Contents and Abstracts of Latest Issue of BBB

(Vol.64 No.2 2000)

Henri CHAHINIAN,a Guillaume VANOT,a Aida IBRIK,a Nathalie RUGANI,b
Louis SARDA,c,・ and Louis-Claude COMEAUa

aLaboratory of Applied Biological Chemistry, Faculty of Sciences and Technology, St-J"er^ome, 13397 Marseille Cedex 20, France
bLaboratory of Microbiology, Faculty of Sciences and Technology, St-J"er^ome, 13397 Marseille Cedex 20, France
cLaboratory of Biochemistry, Faculty of Sciences, St-Charles, University of Provence, P.O. Box 65,
Place Victor Hugo, 13331 Marseille Cedex 3, France

Received May 24, 1999; Accepted October 29, 1999
Penicillium cyclopium, grown in stationary culture, produces a type I lipase specific for triacylglycerols while, in shaken culture, it produces a type II lipase only active on partial acylglycerols. Lipase II has been purified by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-Sephadex. The enzyme exists in several glycosylated forms of 40--43 kDa, which can be converted to a single protein of 37 kDa by enzymatic deglycosylation. Activity of lipase II is maximal at pH 7.0 and 40°C. The enzyme is stable from pH 4.5 to 7.0. Activity is rapidly lost at temperatures above 50°C. The enzyme specifically hydrolyzes monoacylglycerols and diacylglycerols, especially of medium chain fatty acids. The sequence of the 20 first amino acid residues is similar to the N-terminal region of P. camembertii lipase and partially similar to lipases from Humicola lanuginosa and Aspergillus oryzae, but is different from Penicillium cyclopium lipase I. However, it can be observed that residues of valine and serine at positions 2 and 5 in Penicillium cyclopium lipase II are conserved in Penicillium expansum lipase, of which 16 out of the 20 first amino acid residues are similar to Penicillium cyclopium lipase I.
microbial lipases; Penicillium cyclopium; partial acylglycerol lipase; substrate specificity; N-terminal sequence

-2-
Purification and Partial Characterization of a Novel Glucanhydrolase
from Lipomyces starkeyi KSM 22 and its Use for Inhibition
of Insoluble Glucan Formation

RYU, Su-Jin,＊ Doman KIM,＊＊,＊＊＊＊ Hwa-Ja RYU,＊＊＊ Seiya CHIBA,＊＊＊＊＊
Atsuo KIMURA,＊＊＊＊＊ and Donal F. DAY＊＊＊＊＊＊

Department of Biochemical Engineering, ＊Department of Chemical Engineering, ＊＊Research Institute for
Catalysis, and ＊＊＊Department of Fine Chemical Engineering, Chonnam National University,
Kwangju 500-757, Korea
＊＊＊＊Research Center for New-Biomaterials in Agriculture, Seoul National University, Suwon 441-744, Korea
＊＊＊＊＊Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan
＊＊＊＊＊＊The Audubon Sugar Instistute, Louisiana State University, Baton Rouge, LA 70803, USA

Received July 2, 1999; Accepted October 13, 1999
A novel glucanhydrolase from a mutant of Lipomyces starkeyi ATCC 74054 was purified. The single protein (100 kDa) showed either dextranolytic or amylolytic activity. We referred to the glucanhydrolase as a DXAMase. The DXAMase was produced in a starch medium and it was 3.75-fold more active for hydrolysis of the purified insoluble-glucan of Streptococcus mutans than Penicillium funiculosum dextranase. Aggregation of S. mutans cells with dextran and adherence to glass were eliminated by incubating with the DXAMase. The addition of DXAMase (0.1 IU/ml) to the mutansucrase reaction digest with sucrose reduced the formation of insoluble-glucan about 80％. Also the DXAMase (0.5 IU/ml) removed 80％ of the pre-formed sucrose-dependent adherent film. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent.
glucanhydrolase; Lipomyces starkeyi; insoluble-glucan; dextranase; amylase

-3-
Cloning and Characterization of a Gene Complementing the Mutation
of an Ethanol-sensitive Mutant of Sake Yeast

Toyohisa INOUE, Haruyuki IEFUJI, Tsutomu FUJII, Hiroshi SOGA,
and Kaname SATOH

Nishinomiya Syuzo Co., Ltd., 4-57, Yokai, Nishinomiya 662-8521, Japan

Received July 5, 1999; Accepted November 5, 1999
Ethanol-sensitive mutants (es1 to es10) were isolated from sake yeast, Saccharomyces cerevisiae SY-32. These mutants were unable to grow at 7％ ethanol at which the wild type strain SY-32 does grow. The mutants had a variety of fermentation rates and viabilities in the presence of ethanol. The gene ERG6, complementing the ethanol-sensitive mutation of es5, was cloned from an SY-32 gene library. ERG6 encodes S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) in the ergosterol synthetic pathway. Mutant es5 had a reduced ability to synthesize ergosterol. An erg6 disruptant was also ethanol-sensitive. These results suggested that ERG6 plays an important role in the ethanol tolerance of S. cerevisiae.
ethanol-sensitive mutant; ERG6; ergosterol; Saccharomyces cerevisiae

-4-
Serum Glucose and Insulin Response in Rats Administered with Sucrose
or Starch Containing Adenosine, Inosine or Cytosine

Yasunori FUKUMORI,・ Norio MAEDA, Hiroyuki TAKEDA, Shuichi ONODERA,＊
and Norio SHIOMI＊

Department of General Foods, The Hokuren Federation of Agriculture Corporation, Sapporo 060-8651, Japan
＊Department of Food Science, Faculty of Dairy Science, Rakuno Gakuen University, Ebetsu 069-8501, Japan

Received July 8, 1999; Accepted October 12, 1999
Blood glucose and insulin responses and gastric emptying were examined in rats intubated with sucrose or soluble starch that contained adenosine, inosine and cytosine.
The increase in serum glucose and insulin levels in the rats following loading with sucrose (2.5 g/kg of body weight) or soluble starch (1.875 g/kg of body weight) was significantly reduced by the administration of adenosine, inosine and cytosine (0.0625--0.125 g/kg of body weight).
The gastric emptying rates were only marginally affected by the nucleoside administration. The activities of sucrase, maltase, isomaltase and glucoamylase in a crude preparation from the small intestinal mucosa of rats were mildly inhibited by the nucleosides.
The decrease in blood glucose and insulin levels may have been in response to a decrease in glucose absorption caused by the inhibiting effect of the nucleosides on the mucosal enzymes that digest sucrose, maltose, and malto- and isomalto-oligosaccharides.
rats; nucleosides; blood glucose and insulin; α-glucosidase inhibition

-5-
Biosynthesis of Depudecin, a Metabolite of Nimbya scirpicola

Masayasu TANAKA,・ Takane FUJIMORI, and Kensuke NABETA＊

Plant Protection Research Laboratory, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama,
Kanagawa 227-8512, Japan
＊Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro 080-8555, Japan

Received July 27, 1999; Accepted October 19, 1999
Feeding experiments of labeled acetates to Nimbya scirpicola proved the carbon origin of the straight-chain polyketide, depudecin. Differential hydrogen exchange of the 2H label originating from 2H labeled acetate along the polyketide chain occurred. In particular, the deuterium of an epoxide methine at C-3 was lost to a substantial extent in the formation of depudecin.
phytotoxin; Nimbya scirpicola; depudecin; biosynthesis; polyketide

