【作者】 高云；

【导师】 焦炳华； 刘小宇；

【作者基本信息】 第二军医大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 海藻糖是一种稳定的非还原二糖广泛存在于多种生物体内。由于其性质特殊，海藻糖在生物医药、食品、化妆品等多个领域有广泛的应用，但其市场应用由于生产成本过高而受到限制。目前在生物体内已经鉴定了至少5条海藻糖合成酶途径。在这5条途径中，海藻糖合成酶（trehalose synthase, TreS）途径是以低成本的麦芽糖为底物，通过转糖基作用一步法生成海藻糖，具有良好的工业应用前景。海洋是一个复杂的生态大环境，具有高盐、高压、低温、缺氧等一系列复杂的特点，来源于海洋微生物的酶通常具有一些特殊的酶学性质。此外，海藻糖能够在极端环境下对微生物产生保护作用，海洋微生物为了能在海洋中生存需要产生较多的海藻糖来对抗环境压力。因此，从海洋微生物中筛选具有潜在工业应用价值的新型TreS是一个非常有用的途径。目前，虽然已有多篇来源于不同物种的TreS的克隆、表达及性质研究的文献报道，但还没有一个关于海洋微生物来源的TreS的相关报道。在本研究中，通过筛选本课题组的海洋微生物菌株库，我们获取了一个新型的海洋微生物来源的tres基因。我们将该tres基因在大肠杆菌中重组表达并将重组蛋白纯化。此外，我们对该酶可能参与底物结合或催化的重要残基进行了鉴定，并对该酶催化麦芽糖和海藻糖相互转化过程中可能的机制也进行了探讨。第一部分：新型tres基因的获取、克隆、表达及纯化首先，从171株海洋微生物菌株中我们筛选到一个TreS高产菌株P8005。通过设计简并引物与PCR扩增，我们获得了一长648bp的核心序列。随后，通过TAIL-PCR，我们获取了一个长度为3369bp，编码1122aa，理论分子量大小为126kDa的开放式阅读框。通过氨基酸序列比对，我们发现该序列与文献已报道的海藻糖合成酶序列一致在29%－35%之间。该序列已提交至Genbank数据库（GenbankAccession No: JQ951963）。该序列被命名为G526。随后，我们以pET-32a为原核表达载体，构建了重组表达载体pET-32a-G526，并将该重组载体导入大肠杆菌BL21(DE3)中进行了重组表达。经过IPTG诱导后，成功诱导出大量重组蛋白（rG526）。含有重组蛋白的菌体经过超声破碎、离心得到的上清液，用Ni柱进行亲和纯化，纯化后得到的蛋白经蛋白电泳分析为单一条带，大小为141kDa(含亲和标签)，与理论值一致。第二部分：rG526重组蛋白的酶学性质研究我们以麦芽糖和海藻糖为底物对rG526的酶学性质进行研究。rG526能够催化麦芽糖和海藻糖的相互转化，反应达到平衡点时麦芽糖和海藻糖间的比例大约是7:3。此外，该酶在催化麦芽糖生成海藻糖的过程中还生成一定量的葡萄糖（占总产物的5%）。经过动力学参数测定，发现相比海藻糖，rG526对麦芽糖具有较高的亲和力和催化效率（Km/kcat）。rG526的最适反应温度和最适反应pH分别是37℃和pH7.2。该酶在0℃－40℃温育1h能保持85%以上活性，当保温温度超过50℃，酶的活性只能保持原来的10%。不同的金属离子和抑制剂对酶的活性有一定的影响，其中Cu2+和SDS对酶的活性有显著抑制作用。K+浓度对酶的活性有显著影响，K+浓度在20－40mM时，rG526具有最高的催化活性。第三部分：rG526重要功能残基鉴定与催化特性研究通过序列比对，我们发现G526的氨基酸序列与结构已知蛋白的海藻酮糖合成酶MutB（PDB:1_ZJA）的氨基酸序列一致性较高（N端一致性达32%）。我们以MutB的三维结构作为模板，对G526进行同源建模。同源建模结果显示了G526中的一些残基可能参与催化或与底物发生作用。通过定点突变实验，我们发现，D78A、Y81A、H121A、D219A、E261A、H331A和D332A残基的突变会使G526活性大幅降低。此外，我们还对rG526的催化特性进行了研究。通过同位素底物标记实验证实，二氘标记的海藻糖和未标记的海藻糖经过酶的催化反应产生的是二氘标记的二糖以及未标记的二糖。另外，7个氘标记的葡萄糖不会在反应过程中掺入到二糖中，说明该酶的催化机制为分子内转糖基的反应机制。结论：在本研究中我们从海洋假单胞菌中获得了一新型的TreS。该TreS能够催化麦芽糖和海藻糖之间的相互转化并且其平衡点朝向生成海藻糖的方向，该结果表明该酶可能具有潜在工业应用价值。在本研究中还找到一些可能参与催化或底物结合的重要残基：D78、Y81、H121、D219、E261、H331、D332A。这些氨基酸残基可作为候选残基进一步进行结构和功能的研究。同位素底物标记实验表明：该酶在催化底物麦芽糖和海藻糖相互转化过程中采用的是一个彻底的分子内转化机制。更多还原

