食糧　その科学と技術　No.45( )

Transcription

1 J. Eberle Cl. Bernard Rhizomucor miehei X

2

3

4 Rhizopus arrhizus n- n-n- Geotrichum.candicum ph

5 Geotrichum.candidum PEG Rhizopus delemar

6 d Geotrichum candidum Candida antarctica Hydrogenation Fractionation Inter- Intra-esterification Interesterification, Transesterification Interesterification Intraesterification Esterinterchange Transesterification

7 Intermolecular Rearrengement Acidolysis Alcholysis

8 Sn Pb Holmberg Rhizomucor delemar

9 ph n- C sn- C C sn- sn- sn- sn-sn- sn-

10 MCT Random interseterification Na-K

11 sn- sn- Candida rugosa, Geotrichum candidum sn-sn- sn - Rhizomucor miehei Rhizopus arrhizus sn- sn- POP S POS SOS

12 Alcaligenes Rhizomucor EPA DHA

13 Aspergillus niger Candida antarctica Candida cylindracea Candida rugosa Candida sp. Fusarium heterosporum Geotrichum candidum Humicola lanuginosa Mucor miehei Mucor miehei Mucor miehei Mucor miehei Mucor miehei Mucor miehei Rhizopus delemer Rhizopus javanicus Rhizopus riveus Rhizopus sp. Chromobacterium viscosum Pseudomonas fluorescens Pseudomonas sp. Pseudomonas sp. Pseudomonas sp. Pseudomonas sp.

14 PEG sn- sn- sn-sn-c C CMCFA sn- C C sn- CC C

15 Rhizomucor miehei Rhizopus EPA, DHA sn- sn- sn- EPADHA Rhizomucor miehei Candida antarctia Candida cylindracea stereospecificity

16 S C N tert- Candida cylindracea sn-sn-rhizomucor delemar sn- sn- sn- sn- sn-

17 sn-sn- sn- Rhodotorula rubra, Lipomyces lipofer, Molutierella vinacea GLA DGLA AA EPADHA PUFA GLA Mortierella DGLA AA Mortierella alpina AA DGLA AA M.alpina M.heterosporus M.alpina AA AA EPA Alteromonas PUFA EPA AA M.alpina AA EPA DHA PUFA DHA Pseudomonas act

18 Brzozowski,A.M., et al., (1991) Nature (London), 351, 491. Schrag,J.D., and Cygler,M. (1997), Methods Enzymol., 284, 85. Tsuzuki,W., et al. J.Am.Oil Chem. Soc. (1998) 75, 535. Baillargeon,M.W. and Sonnet,P.E. (1988) J.Am. Oil Chem.Soc., 65, Sternby,B., et al. Clinical Nutr. (2002) 21, 395. Tsuzuki,W. et al. (1991) J. Chem. Soc.. Perkin Trans , Baker,R.A. and Wicker,L. (1996) Trends Food Science Technology,7,279. Fomuso,L.B., and Akoh,C.C. (2002) Food Research International, 35, 12. Kimura.Y,. et al. (1983) Eur. J. Appl. Microbiol. Biotechenol., 17, 107. Xu,X.,et al. (2000) J.Am.OilChem.Soc.,77, Tsuzuki,W., (2005) Biosci. Biotechnol. Biochem. 69, 1256.,.

19

20 α α β α β α α α β β α α β β α α CAZy mrs.fr/cazy/ phosphorylase N- - ATP -

21 β Cellvibrio gilvus β Cellvibrio gilvus Euglena gracilis β β

22 β N- β β Clostridium thermocellum YM

23 β β βgg GX XG XX XG NMR β Bacillus halodurans

24 α β α β

25

26

27 N-

28 β α α β N- -N-LNBP GalβGlyNAc GlcNAc -N-or GalNAc -N- α GlyNAc Bifidobacterium bifidum B. bifidum LNBP N Bifidobacterium longum NCC BLBL B. longum NCC B. longum JCM LNBP BL LNBP LNBP BLBL BLBL ABC BL Gal -P UDP Glc B. longum