-6-
Effects of Ethanolamine as a Nitrogen Source on Hydrogen Production
by Rhodobacter capsulatus

Tomohisa KATSUDA,1 Masayuki AZUMA,1 Jyoji KATO,1 Susumu TAKAKUWA,2
and Hiroshi OOSHIMA1,・

1Department of Bioapplied Chemistry, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku,
Osaka 558-8585, Japan
2Department of Natural Science, Kyoto Women′s University, Kyoto 605-0926, Japan

Received July 28, 1999; Accepted November 8, 1999
Ethanolamine was examined as a nitrogen source in the production of hydrogen by Rhodobacter capsulatus ST-410, a hydrogenase-deficient mutant of the strain B-100. It was found that ethanolamine supports cell growth as the sole nitrogen source and permits a large amount of hydrogen evolution, detected at 138 μmol/ml-culture from 3.5 mM ethanolamine and 30 mM DL-malate. The amount corresponded to a stoichiometric yield of 77％ and was close to that obtained from 7.0 mM L-glutamate and 30 mM DL-malate. The hydrogen evolution rate per unit biomass (cells) was higher than that with L-glutamate, and the cells grown with ethanolamine had higher nitrogenase activity than the cells grown with L-glutamate. In terms of bioconversion of cellulosic and hemicellulosic biomass to hydrogen, D-glucose, D-xylose, and D-cellobiose were tested as substrates. The results indicated that those sugars permit a large evolution of hydrogen through cultivation with ethanolamine as a nitrogen source. For instance, the cells grown with 3.5 mM ethanolamine evolved hydrogen of 289 μmol/ml-culture (80％ yield) from 30 mM D-glucose under a controlled pH of 6.4 to 6.9.
ethanolamine; hydrogen production; Rhodobacter capsulatus

-7-
Characterization of the Cellulolytic Complex (Cellulosome)
from Ruminococcus albus

Hiroki OHARA,1 Shuichi KARITA,2 Tetsuya KIMURA,1 Kazuo SAKKA,1 and Kunio OHMIYA1,・

Faculty of Bioresources1 and Center for Molecular Biology and Genetics,2 Mie University, Tsu 514-8507, Japan

Received August 9, 1999; Accepted October 29, 1999
The cellulolytic complex was isolated from the culture supernatant of Ruminococcus albus strain F-40 grown on cellulose by a Sephacryl S-300HR column chromatography. The molecular mass of the cellulolytic complex was found to be larger than 1.5×106 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the cellulolytic complex contained at least 15 proteins with molecular weights from 40 kDa to 250 kDa. Among them, 11 proteins showed endoglucanase and/or xylanase activities on the zymograms. Immunological analysis using an antiserum raised against the dockerin domain of endoglucanase VII of R. albus (DocVII) suggested that at least 7 proteins in the cellulolytic complex contained a dockerin domain immunoreactive with the anti-DocVII antiserum. Furthermore, DocVII was shown to specifically interact with a 40-kDa protein of the cellulolytic complex by Far-Western blot analysis. These results strongly suggest that the cellulolytic complex produced by R. albus resembles the cellulosome specified for the cellulolytic complex of several clostridia such as Clostridium thermocellum and respective components are assembled into the cellulosome by the mechanism common in all of the cellulolytic clostridia, i.e., the cellulosome is formed by the interaction between a dockerin domain of catalytic components and a cohesin domain of a scaffolding protein.
Ruminococcus albus; dockerin domain; cellulosome

-8-
An Organic Solvent Resistant Tyrosinase from Streptomyces sp. REN-21:
Purification and Characterization

Masaaki ITO1 and Kohei ODA2

1Central Laboratory, Rengo Co., Ltd. Fukushima-ku, Osaka 553-0007, Japan
2Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku,
Kyoto 606-8585, Japan

Received August 9, 1999; Accepted October 20, 1999
We found a tyrosinase, which has high activity in the presence of organic solvents, in the culture filtrate of Streptomyces sp. REN-21. The organic solvent resistant tyrosinase (OSRT) was purified from the culture filtrate by three column chromatographies. About 1.2 mg of purified OSRT was obtained from 5.6 liters of the culture filtrate with a yield of 26.0％. The purified enzyme had a single polypeptide chain with a molecular mass of about 32,000 Da. The optimum pH and temperature of OSRT were pH 7.0 and 35°C using L-β-(3,4-dihydroxyphenyl)alanine (L-DOPA) as substrate. OSRT showed stereospecificity toward L-, DL-, and D-enantiomers of DOPA or tyrosine. OSRT had 44％ of the activity of the control even in the presence of 50％ ethanol, while a mushroom tyrosinase showed only 6％ activity under the same conditions. Moreover, OSRT retained its original activity even after 20 h of incubation at 30°C in the presence of 30％ ethanol.
tyrosinase; organic solvent resistance; Streptomyces; purification

-9-
Activation of Maturation Promoting Factor and 26S Proteasome Assembly
Accelerated by a High Concentration of 1-Methyladenine in Starfish Oocytes

Chikako MORINAGA,1 Kazuo IZUMI,1 Hitoshi SAWADA,2 and Michiko TAKAGI SAWADA1,・

1Bioscience and Chemistry Division, Hokkaido National Industrial Research Institute, AIST, MITI,
Sapporo 062-8517, Japan
2Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University,
Sapporo 060-0812, Japan

Received August 9, 1999; Accepted October 25, 1999
In the oocyte maturation process of the starfish Asterina pectinifera, the extent of inhibition of germinal vesicle breakdown (GVBD) by the proteasome inhibitor MG115 (benzyloxycarbonyl-leucyl-leucyl-nor-
valinal), as well as the timing of activation of pre-MPF (inactive maturation promoting factor) and 26S proteasome assembly, were found to be dependent on the concentration of the maturation-inducing hormone 1-methyladenine (1-MeAde). Activation of pre-MPF was accelerated by increasing the concentration of 1-MeAde, while there was little effect on the time required for GVBD. Assembly of the 26S proteasome was also accelerated by increasing the concentration of 1-MeAde. These results indicate that a higher concentration of 1-MeAde triggers acceleration of the assembly and increase in the activity of the 26S proteasome, which results in activation of pre-MPF, although there is little effect on the timing of GVBD. It was also clarified that the timing of GVBD is controlled by a rate-liming step after MPF-activation.
proteasome; 1-methyladenine; oocyte maturation; starfish; cell cycle

-10-
Interspecific Transformation of Bacillus subtilis Competent Cells
by Chromosomal DNA in Lysates of Protoplasts
of Bacillus amyloliquefaciens

Takashi AKAMATSU・ and Hisataka TAGUCHI

Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda 4-22-1,
Kumamoto 860-0082, Japan

Received August 9, 1999; Accepted November 7, 1999
Competent cells of Bacillus subtilis were transformed with chromosomal DNA in lysates of protoplasts of B. subtilis or B. amyloliquefaciens. The interspecific transformation frequency of B. subtilis by cysA in a conserved region was 3.1×104 transformants per μg DNA, 60 times higher than that for conventional transformation using purified DNA. Increased interspecific transformation frequencies of B. subtilis were also observed for arg-1, lys-1, leuB, aroG, thr-5, hisH, or metC markers outside the conserved region (3.1×10~5.2×102 transformants per μg DNA). An interspecific cotransformation ratio (33--50％) as high as an intraspecific one (46％) using purified DNA was also detected between cysA and rpsL markers, which are separated by 16 kb on the B. subtilis chromosome. Interspecific double transformation of the cysA-arg-1 or cysA-metC marker was observed, which have not been detected for conventional transformation. The involvement of mutS in the interspecific transformation was not significant.
Bacillus subtilis; competence; interspecific; transformation

