Contents and Abstracts of Latest Issue of BBB

(Vol.63 No.12 1999)

Synthesis of (±)-Methyl Tuberonate, a Potato Tuber-forming Substance,
and Its Epimer
Hiromasa KIYOTA,・ Daisuke NAKASHIMA, and Takayuki ORITANI

Antioxidative Effects of Turmeric, Rosemary and Capsicum Extracts
on Membrane Phospholipid Peroxidation and Liver Lipid Metabolism in Mice
Akira ASAI,・ Kiyotaka NAKAGAWA, and Teruo MIYAZAWA

Local Antibody Response in Peyer′s Patches to the Orally Administered
Dietary Protein Antigen
Michiko SHIMODA, Yuri INOUE, Norihiro AZUMA, and Choemon KANNO

A Fibrinolytic Metalloprotease from the Fruiting Bodies of an Edible
Mushroom,
Armillariella mellea
Jun-Ho KIM and Yang Sun KIM

Crystallization and Properties of NADPH-Dependent L-Sorbose Reductase
from Gluconobacter melanogenus IFO 3294
Osao ADACHI,＃ Yoshitaka ANO, Duangtip MOONMANGMEE,・ Emiko SHINAGAWA,＊

Improvement of the Physical Properties of Pepsin-Solubilized
Elastin-Collagen Film by Crosslinking
Koji TAKAHASHI,・ Yoshikadzu NAKATA, Kenji SOMEYA, and Makoto HATTORI

Lipase-catalyzed Kinetic Resolution of (±)-trans- and cis-2-Azidocycloalkanols
Ei′ichi AMI and Hiroshi OHRUI・

Analysis of a Catalytic Acidic Pair in the Active Center of Cellulase
from Aspergillus aculeatus
Akira OHNISHI,1 Toshihiko OOI,1 Shinichi KINOSHITA,1,・ Hirotaka TOMATSURI,2
Kazuyuki UMEDA,2 Shunsaku UEDA,2 Yasuo HATA,3 and Motoo ARAI4

Dietary Effect of Guar Gum and its Partially Hydrolyzed Product
on the Lipid Metabolism and Immune Function of Sprague-Dawley Rats
Koji YAMADA,・ Yoko TOKUNAGA, Atsushi IKEDA, Ken-ichi OHKURA, Soichi MAMIYA
,＊
Shihoko KAKU,＊＊ Michihiro SUGANO,＊＊ and Hirofumi TACHIBANA

Production of D-Glutamate from L-Glutamate with Glutamate Racemase
and L-Glutamate Oxidase
Tadao OIKAWA,1,2,・ Mayumi WATANABE,1 Hidemi MAKIURA,1 Hitoshi KUSAKABE,3
Kazuhiro YAMADE,1,2 and Kenji SODA1,2

Cloning and Sequence Analysis of the Gene for Glucodextranase
from Arthrobacter globiformis T-3044 and Expression in Escherichia coli
Cells
Tetsuya OGUMA, Toshiko KUROKAWA, Kouichiro TOBE,＊ Satoshi KITAO,＊
and Mikihiko KOBAYASHI＊＊

Permeation of Hesperidin Glycosides Across Caco-2 Cell Monolayers Via
the Paracellular Pathway
Minji KIM, Takashi KOMETANI,＊ Shigetaka OKADA,＊ and Makoto SHIMUZU・

Functional Analysis of the DXDDTA Motif in Squalene-Hopene Cyclase
by Site-directed Mutagenesis Experiments: Initiation Site
of the Polycyclization Reaction and Stabilization Site
of the Carbocation Intermediate of the Initially Cyclized A-Ring
Tsutomu SATO and Tsutomu HOSHINO・

Note
Subsite Structure of α-Amylase II from Thermoactinomyces vulgaris R-47
Yoichiro SHIMURA, Qiong WANG, and Yoshiyuki SAKANO・

Note
HIV-1 Protease Inhibition and Anti-HIV Effect of Natural
and Synthetic Water-soluble Lignin-like Substances
Toshiaki ICHIMURA,・ Toru OTAKE,1 Haruyo MORI,1 and Susumu MARUYAMA

Note
High-level Expression of a Recombinant Thermostable Phytase in Bacillus
subtilis
Young-Ok KIM, Jung-Kee LEE, Byung-Chul OH, and Tae-Kwang OH・

Note
Competing Effect of Polyols on the Thermal Stability
and Gelation of Soy Protein
Kunihiko GEKKO,＊,・ Xuan LI, and Shio MAKINO

Note
Optical Resolution by the Replacing Crystallization of DL-Threonine
with L-Alanine as an Optically Active Cosolute
Tadashi SHIRAIWA,＊,・ Motoki KUBO, Keiji FUKUDA, and Hidemoto KUROKAWA

Note
Site-directed Mutagenesis of Two Zinc-binding Centers
of the NADH-dependent Phenylacetaldehyde Reductase
from Styrene-assimilating Corynebacterium sp. Strain ST-10
Jiu-Cun WANG, Mikio SAKAKIBARA, Michiko MATSUDA,＊ and Nobuya ITOH＊,・

Note
Activity of Glucose-6-phosphate 1-Dehydrogenase in Hair Follicles
with Male-pattern Alopecia
Kuniaki ADACHI,1 Yasusi WATANABE,2 and Kuniyo INOUYE3,・

Note
Cloning and Expression in Escherichia coli of 2-Hydroxypropylphosphonic
Acid Epoxidase from the Fosfomycin-producing Organism,
Pseudomonas syringae PB-5123
Tomohisa KUZUYAMA, Takayuki SEKI, Seiji KOBAYASHI, Tomomi HIDAKA,＊
and Haruo SETO

Note
Nucleotide Sequence of cDNA Coding the Mitochondrial Precursor Protein
of the ATPase Inhibitor from Humans
Naoki ICHIKAWA,・ Saori USHIDA, Miho KAWABATA, and Yukiko MASAZUMI

Note
Analysis of mfbA Alleles of the Eubasidiomycete Lentinus edodes
Toru YASUDA・ and Kazuo SHISHIDO・・

Note
Arginine-55 in the β-Arm is Essential for the Activity of DNA-Binding Prot
ein HU from Bacillus stearothermophilus
Fumiyo SAITOH,1 Shunsuke KAWAMURA,1 Nobuyuki YAMASAKI,1
Isao TANAKA,2 and Makoto KIMURA1,・

Note
Antibacterial Activities of Cryptotanshinone and Dihydrotanshinone I
from a Medicinal Herb, Salvia miltiorrhiza Bunge
Dong-Sun LEE,1,2,・ Sang-Han LEE,1 Jae-Geun NOH,3 and Soon-Duck HONG1

Note
Promotive Effects of the Peptidyl Plant Growth Factor, Phytosulfokine-α,
on the Growth and Chlorophyll Content of Arabidopsis Seedlings
under High Night-time Temperature Conditions
Seiyei YAMAKAWA,1 Yoshikatsu MATSUBAYASHI,2 Youji SAKAGAMI,2
Hiroshi KAMADA,1 and Shinobu SATOH1,・

Note
Cloning of L-Amino Acid Deaminase Gene from Proteus vulgaris
Eiji TAKAHASHI,・ Kiyoshi ITO,＊ and Tadashi YOSHIMOTO＊

Note
Synthesis and Characterization of Intermediate and Transition-State
Analogue Inhibitors of γ-Glutamyl Peptide Ligases
Makoto INOUE, Jun HIRATAKE,・ and Kanzo SAKATA

Preliminary Communication
Interaction of Tea Catechins with Lipid Bilayers Investigated
with Liposome Systems
Toshihiko HASHIMOTO, Shigenori KUMAZAWA, Fumio NANJO,＊
Yukihiko HARA,＊ and Tsutomu NAKAYAMA

Preliminary Communication
Soybean Curd Refuse Alleviates Experimental Tumorigenesis in Rat Colon
Naoyuki AZUMA, Hitoshi SUDA, Hiroyuki IWASAKI, Rhyuhei KANAMOTO,
and Kimikazu IWAMI＊


-1-
Enhancement of Swimming Endurance in Mice
by Highly Branched Cyclic Dextrin

Hiroshi TAKII,・ Kengo ISHIHARA,＊ Takashi KOMETANI,
Shigetaka OKADA, and Tohru FUSHIKI＊