29 N N N- N- LNBP N-

30 Kitaoka, M. and Hayashi, K., Carbohydrate processing phosphorolytic enzymes. Trends Glycosci. Glycotechnol. 14, (2002). Kitaoka, M., Taniguchi, H. and Sasaki, T., Production of glucosyl xylose using Cellvibrio gilvus cells and its properties. Kitaoka et al., Appl. Microb. Biotechnol., 34, (1990). Kitaoka,M.,Ogawa,S.andTaniguchi, H., A cellobiose phosphorylase from Cellvibrio gilvus recognizes only the β D form of 5a carba glucopyranose., Carbohydr. Res., 247, (1993). Tariq, M. A., Hayashi, K., Tokuyasu, K., and Nagata, T., Synthesis and structural analysis of disaccharides of 4 O β D glucopyranosyl D glucosamine and 4 O β D glucopyranosyl 2 deoxy D glucose. Carbohydr. Res., 275, (1995). Tariq, M. A. and Hayashi, K., Synthesis of three hetero disaccharides, 4 O β D glucopyranosyl D 6 deoxy D glucose, 4 O β D glucopyranosyl D mannosamine, and 4 O β D glucopyranosyl D mannose, and confirmation of their structures by C 13 NMR and MS. Biochem. Biophys. Res. Commun., 214, (1995). Percy, A., Ono, H., Watt, D. and Hayashi, K. Synthesis of β D glucopyranosyl (1 4) D arabinose, β D glucopyranosyl (1 4) L fucose, and β D glucopyranosyl (1 4) D altrose catalysed by cellobiose phosphorylase from Cellvibrio gilvus. Carbohydr. Res., 305, (1998). Percy, A., Ono, H. and Hayashi, K., Acceptor specificity of cellobiose phosphorylase from Cellvibrio gilvus: synthesis of three branched trisaccha-

31 rides. Carbohydr. Res., 308, (1998). Kitaoka, M., Nomura, S., Yoshida, M. and Hayashi, K., Reaction on D glucal by an inverting phosphorylase to synthesize derivatives of 2 deoxy β D arabino hexopyranosyl (1 4) D glucose (2 II deoxycellobiose). Carbohydr. Res., 341, (2006). Kitaoka, M., Sasaki, T. and Taniguchi, H.,Synthesis of laminarioligosaccharides using crude extract of Euglena gracilis z cells. Agric. Biol. Chem., 55, (1991). Shintate, K., Kitaoka, M., Kim, Y. K. and Hayashi, K., Enzymatic synthesis of a library of β (1 4) hetero D glucose and D xylose based oligosaccharides employing cellodextrin phosphorylase. Carbohydr. Res., 338, (2003). Honda, Y. and Kitaoka, M., A family 8 glycoside hydrolase from Bacillus halodurans C 125 (BH2105) is a reducing end xylose releasing exo oligoxylanase. J. Biol. Chem., 279, (2004). Murao,S.,Nagano,H.,Ogura,S.andNishino,T.,Enzymaticsynthesisof trehalose from maltose. Agric. Biol. Chem., 49, (1985) Kitaoka, M., Sasaki, T. and Taniguchi, H., Conversion of sucrose into cellobiose using sucrose phosphorylase, xylose isomerase and cellobiose phosphorylase. Denpun Kagaku, 39, (1992). Kitaoka, M., Sasaki, T. and Taniguchi, H., Conversion of sucrose into laminaribiose using sucrose phosphorylase, xylose isomerase and laminaribiose phosphorylase. Denpun Kagaku, 40, (1993). Saito, K., Kase, T., Takahashi, E., Takahashi, E. and Horinouchi, S., Purification and characterization of a trehalose synthase from the basidiomycete Grifola frondosa. Appl. Environ. Microbiol., 64, (1998). Derensy Dron, D., Krzewinski, F., Brassart, C. and Bouquelet, S., β 1,3 Galactosyl N acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082: characterization, partial purification and relation to mucin degradation. Biotechnol. Appl. Biochem., 29, 3 10 (1999). Kitaoka, M., Tian, J. and Nishimoto, M., A novel putative galactose operon involving lacto N biose phosphorylase found in Bifidobacterium longum. Appl. Environ. Mocrobiol., 71, (2005). Park, J. K., Keyhani, N. O. and Roseman, S., Chitin catabolism in the