-11-
Thermostable Lipase of Bacillus stearothermophilus: High-level Production,
Purification, and Calcium-dependent Thermostability

Myung-Hee KIM, Hyung-Kwoun KIM,・ Jung-Kee LEE, Sun-Yang PARK, and Tae-Kwang OH

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

Received August 11, 1999; Accepted November 5, 1999
An efficient expression system was developed for the production of the thermostable lipase from Bacillus stearothermophilus L1 in an Escherichia coli system. A structural gene corresponding to mature lipase was subcloned in the pET-22b(＋) expression vector and its expression was induced by IPTG at 30°C in E. coli cells. The lipase activity in a cell-free extract was as high as 448,000 units/g protein, which corresponds to as much as 26％ of the total cellular protein and is 77 times higher than that of E. coli RR1/pLIP1. Based on its pI (7.4) and pH stability data reported previously, the L1 lipase was efficiently purified to homogeneity with CM (at pH 6.0) and DEAE (at pH 8.8) column chromatographies with a recovery yield of 62％. The specific activity of the purified enzyme was 1700 units/mg protein when olive oil emulsion was used as a substrate. Its optimum temperature for the hydrolysis of olive oil was 68°C and it was stable up to 55°C for 30 min-incubation. The thermostability increased by about 8--10 degrees in the presence of calcium ions. This calcium-dependent thermostability was confirmed by the tryptophan fluorescence emission kinetics showing that the enzyme starts to unfold at 66°C in the presence of calcium ions but at 58°C in the absence of calcium ions, implying that the calcium ions bind to the thermostable enzyme and stabilize the protein tertiary structure even at such high temperatures.
lipase; Bacillus stearothermophilus; tryptophan fluorescence emission; calcium-dependent thermostability

-12-
Inhibiting Effects of Theanine on Caffeine Stimulation Evaluated
by EEG in the Rat

Takami KAKUDA, Ayumu NOZAWA, Tomonori UNNO, Noritaka OKAMURA,＊
and Osamu OKAI＊＊

Central Research Institute, Itoen Ltd., 21 Megami, Sagara, Haibara, Shizuoka 421-0516, Japan
＊Department of Medical Laboratory, Ehime College of Health Science, 543 Takouda, Tobe, Iyo,
Ehime 791-2101, Japan
＊＊Department of Physiology, School of Health Science, Kyorin University, 476 Miyashita, Hachioji,
Tokyo 192-0005, Japan

Received August 11, 1999; Accepted November 9, 1999
In this study, the inhibiting action of theanine on the excitation by caffeine at the concentration regularly associated with drinking tea was investigated using electroencephalography (EEG) in rats. First, the stimulatory action by caffeine i.v. administration at a level higher than 5 μmol/kg (0.970 mg/kg) b.w. was shown by means of brain wave analysis, and this level was suggested as the minimum dose of caffeine as a stimulant. Next, the stimulatory effects of caffeine were inhibited by an i.v. administration of theanine at a level higher than 5 μmol/kg (0.781 mg/kg) b.w., and the results suggested that theanine has an antagonistic effect on caffeine′s stimulatory action at an almost equivalent molar concentration. On the other hand, the excitatory effects were shown in the rat i.v. administered 1 and 2 μmol/kg (0.174 and 0.348 mg/kg) b.w. of theanine alone. These results suggested two effects of theanine, depending on its concentration.
theanine; caffeine; stimulant action; EEG; tea

-13-
α-Glucosidase Inhibitors from Clove (Syzgium aromaticum)

Miou TODA, Jun KAWABATA,・ and Takanori KASAI

Laboratory of Food Biochemistry, Division of Applied Bioscience, Research School of Agriculture,
Hokkaido University, Kitaku, Sapporo 060-8589, Japan

Received August 12, 1999; Accepted October 23, 1999
The ellagitannins, casuarictin and eugeniin, were isolated as rat intestinal maltase inhibitors from methanol extracts of clove (Syzgium aromaticum). Eugeniin showed inhibitory activity with an IC50 value of 10−3 M. A structure-activity relationship study among the isolates and their related compound, penta-O-galloyl-β-D-glucose, indicates that an increasing number of galloyl units in the molecule might lead to an increase in the inhibitory activity. Eugeniin also inhibited maltase activity toward the human intestinal epithelial cell line, Caco-2.
α-glucosidase; inhibitor; clove (Syzgium aromaticum); ellagitannin; Caco-2

-14-
1-Aminocyclopropane-1-carboxylate (ACC) Deaminase Induced
by ACC Synthesized and Accumulated in Penicillium citrinum
Intracellular Spaces

Yan-Jun JIA,・ Hiroyuki ITO, Hirokazu MATSUI, and Mamoru HONMA

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received August 12, 1999; Accepted October 4, 1999
We have already described how 1-aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of the plant hormone ethylene, is synthesized in Penicillium citrinum through the same reaction by the catalysis of ACC synthase [EC 4.4.1.14] as in higher plants. In addition, ACC deaminase [EC 4.1.99.4], which degrades ACC to 2-oxobutyrate and ammonia, was also purified from this strain. To study control of induction of ACC deaminase in this organism, we have isolated and analyzed the cDNA of P. citrinum ACC deaminase and studied the expression of ACC deaminase mRNA in P. citrinum cells. By the analysis of peptides from the digests of the purified and modified ACC deaminase with lysylendopeptidase, 70％ of its amino acid sequences were obtained. These amino acid sequences were used to identify a cDNA, consisting of 1,233 bp with an open reading frame of 1,080 bp encoding ACC deaminase with 360 amino acids. The deduced amino acids from the cDNA are identical by 52％ and 45％ to those of enzymes of Pseudomonas sp. ACP and Hansenula saturnus. Through Northern blot analysis, we found that the mRNA of ACC deaminase was expressed in P. citrinum cells grown in a medium containing 0.05％ L-methionine. These findings suggest that ACC synthesized by ACC synthase and accumulated in P. citrinum intracellular spaces can induce the ACC deaminase that degrades the ACC.
1-aminocyclopropane-1-carboxylic acid (ACC); ACC deaminase; ACC synthase; Penicillium citrinum

-15-
Studies on the 1,1-Diphenyl-2-picrylhydrazyl Radical Scavenging Mechanism
for a 2-Pyrone Compound

Naoki ABE,・ Aya NEMOTO, Yukiko TSUCHIYA, Hiroshi HOJO, and Akira HIROTA

Laboratory of Applied Microbiology, School of Food and Nutritional Sciences, University of Shizuoka,
52-1 Yada, Shizuoka 422-8526, Japan

