Contents and Abstracts of Latest Issue of BBB

(Vol.62 No.9 1998)

Chemical Structure of Antithrombin-active Rhamnan Sulfate from Monostrom nitidum
Naoki HARADAa and Masaakira MAEDAb　1647

Hinokitiol Production in a Suspension Culture of Calocedrus formosana Florin
Midori ONO,1,＊ Tsunetomo ASAI,1,＊＊ and Hitoshi WATANABE2　1653

Pheophorbide a Content and Chlorophyllase Activity in Green Tea
Katsunori KOHATA,1 Kouichi HANADA,2 Yuji YAMAUCHI,1 and Hideki HORIE1　1660

Evaluation of Spray Cycle Bioreactor Using L-Alanine Degradation by Candida maltosa
Hideki SAKASHITA, Masayuki AZUMA, Hiroshi OOSHIMA, and Jyoji KATO　1664

Increase in Thermostability of N-Carbamyl-D-Amino Acid Amidohydrolase on Amino Acid Substitutions
Yasuhiro IKENAKA, Hirokazu NANBA, Kazuyoshi YAJIMA, Yukio YAMADA,
Masayuki TAKANO, and Satomi TAKAHASHI　1668

Relationship Between an Increase in Thermostability and Amino Acid Substitutions in N-Carbamyl-D-Amino Acid Amidohydrolase
Yasuhiro IKENAKA, Hirokazu NANBA, Kazuyoshi YAJIMA, Yukio YAMADA,
Masayuki TAKANO, and Satomi TAKAHASHI　1672

Xylose Transport Insensitivity to Catabolite Inhibition by Phosphoenolpyruvate:Sugar Phosphotransferase System in Tetragenococcus halophila
Keietsu ABE,＊,1 Takeshi HIGUCHI,1 and Ichiro YAMATO,2　1676

Isolation and Characterization of a Hydrogen Sulfide-Removing Bacterium, Pseudomonas sp. Strain DO-1
Tsutomu HONMA＊ and Toshiro AKINO　1684

Effects of Soy Milk and Bifidobacterium Fermented Soy Milk on Lipid Metabolism in Aged Ovariectomized Rats
Hiroko KIKUCHI-HAYAKAWA, Norie ONODERA, Satoshi MATSUBARA, Emi YASUDA,
Osamu CHONAN, Rie TAKAHASHI, and Fumiyasu ISHIKAWA　1688

Synthesis and Antifungal Activities of Furoxan Derivatives Designed as Novel Fungicide
Kwang-Jin HWANG,・ Young C. PARK, Hyung Jin KIM,＊ and Jae Hyun LEE＊＊　1693

Dietary Docosahexaenoic Acid Dose Not Promote Lipid Peroxidation in Rat Tissue to the Extent Expected from Peroxidizability Index of the Lipids1
Kazuhiro KUBO,2,3 Morio SAITO,2,4 Tadahiro TADOKORO,5 and Akio MAEKAWA5　1698

Restricted Transcription from Sigma H or Phosphorylated Spo0A Dependent
Promoters in the Temperature-sensitive secA341 Mutant of Bacillus subtilis
Kei ASAI,＊1,・1 Masaya FUJITA,＊2 Fujio KAWAMURA,＊3,・2 Hideo TAKAHASHI,＊4
Yasuo KOBAYASHI,＊5 and Yoshito SADAIE,＊6,・3　1707

Relationship between Suspension Properties and Fibril Structure of Disintegrated Bacterial Cellulose
Hiroshi OUGIYA,・ Kunihiko WATANABE,・・ Takayuki MATSUMURA,・・・
and Fumihiro YOSHINAGA　1714

Hyperexpression of the Gene for a Bacillus α-Amylase in Bacillus subtilis Cells:
Enzymatic Properties and Crystallization of the Recombinant Enzyme＊
Kaori IKAWA, Hiroyuki ARAKI, Yukiharu TSUJINO, Yasuhiro HAYASHI,
Kazuaki IGARASHI,＊＊ Yuji HATADA, Hiroshi HAGIHARA, Tadahiro OZAWA,
Katsuya OZAKI, Tohru KOBAYASHI, and Susumu ITO　1720

Stereoselective Synthesis of (2R, 3S)-2-Benzyl-2-hydroxy-3-
(3,4-methylenedioxybenzyl)-γ-butyrolactone from L-(＋)-Arabinose
Satoshi YAMAUCHI＊ and Yoshiro KINOSHITA　1726

Molecular Cloning and Sequence Analysis of Two Endoinulinase Genes from Aspergillus niger
Kazuyoshi OHTA,・ Hidetoshi AKIMOTO, Shusaku MATSUDA,
Daisuke TOSHIMITSU, and Toyohiko NAKAMURA　1731

Purification and Properties of a Novel Sulfatase from Pseudomonas testosteroni That Hydrolyzed 3β-Hydroxy-5-cholenoic Acid 3-Sulfate
Yasuhiko TAZUKE, Kumiko MATSUDA, Kenichi ADACHI, and Yoji TSUKADA　1739

Further Study on the Two Pivotal Parts of Hlg2 for the Full Hemolytic Activity
of Staphylococcal γ-Hemolysin
Kenji YOKOTA, Noriko SUGAWARA, Hirofumi NARIYA, Jun KANEKO,
Toshio TOMITA, and Yoshiyuki KAMIO・　1745

Endo-β-Glucanase Secreted by a Psychrotrophic Yeast: Purification and Characterization
Tadao OIKAWA,1,2・ Yasuyuki TSUKAGAWA,1 and Kenji SODA1,2　1751

Studies on the Carbohydrate Binding Sites of the Hemolytic Lectin CEL-III
Isolated from the Marine Invertebrate Cucumaria echinata
Imre SALLAY, Tomomitsu HATAKEYAMA,＊ and Nobuyuki YAMASAKI＊＊　1757

Production of Ethyl (R)-2-Hydroxy-4-phenylbutanoate via Reduction of
Ethyl 2-Oxo-4-phenylbutanoate in an Interface Bioreactor
Shinobu ODA,＊ Yuichi INADA, Atsuko KOBAYASHI, and Hiromichi OHTA＊＊　1762

Note
Scavenging Effect of Various Tea Extracts on Superoxide Derived from the Metabolism of Mutagens
Gow-Chin YEN・ and Hui-Yin CHEN　1768

Note
Evaluation of the Cytotoxic Activity of Chiral (E)-13-Hydroxy-10-oxo-11-
octadecenoic Acid and Its Lactone
Yoshiki HAYASHI,・ Yasushi NISHIKAWA, Hirotaka MORI, Yoh-ichi MATSUSHITA,＊
Kazuhiro SUGAMOTO,＊ and Takanao MATSUI＊ 1771

Note
Molecular Cloning, Sequencing and Characterization of the Genes
for Adenosylcobalamin-dependent Diol Dehydratase of Klebsiella pneumoniae
Takamasa TOBIMATSU, Muneaki AZUMA, Shinji HAYASHI, Kyou-ichi NISHIMOTO,
and Tetsuo TORAYA・ 1774

Note
Increased Cellulose Production from Sucrose by Acetobacter after Introducing
the Sucrose Phosphorylase Gene
Naoto TONOUCHI,・ Sueharu HORINOUCHI,＊ Takayasu TSUCHIDA, and Fumihiro YOSHINAGA 1778

Note
Chick Skeletal Muscle Proteolysis in vitro Increased by Corticosterone
Akira OHTSUKA,・ Hiroko TANAKA, Ko HIZAKI, and Kunioki HAYASHI 1781

Note
secA341 Mutation Inhibition of Expression of the Bacillus subtilis Protease Gene, aprE
Yoshito SADAIE 1784

