exhibited strong EOH-scavenging activity. In particular, 3 varieties of mustard had above 90% EOH-scavenging activity with a 1 สg/ml concentration of their extracts. The EOH scavenger of Brassica nigra (brown mustard) was isolated and purified by XAD-2 column chromatography and preparative HPLC, and was identified as a 3,5-dimethoxy-4-hydroxycinnamic acid methyl ester by MS, 1H-NMR, and 13C-NMR. The 3,5-dimethoxy-4-hydroxycinnamic acid methyl ester was prepared by methylating of sinapic acid with diazomethane.
Key words: spices; EOH-scavenging effect; Brassica nigra; 3,5-dimethoxy-4-hydroxycinnamic acid methyl ester
-22-
A New Staining Method for Lyases
Catalyzing Cleavage of a C-S Bond in Sulfur-containing Compounds after
Polyacrylamide Gel Electrophoresis
Koji Ukai๕ and Jiro Sekiya
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan
Received July 25, 1996
@We developed a rapid and simple staining method for lyases catalyzing cleavage of a C-S bond in sulfur-containing compounds after polyacrylamide gel electrophoresis. This method was based on the finding that 3-(4',5'-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was readily reduced by various thiol compounds in the presence of phenazine methosulfate to yield formazan. Cystathionine ม-lyase [EC 4.4.1.1] and cystine lyase were quickly detected by this staining method after polyacrylamide gel electrophoresis. The staining method developed here may be applicable to a wide range of lyases catalyzing cleavage of a C-S bond as a quick and simple method for detection of their activities on polyacrylamide gels.
Key words: C-S lyase; activity staining; tetrazolium salt; phenazine methosulfate; thiol compounds
-23-
Concise Synthesis of a Racemic
and Diastereomeric Mixture of the Sex Pheromones of Matsucoccus Pine Scales
Hidenori Watanabe, Takeru Watanabe, Takeshi Kitahara, and Kenji Mori *
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
* Department of Chemistry, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162, Japan
Received July 26, 1996
@A short (3-5 steps) synthesis of a racemic and diastereomeric mixture of Matsucoccus sex pheromones (1-3) is described. The key reaction is Lewis acid-mediated cyanation of symmetric tertiary alcohol 6 to afford common intermediate 7.
Key words: sex pheromone; pine scale; Matsucoccus ; cyanation of allylic alcohol
-24-
Purification and Properties
of 3ฟ-Hydroxysteroid Dehydrogenase from Pseudomonas putida
Osao Adachi,๕ Hirohide Toyama, and Kazunobu Matsushita
Laboratory of Applied Microbiology, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan
Received July 29, 1996
@A Gram negative bacterial strain that can use cholate or deoxycholate as the sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida. The strain formed 3ฟ-hydroxysteroid dehydrogenase (EC 1.1.1.50) in the cytosolic fraction. The enzyme was purified and crystallized from the cell-free extract by a procedure involving chromatographic separation and ammonium sulfate fractionation. The crystallized enzyme showed a single protein band on both polyacrylamide gel electrophoresis and SDS-PAGE, giving a molecular weight of 25,000. An alternative molecular weight measurement by gel filtration also indicated that the enzyme has such a low molecular weight. This is the lowest molecular weight among the related enzymes so far reported. In addition to high enzyme content in the cell-free extract, since the enzyme was stable in solution for a long period and tended to be crystallized readily, a simple purification method for the enzyme with high recovery was proposed that is convenient for a large scale rapid enzyme preparation. The enzyme required NAD specifically as the primary electron acceptor but not NADP or other compounds. The apparent Michaelis constant, Km, for cholate was 15.0 สM. The enzyme also showed high substrate specificity to other 3ฟ-hydroxysteroids. Besides cholate, deoxycholate, androsterone, glycocholate, taurocholate, and ursodeoxycholate were readily oxidized by the enzyme and showed a similar order of kinetic parameters. Judging from the enzymatic properties obtained, it is reasonable to propose that the enzyme can be available for enzymatic diagnosis of bile acids, though the enzyme reaction is virtually reversible.