Received August 12, 1999; Accepted October 18, 1999
The radical scavenging mechanisms for the 2-pyrone compound, 4-hydroxy-3,6-dimethyl-2H-pyrane-2-one (1), and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (4) in several solvent systems were evaluated by the quantitative change in compounds detected at 270 nm and subsequent HPLC analyses. The HPLC profile for each condition suggested that the reaction proceeded by a different mechanism in each solvent system. In organic solvents (CHCl3, iso-propanol, and EtOH), 1-[4-(3,4-dihydro-3,6-dimethyl-2,4-dioxo-2H-pyran-3-yl) phenyl]-1-phenyl-2-picrylhydrazine (2) was produced as an adduct of the DPPH radical and 1. On the other hand, the reaction in a buffer solution (an acetate buffer at pH 5.5) gave several degradation products with 1-[4-(2,3-dihydro-2,5-dimethyl-3-oxo-fur-2-yl) phenyl]-1-phenyl-2-picrylhydrazine (5), this being structurally elucidated by spectroscopic analyses. The decrease of the DPPH radical in each reaction system suggests that compound 1 could scavenge about 1.5--1.8 equivalents of the radical in organic solvents and about 3.5--3.9 in the buffer solution.
1,1-diphenyl-2-picrylhydrazyl; radical scavenger; radical scavenging mechanism; 2-pyrone

-16-
Role of Basic Residues of Human Lactoferrin in the Interaction
with B Lymphocytes

Yoshihiro KAWASAKI,・ Kaoru SATO, Hiroshi SHINMOTO, and Shun′ichi DOSAKO

Technical Research Institute, Snow Brand Milk Products Co. Ltd., 1-1-2 Minamidai, Kawagoe 350-1164, Japan

Received August 16, 1999; Accepted October 12, 1999
We have previously demonstrated that lactoferrin was incorporated into B lymphocytes and that a trypsin treatment for a short period reduced the number of lactoferrin molecules incorporated into B lymphocytes. An N-terminal sequence analysis revealed that the mild trypsin treatment had cleaved the three N-terminal amino acids, Gly1-Arg2-Arg3. Chemical conjugation of lost sequence analogue Gly-Arg-Arg-Gly with the mildly digested lactoferrin recovered the interaction with B lymphocytes, while conjugation of acetyl-Arg-Arg-Gly, a deamino analogue of Gly-Arg-Arg-Gly, did not recover the interaction. This shows that the N-terminal basic region containing N-terminal Gly played an important role in the interaction with B lymphocytes. Acylation of the amino groups of lactoferrin also significantly reduced the interaction with B lymphocytes, and an O-methylisourea treatment of the amino groups, which preserved the positive charge, hardly affected the interaction. These results suggest that both the N-terminal basic region and the basic characteristics of the whole molecule contributed to its interaction with B lymphocytes.
lactoferrin; peptide conjugation; B lymphocytes; amino groups acylation

-17-
Subsite Structure and Catalytic Mechanism of a New Glycosyltrehalose-
producing Enzyme isolated from the Hyperthermophilic Archaeum,
Sulfolobus solfataricus KM1・

Masaru KATO,・・ Kyoko TAKEHARA, Masako KETTOKU, Kazuo KOBAYASHI,＊
and Toshiyuki SHIMIZU＊＊

Applied Bioresearch Center and ＊＊Pharmaceutical Research Laboratories, Kirin Brewery Co., Ltd., 3,
Miyahara, Takasaki-shi, Gunma 370-1295, Japan, ＊Central Laboratories for Key Technology,
Kirin Brewery Co., Ltd., 1-13-5, Fukuura, Kanazawa-ku, Yokohama-shi,
Kanagawa 236-0004, Japan

Received August 20, 1999; Accepted November 16, 1999
A glycosyltrehalose-producing enzyme from Sulfolobus solfataricus KM1 catalyzes a conversion of maltooligosaccharides to glycosyltrehaloses and also hydrolyzes maltooligosaccharides to liberate glucose, as a side reaction. From the sum of the conversion and hydrolysis reaction rates, the rate parameters involved in the ``splitting′′ of the α-1,4 glucosidic linkage were calculated. From the data obtained, the subsite structure for maltooligosaccharides was identified. From the analysis of the hydrolysate of maltotriose in [18O] labeled H2O, the hypothesis of the C1-O bond splitting and the formation of a glycosyl (maltosyl)-enzyme intermediate was strongly supported. From the analysis of the reaction product in the presence of [3H] labeled glucose, the occurrence of intermolecular transglycosylation was confirmed. These data strongly support the suggestion that the catalytic mechanism of this enzyme is a transglycosylation.
trehalose; glycosyltransferase; subsite structure; catalytic mechanism; Sulfolobus solfataricus

-18-
Apoptosis Induced by Nicotinamide-related Compounds and Quinolinic Acid
in HL-60 Cells

Shin OGATA,1 Masayo TAKEUCHI,1 Hiroaki FUJITA,1 Katsumi SHIBATA,2
Katsuzumi OKUMURA,1 and Hiroshi TAGUCHI1,＊

1Laboratory of Biological Chemistry, Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
2Course of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures,
The University of Shiga Prefecture, Hikone, Shiga 522-8533, Japan

Received August 20, 1999; Accepted October 4, 1999
In our previous paper, we have reported that niacin-related compounds, particularly picolinic acid, dipicolinic acid, and isonicotinamide, induced apoptosis in HL-60 cells but that niacin did not. Moreover, picolinamide, N1-methylnicotinamide, 6-aminonicotinamide, quinolinic acid, and cinchomeronic acid also had the function of DNA fragmentation in HL-60 cells analyzed by flow cytometry, the ratio of DNA fragmentation finally being about 40％ after treatment with these compounds at 10 mM for 24 h. In this study, we
found that these compounds also induced apoptosis in HL-60 cells. The wide-spectrum caspase inhibitors prevented DNA fragmentation induced by these compounds. Interestingly, 6-aminonicotinamide induced apoptosis at a comparatively low concentration, while picolinic acid, dipicolinic acid, and isonicotinamide did not at 1 mM. Our results suggest that both NAD metabolism and NAD biosynthesis may be related to the process of apoptosis induced by niacin-related compounds.
niacin; NAD; apoptosis; HL-60; flow cytometry

-19-
Purification and Some Properties of a β-Glucosidase
from Flavobacterium johnsonae

Katsuyuki OKAMOTO,1,・ Hirofumi NAKANO,2 Tsunneya YATAKE,1 Taro KISO,2
and Sumio KITAHATA2

1Showa Sangyo Co., Ltd., 16, Sakura 1-chome, Tukubashi, Ibaraki, 350-0003, Japan
2Osaka Municipal Technical Research Inst., 6-50, Morinomiya 1-chome, Joto-ku, Osaka 536-8553, Japan

Received August 23, 1999; Accepted October 20, 1999
Flavobacterium johnsonae was isolated as a microorganism that produced a β-glucosidase with hydrolytic activity of β-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point of pI 8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature was 45°C, and the enzyme was stable below 35°C. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad β-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl β-glucosides such as p-nitrophenyl β-glucoside, phenyl β-glucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg2＋, and reduced by Fe3＋, Cu2＋, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9--84.8％). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl β-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.
β-glucosidase; Flavobacterium johnsonae; steviol glycosides; β-glucoysyl ester linkage

-20-
Synthesis of Four Stereoisomers of 1,4-Thiazane-3-carboxylic Acid 1-Oxide via
the Asymmetric Transformation (combined isomerization-preferential
crystallization) of 1,4-Thiazane-3-carboxylic Acid

Tadashi SHIRAIWA,・ Kohya TADOKORO, Joji ISHIKAWA, Haruyuki TANAKA,
Tooru MASAKI, and Hidemoto KUROKAWA

