【作者】 余继刚；

【导师】 张学成；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 α-淀粉酶广泛分布于动物、植物和微生物中,能水解淀粉产生糊精、麦芽糖、低聚糖和葡萄糖等,是工业中应用最为广泛的酶制剂之一。构成α-淀粉酶家族的水解酶和转移酶是多结构域蛋白,他们共同的特点是都包含一个（α/β）8-barrel式催化结构单元,且活性位点位于p-折叠链的C末端。虽然α-淀粉酶都有一个共同的催化结构域及催化机制,但蛋白序列同源性分析发现α-淀粉酶可以被归属于3个不同的糖苷转移酶及水解酶家族—GH13、GH70和GH77。每个α-淀粉酶都包含有一个谷氨酸和两个天冬氨酸作为其必须的活性位点残基,同时其中大部分都有两个组氨酸保证其构象变换过渡态的稳定性。这五个氨基酸残基在整个a-淀粉酶家族中保守地存在于4段短的氨基酸序列中,且酶的特异性也都与这些氨基酸残基相关。本课题所研究的AmyP是从海洋微生物宏基因组中获得的一个能降解生淀粉的α-淀粉酶,它同已知的糖苷水解酶序列一致性和相似性很低,被归为一个新的糖苷水解酶亚家族GH13₃7。值得注意的是AmyP表现出优先降解生大米淀粉颗粒的特性,这是α-淀粉酶家族中第一次报道的有此特性的酶。在本课题中,全长AmyP基因已被成功克隆重组至表达载体并转化入大肠杆菌异源表达。纯化后的重组AmyP在底物类似物β-环糊精存在的条件下进行了结晶,对所获单晶进行X-射线衍射得到了分辨率达2.1A的数据。初步晶体学分析结果显示,AmyP可能以二聚体形式行使功能,AmyP/β-环糊精晶体结构的空间群为P21212,晶胞参数为：a=129.824A,b=215.534A,c=79.699A,α=90°,β=90°,γ=90°。在α-淀粉酶家族中有约10%的成员包含一个非催化模块,这个模块有助于酶结合和降解生淀粉。此类酶除了有一个共同的（α/β）8-barrel式催化结构域外,在蛋白的C末端还有一个β链组成的淀粉结合结构域SBD（starch binding domain）,此结构域为水解生淀粉所必需的特异性结构域。淀粉是碳水化合物中的一种,因此SBD属于碳水化合物结合模块CBM（carbohydrate binding module）。所有的碳水化合物结合模块可被分为多个CBM家族,在基于序列同源性的分类中,SBD被归为7大类的CBM家族：CBM20、CBM21、CBM25、CBM26、CBM34、 CBM41、CBM45。其中CBM20家族包含微生物α-淀粉酶的C末端SBD,是已知研究最为清楚的SBD。一些SBD的三维结构已经通过X-射线晶体学或核磁共振波谱学方法得到了解析。同全长α-淀粉酶的结构特点类似,虽然SBD的氨基酸序列有些差异很大,但其三维结构很保守,都是β折叠片堆叠形成的β-barrel式结构,构成活性位点的几个芳香族氨基酸也都相对保守。为揭示AmyP蛋白的功能机制特别是其降解生淀粉颗粒的特性,我们对其推定的C末端SBD结构域的结构与功能进行了初步的研究。在本课题中,我们重组表达和纯化了AmyP-SBD,并用圆二色谱（CD）、等温滴定微量热（ITC）和核磁共振（NMR）等技术对AmyP-SBD的结构与功能进行了初步的研究。CD和NMR实验结果显示Amy-SBD是一个类溶球态。等温滴定微量热和核磁共振实验则证实了AmyP-SBD与底物类似物p-环糊精的相互作用。值得注意的是,NMR化学位移扰动实验揭示：AmyP-SBD在结合p-环糊精前后经历了从无序向有序的构象转变。AmyP-SBD的这一“结合偶联折叠”性质在其它SBD上很罕见,对它的进一步考察对于我们深入理解AmyP蛋白的某些特性如优先降解生淀粉的机制及进一步的应用打下基础。更多还原

【Abstract】 a-amylase are universally distributed throughout the animal, plant and microbial kingdoms. They can hydrolyse starch molecules to give diverse products including dextrins and progressively smaller polymers composed of glucose units, a-amylase are one of the most popular and important form of industrial amylases.The hydrolases and transferases in the a-amylase family are multidomain proteins which has a catalytic domain of a (α/β)8-barrel fold, with the active site at the C-terminal end of the β-strands. Although all the a-amylase enzymes contain similar catalytic domains and share a common functioning mechanism, they have been assigned to three separate glycoside hydrolase/transferase families GH13, GH70and GH77based on amino acid sequence similarity. Every a-amylase need one glutamic acid and two aspartic acid residues for its activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilization. Those five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity.AmyP is a raw starch-degrading a-amylase newly identified from a marine metagenome library. It shares low sequence similarity with characterized glycoside hydrolases and was classified into a new subfamily of GH13. Especially, it showed preferential degradation to raw rice starch. In the present study, full length AmyP was cloned and overexpressed in Escherichia coli, then purified and crystallized in the presence of its substrate analogue β-cyclodextrin. Single crystals were successfully obtained, and X-ray diffraction on one of the single crystals gained data to a resolution of2.1A. Preliminary analysis on the data indicated that AmyP might be a dimmer in the crystal, and the crystal belonged to space group P21212, with unit-cell parameters a=129.824A, b=215.534A, c=79.699A, α=90°,β=90°,γ=90°Only10%of amylases contain a distinct non-catalytic module that is known to facilitate binding and degradation of raw starch. Besides the common (α/β)8-barrel catalytic domain, these enzymes contain another β-barrel domain nominated starch binding domain (SBD) at the C-terminus, which is critical for the degradation of raw starch granules. SBDs belong to carbohydrate binding modules (CBMs), which have been classified into various CBM families. In the sequence-based classification, SBDs have been grouped into seven CBM families:CBM20, CBM21, CBM25, CBM26, CBM34, CBM41and CBM45. The SBDs in the family CBM20, including classical C-terminal SBDs of microbial amylases, are the most thoroughly studied SBDs. The three-dimensional structures of some SBDs have already been determined by X-ray crystallography or nuclear magnetic resonance. Similar to full length a-amylases, though the sequences of SBDs varies from each other, their three-dimensional structures are conserved. All SBDs adopt a β-barrel fold, with the activity center composed of a few conserved aromatic residues.To understand the functioning mechanism of AmyP especially its preferential activity to degrade raw starch granules, we further investigated the structure and function of the C-terminal putative starch binding domain of AmyP. In this study, AmyP-SBD was recombinantly expressed, purified, and characterized in structure and function by using circular dichroism (CD), nuclear magnetic resonance (NMR), and isothermal titration calorimetry (ITC). CD and NMR results indicated that AmyP-SBD adopted a molten globule like state. ITC and NMR data confirmed its substrate binding activity using P-cyclodextrin as substrate analogue. Intriguingly, NMR chemical shift perturbation experiments showed that AmyP-SBD experienced a conformational change from disorder to order when binding to P-cyclodextrin. This substrate binding coupled folding property was rarely observed on other SBDs. Further in depth study on the binding coupled folding of AmyP-SBD may be helpful to elucidate the functioning mechanism of AmyP and its application.更多还原