Note
Germinated Barley Foodstuff Improves Constipation Induced by Loperamide in Rats
Osamu KANAUCHI,・ Yoshitaka HITOMI, Kazue AGATA, Tomohiko NAKAMURA,
and Tohru FUSHIKI＊ 1788

Note
Structures of Novel Acidic Galactooligosaccharides Synthesized by Bacillus circulans β-Galactosidase
Shuichi YANAHIRA,・ Yukiko YABE, Masamichi NAKAKOSHI,＊ Susumu MIURA,
Noritaka MATSUBARA, and Hidetoshi ISHIKAWA 1791

Note
Cloning, Sequencing, and Expression of an Endoglucanase Gene＊
from the Rumen Anaerobic Fungus Neocallimastix frontalis MCH3
Yuko FUJINO,＊＊ Koretsugu OGATA,＊＊＊ Takafumi NAGAMINE,＊＊＊ and Kazunari USHIDA＊＊＊＊ 1795

Note
Facile Syntheses of AM-toxins and Analogs as Cyclic Depsipeptides by the Solid-phase Method
Masahiro MIYASHITA, Tomoko NAKAMORI, Takahiro MURAI, Hisashi MIYAGAWA,
Miki AKAMATSU, and Tamio UENO 1799

Note
Expression of the Extracellular Levansucrase and Invertase Genes
from Zymomonas mobilis in Escherichia coli Cells
Hideshi YANASE,・ Junichi FUJIMOTO, Masahiro MAEDA, Kenji OKAMOTO,
Keiko KITA, and Kenzo TONOMURA＊ 1802

Note
Stability of Thermostable Enzyme, Aqualysin I ; a Subtilisin-type Serine Protease from Thermus aquaticus YT-1
Terumichi TANAKA,・ Hiroshi MATSUZAWA, and Takahisa OHTA＊ 1806

Note
Expression and Purification of Cytokine Receptor Homology Domain of Human Granulocyte-Colony Stimulating Factor Receptor in Escherichia coli
Ryoichi TANAKA, Hiroko TOKUNAGA, Shinichi HARA,＃ Tsutomu ARAKAWA,＃
and Masao TOKUNAGA・ 1809

Note
Detection of Proteins Binding to the Promoter Region DNA Using a Nonradioactive Gel-Retardation Assay
Shin-ichiro MITSUNAGA,・ Kiichi FUKUI,＊ Hideo OHYAMA, and Junji YAMAGUCHI＊＊ 1812

Note
Structural Confirmation of Cotylenin A, a Novel Fusicoccane-diterpene Glycoside with Potent Plant Growth-regulating Activity from Cladosporium Fungus sp. 501-7W
Takeshi SASSA,・ Takahiro OOI, Manabu NUKINA, Michimasa IKEDA, and Nobuo KATO＊ 1815

Note
Effects of β-Lactoglobulin on the Tight-junctional Stability of Caco-2-SF Monolayer
Kei HASHIMOTO,・ Tsutomu NAKAYAMA,＊ and Makoto SHIMIZU＊＊ 1819

Note
Study on the Application of the (S)-2-tert-Butyl-2-methyl-1,3-benzodioxole-
4-carboxylic acid-based On-line HPLC-Exciton CD Analysis to Acyclic 1,3-Diols
Syun-ichi SATO, Kumi SHIMIZU, Jeong-Hwan KIM, Eiichi AMI,
Kazuaki AKASAKA, and Hiroshi OHRUI・ 1822

Note
A Rapid Method for Disrupting Genes in the Escherichia coli Genome
Chinatsu KATO, Ryusuke OHMIYA, and Takeshi MIZUNO＊ 1826

Note
Identification and Characterization of Acinetobacter sp. CNU961 Able to Grow with Phenol at High Concentrations
Kwang Cheol JEONG, Eun Ye JEONG, Tay Eak HWANG,1 and Sang Ho CHOI＊ 1830

Preliminary Communication
Phosphorylation of LukS by Protein Kinase A is Crucial for the LukS-Specific Function of the Staphylococcal Leukocidin on Human Polymorphonuclear Leukocytes
Akihito NISHIYAMA, Hirofumi NARIYA, and Yoshiyuki KAMIO＊ 1834


-1-
Chemical Structure of Antithrombin-active Rhamnan Sulfate
from Monostrom nitidum

Naoki HARADAa and Masaakira MAEDAb

Department of Biochemistry, Saitama University Urawa, Saitama 338-0825, Japan

Received October 6, 1997
Rhamnan sulfate was extracted with boiling water from the cell wall of Monostroma nitidum, and the resulting extract was purified by ion-exchange and size-exclusion chromatography. The polysaccharide, which is regarded as a homopolysaccharide, was 6-fold more antithrombin-active than the heparin standard. The antithrombin activity was decreased, by desulfation, although the non-active product still retained 8％ of the sulfate ester. A comparative structural study of the intact rhamnan sulfate and rhamnan, its desulfated product, by periodate oxidation, Smith degradation and methylation analysis revealed the rhamnan sulfate to consist of α-1,3-linked L-rhamnose residues, some of which were substituted with sulfate groups mainly at position O-2. Minor amounts of internal 1,2-linked rhamnose and branched rhamnose linkages were also detected.
Monostroma nitidum; Chlorophyta; sulfated cell-wall polysaccharides; rhamnan sulfate; antithrombin activity

-2-
Hinokitiol Production in a Suspension Culture of Calocedrus formosana Florin

Midori ONO,1,＊ Tsunetomo ASAI,1,＊＊ and Hitoshi WATANABE2

1Vitamin and Food Research and Development Department, Takeda Chemical Industries, Ltd., 2-17-85,
Juso-honmachi, Yodogawa-ku, Osaka 532-8686, Japan
2Herbal Garden, Takeda Chemical Industries, Ltd., 11, Ichijoji-takenouch-cho,
Sakyo-ku, Kyoto 606-8134, Japan

Received December 3, 1997
A suspension culture of Calocedrus formosana Florin was studied as a material for efficient production of hinokitiol. Murashige-Skoog′s medium containing 3％ sucrose and 1 mg/l 1-naphthylacetic acid was most desirable for cell growth. Cell growth, expressed as fresh cell weight, showed a 20-fold increase after 4 weeks of culture in this medium. Adding potassium acetate or chitosan to the medium increased hinokitiol production. The highest hinokitiol yield, 1700 μg/g fresh cells, was obtained when cells were cultured in the growth medium with chitosan.
hinokitiol (β-thujaplicin); Calocedrus formosana Florin; suspension culture; chitosan

-3-
Pheophorbide a Content and Chlorophyllase Activity in Green Tea

Katsunori KOHATA,1 Kouichi HANADA,2 Yuji YAMAUCHI,1 and Hideki HORIE1

1National Research Institute of Vegetables, Ornamental Plants and Tea (NIVOT), 2769 Kanaya,
Shizuoka 428-8501, Japan
2Itochu Sugar Co. Ltd., 3 Hekinan, Aichi 447-0834, Japan

Received December 22, 1997
We investigated the total content of pheophorbide a (PB a), which is sum of the contents of newly produced PB a, including PB a initially present and that converted from chlorophyllide a (Chd a) by the chlorophyllase reaction during incubation, in green tea samples, and found that the total content of PB a markedly increased in both Sencha and Matcha, compared with the initially present PB a content in each. This result demonstrates that chlorophyllase activity still remains in green tea, even after processing fresh green leaves. A comparison of the total contents of PB a produced during the incubation of chlorophyll a (Chl a) with Sencha and fresh green leaf acetone powder indicates that the ratio of chlorophyllase activity in Sencha and in fresh green leaves was about 1:20.
chlorophyllase; green tea; pheophorbide a; chlorophyllide; chlorophyll

