(Vol.62 No.2 1998)
Antioxidative Activity of Carp Blood Plasma on Lipid Peroxidation
Changhu XUE,1 Guangli YU1, Takashi HIRATA,2 Morihiko SAKAGUCHI2 and Junji
TERAO3
Antioxidative Activities of Several Marine Polysaccharides
Evaluated in a Phosphatidylcholine-liposomal Suspension and Organic Solvents
Changhu XUE,1 Guangli YU,1 Takashi HIRATA,2 Junji TERAO,3 and Hong LIN1
Mass Production of Bacterial 2,6-Sialyltransferase and
Enzymatic Syntheses of Sialyloligosaccharides
Takeshi YAMAMOTO, Hideki NAGAE, Yasuhiro KAJIHARA, and Ichiro TERADA
Evidence for Structural Differences between the Two Highly
Homologous Actin-regulatory Proteins, Destrin and Cofilin
Kazunari ARIMA,{ Minako IMANAKA, Seigou OKUZONO, Yasuaki KAZUTA, and Susumu
KOTANI
Enzymatic Synthesis of a New Derivative of Thiamin, O--Glucosylthiamin
Kei UCHIDA and Yukio SUZUKI{
Biosynthesis of 2H-Labeled Phenylalanine by a New Methylotrophic
Mutant Brevibacterium methylicum
Oleg V. MOSIN,1 Dimitry A. SKLADNEV,2 and Vitalyi I. SHVETS1{
Correlation of Polar Lipid Composition with 16S rRNA Phylogeny
in Methanogens. Further Analysis of Lipid Component Parts
Yosuke KOGA, Hiroyuki MORII, Masayo AKAGAWA-MATSUSHITA,{ and Mami OHGA
Structure of Genes for Cecropin A and an Inducible Nuclear
Protein That Binds to the Promoter Region of the Genes from the Silkworm,
Bombyx mori
Yoshiaki YAMANO, Masahito MATSUMOTO, Kiyomi SASAHARA, Emi SAKAMOTO,
and Isao MORISHIMA
Alanine Racemase from an Acidophile, Acidiphilium organovorum:
Purification and Characterization
Teck Keong SEOW, Kenji INAGAKI,{ Takashi TAMURA, Kenji SODA,
and Hidehiko TANAKA
Interaction between Potato Starch and Sucrose-lipid Monoesters
Studied by Differential Scanning Calorimetry
Fumiko NAKAZAWA, Junko TAKAHASHI, and Masako TAKADA
Enzymatic Properties of a Ginkgo biloba Endo--N-acetylglucosaminidase
and N-Glycan Structures of Storage Glycoproteins in the Seeds{
Yoshinobu KIMURA,{{ Sayuri MATSUO, and Shigeaki TAKAGI
Continuous Hydrolysis of Pectate by Immobilized Endo-polygalacturonase
in a Continuously Stirred Tank Reactor
Ken-ichi IWASAKI, Masako INOUE, and Yasuhito MATSUBARA
A Cysteine-Dependent Serine Protease Associated with the
Dormant Spores of Bacillus cereus: Purification of the Protein and Cloning
of the Corresponding Gene
Ryuichi MORIYAMA,{ Kazuhiro SUGIMOTO, Haishuo ZHANG, Toshihiko INOUE,
and Shio MAKINO
Generation of Nitric Oxide from Spin-trapping Agents under
Oxidative Conditions
Kieko SAITO, Toyohiko ARIGA, and Hisashi YOSHIOKA
Purification and Characterization of NADPH-Dependent Carbonyl
Reductase, Involved in Stereoselective Reduction of Ethyl 4-Chloro-3-oxobutanoate,
from Candida magnoliae
Masaru WADA,{{ Michihiko KATAOKA, Hiroshi KAWABATA, Yoshihiko YASOHARA,
Noriyuki KIZAKI, Junzo HASEGAWA, and Sakayu SHIMIZU{
Identification of the Promoter Region and the Transcriptional
Regulatory Sequence of the evgAS Operon of Escherichia coli
Hiroyuki TANABE, Katsuhide YAMASAKI, Akinori KATOH, Sachiko YOSHIOKA, and
Ryutaro UTSUMI{
Solubilization and Chemical Characterization of an Insoluble
Matrix Protein in the Gastroliths of a Crayfish, Procambarus clarkii
Katsuaki ISHII,, Naoaki TSUTSUI, Toshiki WATANABE, Tadashi YANAGISAWA,
and Hiromichi NAGASAWA,{
Non-thermal Effect of a Ceramics Radiation on a Yeast
Glucose-6-phosphate dehydrogenase
Masahiro KOHASHI,{ Yuka OHTA, and Tatsuo WATANABE
Purification and Properties of an Amylopullulanase, a
Glucoamylase, and an -Glucosidase in the Amylolytic Enzyme System of
Thermoanaerobacterium thermosaccharolyticum
Dirk GANGHOFNER,{ Josef KELLERMANN,1 Walter L. STAUDENBAUER,{{ and Karin
BRONNENMEIER
Overproduction of 1,2--Mannosidase, a Glycochain Processing
Enzyme, by Aspergillus oryzae
Takashi YOSHIDA,1 Tasuku NAKAJIMA, and Eiji ICHISHIMA
Reptile Lysozyme: The Complete Amino Acid Sequence of
Soft-Shelled Turtle Lysozyme and Its Activity
Tomohiro ARAKI,{ Takaki YAMAMOTO, and Takao TORIKATA
Diisopropylfluorophosphate (DFP) Inhibits Ricin-induced
Apoptosis of MDCK Cells
Tatsuya ODA, Nobukazu KOMATSU, and Tsuyoshi MURAMATSU
Trehalose as osmoprotectant in Rhodobacter sphaeroides
f. sp. denitrificans IL106
Xiaoyuan XU, Mitsuru ABO, Akira OKUBO, and Sunao YAMAZAKI
Two Peaks in pH Dependence of Renin-angiotensinogen Reaction
Uddin Mohammad NASIR, Kohji TAKAHASHI, Takao NAGAI, Tsutomu NAKAGAWA,
Fumiaki SUZUKI, and Yukio NAKAMURA,{
Influence of Phytate Removal and Structural Modification
on the Calcium-binding Properties of Soybean Globulins
Hitomi KUMAGAI,{ Yoshiharu SHIZAWA, Hidetoshi SAKURAI, and Hitoshi KUMAGAI
Molecular Cloning and Expression of a Gene Encoding a
Novel Sorbitol Oxidase from Streptomyces sp. H-7775
Kazumi HIRAGA, Takashi ETO, Issei YOSHIOKA, and Kohei ODA1
Molecular Properties and Activity of Amino-Terminal Truncated
Forms of Lipase Activator Protein
Hiroyuki SHIBATA, Hiroaki KATO, and Junichi ODA{
Note
Cloning Genomic DNA Encoding Apple Polyphenol Oxidase and Comparison of
the Gene Product in Escherichia coli and in Apple
Miyoshi HARUTA, Masatsune MURATA,,{ Ayami HIRAIDE,,{{ Hiroshi
KADOKURA, Makari YAMASAKI, Masaaki SAKUTA, Seki SHIMIZU,
and Seiichi HOMMA
Note
6-Methylsulfinylhexyl Isothiocyanate and Its Homologues as Food-originated
Compounds with Antibacterial Activity against Escherichia coli and Staphylococcus
aureus
Haruhiro ONO, Shoko TESAKI, Soichi TANABE, and Michiko WATANABE
Note
Effects of Germinated Barley Foodstuff in Preventing Diarrhea and Forming
Normal Feces in Ceco-colectomized Rats
Osamu KANAUCHI,{ Tomohiko NAKAMURA, Kazue AGATA, Tohru FUSHIKI, and
Hiroshi HARA
Note
Protein Disulfide Isomerase Activity of Some Plant Seeds
Ken KAINUMA, Tetsuya OOKURA, Akiko OKAMOTO, Kazumi KITTA, Mariko
MANABE, and Yukio KAWAMURA
Note
Overproduction and Substrate Specificity of 3-Isopropylmalate Dehydrogenase
from Thiobacillus ferrooxidans
Hideyuki MATSUNAMI, Hiroshi KAWAGUCHI, Kenji INAGAKI,{ Tadashi EGUCHI,
Katsumi KAKINUMA, and Hidehiko TANAKA
Note
Lack of Light/Dark Regulation of Enzymes Involved in the Photosynthetic
Carbon Reduction Cycle in Cyanobacteria, Synechococcus PCC 7942 and Synechocystis
PCC 6803
Masahiro TAMOI, Akiko MURAKAMI, Toru TAKEDA, and Shigeru SHIGEOKA{
Note
Detection of Antitumor Promoting Activity in Raji Cells Carrying Epstein-Barr
Virus Genome by Immunoblotting Analysis
Akira KONDO, Tetsuhiro MORIMOTO, and Katsuichiro OKAZAKI{
Note
Detection of Potato Virus Y P1 Protein in Infected Cells and Analysis of
Its Cleavage Site
Li Jun YANG, Makoto HIDAKA,{ Haruhiko MASAKI, and Takeshi UOZUMI
Note
Identification of the Aspartic Acid Residue Located at or near Substrate-binding
Site of Rye Seed Chitinase-c{
Takeshi YAMAGAMI and Gunki FUNATSU
Note
Complete Amino Acid Sequence of Chitinase-a from Bulbs of Gladiolus (Gladiolus
gandavensis)
Takeshi YAMAGAMI, Yoichiro MINE, and Masatsune ISHIGURO
Laboratory of Protein Chemistry Engineering, Faculty of Agriculture,
Kyushu
Key words: chitinase; plant bulb; gladiolus; amino acid sequence
Note
Multidrug Resistance Phenotype Conferred by Overexpressing bfr2{/pad1{/sks1{
or pap1{ Genes and Mediated by bfr1{ Gene Product, a Structural and Functional
Homologue of P-Glycoprotein in Schizosaccharomyces pombe
