Studies on enzymatic mechanism of beta-amylase in sweet potato and its possible cooperation with starch phosphorylase

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。 VSeveral 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. VIAbbreviation…………………………………………………………………………...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…………………………………………………………………………….7