Biochemical Research Laboratory, Ezaki Glico Co. Ltd., 4-6-5 Utajima,
Nishiyodogawa-ku,
Osaka 555-8502, Japan
＊Laboratory of Nutrition Chemistry, Division of Applied Life Sciences, Gra
duate School of Agriculture,
Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan

Received October 5, 1998; Accepted August 11, 1999
We investigated the ergogenic effect in mice of administering highly
branched cyclic dextrin (HBCD), a new type of glucose polymer, on the
swimming endurance in an adjustable-current swimming pool. Male Std ddY
mice were administered a HBCD, a glucose solution or water via a stomach
sonde 10 min before, 10 min after or 30 min after beginning swimming
exercise, and were then obliged to swim in the pool. The total swimming
period until exhaustion, an index of the swimming endurance, was measured.
An ergogenic effect of HBCD was observed at a dose of 500 mg/kg of body
weight, whereas it had no effect at a dose of 166 mg/kg of body wt
(p<0.05). The mice administered with the HBCD solution 10 min after
starting the exercise were able to swim significantly longer (p<0.05) than
the mice who had ingested water or the glucose solution. The rise in mean
blood glucose level in the mice administered with HBCD, which was measured
20 min after starting swimming, was significantly lower (p<0.05) than that
in the mice administered with glucose, although it was significantly higher
(p<0.05) than that in the mice administered with water. The mean blood
insulin rise in the mice given HBCD was significantly lower (p<0.05) than
that in the mice given glucose. The mice administered with HBCD 30 min
after starting the exercise swam significantly longer (p<0.05) than the
mice who had ingested water, although the enhancement of swimming time was
similar to that of the glucose-ingesting mice. The gastric emptying rate of
the HBCD solution was significantly faster (p<0.05) than that of the
glucose solution. However, this glucose polymer must have spent more time
being absorbed because it has to be hydrolyzed before absorption,
reflecting a lower and possibly longer-lasting blood glucose level. We
conclude that the prolongation of swimming endurance in mice administered
with HBCD depended on its rapid and longer-lasting ability for supplying
glucose with a lower postprandial blood insulin response, leading to a
delayed onset of fatigue.
highly branched cyclic dextrin (HBCD); glucose; swimming endurance;
current swimming pool; gastric emptying rate

-2-
Catabolite Repression of the Xylanase Gene (xynA) Expression
in Bacillus stearothermophilus No. 236 and B. subtilis

Ssang-Goo CHO and Yong-Jin CHOI・

Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea

Received April 5, 1999; Accepted August 23, 1999
Catabolite repression of the Bacillus stearothermophilus No. 236 xynA
gene, encoding an extracellular xylanase, was investigated in this work.
Expression of the xynA gene in the B. stearothermophilus strain was found
to be subject to glucose catabolite repression, and the level of repression
was about 50-fold when the relative amounts of xynA transcript synthesized
on different carbon sources were analyzed. The experiments with the B.
subtilis MW15 strains carrying plasmids containing the xynA::aprA fusion
gene showed that the cloned xynA gene did not require any specific carbon
source for its induction. Nevertheless, the expression of the cloned gene
was repressed by the presence of glucose. From the nucleotide sequence of
the cloned xynA gene, we found two potential catabolite responsive elements
(cre) within its reading frame region (cre-1: nucleotides ＋160 to ＋173 and
cre-2: ＋173 to ＋186). Furthermore, by using various deletion derivatives o
f the xynA::aprA fusion plasmid (pMGW23), we suggested that only the cre-2
element might play a role in the glucose catabolite repression. Repression
level of the xynA gene expression in the recombinant B. subtilis strain was
estimated to be about 3-fold by analysis of the amounts of xynA transcript.
catabolite repression; xylanase; xynA; Bacillus stearothermophilus;
Bacillus subtilis

-3-
Structural Characterization of the 16-kDa Allergen, RA17, in Rice Seeds.
Prediction of the Secondary Structure and Identification of Intramolecular
Disulfide Bridges

Hidehiko IZUMI,1,2 Minoru SUGIYAMA,2 Tsukasa MATSUDA,2,・ and Ryo NAKAMURA3

1Department of Food and Nutrition, Nagoya College of Nutrition, Naka,
Nagoya 460-0007, Japan
2Department of Applied Biological Sciences, School of Agricultural
Sciences, Nagoya University,
Chikusa, Nagoya 464-8601, Japan
3Department of Food Science and Technology, Nihon University, Setagaya
154-0002, Japan

Received April 22, 1999; Accepted August 3, 1999
The 16-kDa rice allergen, RA17, belonging to the α-amylase/trypsin inhibit
or family was isolated from rice seed and structurally characterized by
identifying cystine-containing peptides and predicting the secondary
structure and hydrophobic regions. Eight peptides, which constitute three
sets of cystine-containing peptides, were purified by HPLC from a
thermolytic digest of RA17 and identified by their amino acid sequence and
composition, indicating five intramolecular disulfide bridges: Cys34-Cys94,
Cys26-(Cys50 or Cys51)-Cys110 and Cys12-(Cys62 or Cys64)-Cys122. Analyses
of the CD spectrum and the Chou-Fasman prediction suggested that RA17 had
some helical- and sheet-structure regions. Based on these experimental and
predicted data, RA17 is proposed to be a globular molecule with a small
hydrophobic core having folding restricted by five intramolecular disulfide
bridges.
rice; allergen; food allergy; α-amylase/trypsin inhibitor; disulfide bridg
e

-4-
Production of an Extracellular Polysaccharide Bioflocculant
by Klebsiella pneumoniae

Kuniho NAKATA・,＊ and Ryuichiro KURANE＊＊

＊Central Research Laboratories, Mercian Corporation, 4-9-1 Johnan, Fujisaw
a 251-0057, Japan
＊＊National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsu
kuba 305-8565, Japan

Received May 11, 1999; Accepted August 27, 1999
Klebsiella pneumoniae H12 produced a newly identified extracellular
polysaccharide in an ethanol medium with a yield of 3.0 g/l. The molar
composition of the polysaccharide was 56.04％ galactose, 25.92％ glucose, 10
.92％ galacturonic acid, 3.71％ mannose, and 3.37％ glucuronic acid. The add
ition of 0.5％--1.5％ NaCl increased production. The polysaccharide floccula
ted with kaolin clay in suspension at the concentration of 1 ppm in a
300-ppm solution of CaCl2. Almost all bacterial species cells aggregated in
the polysaccharide solution. The ability to flocculate with kaolin clay
changed with the pH and with the concentrations of coexisting cation and
anion species. The polysaccharide flocculant may participate in in vivo
bacterial aggregation or adherence to host organisms.
Klebsiella; polysaccharide; flocculant; ethanol

-5-
Amino Acid Sequence Analysis of Bitter Peptides from a Soybean Proglycinin
Subunit Synthesized in Escherichia coli

Mi-Ryung KIM, Sang-Yun CHOI,・ Chan-Shick KIM,1 Chan-Wha KIM,
Shigeru UTSUMI,2 and Cherl-Ho LEE

Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea
1Department of Agricultural Chemistry, Cheju National Univesity, Cheju
690-756, Korea
2The Research Institute for Food Science, Kyoto University, Uji, Kyoto
611-0011, Japan

Received May 14, 1999; Accepted August 31, 1999
The cDNA encoding A1aB1b proglycinin was expressed in E. coli, for the
efficient isolation of a single peptide responsible for the bitterness. The
55-kD proglycinin was highly purified, hydrolyzed, and further purified
through a series of chromatographic steps to yield fractions with the major
bitter peptides. The most bitter-tasting fractions contained peptides with
average molecular weights lower than 1,700 Da. An analysis of the amino
acid sequences indicated that many small bitter peptides (<1,000 Da) are
composed of uncharged polar amino acids as well as hydrophobic amino acids,
with a charged residue often being present at either end. This suggests the
involvement of a certain structural requirement in taste perception.
proglycinin; soybean protein hydrolysate; bitter peptide

-6-
Rheological Properties of a Heat-reversible Gel of Water-soluble Soybean
Polysaccharide Extracted under Acidic Conditions

Hitoshi FURUTA,・ Junko TOBE, Ryosuke KIWATA, and Hirokazu MAEDA＊

Novelty Materials Research Institute, Tsukuba R＆D Center, Fuji Oil Co. Ltd
., 4-3 Kinunodai,
Yawara-mura, Tsukuba-gun, Ibaraki 300-2497, Japan
＊Protein Development Dept. II, Hannan R＆D Center, Fuji Oil Co. Ltd., 1 Su
miyoshi-cho, Izumisano,
Osaka 598-0061, Japan