-4-
Evaluation of Spray Cycle Bioreactor Using L-Alanine Degradation by Candida maltosa

Hideki SAKASHITA, Masayuki AZUMA, Hiroshi OOSHIMA, and Jyoji KATO

Department of Bioapplied Chemistry, Osaka City University, Sugimoto, Sumiyoshi-ku,
Osaka 558-8585, Japan

Received January 23, 1998
The oxygen-supply capability of a spray cycle reactor was evaluated by using it for oxidative degradation of L-alanine. The volumetric oxygen transfer coefficient, kLa, was evaluated as a parameter for the oxygen supply. The liquid circulation rate in the spray cycle reactor was represented in terms of the number of circulations. The kLa increased with the number of circulations, especially by stirring in the reservoir vessel, reaching 272/h at 4.4/min of circulation numbers. This value was 1.4 times higher than that without stirring. The L-alanine degradation rate increased as the cell growth was promoted, and as the circulation numbers increased. Finally, the spray cycle reactor was evaluated by the specific degradation rate. This rate increased in proportion to the kLa, and was 8.8 times higher than that in the jar fermentor, suggesting that the spray cycle reactor is superior for oxygen-demanding fermentation.
spray cycle reactor; L-alanine degradation; volumetric oxygen transfer coefficient; Candida maltosa

-5-
Increase in Thermostability of N-Carbamyl-D-Amino Acid Amidohydrolase on Amino Acid Substitutions

Yasuhiro IKENAKA, Hirokazu NANBA, Kazuyoshi YAJIMA, Yukio YAMADA,
Masayuki TAKANO, and Satomi TAKAHASHI

Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan

Received February 6, 1998
To improve the production of D-amino acids using an immobilized N-carbamyl-D-amino acid amidohydrolase, the enzyme gene of Agrobacterium sp. KNK712 was mutagenized randomly to increase its thermostability. The gene was inserted into M13mp19, mutagenized with hydroxylamine, ligated into pUC19 after restriction endonuclease digestion, and then used to transform Escherichia coli. The resultant transformants were screened by a newly developed colorimetric enzyme assay method, and the candidate colonies corresponding to red spots were separated from the master plates. Using cell-free extracts of these clones, the properties of the enzymes produced were investigated, it being proved that these enzymes had almost the same activity and improved thermostability by about 5°C compared with those of the native enzyme. As found on enzyme gene analysis of these mutants, the 57th amino acid, histidine, of the enzyme was changed to tyrosine, or the 203rd amino acid, proline, to leucine or serine.
N-carbamyl-D-amino acid amidohydrolase; thermostabilized enzyme; D-amino acid

-6-
Relationship Between an Increase in Thermostability and Amino Acid
Substitutions in N-Carbamyl-D-Amino Acid Amidohydrolase

Yasuhiro IKENAKA, Hirokazu NANBA, Kazuyoshi YAJIMA, Yukio YAMADA,
Masayuki TAKANO, and Satomi TAKAHASHI

Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago,
Hyogo 676-8688, Japan

Received March 9, 1998
For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10°C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.
N-carbamyl-D-amino acid amidohydrolase; thermostabilized enzyme; D-amino acid

-7-
Xylose Transport Insensitivity to Catabolite Inhibition by Phosphoenolpyruvate:Sugar Phosphotransferase System in Tetragenococcus halophila

Keietsu ABE,＊,1 Takeshi HIGUCHI,1 and Ichiro YAMATO,2

1Microbiology Group, Research and Development Division, Kikkoman Corporation,
399 Noda, Noda-shi, Chiba 278-0037, Japan
2Department of Biological Science and Technology, Science University of Tokyo,
2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan

Received February 9, 1998
Tetragenococcus halophila accumulates glucose and 2-deoxyglucose (dGlc) via the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and pentoses, maltose, and glycerol via non-PTS carriers. Based on the discovery that xylose metabolism in T. halophila was subject to catabolite repression but not to catabolite inhibition, we designed a selection protocol for thermosensitive mutants pleiotropically unable to use sugars. One such mutant was a ptsI mutant with a thermosensitive enzyme I (EI) of the PTS (leaky at 30°C). Using this ptsI mutant, catabolite inhibition was studied. dGlc was more strongly inhibitory of glycerol uptake in the mutant than in the parent because of the leaky ptsI mutation. Thermoinactivation of EI at 42°C resulted in the total loss of uptake of PTS sugars and in the virtual abolishment of glycerol uptake. However, xylose uptake of the ptsI mutant was scarcely inhibited by dGlc even after thermoinactivation of EI. These results suggest that sensitivities of non-PTS uptakes to PTS-mediated inhibition vary among non-PTS sugars.
Tetragenococcus halophila; Pediococcus halophilus; xylose transport; glycerol transport; phosphotransferase

-8-
Isolation and Characterization of a Hydrogen Sulfide-Removing Bacterium,
Pseudomonas sp. Strain DO-1

Tsutomu HONMA＊ and Toshiro AKINO

Central Research Laboratory, Godo Shusei Co., Ltd., 250, Nakahara, Kamihongo, Matsudo,
Chiba, 271-0064, Japan

Received February 9, 1998
Five microbial strains that removed hydrogen sulfide (H2S) or methylmercaptan (CH3SH) gas were newly isolated from soil samples. Strain DO-1, one of the isolates, was identified as a member of Pseudomonas sp., and it′s immobilized cells removed 1 or 10 ppm of H2S gas within 2 hours. When strain DO-1 was cultured aerobically in a flask containing nutrient broth medium, the deodorizing activity increased, depending on the growth of the culture, and the maximum activity was obtained after 48 hours. Even though the immobilized cells were stored at 4 or 25°C in sealed bottles for 6 months, the deodorizing activity remained. Throughout this study, strain DO-1 removed H2||S gas without preliminary feeding or exposure to sulfur com||pounds as growth substrates or inducers. These characteristics are advantageous for the deodorization of the malodorous gases surrounding us in daily life.
biological deodorization; sulfur-containing odor; hydrogen sulfide; methylmercaptan; Pseudomonas

-9-
Effects of Soy Milk and Bifidobacterium Fermented Soy Milk
on Lipid Metabolism in Aged Ovariectomized Rats

Hiroko KIKUCHI-HAYAKAWA, Norie ONODERA, Satoshi MATSUBARA, Emi YASUDA,
Osamu CHONAN, Rie TAKAHASHI, and Fumiyasu ISHIKAWA

Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan

Received February 16,1998
The effects of soy milk and fermented soy milk on lipid metabolism were studied in aged ovariectomized rats. Twenty 8- mo-old Wistar rats were randomly assigned to four treatment groups: sham-operated＋control diet (sham-C); ovariectomized (OVX)＋control diet (OVX-C); OVX＋soy milk diet (OVX-SM); and OVX＋fermented soy milk diet (OVX-FSM). The rats were fed on these diets for 6 weeks. Ovariectomy induced an increase in the plasma cholesterol level by 40％. The plasma total cholesterol level of the OVX-FSM rats was decreased by 20％ compared to that of the OVX-C rats. The plasma total cholesterol level of the OVX-SM group was not significantly different from that of the OVX-C and sham-C rats. The plasma triglyceride level of the OVX-FSM rats was lower than that of the sham-C rats. The liver cholesterol content in OVX-SM and OVX-FSM rats was lower than that of the OVX-C rats. The liver triglyceride contents of the sham-C, OVX-SM, and OVX-FSM groups were lower than that of the OVX-C group. Fecal steroid excretion did not differ among the groups. Ovariectomy decreased the uterus weight. The OVX-SM and OVX-FSM groups had the same uterus weights as those of the OVX-C group.
Thus, the diet including fermented soy milk prevented the cholesterol elevation induced in rats by ovarian hormone deficiency.
soy milk; cholesterol; Bifidobacterium; ovariectomy; isoflavones

