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1)Holman, R.T.,et al.,Amer.Orchid Soc.,Bull.42,678(1973)
2)Ono,T.,et al.,Natural Product Letters,13,53(1999)
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1. Fall, D.; Duval, R. A.; Gleye, C.; Laurens, A.; Hocquemiller, R. J.
Nat. Prod. 2004, 67, 1041-1043.
2. Hoye, T. R.; Ye, Z. J. Am. Chem. Soc. 1996, 118, 1801-1802.

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1) K. Swireczek et al., J. Med. Chem., 46, 3703 (2003).
2) M. Pal et al., Indian J. Chem., 42B, 593 (2003).

- 43 -

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Yamashita, M. et al. PNAS 2004, 101, 4346.
Blanchard, N. et al. Org. Lett. 2007, 9, 1485.

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Cloning, Expression and Biochemical
Characterization of Novel
Dihydrolipoamide Dehydrogenase Gene
from Clostridium kluyveri
fSaikat Chakraborty, Hiroshi Nonaka*,
B-2
Masayuki Inui*, Hideaki Yukawa*,
Makiko Sakka, TetsuyaKimura, andKazuo Sakka (Grad. Sch. Bioresources,
Mie Univ., *RITE)
D ih y d r o lip o a m id ed eh y d r o g en as eo f t enter m ed a s “D i ap h o r a se ” f o r i t s ch ar a c te r i st i cf lav in m o ie ty an d N A D H o x id as e ac t iv i t y.
D ih y d r o lip o a mid e d eh y d r o g en as e catalyzes theoxidationof dihydrolipoamide: dihydrolipoamide +NAD+ -> lipoamide + NADH + H+. In case of bacteria
and eucarya, this enzyme normally functions as anintegral component of the pyruvate, 2-oxoglutarate, andbranched chain 2-oxoacid dehydrogenase multienzyme
complex or the glycine cleavage system. A novel1368-bp Clostridium kluyveri gene codes for ~ 50 kDa
protein was cloned and over expressed in E.coli systemand further biochemical and spectral studies were sought
to investigate its identity. The recombinant protein
showed very strong dihydrolipoamide dehydrogenase
and diaphorase activity. Non-covalantly attached FAD
was released at boiling temperature and quantified(7.2M). UV-visible absorption spectra showed two
peaks at 453nm and 270nm and the ratio of A280/450 =4.98. Although the optimum temperature was 40ºC at pH
7.0, the protein showed some stability as high as 70ºC
and 6.0-9.0 pH range. Fluorescent spectral study
revealed the formation of NADH in forward reaction inpresence of reduced lipoamide, in turn confirmed
dihydrolipoamide dehydrogenase activity. Unlike otheranaerobic bacterial dihydrolipoamide dehydrogenase
enzyme, this enzyme had specificity to only NADH as
coenzyme, where NADPH did not react at all.

- 45 -

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ũnŔƶĺĢĩĚěĚė ŹæŔŚłŤƶőŶųťơƪƋƥƖŹ       Ē īġĦĦĒ ęĜĿęĚĠěĝ ũPŔŚaƶpp¿ŤŲŴƶ   ũhœŗƶ¨ ťêPŹ ŭŚłŬŚƶƇƯƕƮƱƬšŔşƶ    ú­ťŭƶŧŤƶĺĢĩĚěĚ Źmŕŵ    ŹŰhœŗƶ¨ ťêPŹ ŭŚłœųŤƶőŶų¨ ŋųťƂƬ
ƋêPŤ^ōÎŇşƶKáťêPŹ ŭŚłńÌŅpp¿ŤŲŴƶ/     ŹhœŗŚÌƶŘŶřŶŋųtť ýŕŵ¨ ŹbŚłœųŤƶőŶųť¨ Ź ħıķĲĹĻĵėĢĞ ?=ƴĚĒμīĒĬġġƶĚęĒμīĒĝėģĮĮİ
Ź3ŮƵũÕ\ŔƶěğŀƶĚğ zþy~Š?čŔŚÌƶÐ(ŔŚƂƬƋŹbŚłœųŤőťƂƬƋŹ1Êiť?=ũÕ\Ŕƶ?čŔŚÌƶ ŌPœŶşŇŵšfœŶŵƂƬ
ƋŋųKáŹbŚłĒ