Received May 14, 1999; Accepted August 13, 1999
We studied the rheological properties of a heat-reversible gel formed with
water-soluble soybean polysaccharides (RG-SSPS) which had been extracted
from purified polysaccharides of soybean cotyledon meal at pH 2 and 80°C fo
r 90 min. It was found that the gelling point and melting point of this
heat-reversible gel were at approximately 51°C and 46°C, respectively. Gel
ation was observed in aqueous solutions with a concentration of over 3％, bu
t not at 1％, at 5°C. The influence of pH on the gel strength of RG-SSPS wa
s strong. Especially on the acidic side, the G′ and G″ values, which were
highest at pH 7, were remarkably low and no gel was formed at pH 5. The
addition of EDTA lowered the G′ and G″ values, while the addition of Ca ra
ised them. These findings and the analytical results for the fractions
obtained from RG-SSPS after reheating at pH 5 and 120°C for 90 min, under w
hich conditions the non-gel-forming polysaccharides were extracted, suggest
that gelation was due to weak bonding and cross-linking between polyvalent
cations and low-methoxyl uronic acid regions in the homogalacturonic or
rhamnogalacturonic main chain of the RG-SSPS structure. The results also
suggest that the heat reversibility was due to weakness of the bonding
which did not conform to the egg-box model, like the mechanism for
low-methoxyl pectin or sodium alginate, because the neutral sugar side
chains of the RG-SSPS structure are thought to limit their mutual proximity.
soybean; polysaccharide; rheology; gel

-7-
Encoding of a Cytochrome P450-Dependent Lauric Acid Monooxygenase
by CYP703A1 Specifically Expressed in the Floral Buds of Petunia hybrida

Hiromasa IMAISHI,1 Yuka MATSUMOTO,2 Utako ISHITOBI,2 and Hideo OHKAWA2＊

1Center for Cooperative Research and Development, and 2Department of
Biological and Environmental Science,
Faculty of Agriculture, Kobe University, Rokkodaicho 1-1, Nada-ku, Kobe
657-8501, Japan

Received Jane 7, 1999; Accepted August 31, 1999
The cDNA clone of novel cytochrome P450 CYP703A1 from petunia floral buds
was isolated by RT-PCR. The nucleotide sequences of this cDNA clone
contained the open reading frame that has been predicted to encode
polypeptides consisting of 539 amino acid residues. A significantly high
level of the transcript of the cyp703A1 gene was found in the early stage
of petunia flower buds, but not||in the leaves, stems and roots. The 1041bp
5′-flanking||||sequences of the cyp703A1 gene contained the conserved||||mo
tifs of ATHB-1, AGAMOUS, MYB.Ph3, P and SBF-1||binding boxes. CYP703A1 cDNA
was expressed in yeast Saccharomyces cerevisiae AH22 cells under the
control of an alcohol dehydrogenase I promoter and terminator. The
recombinant yeast microsomes containing the CYP703A1 hemoprotein were found
to metabolize lauric acid. Based on these results, CYP703A1 was
specifically expressed in the early stage of flower development and
appeared to participatie in the monooxygenation of fatty acids.
cytochrome P450; cDNA cloning; floral bud; Saccharomyces cerevisiae;
Petunia hybrida

-8-
Cloning and Expression of the N,N-Dimethylformamidase Gene
from Alcaligenes sp. Strain KUFA-1

Yoshie HASEGAWA,・ Tai TOKUYAMA, and Hiroaki IWAKI

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

Received June 14, 1999; Accepted August 30, 1999
N,N-Dimethylformamidase (DMFase) from Alcaligenes sp. strain KUFA-1, a
bacterium that can grow on N,N-dimethylformamide (DMF) as the sole carbon
and nitrogen source, catalyzes the first step of the DMF degradation. The
DMFase gene dmfA1A2 was cloned in Escherichia coli, and its nucleotides
were sequenced. The deduced amino acid sequence of the enzyme consisted of
two α- and two β-subunits with 132 and 762 amino acids, respectively, and
had little similarity to sequences in protein databases, including various
amidases. The protein may be a new kind of amidase. DMFase activity was
detected in E. coli cells transformed with an expression plasmid of the
cloned DMFase gene. The properties of recombinant DMFase purified from E.
coli were identical to those of Alcaligenes DMFase.
Alcaligenes sp.; N,N-dimethylformamidase gene; gene cloning; nucleotide
sequencing

-9-
The Expression of proUK in Escherichia coli: The vgb Promoter Replaces
IPTG and Coexpression of argU Compensates for Rare Codons
in a Hypoxic Induction Model

Lan JIANG,1,2,・ Yonghua YANG,1 Shampa CHATTERJEE,2 Bertolt SEIDEL,2
Gerald WOLF,2 and Shengli YANG1

1Shanghai Research Center of Biotechnology, Chinese Academy of Sciences,
Shanghai, 200233, P.R.China
2Institute for Medical Neurobiology, Otto-von-Guericke University,
Leipziger Str. 44, 39120-Magdeburg, Germany

Received June 16, 1999; Accepted August 20, 1999
The expression of the proUK gene was improved by the coexpression of the
argU gene cloned in a moderate copy number vector. As the proUK gene
contains 2％ AGG/AGA condons, which is much higher than the normal frequency
in E. coli, about 0.14％--0.21％, the argU gene cloned in a multicopy plasmi
d was coexpressed with the proUK expression vector in our experiments. In
E. coli strain BL21(DE3), IPTG is known to induce the expression of T7 RNA
polymerase gene and this enzyme can transcribe the proUK gene under the
control of the T7 promoter leading to expression of proUK. To replace IPTG
by a cheaper alternative on a large scale, we constructed a plasmid in
which the vgb promoter---which is known to be activated by the onset of
hypoxic conditions---controls the T7RNA polymerase gene expression. Low
oxygen conditions were then used to activate the vgb promoter causing T7RNA
polymerase gene expression and finally leading to the expression of proUK
as inactive inclusion bodies. Our experiments on a large scale in a
bioreactor show that the expression of proUK accounts for about 30％ of tota
l protein after about 6 h of anaerobic cultivation, so the presented model
represents an economical alternative to IPTG induction.
pro-urokinase expression; vgb promoter; argU coexpression; anaerobic
induction

-10-
Enzymes Responsible for Acetate Oxidation by Acetic Acid Bacteria＃

Akihiko SAEKI, Kazunobu MATSUSHITA,＊ Seiki TAKENO,＊ Mariko TANIGUCHI,＊
Hirohide TOYAMA,＊ Gunjana THEERAGOOL,＊＊ Napha LOTONG,＊＊ and Osao ADACH
I＊,＊＊＊

Food ＆ Biotechnology Department, Yamaguchi Prefectural Industrial Technolo
gy Institute,
Asutopia, Ube 755-0151, Japan
＊Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi Uni
versity, Yoshida,
Yamaguchi 753-8515, Japan
＊＊Department of Microbiology, Faculty of Science, Kasetsart University, P
haholyotin Road.,
Bangkok 10900, Thailand

Received June 17, 1999; Accepted August 26, 1999
Several acetic acid bacteria of the genus Acetobacter oxidize much acetate
oxidation, which is not desired in vinegar manufacturing. Acetobacter
rancens SKU 1111, a strong acetate oxidant, grew rapidly with a biphasic
growth curve while consuming acetate in the second growth phase.
Acetobacter aceti IFO 3284 did not show extensive acetate oxidation.
Addition of glycerol to the culture medium of Acetobacter rancens SKU 1111
increased acetate oxidation and resulted in more biomass in the second
growth phase than when glycerol was not added. Enzyme activities of
acetyl-CoA synthetase and phosphotransacetylasein the organism were high
during acetate oxidation. The activity of phosphoenolpyruvate carboxylase
was most stimulated by a trace amount of acetyl-CoA among the enzymes of
glycerol catabolism. Phosphoenolpyruvate carboxylase in A. rancens SKU 1111
showed a sigmoidal saturation curve with acetyl-CoA. This finding suggested
that strong acetate oxidation caused by acetyl-CoA synthetase or
phosphotransacetylase activity, together with phosphoenolpyruvate
carboxylase, increased the biomass.
acetate oxidation; acetic acid bacteria; acetyl-CoA synthetase,
phosphoenolpyruvate carboxylase; vinegar fermentation

-11-
Synthesis of (±)-Methyl Tuberonate, a Potato Tuber-forming Substance,
and Its Epimer