Manabu ARIOKA, Mutsuo KOUHASHI, Koji YODA, Akira TAKATSUKI, Makari
YAMASAKI, and Katsuhiko KITAMOTO
Note
Extracellular Dextran-induced p-Nitrophenyl--D-glucoside-hydrolyzing
Enzyme of Bacillus circulans KA-304: A Producer of Schizophyllum commune-lytic
Enzyme
Katsushige MIZUNO and Takashi TACHIKI{
Note
Syntheses of Highly Oxygen-functionalized Derivatives of Dihydrodihydroxyphthalic
Acid in Enantiomerically Enriched Forms
Hajime IKEDA, Takeshi SUGAI,{ Hiroyuki HOSOMI, Shigeru OHBA, and Hiromichi
OHTA
Note
Binding of the Protein from Thermus aquaticus ISLtaq1 to Its Inverted Repeat
in Vitro
Hiroyuki TANABE, Yoshihiko KAKUTA, Sachiko YOSHIOKA, and Ryutaro UTSUMI{
Note
Purification and Properties of Three -N-Acetylglucosaminidases from Lactobacillus
casei ATCC 27092
Mio SENBA, Nobuhiro KASHIGE, Fumio MIAKE, and Kenji WATANABE
Rapid paper
Overexpression of Squalene-Hopene Cyclase by the pET Vector in Escherichia
Coli and First Identification of Tryptophan and Aspartic Acid Residues
inside the QW Motif as Active Sites
Tsutomu SATO,1 Yoshinori KANAI,2 and Tsutomu HOSHINO1,2{
Rapid paper
A New Peptide-N4-(acetyl--glucosaminyl)asparagine Amidase from Soybean
(Glycine max) Seeds: Purification and Substrate Specificity{
Yoshinobu KIMURA{{ and Akira OHNO
-1-
Effects of Lactic Acid Bacteria on Binding and Absorption
of Mutagenic Heterocyclic Amines
Masaki TERAHARA, Sachiko MEGURO, and Tsutomu KANEKO
Central Research Institute, Meiji Milk Products Co., Ltd., 1-21-3 Sakae-Cho, Higashimurayama, Tokyo 189, Japan
Received January 23, 1997
Effects of binding heterocyclic amines to cells of lactic acid bacteria
on theirs absorption were investigated. Cells of Lactobacillus delbrueckii
subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 bind both 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole
(Trp-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). The
binding of strain 1131 cells to Trp-P-1 was maximum in the pHs from 4 to
8, but strain 2038 cells bound Trp-P-1 and MeIQx only slightly at pH 7.
We investigated the absorption of heterocyclic amines by the small intestine
of F344 rats in the presence of these bacterial cells, using an in situ
loop technique. The absorption of Trp-P-1 by the small intestine was significantly
lower in the presence of strain 1131 cells than in the absence of the cells,
but the presence of strain 2038 cells had no effect on Trp-P-1 absorption.
Perhaps strain 1131 cells bind to Trp-P-1 at the same pH as that of the
small intestine (pH 6-7) and thus decrease its absorption.
Key words: Lactobacillus delbrueckii subsp. bulgaricus 2038; Streptococcus
thermophilus 1131; small intestine
-2-
Antioxidative Activity of Carp Blood Plasma on Lipid Peroxidation
Changhu XUE,1 Guangli YU1, Takashi HIRATA,2 Morihiko SAKAGUCHI2 and Junji TERAO3
O{1}Department of Food Science and Technology, Faculty of Fisheries,
Ocean University of Qingdao, China
O{2}Department of Fisheries, Faculty of Agriculture, Kyoto University,
Japan
O{3}National Food Research Institute, Ministry of Agriculture, Forestry,
and Fisheries, Japan
Received March 27, 1997
Antioxidative activity of carp blood plasma was estimated by measuring
hydroperoxides formed by liposome peroxidation during the exposure of liposomes
to AAPH. Ascorbic acid of high concentration, uric acid of low content,
and tocopherol formed special protective system against lipid poroxidation
in fish plasma. The decrease of uric acid, ascorbic acid, and tocopherol
showed synergism of ascorbic acid and tocopherol, uric acid, and tocopherol.
Carp blood plasma with a low concentration of protein (about 2) and SH
groups (88 M) had a great effect on the antioxidative activity, as the
effects of ascorbic acid, uric acid, and tocopherol were dramatically extended.
Dialysed carp protein also displayed a very strong antioxidative activity
on lipid peroxidation of a multilayer liposome system.
Key words: antioxidative activity; carp blood plasma; tocopherol: ascorbic
acid; uric acid
-3-
Antioxidative Activities of Several Marine Polysaccharides
Evaluated in a Phosphatidylcholine-liposomal Suspension and Organic Solvents
Changhu XUE,1 Guangli YU,1 Takashi HIRATA,2 Junji TERAO,3 and Hong LIN1
1Department of Food Science and Technology, Faculty of Fisheries, Ocean
University of Qingdao, China
2Department of Fisheries, Faculty of Agriculture, Kyoto University, Japan
3National Food Research Institute, Ministry of Agriculture, Forestry and
Fisheries, Japan
Received June 18, 1997
The antioxidative activities of several water-soluble marine polysaccharides,
alginate (ALG), alginate sulfate (SALG), propylene glucolalginate sodium
sulfate (PSS), propylene glucol mannuronate sulfate (PGMS), the oligosaccharide
of chitosan (OLC), N,O-carboxymethyl chitosan (NOCC) and hydroxypropylated
chitosan (HPC), were examined in a phosphatidylcholine (PC)-liposomal suspension
containing the water-soluble radical emitter, 2,2-azobis (2-amidinopropane)
dihydrochloride. In the suspensions containing OLC and SALG, the initial
rates of PC-OOH accumulation were 2.78~10|8 Ms|1 and 2.88~10|8 Ms|1,
respectively, while all the polysaccharides tested showed antioxidative
activity.
@Liposoluble marine polysaccharides, hexanoyl chitin (HCH) and an N-benzoylhexanoyl
chitosan (NBHC) solution, also retarded the hydroperoxide accumulation
of methyl linoleate by effectively trapping peroxide radicals in organic
solvents when the radical chain reaction had been initiated by 2,2-azobis
(2,4-dimethylvaleronitrile).
@The kinetic data presented indicate that the alginate and chitin derivatives
can be expected to play a role in the antioxidative mechanism of biological
systems.
Key words: antioxidant; liposome; polysaccharide; alginate; chitosan
-4-
Mass Production of Bacterial 2,6-Sialyltransferase and
Enzymatic Syntheses of Sialyloligosaccharides
Takeshi YAMAMOTO, Hideki NAGAE, Yasuhiro KAJIHARA, and Ichiro TERADA
Sea Water Science Research Laboratory, Japan Tobacco Inc., 4-13-20,
Sakawa, Odawara, Kanagawa 256, Japan
Department of System Function, Faculty of Science, Yokohama City University,
22-2 Seto, Kanazawa-ku, Yokohama 236, Japan
Received June 25, 1997
To supply 2,6-sialyltransferase for the large-scale synthesis of sialoside,
we investigated culture conditions for the production of sialyltransferase
0160.
@The addition of galactose and beef extract, and control of the pH of
the culture medium were effective on the production of sialyltransferase
0160. The maximal enzyme productivity reached 550 units/L.
@Using a crude extract of Photobacterium damsela JT0160 cells as an enzyme
source, enzymatic syntheses were performed with mono- and di-saccharides
as the sialyl acceptors. It was clarified that a crude extract of P. damsela
JT0160 cells can be used as an synthetic catalyst for the enzymatic synthesis
of sialyloligosaccharides. Furthermore, the enzyme assay showed that sialyltransferase
0160 could transfer NeuAc to not only N-linked but also O-linked carbohydrate
chains.
@These results indicated that an abundant supply of sialyltransferase
0160 and its broad specificity make possible the synthesis of sialoside
on a large scale.