Key words: cholate dehydrogenase; diagnostic enzyme for bile acids; 3ฟ-hydroxysteroid dehydrogenase; oxidation of steroids; Pseudomonas putida
-25-
Development of Thermotolerant
Acetic Acid Bacteria Useful for Vinegar Fermentation at Higher Temperatures๕
Akihiko Saeki, Gunjana Theeragool,* Kazunobu Matsushita,** Hirohide Toyama,** Napha Lotong,* and Osao Adachi**,๕๕
Department of Bioindustry, Industrial Technology Institute, Yamaguchi Prefectural Government, Yamaguchi 753, Japan
* Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
** Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan
Received July 29, 1996
@Thermotolerant acetic acid bacteria that can grow at 37 to 40C were collected from places all over Thailand. They were divided into several groups according to their taxonomic and physiological properties, such as rapid ethanol oxidation, rapid acetate oxidation, cellulosic biopolymer formation, growth at 40C, growth in 3% acetic acid, growth in 8% ethanol, formation of thermotolerant alcohol, and aldehyde dehydrogenases, etc. Though the complete taxonomic analysis has not been completed with all the strains, the majority of the acetic acid bacteria isolated have been confirmed to be classified as Acetobacter rancens subsp. pasteurianus, A. lovaniensis subsp. lovaniensis, A. aceti subsp. liquefaciens, and A. xylinum subsp. xylinum. They produced acetic acid at high temperatures such as 38 to 40C. Even when acetic acid was initially added to 4%, they still oxidized ethanol to accumulate acetic acid, while 2% of the initial acetic acid was the upper limit for mesophilic strains*1 at higher temperatures. They oxidized higher concentrations of ethanol up to 9% without any appreciable lag time, while alcohol oxidation with mesophilic strains was delayed or became almost impossible under such conditions. Fermentation efficiency in vinegar production with the thermotolerant strains at 38 to 40C was almost the same as that of mesophilic strains at 30C. However, the thermotolerant strains worked rapidly with a higher fermentation rate at higher temperatures, which the mesophilic strains were unable to do. Vinegar fermentation at higher temperatures was successful in submerged culture as well as static culture. The fermentation rate as well as fermentation efficiency in continuous vinegar fermentation at higher temperatures by the thermotolerant strains in a jar fermentor was also more than 2 to 3 times that with mesophilic strains at 30C. Thus, thermotolerant acetic acid bacteria are useful for vinegar fermentation at higher temperatures, which may reduce cooling water expenses.
Key words: acetic acid bacteria; Acetobacter lovaniensis; thermotolerant acetic acid bacteria; vinegar fermentation at higher temperatures
-26-
Purification and Characterization
of Dipeptidyl Aminopeptidase from Aureobacterium sp. WO26
Wataru Ogasawara, Noriyuki Inanobe, Keiko Ochiai,* Katsuhiko Ando,* Hirofumi Okada, and Yasushi Morikawa๕ Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-21, Japan * Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahimachi, Machida, Tokyo 194, Japan Received August 12, 1996 We isolated a bacterial strain with an enzyme which releases dipeptide from Gly-Arg-p-nitroanilide. The bacterium was tentatively identified as Aureobacterium sp. The enzyme, named AuDAP, was purified and characterized. It was homogenous by SDS-PAGE and IEF, and had a molecular mass of 90,000 Da by SDS-PAGE and 88,000 Da by gel filtration, so it may be a monomer. The isoelectric point was 3.8 and the optimum pH was 10.0. The purified enzyme hydrolyzed Gly-Arg-pNA, a model substrate for DAP I, and Arg-Arg-MNA, a model substrate for DAP III. However, this enzyme did not hydrolyze Gly-Phe pNA, also a model substrate for DAP I. These results suggested that this enzyme did not fall under the classification of mammalian DAPs and was similar to DAP BI from Pseudomonas sp. WO24 and dDAP from Dictyostelium discoideum, although several differences were observed between them. The N-terminal amino acid sequence of this enzyme showed no significant homology to any enzyme and protein, except only for DAP BI. Key words: dipeptidyl aminopeptidase; DAP BI; proteolytic enzyme; Aureobacterium
-27-
A Novel NADP+-dependent Serine
Dehydrogenase from Agrobacterium tumefaciens
Emran Kabir Chowdhury, Kazuhiko Higuchi, Shinji Nagata, and Haruo Misono๕
Laboratory of Applied Microbiology, Department of Bioresources Science, Kochi University, Nankoku, Kochi 783, Japan
Received August 14, 1996
@NADP+-dependent serine dehydrogenase [EC 1.1.1.-], which catalyzes the oxidation of the hydroxyl group of serine to form 2-aminomalonate semialdehyde, was purified to homogeneity from a crude extract of Agrobacterium tumefaciens ICR 1600. The enzyme had a molecular mass of about 100 kDa and consisted of four identical subunits. In addition to L-serine, D-serine, L-glycerate, D-glycerate, and 2-methyl-DL-serine were substrates. However, O-methyl-DL-serine and L-threonine were inert. The enzyme showed maximal activity at about pH 9 for the oxidation of L-serine. The enzyme required NADP+ as a coenzyme, NAD+ was inert. The enzyme was not inhibited by EDTA, o-phenanthroline, or ฟ,ฟ'-dipyridyl, but was inhibited by HgCl2, p-chloromercuribenzoate, L-cysteine, D-cysteine, malonate, 2-methylmalonate, and tartronate. The Michaelis constants for L-serine, D-serine, and NADP+ were 42, 44, and 0.029 mM, respectively.