- 51 -

C-10

        M9,
1¡h =P8k' C 1 Putu Suparthana1 EBr
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Agrobacterium tumefaciens Ĝ 2 8®Ę LBA4404 ®ė in planta K_ČĒĽŅ(Fagopyrum esculentum, T1 )ěăĈĪÀ ={ĮqČĒûý d  þ {  À = Ę Č Ė Ľ Ņ Ĝ  1 À = (acc No. AB327276,
U-gene Ę )Į¿Ğútargeting vector ĝ U-gene Ĝ<ě pBluescriptĜ multi-cloning site Į.Ĥ 445 bp ĜbĮ]ČúpIG121-Hm binary
vector Ĝ GUS À =ÁĘ¨_čĪĉĘěħĩy ČĒûĉĜ targeting
vector Į A. tumefaciens ŚřĘ M-31 8®%Ğ LBA4404 ®ěDČĒûĉĜ A. tumefaciens ®ĮĀúYüĜÈČĒ in planta K_ėĽŅŗ 1 'ŘĮK_Čúě¿įē T1 wěăĈĪÀ ={ĜX-Įú´K úSouthern q%Ğ PCR ė¶ČĒûý¥rþ3 ®tĜ A. tumefaciens ėK_ČĒĐīđīĜ T1 Ĝtransformants Ĝ´K ĝ2 ėúĀĒ®těħĩĚĔĖĀĒûĉ
Ĝ¥rĝ®těħĩ targeting construct Ĝ genome Ĝ]ÁąĚĔĖĀĪĉĘĮ0ČĖĀĪûSouthern qěeĀĖúM-1 Ę M-31 8®ěħĪ transformants ė1 U-gene Ĝ band Ĝ¨ą#ČĒûĉĜ
¥rĝ targeting construct ą1 U-gene ĜÁĄĐĜĊć¹ě]
ċīĒĉĘĮJć0ČĒûTargeting construct Ĝ54ÂĘ U-geneĜÌ^ÁĜ54ÂĮ primers ĘČĖĀĒ PCR Į²ĔĒĘĉĬú
M-1 ®ěħĪ transformants ėĝmMºĩĜ 3’-end flanking DNA ą
M-31 ®ěħĪ transformants ėĝmMºĩĜ 5’-end flanking DNA ąÔ
ÑIė6HċīĒû Ĝ¥rħĩúM-1 Ę M-31 ®ėK_čĪĘÔÑIė”non-precise”Õ gene targeting ą·ĆĪĉĘą0ċīĒû

C-12

Research purposeCold stress-related Mitogen-ActivatedProtein Kinase (MAPK) pathway has been extensively studied
in Arabidopsis, tobacco and maize. We have shown that
OsMEK1 (OsMKK1) and OsMAP1 (OsMPK3) are induced
during a moderate chilling (12°C) stress in rice. Here report
identification of rice MAPK components specifically interact
with OsMEK1, and possible involvement of thioredoxin h in
the regulation of this MAPK signaling pathway.
MethodsYeast Two-hybrid system and PXG and ONPG
methods were used in evaluating the physical interaction. In
vitro kinase assay was carried out with myelin basic protein(MBP) as a substrate. Northern blots were done with plants
treated for 12°C stress.
ResultsTwo-hybrid screening identified specific and strong
interactions of OsMEK1 with OsMPK3 and OsMPK6,
respectively. In vitro, OsMEK1 directly phosphorylated
OsMPK3. A constitutive active OsMEK1 (OsMEK1DD)
strongly enhanced kinase activity of OsMPK3. These data
indicated that OsMEK1 and OsMPK3 are the components ofthe same signaling pathway for moderate chilling stress. Twohybrid assay also identified thioredoxin h as an interactor of
OsMPK3. Thioredoxin h inhibited MBP kinase activities of
OsMPK3 and OsMPK6 in vitro. Interestingly, thioredoxin h is
up-regulated in response to moderate chilling stress. Therefore
it was suggested that thoredoxin h is a negative regulator of
OsMEK1-OsMPK3 and OsMEK1-OsMPK6 pathways.