Key words: bacterial 2,6-sialyltransferase; mass production; enzymatic
synthesis; Photobacterium damsela
-5-
Evidence for Structural Differences between the Two Highly
Homologous Actin-regulatory Proteins, Destrin and Cofilin
Kazunari ARIMA,{ Minako IMANAKA, Seigou OKUZONO, Yasuaki KAZUTA, and Susumu KOTANI
Department of Biochemical Engineering and Science, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka-shi, Fukuoka 820-8502, Japan
Received June 26, 1997
The amino acid sequences of destrin and cofilin are very similar (84
homology) throughout the entire range of proteins, but they have different
functions. In this study, we constructed a new cofilin expression plasmid,
which had high expression frequency, and the structures of destrin and
cofilin were analyzed by limited proteolysis and circular dichroism (CD).
When destrin was digested by trypsin, two fragments of 17.0 kDa and 9.2
kDa were obtained, whereas only one 8.4 kDa fragment was obtained from
cofilin. In spite of the overall sequence homology, an N-terminal amino
acid sequence analyses of the fragments revealed the cleavage sites on
destrin and cofilin to be different. These results suggest that destrin
and cofilin differ in their overall tertiary folds. Cofilin showed activity
similar to destrin at high pH values, although no pH-dependent structural
change in cofilin was confirmed by using limited proteolysis and CD.
Key words: destrin; cofilin; actin-regulatory protein
-6-
Enzymatic Synthesis of a New Derivative of Thiamin, O--Glucosylthiamin
Kei UCHIDA and Yukio SUZUKI{
Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710, Japan
Received June 30, 1997
A new transglucosylated derivative of thiamin could be synthesized by the
actions of cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus
and glucoamylase from Rhizopus sp., in this order, on a mixture of dextrin
and thiamin. The derivative was isolated in crystalline form and identified
as 5-O-(-D-glucopyranosyl)thiamin by spectroscopy (FAB-MS, UV, 1H-NMR,
and 13C-NMR), thiochrome formation with K3 [Fe(CN)6]-NaOH reagent, and
the hydrolysis products by - and -glucosidases. O--Glucosylthiamin
was odorless and mildly sweet with no tongue-pricking taste, and was more
stable than thiamin hydrochloride in aqueous solutions at pHs 7.0 and 9.3.
Key words: 5-O-(-D-glucopyranosyl)thiamin; O--glucosylthiamin; thiamin
-glucoside; Bacillus stearothermophilus cyclomaltodextrin glucanotransferase
-7-
Biosynthesis of 2H-Labeled Phenylalanine by a New Methylotrophic
Mutant Brevibacterium methylicum
Oleg V. MOSIN,1 Dimitry A. SKLADNEV,2 and Vitalyi I. SHVETS1{
1Department of Biotechnology, M.V. Lomonosov State Academy of Fine Chemical
Technology, Vernadskogo Prospekt 86, 117571, Moscow, Russian Federation
2Laboratory of Genetics of Methylotrophs, Russian State Scientific Centre
for Genetics and Selection of Industrial Microorganisms GNIIGENETIKA, 1-st
Dorozhnij Proezd., 1, 113575, Moscow, Russian Federation
Received July 3, 1997
The biosynthesis of 2H-labeled phenylalanine was done by converse of low
molecular weight substrates ([U-2H]methanol and 2H2O) in a new RuMP facultative
methylotrophic mutant Brevibacterium methylicum. To make the process work,
adapted cells with improved growth characteristics were used on minimal
medium M9 with the maximum content of 2H-labeled substrates. Alanine, valine,
and leucine/isoleucine were produced and accumulated exogeneously in addition
to the main product of biosynthesis. Electron impact mass spectrometry
of methyl esters of the N-Dns-amino acid mixture obtained after the chemical
derivatization of growth medium with dansyl chloride and diazomethane,
was done to calculate the deuterium enrichment of the amino acids synthesized.
The experimental data testified to the character of labeling of amino acid
molecules as heterogeneous; however, high levels of deuterium enrichment
were detected in all presented molecules-for phenylalanine the enrichment
was six, leucine/isoleucine-five, valine-five, and alanine-three deuterium
atoms.
Key words: heavy water; 2H-labeled phenylalanine; biosynthesis; Brevibacterium
methylicum; electron impact mass spectrometry
-8-
Correlation of Polar Lipid Composition with 16S rRNA Phylogeny
in Methanogens. Further Analysis of Lipid Component Parts
Yosuke KOGA, Hiroyuki MORII, Masayo AKAGAWA-MATSUSHITA,{ and Mami OHGA
Department of Chemistry, University of Occupational and Environmental Health, Iseigaoka, Yahatanishi-ku, Kitakyushu, 807, Japan
Received July 4, 1997
Qualitative analyses of lipid component parts (core lipids, phospholipid-polar
head groups, and glycolipid-sugar moieties) without separation of individual
lipids were done for further 14 strains of methanogens. The results confirmed
the conclusion of our previous paper (System. Appl. Microbiol. 16, 342
1993) that the distribution of lipid component parts was characteristic
to taxonomic groups of methanogens at a family or genus level. Our previous
and present analyses of lipid component part distribution of methanogens
supported the division of the order Methanomicrobiales into two new orders
Methanomicrobiales and ``Methanosarcinales proposed by Boone et al.
based on 16S rRNA analyses (Methanogenesis: Ecology, Physiology, Biochemistry,
Genetics, 1993, pp 35--80). The whole results also phenotypically supported
the establishment of new families ``Methanocaldococcaceae and ``Methanosaetaceae
and new genera ``Methanothermococcus, ``Methanocaldococcus, ``Methanoignis,
and ``Methanosalsus proposed by Boone et al.
Key words: methanogen; taxonomy; core lipid; phospholipid; glycolipid
-9-
Structure of Genes for Cecropin A and an Inducible Nuclear
Protein That Binds to the Promoter Region of the Genes from the Silkworm,
Bombyx mori
Yoshiaki YAMANO, Masahito MATSUMOTO, Kiyomi SASAHARA, Emi SAKAMOTO, and Isao MORISHIMA
Laboratory of Metabolic Biochemistry, Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University, Tottori 680, Japan
Received July 22, 1997
Cecropins are a family of antibacterial peptide synthesized in insects
as a response to bacterial infection. To study the regulation of the immune
genes in insects, two cecropin A genes were cloned and sequenced from the
silkworm, Bombyx mori. The two genes, CecA1 and CecA2, encoded identical
preprocecropin A, having one intron of 609 bp and 929 bp, respectively.
The 5-upstream regions of the genes contained a NF-B like element and
IL-6-RE Type I element. Electrophoretic mobility shift assay revealed that
a nuclear protein of fat body which specifically bound to the B-like
element was activated by injection of the larvae with peptidoglycan.
Key words: cecropin A gene; silkworm; insect immunity; peptidoglycan; NF-B
-10-
Alanine Racemase from an Acidophile, Acidiphilium organovorum:
Purification and Characterization
Teck Keong SEOW, Kenji INAGAKI,{ Takashi TAMURA, Kenji SODA, and Hidehiko TANAKA
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama
University, 1-1-1 Tsushima-Naka, Okayama-Shi, Okayama 700, Japan
Department of Biotechnology, Faculty of Engineering, Kansai University,
3-3-35 Yamate-Cho, Suita-Shi, Osaka 564, Japan
Received July 22, 1997
An alanine racemase (EC 5.1.1.1) from an acidophilic heterotrophic bacterium,
Acidiphilium organovorum 13H, was purified and characterized. The enzyme
had a dimeric structure with identical subunits of Mr 33,000 each. Although
A. organovorum 13H is an acidophile, the enzyme had its maximum velocity
at pH 9, corresponding to its location in the cytoplasm. Activity was maximum
between 50 and 60C. For an enzyme from a mesophile, it was stable to
heat, showing no loss of activity after a 30-min incubation at 65C. The
enzyme needed pyridoxal 5-phosphate (PLP) as a cofactor for its activity,
as seen from the loss of activity upon dialysis against PLP-free buffer
containing hydroxylamine and its absorption maximum at 420 nm. Activity
was ihhibited by common inhibitors of PLP-dependent enzymes. PLP content
studies found that 1 mole of enzyme contained 2 moles of PLP. The enzyme
catalyzed the symmetric reversible racemization of alanine exclusively.
Key words: alanine racemase; acidophile; Acidiphilium organovorum; pyridoxal
5-phosphate
-11-
Interaction between Potato Starch and Sucrose-lipid Monoesters
Studied by Differential Scanning Calorimetry
Fumiko NAKAZAWA, Junko TAKAHASHI, and Masako TAKADA
Physics Laboratory, Faculty of Home Economics, Kyoritsu Womens University,
1-710 Motohachioji, Hachioji, Tokyo 193, Japan
Department of Preschool Education, St. Cecilia Womens Junior College,
2-6-11 Rinkan, Daiwa, Kanagawa 242, Japan
Received July 25, 1997
The formation and dissociation of complexes composed of potato starch and
sucrose-lipid monoesters (SE: monocaprate, monolaurate, monomyristate,
monopalmitate, and monostearate) were studied by differential scanning
calorimetry (DSC).
@The formation and dissociation temperatures of each complex increased
as the number of carbon atoms in the alkyl chain of SE increased, and as
the content of starch increased, overlapping with the gelatinization temperature.