Key words: serine dehydrogenase; Agrobacterium tumefaciens ; oxidation of serine; short-chain alcohol dehydrogenase
-28-
Note
Bone Resorption Inhibitors
from Hop Extract
Hiroyasu Tobe,๕ Yoshifumi Muraki, Kazuyuki Kitamura, Osamu Komiyama, Yusuke Sato, Tatsuo Sugioka, Hiromi B. Maruyama, Eriko Matsuda,* and Masahiro Nagai*
Pharma Research and Development Division, Drug Discovery Research Laboratories, Hoechst Japan Limited, 3-2 Minamidai 1-chome, Kawagoe-shi, Saitama 350-11, Japan * Hoshi University, Faculty of Pharmaceutical Sciences, 4-41 Ebara 2-chome, Shinagawa-ku, Tokyo 142, Japan
Received February 14, 1996
@We searched hop extract for active component(s) that inhibited bone resorption in the pit formation assay, and isolated xanthohumol and humulone as active ingredients. Especially humulone had extraordinarily strong inhibitory activity and the IC(/)50 (concentration of 50% inhibition) value was 5.9~10-9 M.
Key words: bone resorption inhibitor; hop extract; humulone; xanthohumol
-29-
Note
Protection by Trehalose of DNA
from Radiation Damage
Koichi Yoshinaga,* Hiroe Yoshioka, Hiromu Kurosaki, Miyuki Hirasawa, Masahiro Uritani,* and Kunihiko Hasegawa * Department of Chemistry, Faculty of Science and Radiochemistry Research Laboratory, Shizuoka University, Oya 836, Shizuoka 422, Japan The most serious damage to cells exposed to radiation is attributed mostly to effects on the structure of cellular DNA. We found that trehalose protects DNA from irradiation. In the presence of 10 mM trehalose, DNA can be protected from about 4 times higher doses of ภ- and ม-ray irradiation. The protective effect increases with the amount of the sugar. Other disaccharides, sucrose, and maltose had similar effects. Key words: DNA break; irradiation; radical scavenger; trehalose; tritium
-30-
Note
Neoagarobiose as a Novel
Moisturizer with Whitening Effect
Reijiro Kobayashi, Mikimasa Takisada, Tadashi Suzuki, Kohtaro Kirimura,*,๕ and Shoji Usami *
KOSE Corporation, Basic Research Laboratory, Azusawa 1-18-4, Itabashi-ku, Tokyo 174, Japan
* Department of Applied Chemistry, School of Science and Engineering, Waseda University, Ohkubo 3-4-1, Shinjiku-ku, Tokyo 169, Japan
Received May 22, 1996
@Neoagarobiose, a disaccharide, showed a higher hygroscopic ability than glycerol or hyaluronic acid, typical moisturizing reagents. Beside, neoagarobiose whitened B16 murine melanoma cells, and showed low cytotoxicity. Therefore neoagarobiose was a rare reagent showing both moisturizing and whitening effects.
Key words: B16 murine melanoma cell; hygroscopicity; moisturizing; neoagarobiose; whitening effect
-31-
Note
Syntheses of Alkyl
ภ-D-Mannopyranosides and ภ-1,4-Linked Oligosaccharides Using ภ-Mannosidase
from Rhizopus niveus
Hiroshi Fujimoto, Megumi Isomura, and Katsumi Ajisaka Meiji Institute of Health Science, Naruda Odawara 250, Japan
Received June 10, 1996
@ภ-1,4-Linked mannooligosaccharides were obtained regioselectively when mannose was incubated with ภ-mannosidase from Rhizopus niveus. By use of the obtained ภ-1,4-linked mannobiose as a donor, the transglycosylation reaction was done with the same enzyme, and ภ-linked alkyl glycosides were obtained from various alcohols. In the reaction using the ภ-1,4-linked mannobiose as a donor and acceptor, ภ-1,4-linked mannooligosaccharides were obtained by a transglycosylation mechanism.