-10-
Synthesis and Antifungal Activities of Furoxan Derivatives Designed as Novel Fungicide

Kwang-Jin HWANG,・ Young C. PARK, Hyung Jin KIM,＊ and Jae Hyun LEE＊＊

Department of Industrial Chemistry, Hongik University, Jochiwon, Chungnam 339-701, Korea
＊Department of Chemical Technology, Chonnam National University, Kwangju 500-757, Korea
＊＊Korea Research Institute of Chemical Technology, PO Box 107, Yusong, Daejeon 305-606, Korea

Received February 19, 1998
Furoxan derivatives 3--11 were designed and synthesized as potential fungicides. Symmetrically substituted furoxan derivatives 3a--j were prepared by dimerization of the corresponding nitrile oxide generated from N-hydroxyliminoyl chloride 2 in situ. Further functional group modifications of aldehyde 4, a key intermediate, generated furoxan derivatives 5--11. The fungicidal activities of furoxan derivatives 3--11 were observed over a broad spectrum of plant fungi at 250 ppm.
furazan N-oxide; furoxan; fungicidal activity; wheat leaf rust

-11-
Dietary Docosahexaenoic Acid Dose Not Promote Lipid Peroxidation
in Rat Tissue to the Extent Expected from Peroxidizability Index of the Lipids1

Kazuhiro KUBO,2,3 Morio SAITO,2,4 Tadahiro TADOKORO,5 and Akio MAEKAWA5

2Division of Food Science, The National Institute of Health and Nutrition,
1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan
5Laboratory of Food Chemistry and Nutrition, Department of Agricultural Chemistry,
Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-0054, Japan

Received March 9, 1998
Docosahexaenoic acid (DHA) ingestion enhanced the susceptibility of rat liver and kidney to lipid peroxidation as a function of the dietary DHA level, but did not increase lipid peroxides as assessed by thiobarbituric acid (TBA) values to the level expected from the peroxidizability index of the tissue total lipids. This phenomenon was especially prominent in the liver. In the liver, the higher proportion of DHA in the non-phosphorus lipids might play an important role in lessening the susceptibility of the tissue to lipid peroxidation. In the brain and testis, on the other hand, lipid peroxide levels were decreased when DHA was given to the animals. In the testis, in particular, the proportion of DHA in total lipids was lowest among all tissues examined, even when a relatively high level of DHA had been ingested, and this could be related to the low lipid peroxide level. Therefore, the protection against lipid peroxidation differed from tissue to tissue, even from the viewpoint of the fatty acid composition of the tissue lipids. In addition, changes in the lipid peroxide levels of the liver, kidney, brain and testis, as assessed by TBA values, seemed to be associated with changes in the peroxidizability index of phosphatidylcholine (＋ cardiolipin) in each tissue.
docosahexaenoic acid; lipid peroxidation; peroxidizability index; tissue fatty acid; phospholipid

-12-
Restricted Transcription from Sigma H or Phosphorylated Spo0A Dependent
Promoters in the Temperature-sensitive secA341 Mutant of Bacillus subtilis

Kei ASAI,＊1,・1 Masaya FUJITA,＊2 Fujio KAWAMURA,＊3,・2 Hideo TAKAHASHI,＊4
Yasuo KOBAYASHI,＊5 and Yoshito SADAIE,＊6,・3

＊1,＊3,＊4Institute of Molecular and Cellular Biosciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-0032, Japan
＊2,＊6Radioisotope Center, National Institute of Genetics, Mishima 411-8540, Japan
＊5Laboratory of Molecular Biology and Microbial Chemistry, Tokyo University
of Agriculture and Technology, Fuchu, Tokyo 183-0054, Japan

Received March 19, 1998
The temperature-sensitive secA341 mutation of Bacillus subtilis affects sporulation and sporulation-associated events as well as protein secretion and cell septation. With lacZ or bgaB fusion genes, we examined the expression of the early sporulation genes in the mutant strain. Transcriptional expression of σH dependent kinA, spo0A (Ps), phrC, spoVG, and citG (p2) genes was blocked by the secA341 mutation at 37°C. On the other hand, neither repression of the abrB gene nor induction of the spo0H (σH) gene was affected. Active RNA polymerase containing σH was, however, found to be produced in the mutant cells. Expression of the phosphorylated Spo0A dependent spoIIG operon was also blocked. Thus the secA341 mutation blocks some step(s) or factor(s) required for σH-dependent transcription in vivo.
Bacillus subtilis; secA; σH promoter; sporulation

-13-
Relationship between Suspension Properties and Fibril Structure of Disintegrated Bacterial Cellulose

Hiroshi OUGIYA,・ Kunihiko WATANABE,・・ Takayuki MATSUMURA,・・・
and Fumihiro YOSHINAGA

Bio-Polymer Research Co. Ltd., KSP R＆D Business-park Bldg. B-1015, 3-2-1 Sakato, Takatsu-ku,
Kawasaki, Kanagawa 213-0012, Japan

Received March 24, 1998
A new method evaluating the fibril width and length of disintegrated bacterial cellulose was developed using optical and rheological analysis.
During the early stages of the disintegration process, the bacterial cellulose particles formed loose fibrous aggregates, followed by cutting of the disintegrated fibrils that produced short fibrils. On the other hand, the fibril width decreased steadily throughout disintegration.
The relationships between fibril structure and suspension properties were analyzed by a multiple regression method. The thinner and longer the disintegrated bacterial cellulose fibrils were, the higher the viscosity and water-holding capacity became.
bacterial cellulose; fibril width; fibril length; suspension property; multiple regression

-14-
Hyperexpression of the Gene for a Bacillus α-Amylase in Bacillus subtilis Cells:
Enzymatic Properties and Crystallization of the Recombinant Enzyme＊

Kaori IKAWA, Hiroyuki ARAKI, Yukiharu TSUJINO, Yasuhiro HAYASHI, Kazuaki IGARASHI,＊＊ Yuji HATADA, Hiroshi HAGIHARA, Tadahiro OZAWA, Katsuya OZAKI, Tohru KOBAYASHI, and Susumu ITO

Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan

Received March 27, 1998
We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis [Sumitomo et al., Biosci. Biotech. Biochem., 59, 2172--2175 (1995)]. The structural gene for a novel liquefying semi-alkaline α-amylase from the alkaliphilic Bacillus sp. KSM-1378 was amplified by PCR. It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the α-amylase activity extracellularly, corresponding to approximately 1.0 g (5×106 units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physicochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp. KSM-1378. The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10％ (w/v) ammonium sulfate (pH 6.5).
alkaliphile; Bacillus; α-amylase; cloning; expression

-15-
Stereoselective Synthesis of (2R, 3S)-2-Benzyl-2-hydroxy-3-
(3,4-methylenedioxybenzyl)-γ-butyrolactone from L-(＋)-Arabinose

Satoshi YAMAUCHI＊ and Yoshiro KINOSHITA

College of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790-0905, Japan

Received April 8, 1998
As a model experiment for the stereoselective synthesis of optically active cis-α,β-dibenzyl-α-hydroxy-γ-butyrolactone, (2R, 3S)-2-benzyl-2-hydroxy-3-(3,4-methylenedioxybenzyl)-γ-butyrolactone (3) was stereoselectively synthesized from L-(＋)-arabinose.
lignan; γ-butyrolactone; α,β-dibenzyl-γ-butyrolactone; stereoselective synthesis

-16-
Molecular Cloning and Sequence Analysis of Two Endoinulinase Genes from Aspergillus niger