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C-11

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Ca2+ ¥)ľŔņĴ ĮČúĐĜ£ªE1ĄĨú
CCaP1ŗA. thaliana cytosolic Ca2+-binding proteinŘú
CCaP2úCCaP3 Ę/+ČĒûĉĜ 3 ĕĜľŔņĴ ÉĜİōńÃÂĝÒ TąÔĀûnĝú( CCaP Ĝ
|«Ĝ hĮĘčĪûýdþ( CCaP Ę GFP Ĝ±)ľŔņĴ Į3Ó£ªėċďĪĉĘěħĩú£ªE1Į*?ČĒûĢĒŊœ
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İőľĲŎ PCR ěħĩ?Æ qČĒûňıŁĴœŖŎăħ
ĞĴŐŊŁĴœŖŎÀ =7těăĀĖĦ*zěÀ= qĮ²ĔĒûý¥rþGFP ěħĪ qĄĨúCCaP ĝ£ª ě>1čĪ& TľŔņĴ ėÿĪĉĘąhĨĄěĚĔĒûĢĒ
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Molecular characterization of rice
MAPK components that interact withOsMEK1z rsr `\?©§eR@<¦DgeR¨

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÷øöôòë Ĝ9pľŔņĴ ĮÅtĜ¢ ÞÛã ėĆĪĉĘ
ąhĨĄěĚĔĒûÕ

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- 55 -

D-4

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D-6

Electrochemical
regulationofmetabolism
inPropionibacterium
freudenreichii ET-3
M Yung-Fu Wang, Masaki Masuda, Seiya
Tsujimura, Kenji Kano
Division of Applied Life Sciences, Graduate
School of Agriculture, Kyoto Universityµ{¶± IGuqÑ²Ï²/Ð pH ½A¦IÙAâĄçĄIËØuÄÝÁÌ½Ë¾Ý³ÆÐ pH J=IÏªÄÝÖÑ²E. coli ÎÍ¤ÐËa½×ÛÞÊ¸Ý
½²âÿø¦z¥Ë·Ý Corynebacterium glutamicum ËÑ,%½Î¸³'²#Ï»ÀÝAâąçĄIt-ËÐuÐÖÐ[à{ÌÃÊ² ÐdàÈÇ³µZl¶± .ycÐuÏ»ÀÝ pH 7IÑ²\;*)àw
¸Ç:2*)»ÚÒm*¯ËÔÇ³*)Ð pH Ñ²´ÐùóüáĈàw¸ÊWÃÇ³¢6ÐïĂóõèćê
ĆĈöćëÙïĈìćðÑ8lÏDÈÊÈÇ³µb¶± M´ÑÏ²C. glutamicum ¨uc¼ÛăćòĀ.yÏÚÜAâąçĄIÐ:2*)ËuË¾Î¿ÎÈÇ.yc
à0VEÃÊ,%ÃÇĉH19 > 3 ] 1 Ċ³ÁÞÛ.
ycÐ0¿ÑI ËÑ¨ucÌ#ÏuÃÇ½²pH9àºÝâąçĄIt-ËÑ¨ucÚÜÃ¸uà~ÃÇ³DÈÊ²ÁÞÛ.ycÑâąçĄIONIÏªßÝg½hUÃÇcË·ÝÌºÛÞÇ³_ÐíøĀăäýăĄ¼ÛâąçĄIKIà'FÂÅÝ DNA YÐRàÖÇ³
ÆÐb²!.yc¼Û DNA Y½EÂÞÇ³ïĈìćðÐb²ÁÞÛÐÏÑg{ÏyÎÝÌS8ÂÞÝ´Ð¢6½$ÕÞÊ¸Ç³ÁÐb¼Û²_ÐâąçĄIONIÐÖÑċÉËÑÎ¿²VÐg½ªßÈÊ¸ÝÁÌ½~&ÂÞÇ³µPurpose¶In previous studies, it has been proved that some kindsof bacteria can utilize endogenous mediator for electron transfer tocarbon electrode during substrate oxidation. This metabolism in
microbe should be regulated by controlling the rate of electrontransfer out of cell by adjusting the electrode potential. In thisstudy, P. freudenreichii ET-3 was utilized to verify this purpose.
µMethods¶ P. freudenreichii ET-3 and glucose were used as abiocatalyst and a substrate (electron donor), respectively. All
electrochemical experiments were carried out in a flow-type
electrolysis cell filled with a carbon/graphite-felt electrode and thegrowth medium of P. freudenreichii ET-3. Amperometry was
performed at 37 oC under anaerobic conditions. The profiles ofend products were analyzed for the electrochemical growth
medium under steady state conditions at given applied potentials.
µ Results¶ After seeding bacteria into the electrolysis cell, thesystem produced an oxidation current in the presence of glucose
when positive potentials were applied. This means that the
microbial cells donate the electrons to the electrode during the
glucose oxidation without artificial mediator. At a low applied
potential of -260 mV vs. Ag/AgCl, lactate was the dominant
end-product and reached 18 mM, however it decreased to 5 mM at
a positive potential of 500 mV, in which formate became thedominant one. The concentration of propionate which was one of
the typical end products also decreased when the potentials
changed from -260 to 500 mV. It was considered that electrode athigh applied potentials can work as the electron acceptor for
microbial cells, which leads to the change in the end-productprofile from that obtained under normal anaerobic conditions.