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

Received August 26, 1999; Accepted October 8, 1999
In order to synthesize four stereoisomers of 1,4-thiazane-3-carboxylic acid 1-oxide (TCA・SO), (S)-1,4-thiazane-3-carboxylic acid [(S)-TCA], which is one of the precursors, was prepared by the asymmetric transformation (combined isomerization-preferential crystallization) of (RS)-TCA. This asymmetric transformation was used (2R, 3R)-tartaric acid [(R)-TA] as a resolving agent and salicylaldehyde as the epimerization catalyst in propanoic acid at 110°C to afford a salt of (S)-TCA with (R)-TA in 100％ de with a yield of over 90％. Optically pure (S)-TCA was obtained by treating the salt with triethylamine in methanol in a yield of over 80％, based on (RS)-TCA as the starting material. In addition, asymmetric transformation of (R)-TCA gave (S)-TCA in a yield of 60--70％. (S)-TCA was oxidized by hydrogen peroxide in dilute hydrochloric acid to selectively crystallize (1S, 3S)-TCA・SO. (1R, 3S)-TCA・SO of 70％ de from the filtrate was allowed to form a salt with (R)-TA after a treatment with triethylamine to give (1R, 3S)-TCA・SO as a single diastereoisomer. (1R, 3R)- and (1S, 3R)-TCA・SO were also prepared by starting from (R)-TCA that had been synthesized from L-cysteine.
1,4-thiazane-3-carboxylic acid 1-oxide; 1,4-thiazane-3-carboxylic acid; asymmetric transformation; tartaric acid

-21-
Inhibitory Effects of Bovine Lactoferrin on the Adherence of Enterotoxigenic
Escherichia coli to Host Cells

Yoshihiro KAWASAKI,1,・ Seiki TAZUME,2 Keiko SHIMIZU,2 Hideyuki MATSUZAWA,2
Shun′ichi DOSAKO,1 Hiroko ISODA,1 Mami TSUKIJI,3 Ryuko FUJIMURA,2
Yoko MURANAKA,2 and Hiroshi ISIHIDA4

1Technical Research Institute, Snow Brand Milk Products Co., 1-1-2 Minamidai, Kawagoe 350-1164, Japan
2Tokai University School of Health Sciences, Boseidai, Isehara 259-1193, Japan
3Kanagawa Prefectural College of Medical Technology, 1-5-1 Nakao, Asahi-ku, Yokohama 241-0815, Japan
4Junior College, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-0054, Japan

Received August 31, 1999; Accepted October 22, 1999
Adherence is an essential and prerequisite step for the colonization of mucosal surfaces by enterotoxigenic Escherichia coli (ETEC). We studied the effect of bovine lactoferrin (BLF) on the adherence of ETEC to human epithelial cells in vitro, and to intestinal mucosa of ICR germfree mice in vivo. In the in vitro study, BLF was found to inhibit the adherence of ETEC. This adhesion-inhibiting activity of BLF was found to lessen with decreasing BLF concentration, but the data obtained suggest a positive inhibitory effect of BLF against the adhesion of ETEC cells. In the in vivo study, the counts of adherent bacteria in various sections of the intestinal tract (duodenum, jejunoileum, and large intestine) were lower in the BLF group than in the control group, suggesting the possible action of BLF as an intestinal tract adherence-blocking agent with regards to ETEC.
Escherichia coli adherence; bovine lactoferrin; enterotoxigenic Escherichia coli; germfree mice; human epithelial cell

-22-
Isolation of a cDNA Encoding a 31-kDa, Pathogenesis-related
5/thaumatin-like (PR5/TL) Protein Abundantly Expressed
in Apple Fruit (Malus domestica cv. Fuji)

Dong Ha OH, Kwan Jeong SONG,＊ Yong Uk SHIN,＊ and Won-Il CHUNG・

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusung-dong, Yusung-gu, Taejon 305-701, Korea
＊National Horticultural Research Institute, 475 Imok-dong, Suwon 440-310, Korea

Received September 2, 1999; Accepted November 11, 1999
A fruit-specific and pathogenesis-related 5/thaumatin-like (PR5/TL), 31-kDa protein was isolated by 2D-PAGE from fully-grown apples (Malus domestica cv. Fuji) and named Mdtl1 (Malus domestica thaumatin-like protein 1). Using the N-terminal sequence of the protein, the full-length cDNA encoding Mdtl1 was isolated. The cDNA clone (Mdtl1) consists of 944 bp with an open reading frame (ORF) of 744 bp encoding a protein of 247 amino acids. The deduced amino acid sequence of Mdtl1 shows high similarity to the sequences of PR5/TL proteins. Mdtl1 is a slightly acidic protein with a putative signal peptide and a putative N-glycosylation site, and lacks a C-terminal extension. This suggests that Mdtl1 is an apoplastic glycoprotein. Results of northern blotting indicated that expressions of Mdtl1 are developmentally regulated. Southern blot analysis showed that Mdtl1 may be present as a single copy, and there exist other genes closely related to Mdtl1 in the apple genome.
thaumatin-like; pathogenesis-related; apple (Malus domestica cv. Fuji); fruit-specific; Mdtl1

-23-
Synthesis of Regioselectively Protected Forms of Cytidine Based
on Enzyme-catalyzed Deacetylation as the Key Step

Atsuhito KUBOKI,＊ Takashi ISHIHARA,＊ Eiko KOBAYASHI,＊ Hiromichi OHTA,＊ Takeshi ISHII,＊＊ Ayumu INOUE,＊＊ Satoshi MITSUDA,＊＊ Tatsuo MIYAZAKI,＊＊＊ Yasuhiro KAJIHARA,＊＊＊ and Takeshi SUGAI＊,・

＊Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
＊＊Biotechnology Laboratory, Sumitomo Chemical Co. Ltd., 4-2-1 Takatsukasa, Takarazuka 665-0051, Japan
＊＊＊Department of System Function, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama
236-0027, Japan

Received September 2, 1999; Accepted October 7, 1999
N4-Acetylcytidine (77％) and 2′,3′-O,N4-triacetylcytidine (95％) were obtained from the hydrolysis of a common precursor, the peracetylated form of cytidine with Aspergillus niger lipase (Amano A) and Burkholderia cepacia esterase (SC esterase S), respectively, under very mild conditions. The experimental procedure for the conversion of triacetylcytidine to a corresponding phosphoramidite (82％), an intermediate for sugar nucleotide synthesis, is also elaborated.
cytidine; esterase; lipase; selective deacetylation; phosphorylation

-24-
Cloning and Characterization of EPD2, a Gene Required for Efficient
Pseudohyphal Formation of a Dimorphic Yeast, Candida maltosa

Takanobu NAKAZAWA,・ Maki TAKAHASHI, Hiroyuki HORIUCHI,＊ Akinori OHTA,
and Masamichi TAKAGI

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received September 6, 1999; Accepted October 13, 1999
Candida maltosa is a dimorphic fungus and its pseudohyphal growth is partly induced by additional copies of the centromeric DNA (CEN) region1) or n-alkane. In the course of analyzing the induction mechanism of pseudohyphal growth, we isolated EPD1, which is similar in sequence to PHR1 and PHR2 of Candida albicans. Epd1p could be involved in cell wall maintenance and is essential for pseudohyphal growth induced by CEN and n-hexadecane at pH 4 and by n-hexadecane at pH 7.2) In this paper, we cloned EPD2 of C. maltosa, which is highly similar to EPD1, PHR1, and PHR2. The transcription of EPD2 is induced strongly when cells are grown in SD medium of higher pH (pH 7), but not in SD medium of lower pH (pH 4). This pattern of expression was an inverse of that of EPD1. This alternate expression is similar to that between PHR1 and PHR2. The expression of EPD2 was much higher when C. maltosa was grown on the n-hexadecane solid medium than grown in the n-hexadecane liquid medium. The efficiency of pseudohyphal formation of an epd2 null mutant on n-hexadecane medium at pH 7 or 7.5 was lower than that of the wild-type strain. These results suggest that Epd2p is required for efficient pseudohyphal formation induced by n-hexadecane in the medium at pH 7.
pseudohyphal growth; CEN; Candida maltosa