Hiromasa KIYOTA,・ Daisuke NAKASHIMA, and Takayuki ORITANI

Laboratory of Applied Bioorganic Chemistry, Division of Life Science,
Graduate School of Agricultural Science,
Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai
981-8555, Japan

Received June 23, 1999; Accepted August 26, 1999
Methyl esters of (±)-tuberonic acid and (±)-12-hydroxyjasmonic acid (tran
s-tuberonic acid), the aglycons of strong potato tuber-forming substances,
were synthesized from norbornene via side-chain elongation and
Baeyer-Villiger oxidation as key steps.
methyl tuberonate; methyl 12-hydroxyjasmonate; jasmonic acid; potato
tuberization; total synthesis

-12-
Antioxidative Effects of Turmeric, Rosemary and Capsicum Extracts
on Membrane Phospholipid Peroxidation and Liver Lipid Metabolism in Mice

Akira ASAI,・ Kiyotaka NAKAGAWA, and Teruo MIYAZAWA

Laboratory of Biodynamic Chemistry, Tohoku University Graduate School of
Agriculture,
Sendai 981-8555, Japan

Received June 25, 1999; Accepted August 26, 1999
Phospholipid hydroperoxides (PLOOH) in the plasma, red blood cells (RBC)
and liver of mice were measured after dietary supplementation for one week
(1％ w/w of diet) with a turmeric extract (curcuminoid), hexane extract of r
osemary, and supercritical CO2-extracted capsicum pigment (supplemented
with α-tocopherol to prevent fading). A lower PLOOH level was found in RBC
of the spice extract-fed mice (65--74％ of the non-supplemented control mice
). The liver lipid peroxidizability induced with Fe2＋/ascorbic acid was eff
ectively suppressed by dietary supplementation with the turmeric and
capsicum extracts to mice. While no difference in the plasma lipids was
observed, the liver triacylglycerol concentration of the turmeric
extract-fed mice was markedly reduced to one-half of the level in the
control mice. These findings suggest that these spice extracts could act
antioxidatively in vivo by food supplementation, and that the turmeric
extract has the ability to prevent the deposition of triacylglycerols in
the liver.
antioxidant; spice; erythrocyte; triglyceride; curcuminoid

-13-
Local Antibody Response in Peyer′s Patches to the Orally Administered
Dietary Protein Antigen

Michiko SHIMODA, Yuri INOUE, Norihiro AZUMA, and Choemon KANNO

Department of Applied Biochemistry, Utsunomiya University, 350 Mine-machi,
Utsunomiya,
Tochigi 321-8505, Japan

Received June 28, 1999; Accepted August 23, 1999
To understand local antibody production to dietary protein antigens in the
gut, the reactivity of the monoclonal antibodies (mAbs) from Peyer′s patche
s of BALB/c mice raised against orally administered hen egg lysozyme (HEL)
was studied. These mAbs were of IgG1 (7 clones), IgA (5 clones) and IgM (13
clones) isotypes. Some of the HEL-binding mAbs preferentially reacted with
reduced, carboxy-methylated HEL, rather than with native HEL. MAbs of the
IgA and IgM isotypes had cross-reactivity with other unrelated
environmental antigens such as E. coli, single-strand DNA, and soluble
components of mouse food. In contrast, the IgG1 mAbs did not cross-react
with these antigens. The average of the Kd values for HEL of these mAbs was
in the order of 10−6 M, which is moderately higher than those of mAbs from
the preimmune repertoire. These results suggest that, under normal
physiological conditions, orally administered dietary proteins
predominantly induce the local production of polyreactive IgA/IgM
antibodies cross-reacting with environmental luminal antigens.
Peyer′s patches; polyreactive antibody; mucosal IgA response; lysozyme

-14-
A Fibrinolytic Metalloprotease from the Fruiting Bodies of an Edible
Mushroom,
Armillariella mellea

Jun-Ho KIM and Yang Sun KIM

Department of Chemistry, Sangji University, Wonju 220-702, Korea
＊College of General Education, Semyoung University, Jechon 390-230, Korea

A fibrinolytic metalloprotease has been purified from the fruiting bodies
of the edible honey mushroom (Armillariella mellea). The enzyme has a
molecular weight of 18538.1508, as measured by MALDI-TOF mass spectrometry
and includes Zn2＋ ion as found by ICP/MS. The N-terminal amino acid sequenc
e, XXYNGXTXSRQTTLV, do not match any known protein or open reading frame.
It hydrolyzes fibrinogen as well as fibrin, but does not show any
proteolytic activity for other blood proteins such as thrombin, human
albumin, bovine albumin, human IgG, hemoglobin, or urokinase. This protease
hydrolyzes both Aα and Bβ subunits of human fibrinogen with equal efficien
cy. The enzyme activity was strongly inhibited by EDTA and
1,10-phenanthroline, indicating that the enzyme is a metalloprotease. No
inhibition was found with PMSF, E-64, pepstatin, and 2-mercaptoethanol. The
activity of the purified enzyme was slightly increased by Mg2＋, Zn2＋, and
Co2＋, but the enzyme was totally inhibited by Hg2＋. It has broad substrate
specificity for synthetic peptides, and a pH optimum at 7, suggested that
the purified enzyme was a neutral protease. It was thermally stable up to
60°C and the maximum fibrinolytic activity was at 55°C.
metalloprotease; fruit body; Armillariella mellea; fibrinolytic activity

-15-
Crystallization and Properties of NADPH-Dependent L-Sorbose Reductase
from Gluconobacter melanogenus IFO 3294

Osao ADACHI,＃ Yoshitaka ANO, Duangtip MOONMANGMEE,・ Emiko SHINAGAWA,＊
Hirohide TOYAMA, Gunjana THEERAGOOL,＊＊ Napha LOTONG,＊＊ and Kazunobu MAT
SUSHITA

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi
University,
Yamaguchi 753-8515, Japan
＊Department of Chemical and Biological Engineering, Ube National College o
f Technology,
Tokiwadai, Ube 755-8555, Japan
＊＊Department of Microbiology, Faculty of Science, Kasetsart University, B
angkok 10900, Thailand

Received July 5, 1999; Accepted August 12, 1999
||NADPH-Dependent L-sorbose reductase (SORD, synoni||||mously
NADP-dependent D-sorbitol dehydrogenase) was||purified and crystallized for
the first time from the cytosolic fraction of Gluconobacter melanogenus IFO
3294. The enzyme catalyzed oxidoreduction between D-sorbitol and L-sorbose
in the presence of NADP or NADPH. Affinity chromatography by a Blue-dextran
Sepharose 4B column was effective for purifying the enzyme giving about
770-fold purification with an overall yield of more than 50％. The crystalli
ne enzyme showed a single sedimentation peak in analytical
ultracentrifugation, giving an apparent||sedimentation constant of 3.8 s.
Gel filtration on a Sepha||dex G-75 column gave the molecular mass of 60
kDa to the enzyme, which dissociated into 30 kDa subunit on SDS-PAGE,
indicating that the enzyme is composed of 2 identical subunits. Reduction
of L-sorbose to D-sorbitol predominated in the presence of NADPH with the
optimum pH of 5.0--7.0. Oxidation of D-sorbitol to L-sorbose was observed
in the presence of NADP at the optimum pH of 7.0--9.0. The relative rate of
L-sorbose reduction was more than seven times higher to that of D-sorbitol
oxidation. NAD and NADH were inert for both reactions. D-Fructose reduction
in the presence of NADPH did not occur with SORD. Since the reaction rate
in L-sorbose reduction highly predominated over D-sorbitol oxidation over a
wide pH range, the enzyme could be available for direct enzymatic
measurement of L-sorbose. Even in the presence of a large excess of
D-glucose and other substances, oxidation of NADPH to NADP was highly
specific and stoichiometric to the L-sorbosereduced. Judging from the
enzymatic properties, SORD would contribute to the intracellular
assimilation of L-sorbose incorporated from outside the cells where
L-sorbose is accumulated in huge amounts in the culture medium.
acetic acid bacteria; NADP-dependent D-sorbitol dehydrogenase;
Gluconobacter melanogenus; L-sorbose reductase

-16-
Improvement of the Physical Properties of Pepsin-Solubilized
Elastin-Collagen Film by Crosslinking

Koji TAKAHASHI,・ Yoshikadzu NAKATA, Kenji SOMEYA, and Makoto HATTORI

Department of Applied Biological Science, Faculty of Agriculture,
Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan