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O2 o?Ð«xÑ 0.5% Ï·ÝÁÌà,%ÃÇ³Ã¼Ã²_
Ð O2 wÏÍÐÚ¹Îg½ªßÈÊ¸Ý¼²ÆÐ Ñ[Û¼ËÎ¸³'²_Ð O2 wÏªßÝ¢6ÐRà{Ï ÐdàÈÇ³µZl¶± .ycÐuÏ»ÀÝ¦ 7IÑ²\;*)à
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ìćðÑ8lÏDÈÊÈÇ³µb¶± M´ÑÏ²C. glutamicum ¨uc¼ÛăćòĀ.yÏÚÜ O2ĉ0.5% O2Ċt-ËÐ:2*)ËuË¾Î¿Î
ÈÇ.ycà0VEÃÊ,%ÃÇĉH19 > 3 ] 1
Ċ³ÁÞÛ.ycÐurIàm*¯ËÔÇ³.yc
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ycÑ¦ ÐwÏªßÝg½UÎßÞÇcË·ÝÌºÛÞÇ³_ÐíøĀăäýăĄ¼Û¦ jIà'FÂÅÝ DNA YÐRàÖÇ³ÆÐb²!.yc¼Û
DNA Y½EÂÞÇ³ïĈìćðÐb²ÁÞÛÐÏÑ
g{ÏyÎÝÌS8ÂÞÝVÐ¢6½$ÕÞÊ¸Ç³D-7µ{¶GuqpX­kÑ²Guq½^gqÐ à¹¬ÏvÖÂÞÝ£à­iå÷ąéĈÏ.TÃÊÜÄÁÌ½Ë¾ÝË·Ý³'Ñ {Î5iIÐÉË·Ý¦ Lactococcus lactis à4ÌÃÊGuqpX­kÐ+}{Î|àÈÇ³ëąîĈðà+ ÌÃÊ5i{Ï*¯ÃÇÌ¾²¦Ñ NADH ¼ÛÐ­69ÌÃÊûą
úć¦àwÃ²¦àuLÄÝ³_|ËÑ/­6
9ÌÃÊ­eàw¸²¡BÎ­àÃÊ*¯ÄÝÁÌË²NADH ¼Û­eÓÐ­6à@¾ÁÄÁÌà{ÌÃÇ³ÕÇ²©Ð­6 āôãåĈñĈàuLÄÝÁÌ½, % Â Þ Ê ¸ Ý þ Ć û æ ć ¦  Propionibacterium
freudenreichii ET-3 ÌÐn"*¯à¹ÁÌË²s{Î­6HÐfàPÃÇ³µZl¶黒鉛化処理したカーボンフェルトを作用電極として用い、ポテンシオスタットで電極の電位を制御し、37℃、嫌気条件下で乳酸菌、プロピオン酸菌の培養、および両者の混合培養を行った。また、電極への電子移動を電流として測定した。
µb¶電極電位を一定に保った条件で乳酸菌を培養した
ところ、電流が観察され、生成物の HPLC 分析では、乳酸生成量の減少と酢酸の生成がみられた。このことから、乳酸菌内の NADH から電極への電子移動が起こったと考えられる。また、電子移動の効率を高める目的で培地に少量のメディエーターを添加したところ、より大きな電流が得られ、乳酸の減少と酢酸の増加がより顕著に表れた。次に、乳酸菌とプロピオン酸菌の混合培養を行った
結果、電流値の増加が見られたため、プロピオン酸菌の生成したメディエーターにより、電極への電子移動が促進される可能性が示唆された。