-25-
Identification of α-D-Glucosylglycerol in Sake＊

Fumihito TAKENAKA,＊＊ Hirofumi UCHIYAMA, and the lateTakeshi IMAMURA

Tatsuuma-honke Brewing Co., Ltd., 2-6 Tateishi-cho, Nishinomiya City, Hyogo 662-0943, Japan

Received September 16, 1999; Accepted October 25, 1999
α-D-Glucosylglycerol (GG) was found for the first time in sake (Japanese rice wine) in an amount of about 0.5％. GG was also found in miso and mirin which had been brewed by using koji. GG was hydrolyzed into glucose and glycerol in an equimolar ratio with maltase (EC 3.2.1.20, α-glucosidase from yeast), but not with emulsin (EC 3.2.1.21, β-glucosidase from almond). The retention times and mass spectra of trimethylsilyl derivatives by a GC-MS analysis of GG in sake were comparable to those of various GG samples synthesized by glycol cleavage. It was proven that GG in sake consisted of three components, viz., 2-O-α-D-glucosylglycerol (GG-II), (2R)-1-O-α-D-glucosylglycerol (R-GG-I) and (2S)-1-O-α-D-glucosylglycerol (S-GG-I). The ratio of the three components in GG was 6:66:28 for sake. It is considered that GG was formed by transglucosylation of the glucosyl groups to glycerol by α-glucosidase from koji in the sake mash.
α-D-glucosylglycerol; identification; α-glucosidase; transglucosylation; sake

-26-
An Efficient Method for Production of Uridine
5′-Diphospho-N-Acetylglucosamine

Kiyoshi OKUYAMA, Tomoki HAMAMOTO, Kazuya ISHIGE, Kenji TAKENOUCHI,
and Toshitada NOGUCHI

Biochemicals Division, Yamasa Corporation, Choshi, Chiba 288-0056, Japan

Received September 20, 1999; Accepted November 5, 1999
Uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) has been synthesized by a yeast-based method from 5′-UMP and glucosamine, in which yeast cells catalyze the conversion of 5′-UMP to 5′-UTP and provide enzymes involved in UDP-GlcNAc synthesis using 5′-UTP and glucosamine as substrates. However, this conventional method is not suitable for practical production of UDP-GlcNAc because of the low yield of the product. We found that the yqgR gene product of Bacillus subtilis, which has been identified as a glucokinase, can catalyze the phosphorylation of N-acetylglucosamine (GlcNAc) to give GlcNAc-6-phosphate, an intermediate of UDP-GlcNAc biosynthesis. The addition of the yqgR gene product to the yeast-based reaction system enabled us to synthesize UDP-GlcNAc using GlcNAc in place of glucosamine. The addition of two enzymes, GlcNAc-phosphate mutase and UDP-GlcNAc pyrophosphorylase, increased the yield of UDP-GlcNAc. Using this novel method, UDP-GlcNAc was produced at an amount of 78 mM from 100 mM 5′-UMP and 100 mM GlcNAc.
UDP-GlcNAc; glucokinase; GlcNAc-phosphate mutase; UDP-GlcNAc pyrophosphorylase

-27-
Cloning and Expression of the Momordica charantia Trypsin Inhibitor II Gene
in Silkworm by using a Baculovirus Vector

Shin-ichi SATO, Kaeko KAMEI,・ Mai TANIGUCHI, Hideki SATO, Ryo TAKANO, Hajime MORI, Masatoshi ICHIDA,＊ and Saburo HARA

Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
＊Experimental Farms, Faculty of Textile Science, Kyoto Institute of Technology, Sagaippongi-cho, Ukyo-ku, Kyoto 616-8354, Japan

Received September 20, 1999; Accepted October 27, 1999
MCTI-II (Momordica charantia trypsin inhibitor II) isolated from bitter gourd (Momordica charantia LINN.) seeds is one of the serine protease inhibitors of the squash family. We cloned cDNA that encodes MCTI-II and constructed an expression system for MCTI-II by using a baculovirus vector. The recombinant baculovirus was inoculated to early fifth-instar larvae of the silkworm (strain: Shunrei×Shougetsu). Four days after infection, the hemolymph of silkworm larvae was collected and the recombinant protein was purified. Two kinds of expressed MCTI-II protein were obtained. An amino acid sequence analysis of the two proteins indicates that both were similar to the authentic inhibitor, except for the addition of a tripeptide derived from the vector at the N-terminus. One of the two inhibitors (MCTI-II A) resulted in a single PTH-amino acid in each Edman degradation cycle, while the other (MCTI-II B) resulted in two PTH-amino acids, suggesting the occurrence of cleavage of the reactive site. The inhibitory activities of MCTI-II expressed toward trypsin are examined in terms of the Ki value, these being 6.4×10−10 M for MCTI-II A and 5.2×10−10 M for MCTI-II B.
trypsin inhibitor; squash; expression; baculovirus; silkworm

-28-
Production of Isopropyl cis-6-Hexadecenoate by Regiospecific Desaturation
of Isopropyl Palmitate by a Double Mutant of a Rhodococcus Strain

Kenzo KOIKE,＃ Mikio TAKAIWA, Katsutoshi ARA, Shigeo INOUE, Yoshiharu KIMURA, and Susumu ITO・

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

Received October 4, 1999; Accepted November 1, 1999
Resting cells of a double mutant noted as KSM-MT66, derived from Rhodococcus sp. strain KSM-B-3 by UV irradiation, were found to cis-desaturate isopropyl hexadecanoate, yielding isopropyl cis-6-hexadecenoate. Addition of sodium glutamate (1.0％), MgSO4 (2 mM), and thiamine (2 mM) increased the productivity of the unsaturated product in phosphate buffer. Optimal temperature and pH for the reaction were around 26°C and 7, respectively. Under the optimized conditions, more than 50 g/l of isopropyl cis-6-hexadecenoate was produced after a 3-day incubation by resting cells of the mutant. Thus, cis-6-hexadecenoic acid, the main component of human sebaceous lipids, can be manufactured economically by the rhodococcal bioconversion.
Rhodococcus; desaturase; regiospecificity; cis-6-hexadecenoic acid; sebaceous lipid

-29-
Note
Amplifing Effect of Dietary Taurine on the Induction of Cytochrome P-450
and on the Urinary Excretion of Ascorbic Acid in Rats Fed
on Phenobarbital-containing Diets

Hideki MOCHIZUKI, Jun TAKIDO, Hiroaki ODA,＊ and Hidehiko YOKOGOSHI・

School of Food and Nutritional Sciences, The University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
＊Department of Applied Molecular Biosciences, Nagoya University, Nagoya 464-8601, Japan

Received July 26, 1999; Accepted October 14, 1999
Dietary taurine amplified the induction of cytochrome P-450 and the urinary excretion of ascorbic acid in rats fed on phenobarbital (PB)-containing diets. These facts suggest that taurine could influence the hepatic metabolism of xenobiotics via the induction of drug-metabolizing enzymes (DME) and the ascorbic acid metabolism. Taurine might improve the function of DME exposed by some xenobiotics.
taurine; phenobarbital; cytochrome P-450; ascorbic acid; xenobiotics

