CN117413056A - 用于果寡糖的优化生产的经修饰的果聚糖蔗糖酶 - Google Patents
用于果寡糖的优化生产的经修饰的果聚糖蔗糖酶 Download PDFInfo
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Abstract
本发明涉及包含从由SEQ ID NO:26‑SEQ ID NO:34组成的序列的组中选择的氨基酸序列的经修饰的重氮营养葡糖酸醋酸杆菌(Gluconacetobacter diazotrophicus)果聚糖蔗糖酶(LsdA),以及产生其的经遗传修饰的微生物。该经修饰的酶保持高的比活性,将蔗糖转变为在果糖基连接类型方面具有变化的短链果寡糖,并且不产生果聚糖这种多糖。本发明还提供了包含所述经修饰的果聚糖蔗糖酶的酶制备物,以及用于将蔗糖转变为果寡糖的酶促方法,其中使用包含从由SEQ ID NO:26‑SEQ ID NO:34组成的序列的组中选择的氨基酸序列的经修饰的果聚糖蔗糖酶或者产生其的经遗传修饰的微生物。
Description
技术领域
本发明涉及酶工程和工业生物技术的领域,特别是涉及重氮营养葡糖酸醋酸杆菌(Gluconacetobacter diazotrophicus)果聚糖蔗糖酶(LsdA)的氨基酸序列的修饰,以用于基于果聚糖这种多糖的形成能力的丢失来获得具有高比活性和更大果寡糖(FOS)产率的经修饰的变体。本发明在下述方面具有其主要应用:建立和改进旨在产生不同类型的短链FOS(具有宽广的商业需求的可溶性益生元纤维)的酶促方法。
现有技术
FOS是在功能性食品市场中具有高需求的天然益生元纤维。FOS的消费有利于在结肠中益生微生物(例如,双歧杆菌和乳杆菌)群体的增加,这为人和动物的健康带来相关益处(Singh等人,Appl.Biochem.Biotechnol.184:613-635,2017)。FOS是短链果糖聚糖,其包含2至10个果糖单元,其中β-(2→1)(菊粉类型的FOS)或β-(2→6)(果聚糖类型的FOS)键可以占优势。目前,仅菊粉类型的FOS被商业化。在这些之中,1-蔗果三糖[αglu(1,2)βfru(1,2)βfru]具有最高的商业价值,因为它在近端结肠中发挥有效的益生元作用并且其增甜效力高于包含3个或更多个果糖单元的FOS(Tochio等人,Foods 7:140,2018)。果聚糖类型的FOS具有在人和动物中经证明的高的益生元作用,但是在市场上尚不可得,因此建立使其商业化在经济上可行的工业规模生产方法是特别令人感兴趣的(Porras-Domínguez等人,Carbohydrate Polymers 177:40-48,2017)。
目前,从蔗糖开始的FOS商业生产通过使用真菌来源的β-呋喃果糖苷酶(EC3.2.1.26)或β-果糖基转移酶(EC 2.4.1.100)来进行。在高的蔗糖(GF)浓度(600-800g/L)下这些酶中任一个的温育产生1-蔗果三糖(GF2)、1,1-蔗果四糖(GF3)和1,1,1-蔗果五糖(GF4),其总和达到占该反应混合物中的总碳水化合物的55-60%(w/w)(Flores-Maltos等人,Critical Rev.Biotechnol.36:259-267,2016)。在商业产品和中,GF2:GF3:GF4的比例为大约37:53:10。对于将蔗糖转变为1-蔗果三糖来说特别有吸引力的另一种酶是在巴斯德毕赤酵母(Pichia pastoris)这种酵母中产生的来自高羊茅(Schedonorus arundinaceus)这种植物的蔗糖:蔗糖-1-果糖基转移酶(1-SST,EC2.4.1.99)(Hernández等人,J.Biotechnol.266:59-71,2018)。命名为Sa1-SSTrec的重组酶是稳定的并且将蔗糖转变为以9:1比例的GF2和GF3的混合物,具有55-60%(w/w)的FOS总产率。重组酶Sa1-SSTrec在市场上尚不可得。
