甘藷中beta-澱粉酶之酵素催化機制探討及其與澱粉磷解酶之可能合作關係

Ya-Ting Liu; 劉雅婷

請用此 Handle URI 來引用此文件： http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24366

完整後設資料紀錄

DC 欄位	值	語言
dc.contributor.advisor	楊健志
dc.contributor.author	Ya-Ting Liu	en
dc.contributor.author	劉雅婷	zh_TW
dc.date.accessioned	2021-06-08T05:23:24Z	-
dc.date.copyright	2005-07-26
dc.date.issued	2005
dc.date.submitted	2005-07-25
dc.identifier.citation	Adachi M, Mikami B, Katsube T, Utsumi S (1998) Crystal structure of recombinant soybean beta-amylase complexed with beta-cyclodextrin. J Biol Chem 273: 19859-19865 Alber T, Banner DW, Bloomer AC, Petsko GA, Phillips D, Rivers PS, Wilson IA (1981) On the three-dimensional structure and catalytic mechanism of triose phosphate isomerase. Philos Trans R Soc Lond B Biol Sci 293: 159-171 Albrecht T, Koch A, Lode A, Greve B, Schneider-Mergener J, Steup M (2001) Plastidic (Pho1-type) phosphorylase isoforms in potato (Solanum tuberosum L.) plants: expression analysis and immunochemical characterization. Planta 213: 602-613 Bailey JM, Whelan WJ (1957) The mechanism of carbohydrase action. 3. The action pattern of beta-amylase. Biochem J 67: 540-547 Beck E, Ziegler P (1989) Biosynthesis and degradation of starch in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:95-117 Browner MF, Fletterick RJ (1992) Phosphorylase: a biological transducer. Trends Biochem Sci 17: 66-71 Buchner P, Borisjuk L, Wobus U (1996) Glucan phosphorylases in Vicia faba L.: cloning, structural analysis and expression patterns of cytosolic and plastidic forms in relation to starch. Planta 199: 64-73 Chang TC, Su JC (1986) Starch phosphorylase and its inhibitor in sweet potato. Plant Physiol. 80: 534-538 Cheong CG, Eom SH, Chang C, Shin DH, Song HK, Min K, Moon JH, Kim KK, Hwang KY, Suh SW (1995) Crystallization, molecular replacement solution, and refinement of tetrameric beta-amylase from sweet potato. Proteins 21: 105-117 Davies GJ, Tolley SP, Henrissat B, Hjort C, Schulein M (1995) Structures of oligosaccharide-bound forms of the endoglucanase V from Humicola insolens at 1.9 A resolution. Biochemistry 34: 16210-16220 de Fekete MA, Vieweg GH (1973) The role of phosphorylase in the metabolism of starch. Ann N Y Acad Sci 210: 170-180 Duwenig E, Steup M, Kossmann J (1997) Induction of genes encoding plastidic phosphorylase from spinach (Spinacia oleracea L.) and potato (Solanum tuberosum L.) by exogenously supplied carbohydrates in excised leaf discs. Planta 203: 111-120 French D (1961) Action pattern of beta-amylase. Nature 190: 445-446 French D, Summer R (1956) Action of beta-amylase on branched oligosaccharides. J Biol Chem 222: 469-477 Frydman RB, Slabnik E (1973) The role of phosphorylase in starch biosynthesis. Ann N Y Acad Sci 210: 153-169 Hanes CS (1940) The reversible formation of starch from glucose-1-phosphate catalyzed by potato phosphorylase. Proc Roy Soc B129:174-208 Hehre EJ, Kitahata S, Brewer CF (1986) Catalytic flexibility of glycosylases. The hydration of maltal by beta-amylase to form 2-deoxymaltose. J Biol Chem 261: 2147-2153 Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7: 637-644 Hirata A, Adachi M, Utsumi S, Mikami B (2004) Engineering of the pH optimum of Bacillus cereus beta-amylase: conversion of the pH