32 marine bacterium Vibrio furnissii: identification, molecular cloning, and characterization of a N,N diacetylchitobiose phosphorylase. J. Biol. Chem., 275, (2000). Honda,Y.,Kitaoka,M.andHayashi,K.,Reactionmechanismofchitobiose phosphorylase from Vibrio proteolyticus: Identification of family 36 glycosyltransferase in Vibrio. Biochem. J., 377, (2004). Hidaka,M.,Honda,Y.,Kitaoka,M.,Nirasawa,S.,Hayashi,K.,Wakagi, T., Shoun, H. and Fushinobu, S., Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has an (α/α)6 barrel fold like clan GH L. Structure, 12, (2004). Hidaka, M., Kitaoka, M., Hayashi, K., Wakagi, T., Shoun, H. and Fushinobu, S., Structural dissection of the reaction mechanism of cellobiose phosphorylase. Biochem. J., 398, (2006).

33 CO Iogen Henrissat CAZy website:

34 D β D O LOO D L O p D β β

35 TIM Streptomyces olivaceoviridis E SoXynA SoXynA bp N N C C C

36 X SoXynA β/α C α β γ β SoXynA SoXynA

37

38 O Me C C

39 O Me α O Me α O Me SoXynA SoXynA

40

41 SoXynA Cellulomonas fimi Cex CfXynA SoXynA SoXynA CfXynA SoXynA CfXynA SoXynA CfXynA SoXynA CfXyn A SoXynA CfXynA CfXynA

42

43 CfXynA SoXynA

44 D D

45 SoXynA t t

46 A.Kuno,D.Shimizu,S.Kaneko,Y.Koyama,S.Yoshida,H.Kobayashi,K. Hayashi, K. Taira and I. Kusakabe: PCR cloning and expression of F/10 xylanase gene from Streptomyces olivaceoviridis E 86, J. Ferment. Bioengin. 86, (1998). Z. Fujimoto, A. Kuno, S. Kaneko, S. Yoshida, H. Kobayashi, I. Kusakabe and H. Mizuno: Crystal structure of Streptomyces olivaceoviridis E 86 β xylanase containing xylan binding domain, J. Mol. Biol. 300, (2000). Z. Fujimoto, A. Kuno, S. Kaneko, H. Kobayashi, I. Kusakabe and H. Mizuno: Crystal structure of the sugar complexes of Streptomyces olivaceoviridis E 86 xylanase: Sugar binding structure of family 13 carbohydrate binding module, J. Mol. Biol. 316, (2002). Z. Fujimoto, S. Kaneko, A. Kuno, H. Kobayashi, I. Kusakabe and H. Mizuno: Crystal structures of decorated xylooligosaccharides bound to a family 10 xylanase from Streptomyces olivaceoviridis E 86, J. Biol. Chem. 279, (2004). S.Kaneko,H.Ichinose,Z.Fujimoto,A.Kuno,K.Yura,M.Go,H.Mizuno, I. Kusakabe and H. Kobayashi : Structure and function of a chimeric β xylanase from Streptomyces olivaceoviridis E 86 FXYN and Cellulomonas fimi Cex, J. Biol. Chem., 279, (2004).

47 Aeromonas caviae Vibrio proteolyticus zymogen

48 Aeromonas caviae TapAC N apac Vibrio proteolyticus apvp Vibrio proteolyticus Aeromonas proteolytica apvp apvp X co catalytica apvp kda Heeke apvp apvp kda N C

49 Aeromonas caviae T DNA PCR DNA pasnm pro apacsds PAGE kda pro apac pro apac Aeromonas caviae TPA pro apackda kda PA pro apac apac kdapa pro apac N apac N

50 pro apac PA pro apac pro apac Km kcat Km PA pro apac kcat pro apac pro apac pro apac kcat pro apac pro apac apac kcat Km pro apac kcat kcat Km Km kcat Kmkcat Km pro apac apac kcat Km kcat Km Leu pna kcat Phe Phe Pro Glu Ala Km Km Phe PhePhe Gly kcat Kmkcat Km Leu pna Phe Phe Pro Glu Ala N kcat s Km mm kcat Km s mm pro apac PA pro apac apac

51 pro apac apac pro apac apac ph ph ph ph pro apac pro apac pro apac N

52 pro apac EDTA mm EDTA mm N pasm pro apac N pro apac pro apac PA N N pro apac PA N panm pam SDS PAGE M PA pro apac PA pro apac N