Received July 9, 1999; Accepted September 7, 1999
Pepsin-solubilized elastin (PSE)-conjugated collagen film was prepared
from a collagen matrix with PSE by drying it and crosslinking the
constituents with water-soluble carbodiimide or microbial transglutaminase
to improve the physical properties of the collagen film. The crosslinking
reduced the solubility and improved the thermal stability, the thermal
transition properties, and the elasticity of the control film in water. In
particular, water-soluble carbodiimide strongly influenced these
properties. The PSE-conjugated collagen film showed good permeation by
water-soluble tasting substances such as oligosaccharides and amino acids,
but poor permeation by polysaccharide, protein, and hydrophobic substances
such as retinol and cholesterol.
collagen; elastin; pepsin-solubilized elastin; water-soluble carbodiimide;
transglutaminase

-17-
Lipase-catalyzed Kinetic Resolution of (±)-trans- and cis-2-Azidocycloalka
nols

Ei′ichi AMI and Hiroshi OHRUI・

Division of Life Science, Graduate School of Agricultural Science, Tohoku
University,
Tsutsumidori-Amamiyamachi 1-1, Aoba-ku, Sendai, 981-8555, Japan

Received July 9, 1999; Accepted August 17, 1999
The lipase-catalyzed kinetic resolution of trans- and
cis-2-azidocycloalkanols and the preparation of enantiomerically pure
trans- and cis-2-aminocycloalkanols are described.
Four kinds of lipases were screened for the acetylation of trans- and
cis-2-azidocycloalkanols. Among them, Pseudomonas sp. lipases (lipase PS
and lipase AK, Amamo Pharmaceutical Co.) showed the highest
enantioselectivity. These products were converted to the corresponding
2-aminocycloalkanols to determine their enantiomeric excess (ee) and
absolute configurations by HPLC and CD analyses, using (S)-TBMB carboxylic
acid [(S)-2-tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid] as the
chiral conversion reagent. The results of the CD analysis proved
N,O-bis-(S)-TBMB carboxylated cis-2-aminocycloalkanols to adopt a
predominantly N-equatorial conformation.
The partially resolved trans- and cis-2-aminocycloalkanols, except for
trans-2-aminocyclopentanol, were recrystallized from ethyl acetate to give
enantiomerically pure forms.
lipase-catalyzed kinetic resolution; 2-azidocycloalkanols;
2-aminocycloalkanols; CD detection; (S)-TBMB carboxylic acid

-18-
Analysis of a Catalytic Acidic Pair in the Active Center of Cellulase
from Aspergillus aculeatus

Akira OHNISHI,1 Toshihiko OOI,1 Shinichi KINOSHITA,1,・ Hirotaka TOMATSURI,
2Kazuyuki UMEDA,2 Shunsaku UEDA,2 Yasuo HATA,3 and Motoo ARAI4

1Department of Molecular Chemistry, Graduate School of Engineering,
Hokkaido University, N-13, W-8,
Kita-ku, Sapporo 060-8628, Japan
2Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya
University,
Mine-machi, Utsunomiya 321-8505, Japan
3Division of Molecular Biology and Information, Institute for Chemical
Research, Kyoto University,
Gokasho, Uji, Kyoto 611-0011, Japan
4Faculty of Applied Biological Chemistry, College of Agriculture,
University of Osaka Prefecture,
Gakuen-cho, Sakai, Osaka 593-8531, Japan

Recieved July 13, 1999; Accepted September 3, 1999
Four acidic amino acid residues, Asp97, Asp101, Glu118, and Glu202, were
located in the cleft from the X-ray crystallographic analysis of FI-CMCase,
endo-1,4-β-glucanase (EC: 3.2.1.4) of Aspergillus aculeatus No. F-50.||To i
dentify the catalytic residues of the FI-CMCase, these||residues were
mutated to Glu or Ser from Asp97 and Asp101, and to Asp or Ser from Glu118
and Glu202 by site-directed mutagenesis, and totally 8 single mutant
enzymes experssed in Escherichia coli were prepared: D97E, D97S, D101E,
D101S, E118D, E118S, E202D, and E202S. Mutant enzymes E118S and E202S were
not shown to have any detectable activity. Kinetic parameters of other
mutant enzymes were measured after purification. The Km of mutant enzymes
were not much different from that of wild type FI-CMCase, while the Vmax of
mutant enzymes D97E, D97S, D101E, D101S, E118D, and D202E were much
decreased to 1/50, 1/20, 1/4000, 1/2000, 1/800, and 1/1600 of the wild type
FI-CMCase, respectively. From these results we concluded that Glu118 and
Glu202 were most probable candidates for a catalytic pair of acidic amino
acids in FI-CMCase.
site-directed mutagenesis; active site; cellulase; family 12

-19-
Dietary Effect of Guar Gum and its Partially Hydrolyzed Product
on the Lipid Metabolism and Immune Function of Sprague-Dawley Rats

Koji YAMADA,・ Yoko TOKUNAGA, Atsushi IKEDA, Ken-ichi OHKURA, Soichi MAMIYA
,＊
Shihoko KAKU,＊＊ Michihiro SUGANO,＊＊ and Hirofumi TACHIBANA

Laboratory of Food Chemistry, Department of Bioscience and Biotechnology,
Division of Bioresource
and Bioenvironmental Sciences, Graduate School of Kyushu University,
6-10-1 Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan
＊Department of Research and Development, Nutritional Foods Division, Taiyo
Kagaku Co. Ltd.,
Yokkaichi 510-0844, Japan
＊＊Department of Living Environment Science, Prefectural University of Kum
amoto,
Kumamoto 862-0901, Japan

Received July 15, 1999; Accepted August 26, 1999
The dietary effect of the water-soluble dietary fibers (WSDF), guar gum,
partially hydrolyzed guar gum (PHGG), glucomannan, highly methoxylated (HM)
pectin, on the serum lipid level and immunoglobulin (Ig) production of
Sprague-Dawley rats was compared with that of water-insoluble cellulose.
Although serum total cholesterol and triglyceride levels were significantly
lower in the rats fed with WSDF than in those fed with cellulose, a
decrease in the level of phospholipids was only observed in the rats that
had been fed on guar gum or glucomannan. In addition, all WSDF feeding
enhanced IgA productivity in the spleen and mesenteric lymph node
lymphocytes, although the increase in serum IgA level was only observed in
the rats fed on WSDF, and not on PHGG. When mesenteric lymph node
lymphocytes were cultured in the presence of various concentrations of guar
gum or glucomannan, no significant increase in Ig production was apparent.
These data suggest that WSDF indirectly enhanced the Ig production of
lymphocytes, and that serum lipid reduction and IgA production-enhancing
activities of WSDF were dependent on their molecular sizes.
water-soluble dietary fiber; lipid metabolism; IgA production; guar gum;
PHGG

-20-
Production of D-Glutamate from L-Glutamate with Glutamate Racemase
and L-Glutamate Oxidase

Tadao OIKAWA,1,2,・ Mayumi WATANABE,1 Hidemi MAKIURA,1 Hitoshi KUSAKABE,3
Kazuhiro YAMADE,1,2 and Kenji SODA1,2

1Department of Biotechnology, Faculty of Engineering, Kansai University;
2Kansai University High Technology Research Center, Suita-shi, Osaka
564-8680, Japan
3Yamasa Corporation, Choshi, Chiba 288-0056, Japan

Received July 23, 1999; Accepted September 7, 1999
We studied production of D-glutamate from L-glutamate using a bioreactor
consisting of two columns of sequentially connected immobilized glutamate
racemase (EC 5.1.1.3, from Bacillus subtilis IFO 3336) and L-glutamate
oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6): L-glutamate was
racemized by the glutamate racemase column, and then L-glutamate was
oxidized by the L-glutamate oxidase column. Consequently only D-glutamate
remained, and was easily separated from the α-ketoglutarate formed by anion
-exchange chromatography. Both enzymes were highly stabilized by
immobilization. The pH and temperature optima of immobilized glutamate racemase (pH 8, 40°C) were similar to those of immobilized L-glutamate oxidase
(pH 7, 50°C). Accordingly, we connected the two columns tandemly to do both
enzyme reactions under the same conditions. Actually 4.5 μmol of D-glutamat
e was produced and isolated from 10 μmol of L-glutamate, about 90％ of the
theoretical yield.
glutamate racemase; L-glutamate oxidase; D-glutamate production