Key words: ภ-mannosidase; Rhizopus niveus; ภ-d-mannosyl oligosaccharide; reverse hydrolysis; transglycosylation
-32-
Note
Positional Specificity
and Stereoselectivity of a Lipase Preparation from Oat Seeds Acting on
1,2,3-Trihexanoylglycerol
Yasuhide Ota,๕ Toshio Minesaki, and Aki Oshima๕๕
Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739, Japan
Received June 14, 1996
@Oat seed lipase was extracted with 0.01 M calcium chloride solution containing 0.5% Triton X-100 and precipitated with ammonium sulfate. The precipitate was dissolved in phosphate buffer at pH 6.0 and the supernatant was used as the lipase preparation. The lipase was very selective in the ester positions of 1,2,3-trihexanoylglycerol, hydrolyzing sn-3 most quickly, sn-1 moderately, and sn-2 hardly at all.
Key words: oat; lipase; positional specificity; stereoselectivity; triglyceride
-33-
Note
Isolation and Molecular
Characterization of Four Arginine/Glutamate Rich Polypeptides from the
Seeds of Sponge Gourd (Luffa cylindrica)๕
Hisashi Ishihara, Takahiro Sasagawa, Ritsu Sakai,* Masateru Nishikawa,* Makoto Kimura,* and Gunki Funatsu
Laboratory of Protein Chemistry and Engineering, and * Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-81, Japan
Received June 17, 1996
@Four arginine/glutamate rich polypeptides referred to as 5k-, 6.5k-, 12.5k-, and 14k-AGRPs were purified to homogeneity by gel filtration on Sephadex G-75 followed by CM-cellulose, butyl-Toyopearl 650M, and reverse-phase HPLC from the seed of sponge gourd (Luffa cylindrica). Tricine SDS-PAGE indicated that 5k-and 6.5k-AGRPs are single polypeptides, but 12.5 k- and 14k-AGRPs consist of two polypeptide chains, which are linked by disulfide bond(s). The N-terminal amino acid sequences of four AGRPs were analyzed by a gas-phase sequencer, and the result indicated that they are distinct molecules. Comparison of the sequences with those of proteins in the protein data base demonstrates that 5k- and 6.5k-AGRPs have a significant homology with a basic peptide from pumpkin seeds and with cocoa seed vicilin, respectively, and that 12.5k- and 14k-AGRPs are related to 2S seed storage proteins. Furthermore, it was assumed that the four AGRPs might occur in the protein bodies within cells of the seed.
Key words: arginine/glutamate rich polypeptide; sponge gourd; Luffa cylindrica; seed protein
-34-
Note
Quantitative Analysis
of Alkylpyrazines in Regular- and Low-fat Commercial Peanut Butter Preparations
Kwangjee Joo๕ and Chi-Tang Ho
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, NJ 08903, U.S.A.
Received June 25, 1996
@A simple, modified version of the selective purge and trap method was applied for a quantitative analysis of the trace amount of pyrazines present in both regular- and low-fat commercial peanut butter preparations. A total of 33 volatile compounds were identified, the three most abundant individual pyrazines being 2,5 (or 2,6)-dimethylpyrazine, 2-ethyl-6-methylpyrazine, and pyrazine which comprised 55-79% of the total pyrazine concentration. Minor or trace quantities of thiazoles, oxazoles, pyrroles, and pyridine were also identified in the samples.
Key words: alkylpyrazines; quantification; regular-fat; low-fat; peanut butter
-35-
Note
Isolation and Characterization
of Chitinase from a Flake-chitin Degrading Marine Bacterium, Aeromonas
hydrophila H-2330
Kazumi Hiraga, Lee Shou, Mitsunori Kitazawa, Saori Takahashi, Masahiko Shimada,* Ryoichi Sato,* and Kohei Oda
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606, Japan
* Central Research Institute, Maruha, Tsukuba 300-42, Japan
Received July 2, 1996
@A flake-chitin degrading marine bacterium was isolated and identified as Aeromonas hydrophila. This strain secreted five chitinases and an ภ-N-acetylglucosaminidase. The main chitinase (Chi-A) was purified and characterized. The optimum pH of Chi-A was 5-8, and the activity was inhibited by Hg2+ and Fe3+. Chi-A was different from chitinases of other Aeromonas species with respect to molecular weight (62,000) and insensitivity to monoiodoacetate. The amino-terminal amino acid sequence showed extensive similarity with chitinases from Gram-negative bacteria.