Therefore, the DSC curves for starch gelatinization differed according
to the added SE and water content. The completion temperature for the dissociation
of each starch-SE complex depended on the water content, similar to that
for the gelatinization of starch.
@The heat of fusion of the starch-SE (monopalmitate) complex obtained
from the completion temperature was nearly twice that of the original starch,
140 kJ/mol glucose unit.
@It is suggested that a stable conformation of each complex was not formed
during the gelatinization of the starch granules, but during cooling from
a temperature higher than the dissociation temperature of the complex which
had formed during the gelatinization process.
Key words: starch; sucrose-lipid monoester; starch complex; heat of fusion;
differential scanning calorimetry
-12-
Enzymatic Properties of a Ginkgo biloba Endo--N-acetylglucosaminidase
and N-Glycan Structures of Storage Glycoproteins in the Seeds{
Yoshinobu KIMURA,{{ Sayuri MATSUO, and Shigeaki TAKAGI
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-Naka 1-1-1, Okayama 700, Japan
Received July 29, 1997
An endo--N-acetylglucosaminidase has been purified to homogeneity from
mature seeds of Ginkgo biloba. The molecular mass of the endo--N-acetylglucosaminidase,
named Endo-GB, was estimated to be around 63 kDa by SDS-PAGE and around
62 kDa by Hiprep S-200 chromatography, respectively. The substrate specificity
has been explored with regard to the pyridylaminated N-glycans. Several
high mannose-type sugar chains bearing -1,2-mannosyl residue(s), Man9--6GlcNAc2-PA,
were the most favored substrates followed by Man5GlcNAc2-PA and a typical
hybrid-type structure (GlcNAc1Man5GlcNAc2-PA) which does not bear an -1,2-mannosyl
residue. On the contrary, endo-GB could hardly hydrolyze the common core
pentasaccharide of N-glycan (Man3GlcNAc2-PA) and the xylose-containing
sugar chains (Man4--3Xyl1GlcNAc2-PA, Man3Fuc1Xyl1GlcNAc2-PA) being widely
distributed in plant glycoproteins. Furthermore, we analyzed the structures
of N-glycans conjugated to storage glycoproteins in the mature Ginkgo seeds
to see the occurrence of endogenous substrates for Endo-GB. The structural
analysis showed, however, only xylose-containing type N-glycans (Man3Fuc1Xyl1GlcNAc2
(95) and Man3Xyl1GlcNAc2 (5)), which can not be substrate for Endo-GB,
predominantly occur in the storage glycoproteins.
Key words: endo--N-acetylglucosaminidase; N-glycan releasing enzyme;
N-glycan structure; plant glycoprotein; Ginkgo biloba
-13-
Continuous Hydrolysis of Pectate by Immobilized Endo-polygalacturonase
in a Continuously Stirred Tank Reactor
Ken-ichi IWASAKI, Masako INOUE, and Yasuhito MATSUBARA
Food Research Institute, Kagawa Prefectural Government, Goto, Takamatsu,
Kagawa 761, Japan
Kagawa Prefectural Fermentation and Food Experimental Station, Uchinomi,
Kagawa 761-44, Japan
Received July 31, 1997
Enzymatic hydrolysis of pectate was carried out continuously to produce
pectate oligosaccharides by immobilized endo-polygalacturonase in a continuous
stirred tank reactor (CSTR) with high efficiency. The enzyme was immobilized
on to chitosan beads by the absorption method, and the reaction was performed
with an initial pectate concentration of 10 gl|1 at 35C and pH 4.0 at
a dilution rate of 0.87--2.8 h|1. The hydrolysis products mainly consisted
of mono-, di-, tri-, tetra-, penta-, hexa- and heptasaccharides, with the
highest conversion being 0.78. A higher volumetric production rate of the
total hydrolyzate, which was dependent on the dilution rate, was obtained
than that by a batch reaction. The hydrolysis process was mathematically
modeled from the basic material balance and rate equations, and showed
agreement between the simulated and experimental results. This reactor
system was found to be effective for obtaining pectate oligosaccharides
with a high production rate.
Key words: continuous hydrolysis; pectin; polygalacturonase; oligosaccharide;
continuous stirred tank reactor (CSTR)
-14-
A Cysteine-Dependent Serine Protease Associated with the
Dormant Spores of Bacillus cereus: Purification of the Protein and Cloning
of the Corresponding Gene
Ryuichi MORIYAMA,{ Kazuhiro SUGIMOTO, Haishuo ZHANG, Toshihiko INOUE, and Shio MAKINO
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan
Received July 31, 1997
@Subtilisin-like serine protease, which is associated with the dormant
spores of Bacillus cereus, was solubilized by washing the spores with 2
M KCl and purified to homogeneity by carbobenzoxy-D-phenylalanine-liganded
affinity column chromatography and hydrophobic interaction column chromatography.
Enzyme activity was completely inhibited by reagents for sulfhydryl groups
such as HgCl2 as well as by conventional subtilisin inhibitors, suggesting
the enzyme to be cysteine-dependent. The enzyme retained activity in 5
M urea at 4C for at least 2 months, and the specific activity was 50
times that of subtilisin BPN when measured for a common chromogenic substrate,
carbobenzoxy-glycyl-glycyl-L-leucine p-nitroanilide. The gene encoding
this protease was cloned in Escherichia coli, and its nucleotide sequence
was analyzed. The deduced amino acid sequence suggested that the protease
is produced as a precursor comprising three portions; a signal sequence
(28 amino acid residues), a prosequence (80 amino acid residues) and a
mature enzyme (289 amino acid residues). The mature region of the enzyme
had high similarity with a thermitase from Thermoactinomyces vulgaris (72
identity) and a thermostable alkaline protease from Thermoactinomyces sp.
E79 (66 identity), which have the N-terminal sequence showing scarcely
noticeable similarity with corresponding stretches of subtilisins and mercuric
ion-sensitive free cysteine in the equivalent position of the primary structure.
Key words: Bacillus cereus; bacterial spores; serine protease; purification;
subtilisin family
-15-
Generation of Nitric Oxide from Spin-trapping Agents under
Oxidative Conditions
Kieko SAITO, Toyohiko ARIGA, and Hisashi YOSHIOKA
Graduate School of Nutritional and Environmental Sciences, University
of Shizuoka, 52-1 Yada, Shizuoka-shi 422, Japan
College of Bioresource Sciences, Nihon University, 3-34-1 Shimouma, Setagaya,
Tokyo 154, Japan
Received July 31, 1997
Nitric oxide (NO) generation from the spin-trapping agents, phenyl-tert-butylnitrone
(PBN), -(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimethyl-1-pyrroline
N-oxide (DMPO), under UV irradiation in the presence of dissolved oxygen
and by oxidation with the Fenton reagent was examined by using ESR spin-trapping
and spectrophotometric methods. A triplet signal at g2.041 was observed
after the ferrous complex of dithiocarbamate [Fe(MGD)2] had been added
to a solution of these trapping agents treated with UV irradiation and
the Fenton reagent, showing that NO was trapped with Fe(MGD)2. The concentration
of nitrite induced from NO was determined via the Griess reaction to increase
with the time of the treatment. It is speculated by reference to the ESR
signal observed at the position around g2.006 that the CN double bond
might have been cleaved by oxidation, resulting in the formation of a nitroso
compound, and that NO was then generated by the fission of the C--N bond
of the nitroso compound. NO generated in this way activated guanylate cyclase,
from which it can be expected that a spin-trapping agent acts as an NO
generator in vivo as well as a free radical scavenger.
Key words: nitric oxide (NO); phenyl-tert-butylnitrone (PBN); 5,5-dimethyl-1-pyrroline
N-oxide (DMPO); oxidative stress; cyclic GMP (cGMP)
-16-
Purification and Characterization of NADPH-Dependent Carbonyl
Reductase, Involved in Stereoselective Reduction of Ethyl 4-Chloro-3-oxobutanoate,
from Candida magnoliae
Masaru WADA,{{ Michihiko KATAOKA, Hiroshi KAWABATA, Yoshihiko YASOHARA, Noriyuki KIZAKI, Junzo HASEGAWA, and Sakayu SHIMIZU{
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto
University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
Fine Chemical Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi,
Takasago-cho, Takasago 676, Japan
Received August 8, 1997
A NADPH-dependent carbonyl reductase was purified to homogeneity from Candida
magnoliae AKU4643 through four steps, including Blue Sepharose affinity
chromatography. The enzyme catalyzed the stereoselective reduction of ethyl
4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a 100 enantiomeric
excess, which is a useful chiral building block for the chemical synthesis
of pharmaceuticals. The relative molecular mass of the enzyme was estimated
to be 76,000 on high performance gel filtration chromatography and 32,000
on SDS polyacrylamide gel electrophoresis. The enzyme reduced -, -keto
esters and conjugated diketones in addition to ethyl 4-chloro-3-oxobutanoate.
The enzyme activity was inhibited by quercetin and HgCl2, but not by EDTA.
The N-terminal amino acid sequence of the enzyme showed no apparent similarity
with those of other oxidoreductases.