作为共同的缺点,真菌和植物的果糖基转移酶具有内在的外切果糖聚糖酶活性并且水解其自己的FOS产物,随着底物蔗糖被耗尽(Hernández等人,J.Biotechnol.266:59-71,2018)。在为了商业目的的使用这些酶的方法中,当FOS的产率达到55-60%(w/w)并且蔗糖尚在糖类总组成的15和25%(w/w)之间波动时,需要停止反应。在反应混合物(FOS-55%糖浆)中存在的剩余蔗糖和单糖(葡萄糖和果糖)是能量污染物,其应当在旨在用于糖尿病的低热量产品中被消除。
目前,直至95%(w/w)的纯度的FOS的工业纯化通过基于分子排阻原理的色谱法技术来进行,其中蔗糖这种二糖的去除是在回收1-蔗果三糖这种三糖中的主要损失因素。还不存在有关于允许在真核生物果糖基转移酶反应混合物中的剩余蔗糖的完全转变而不导致产物FOS水解的酶促程序的报道。
在从蔗糖开始的不同类型的FOS的产生中可以使用由某些细菌分泌的果聚糖蔗糖酶(EC 2.4.1.10)和菊粉蔗糖酶(EC 2.4.1.9)(Díez-Municio等人,Appl.Microbio.IBiotechnol.100:6251-6263,2016;Kirtel等人,Appl.Microbiol.Biotechnol.102:9207-9220,2018)。键的类型、聚合的程度和FOS的产率依赖于酶的来源和反应条件而变化(等人,Biotechnol.Advances 34:827-844,2016)。通常,细菌果糖基转移酶将少于一半的在反应中存在的初始蔗糖含量(50-70%,w/v)转化为短链FOS。其余的底物经由直接水解(转化酶活性)和在很大程度上通过产物FOS的持续聚合(聚合酶活性)而被消耗。除了降低短链FOS的最终产率外,高分子量的果聚糖或菊粉这些多糖的形成还造成反应混合物的粘度的显著增加,这使生物催化剂的操作过程和通过色谱法的产物分开变得复杂。由于这样的原因,细菌果聚糖蔗糖酶和菊粉蔗糖酶被认为是对于FOS的工业生产来说很少有吸引力的候选物(/>等人,Biotechnol.Advances 34:827-844,2016)。
从天然细菌的培养物上清液中获得(Hernández等人,Biochemical J.309:113-118,1995)或在巴斯德毕赤酵母这种酵母中重组产生(Trujillo等人,Enzyme MicrobialTechnol.28:139-144,2001)的重氮营养葡糖酸醋酸杆菌果聚糖蔗糖酶(名为LsdA;GenBank:L41732.5)特别地通过在蔗糖转化过程期间积累产物1-蔗果三糖和1,1-蔗果四糖[αglu(1,2)βfru(1,2)βfru(1,2)βfru]而相区别。LsdA还合成6-蔗果三糖[αglu(1,2)βfru(6,2)βfru],它是作为形成果聚糖这种多糖的直接前体起作用的三糖。该同聚体达到104个果糖单元,并且以β-(2→6)连接的主链来构成,具有β-(2→1)分支点。果聚糖这种多糖的形成通过持续机制而发生,其中所述酶可以与果糖基化产物相结合,这有利于其继续延伸而不介导中间的寡果糖聚糖的释放(Raga-Carbajal等人,Glycobiology 26:377-385,2016)。在底物蔗糖不存在下,LsdA显示出外切果聚糖酶活性,但是不水解菊粉类型的果糖聚糖(Hernández等人,Biochemical J.309:113-118,1995)。