53 Vibrio proteolyticus DNA PCR DNA pvsnmc pvsnm C pro apvp C pro apvpsds PAGE kda pro apvp kda C pro apvp pro apvp C pro apvp PA kda PA pro apvp C pro apvp apvp kda

54 pro apvp C pro apvp PA pro apvp C pro apvp apvp Km kcat Km PA pro apvp C pro apvp kcat pro apvp C pro apvp apvp pro apvp C pro apvp kcat N C pvsmc pvsm pro apvp C pro apvp N pro apvp N C pro apvp kcat s Km mm kcat Km s mm pro apvp PA pro apvp C pro apvp PA C pro apvp apvp

55 pro apvp C pro apvp PA N N pro apvp N C pro apvp PA N pvnmc pvnm pvmc pvm SDS PAGE M PA pro apvp C pro apvp PA pro apvp C pro apvp N N C pro apvp N N C pro apvp N N N A. caviae T DEAE Mono Q PA units/mg SDS PAGE PA N TAIL PCR

56 V.proteolyticus PA DNA DNA bp ORF N N Met HisHis Glu N Perlman Halvorson Vonheijine AlsAla Met Ala N Ala HisGlu TyrkDa kda kda PA kda C kda PA EMBL SWISS PROT NFBRS PA Vibrio proteolyticus Vibrio cholerae Vibrio vulnificus Bibrio anguil-

57 larum Pseudomonas aeruginosa Legionella pneumophila Legionella longbeachae PA N Cys T lac promotor pelb PA N PA ppsnmc ppsmc BL DE PA N PA SDS PAGE PA N PA BL DE N BLDE ppsnmc kda PA kda kda PA N N Gln Asp Ala Thr Gly A. caviae T PA N PA N C pro apac PA N U µgu µg U µg PA PA U µg U µg PA

58 pro apac N A A X apac apvp N N N apvp C apac apvp apvp N N N apvp C Xanthomonas campestris Vibrio alginolyticus Xanthomonas camp-

59 estris Vibrio alginolyticus C pro apac ph N Porcine A B kcat Km pro cathepsin B pro cathepsin D α B A X pro apac Leu pna Met pna Val pna Leu Phe Met Phe Ala Phe Phe Phe Phe Leu Phe Met Phe Gly Phe Ala Phe Phe Bovine A apac apac N N pro apac ph apvp N C pro apac pro apvp PA pro aminopeptidase processing protease

60 Habibi Najafi, M.B., and Lee, B. H., Bitterness in cheese. Crit. Rev. Food Sci. Nutr., 36, (1996). Izawa, N., Tokuyasu, K., and Hayashi, K., Debittering of protein hydrolysates using Aeromonas caviae aminopeptidase. J. Agric. Food Chem., 45, (1997). Izawa, N., Ishikawa, S., Tanokura, T., Ohta, K., and Hayashi, K., Purification and characterization of Aeromonas caviae aminopeptidase possessing debittering activity. J. Agric. Food Chem., 45, (1997). Izawa, N., and Hayashi, K., Cloning and nucleotide sequencing of the aminopeptidase gene from Aeromonas caviae T 64. J. Ferment. Bioeng., 82, (1996). Chevrier, B., Schalk, C., D Orchymont, H., Rondeau, J. M., Moras, D., and Tarnus, C., Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical member of the co catalytic zinc enzyme family. Structure 15, (1994) Van Heeke, G., Denslow, S., Watkins, J. R., Wilson, K. J., and Wagner, F. W., Cloning and nucleotide sequence of the Vibrio proteolyticus aminopeptidase gene. Biochim. Biophys. Acta 1131, (1992) Nirasawa, S., Nakajima, Y., Zhang, Z., Yoshida, M., and Hayashi, K., Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase. Biochem. J., 341, 25 31(1999). Zhang, Z., Nirasawa, S., Nakajima, Y., Yoshida, M. Kusakabe, I., and Hayashi, K., Characterization of the pro aminopeptidase from Aeromonas caviae T 64. Biosci Biotechnol Biochem., 65, (2001). Zhang, Z., Nirasawa, S., Nakajima, Y., Yoshida, M., and Hayashi, K., Function of the N terminal propeptide of an aminopeptidase from Vibrio proteolyticus. Biochem. J. 350, (2000). Nirasawa, S., Nakajima, Y., Zhang, Z., Yoshida, M., and Hayashi, K., Molecular cloning and expression in E. coli of the extracellular endoprotease of Aeromonas caviae T 64, a pro aminopeptidase processing enzyme. Biochim. Biophys. Acta 1433, (1999).