Key words: carbonyl reductase; stereoselective reduction; Candida magnoliae
-17-
Identification of the Promoter Region and the Transcriptional
Regulatory Sequence of the evgAS Operon of Escherichia coli
Hiroyuki TANABE, Katsuhide YAMASAKI, Akinori KATOH, Sachiko YOSHIOKA, and Ryutaro UTSUMI{
Department of Agricultural Chemistry, Kinki University, Naka-machi, Nara 631, Japan
Received August 8, 1997
The evgAS operon of Escherichia coli encodes the EvgA response regulator
and the EvgS sensory kinase, which are members of one of the two-component
signal transduction systems of Escherichia coli. In this study, we identified
the evg promoter and the EvgA-responsive element. Primer extension analysis
found two evg transcritional initiation sites, designated P1 ({1) and
P2 (|10), and placed them 114 bp and 124 bp upstream of evgA, respectively.
A gel retardation assay demonstrated that EvgA specifically bound to an
inverted repeat located between |102 and |128 counting from P1. We also
did a -galactosidase induction experiment using a promoter-probing vector
and found that the EvgA-binding sequence was important to stimulate the
evg promoter. These results suggest that the expression of evgAS is positively
regulated by its own product, EvgA.
Key words: EvgA; evgAS; transcription factor; two-component system; signal
transduction
-18-
Solubilization and Chemical Characterization of an Insoluble
Matrix Protein in the Gastroliths of a Crayfish, Procambarus clarkii
Katsuaki ISHII,, Naoaki TSUTSUI, Toshiki WATANABE, Tadashi YANAGISAWA, and Hiromichi NAGASAWA,{
Ocean Research Institute, University of Tokyo, Nakano-ku, Tokyo 164,
Japan
Department of Applied Biochemistry, Faculty of Agriculture, Utsunomiya
University, Utsunomiya, Tochigi 321, Japan
Received August 11, 1997
The gastrolith of the crayfish Procambarus clarkii contains a small amount
of an organic matrix that is mainly chitin and proteins, together with
a large amount of calcium carbonate. As the first step to understand the
mechanism of calcification, we tried to characterize matrix proteins in
the gastrolith. An insoluble matrix protein, referred to as gastrolith
matrix protein, was made soluble with 1 SDS containing 10 mM dithiothreitol,
and was purified by reverse-phase high-performance liquid chromatography.
The protein had a molecular weight of about 50,500 and a blocked amino
terminus. By enzymatic digestion and microsequencing, five partial amino
acid sequences with a total of 225 amino acid residues were identified
and found to include a repetitive sequence not reported previously.
Key words: crayfish; Procambarus clarkii; gastrolith; matrix protein; calcification
-19-
Non-thermal Effect of a Ceramics Radiation on a Yeast
Glucose-6-phosphate dehydrogenase
Masahiro KOHASHI,{ Yuka OHTA, and Tatsuo WATANABE
School of Food and Nutritional Sciences, University of Shizuoka, 52-1, Yada, Shizuoka 422, Japan
Received August 12, 1997
Non-thermal effect of a ceramics radiation on glucose-6-phosphate dehydrogenase
has been investigated using the enzyme, glucose-6-phosphate and NADP{
separately irradiated at 10C by a ceracompo R plate and a ceramics un-sewed
cloth (sheet). The Km for glucose-6-phosphate was increased 20 after
6 h of irradiation by the plate, but the Vmax/Km was decreased 24. After
3 h of irradiation by the sheet, the Km was increased 17, but after 6
h of irradiation it was decreased 11. The 3 h of irradiation by the sheet
slightly increased both enthalpy and entropy changes of the reaction, but
the 6 h of irradiation significantly decreased them. Both thermodynamic
parameters in the activated state were increased by the sheet irradiation.
The promotion energy for both formations of the enzyme-substrate and their
activated complex depended on enthalpy. The different effects of two ceramics
radiators on G6PDH activity were discussed.
Key words: ceramics radiation; glucose-6-phosphate dehydrogenase; far-infrared;
hydration; thermodynamics
-20-
Purification and Properties of an Amylopullulanase, a
Glucoamylase, and an -Glucosidase in the Amylolytic Enzyme System of
Thermoanaerobacterium thermosaccharolyticum
Dirk GANGHOFNER,{ Josef KELLERMANN,1 Walter L. STAUDENBAUER,{{ and Karin BRONNENMEIER
Institute for Microbiology, Technical University of Munich, Arcisstrasse
21, D-80290 Munchen, Germany
1Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried,
Germany
Received August 12, 1997
Thermoanaerobic bacteria are of considerable interest as producers of thermostable
amylolytic enzymes. The soluble amylolytic enzyme system of Thermoanaerobacterium
thermosaccharolyticum DSM 571 was fractionated into a pullulanase, a glucoamylase,
and an -glucosidase. The enzymes were purified to homogeneity and their
physical and catalytic properties were studied. The pullulanase, which
cleaved both -1,4- and -1,6-glucosidic bonds, was an amylopullulanase
closely related to similar enzymes from other thermoanaerobic bacteria.
Partial amino acid sequences of the glucoamylase were identical with the
corresponding sequences deduced from the cga gene encoding the glucoamylase
from Clostridium sp. strain G0005. The -glucosidase was identified as
an isomaltase belonging to a group of structurally related exo--1,4-glucosidases
and oligo-1,6-glucosidases from bacilli. Comparison of enzyme activities
indicated that the glucoamylase had the major amylolytic activity of T.
thermosaccharolyticum, with amylopullulanase and -glucosidase assisting
in the cleavage of -1,6-glucosidic bonds.
Key words: -glucosidase; glucoamylase; amylopullulanase; thermophilic
enzymes; Thermoanaerobacterium thermosaccharolyticum
-21-
Overproduction of 1,2--Mannosidase, a Glycochain Processing
Enzyme, by Aspergillus oryzae
Takashi YOSHIDA,1 Tasuku NAKAJIMA, and Eiji ICHISHIMA
Department of Applied Biological Chemistry, Faculty of Agriculture,
Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aobaku, Sendai 981, Japan
Department of Bioengineering, Faculty of Engineering, Soka University,
1-236 Tanki-cho, Hachioji, Tokyo 192, Japan
Received August 22, 1997
A recombinant strain of Aspergillus oryzae has been constructed in which
1,2--mannosidase, an intracellular glycochain processing enzyme with
specificity toward 1,2--mannosidic linkages, has been overexpressed.
For the construction, the N-terminal signal-encoding sequence of the 1,2--mannosidase
gene (msdC) from Penicillium citrinum was replaced with that of the aspergillopepsin
I signal, and the fused gene was inserted between amyB promoter-terminator
elements in the expression plasmid pTAPM1. A transformant of A. oryzae
(the strain PM-1) secreted a great deal of heterogeneous 1,2--mannosidase
into the culture media, which was purified by CM ion-exchange chromatography.
Approximately 21 mg of the purified enzyme was obtained per liter of culture.
N-terminal amino acid analysis indicated that the signal peptide was removed
from the secreted enzyme. The Penicillium 1,2--mannosidase expressed
in A. oryzae did not show any notable difference from the enzyme from P.
citrinum in such properties as Mr, specific activity, CD spectra, or kinetic
parameters. Man7GlcNAc2 accumulated temporarily during the degradation
of Man9GlcNAc2 to Man5GlcNAc2 by fungal 1,2--mannosidase.
Key words: -mannosidase; expression; fungi; Penicillium; Aspergillus
-22-
Reptile Lysozyme: The Complete Amino Acid Sequence of
Soft-Shelled Turtle Lysozyme and Its Activity
Tomohiro ARAKI,{ Takaki YAMAMOTO, and Takao TORIKATA
Department of Biochemistry, School of Agriculture, Kyushu Tokai Universiry. Cyoyo, Aso, Kumamoto 869-1404, Japan
Received August 22, 1997
Soft-shelled turtle egg-white lysozyme was purified and sequenced. Lysozyme
was reduced and carboxymethylated to fragment it with trypsin, V8 protease
and CNBr. The peptides yielded were purified by RP-HPLC and sequenced.
Every trypsin peptide was overlapped by V8 protease peptides and CNBr fragment.
The amino acid sequence was compared with other lysozymes. This lysozyme
has an extra Gly residue at N-terminus, which was found in pheasant lysozyme.
Further, this lysozyme has an insertion of a Gly residue between 47 and
48 residues when compared with chicken lysozyme, as found in human lysozyme,
therefore it proved that this lysozyme has the largest number of amino
acids (131 aa) in chicken type lysozymes. The amino acid substitutions
were found at subsites E and F. Namely Phe34, Arg45, Thr47, and Arg114
were replaced by His, Tyr, Arg, and Tyr, respectively. The time course
using N-acetylglucosamine pentamer as a substrate showed a reduction of
the rate constant for glycosidic cleavage and increase of binding free
energy for subsites E and F, which proved the contribution of amino acids
mentioned above for substrate binding at subsites E and F.