目前,在具有底物蔗糖的完全转变的FOS的商业生产方法中还没有可以使用的任何来源的经修饰的果糖基转移酶,无论是单个酶还是互补酶。产物FOS连续延伸从而形成果聚糖这种多糖是酶LsdA所具有的对于其在FOS的直接产生中或者在真核生物果糖基转移酶的反应混合物中的剩余蔗糖的转变中的工业使用来说的主要催化障碍。
发明描述
本发明通过下述方式而解决了上面提到的问题:提供经修饰的重氮营养葡糖酸醋酸杆菌果聚糖蔗糖酶(LsdA),其特征在于,其包含从由SEQ ID NO:26-SEQ ID NO:34组成的序列的组中选择的氨基酸序列。LsdA的该经修饰的变体合成短链FOS(其显示出果糖基连接类型的变化),并且不形成果聚糖这种多糖。此外,它不水解1-蔗果三糖,也不水解1,1-蔗果四糖,其是在蔗糖转化过程期间倾向于积累的产物。
通过使用随机诱变的化学方法作为初始工作工具,以及与之相结合的大肠杆菌(Escherichia coli)重组克隆文库的通过表型的大规模筛选,使得能够鉴定出在聚合反应(果聚糖这种聚合物的合成)中所牵涉的LsdA的活性位点的几个氨基酸。这些氨基酸中的几个在其他细菌果糖基转移酶中具有等价的结构,但是其功能还有待探索。在所鉴定出的氨基酸中,选择R141、H142、F274、N276和R304(编号是指成熟酶的序列,即不考虑形成在前体蛋白中存在并且在分泌期间被切掉的信号肽的前30个氨基酸)作为用于通过饱和诱变的定向进化工作的靶标。通过该途径,构建了LsdA变体文库,其包含在与果糖基残基的供者底物和/或接受者底物直接或间接相互作用的这五个氨基酸处的所有可能置换。靶氨基酸变化的优化使得能够获得具有高比活性的经修饰的LsdA的组,其不产生果聚糖这种多糖并且在产物FOS的产率和谱方面相互不同。
在本发明中鉴定出25个在果聚糖这种多糖的形成(聚合反应)中直接或间接牵涉的散布在LsdA序列中的氨基酸。为了取得该结果,进行质粒pALS200的构建,其在组成性表达启动子nptII之下表达编码与6xHis尾相融合的LsdA前体蛋白的嵌合基因。由宿主大肠杆菌进行的LsdA信号肽的识别和蛋白水解加工使得能够向细胞周质分泌成熟蛋白质(没有在前体蛋白中存在的前30个氨基酸的序列),在细胞周质中外源蔗糖渗透进来并且充当该重组酶的底物。在周质间隙中未突变的LsdA的活性赋予宿主细菌以利用培养基的蔗糖的能力(Sac+表型),并且因此菌落获得与果聚糖这种多糖的形成相关联的粘液状外观(Lev+表型)。将质粒pALS200与羟胺水溶液一起在非剧烈诱变条件下进行温育,目的是生成在该蛋白质的序列的随机位置处具有单个氨基酸置换的LsdA变体的文库。对从具有糖解活性并且有很少或无粘液性(Lev-表型)的每个大肠杆菌转化体菌落回收的质粒进行测序使得能够鉴定在聚合反应中具有可能的直接功能的位于活性位点的空穴中或其附近的14个氨基酸的点变化。如此还鉴定出11个在远离活性位点的位置处的氨基酸,其表面上的功能是维持酶折叠的稳定性。在所有情况下,随机突变引起通过葡萄糖释放所测量的酶促活性降低,相比于未突变的LsdA而言。选择活性位点的氨基酸R141和H142(亚位点-1)、F274和N276(亚位点+2)和R304(亚位点+3)作为用于通过饱和诱变的定向进化实验的靶标。这五个氨基酸位于与供者底物的果糖(亚位点-1)或者与果糖基残基的接受者分子的第二(亚位点+2)或第三(亚位点+3)个单糖相互作用的蛋白质区域中。
在本发明中,定向诱变实验使得能够确立在位置141、142、274、276和304中的哪种氨基酸置换是最佳的,以便获得不产生果聚糖这种多糖并且具有类似于或高于未突变的酶的比活性的经修饰的LsdA变体。从大肠杆菌的细胞提取物开始纯化在所述五个突变体文库中的每一个之内最有活性的经修饰的变体,并且研究其催化特性。在与蔗糖一起进行温育后的产物特性谱分析揭示了在FOS的产率和组成方面在经修饰的LsdA变体之间的差异行为。果糖基化活性与水解活性之比以及产物1-蔗果三糖与6-蔗果三糖之比依赖于被置换的氨基酸的位置和性质而变化。