optimum from a bacterial type to a higher-plant type. Biochemistry 43: 12523-12531 Kang YN, Adachi M, Mikami B, Utsumi S (2003) Change in the crystal packing of soybean beta-amylase mutants substituted at a few surface amino acid residues. Protein Eng 16: 809-817 Kang YN, Adachi M, Utsumi S, Mikami B (2004) The roles of Glu186 and Glu380 in the catalytic reaction of soybean beta-amylase. J Mol Biol 339: 1129-1140 Kang YN, Tanabe A, Adachi M, Utsumi S, Mikami B (2005) Structural analysis of threonine 342 mutants of soybean beta-amylase: role of a conformational change of the inner loop in the catalytic mechanism. Biochemistry 44: 5106-5116 Kaplan F, Guy CL (2004) beta-Amylase induction and the protective role of maltose during temperature shock. Plant Physiol 135: 1674-1684 Kitahata S, Chiba S, Brewer CF, Hehre EJ (1991) Mechanism of maltal hydration catalyzed by beta-amylase: role of protein structure in controlling the steric outcome of reactions catalyzed by a glycosylase. Biochemistry 30: 6769-6775 Lilley RM, Chon CJ, Mosbach A, Heldt HW (1977) The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro. Biochim Biophys Acta 460: 259-272 Mikami B, Adachi M, Kage T, Sarikaya E, Nanmori T, Shinke R, Utsumi S (1999) Structure of raw starch-digesting Bacillus cereus beta-amylase complexed with maltose. Biochemistry 38: 7050-7061 Mikami B, Degano M, Hehre EJ, Sacchettini JC (1994) Crystal structures of soybean beta-amylase reacted with beta-maltose and maltal: active site components and their apparent roles in catalysis. Biochemistry 33: 7779-7787 Mikami B, Hehre EJ, Sato M, Katsube Y, Hirose M, Morita Y, Sacchettini JC (1993) The 2.0-A resolution structure of soybean beta-amylase complexed with alpha-cyclodextrin. Biochemistry 32: 6836-6845 Mikami B, Nomura K, Morita Y (1994) Two sulfhydryl groups near the active site of soybean beta-amylase. Biosci Biotechnol Biochem 58: 126-132 Mikami B, Yoon HJ, Yoshigi N (1999) The crystal structure of the sevenfold mutant of barley beta-amylase with increased thermostability at 2.5 A resolution. J Mol Biol 285:1235-1243 Mori H, Tanizawa K, Fukui T (1991) Potato tuber type H phosphorylase isozyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli. J Biol Chem 266: 18446-18453 Mori H, Tanizawa K, Fukui T (1993) A chimeric alpha-glucan phosphorylase of plant type L and H isozymes. Functional role of 78-residue insertion in type L isozyme. J Biol Chem 268: 5574-5581 Nakamura M (1953) Effect of added primer on lima bean phosphorylase. Nature 171: 795-796 Newgard CB, Hwang PK, Fletterick RJ (1989) The family of glycogen phosphorylases: structure and function. Crit Rev Biochem Mol Biol 24: 69-99 Niittyla T, Messerli G, Trevisan M, Chen J, Smith AM, Zeeman SC (2004) A previously unknown maltose transporter essential for starch degradation in leaves. Science 303: 87-89 Oyama T, Kusunoki M, Kishimoto Y, Takasaki Y, Nitta Y (1999) Crystal structure of beta-amylase from Bacillus cereus var. mycoides at 2.2 A resolution. J Biochem (Tokyo) 125: 1120-1130 Oyama T, Miyake H, Kusunoki M, Nitta Y (2003) Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents. J Biochem (Tokyo) 133: 467-474 Pan SM, Chang TC, Juang RH, Su, JC (1988) Starch phosphorylase inhibitor is b-amylase. Plant physiol. 