Key words: lysozyme; turtle; egg white; amino acid sequence
-23-
Diisopropylfluorophosphate (DFP) Inhibits Ricin-induced
Apoptosis of MDCK Cells
Tatsuya ODA, Nobukazu KOMATSU, and Tsuyoshi MURAMATSU
Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi, Nagasaki 852, Japan
Received August 22, 1997
Diisopropylfluorophosphate (DFP), a general serine protease inhibitor,
inhibited the DNA fragmentation and cell death in MDCK cells treated with
ricin, modeccin, Pseudomonas toxin, or diphtheria toxin. A trypsin-like
serine protease inhibitor, N-tosyl-L-lysine chloromethyl ketone (TLCK)
also prevented ricin-induced DNA fragmentation and cell death, albeit less
effectively than DFP. Microscopic observation showed that the morphological
changes of MDCK cells induced by ricin were prevented by DFP. DFP did not
affect the binding, internalization, or subsequent excretion of ricin,
but reduced the degradation of ricin in MDCK cells, suggesting that DFP
inhibits at least the cellular protease that may be involved in the degradation
of internalized ricin. In addition, SDS-PAGE analysis of cytosolic proteins
suggested that DFP-sensitive endogenous proteases are activated in the
ricin-treated cells. In the cells treated with DFP, the protein synthesis
inhibitory activity of ricin was increased rather than inhibited. The activities
of modeccin and Pseudomonas toxin were also slightly increased by DFP,
but no effect of DFP on the activity of diphtheria toxin was observed.
Therefore, these results suggest that protein toxins have a DFP-sensitive
common pathway leading to apoptosis that is distinct from the pathway leading
to the inhibition of cellular protein synthesis.
Key words: ricin; protein toxin; apoptosis; protease inhibitor; diisopropylfluorophosphate
-24-
Trehalose as osmoprotectant in Rhodobacter sphaeroides
f. sp. denitrificans IL106
Xiaoyuan XU, Mitsuru ABO, Akira OKUBO, and Sunao YAMAZAKI
Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received August 25, 1997
The photosynthetic bacterium Rhodobacter sphaeroides f. sp. denitrificans
IL106 can grow in high osmolarity. The accumulation of intracellular inorganic
ions and organic solutes in cells grown in a synthetic medium containing
different concentrations of NaCl was examined. Together with potassium
ion, trehalose was the major organic osmoprotectant and its accumulation
depended on the external salt concentration. Intracellular levels of glycine
betaine and other osmoprotectants such as proline did not change when osmolarity
increased.
Key words: Rhodobacter sphaeroides f. sp. denitrificans IL106; trehalose;
osmoprotectant; salt stress; glycine betaine
-25-
Two Peaks in pH Dependence of Renin-angiotensinogen Reaction
Uddin Mohammad NASIR, Kohji TAKAHASHI, Takao NAGAI, Tsutomu NAKAGAWA, Fumiaki SUZUKI, and Yukio NAKAMURA,{
United Graduate School of Agricultural Science, Department of Biotechnology, Faculty of Agriculture, and Molecular Genetics Research Center, Gifu University, 1-1 Yanagido, Gifu 501-11, Japan
Received August 29, 1997
The pH dependence of the reaction of purified recombinant human renin with
purified recombinant sheep angiotensinogen was not a typical bell-shape
but had two peaks, pH 6.4 and 8.9. The pH dependence of the reaction of
human renin with human, hog, and rat angiotensinogens partially purified
from plasma had a peak with a shoulder containing two peaks close together.
These findings indicate that the reaction of renin with angiotensinogen
involves at least two amino acid residues other than the two aspartic acid
residues known to be involved and occurs at acidic pH and basic pH by two
different pairs of these amino acid residues.
Key words: renin-angiotensinogen reaction; two-peak pH dependence; pH dependence
of renin
-26-
Influence of Phytate Removal and Structural Modification
on the Calcium-binding Properties of Soybean Globulins
Hitomi KUMAGAI,{ Yoshiharu SHIZAWA, Hidetoshi SAKURAI, and Hitoshi KUMAGAI
Department of Agricultural and Biological Chemistry, College of Bioresource
Sciences, Nihon University, 3-34-1 Shimouma, Setagaya-ku, Tokyo 154, Japan
Department of Applied Biological Chemistry, Division of Agriculture and
Agricultural Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113, Japan
Received September 4, 1997
The calcium-binding properties of soybean globulins that have been deamidated
or enzymatically hydrolyzed after the removal of phytate were physicochemically
investigated. The level of calcium was reduced from 0.32 to 0.013 and
that of phosphorus was reduced from 1.1 to 0.050 by treating with cation-
and anion-exchange resins. The calcium-binding properties of soybean globulins
were described by the Langmuir equation, the maximum amount of bound calcium
(N) and the affinity parameter for calcium (K) being obtained for each
sample. The value of N was decreased by the removal of phytate, while the
deamidation caused the value of N to increase. As hydrolysis proceeded,
the value of N increased to a degree of hydrolysis of 32, and then decreased.
Based on this result, there seems to be an optimum molecular weight of
hydrolyzed soybean globulins for the amount of bound calcium. In addition,
the value of K for every soybean globulin sample was much lower than that
of phytic acid, indicating that the globulins had proper calcium-binding
properties for calcium absorption in the small intestine.
Key words: calcium-binding property; soybean globulin; phytate removal;
deamidation; enzymatic hydrolysis
-27-
Molecular Cloning and Expression of a Gene Encoding a
Novel Sorbitol Oxidase from Streptomyces sp. H-7775
Kazumi HIRAGA, Takashi ETO, Issei YOSHIOKA, and Kohei ODA1
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute
of Technology, Sakyo-ku, Kyoto 606, Japan
Asahi Chemical Industry, Mifuku, Ohito-cho, Tagata-gun, Shizuoka 410-23,
Japan
Received September 8, 1997
A gene encoding a novel intracellular sorbitol oxidase of a soil bacterium,
Streptomyces sp. H-7775, was cloned and sequenced. The gene consists of
an open reading frame of 1,260-bp encoding a protein of 420 amino acids
with a molecular weight of 45,148. Deduced amino acid sequence of the gene
has 25.3 identity and 68.1 similarity to that of rat L-gulonolactone
oxidase at the overall amino acids. Nucleotide-binding motifs were not
found in the deduced amino acid sequence of SOX protein. We succeeded in
expressing recombinant sorbitol oxidase with covalently bound FAD in E.
coli at about a 4,000-fold higher total enzyme activity than that of the
Streptomyces sp. H-7775. The enzymatic properties of the recombinant SOX
were similar to those of the enzyme from Streptomyces sp. H-7775. This
is the first report of the cloning and expression of a newly categorized
enzyme, sorbitol oxidase, from Streptomyces sp.
Key words: oxidase; sorbitol; sorbitol oxidase; cloning; expression
-28-
Molecular Properties and Activity of Amino-Terminal Truncated
Forms of Lipase Activator Protein
Hiroyuki SHIBATA, Hiroaki KATO, and Junichi ODA{
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan
Received September 25, 1997
Two mutant forms, which had truncated N-terminals, of lipase activator
protein (LipB) from Pseudomonas aeruginosa TE3285 were prepared, and their
molecular properties and activity were compared with those of the full-length
form. A truncated LipB lacking its hydrophobic N-terminal 21 residues was
dispersed homogeneously in solution, and could reactivate the stoichiometric
amount of denatured lipase. In contrast, full-length LipB formed soluble
aggregates, and reactivated less than an equimolar amount of the lipase
even under the most suitable conditions. These findings suggest that some
or all of the N-terminal 21 residues caused aggregation of the protein
molecules, and prevented LipB from fully stoichiometric reactivation. A
truncated LipB lacking the N-terminal 61 residues also reactivated denatured
lipase, suggesting that the N-terminal 61-residue region of LipB is not
involved in reactivation.
Key words: lipase activator protein; amino-terminal region; truncated mutant;
aggregation; stoichiometry
-29-
Note
Cloning Genomic DNA Encoding Apple Polyphenol Oxidase and Comparison of
the Gene Product in Escherichia coli and in Apple
Miyoshi HARUTA, Masatsune MURATA,,{ Ayami HIRAIDE,,{{ Hiroshi KADOKURA, Makari YAMASAKI, Masaaki SAKUTA, Seki SHIMIZU, and Seiichi HOMMA
Department of Nutrition and Food Science
Department of Biotechnology, University of Tokyo, Tokyo 113, Japan
Department of Biology, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku,
Tokyo 112, Japan
Received June 12, 1997
Two PCR-amplified genomic DNA fragments encoding apple (cv. Fuji) polyphenol
oxidase (PPO) were cloned and sequenced. A comparison of genomic DNA with
cDNAs revealed that the PPOs lacked introns. Both PPO DNAs appear to encode
a 66-kDa precursor protein consisting of a 56-kDa mature protein and a
N-terminal transit peptide of 10-kDa N-terminal transit peptide. Apple
PPO DNA was expressed in Escherichia coli, and the gene product (56 kDa)
without a transit peptide was immunochemically detected and was the same
size (ca. 65 kDa) as the main PPO of apple fruit by SDS-PAGE.