H142的饱和突变揭示,将FOS聚合为果聚糖的能力的保留或丢失根据在该位置处所掺入的氨基酸的类型而变化。经修饰的变体H142A、H142G和H142N形成高分子量果聚糖,与未突变的酶一样。经修饰的变体H142E和H142P以高于未突变的LsdA的比活性经由水解转化底物蔗糖。经修饰的变体H142S(也是高活性的)未显示出对于FOS产率的显著影响,但是未完全取消果聚糖这种多糖的合成。本发明还揭示了亚位点-1的经双重修饰的变体,其中饱和置换氨基酸R141并且保持突变H142S不变。通过这个途径,获得了具有高比活性的经修饰的酶R141K-H142S、R141Y-H142S和R141F-H142S,其在形成β-(2→6)连接的能力方面受到影响,但是的确合成1-蔗果三糖,尽管以低于蔗糖水解反应的比例。
氨基酸F274和N276(亚位点+2)以及R304(亚位点+3)位于暴露于活性位点空穴表面的环中,并且专门与果糖基残基的接受者底物相互作用。F274通过其他芳香族氨基酸(Y或W)进行的变化使LsdA的聚合酶活性保持完整。经修饰的酶F274A和F274N合成短链FOS,并且不将它们聚合为果聚糖。氨基酸N276的饱和突变体文库的生物化学分析揭示,在位置276处的Asn(N)通过带正电荷的氨基酸例如His(H)、Lys(K)或Arg(R)的点置换提供不形成果聚糖这种多糖并且在FOS产物的谱方面不同的经修饰的变体。N276R这一置换因合成6-蔗果三糖作为主要FOS而突出,这不同于未突变的LsdA,其主要经由β-(2→1)连接来使蔗糖果糖基化。另一方面,在位置304处的Arg(R)通过Lys(K)进行的变化导致具有高比活性的变体。该经修饰的酶合成1-蔗果三糖和1,1-蔗果四糖作为主要FOS,以及还有β-(2→6)连接的线性FOS(GF2-15)的浓度下降的阶梯,后者的链通过非持续方式进行延伸。具有双重置换N276H-R304K、N276K-R304K和N276R-R304K的LsdA变体具有高比活性,以不同的比例合成1-蔗果三糖和6-蔗果三糖,并且不形成果聚糖这种多糖。
在本发明的一个实施方案中,所述包含从由SEQ ID NO:26-SEQ ID NO:34组成的序列的组中选择的氨基酸序列的经修饰的酶LsdA在经遗传修饰的宿主微生物中产生。在一个特别的实施方案中,所述经遗传修饰的宿主微生物为巴斯德毕赤酵母这种酵母或大肠杆菌这种细菌。
因此,本发明的目标为经遗传修饰的微生物,其特征在于,其产生包含从由SEQ IDNO:26-SEQ ID NO:34组成的序列的组中选择的氨基酸序列的经修饰的变体。在本发明的一个实施方案中,这提供了巴斯德毕赤酵母这种非糖解酵母的重组克隆,其分泌高水平的不产生果聚糖这种多糖的九个经修饰的LsdA变体(亚位点-1、+2和+3),它们是基于更高的比活性和在产物FOS的谱方面的差异的标准而选择的。巴斯德毕赤酵母这种非糖解酵母,与大肠杆菌这种细菌一样,满足了不具有可以与底物蔗糖或产物FOS进行反应的内源性酶的要求。在所述重组酶中N-糖基化的出现不引起其催化特性的变化,并且的确增加其热稳定性。所采用的组成性表达系统不需要向生长培养基添加诱导物,因此适合于产生在食品加工中具有应用的酶。这一点与在酵母培养物上清液中重组蛋白质的高初始纯度一起使得可能获得满足关于其商业应用的技术和监管要求的酶制备物。因此,本发明还提供了酶制备物,其特征在于,其包含有包含从由SEQ ID NO:26-SEQ ID NO:34组成的序列的组中选择的氨基酸序列的经修饰的酶LsdA。在重组酵母的培养物上清液中每种经修饰的蛋白质的高初始纯度使得能够获得具有高比活性的稳定的酶粗制品或制备物。将制备酶粗制品或制备物的方法按比例放大到工业水平是可行的,包括该技术领域中的熟练技术人员众所周知的离心、过滤、浓缩、透析和冻干的步骤。在本发明的一个实施方案中,所述酶制备物的特征在于,其为液体或冻干物。