88: 1154-1156 Parrish FW, Smith EE, Whelan WJ (1970) Actions of starch carbohydrases on chemically modified maltodextrins. Arch Biochem Biophys 137: 185-189 Porter HK (1950) The inhibition of plant phhosphorylase by β-amylase and the detection of phosphorylase in barley. Biochem. 47: 476-482 Preiss J, Okita TW, Greenberg E (1980) CHarization of the spinach leaf phosphorylase. Plant Physiol 66: 864-869 Ray RR, Nanda G (1996) Microbial beta-amylases: biosynthesis, characteristics, and industrial applications. Crit Rev Microbiol 22: 181-199 Ritte G, Steup M, Kossmann J, Lloyd JR (2003) Determination of the starch-phosphorylating enzyme activity in plant extracts. Planta 216: 798-801 Schupp N, Ziegler P (2004) The relation of starch phosphorylases to starch metabolism in wheat. Plant Cell Physiol 45: 1471-1484 Servaites JC, Geiger DR (2002) Kinetic characteristics of chloroplast glucose transport. J Exp Bot 53: 1581-1591 Slabnik E, Frydman RB (1970) A phosphorylase involved in starch biosynthesis. Biochem Biophys Res Commun 38: 709-714 Sivak MN, Tandecarz JS, Cardini CE (1981) Studies on potato tuber phosphorylase-catalyzedreaction in the absence of an exogenous acceptor. I. Characterization and properties of the enzyme. Arch Biochem Biophys 212: 525-536 Smith AM, Denyer K, Martin C (1997) The Synthesis of the Starch Granule. Annu Rev Plant Physiol Plant Mol Biol 48: 67-87 Smith AM, Zeeman SC, Smith SM (2005) Starch Degradation. Annu Rev Plant Biol 56: 73-98 Sonnewald U, Basner A, Greve B, Steup M (1995) A second L-type isozyme of potato glucan phosphorylase: cloning, antisense inhibition and expression analysis. Plant Mol Biol 27: 567-576 Steup M (1981) Purification of chloroplast alpha-1,4-glucan phosphorylase from spinach leaves by chromatography on Sepharose-bound starch. Biochim Biophys Acta 659: 123-131 Steup M, Gerbling KP (1983) Multiple forms of amylase in leaf extracts: electrophoretic transfer of the enzyme forms into amylose-containing polyacrylamide gels. Anal Biochem 134: 96-100 Steup M, Schachtele C (1986) Alpha-1,4 glucan phosphorylase forms from leaves of spinach spinacia-oleracea II peptide patterns and immunoligical properties a comparison with other phosphorylase forms. Planta 168: 222-231 Steup M (1988) Starch degradation in 'The biochemistry of plants' Academia Press, New York 14: 255-296 Steup M, Peavey DG, Gibbs M (1976) The regulation of starch metabolism by inorganic phosphate. Biochem Biophys Res Commun 72: 1554-1561 Stitt M, Rees TA (1980) Carbohydrate breakdown by chloroplasts of Pisum sativum. Biochim Biophys Acta 627: 131-143 Stitt M, Wirtz W, Heldt HW (1980) Metabolite levels during induction in the chloroplast and extrachloroplast compartments of spinach protoplasts. Biochim Biophys Acta 593: 85-102 Su YC, Chiu RJ, Yu N, Chang WR (1984) The microbial production of amylase inhibitor and its application. I. Isolation and cultivation of Streptomyces nigrifaciens NTU-3314. Proc Natl Sci Counc Repub China B 8: 292-301 Taussky H, Shorr E (1953) A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 202(2):675-85. Tanaka N, Kajimoto S, Mitani D, Kunugi S (2002) Effects of guanidine hydrochloride and high pressure on subsite flexibility of beta-amylase. Biochim Biophys