Kazuyoshi OHTA,・ Hidetoshi AKIMOTO, Shusaku MATSUDA,
Daisuke TOSHIMITSU, and Toyohiko NAKAMURA

Department of Biological Resource Sciences, Faculty of Agriculture, Miyazaki University, 1-1
Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan
Received April 13, 1998
Two genomic DNAs encoding endoinulinase from Aspergillus niger 12 were cloned and sequenced. Open reading frames (ORFs) of the endoinulinase genes, inuA and inuB, both consisted of 1,548 nucleotides encoding 516 amino acids. It was suggested that the coding regions were not interrupted by introns. The ORFs differed from each other by 23 nucleotide substitutions or by eight amino acid replacements, indicating that the inuA and inuB genes arose by gene duplication. Each mature enzyme of 493 amino acids was preceded by a hydrophobic signal peptide of 23 amino acids. The enzymes contained two Cys residues and five potential sites for N-linked glycosylation. Partial amino acid sequences of the secreted enzyme suggested that it originated from the inuB gene product. The deduced amino acid sequences of the mature A. niger enzymes showed 73％ identity with that of the Penicillium purpurogenum endoinulinase.
Aspergillus niger; endoinulinase; fungal genome; gene duplication; inulin

-17-
Purification and Properties of a Novel Sulfatase from Pseudomonas testosteroni That Hydrolyzed 3β-Hydroxy-5-cholenoic Acid 3-Sulfate

Yasuhiko TAZUKE, Kumiko MATSUDA, Kenichi ADACHI, and Yoji TSUKADA

Kyoto Research Laboratories, Marukin Shoyu Co., Ltd., 27 Monnomae, Todo, Uji, Kyoto 611-0013, Japan

Received April 13, 1998
A novel sulfatase hydrolyzing the sulfate ester bond in 3β-hydroxy-5-cholenoic acid 3-sulfate (Δ5-3β-sulfate) was purified from Pseudomonas testosteroni ATCC 11996 as the second bile acid sulfatase. The molecular weight was 95,000 and the molecule was composed of a homodimer of a subunit of which the molecular weight was 46,000. This sulfatase hydrolyzed Δ5-3β-sulfate to 3α-hydroxy-5-cholenoic acid and sulfuric acid with inversion of β- to α-configuration of the hydroxyl group at the C-3 position of the substrate. The optimum pH and the stable pH of the enzyme were 8.5 and 6.5--9.7, respectively. 3β-Sulfate ester bonds of steroids such as isolithocholic acid, pregnenolone, and epiandrosterone, in which the side chain of the steroid ring was shorter than cholesterol, were also hydrolyzed to 3α-hydroxyl compounds corresponding to each steroid compound and sulfuric acid. We tentatively named this novel enzyme bile acid 3β-sulfate sulfohydrolyase (β-BSS).
sulfatase; bile acid sulfate sulfatase; bile acid 3β-sulfate sulfatase; sulfatase hydrolyzing 3β-hydroxy-5-cholenoic acid 3-sulfate; sulfatase of Pseudomonas testosteroni

-18-
Further Study on the Two Pivotal Parts of Hlg2 for the Full Hemolytic Activity
of Staphylococcal γ-Hemolysin

Kenji YOKOTA, Noriko SUGAWARA, Hirofumi NARIYA, Jun KANEKO,
Toshio TOMITA, and Yoshiyuki KAMIO・

Department of Applied Microbiology, Graduate School of Agricultural Science, Tohoku University,
1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan

Received April 15, 1998
Staphylococcal γ-hemolysin consists of LukF of 34 kDa and Hlg2 (or HγII) of 32 kDa, which cooperatively lyse human and rabbit erythrocytes. Our previous data showed that the 5-residue segment K23R24L25A26I27 of Hlg2 is pivotal for the hemolytic activity [Nariya, H. and Kamio, Y., Biosci. Biotechnol. Biochem., 59, 1603--1604 (1997)]. Here, we identify an additional amino acid residue in Hlg2 necessary for the full γ-hemolysin activity by measuring the toxin activity of Hlg2 mutants in the presence of LukF. The data obtained showed that Arg217 of Hlg2 is an additional pivotal amino acid residue besides the KRLAI segment for the full Hlg2-specific function in γ-hemolysin. We also report evidence that the Hlg2 mutants showing a low or null hemolytic activity in the presence of LukF towards human erythrocytes had low or no binding activity to the cells, resulting in failure of formation of the ring-shaped pore-forming complex on the erythrocytes.
Staphylococcal γ-hemolysin; bi-component cytotoxin; LukS; Hlg2

-19-
Endo-β-Glucanase Secreted by a Psychrotrophic Yeast: Purification and Characterization

Tadao OIKAWA,1,2・ Yasuyuki TSUKAGAWA,1 and Kenji SODA1,2

1Department of Biotechnology, Faculty of Engineering, Kansai University
2Kansai University High Technology Research Center, Suita-shi, Osaka 564-8680, Japan

Received April 15, 1998
A psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, isolated from soil, effectively produced an extracellular endo-β-glucanase (EC 3.2.1.4). The enzyme was monomeric, and the molecular mass was about 40,000 Da. The N-terminal amino acid sequence was H-Ser-Leu-Pro-||Lys - Leu - Gly - Gly - Val - Asp - Leu - Ala - Gly - Leu - Asp - Ile - Gly-||Lys-Asp-Lys-Asn-. α-Helix content was calculated to be about 32.6％. The isoelectric point was 8.57. The activation energy was 20.9 kJ/mol, which was much smaller than that of mesophilic enzymes. The enzyme was active at temperatures from 0 to 70°C, with a highest initial velocity at 50°C similar to other psychrotrophic enzymes. The enzyme was inhibited by Hg2＋. The enzyme catalyzed hydrolysis of carboxymethyl cellulose with an apparent Km of 1.1％ and||Vmax of 556 μmol/min/mg. Products from the enzymatic||hydrolysis of carboxymethyl cellulose by the enzyme were glucose, cellobiose, and cellotriose. The enzyme also catalyzed the transglycosylation of p-nitrophenyl-β-cellotrioside to cellotetraose.
psychrotrophic yeast; endo-β-glucanase; Rhodotorula glutinis; transglycosylation

-20-
Studies on the Carbohydrate Binding Sites of the Hemolytic Lectin CEL-III
Isolated from the Marine Invertebrate Cucumaria echinata

Imre SALLAY, Tomomitsu HATAKEYAMA,＊ and Nobuyuki YAMASAKI＊＊

Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan

Received April 22, 1998
The binding of carbohydrates to the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata was studied. Equilibrium dialysis data suggest that CEL-III has two carbohydrate-binding sites with equal affinity. The binding of specific carbohydrates to CEL-III induces a decrease in the fluorescence intensity at 339 nm and the shift of the fluorescence emission maximum to a wavelength shorter by 3 nm, owing to the change in the environment of tryptophan. By analyzing the change in the fluorescence intensity at 339 nm as a function of the concentration of carbohydrates, the association constants for||binding of individual carbohydrates to CEL-III were calcu||lated. The results indicate that GalNAc, lactulose, and lactose are bound by CEL-III with fairly high affinity among the carbohydrates tested. The pH-dependence profile of the association constant of lactose suggests that CEL-III binds carbohydrates with highest affinity around pH 5.0. Modification of CEL-III with N-bromosuccinimide produces an oxidized derivative, in which four tryptophan residues/mol were oxidized and had no hemolytic activity. However, two out of these four tryptophans escaped from the modification in the presence of specific saccharides and the resulting derivative retained fairly high hemolytic activity.
marine invertebrate lectin; Cucumaria echinata; Hemolytic lectin; carbohydrate binding site; tryptophan