在另一个方面,本发明揭示了用于将蔗糖转变为FOS的酶促方法,其包括使用包含从由SEQ ID NO:26-SEQ ID NO:34组成的序列的组中选择的氨基酸序列的经修饰的酶LsdA或者产生其的经遗传修饰的微生物。
本发明还揭示了用于将蔗糖转变为FOS的双酶促方法,其中在该方法的第一步中使用具有果糖基转移酶活性的酶,和在第二步中使用包含从由SEQ ID NO:26-SEQ ID NO:34组成的序列的组中选择的氨基酸序列的经修饰的果聚糖蔗糖酶、未突变的果聚糖蔗糖酶或产生包含从由SEQ ID NO:26-SEQ ID NO:34组成的序列的组中选择的氨基酸序列的经修饰的果聚糖蔗糖酶的经遗传修饰的微生物。在一个特别的实施方案中,在第一步中所使用的酶是蔗糖:蔗糖-1-果糖基转移酶(1-SST)类型的。以两个步骤的级联反应双酶促方法的建立是本发明的一个新方面。这通过下述方式对于目前的获得FOS糖浆的方法的主要限制提供了解决办法:允许底物蔗糖的完全转变,产生具有不同类型的连接的短链FOS的混合物,和增加总FOS的产率。在本发明的一个特别的实施方案中,整个方法在具有温度控制的搅拌罐中进行,这是规模可改变的并且容易在工业上实施。以举例说明所述方法如何发生而不构成对于本发明的限制的方式,在所述方法的第一步中,重组酶Sa1-SSTrec催化浓度为700g/L的蔗糖向具有β-(2→1)连接的FOS的初始转变。在产物1-蔗果三糖的水解开始的时刻,通过将反应器的温度从45℃逐渐增加至80℃来进行所述酶的不可逆失活。在所述方法的第二步中,经修饰的LsdA变体催化剩余蔗糖的转化,扩大短链FOS的谱,并且增加在反应混合物中的总FOS含量。与产物特性谱的该改善相结合的是重要的技术优势。通过酶促去除在反应混合物中的蔗糖,消除了通过色谱分离技术的FOS工业纯化方法的主要污染物。
以例示本发明的方法的方式,在停止Sa1-SSTrec反应的时刻,在剩余底物蔗糖的转变中采用具有不同催化特性的高活性的经修饰的LsdA变体的选择。该酶合成1-蔗果三糖和1,1-蔗果四糖作为唯有的果糖基化产物。FOS产率的增加幅度取决于为第二个反应所选择的LsdA变体的果糖基化活性与水解活性之比。通过经修饰的变体H142E和R141K-H142S(亚位点-1)进行的剩余蔗糖的转变主要经由水解而发生,而不改变在第一个反应中获得的菊粉类型的FOS的含量和组成。在第二个反应中使用具有在位置F274、N276(亚位点+2)和/或R304(亚位点+3)处的置换的经修饰的变体增加了FOS的最终产率,而1-蔗果三糖和1,1-蔗果四糖与具有β-(2→6)连接的FOS之比依赖于所置换的氨基酸的性质而变化。
本发明还揭示了包含从由SEQ ID NO:26-SEQ ID NO:34组成的序列的组中选择的氨基酸序列的经修饰的果聚糖蔗糖酶或者产生其的经遗传修饰的微生物在用于将蔗糖转变为FOS的酶促方法中的用途。
附图简述
图1.在用于产生FOS的两步反应双酶促方法中获得的糖浆样品的高效液相色谱(HPLC)分析。图版A和C的色谱图显示了在将酶Sa1-SSTrec与蔗糖溶液(700g/L;pH 5.5和温度45℃)一起进行温育(步骤1)后获得的中间糖浆的产物特性谱。图版B的色谱图显示了在添加具有突变R141K-H142S的经修饰的LsdA的可溶性制备物(步骤2)后获得的最终糖浆的产物特性谱。图版D的色谱图显示了在添加在周质中表达具有突变R141K-H142S的经修饰的酶LsdA的固定化巴斯德毕赤酵母细胞(步骤2)后获得的最终糖浆的产物特性谱。图例:葡萄糖(G)、果糖(F)、蔗糖(GF)、蔗果三糖(GF2)、蔗果四糖(GF3)和蔗果五糖(GF4)。