-30-
Note
Purification and Some Properties of High-molecular-weight Xylanases,
the Xylanases 4 and 5 of Aeromonas caviae W-61

Narayan ROY,1,＃ Naoko OKAI,1 Toshio TOMITA,1 Koji MURAMOTO,2 and Yoshiyuki KAMIO1,＊

1Laboratory of Applied Microbiology and 2Laboratory of Applied Phytochemistry, Tohoku University Graduate School of Agricultural Science, Aoba-ku, Sendai 981-8555, Japan

Received July 30, 1999; Accepted October 27, 1999
Aeromonas caviae W-61 produces multiple extracellular xylanases, the xylanases 1, 2, 3, 4, and 5 [Nguyen, V. D. et al., Biosci. Biotechnol. Biochem., 56, 1708--1712 (1993)]. Here we purified and characterized high-molecular-weight xylanases, the xylanases 4 and 5 from the culture fluids of the bacterium. The purified xylanases 4 and 5, which had molecular masses of 120 and 140 kDa, respectively, were endo-β-1,4-xylanases with similar enzymatic properties except for transxylosidase activity. The xylanase 4 showed a prominent transxylosidase activity when xylotriose and xylotetraose were used as the substrates, while the xylanase 5 had little transxylosidase activity under the same conditions. Protein sequencing indicated that the xylanase 4 was a C-terminally-truncated xylanase 5, suggesting that the C-terminal truncation of the xylanase 5 may endow the enzyme with transxylosidase activity.
Aeromonas caviae; xylanase; purification; enzyme characteristics; transxylosidase

-31-
Note
Antimutagenicity of the Purple Pigment, Hordeumin, from Uncooked Barley
Bran-Fermented Broth

Tomoaki DEGUCHI, Makoto YOSHIMOTO,＊ Riichiro OHBA, and Seinosuke UEDA

Department of Applied Microbial Technology, Faculty of Engineering, Kumamoto Institute of Technology, 4-22-1 Ikeda, Kumamoto 860-0082, Japan
＊Department of Upland Farming, Kyushu National Agricultural Experiment Station, 6644 Yokoichi-cho, Miyakonojo, Miyazaki, 885-0091, Japan

Received August 9, 1999; Accepted November 15, 1999
The novel purple pigment hordeumin, an anthocyanin-tannin pigment, was produced from barley bran-fermented broth. The mutagenicity or antimutagenicity of hordeumin was investigated according to the Ames method, an indication of the safety of food, using Salmonella typhimurium TA98. Despite the presence of S-9 mix, hordeumin was not mutagenic. On the other hand, hordeumin effectively decreased a reverse mutation from Trp-P-1, Trp-P-2, IQ, and B[a]P. Furthermore, hordeumin also decreased the reverse mutation from dimethyl sulfoxide extracts of grilled beef.
hordeumin; barley bran; antimutagenicity; Salmonella typhimurium TA98

-32-
Note
Effect of a Polysaccharide (TAP) from the Fruiting Bodies of Tremella aurantia
on Glucose Metabolism in Mouse Liver

Tadashi KIHO,・ Hotaka MORIMOTO, Takashi KOBAYASHI, Shigeyuki USUI, Shigeo UKAI,
Koichi AIZAWA,＊ and Takahiro INAKUMA＊

Department of Pharmaceutics, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan
＊Research Institute, Kagome Co., Ltd., 17 Nishitomiyama, Nishinasuno-machi, Nasu-gun,
Tochigi 329-2762, Japan

Received August 9, 1999; Accepted November 2, 1999
An acidic polysaccharide (TAP) obtained from the fruiting bodies of Tremella aurantia significantly increased the activities of glucokinase, hexokinase, and glucose-6-phosphate dehydrogenase, and decreased the activity of glucose-6-phosphatase in normal and diabetic mouse liver after intraperitoneal administration, while the glycogen content in the liver was reduced. Furthermore, TAP lowered the plasma cholesterol level in normal and diabetic mice.
Tremella aurantia; polysaccharide; glucokinase; glucose-6-phosphatase; cholesterol

-33-
Note
Coumarin-related Compounds as Plant Growth Inhibitors from Two Rutaceous Plants in Thailand

Suratwadee JIWAJINDA,1,・ Vilai SANTISOPASRI,2 and Hajime OHIGASHI3

1Central Laboratory and Greenhouse Complex, Kasetsart University, Kamphaengsaen Campus,
Nakhon-Pathom 73140, Thailand
2Department of Biochemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900, Thailand
3Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received August 11, 1999; Accepted October 9, 1999
Chemical investigation of naturally occurring plant growth inhibitors from Rutaceous plants in Thailand led us to identify five 7-methoxycoumarins and one 5,7-dimethoxycoumarin from Murraya paniculata, and six furanocoumarins from Citrus aurantifolia. Of these compounds, murranganon senecioate (1) is a new natural compound found in M. paniculata. Minumicrolin (6) was found to be highly active against the 2nd leaf sheath elongation of rice seedlings.
Murraya paniculata; Citrus aurantifolia; coumarin; furanocoumarin; plant growth inhibitor

-34-
Note
A New Type of Glycoglycerolipids from Corynebacterium aquaticum

Hidekuni YANAGI, Motoko MATSUFUJI,1 Kuniho NAKATA,1 Yasunori NAGAMATSU,
Shinji OHTA,2 and Akihiro YOSHIMOTO・

Faculty of Applied Biological Science, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima 739-8528, Japan
1Central Research Laboratories, Mercian Corporation, Johnan 4-9-1, Fujisawa 251-0047, Japan
2Instrument Center for Chemical Analysis, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8526, Japan

Received August 16, 1999; Accepted October 18, 1999
A new type of glycoglycerolipids, S361A and S365A, were obtained from Corynebacterium aquaticum strains, S361 and S365, newly isolated from soils, and were identified as (2R)-1-[α-glucopyranosyl-(1α-3)-(6-O-acyl-α-mannopyranosyl)]-3-O-acylglycerol and (2R)-1-[α-mannopyranosyl-(1α-3)-(6-O-acyl-α-mannopyranosyl)]-3-O-acylglycerol, respectively. S365A was identical to a novel glycoglycerolipid recently isolated from some bacteria, but S361A was a new analog having a glucosylmannosyl in place of the dimannosyl group. Our results indicate that this sn-2 lysotype of glyceroglycolipids may be widely distributed in bacteria.
glycoglycerolipid; sn-2-lysoglycero-
glycolipid; diglycosyl monoacylglycerol; Corynebacterium aquaticum

-35-
Note
Cloning and Sequencing of the Maltohexaose-producing Amylase Gene
of Klebsiella pneumoniae

Mitsuru MOMMA・

Department of Biotechnology, National Institute of Agrobiological Resources, P.O. Box 12, Tsukuba Norin, Ibaraki 305-8602, Japan

Received August 17, 1999; Accepted October 19, 1999
The molecular characterization of the maltohexaose-producing amylase gene of Klebsiella pneumoniae revealed an open reading frame in which 2,031 base pairs encode a protein of 677 amino acids with a calculated molecular weight of 75,921. The amylase gene had high similarities of 73.6％ in DNA sequence and 79.3％ in deduced amino acid sequence with the periplasmic α-amylase MalS gene of Escherichia coli.
gene cloning; amylase; maltohexaose; Klebsiella pneumoniae