-21-
Production of Ethyl (R)-2-Hydroxy-4-phenylbutanoate via Reduction of
Ethyl 2-Oxo-4-phenylbutanoate in an Interface Bioreactor

Shinobu ODA,＊ Yuichi INADA, Atsuko KOBAYASHI, and Hiromichi OHTA＊＊

Technical Research Laboratory, Kansai Paint Co., Ltd., Higashi-Yawata 4-17-1, Hiratsuka,
Kanagawa 254-8562, Japan
＊＊Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1,
Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan

Received May 12, 1998
Ethyl (R)-2-hydroxy-4-phenylbutanoate [(R)-EHPB], a||useful intermediate for the synthesis of various anti-hyper||||tension drugs, was produced via microbial reduction of||ethyl 2-oxo-4-phenylbutanoate [EOPB] in an interface bioreactor. Rhodotorula minuta IFO 0920 and Candida holmii KPY 12402 were selected as the best type culture and isolated yeasts, respectively. The highest enantiomeric excess of (R)-EHPB produced by R. minuta and C. holmii were 95 and 94％, respectively. C. holmii was used for the reduction of EOPB in a pad-packed interface bioreactor (inner volume, 3 liter). After incubation for 4 days, 4.4 g of (R)-EHPB was obtained via extraction with methanol followed by column chromatography. The overall yield, chemical purity, and enantiomeric excess of (R)-EHPB were 58％, 99.1％, and 90％, respectively.
interface bioreactor; stereoselective reduction; non-aqueous bioreactor; microbial transformation; bioconversion

-22-
Note
Scavenging Effect of Various Tea Extracts on Superoxide Derived from the Metabolism of Mutagens

Gow-Chin YEN・ and Hui-Yin CHEN

Department of Food Science, National Chung Hsing University, 250 Kuokuang Road,
Taichung 402, Taiwan, Republic of China

Received January 13, 1998
We determine the superoxide formed in the self-degradation of mutagens activated by cytochrome enzymes and evaluated the scavenging effect of various tea extracts. Benzo[a]pyrene (B[a]P) and 2-amino-6-methyldipyrido(1,2-a:3′,2′-d)imidazole (Glu-P-1) each produced a large amount of superoxide after activation by cytochrome enzymes. However, 2-amino-3-methyl-imidazo(4,5-f)quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyridol(4,3-b)indole (Trp-P-1) and alfatoxin B1 (AFB1) failed to generate a significant amount of superoxide. The addition of a tea extract to the reaction system marked inhibited the derivation of superoxide from Glu-P-1. However, the tea extracts showed weaker inhibition of the B[a]P-mediated formation of superoxide. Among the four teas tested, the oolong tea extract tended to exhibit the strongest inhibitory effect. Our results suggest that the chemopreventive efficacy of a tea extract is partly associated with its antioxidative activity.
tea extract; superoxide; scavenging effect; mutagen

-23-
Note
Evaluation of the Cytotoxic Activity of Chiral (E)-13-Hydroxy-10-oxo-11-
octadecenoic Acid and Its Lactone

Yoshiki HAYASHI,・ Yasushi NISHIKAWA, Hirotaka MORI, Yoh-ichi MATSUSHITA,＊
Kazuhiro SUGAMOTO,＊ and Takanao MATSUI＊

Research Laboratories II, Tamanoi Vinegar Co.Ltd., 1-32, 1-Cho, Kurumano-Cho Nishi, Sakai,
Osaka 590-0940, Japan
＊Faculty of Engineering, Miyazaki University, Gakuen-Kibanadai-Nishi, Miyazaki 889-2155, Japan

Received February 17, 1998
Both the S and R enantiomers of (E)-13-hydroxy-10-oxo-11-octadecenoic acid (1) and (E)-10-oxo-11-octadecen-13-olide (2) had similar IC50 values against P388 mouse leukemia cells; i.e. the stereochemistry of the asymmetric center of 1 and 2 had no influence on the cytotoxic activity. Bioassay results of various compounds related to 1 and 2 suggests that the 10-oxo and lactone moieties of 2 were important for enhancing the cytotoxicity.
corn; cytotoxic activity; fatty acid; (E)-13-hydroxy-10-oxo-11-octadecenoic acid; (E)-10-oxo-11-octadecen-13-olide

-24-
Note
Molecular Cloning, Sequencing and Characterization of the Genes for Adenosylcobalamin-dependent Diol Dehydratase of Klebsiella pneumoniae

Takamasa TOBIMATSU, Muneaki AZUMA, Shinji HAYASHI, Kyou-ichi NISHIMOTO,
and Tetsuo TORAYA・

Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University,
Tsushima-Naka, Okayama 700-8530, Japan

Received February 23, 1998
Klebsiella pneumoniae and some of the other Enterobacteriaceae form both diol dehydratase and glycerol dehydratase in response to growth substrates. To compare these enzymes produced by the same bacterium, the pdd genes of K. pneumoniae encoding adenosylcobalamin-dependent diol dehydratase were cloned and sequenced. The sequential three open reading frames (pddA, pddB, and pddC genes) encoded polypeptides of 554, 228, and 174 amino acid residues with predicted molecular weights of 60,379(α), 24,401(β), and 19,489(γ), respectively. The deduced amino acid sequences of the subunits were 84--100％ and 54--71％ identical with those reported for diol dehydratases and glycerol dehydratases, respectively.
diol dehydratase; adenosylcobalamin; Klebsiella pneumoniae; nucleotide sequence; glycerol dehydratase

-25-
Note
Increased Cellulose Production from Sucrose by Acetobacter after Introducing
the Sucrose Phosphorylase Gene

Naoto TONOUCHI,・ Sueharu HORINOUCHI,＊ Takayasu TSUCHIDA, and Fumihiro YOSHINAGA
Bio-Polymer Research Co., Ltd., 3-2-1 Sakato, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan
＊Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received March 11, 1998
A sucrose phosphorylase (SPase) gene derived from Leuconostoc mesenteroides was introduced into a cellulose-producing Acetobacter strain and expressed under the lac promoter. The activity of the SPase was detected in extracts of the transformed cells and cellulose production from sucrose by the cells was found to have increased, which strongly suggests that the increase was the result of the new metabolizing pathway. Furthermore, the level of SPase expression was increased by altering the length of the lac promoter.
sucrose phosphorylase; Acetobacter; cellulose; gene introduction

-26-
Note
Chick Skeletal Muscle Proteolysis in vitro Increased by Corticosterone

Akira OHTSUKA,・ Hiroko TANAKA, Ko HIZAKI, and Kunioki HAYASHI

Faculty of Agriculture, Department of Biochemical Science and Technology, Kagoshima University,
Kagoshima 890-0065, Japan

Received March 16, 1998
Six-day-old chick skeletal muscle (extensor digitorum longus) was incubated in the presence of corticosterone (CTC; 0, 3, 30, and 300 ng/ml) for 2 h at 37°C. Tyrosine and Nτ-methylhistidine releases, as indices of total muscle and myofibrillar proteolysis, were increased by CTC but with different dose responses, indicating an independent regulation of myofibrillar and non-myofibrillar protein degradation.
corticosterone; skeletal muscle proteolysis; tyrosine; Nτ-methylhistidine

-27-
Note
secA341 Mutation Inhibition of Expression of the Bacillus subtilis Protease Gene, aprE

Yoshito SADAIE

Radioisotope Center, National Institute of Genetics, Mishima 411-8540, Japan

Received March 19, 1998
The production of extracellular protease(s) of Bacillus subtilis is induced in the post-exponential growth phase, and is severely reduced in a strain carrying a temperature sensitive mutation (secA341ts) in the secA gene, the product of which is required for protein secretion. The expression of the extracellular serine protease gene, aprE, as monitored by the β-galactosidase activity of an aprE-lacZ translational fusion, was inhibited in the secA341 mutant cell and restored by the introduction of the degU32(Hy) mutation, which makes the phosphorylated DegU response regulator of a two-component system refractory to dephosphorylation and causes the cell to become a strong producer of AprE. The secA341 mutation appears to block some activation step required for aprE expression.
Bacillus subtilis; aprE expression; secA