61 PCR γ Bacillus subtilis natto Bacillus subtilis

62 Bacillus subtilis Bacillus subtilis 168 γ Insertion sequence γ L D α γ Bacillus Bacillus subtilis Bacillus licheniformis Bacillus anthracis Bacillus megaterium Umamiγ

63 γ γ γ γ γ γ γ γ Cap- BCA γ γ comqxpa γ ComX ComX ComP ComP ComP Pi Pi ComA ComA Pi Two component system ComQ ComX ComX DNA

64 γ γ γ ComP ComA comqxpa ComP

65 comqxpa γ ComA γ capbca pgsbca ywscywtab γ ComP ComA capbca cap- BCA γ ComA CapBCA γ comqxpa degs degu degq yvzd/swra degq degq ComX ComQ Bacillus subtilis comx comp N ComX ComX ComX ComX ComX γ γ γ

66 γ capbca pgsbca ywscywtab γ mucoid CapB CapC CapA CapB CapB CapB CapB CapB ADP forming MurD and folyl gamma glutamate ligase family ATP L L D D Mn D CapA CapC CapC γ porecapbca B C A Bacillus anthracis γ D B. anthracis CapBCA T Candela CapD GGT YwrD

67 cap γ B. subtilis B. licheniformis cap CapD B. anthracis ywtd cap B. subtilis B. licheniformis γ YwtD γ B. anthracis γ D B. anthracis cap γ Ashiuchi L γ Natrilba aegyptiaca γ B. licheniformis Troy ATP transamidation B. anthracis CapD γ

68 Bacillus subtilis γ CapB D D MurD γ γ γ gamma glutamyltransferase GGT γ γ γ GTP γ GTP GGT γ GGT γ N D L GGT γ glutamyl p nitoroanilide Km µm GGT µm µm GGT γ

69 γ mg ml µm GGT γ Km µm µm γ YwrD GGT GGT γ MDa MDa YwrD YwrD γ YwrD GGT γ γ GGT YwrD GGT MDa YwrD γ GGT YwrD γ MDa γ complementation test

70 γ polar effectsggt YwrD γ γ GGT γ γ GGT GGT YwrD γ L L D

71 D L D race yrpc

72 RacE YrpC γ γ GGT YwrDGGT YwrD γ MDa GGT MDa γ GGT YwrD GGT γ γ γ D DD D D D γ γ γ ΦNIT γ PghP PghP γ γ PghP Poly gamma glutamate hydrolase PPghP PghP γ Ackermann PghP PghP

73 γ PghP γ γ PghP γ Bacillus subtilis Bacillus subtilis natto generally accepted as safe Bacillus sub-

74 tilis Tran Phan Lam-Son γ γ Bacillus subtilis Bacillus subtilis natto Quorum DNA DNA

75 N N β K mol LKm IS L D D L D L -L -D L-L-D -L -L-L Kimura, K. and Itoh, Y. Characterization of poly γ glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly γ glutamate. Appl. Environ. Microbiol., 69, (2003). Nagai, T., Koguchi, K., and Itoh, Y., Chemical analysis of poly γ glutamic acid produced by plasmid free Bacillus subtilis (natto): evidence that plas-