Key words: chitinase; flake-chitin; purification; marine bacterium; Aeromonas hydrophila
-36-
Note
Isolation and Identification
of ฟ-Glucosidase Inhibitors from Tochu-cha@(Eucommia ulmoides )
Jun Watanabe, Jun Kawabata,๕ Hideyuki Kurihara,* and Ryoya Niki
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060, Japan
* Department of Marine Bioresources Chemistry, Faculty of Fisheries, Hokkaido University, Minato-machi, Hakodate 041, Japan
Received July 8, 1996
@ฟ-Glucosidase inhibitory activity was found in aqueous methanol extracts of tochu-cha, dried leaves of Eucommia ulmoides (Eucommiaceae). Five active principles against yeast enzyme were isolated and characterized. Among them, quercetin (1, Ki: 8.5~10-6 M) was considered to contribute mostly to the activity of the tochu leaves. In regard to an animal ฟ-glucosidase, rat intestinal sucrase activity was also inhibited by 1.
Key words: ฟ-glucosidase inhibitor; quercetin; tochu-cha; Eucommia ulmoides
-37-
Note
Rapid Detection Method for Bacteriocin
and Distribution of Bacteriocin-producing Strains in Lactobacillus acidophilus
Group Lactic Acid Bacteria Isolated from Human Feces
Yasushi Kawai, Tadao Saito, Junko Uemura, and Takatoshi Itoh
Laboratory of Animal Products Chemistry, Department of Applied Bio-Science, Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981, Japan
Received July 8, 1996
@A new screening method, using micro plate wells, was developed for detecting the bacteriocin producer in lactic acid bacteria. The method was applied for screening the bacteriocin producer in L. acidophilus group lactic acid bacteria isolated from human feces with 19 bacteria used as indicator strains. Of the 98 strains, 62 were finally selected as the bacteriocin producers, including 39 strains positive against at least one of food-borne pathogenic bacteria.
Key words: bacteriocin; Lactobacillus acidophilus ; lactic acid bacteria; micro-plate
-38-
Note
Isolation of Isoeugenitin as
a Fruiting Body Inducer for Stigmatella aurantiaca from a Soil Fungus Papulaspora
sp.
Ryosuke Fudo, Toshihiko Ando, Seiichi Sato, Toshiyuki Kameyama, and Shigeru Yamanaka Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki, Kanagawa 210, Japan
Received July 10, 1996
@A myxobacterium, Stigmatella aurantiaca, requires light for the efficient formation of its fuiting body. Isoeugenitin was isolated from a culture of the fungus as a regulatory substance that induces morphological development in S. aurantiaca. At a concentration of about 1-40 สM, the compound induced development of stalks on which myxospore-containing sporangia are formed without light irradiation.
Key words: myxobacteria; Stigmatella aurantiaca; isoeugenitin; fruiting body; Papulaspora sp.
-39-
Note
Mechanism of Stereospecific
Conversion of DL-5-Substituted Hydantoins to the Corresponding L-Amino
Acids by Pseudomonas sp. Strain NS671
Takahiro Ishikawa, Ken Watabe, Yukuo Mukohara, and Hiroaki Nakamura Odawara Research Center, Nippon Soda Co., Ltd., 345 Takada, Odawara, Kanagawa 250-02, Japan
Received July 12, 1996
@The mechanism of stereospecific conversion of DL-5-substituted hydantoins to the corresponding L-amino acids by Pseudomonas sp. strain NS671 was studied. The results indicated that the hydantoinase catalyzed the hydrolysis reaction of both D- and L-5-(2-methylthioethyl)hydantoin, and that the hydrolysis of the L-enantiomer proceeded preferentially compared with that of the D-enantiomer. On the basis of these findings, the mechanism was speculated to be as follows: DL-5-substituted hydantoins are converted exclusively to the L-forms of the corresponding N-carbamyl-amino acids by the hydantoinase in combination with hydantoin racemase. The N-carbamyl-L-amino acids are then converted to L-amino acids by N-carbamyl-L-amino acid amidohydrolase.