-36-
Note
Lysyl-tRNA Synthetase of Bacillus stearothermophilus Molecular Cloning
and Expression of the Gene1

Teisuke TAKITA,＊ Naofumi SHIMIZU,＊＊ Tokuo SUKATA,＊＊ Satoshi HASHIMOTO,＊＊
Eriko AKITA,＊＊ Takuya YOKOTA,＊＊ Nobuyoshi ESAKI,＊＊＊ Kenji SODA,＊＊＊ Kuniyo INOUYE,＊
and Ben′ichiro TONOMURA＊＊,2,・

＊Division of Applied Life Sciences, Graduate School of Agriculture, and ＊＊Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan
＊＊＊Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

Received August 23, 1999; Accepted October 27, 1999
The gene of the lysyl-tRNA synthetase of Bacillus stearothermophilus NCA1503 was cloned and sequenced. The gene consists of 1485 bp nucleotides commencing with an ATG start codon and ending with a TAA stop codon, and encodes a polypeptide of 493 amino acids. The recombinant enzymes were expressed in E. coli using an expression plasmid containing the T7 RNA polymerase/promoter.
Bacillus stearothermophilus; gene cloning; gene expression; lysyl-tRNA synthetase

-37-
Note
Basidiomycete Fungal Gene Encoding a Regulatory Subunit A Homologue
of Protein Phosphatase 2A

Takayuki ISHIZAKI, Hiroshi YOKOYAMA, Susumu KAJIWARA, 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 August 30, 1999; Accepted October 18, 1999
A gene, Le.paa, encoding a regulatory subunit A (PR65) homologue of protein phosphatase 2A was isolated from the basidiomycete mushroom Lentinus edodes. The deduced Le.paa gene product (Le.PR65) had the highest sequence similarity to the Schizosaccharomyces pombe PR65 protein (54.1％ similarity). The Le.paa gene was shown to be transcribed more actively during the late stages of fruiting development of the fungus. Gill tissue in which basidiospores are formed contained abundant Le.paa transcript as compared with gill-depleted pileus and stipe.
basidiomycete; Northern blotting; protein phosphatase 2A; recombinant DNA; regulatory subunit A

-38-
Note
Isolation and Identification of the Probing Stimulants in the Rice Plant
for the White-back Planthopper, Sogatella furcifera
(Homoptera: Delphacidae)

Francis ADJEI-AFRIYIE, Chul-Sa KIM,・ Masami TAKEMURA, Masahiro ISHIKAWA,
and Michio HORIIKE

Department of Bioresources Science, Faculty of Agriculture, Kochi University, B200 Monobe,
Nankoku 783-8502, Japan

Received September 1, 1999; Accepted October 18, 1999
Adult females of the white-back planthopper, Sogatella furcifera, showed characteristic behavior of stylet sheath deposit on a parafilm membrane when fed on a 2％ aqueous crude rice leaf and stem extract containing 15％ sucrose. Subsequent bioassays revealed that the butanol-soluble fraction of the extract was highly active against the insects. When the butanol fraction was chromatographed on an ODS open column and eluted in sequence with a mixture of an increasing concentration of methanol in water, the 40％ methanol fraction was separated as the most active. A further bioassay of the HPLC components in the active fraction revealed that two major components (1 and 3) stimulated the high probing activity of the white-back planthopper only when they were combined. Of the active components, one component (3) was identified to be tricin 5-O-glucoside by spectroscopic analyses.
Sogatella furcifera; probing stimulant; multi-component system; tricin 5-O-glucoside; rice plant

-39-
Note
Purification and Characterization of a Family G/11 β-Xylanase
from Streptomyces olivaceoviridis E-86

Satoshi KANEKO,1,・ Atsushi KUNO,2,3 Mizuho MURAMATSU,4 Shinnosuke IWAMATSU,2
Isao KUSAKABE,4 and Kiyoshi HAYASHI1

1National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2Department of Material and Biological Chemistry, Faculty of Science, Yamagata University,
Yamagata 990-8560, Japan
3National Institute for Advanced Interdisciplinary Research, 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan
4Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoodai, Tsukuba,
Ibaraki 305-8572, Japan

Received September 2, 1999; Accepted October 18, 1999
A β-xylanase (GXYN) was purified from the culture filtrate of Streptomyces olivaceoviridis E-86 by successive chromatography on DE-52, CM-Sepharose and Superose 12. The molecular mass of the xylanase was estimated to be 23 kDa, indicating that the enzyme consists of a catalytic domain only. The enzyme displayed an optimum pH of 6, a temperature optimum of 60°C, a pH stability range from 2 to 11 and thermal stability up to 40°C. The N-terminal amino acid sequence of GXYN was A-T-V-I-T-T-N-Q-T-G-T-N-N-G-I-Y-Y-S-F-W-, and sharing a high degree of similarity with the N-terminal sequence of xylanases B and C from Streptomyces lividans, indicating GXYN belongs to family G/11 of glycoside hydrolases. GXYN was inferior to xylanase B from Streptomyces lividans in the hydrolysis of insoluble xylan because of its lack of a xylan binding domain.
family G/11 xylanase; purification; Streptomyces olivaceoviridis; Streptomyces lividans; xylan binding domain

-40-
Note
Identification of a Methylated Tea Catechin as an Inhibitor of Degranulation
in Human Basophilic KU812 Cells

Hirofumi TACHIBANA,・ Yousuke SUNADA, Toshio MIYASE,＊ Mitsuaki SANO,＊
Mari MAEDA-YAMAMOTO,＊＊ and Koji YAMADA

Division of Bioresources and Bioenvironmental Science, Graduate School, Kyushu University, Hakozaki
6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan
＊School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
＊＊National Research Institute of Vegetables, Ornamental Plants and Tea, Ministry of Agriculture,
Forestry and Fisheries, 2769 Kanaya, Shizuoka 428-8501, Japan

Received September 6, 1999; Accepted October 21, 1999
We examined the constituents of tea that had an inhibitory effect on the degranulation process in the human basophilic cell line, KU812. Among the constituents purified from a extract of `Benihomare′ oolong tea by column chromatography, a methylated (−)-epigallocatechin gallate ((−)-epigallocatechin 3-O-(3-O-methyl) gallate) was found to inhibit the degranulation of KU812 cells that had been stimulated with calcium ionophore A23187. The inhibitory effect of this methylated (−)-epigallocatechin gallate on degranulation was greater than that of (−)-epigallocatechin gallate. This result indicates that methylation of (−)-epigallocatechin gallate may be an effective modification for the catechin to inhibit degranulation from human basophils.
tea catechin; basophil; degranulation; methylation; (−)-epigallocatechin gallate

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Note
A Simple and Rapid Method for the Preparation of a Cell-free Extract
with CCAAT-Binding Activity from Filamentous Fungi

Masashi KATO,・ Kojiro HAYASHI, Tetsuo KOBAYASHI, and Norihiro TSUKAGOSHI

Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan

Received Septmber 16, 1999; Accepted October 27, 1999
A simple and rapid method for the preparation of a cell-free extract with the CCAAT-binding activity was established with Aspergillus nidulans as a model fungus. Proteins were extracted with 6 M guanidine hydrochloride directly from mycelia and renatured by dialysis. This method was found applicable to other filamentous fungi such as Aspergillus oryzae and Trichoderma viride.
filamentous fungi; CCAAT-binding protein; cell free extracts; electrophoretic mobility shift assays