Key words: apple (Malus pumila); enzymatic browning; polyphenol oxidase;
cloning genomic DNA; expression in E. coli
-30-
Note
6-Methylsulfinylhexyl Isothiocyanate and Its Homologues as Food-originated
Compounds with Antibacterial Activity against Escherichia coli and Staphylococcus
aureus
Haruhiro ONO, Shoko TESAKI, Soichi TANABE, and Michiko WATANABE
Food Science Laboratory, Faculty of Education, Tokyo Gakugei University,
Koganei-shi, Tokyo 184, Japan
The Skylark Food Science Institute, Chiba-shi, Chiba 261-01, Japan
Received June 17, 1997
Cruciferae plants, banana and coriander each showed antibacterial activity.
The highest activity among the foodstuffs tested was found in the stems
of wasabi. An ethereal extract from wasabi stems had potent antibacterial
activity and we isolated the active compound from the extract. Instrumental
analysis identified the compound as 6-methylsulfinylhexyl isothiocyanate.
Some homologues of 6-methylsulfinylhexyl isothiocyanate were also active
against Escherichia coli and Staphylococcus aureus.
Key words: antibacterial activity; Escherichia coli; Staphylococcus aureus;
methylsulfinylalkyl isothiocyanate
-31-
Note
Effects of Germinated Barley Foodstuff in Preventing Diarrhea and Forming
Normal Feces in Ceco-colectomized Rats
Osamu KANAUCHI,{ Tomohiko NAKAMURA, Kazue AGATA, Tohru FUSHIKI, and Hiroshi HARA
Applied Bioresearch Center, Corporate Research and Development Division
Kirin Brewery Co. Ltd., 3 Miyaharacho, Takasakishi, Gunma 370-12, Japan
Department of Food Science and Technology, Faculty of Agriculture, Kyoto
University, Oiwakemachi, Kitashirakawa, Sakyo-ku, Kyoto 606, Japan
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido
University, Kita 9 Nishi 9 Kita-ku, Sapporo 060, Japan
Received July 2, 1997
Germinated barley foodstuff (GBF) derived from the aleurone and scutellum
fractions of germinated barley was rich in glutamine and low-lignified
hemicellulose. The diarrhea caused by ceco-colectomy could be prevented
by feeding GBF to rats. GBF could also increase the protein content and
sucrase activity of small intestinal mucosa in this model. This diarrhea-preventive
effect of GBF would be based on the water-holding capacity and bulging
force under alkaline conditions, e.g. in the small intestine.
Key words: intestines; dietary fiber; germination; diarrhea; barley
-32-
Note
Protein Disulfide Isomerase Activity of Some Plant Seeds
Ken KAINUMA, Tetsuya OOKURA, Akiko OKAMOTO, Kazumi KITTA, Mariko MANABE, and Yukio KAWAMURA
National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan
Received July 23, 1997
The activity of protein disulfide isomerase, in the extracts of several
dormant seeds including soybean, rice, wheat, and maize was assayed. The
activity was higher in the extracts of beans than in those of the other
seeds. A correlation was significant (R0.95 and 0.93; p<0.01) between
the PDI activity and the concentration of protein soluble in a salt solution.
Key words: PDI; protein disulfide isomerase; folding; seed
-33-
Note
Overproduction and Substrate Specificity of 3-Isopropylmalate Dehydrogenase
from Thiobacillus ferrooxidans
Hideyuki MATSUNAMI, Hiroshi KAWAGUCHI, Kenji INAGAKI,{ Tadashi EGUCHI, Katsumi KAKINUMA, and Hidehiko TANAKA
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama
University, Okayama 700, Japan
Department of Chemistry, Tokyo Institute of Technology, Yokohama 227,
Japan
Received August 14, 1997
We constructed an overexpression system in Escherichia coli of the leuB
gene coding for 3-isopropylmalate dehydrogenase in Thiobacillus ferrooxidans.
E. coli harboring the plasmid we constructed, pKK leuB1, produced 17-fold
the enzyme protein of the expression system previously used for purification.
The substrate specificity of the enzyme was analyzed with synthetic (2R,
3S)-3-alkylmalates. The 3-isopropylmalate dehydrogenase of Thiobacillus
ferrooxidans had broad specificity toward the alkylmalates.
Key words: Thiobacillus ferrooxidans; 3-isopropylmalate dehydrogenase;
leuB; overproduction; substrate specificity
-34-
Note
Lack of Light/Dark Regulation of Enzymes Involved in the Photosynthetic
Carbon Reduction Cycle in Cyanobacteria, Synechococcus PCC 7942 and Synechocystis
PCC 6803
Masahiro TAMOI, Akiko MURAKAMI, Toru TAKEDA, and Shigeru SHIGEOKA{
Department of Food and Nutrition, Kinki University, Nara 631, Japan
Received August 15, 1997
During the photosynthetic carbon reduction (PCR) cycle of Synechococcus
PCC 7942 and Synechocystis PCC 6803, fructose-1,6-bisphosphatase, NADP{-glyceraldehyde-3-phosphate
dehydrogenase, and ribulose-5-phosphate kinase were not sensitive to treatment
with dithiothreitol (DTT), a reducing agent, in vitro and were not regulated
by light in vivo, unlike the chloroplastic enzymes of higher plants. These
results indicate that the PCR cycle in the cyanobacterial cells may not
be actually regulated by light in vivo even if the ferredoxin/thioredoxin
system is present.
Key words: Synechococcus PCC7942; Synechocystis PCC 6803; thiol-modulated
enzyme; light/dark regulation
-35-
Note
Detection of Antitumor Promoting Activity in Raji Cells Carrying Epstein-Barr
Virus Genome by Immunoblotting Analysis
Akira KONDO, Tetsuhiro MORIMOTO, and Katsuichiro OKAZAKI{
Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan
Received August 22, 1997
Extract of Raji cells treated with sodium n-butyrate (1 mM) and a tumor
promoter, 12--O--tetradecanoylphorbol-13-acetate (TPA, 40 ng/ml), was analyzed
by immunoblotting using ten human sera with different antibody titers against
Epstein-Barr virus early antigens. Two human sera reacted with one induced
polypeptide of 48 kDa and its induction was inhibited by curcumin (4 g/ml),
an antitumor promoter from turmeric. A mouse antiserum against P3HR-1 cells
treated with TPA and sodium n-butyrate also detected the 48-kDa polypeptide
in Raji cells treated with TPA at concentrations of 2.5 to 80 ng/ml. These
results indicate that the immunoblotting analysis can be used in a confirmation
test for detection of antitumor promoting activity.
Key words: antitumor promoter; Epstein-Barr virus early antigen; Raji cells;
P3HR-1 cells; TPA
-36-
Note
Detection of Potato Virus Y P1 Protein in Infected Cells and Analysis of
Its Cleavage Site
Li Jun YANG, Makoto HIDAKA,{ Haruhiko MASAKI, and Takeshi UOZUMI
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan
Received August 22, 1997
The P1 protein is liberated from the N-terminal region of the potyviral
polyprotein by cleavage depending on its own autoproteolytic activity.
Existence of the 32-kDa P1 protein in tobacco plants infected with potato
virus Y ordinary strain (PVY-O) was detected by an antiserum against a
recombinant PVY-O P1 protein. In vivo analysis using tobacco protoplasts
confirmed that the Phe284--Ser285 was the cleavage site separating the
P1 protein from the PVY-O polyprotein. Phe284 was indispensable for proteolysis
and Ser285 was needed for optimal cleavage susceptibility.
Key words: potato virus Y; P1 protein; proteolytic activity; in vivo processing
-37-
Note
Identification of the Aspartic Acid Residue Located at or near Substrate-binding
Site of Rye Seed Chitinase-c{
Takeshi YAMAGAMI and Gunki FUNATSU
Laboratory of Protein Chemistry Engineering, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Received August 25, 1997
Carboxyl groups of rye seed chitinase-c (RSC-c) were modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) and glycine ethyl ester (GEE) at pH 5.5 and 5C in the presence
and absence of (GlcNAc)4. In the absence of (GlcNAc)4, 5.2 carboxyl groups
were modified by 90 min-reaction and the chitinase activity was reduced
to 2.0, while in the presence of (GlcNAc)4, 4.6 carboxyl groups were
modified and 72 of the activity was retained. To identify the carboxyl
group protected by (GlcNAc)4 from the modification, RSC-c was first modified
with EDC and GEE in the presence of (GlcNAc)4 and then radiolabeled with
EDC and [14C]GEE in the absence of (GlcNAc)4. Analyses of the radioactive
peptides from the tryptic and chymotryptic digests of radiolabeled RSC-c
showed that the main radiolabeled carboxyl group is that of Asp95, suggesting
that Asp95 is located at or near substrate-binding site of RSC-c.