-28-
Note
Germinated Barley Foodstuff Improves Constipation Induced by Loperamide in Rats

Osamu KANAUCHI,・ Yoshitaka HITOMI, Kazue AGATA, Tomohiko NAKAMURA, and Tohru FUSHIKI＊

Applied Bioresearch Center, Corporate Research and Development Division Kirin Brewery Co. Ltd.,
Miyaharacho 3, Takasaki, Gunma 370-1295, Japan
＊Department of Food Science and Technology, Faculty of Agriculture, Kyoto University,
Oiwakemachi, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

Received March 27, 1998
The effects of germinated barley foodstuff (GBF) derived from the aleurone and scutellum fractions of germinated barley low-lignified hemicellulose were examined in Sprague-Dawley rats with constipation induced by loperamide by addition to the diet (2 mg/kg body weight). Bowel movements were higher in the GBF-fed rats than in the cellulose-fed rats used as a control. Fecal water content was also higher in the GBF-fed rats. The concentration of short chain fatty acids in cecal content, especially butyrate, was significantly higher in the GBF-fed rats than in the cellulose-fed rats. These findings suggested that GBF helps normalize defecation not only in diarrhea but also constipation.
germinated barley; butyrate; constipation; dietary fiber; rat

-29-
Note
Structures of Novel Acidic Galactooligosaccharides Synthesized by
Bacillus circulans β-Galactosidase

Shuichi YANAHIRA,・ Yukiko YABE, Masamichi NAKAKOSHI,＊ Susumu MIURA,
Noritaka MATSUBARA, and Hidetoshi ISHIKAWA

Technology and Research Institute, Snow Brand Milk Products Co. Ltd., 1-1-2 Minamidai, Kawagoe,
Saitama 350-1165, Japan
＊Research Institute of Life Science, Snow Brand Milk Products Co. Ltd., 519 Shimoishibashi, Ishibashi,
Shimotsuga-gun, Tochigi 329-0512, Japan

Received April 7, 1998
The structures of acidic oligosaccharides synthesized by a transglycosylation reaction by Bacillus circulans β-galactosidase, using lactose as the galactosyl donor, and N-acetylneuraminic acid (NeuAc) and glucuronic acid (GlcUA) as the acceptors were investigated. Acidic oligosaccharides thus synthesized were purified by anion exchange chromatography and charcoal chromatography. The MS and NMR studies indicated that the acidic oligosaccharides from NeuAc were Galβ-(1→8)-NeuAc, Galβ-(1→9)-NeuAc, and Galβ-(1→3)-Galβ-(1→8)-NeuAc, and those from GlcUA were Galβ-(1→3)-GlcUA and Galβ-(1→4)-Galβ-(1→3)-GlcUA. These are novel acidic galactooligosaccharides.
Bacillus circulans; β-galactosidase; N-acetylneuraminic acid; glucuronic acid; acidic galactooligosaccharides

-30-
Note
Cloning, Sequencing, and Expression of an Endoglucanase Gene＊
from the Rumen Anaerobic Fungus Neocallimastix frontalis MCH3

Yuko FUJINO,＊＊ Koretsugu OGATA,＊＊＊ Takafumi NAGAMINE,＊＊＊ and Kazunari USHIDA＊＊＊＊

Laboratory of Animal Science, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522 Japan
＊＊＊STAFF Institute, Tukuba 305-0850, Japan

Received April 7, 1998
A cDNA clone encoding an endo-1,4-β-glucanase from a rumen fungus, Neocallimastix frontalis MCH3, was isolated. The nucleotide sequence showed that the gene, celA, encoded a multidomain enzyme containing a family 5 catalytic domain and a reiterated sequence that is involved in the association of a multienzyme complex, the cellulosome. The enzyme expressed in Escherichia coli showed the highest activity against carboxymethylcellulose at 40°C and pH 8.5.
anaerobic fungi; Neocallimastix frontalis; endoglucanase; dockerin domain

-31-
Note
Facile Syntheses of AM-toxins and Analogs as Cyclic Depsipeptides by the Solid-phase Method

Masahiro MIYASHITA, Tomoko NAKAMORI, Takahiro MURAI, Hisashi MIYAGAWA,
Miki AKAMATSU, and Tamio UENO

Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received April 10, 1998
The cyclic depsipeptides, AM-toxins I and II and AM-toxin I analogs, were efficiently and rapidly prepared by the Fmoc-based solid-phase method for the synthesis of linear depsipeptides, with N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) being used for their subsequent cyclization.
AM-toxin; host-specific toxin; cyclic depsipeptide; solid-phase synthesis; HATU

-32-
Note
Expression of the Extracellular Levansucrase and Invertase Genes
from Zymomonas mobilis in Escherichia coli Cells

Hideshi YANASE,・ Junichi FUJIMOTO, Masahiro MAEDA, Kenji OKAMOTO, Keiko KITA, and Kenzo TONOMURA＊

Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-0945, Japan
＊Department of Food Technology, Faculty of Engineering, Fukuyama University,
Fukuyama 721-0000, Japan

Received April 13, 1998
We investigated the expression and localization in Escherichia coli of sucZE2 and sucZE3, encoding Zymomonas mobilis extracellular levansucrase and invertase, respectively, and lacking a typical N-terminal secretion signal. Levansucrase and invertase were expressed efficiently under the lac and tac promoters in E. coli cells, and some of the levansucrase produced was localized in the periplasmic space. The sucZE2 expression was not lethal to E. coli in the presence of 5％ sucrose, and led to the accumulation of levan from sucrose.
extracellular levansucrase; extracellular invertase; secretion; signal peptide-independent pathway; Zymomonas mobilis

-33-
Note
Stability of Thermostable Enzyme, Aqualysin I ; a Subtilisin-type Serine Protease from Thermus aquaticus YT-1

Terumichi TANAKA,・ Hiroshi MATSUZAWA, and Takahisa OHTA＊

Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Received in April 14, 1998
We characterized the heat stability and detergent stabilities of aqualysin I, produced by Thermus aquaticus YT-1, and compared them with those of fungal proteinase K and Bacillus subtilisin.
Aqualysin I displayed excellent heat and detergent stabilities. Proteinase K, another Cys-containing enzyme, was less stable than aqualysin I. All these enzymes maintained activities in the presence of urea or Tween-20.
alkaline serine protease; subtilisin-type protease; Thermus aquaticus YT-1; detergents

-34-
Note
Expression and Purification of Cytokine Receptor Homology Domain of Human Granulocyte-Colony Stimulating Factor Receptor in Escherichia coli

Ryoichi TANAKA, Hiroko TOKUNAGA, Shinichi HARA,＃ Tsutomu ARAKAWA,＃ and Masao TOKUNAGA・

Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture,
Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan
＃Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1789, U.S.A.