Key words: l-amino acid production; hydantoinase; Pseudomonas sp. strain NS671; 5-substituted hydantoin
-40-
Note
Depolymerization of Hyaluronic
Acid by Low-molecular-weight Amadori-rearrangement Products and Glycated
Polylysine
Jian Zu, Shiro Nishikawa, and Naoki Kashimura๕
Laboratory of Biofunctional Chemistry, Department of Bioscience, Faculty of Bioresources, Mie University, Kamihamacho, Tsu, Mie 514, Japan
Received July 12, 1996
@Depolymerization of hyaluronic acid (HA) by low-molecular-weight Amadori-rearrangement products in the presence of Cu2+ was studied as an in vitro model for the glycated protein-mediated degradation of biopolymers. This oxygen radical-mediated depolymerization was found to be specifically accelerated by Cu2+, and significantly inhibited by catalase, hydroxyl radical scavengers, and metal ion chelators. Glycated polylysine also depolymerized HA. The difference in depolymerization rate between low- and high-molecular-weight Amadori products is discussed.
Key words: hyaluronic acid; oxidative depolymerization; Amadori product; glycated polylysine
-41-
Note
Coulometric Electrochemical
Detection of Phospholipid Hydroperoxides by High-performance Liquid Chromatography
Hirofumi Arai, Satoshi Mohri,* Tetsuya Suzuki, Kozo Takama, and Junji Terao**,๕ Department of Marine Bioresources Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, Japan
* Miyagi Prefectural Institute of Technology, Sendai, Miyagi 982, Japan
** National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan
Received July 23, 1996
@A technique for the sensitive and convenient determination of phosphatidylcholine hydroperoxides was developed that uses coulometric electrochemical detection in a high-performance liquid chromatographic analysis. The detection limit for phosphatidylcholine hydroperoxides by this method was 20 pmol at a signal-to-noise ratio of 3. The glutathione-dependent phospholipid hydroperoxide-reducing activity of fresh human plasma was ascertained by this method.
Key words: HPLC; coulometric electrochemical detection; phospholipid hydroperoxide
-42-
Note
Enzymatic Resolution of 2,2,2-Trifluoro-1-(1-pyrenyl)ethanol
with Lipases
Katsuya Kato,๕ Masato Katayama, Shozo Fujii, and Hiroshi Kimoto Department of Chemistry, National Industrial Research Institute of Nagoya, Hirate-cho, Kita-ku, Nagoya 462, Japan
Received July 26, 1996
@The optical resolution of (})-2,2,2-trifluoro-1-(1-pyrenyl)ethanol was achieved by using lipases. In particular, Pseudomonas aeruginosa lipase (lipase LIP) showed high enantioselectivity (E=>100) and reactivity in the alcoholysis of the chloroacetyl ester of the title compound. The reactivity of the lipase LIP-catalyzed enantioselective alcoholysis of the chloroacetate with 1-hexanol was much higher than that of the acetylating the alcohol with vinyl acetate.
Key words: lipase; enantioselective alcoholysis; enzymatic resolution
-43-
Note
Increased Stability of PEG-PPG
Conjugated Human Urokinase against Autolysis
Jun-ichi Kajihara, Kozue Shibata, and Kazuo Kato Development and Research Laboratories, JCR Pharmaceuticals Co., Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-22, Japan
Received July 26, 1996
@Human urokinase (UK) was easily degraded during the incubation at 37C in a time-dependent manner. The degradation was also observed in the presence of trypsin inhibitors, suggesting that UK was not degraded by exogeneous trypsin-like serine protease but by autolysis. In this cases, the A-chain of UK was selectively degraded. Polyethylene glycol-polypropylene glycol conjugated urokinase (PEG-PPG-UK) was not degraded after prolonged incubation at 37C. These results demonstrated that PEG-PPG modification completely blocked the degradation of UK by autolysis.