Key words: chitinase; rye seeds; active site; substrate-binding site; chemical
modification
-38-
Note
Complete Amino Acid Sequence of Chitinase-a from Bulbs of Gladiolus (Gladiolus
gandavensis)
Takeshi YAMAGAMI, Yoichiro MINE, and Masatsune ISHIGURO
Laboratory of Protein Chemistry Engineering, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Received October 8, 1997
The complete amino acid sequence of gladiolus bulb chitinase-a (GBC-a)
was determined. First the tryptic peptides from GBC-a after it was reduced
and S-carboxymethylated were sequenced and then the peptides were further
studied by chemical cleavage of the enzyme. GBC-a consisted of 274 amino
acid residues and had a molecular mass of 30,714 Da. Two consensus sequences
essential for chitinase activity by plant class III chitinases were conserved
in GBC-a, although its sequence similarity with plant class III chitinases
was less than 20. Sequence comparison of GBC-a with sequences of other
proteins in a protein identification resource (PIR) showed that the GBC-a
sequence was 33 similar to that of narbonin, a seed storage 2S globulin
from narbon beans.
Key words: chitinase; plant bulb; gladiolus; amino acid sequence
-39-
Note
Multidrug Resistance Phenotype Conferred by Overexpressing bfr2{/pad1{/sks1{
or pap1{ Genes and Mediated by bfr1{ Gene Product, a Structural and Functional
Homologue of P-Glycoprotein in Schizosaccharomyces pombe
Manabu ARIOKA, Mutsuo KOUHASHI, Koji YODA, Akira TAKATSUKI, Makari YAMASAKI, and Katsuhiko KITAMOTO
Department of Biotechnology, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku,
Tokyo 113-8657, Japan
Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi,
Saitama 351-0198, Japan
Present address: College of Bioresource Sciences, Department of Food
Science and Technology, Nihon University, Tokyo 154-0002, Japan
Received August 27, 1997
We investigated the mechanism of multidrug resistance conferred by overexpression
of bfr2{/pad1{/sks1{ or pap1{ genes of Schizosaccharomyces pombe. Overexpression
of bfr2{ did not confer multidrug resistance on a pap1-disrupted strain.
In a mutant with bfr1{ (a putative membrane transporter which belongs
to the ATP-binding cassette superfamily) disrupted, overexpression of either
bfr2{ or pap1{ did not confer multidrug resistance. These findings suggest
that bfr1{ acts as the most downstream effector of the multidrug resistance
conferred by bfr2{ and pap1{ genes.
Key words: ATP-binding cassette (ABC) superfamily; brefeldin A; multidrug
resistance (MDR); bfr1{ gene; pad1{ gene
-40-
Note
Extracellular Dextran-induced p-Nitrophenyl--D-glucoside-hydrolyzing
Enzyme of Bacillus circulans KA-304: A Producer of Schizophyllum commune-lytic
Enzyme
Katsushige MIZUNO and Takashi TACHIKI{
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu 525-77, Japan
Received August 27, 1997
p-NP--D-Glucoside-hydrolyzing activity in the culture filtrate of Bacillus
circulans KA-304, a producer of Schizophyllum commune cell-wall lytic enzyme,
increased remarkably when the bacterium was grown on dextran as a carbon
source. It was suggested that the increase of the activity was caused by
increases of two major species, -D-glucosidase I and -D-glucosidase
II. -D-Glucosidase I, which showed a certain reactivity toward dextran,
was isolated from the filtrate (MW 70 kDa, 35-fold, 10 recovery). The
enzyme was stable around pH 6.5--7.5 and showed its highest activity at
pH 6.5. The enzyme preparation inactivated with p-chloromerucuribenzoic
acid recovered its activity by incubating with ditiothereitol. Its substrate
specificity suggested that the enzyme was an exo-type enzyme with certain
affinity toward -1,6-glucosidic linkage.
Key words: Schizophyllum commune; p-NP--D-glucosidase; Bacillus circulans
KA-304
-41-
Note
Syntheses of Highly Oxygen-functionalized Derivatives of Dihydrodihydroxyphthalic
Acid in Enantiomerically Enriched Forms
Hajime IKEDA, Takeshi SUGAI,{ Hiroyuki HOSOMI, Shigeru OHBA, and Hiromichi OHTA
Department of Chemistry, Keio University 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223, Japan
Received August 27, 1997
Starting from dihydrodihydroxyphthalic acid (DDP), (1R, 2S, 5R, 6S)-(-)-3,4-bis(benzyloxymethyl)-5-hydroxy-8,8-dimethyl-6,8-dioxabicyclo[4.3.0]non-3-en-2-yl
chloroacetate (>95e.e.), a highly oxygen-functionalized derivative,
was prepared by a combination of chemical and enzymatic reactions. The
key step for asymmetrization was hydrolysis of the corresponding meso bis-chloroacetate
with pig pancreatic lipase.
Key words: dihydrodihydroxyphthalic acid; photooxygenation; pig pancreatic
lipase; meso substrate; hydrolysis
-42-
Note
Binding of the Protein from Thermus aquaticus ISLtaq1 to Its Inverted Repeat
in Vitro
Hiroyuki TANABE, Yoshihiko KAKUTA, Sachiko YOSHIOKA, and Ryutaro UTSUMI{
Department of Agricultural Chemistry, Kinki University, Naka-machi, Nara 631, Japan
Received September 1, 1997
We have isolated from Thermus aquaticus an insertion-sequence-like genetic
element (ISLtaq1) that induces thermotolerance and has a high sequence
similarity to IS150 belonging to the IS3 family. An open reading frame
on ISLtaq1, termed ORF1, encodes the ORF1 protein, which carries a DNA-binding
motif. In this study, we found an imperfect inverted repeat in ISLtaq1.
We next overproduced and purified a His-tagged ORF1 protein. Gel retardation
analysis demonstrated that this protein specifically bound to an DNA fragment
containing the inverted repeat in ISLtaq1. These results suggest that ISLtaq1
and the ORF1 protein are an insertion sequence and part of the transposase
encoded by ISLtaq1, respectively.
Key words: ISLtaq1; inverted repeat; insertion sequence; transposon; Thermus
aquaticus
-43-
Note
Purification and Properties of Three -N-Acetylglucosaminidases from Lactobacillus
casei ATCC 27092
Mio SENBA, Nobuhiro KASHIGE, Fumio MIAKE, and Kenji WATANABE
Microbiology Laboratory, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-80, Japan
Received September 1, 1997
Three -N-acetylglucosaminidases, GlcNAcase A, B, and C, were purified
from the culture fluid of Lactobacillus casei ATCC 27092, and the molecular
weights of these enzymes were estimated to be 54,000, 51,000, and 44,000,
respectively, by SDS-PAGE. The production of these GlcNAcases was accelerated
by the addition of N-acetylglucosamine to the culture. These enzymes had
pIs of about 5.2, an optimum pH of 5.0--5.5, and an optimum temperature
of 37--40C. The Km values of GlcNAcase A, B, and C for p-nitrophenyl--N-acetylglucosamine
were 0.85, 1.30, and 1.04 mM and those for p-nitrophenyl--N-acetylgalactosamine
were 39.6, 57.7, and 60.8 mM, respectively.
Key words: -N-acetylglucosaminidase; Lactobacillus casei; N-acetylglucosamine
-44-
Rapid paper
Overexpression of Squalene-Hopene Cyclase by the pET Vector in Escherichia
Coli and First Identification of Tryptophan and Aspartic Acid Residues
inside the QW Motif as Active Sites
Tsutomu SATO,1 Yoshinori KANAI,2 and Tsutomu HOSHINO1,2{
1Graduate School of Science and Technology, and
2Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata
University, Ikarashi, Niigata 950-21, Japan
Received September 12, 1997
An overexpression system for squalene-hopene cyclase (SHC) was constructed
by using the pET3a vector, which is responsible for high expression with
help from the strong T7 promoter when incorporated into E. coli BL21(DE3).
Site-directed mutagenesis experiments prove that two amino acid residues
of tryptophan and aspartic acid inside the QW-motif 5 resided as active
sites.
Key words: squalene; hopene; squalene cyclase; Alicyclobacillus acidocaldarius;
site-directed mutagenesis
-45-
Rapid paper
A New Peptide-N4-(acetyl--glucosaminyl)asparagine Amidase from Soybean
(Glycine max) Seeds: Purification and Substrate Specificity{
Yoshinobu KIMURA{{ and Akira OHNO
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-Naka 1-1-1, Okayama 700, Japan
Received October 9, 1997
We report here the isolation and characterization of a peptide-N4-(acetyl--glucosaminyl)
asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds.
The enzyme was purified to homogeneity with 6.5 yield from defatted soybean
meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite
chromatography, and hydrophobic chromatography. The purified enzyme, designated
PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90
kDa by gel filtration, indicating this PNGase is a monomeric protein. The
enzyme showed maximal activity at pH 4.5--5.0. PNGase-GM was capable of
hydrolyzing the -aspartylglycosylamine linkage (GlcNAc1Asn) of various
glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing
plant complex type N-glycan units, while this amidase was far less active
on the glycopeptides bearing sialylated animal complex-type glycans.
Key words: peptide: N-glycanase; glycoamidase; N-glycan releasing enzyme;
free N-glycan; Glycine max