Acta 1596: 318-325 Tanaka N, Mitani D, Kunugi S (2001) Pressure-induced perturbation on the active site of beta-amylase monitored from the sulfhydryl reaction. Biochemistry 40: 5914-5920 Thoma JA (1974) Interaction of beta-amylase with substrates and inhibitors with comments on Koshland's Induced-Fit Theory. A reply. Eur J Biochem 44: 139-142 Totsuka A, Fukazawa C (1993) Expression and mutation of soybean beta-amylase in Escherichia coli. Eur J Biochem 214: 787-794 Totsuka A, Fukazawa C (1996) Functional analysis of Glu380 and Leu383 of soybean beta-amylase. A proposed action mechanism. Eur J Biochem 240: 655-659 Walters RG, Ibrahim DG, Horton P, Kruger NJ (2004) A mutant of Arabidopsis lacking the triose-phosphate/phosphate translocator reveals metabolic regulation of starch breakdown in the light. Plant Physiol 135: 891-906 Wang Q, Monroe J, Sjolund RD (1995) Identification and characterization of a phloem-specific beta-amylase. Plant Physiol 109: 743-750 Weise SE, Kim KS, Stewart RP, Sharkey TD (2005) beta-Maltose is the metabolically active anomer of maltose during transitory starch degradation. Plant Physiol 137: 756-761 Yang Y, Steup M (1990) Polysaccharides fraction from higher plants which strongly interacts with cytosolic phosphorylase isozyme. Plant Physiol. 94:960-69 Yoshida N, Nakamura K (1991) Molecular cloning and expression in Escherichia coli of cDNA encoding the subunit of sweet potato beta-amylase. J Biochem (Tokyo) 110: 196-201 Zeeman SC, Thorneycroft D, Schupp N, Chapple A, Weck M, Dunstan H, Haldimann P, Bechtold N, Smith AM, Smith SM (2004) Plastidial alpha-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress. Plant Physiol 135: 849-858 莊榮輝 (1989) 水稻蔗糖合成酶之生化及免疫研究。國立台灣大學農業化學研究所博士論文潘素美 (1989) 水稻澱粉磷解酶的生化性質研究。國立台灣大學農業化學研究所博士論文王恆隆 (1992) 澱粉磷解酶及β-澱粉酶在甘藷癒創組織內的表現。國立台灣大學農業化學研究所碩士論文陳師瑩 (1997) β-澱粉酶阻礙澱粉磷解酶的分子機轉。國立台灣大學農業化學研究所博士論文林泰元 (1998) 甘藷塊根澱粉酶基因表現、免疫組織定位與生化性質研究。國立台灣大學農業化學研究所博士論文陳韋琮 (1999) 甘藷β-澱粉酶在大腸桿菌之表現及以原位雜交法探討其在甘薯塊根內之分佈。國立台灣大學農業化學研究所碩士論文張弘儒 (2003) β-澱粉酶基質結合區與催化機制的探討。國立台灣大學農業化學研究所碩士論文葉昭圻 (2005) 甘藷塊根澱粉磷解酶高溫下階段式降解之探討
dc.identifier.uri	http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24366	-
dc.description.abstract	先前研究顯示，在甘藷 β-澱粉酶的催化機制中，參與基質結合區中的許多胺基酸可能協助催化反應。因此，張 (2003) 分別將與第一個糖及第四個糖作用的相關胺基酸進行點突變，這些突變株分別喪失不同程度的活性。在不同 pH 值的酵素動力學結果分析，發現與第一個糖作用的胺基酸中，His 94 對於催化作用扮演最重要的角色；而在與第四個糖作用的胺基酸中，疏水性的胺基酸對於整體催化作用影響較大。先前研究已知 β-澱粉酶為澱粉磷解酶的非競爭型抑制劑。將帶有不同程度活性之 β-澱粉酶突變株與澱粉磷解酶進行反應，發現澱粉磷解酶的活性受到抑制，但是抑制的程度並不與 β-澱粉酶本身的活性成正比。另一方面，β-澱粉酶的活性可能透過澱粉磷解酶的作用而被調節，並且利用薄膜色層分析法發現在澱粉磷解酶的存在下，β-澱粉酶會失去其多重攻擊的特性。不同pH 值環境下的酵素動力學分析顯示，在澱粉磷解酶的存在下，β-澱粉酶的活性在較低pH值環境下 (pH 4 以下) 下有上升的趨勢;但在較高的pH值環境下 (pH 4 以上)，β-澱粉酶的活性則迅速的降低。近來研究中顯示夜晚時，阿拉伯芥葉中 β-澱粉酶為負責澱粉降解過程中的重要酵素。因此，我們推測 β-澱粉酶的活性可能透過澱粉磷解酶的作用來進行調節。 V	zh_TW
dc.description.abstract	Several β-amylase mutants with substitution at substrate binding site, in particular subsite 1 and 4, have been generated (Chang 2003). Most of the mutants exhibited higher affinity to soluble starch or reduced their enzyme activity, reflecting these amino acids contribute to the enzyme catalysis or multiple attack mechanism. Analysis of the His 94 mutants at different pH indicated its role in binding and catalysis. Furthermore, the hydrophobic interaction at subsite 4 was demonstrated to be important