-21-
Cloning and Sequence Analysis of the Gene for Glucodextranase
from Arthrobacter globiformis T-3044 and Expression in Escherichia coli
Cells

Tetsuya OGUMA, Toshiko KUROKAWA, Kouichiro TOBE,＊ Satoshi KITAO,＊
and Mikihiko KOBAYASHI＊＊

Noda Institute for Scientific Research, 399 Noda, Noda-Shi, Chiba
278-0037, Japan
＊Research and Development Division, Kikkoman Corporation, 399 Noda, Noda-S
hi, Chiba 278-0037, Japan
＊＊National Food Research Institute, Ministry of Agriculture, Forestry, an
d Fisheries, 2-1-2 Kannondai,
Tsukuba, Ibaraki 305-8642, Japan

Received July 27, 1999; Accepted September 13, 1999
The gld gene for glucodextranase from Arthrobacter globiformis T-3044 was
cloned by using a combination of gene walking and probe methods and
expressed on the recombinant plasmid pGD8, which was constructed with
pUC118, in Escherichia coli cells. The enzyme gene consisted of a unique
open reading frame of 3,153 bp. The comparison of the DNA sequence data
with the N-terminal and 6 internal amino acid sequences of the purified
enzyme secreted from A. globiformis T-3044 suggested the enzyme was
translated from mRNA as a secretory precursor with a signal peptide of 28
amino acids residues. The deduced amino acids sequence of the mature enzyme
contained 1,023 residues, resulting in a polypeptide with a molecular mass
of 107,475 daltons. The deduced sequence showed about 38％ identity to that
of the glucoamylase from Clostridium sp. G0005. The glucodextranase
activity of transformant harboring pGD8 was about 40 mU/ml at 30°C for a 16
-h culture. Although the GDase that was produced from the transformant was
shorter than authentic GDase by 2 amino acid residues at the N-terminal end
side, its enzymatic properties were almost same as the authentic one.
Two kinds of genes, dex1 and dex2, for endo-dextranases from A.
globiformis T-3044 were also cloned into Escherichia coli cells. The
N-terminal of the purified endo-dextranase from A. globiformis T-3044
agreed with the deduced amino acid sequence, after the 33rd alanine
residue, of only the dex1 gene for endo-dextranase. This result suggests
that the endo-dextranase is translated from mRNA as a secretory precursor
with a signal peptide of 32 amino acids residues. The deduced sequence of
endo-dextranase 1 and endo-dextranase 2 showed about 93％ and 65％ identity
with that of known endo-dextranase from Arthrobacter sp. CB-8, respectively.
glucodextranase; cloning; Arthrobacter; glucoamylase; Clostridium

-22-
Permeation of Hesperidin Glycosides Across Caco-2 Cell Monolayers Via
the Paracellular Pathway

Minji KIM, Takashi KOMETANI,＊ Shigetaka OKADA,＊ and Makoto SHIMUZU・

Department of Applied Biological Chemistry, Graduate School of
Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
＊Biochemical Research Laboratory, Ezaki Glico Co. Ltd., Utajima 4-6-5, Nis
hiyodogawa, Osaka 555-8502, Japan

Received July 27, 1999; Accepted August 26, 1999
The intestinal permeability to hesperidin glycosides was investigated by
using a cultured monolayer of Caco-2 as a model for the small intestinal
epithelium. Hesperidin glycosides were added to the apical side of the
monolayer, and the substances that permeated to the basolateral side were
determined by HPLC. Whereas hesperidin did not permeate across the Caco-2
monolayer, probably owing to its low solubility, the hesperidin glycosides
did permeate. The transepithelial transport of hesperidin glycosides
occurred in time- and dose-dependent manners. The transport was observed to
be energy-independent, and was inversely correlated with the
transepithelial electrical resistance (TEER) of the monolayer. These
results suggest that hesperidin glycosides permeate across the Caco-2 cell
monolayer via the paracellular pathway.
hesperidin; Caco-2; paracellular pathway; permeation

-23-
Functional Analysis of the DXDDTA Motif in Squalene-Hopene Cyclase
by Site-directed Mutagenesis Experiments: Initiation Site
of the Polycyclization Reaction and Stabilization Site
of the Carbocation Intermediate of the Initially Cyclized A-Ring

Tsutomu SATO and Tsutomu HOSHINO・

Department of Applied Biological Chemistry, Faculty of Agriculture, and
Graduate School of Science
and Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan

Received July 30, 1999; Accepted September 2, 1999
In order to clarify the function of the DXDDTA motif in squalene-hopene
cyclase and to identify the acidic amino acid residues crucial for the
catalysis, site-directed mutagenesis experiments were carried out. The
following results were found: (1) residues D374 and D376 work for the
initiation of polyolefin cyclization which arises from the proton attack on
the terminal double bond; (2) residue D377 stabilizes C-10 carbocation of
the initially cyclized A-ring intermediate, leading to subsequent B-ring
closure, which was further verified by isolating the partially cyclized
monocyclic product; (3) residues D313 and D447 outside the DXDDTA motif
were identified as new active sites; (4)the H451 residue is likely to work
in the protonated form to enhance the acidity of the carboxyl groups of
D374 and/or D376.
squalene; hopene; DXDDTA motif; Alicyclobacillus acidocaldarius;
oxidosqualene

-24-
Note
Subsite Structure of α-Amylase II from Thermoactinomyces vulgaris R-47

Yoichiro SHIMURA, Qiong WANG, and Yoshiyuki SAKANO・

Department of Applied Biological Science, Faculty of Agriculture, Tokyo
University of Agriculture
and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan

Reviced May 11, 1999; Accepted September 6, 1999
We estimated the subsite structure of α-amylase II (TVA II) from Thermoact
inomyces vulgaris R-47 expressed in Escherichia coli. TVA II has eight
subsites, and the catalytic site is between the 5th and 6th subsite from
the non-reducing end side. The subsite affinities, A−5, A−4, A−3, A−2, (
A−1＋A＋1), A＋2, and A＋3, were calculated to be −0.35, 0.93, 0.55, 2.56,
1.18, 1.71, and 0.01 kcal mol−1, respectively.
subsite structure; α-amylase; Thermoactinomyces vulgaris

-25-
Note
HIV-1 Protease Inhibition and Anti-HIV Effect of Natural
and Synthetic Water-soluble Lignin-like Substances

Toshiaki ICHIMURA,・ Toru OTAKE,1 Haruyo MORI,1 and Susumu MARUYAMA

National Institute of Bioscience and Human-Technology, Agency of
Industrial Science and Technology,
1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
1Osaka Prefectural Institute of Public Health, 1-3-69 Nakamichi,
Higashinari-ku, Osaka 537-0025, Japan

Received May 24, 1999; Accepted August 4, 1999
Water-soluble lignin extracted from natural sources and dehydrogenated
polymers of p-coumaric acid and ferulic acid inhibited HIV-1 protease
activity. The dehydrogenated polymers, which are thought to be model
compounds for lignin, were synthesized and fractionated into four ranges of
molecular mass by ultra-filtration: i.e., over 30 kDa, 30--10 kDa, 10--1
kDa and 1 kDa--500 Da. All of these fractions had HIV-1 protease inhibitory
activity. The anti-HIV-1 effect of the smallest mass fractions of the
dehydrogenated polymers (1 kDa--500 Da) was also tested, and it was found
that these fractions inhibited the replication of HIV-1 in MT-4 cells.
HIV-1 protease; lignin; MT-4 cells

-26-
High-level Expression of a Recombinant Thermostable Phytase in Bacillus
subtilis

Young-Ok KIM, Jung-Kee LEE, Byung-Chul OH, and Tae-Kwang OH・

Microbial enzyme RU, Korea Research Institute of Bioscience ＆ Biotechnolog
y (KRIBB), P.O. Box 115,
Yusong, Taejon 305-600, Korea

Received June 8, 1999; Accepted August 16, 1999
An efficient expression system was developed in Bacillus subtilis for the
large scale production of phytase. The phytase gene with a native promoter
derived from Bacillus amyloliquefaciens was cloned in the Bacillus
expression vector pJH27 under a strong BJ27 promoter and its expression was
optimized. The expression of the phytase gene occurred during late
exponential growth and the extracellular phytase production was 2.0
units/ml, which constituted over 90％ of the total protein. The yield was 10
0-fold higher than wild type Bacillus amyloliquefaciens DS11.
Bacillus subtilis; phytase; phytate; overexpression