【Abstract】 Trehalose is a stable and non-reducing disacarides showing widespread occurrencein many organisms. Trehalose has many applications in the pharmaceutical, food andcosmetics industries due to its special characteristics. However, its applications arelimited by the high cost of production. Until now, At least five biological pathwayswere found in organisms for trehalose production. Among them, Trehalose synthase(TreS) utilizes the low cost substrate of maltose and convert it to trehalose in one step.This pathway is prospective for the industrial production of trehalose.The ocean is a complicated environment which has the characteristics of salinity,high pressure, cold and hypoxia. In addition, trehalose can protect microorganismsagainst environmental pressures. Marine microorganisms, to achieve tolerance, willincrease the level of cytosolic trehalose to cope with external stress. Therefore,screening TreS-producing strains from the ocean and isolating the effective enzyme isprespective for the production of trehalose.Although cloning, expression and characterization of several tres genes from manydifferent strains have been reported, tres genes from marine microorganisms have notbeen investigated until now. In this study, a novel tres gene was obtained from a marinePseudomonas. This new gene was cloned and expressed in Escherichia coli.(E.coli)and the recombinant enzyme was purified and characterized. Several importantresidues that might be involved in catalysis or substrate binding were identified bysite-directed mutagenesis. In addition, possible mechanism concerning the aglyconerearrangement during the conversion between maltose and trehalose catalyzed by thisenzyme was also investigated in this study. Section I: Isolation, cloning, expression and purification of this novel tres genePseudomonas sp. P8005, which demonstrated the highest activity of trehaloseproduction, was isolated from hundreds of marine bacteria collected by this laborary. Acore region of648bp was obtained by degenerate PCR. Subsequently, an open readingframe (ORF) with a length of3369bp, encoding1122amino acids and with a predictedmolecular weight of126kDa was obtained by thermal asymmetric interlaced PCR（TAIL-PCR）. The amino acids sequence identities between this TreS and otherreported TreS was relatively low (29%-35%). The new sequence was submitted toGenbank database (Genbank Accession No: JQ951963). The novel tres gene wasnamed G526.Then the recombinant expression vector pET-32a-G526was constructed andtransformed into E. coli strain BL21(DE3). The recombinant TreS (rG526) wasproduced after induction. After ultrasonication and centrifugation, the purified rG526was obtained by using the Ni sepharose affinity chromatography. After purification,one major protein band at about141kDa which corresponded to the theoretical valuecould be found in SDS-PAGE.Section II Enzymatic characterization of rG526The recombinant enzyme can catalyze the interconversion between maltose andtrehalose. The ratio of maltose to trehalose at equilibrium point is about3:7. In additionto trehalose, a small amount of glucose (about5%of the total yield) is produced duringthe reaction. The novel enzyme has a higher affinity and a higher catalytic efficiency(Km/kcat) towards maltose than towards trehalose. The optimal pH and temperature ofrG526was pH7.2and37℃, respectively. It can remain over80%of its initial activityafter pre-incubated at temperature below40℃for1h. When the pre-incubationtemperature is over50℃，the residual activity is less than10%of its initial activity.Reagents such as Cu2+and SDS had strong inhibition on the enzymatic activity ofrG526. Adequate concentration of K+was necessary for the enzymatic activity. This enzyme demonstrated the highest activity at20-40mM of K+.Section III Identification of functional important residues and investigation ofpossible catalytic mechanisms of rG526By searching the PDB database, the amino acids sequence was shown to have highidentities with the structural solved protein, trehalulose synthase MutB (PDB:1_ZJA).Several important residues involved in catalysis or substrate binding have already beenidentified in the crystal structure of MutB. By sequence alignment, severalcorresponding residues were also found in G526(D78, Y81, H121, D219, E261, H331and D332). These residues were individually replaced by alanine. The dramaticallydecrease of each mutant enzyme implied that these residues might be important incatalysis or substrate binding. By using isotope-labeled substrate, it was demonstratedthat [2H2] trehalose combined with unlabeled trehalose could not convert to [2H]trehalose or [2H] maltose and no incorporation of [2H7] glucose into maltose ortrehalose happened during the reaction catalyzed by rG526.ConclusionA novel TreS was obtained from marine Pseudomonas in this study. This TreS cancatalyze the interconversion between maltose and trehalose and the equilibrium point istowards production of trehalose indicating its potential application in industry. Severalresidues that might be important in catalysis or substrate binding was also identified inthis study, including: D78, Y81, H121, D219, E261, H331and D332. Isotope-labeledsubstrate experiments demonstrated that this enzyme involved a completelyintramolecular mechanism.更多还原