to catalysis through kinetic analysis and fluorescence spectroscopy. β-amylase has been reported to be a non-competitive inhibitor of starch phosphorylase. We are interested in the effect of the β-amylase with altered characters on starch phosphorylase. It seemed that the β-amylase with reduced activity still exerted inhibition to starch phosphorylase, but the inhibition level was not proportional to the degree of reduced activity. However, it is interesting to note the changes of β-amylase’s behavior in the presence of starch phosphorylase. Like most β-amylase mutants, the recombinant wild-type β-amylase also lost its multiple attack of a single chain glucan in the presence of starch phosphorylase. Besides, the activity of β-amylase mutants with reduced activity at lower pH seems to be enhanced in the presence of starch phosphorylase. β-amylase has been demonstrated to be one of the major enzymes responsible for starch breakdown in the leaves of Arabidopsis during the night. We speculated that the activity of β-amylase may be regulated through starch phosphorylase. VI	en
dc.description.provenance	Made available in DSpace on 2021-06-08T05:23:24Z (GMT). No. of bitstreams: 1 ntu-94-R92b47210-1.pdf: 4148389 bytes, checksum: 0f842dbe980e892632ad44a78656c47a (MD5) Previous issue date: 2005	en
dc.description.tableofcontents	Abbreviation…………………………………………………………………………...IV 中文摘要………………………………………………………………………………V Abstract………………………………………………………………………………VI Chapter 1 Introduction…………………………………………………………....1 1.1 General properties of β-amylase………………………………………………...1 1.1.1 The reaction catalyzed by β-amylase……………………………………………1 1.1.2 The comparison of β-amylase between higher plants and microorganisms.........1 1.1.3 The tertiary structure of β-amylase……………………………………………...2 1.1.4 Substrate binding site (subsite) of β-amylase…………………………………...2 1.1.5 Residues in the subsites of β-amylase…………………………………………..3 1.1.6 The loops of β-amylase in substrate binding……………………………………5 1.1.7 Single and multiple attack mechanism of β-amylase…………………………...6 1.2 General properties of starch phosphorylase……………………………………8 1.2.1 The discovery of starch phosphorylase………………………………………….9 1.2.2 The classification of starch phosphorylase…………………………………….10 1.2.3 The relationship between the conformation and the activity of starch phosphorylase………………………………………………………………….10 1.3 Starch degradation……………………………………………………………10 1.3.1 The role of β-amylase in starch degradation in Arabidopsis…………………11 1.3.2 The role of starch phosphorylase in starch degradation………………………..12 1.4 Experimental background……………………………………………………13 1.5 Motivation………………………………………………………………………15 Chapter 2 Material and Method……………………………………………16 2.1 Materials………………………………………………………………………...16 2.1.1 Materials………………………………………………………………….........16 2.1.2 Reagents………………………………………………………………………..16 2.1.3 Equipment……………………………………………………………………...19 2.2 Methods………………………………………………………………………….20 2.2.1 Expression of recombinant β-amylase…………………………………………20 2.2.2 Purification of recombinant β-amylase………………………………………...20 2.2.3 Purification of starch phosphorylase…………………………………………...21 2.2.4 Protein concentration determination…………………………………………...22 2.2.5 Electrophoresis…………………………………………………………………22 2.2.6 Coomassie brilliant blue