- 56 -

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D-10

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- 57 -

D-12¹¤¯¼pJK-UG_     ~        {{|  }C\ZpW/1÷č¨ĎgČīęĸĮĥĩ Mortierella alpina 1S-4 ,ĬğĪÕ
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E-2

A dipeptide YY derived from royal jelly
proteins inhibits the renin activity.
pAfroza Sultana1), A.H.M. Nurun Nabi2,
Uddin M. Nasir2, Hiroe Maruyama3,
Kazu-michi Suzuki3, Satoshi Mishima3,
and Fumiaki Suzuki,2.1) (1)United
Graduate School of Agric. Sci. and 2)Fac.
Appl. Biol. Sci., Gifu University,
3)
Nagaragawa Res. Ctr,
API Co. Ltd.)

Objective: Royal jelly has been recently reported toposses the antihypertensive effect by the inhibition of
ACE in the spontaneously hypertensive rats (SHR).
Renin is the key enzyme in the renin angiotensin
system. In this study, we investigated the renin
inhibition of royal jelly protein and its derived
dipeptide YY.
Methods: A dipeptide YY was isolated from digestive
fraction of royal jelly protein fraction by HPLC. The
renin activity was measured using recombinant sheep
angiotensinogen at pH 7.0 under the standard assay
conditions.
Results and Conclusion: Inhibitory effects of renin
activity were observed by the protein fraction anddipeptide YY. The Km value of the substrate;angiotensinogen was 0.15 M and the Ki of YY was
10 M. Besides this, the dipeptide YY showed thesystolic blood pressure lowering ability after the oral
administration of it (10 mg/kg) in the SHR byapproximately 12 mmHg.
Therefore it can be assumed that the dipeptide YY
could be beneficial for the supplement ofanti-hypertension and prevention for its related
complications.

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w[/9ğ mRNA YĆBęćēü

E-4

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(1) Sugimura et al. J. Biol. Chem. 281, 17699 (2006)

C. ŰŶŲųŴűŵŷ et al. (2007) Lett. Drug. Design Discov. 4, 239-245.

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1) Shimomura et al., Neurosci. Lett. 363, 195-198 (2004).
2) Shimomura et al., Neurosci. Lett. 385,168-272 (2005).

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日本農芸化学会
2007 年度（平成 19 年度）関西・中部支部合同大会
御寄贈会社名本大会の開催に当たり下記 4 社より御芳志を頂きました。
この場をかりて篤くお礼申し上げます。

株式会社ミツカン

グループ本社

アピ株式会社株式会社

ポッカコーポレーション

愛知県酒造組合連合会（順序不同）

2007 年度日本農芸化学会実行委員長：小林総

関西・中部支部合同大会実行委員会

猛（中部大学 応用生物学部）

務：森山龍一（中部大学 応用生物学部）〒487-8501 愛知県春日井市松本町 1200
中部大学応用生物学部
TEL & FAX: 0568-51-6084（総務）

日本農芸化学会中部支部〒464-8601 名古屋市千種区不老町
名古屋大学大学院生命農学研究科内支部長：前島正義
庶務幹事：小田裕昭
Tel. 052-789-4124, Fax: 052-789-5050（庶務幹事）
http://www.agr.nagoya-u.ac.jp/~jsbba/

日本農芸化学会関西支部〒606-8502 京都市左京区北白川追分町
京都大学大学院生命科学研究科内支部長：山本憲二
庶務幹事：中川好秋
TEL: 075-753-6117, FAX: 075-753-6123（庶務幹事）
http://jsbba-kansai.kir.jp/