-27-
Note
Competing Effect of Polyols on the Thermal Stability
and Gelation of Soy Protein

Kunihiko GEKKO,＊,・ Xuan LI, and Shio MAKINO

＊Department of Mathematical and Life Sciences, Graduate School of Science,
Hiroshima University,
Higashi-Hiroshima 739-8526, Japan

Department of Molecular Biosciences, Graduate School of Bioagricultural
Sciences, Nagoya University,
Nagoya, Aichi 464-8601, Japan
Received June 10, 1999; Accepted July 30, 1999
The thermal denaturation temperature of a soy protein isolate was
increased, but its gel-melting temperature was decreased by the addition of
polyols with increasing concentration and number of hydroxyl groups of the
polyols. This inverse stabilizing effect of polyols on the protein
structure and gel is discussed in terms of the competing solvent effects on
intra- and intermolecular hydrophobic interactions and on the
peptide-peptide hydrogen bonds of the protein.
soy protein; thermal stability; gelation; polyol effect

-28-
Note
Optical Resolution by the Replacing Crystallization of DL-Threonine
with L-Alanine as an Optically Active Cosolute

Tadashi SHIRAIWA,＊,・ Motoki KUBO, Keiji FUKUDA, and Hidemoto KUROKAWA

＊Chemical Branch, Faculty of Engineering and Kansai University High Techno
logy Research Center,
Kansai University, Yamate-cho, Suita, Osaka 564-8680, Japan

Received June 14, 1999; Accepted July 30, 1999
DL-Threonine (DL-Thr) was optically resolved by replacing crystallization
with L-alanine (L-Ala) as an optically active cosolute. D-Thr was
preferentially crystallized from a supersaturated aqueous solution of
DL-Thr in the presence of L-Ala. Optical resolution was successfully
achieved to afford D-Thr with an optical purity of 96--98％ and L-Thr of 91-
-95％. The partially resolved D- and L-Thr were recrystallized from water, t
aking account of the solubility of DL-Thr, to efficiently yield both
enantiomers in an optically pure form.
threonine; optical resolution; replacing crystallization; L-alanine

-29-
Note
Site-directed Mutagenesis of Two Zinc-binding Centers
of the NADH-dependent Phenylacetaldehyde Reductase
from Styrene-assimilating Corynebacterium sp. Strain ST-10

Jiu-Cun WANG, Mikio SAKAKIBARA, Michiko MATSUDA,＊ and Nobuya ITOH＊,・

Department of Applied Chemistry and Biotechnology, Faculty of Engineering,
Fukui University,
Bunkyo 3-9-1, Fukui 910-8507, Japan
＊Biotechnology Research Center, Toyama Prefectural University, Kurokawa 51
80, Kosugi,
Toyama 939-0398, Japan

Received June 18, 1999; Accepted August 16, 1999
Phenylacetaldehyde reductase (PAR) with a unique and wide substrate range
from styrene-assimilating Corynebacterium sp. strain ST-10, which is a
useful biocatalyst producing chiral alcohols, has been found to belong to a
family of zinc-containing, long-chain alcohol dehydrogenases (ADHs) on the
basis of the primary structure similarity. The enzyme contains 2 moles of
zinc per mole of subunit. The amino acid residues assumed to be three
catalytic and four structural zinc-binding ligands were characterized by
site-directed mutagenesis, compared with other zinc-containing, long-chain
ADHs. Sixteen PAR mutants gave measurable but rather low activities toward
phenylacetaldehyde, n-hexyl aldehyde, and 2-heptanone, although they
maintained the activities of 8 to 16％ of that of wild-type PAR for an aceto
phenone substrate except that the D153N mutant showed quite low activity.
The results suggested that the seven residues present in PAR were probably
zinc-binding ligands, and mutation in these residues caused a change in
activities for some substrates.
phenylacetaldehyde reductase; zinc-containing; long-chain alcohol
dehydrogenase; zinc-binding site; site-directed mutagenesis

-30-
Note
Activity of Glucose-6-phosphate 1-Dehydrogenase in Hair Follicles
with Male-pattern Alopecia

Kuniaki ADACHI,1 Yasusi WATANABE,2 and Kuniyo INOUYE3,・

1Research and Development Headquarters, Lion Corporation, Odawara,
Kanagawa 256-0811, Japan
2Watanabe Dermatological Clinic, Shinjuku-ku, Tokyo 160-0022, Japan
3Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto
University,
Sakyo-ku, Kyoto 606-8502, Japan

Received June 23, 1999; Accepted September 13, 1999
Activity of glucose-6-phosphate 1-dehydrogenase (G6PDH) in human hair
follicles was measured. A good relationship has been demonstrated between
the activity and the ratio of the number of the anagen hairs to that of all
the plucked hairs in the frontal-parietal region of the scalp with
male-pattern alopecia. As the ratio becomes lower so that the advancing
degree of alopecia is higher, the G6PDH activity becomes lower.
alopecia; glucose 6-phosphate 1-dehydrogenase; hair follicle; pentose
phosphate pathway

-31-
Note
Cloning and Expression in Escherichia coli of 2-Hydroxypropylphosphonic
Acid Epoxidase from the Fosfomycin-producing Organism,
Pseudomonas syringae PB-5123

Tomohisa KUZUYAMA, Takayuki SEKI, Seiji KOBAYASHI, Tomomi HIDAKA,＊
and Haruo SETO

Institute of Molecular and Cellular Biosciences, University of Tokyo,
Bunkyo-ku, Tokyo 113-0032, Japan
＊Center for Gene Research, Ehime University, Tarumi, Matsuyama, Ehime 790-
8566, Japan

Received June 23, 1999; Accepted August 11, 1999
The fosfomycin resistance gene, fosC, has been cloned from the
fosfomycin-producing organism, Pseudomonas syringae PB-5123. Sequence
analysis upstream of this gene found a new ORF showing significant homology
to 2-hydroxypropylphosphonic acid epoxidase from fosfomycin-producing
Streptomyces wedmorensis. The purified recombinant protein of this ORF
converted 2-hydroxypropylphosphonic acid to fosfomycin. This result clearly
showed the ORF to encode 2-hydroxypropylphosphonic acid epoxidase in
PB-5123.
fosfomycin; biosynthesis; Pseudomonas syringae; 2-hydroxypropylphosphonic
acid epoxidase

-32-
Note
Nucleotide Sequence of cDNA Coding the Mitochondrial Precursor Protein
of the ATPase Inhibitor from Humans

Naoki ICHIKAWA,・ Saori USHIDA, Miho KAWABATA, and Yukiko MASAZUMI

Department of Food and Nutrition, Faculty of Human Life Science, Osaka
City University,
3-138 Sugimoto, Sumiyoshiku, Osaka 558-8585, Japan

Received July 5, 1999; Accepted August 31, 1999
Mitochondrial ATP synthase (F1Fo-ATPase) is regulated by an intrinsic
ATPase inhibitor protein. In the present study, cDNA coding the human
homolog of the inhibitor protein was isolated and sequenced. The deduced
protein sequence shows that the protein was composed of 106 amino acids and
had a molecular weight of 12248. The structural features of the protein
show that the cDNA isolated in this study codes the human ATPase inhibitor.
ATPase inhibitor (IF1); ATP synthase; F1Fo-ATPase; mitochondria

-33-
Note
Analysis of mfbA Alleles of the Eubasidiomycete Lentinus edodes

Toru YASUDA・ and Kazuo SHISHIDO・・

Department of Life Science, Graduate School of Bioscience and
Biotechnology, Tokyo Institute of Technology,
Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

Received July 5, 1999; Accepted September 3, 1999
A fruiting-body-specific mfbA cDNA derived from Lentinus edodes FMC2 has
been shown to encode a high-molecular-weight protein, MFBA, containing the
cell-adhesion-promoting Arg-Gly-Asp (RGD) sequence. Southern-blot analysis
showed that all L. edodes strains tested have the mfbA gene (homologue).
Nucleotide sequence analysis of the 1-kb mfbA fragments containing the
RGD-coding sequence showed that each L. edodes strain has two types of mfbA
homologues. It was found in FMC2 that two mfbA homologues are derived from
different nuclei and these mfbA alleles are transcribed with similar
frequencies in the fruiting bodies.
allele; Arg-Gly-Asp (RGD) sequence; fruiting body; Lentinus edodes;
transcriptional expression

-34-
Note
Arginine-55 in the β-Arm is Essential for the Activity of DNA-Binding Prot
ein HU
from Bacillus stearothermophilus