Key words: human urokinase; polyethylene glycol-polypropylene glycol; chemical modification; autolysis
-44-
Note
Changes of Lipoxygenase and Fatty
Acid Hydroperoxide Lyase Activities in Bell Pepper Fruits during Maturation
Kenji Matsui, Yasuhi Shibata, Hideki Tateba,* Akikazu Hatanaka,** and Tadahiko Kajiwara Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan
* Flavor Institute, Ogawa Perfume Ltd., 6-32-9 Akabanenishi, Kita-ku, Tokyo 115, Japan
** Department of Life Science, Graduate School of Integrated Arts and Science, University of East Asia, Shimonoseki 751, Japan
Received July 26, 1996
Developmental changes in fatty acid hydroperoxide lyase (HPO lyase) and lipoxygenase (LOX) during the maturation of bell pepper fruits (Capsicum annuum L. cv. Kyonami) were examined by means of activity measurements, immunological detection of both the enzymes, and analysis of the volatile compounds formed upon homogenization of the fruits. Both the enzyme activities decreased with maturation, and immunological studies showed that the amounts of the enzymes concomitantly decreased. The amounts of six-carbon aldehydes and alcohols formed from bell pepper fruits upon homogenization also decreased during maturation, and with the fully ripened red fruits, these volatile compounds were hardly detectable. These results suggest that the major factor contributing to the changes in the composition of volatile compounds during the maturation of bell pepper fruits was changes in the amounts of HPO lyase and LOX.
Key words: fatty acid hydroperoxide lyase; lipoxygenase; volatile compounds
-45-
Note
High Efficiency Transformation
of Bacillus brevis by Electroporation
Akiko Okamoto, Akihiko Kosugi, Yukimichi Koizumi, Fujiharu Yanagida, and Shigezo Udaka Department of Brewing and Fermentation, Tokyo University of Agriculture, Sakuragaoka, Setagaya, Tokyo 156, Japan
Received July 29, 1996
@Various conditions in the Bacillus brevis transformation by electroporation were investigated to increase the efficiency. Competent cell volume and presence of polyethylene glycol 6000 and single stranded DNA on pulsing were critical for the efficiency. As a result, we established the improved method by which 107-108 transformants/สg plasmid DNA could be obtained.
Key words: Bacillus brevis; electroporation; transformation; single-stranded DNA; polyethylene glycol 6000
-46-
Note
Inhibitory Effect of Green
Tea on Injury to a Cultured Renal Epithelial Cell Line, LLC-PK1
Takako Yokozawa, Erbo Dong, Hae Young Chung,* Hikokichi Oura, and Hitomi Nakagawa**
Research Institute for Wakan-Yaku, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-01, Japan
* College of Pharmacy, Pusan National University, Gum Jung-Ku, Pusan 609-735, Korea
** Faculty of Education, Toyama University, Gofuku, Toyama 930, Japan
Received August 9, 1996
@When cells from a cultured renal epithelial cell line, LLC-PK1, were cultured under hypoxic conditions (oxygen concentration of 2% or less) before reoxygenation was applied (95% air, 5% CO2), the leakage of lactate dehydrogenase (LDH) into the medium increased. This phenomenon was inhibited in the presence of dimethyl sulfoxide, a hydroxyl radical scavenger, suggesting the involvement of free radicals. Such oxidative stress was significantly inhibited by a green tea extract, and more potently by a tannin mixture. On the other hand, under ordinary culture conditions (95% air, 5% CO2), there was cell injury, although the LDH leakage was less than that under hypoxia/reoxygenation, and such injury was inhibited by the green tea extract and the tannin mixture.
Key words: green tea; renal epithelial cell; LLC-PK1; culture; lactate dehydrogenase
-47-
Rapid Paper
Increase in Lysophosphatidylethanolamine
in the Cell Membrane upon the Regulated Exocytosis of Pancreatic Acinar
AR42J Cells
Yoshiki Ikeda, Shin-Ichi Fukuoka, and Makoto Kito ๕ Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan
Received August 8, 1996 A specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), was found to inhibit the regulated exocytosis of pancreatic acinar AR42J cells. When AR42J cells were stimulated with cholecystokinin octapeptide (CCK-8), the regulated exocytosis monitored by amylase release was rapidly activated and increased by 2.5-fold during one hour. After AR42J cells were treated with AACOCF3, amylase release by CCK-8 remained at the basal level. Thus, changes in the composition of membrane phospholipids before and after stimulation were investigated. Within 1 min after CCK-8 stimulation, lysophosphatidylethanolamine (lysoPE) in the cellular membranes of AR42J was increased while lysophosphatidylcholine stayed unchanged. In the presence of AACOCF3, lysoPE was not increased by CCK-8. Those results indicate that the increment of lysoPE is linked to the regulated exocytosis. Key words: lysophosphatidylethanolamine; membrane fusion; exocytosis; pancreas