76 mids are not involved in poly γ glutamic acid production. J. Gen. Appl. Microbiol., 43, (1997). Tran, L. S. P., Nagai, T., and Itoh, Y., Divergent structure of the Com- QXPA quorum sensing components: molecular basis of strain specific communication mechanism in Bacillus subtilis. Mol. Microbiol., 37, (2000). Stanley, N. R. and Lazazzera, B. A., Defining the geneticdifferences between wild and domestic strains of Bacillus subtilis that affect poly γ DL glutamic acid production and biofilm formation. Mol. Microbiol., 57, (2005). Urushibata, Y., Tokuyama, S., and Tahara, Y., Characterization of the Bacillus subtilis ywsc gene, involved in γ polyglutamic acid production. J. Bacteriol., 184, (2002). Kimura,K.,Tran,L. S. P., and Itoh, Y., Roles and regulation of the glutamate racemase isogenes, race and yrpc, in Bacillus subtilis. Microbiology, 150, (2004). Candela, T. and Fouet, A., Bacillus anthracis CapD, belonging to the γ glutamyltranspeptidase family, is required for the covalent anchoring of capsule to peptidoglycan. Mol. Microbiol., 57, (2005). Troy, F. A. Chemistry and biosynthesis of the poly(γ D glutamyl)capsule in Bacillus licheniformis. I. Properties of the membrane mediated biosynthetic reaction. J. Biol. Chem., 248, (1973). Kimura,K.,Tran,L. S. P., Uchida, I., and Itoh, Y., Characterization of Bacillus subtilis γ glutamyltransferase and its involvement in the degradation of capsule poly γ glutamate. Microbiology, 150, (2004). γ γ Kimura, K., Inatsu, Y., and Itoh, Y., Frequency of the insertion sequence IS4Bsu1 amongbacillus subtilis strains isolated from fermented soybean foods in southeast Asia. Biosci. Biotechnol. Biochem., 66, (2002).

77 Aspergillus Herman Ahlburg Eurotium oryzae Aspergillus oryzae Ahlburg Cohn

78

79 Aspergillus flavus FDA GRAS Generally Recognized As Safe DNA ACGT RNA mrna DNA DNA RNA DNA

80 EST EST cdna DNAEST:expressed sequence tags mrna cdna EST cdna EST

81 EST cdna cdna EST EST EST EST Aspergillus oryzae RIB mrna cdna EST EST EST EST EST NEDO EST EST DDBJ/GenBank/EMBL html DNA atf B Aohsp

82 EST NITE A. oryzae RIB WGS kb DNA NITE DNA AT WGS BAC WGS Mb WGS

83 DNA GeneDecoder Mb Mb

84 Aspergillus oryzae _ID=ao A. nidulans / www. broad. mit. edu / annotation / genome / aspergillus _ nidulans / Home.htmlA. fumigatus Aspergillus A. oryzae Mb A. nidulans Mb A. fumigatus Mb A. nidulans A. fumigatus A. oryzae A. fumigatus A. nidulans A. Table 1 Comparison of genome characteristics Genome characteristic A. nidulans A. fumigatus A. oryzae General Assembly size bp G+C Protein coding genes Protein coding genes amino acids Predicted protein coding sequences amino acids Coding Gene density gene every n bp Median gene length mean Average number of exons per gene

85 Table Redundancy of secretory proteolytic enzyme genes A. oryzae A. nidulans A. fumigatus Exopeptidase Aminopeptidase Dipeptidae Dipeptidyl or tripeptidyl peptidase Serine type carboxypeptidase Metallocarboxypeptidase unknown peptidase EC EC EC EC EC Total Endopeptidase Oryzin Aorsin Kexin ATP dependent proteinase Cysteine proteinase Ubiquitin specific proteinase PalB Aspartic proteinase Pepstatin insensitive proteinase Saccharolysin Metalloprotease Insulinase unknown EC EC EC EC EC EC EC EC EC EC EC EC EC Total Exopeptidases + Endopeptidases nidulans A. fumigatus P

86 DNA A. nidulans A. niger

87 Mb DNA EST cdna DNA bp bp kb Mb

88 EST Goffeau,A.,Barrell,BG.,Bessey,H.,Davis,RW.,Dujon,B.,Feldmann,H., Galigert, F., Hoheisel, JD., Jacq, C., Johnston, M., Louis, EJ., Mewes, HW., Murakami, Y., Phillippsen, P., Tettelin, H., Oliver, SG., Life with 6000 genes. Science, 274, (1997) Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V., Bertero, M.G., Bessieres, P., Bolotin, A., Borchert, S., Borriss, R. et al., The complete genome sequence of the Gram positive bacterium Bacillus subtilis. Nature, 390, (1997) pp EST Sasaki T., Song J., Koga Ban Y., Matsui E., Fuang F., Higo H., Nagasaki H., Mori M., Miya M., Murayama Kayano E., Takiguchi T., Takasauga A., Niki T., Ishimaru K., Ikeda H., Yamamoto Y., Mukai Y., Ohta I., Miyadera N., Havukkaia, I. and Minobe, Y., Toward cagaloguing all rice genes: large scale sequencing of randomly chosen rice cdnas from a callus cdna library, Plant J., 6, (1994) p A. oryzae atfb p Aspergillus oryzae AoHSP p