Fumiyo SAITOH,1 Shunsuke KAWAMURA,1 Nobuyuki YAMASAKI,1
Isao TANAKA,2 and Makoto KIMURA1,・

1Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University,
Fukuoka 812-8581, Japan
2Division of Biological Science, Graduate School of Science, Hokkaido
University, Sapporo 060-0810, Japan

Received July 6, 1999; Accepted August 12, 1999
DNA-binding protein HU (BstHU) from Bacillus stearothermophilus is a
homodimeric protein which binds to DNA in a sequence-nonspecific manner. In
order to identify the Arg residues essential for DNA binding, four Arg
residues (Arg-53, Arg-55, Arg-58, and Arg-61) within the β-arm structure we
re replaced either by Gln, Lys, or Glu residues, and the resulting mutants
were characterized with respect to their DNA-binding activity by a
filter-binding analysis and surface plasmon resonance analysis. The results
indicate that three Arg residues (Arg-55, Arg-58, and Arg-61) play a
crucial role in DNA binding as positively charged recognition groups in the
order of Arg-55>Arg-58>Arg-61 and that these are required to decrease the
dissociation rate constant for BstHU-DNA interaction. In contrast, the
Arg-53 residue was found to make no contribution to the binding activity of
BstHU.
Bacillus stearothermophilus; DNA-binding protein HU; site-directed
mutagenesis; surface plasmon resonance

-35-
Note
Antibacterial Activities of Cryptotanshinone and Dihydrotanshinone I
from a Medicinal Herb, Salvia miltiorrhiza Bunge

Dong-Sun LEE,1,2,・ Sang-Han LEE,1 Jae-Geun NOH,3 and Soon-Duck HONG1

1Department of Microbiology, College of Natural Science, Kyungpook
National University, Taegu, 702-701, Korea
2Biophysical Chemistry Laboratory, The Institute of Physical and Chemical
Research (RIKEN),
RIKEN Harima Institute, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148
Japan
3Exotic Nano-material Laboratory, The Institute of Physical and Chemical
Research (RIKEN),
Wako-shi, Saitama 351-01, Japan

Received July 7, 1999; Accepted September 7, 1999
Cryptotanshinone and dihydrotanshinone I, constituents of a medicinal
plant, Salvia miltiorrhiza Bunge, had antibacterial activity against a
broad range of Gram positive bacteria. These compounds generated superoxide
radicals in Bacillus subtilis lysates. A recombination-deficient mutant
strain of B. subtilis was 2- to 8-fold more sensitive than a wild strain,
and this hypersensitivity was reduced in the presence of dithiothreitol as
an antioxidant. DNA, RNA, and protein syntheses in B. subtilis were
non-selectively inhibited by these compounds. These results suggest that
superoxide radicals are important in the antibacterial actions of the
agents.
cryptotanshinone; dihydrotanshinone I; antibacterial activity; superoxide
radical; Salvia miltiorrhiza Bunge

-36-
Note
Promotive Effects of the Peptidyl Plant Growth Factor, Phytosulfokine-α,
on the Growth and Chlorophyll Content of Arabidopsis Seedlings
under High Night-time Temperature Conditions

Seiyei YAMAKAWA,1 Yoshikatsu MATSUBAYASHI,2 Youji SAKAGAMI,2
Hiroshi KAMADA,1 and Shinobu SATOH1,・

1Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki
305-8572, Japan
2Department of Biological Sciences, Nagoya University, Chikusa, Nagoya
464-01, Japan

Received July 8, 1999; Accepted September 1, 1999
In order to investigate the function of the peptidyl plant growth factor,
phytosulfokine-α (PSK-α), in plants, we examined the effect of PSK-α on t
he growth and chlorophyll content of Arabidopsis seedlings under high
night-time temperature conditions. Although exposure to high night-time
temperatures markedly reduced the fresh weight and chlorophyll content of
the seedlings, these parameters in the plants supplied with PSK-α remained
at the same levels as those of non-treated controls. These effects were not
apparent when [2--5]PSK, Tyr-SO3H and kinetin were similarly supplied. The
results suggest thatPSK-α not only promotes cell proliferation, but may aid plants in their tol
erance of heat stress.
phytosulfokine; peptidyl growth factor; DIF; cucumber; chlorophyll
formation

-37-
Note
Cloning of L-Amino Acid Deaminase Gene from Proteus vulgaris

Eiji TAKAHASHI,・ Kiyoshi ITO,＊ and Tadashi YOSHIMOTO＊

Basic Technology Department, Discovery Research Laboratory, Tanabe Seiyaku
Co. Ltd.,
2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan
＊School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi
, Nagasaki 852-8521, Japan

Received July 30, 1999; Accepted August 30, 1999
The L-amino acid degrading enzyme gene from Proteus vulgaris was cloned
and the nucleotide sequence of the enzyme gene was clarified. An open
reading frame of 1,413 bp starting at an ATG methionine codon was found,
which encodes a protein of 471 amino acid residues, the calculated
molecular weight of which is 51,518. The amino acid sequence of P. vulgaris
was 58.6％ identical with the L-amino acid deaminase of P. mirabilis. A sign
ificantly conserved sequence was found around the FAD-binding sequence of
flavo-proteins. The partially purified wild and recombinant enzymes had the
same substrate specificity for L-amino acids to form the respective
keto-acids, however not for D-amino acids.
L-amino acid deaminase; gene cloning; Proteus vulgaris

-38-
Note
Synthesis and Characterization of Intermediate and Transition-State
Analogue Inhibitors of γ-Glutamyl Peptide Ligases

Makoto INOUE, Jun HIRATAKE,・ and Kanzo SAKATA

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011,
Japan

Received August 9, 1999; Accepted August 31, 1999
The phosphonodifluoromethyl ketone and phosphonofluoridate derivatives of
L-glutamic acid were synthesized and characterized as analogues of the γ-gl
utamyl phosphate intermediate and the tetrahedral transition state,
respectively, for the inhibition of γ-glutamylcysteine synthetase and gluta
mine synthetase. The former served as a poor inhibitor of both enzymes, but
the latter inhibited glutamine synthetase with a Ki of 59 μM and partially
inactivated the enzyme in an NH3- and ATP-dependent manner.
γ-glutamyl peptide ligase; intermediate analogue; transition-state analogu
e; phosphonodifluoromethyl ketone; phosphonofluoridate

-39-
Preliminary Communication
Interaction of Tea Catechins with Lipid Bilayers Investigated
with Liposome Systems

Toshihiko HASHIMOTO, Shigenori KUMAZAWA, Fumio NANJO,＊
Yukihiko HARA,＊ and Tsutomu NAKAYAMA

School of Food and Nutritional Sciences, University of Shizuoka, 52-1
Yada, Shizuoka 422-8526, Japan
＊Food Research Laboratories, Mitsui Norin Co., Ltd., 223-1 Miyahara, Fujie
da 426-0133, Japan

Received July 23, 1999; Accepted October 19, 1999
Interaction of tea catechins with lipid bilayers was investigated with
liposome systems, which enabled us to separate liposomes from the external
medium by centrifugation. We found that epicatechin gallate had the highest
affinity for||lipid bilayers, followed by epigallocatechin gallate,
epicate||||chin, and epigallocatechin. Epicatechin gallate and
epi||gallocatechin gallate in the surface of lipid bilayer perturbed the
membrane structure.
catechins; tea; EGCg; liposome; lipid bilayer

-40-
Preliminary Communication
Soybean Curd Refuse Alleviates Experimental Tumorigenesis in Rat Colon

Naoyuki AZUMA, Hitoshi SUDA, Hiroyuki IWASAKI, Rhyuhei KANAMOTO,
and Kimikazu IWAMI＊

Department of Biological Resource Chemistry, Kyoto Prefectural University,
Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan

Received August 9, 1999; Accepted October 13, 1999
Adult Fischer-344 rats which underwent administration of azoxymethane were
fed diets containing soybean curd refuse (SCR) or a high-molecular-weight
fraction of soy protein digest (HMF), or Hammarsten casein (CAS) as a
protein source over a period of 34 weeks. All the living rats of each group
at 22, 28 or 34 weeks were endoscopically inspected for tumor incidence in
the colon. SCR turned out to be comparable to HMF in anti-tumorigenicity,
or rather better than HMF.
soybean curd refuse; Fischer-344 rats; tumor incidence; per-anal
endoscopy; anti-tumorigenicity