Received April 17, 1998
In an attempt to generate a stable non-glycosylated cytokine receptor homology (CRH) domain (Tyr97-Ala309) of human granulocyte-colony stimulating factor (G-CSF) receptor, two free cysteines in the CRH domain were converted to serine by site-directed mutagenesis. Taking advantage of the tight regulation for the expression of T7 RNA polymerase, the mutated CRH domain was successfully expressed in Escherichia coli (E. coli) with a pelB signal sequence at its NH2-terminus and with a His tag at its COOH-terminus. The processed and secreted CRH domain after solubilization and in vitro refolding retained G-CSF binding activity, and its yield (~40 μg/30 ml culture) was more than 100-fold higher than that of the mouse CRH domain expressed by the MalE fusion system in E. coli.
cytokine receptor homology domain; human G-CSF receptor; pelB leader; His tag

-35-
Note
Detection of Proteins Binding to the Promoter Region DNA Using a Nonradioactive Gel-Retardation Assay

Shin-ichiro MITSUNAGA,・ Kiichi FUKUI,＊ Hideo OHYAMA, and Junji YAMAGUCHI＊＊

Department of Life and Health Science, Joetsu University of Education, 1 Yamayashiki, Joetsu,
Niigata 943-8512, Japan
＊Laboratory of Rice Genetics Engineering, Hokuriku National Agricultural Experiment Station,
1-2-1 Inada, Joetsu, Niigata 943-0193, Japan
＊＊BioScience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Received April 20, 1998
Binding of nuclear proteins to the promoter region was studied by a nonradioactive gel-retardation assay. The procedure uses biotinylated oligonucleotides in combination with streptavidin and biotin-conjugated alkaline phosphatase. This method offers sensitivity comparable to radioactive detection, and the advantage of the high stability of probes. Moreover the hazards of usage associated with radiation are avoided.
DNA-binding protein; nonradioactive; gel-retardation assay

-36-
Note
Structural Confirmation of Cotylenin A, a Novel Fusicoccane-diterpene Glycoside with Potent Plant Growth-regulating Activity from Cladosporium Fungus sp. 501-7W

Takeshi SASSA,・ Takahiro OOI, Manabu NUKINA, Michimasa IKEDA,
and Nobuo KATO＊

Department of Bioresources, Faculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan
＊Institute of Advanced Material Study, Kyushu University, Kasuga 816-8580, Japan

Received April 20, 1998
The structure of cotylenin A, a potent plant-growth stimulant with the most complex molecule in cotylenins from Cladosporium fungus sp. 501-7W, was analyzed again by HMBC experiments and X-ray crystallography of its diacetyl-dihydroderivative. The previously proposed stereostructure of cotylenin A was thus completely confirmed.
cotylenin A; stereostructure; NMR assignment; X-ray crystallography

-37-
Note
Effects of β-Lactoglobulin on the Tight-junctional Stability of Caco-2-SF Monolayer

Kei HASHIMOTO,・ Tsutomu NAKAYAMA,＊ and Makoto SHIMIZU＊＊

Department of Bioproductive Science, Utsunomiya University, 350 Mine-machi, Utsunomiya 321-8505, Japan
＊School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
＊＊Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Received April 23, 1998
The mechanisms for tight-junction (TJ) stabilization by β-lactoglobulin (β-Lg) were studied. Treatment of Caco-2-SF cells with inhibitors for some enzymes in the intracellular signal transduction pathways and a cytoskeleton-disturbing agent (cytochalasin D) reduced the TJ-stabilizing activity of β-Lg. So β-Lg is suggested to modulate the cytoskeletal structure through the activation of phospholipase C and protein kinase C, resulting in the TJ stabilization.
Caco-2; intestinal epithelial cell; tight junction; β-lactoglobulin

-38-
Note
Study on the Application of the (S)-2-tert-Butyl-2-methyl-1,3-benzodioxole-
4-carboxylic acid-based On-line HPLC-Exciton CD Analysis to Acyclic 1,3-Diols

Syun-ichi SATO, Kumi SHIMIZU, Jeong-Hwan KIM, Eiichi AMI, Kazuaki AKASAKA, and Hiroshi OHRUI・

Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University,
1-1 Tsutsumidori Amamiya, Aoba-ku, Sendai 981-8555, Japan

Received April 27, 1998
The application of the on-line HPLC-exciton CD analysis with (S)-TBMB-carboxylic acid that simultaneously determined the enantiomeric composition and absolute configuration of vicinal diols, diamines and amino alcohols to 1,3-diols was studied. All optical isomers of di--O--(S)-TBMB-carbonyl-1,3-butanediols and 2,4-pentanediols were separated and their absolute configurations were determined without reference samples on line by HPLC.
on-line HPLC-exciton CD analysis; (S)-TBMB-carboxylic acid; 1,3-diols; fluorometry; exciton CD monitoring

-39-
Note
A Rapid Method for Disrupting Genes in the Escherichia coli Genome

Chinatsu KATO, Ryusuke OHMIYA, and Takeshi MIZUNO＊

Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University,
Chikusa-ku, Nagoya 464-8601, Japan

Received April 28, 1998
The entire genomic sequence of Escherichia coli has recently been completed. To gain insight into the function of the vast array of yet uncharacterized open reading frames (ORFs), a variety of new genetic tools will be required. Here we examined a genetic system, using an integration||plasmid vector (named pINT007), for rapid construction of||disruption mutants of any ORF in E. coli. It was found that the vector allows us to rapidly construct a disruption mutant for any gene on the chromosome as a cointegrate, furthermore, resolution of the resulting cointegrate can be surely accomplished by using a pair of the bla (ampicillin resistant) genes on the vector as a positive-selection marker.
E. sherichia coli; gene disruption; genome analysis; plasmid vector

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Note
Identification and Characterization of Acinetobacter sp. CNU961 Able to Grow with Phenol at High Concentrations

Kwang Cheol JEONG, Eun Ye JEONG, Tay Eak HWANG,1 and Sang Ho CHOI＊

Department of Food Science and Technology, Institute of Biotechnology,
Center for Specialized Agricultural Sciences, Division of Biological Resources,1
Chonnam National University, Kwang-Ju 500-757, Korea

Received May 11, 1998
An Acinetobacter sp., strain CNU961, with a higher tolerance to phenol was isolated, and identified through a set of taxonomic studies and a genetic complementation test. Enzymatic and mutagenic studies found that the strain dissimilate phenol by hydroxylation to catechol followed by an ortho-ring cleavage pathway to further mineralize it. The phenol hydroxylase, which is an inducible enzyme and requires NADPH for optimum activity, was not inhibited by phenol at concentrations up to 0.5 mM. The different kinetic behaviors of the enzyme activities on NADPH and on phenol reflected that the phenol hydroxylase of strain CNU961 is a multisubunit allosteric enzyme consisting of heterogeneous polypeptides.
Acinetobacter sp.; phenol tolerance; catechol production; ortho-pathway; phenol hydroxylase

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Preliminally Communication
Phosphorylation of LukS by Protein Kinase A is Crucial for the LukS-Specific Function of the Staphylococcal Leukocidin on Human Polymorphonuclear Leukocytes

Akihito NISHIYAMA, Hirofumi NARIYA, and Yoshiyuki KAMIO＊

Department of Applied Microbiology, Graduate School of Agricultural Science, Tohoku University,
1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan

Received May 29, 1998
Staphylococcal leukocidin (Luk) consists of two protein components, LukF and LukS, which cooperatively lyse human and rabbit polymorphonuclear leukocytes. Here, we demonstrate that the phosphorylation of LukS by protein kinase A is crucial for the LukS-specific leukocytolytic function of Luk on HPMNLs by using N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), which is a potent and selective inhibitor of protein kinase A. At 0.5 μM H-89 completely prevented the Luk-induced cell lysis accompanied by blocking of the incorporation of exogenous 32P-H3PO4 into LukS on HPMNLs. However, with LukS and LukF together, 0.5 μM H-89 did not inhibit the cell swelling which takes place before the cell lysis. HPMNLs also became swollen upon treating with both LukF and LukS mutants which could not be phosphorylated.
staphylococcal leukocidin; bi-component cytolysin; LukS; toxin phosphorylated by protein kinase A; protein kinase A inhibitor H-89

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