89 p Machida, M., Asai, K., Sano, M., Tanaka, T., Kumagai, T., Terai, G., Kusumoto, K., Arima, T., Akita, O., Kashiwagi, Y., Abe, K., Gomi, K., Horiuchi, H., Kitamoto, K., Kobayashi, T., Takeuchi, M., Denning, D.W., Galagan, J. E., Nieman, W.C., Yu, J., Archaer, D.B., Bennett, J.W., Bhatnagar, D., Cleaveland, T.E., Fedorova, N.D., Gotho, O., Horikawa, H., Hosoyama, A., Ichinomiya,M.,Igarash,R.,Iwashita,K.,Juvvadi,P.R.,Kato,M.,Kato, Y., Kin, T., Kokubun, A., Maeda, H., Maeyama, N., Maruyama, J., Nagasaki, H., Nakajima, T., Oda, K., Okada, K., Paulsen, I., Sakamoto, K., Sawano, T., Takahashi, M., Takase, K., Terabayashi, Y., Wortman, J.R., Yamada, O., Yamagata, Y., Anazawa, H., Hata, Y., Koide, Y., Komori, T., Koyama, Y., Minetoki, T., Suharnan, S., Tanaka, A., Isono, K., Kuhara, S., Ogasawara, N., Kikuchi, H., Genome sequence and analysis of Aspergillus oryzae. Nature, 438, (2005)

90 A RIN rin rin

91 PG PG PG PG

92 high pigmenthp 1 hp 2 β non ripening norripening inhibitor rin Never ripe Nr Never ripe nor rin rin rin rin

93 LeMADS RIN Vrebarov MADS rin LeMADS RIN mrna rin rin/rin RIN/rin BRIN/rin RIN/RINrin/rin FRIN/rin rin RIN/rin F RIN/rin F RIN rin rin RIN/rin F F

94 rin F a Kc PK TK Kc PK PK Kc PK PK Kc PK PK Kc PK PK Kc F PK Kc PK PK Kc PK PK CIE rin a F LeMADS RIN Kc KcLeMADS RIN F F KcKGM A KGM RIN/rin F KGM LeMADS RIN rin KGM RIN/rin

95 A F B F F Mature Green; G Breaker; BPink; PRed ripe; R rin P R Psy Zds Pds Zds Pds rin Psy rin B F F Psy

96 β F A PGβ TBG4 LeEXP1 F B F S

97 aminocyclopropanecarboxylic acid ACC ACC BACC ACSACC ACO LeACS2 LeACS4 LeACO1 rin F A F ACC ACC

98 LeACS2 LeACS4 LeACO1 mrna F LeACS2 LeACS4 LeACO1 mrna ACS ACO mrna CF mrna C F ACS ACO LeMADS RIN RIN/rin ACS ACS F rin RIN/rin rin F F RIN/rin F RIN RIN RIN RIN

99 rin Oral allergy syndrome: OAS RIN rin RIN/rin F rin Cornell Boyce Thompson cdna rin F minor allergen beta fructofuranosidase precursor β

100 rin F F PG PG B F β PG β β PG PCR β rin AF PG F A

101 β β β SDS PAGE B kda akda c β kda b PG F F F IgE kda F F RIN rin F

102 rin F rin rin Cryj PGA

103 rin rin rin RIN/rin F rin RIN/rin Kitagawa, M., Moriyama, T., Ito, H., Ozasa, S., Adachi, A., Yasuda, J., Ookura, T., Inakuma, T., Kasumi, T., Ishiguro, Y. and Y., I., Reduction of allergic proteins by the effect of the ripening inhibitor (rin) mutant gene in F1 hybrid of the rin mutant tomato. Biosci Biotechnol Biochem, 70, (2006).

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