staining………………………………........................24 2.2.7 Enzyme activity staining………………………………………………….........24 2.2.8 Enzyme activity assay…………………………………………………….........25 2.2.9 Comment of enzyme kinetic assay……………………………………….........27 2.2.10 Carbohydrate analysis by TLC development…………………………………28 2.2.11 Circular dicroism spectrum detection………………………………………...28 2.2.12 Fluorescence spectrum detection……………………………………………..28 2.2.13 Pull down assay………………………………………………………….........28 Chapter 3 Results and Discussion………………………………………......30 3.0 The amino acids in sweet potato corresponding to the ones in soybean…….30 3.1 The enzyme kinetic analysis of β-amylase…………………………………….30 3.1.1 The enzyme kinetic parameters at the subsite 1……………………………30 3.1.2 The enzyme kinetic parameters at the subsite 4…………………………….31 3.2 The pH profile of β-amylase……………………………………………………31 3.2.1 The pH profile at the subsite 1……………………………………………...32 3.2.1.1 The pH profile of eBA………………………………………………………………..32 3.2.1.2 The pH profile of D54A……………………………………………………………...32 3.2.1.3 The pH profile of H94A…………………………………………...............................32 3.2.1.4 The pH profile of H94S…………………………………………................................32 3.2.1.5 The pH profile of R421A……………………………………………………………..33 3.2.2 The pH profile at the subsite 4…………………………………………............33 3.2.2.1 The pH profile of F201A……………………………………………………………33 3.2.2.2 The pH profile of H302AW303A…………………………………………………….34 3.2.2.3 The pH profile of W303A……………………………………………………………34 3.2.2.4 The pH profile of H302Y…………………………………………………………….34 3.3 The enzyme kinetic analysis of starch phosphorylase in the presence of β-amylase……………………………………………………………………….35 3.4 The enzyme kinetic analysis of β-amylase in the presence of starch phosphorylase at different pH………………………………………………...36 3.5 Analysis of multiple attack of β-amylase by thin layer chromatography…...36 3.6 Pull down assay…………………………………………………………………37 3.7 Analysis by Circular Dichroism Spectrum……………………………………37 3.8 Analysis by Fluorescence Spectrum…………………………………………..38 3.8.1 The intrinsic fluorescence contributed by Tryptophan………………………38 3.8.2 The extrinsic fluorescence……………………………………………………..38 3.9 Summary and Discussion………………………………………………………39 3.9.1 The catalytic mechanism of β-amylase………………………………………...39 3.9.2 Possible cooperation between β-amylase and starch phosphorylase…………..40 Chapter 4 Future work………………………………………………………42 Figures………………………………………………………………………………43 Reference……………………………………………………………………………74 Appendix…………………………………………………………………………….79
dc.language.iso	en
dc.subject	澱粉&#37238	zh_TW
dc.subject	澱粉磷解&#37238	zh_TW
dc.subject	amylase	en
dc.subject	starch phosphorylase	en
dc.title	甘藷中beta-澱粉酶之酵素催化機制探討及其與澱粉磷解酶之可能合作關係	zh_TW
dc.title	Studies on enzymatic mechanism of beta-amylase in sweet potato and its possible cooperation with starch phosphorylase	en
dc.type	Thesis
dc.date.schoolyear	93-2
dc.description.degree	碩士
dc.contributor.oralexamcommittee	蘇仲卿,靳宗洛,吳素幸,陳佩燁
dc.subject.keyword	澱粉&#37238,澱粉磷解&#37238,	zh_TW
dc.subject.keyword	amylase,starch phosphorylase,	en
dc.relation.page	86
dc.rights.note	未授權
dc.date.accepted	2005-07-25
dc.contributor.author-college	生命科學院	zh_TW
dc.contributor.author-dept	微生物與生化學研究所	zh_TW
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