实施方案的详细描述/实施例
实施例1.重氮营养葡糖酸醋酸杆菌果聚糖蔗糖酶基因(lsdA)的随机诱变以及在果聚糖这种多糖的形成中所牵涉的氨基酸的鉴定
重氮营养葡糖酸醋酸杆菌果聚糖蔗糖酶(名为LsdA)包含具有30个氨基酸的信号肽,其在向周质运输期间被切掉(Hernández等人,Current Microbiology 39,146-152,1999)。成熟酶(没有前30个氨基酸的序列)向外部环境的转运通过II型分泌机制来介导,并且不牵涉蛋白水解加工(Arrieta等人,J.Bacteriol.186:5031-5039,2014)。
将包含在组成型启动子nptII之下的编码前体蛋白(氨基酸1-584)的lsdA基因的质粒pALS200(25μg)与在0.5M磷酸钠缓冲液(pH6)中的0.5M羟胺-HCl和5mM EDTA的溶液一起在70℃下温育30分钟。将经诱变的质粒通过电穿孔引入到大肠杆菌DH5α菌株中。在具有补充有0.25%(v/v)甘油、5%(w/v)蔗糖和抗生素氨苄青霉素(100μg mL-1)的LB固体培养基(pH 7)的培养皿上进行转化体的选择。在30℃下温育4-5天后,评价转化体的表型。
在总共大约60,000个对于选择抗生素具有抗性的菌落中,340个显示出粘液状外观(其与由于分泌至周质的成熟LsdA的作用的果聚糖这种多糖的形成相关联)的丧失或减少。将具有粘液状表型影响的菌落独立地接种在具有补充有0.25%(v/v)甘油、5%(w/v)蔗糖和和0.025%(w/v)溴麝香草酚蓝的LB固体培养基(pH 7)的24-孔平板中,并且在30℃下温育4天。总共47个包含经诱变的质粒的菌落和阳性对照(具有未用羟胺进行处理的质粒pALS200的大肠杆菌)显示出糖解活性(Sac+表型),其通过培养基的酸化来证明。在这些情况下,培养基的颜色从初始的绿色变为黄色。其余的非粘液状重组菌落和阴性对照(具有没有lsdA基因的质粒的大肠杆菌)不能利用蔗糖作为碳源。其以酵母提取物和胰蛋白胨为代价的生长引起铵的释放和随之而来的培养基的碱化。在这些情况下,培养基的颜色从初始的绿色变为蓝色。pH指示剂溴麝香草酚蓝的变色区间为黄色(pH<6.0)、绿色(pH 6.0-7.6)和蓝色(pH>7.6)。
为了区分对于47个表达有活性的经修饰的LsdA变体的菌落的果聚糖这种多糖的形成的影响程度,建立了0(非粘液状菌落)至5(非常粘液状菌落)的菌落的粘液性值量表。产生未突变的LsdA的大肠杆菌菌落具有粘液性程度5。从每个具有Sac+表型和0至3的粘液性程度(Lev表型)的菌落中纯化质粒,并且对相应于lsdA基因的区域进行测序。脱氧核糖核酸(DNA)测序结果的分析揭示获得了26个独立的经修饰的变体,其在蛋白质的一级结构中具有单个氨基酸置换。在一种情况下,被置换的氨基酸一致,但是置换物氨基酸发生变化(R141C/H)。将25个通过随机诱变而鉴定出的在果聚糖这种多糖的形成中所牵涉的氨基酸映射至在数据库(PDB:1W18)中可得的LsdA的3D结构。结构建模定位了在活性位点的空穴中或附近的14个氨基酸(R141、H142、S198、R199、T213、F274、N276、E284、E297、R304、A339、D368、H389和G402)和在不直接牵涉催化过程的该蛋白质的其他区域中的11个氨基酸(S179、H255、G263、M294、G315、E324、P359、A394、R412、S413和P417)。
为了评价每个突变对于酶的总活性的效应,进行未突变的LsdA以及26个在大肠杆菌中产生的经修饰的变体的纯化(表1)。为此,使每个大肠杆菌重组克隆在30℃下在旋转筛上在具有100mL的补充有2%(v/v)甘油的LB培养基的瓶中进行生长直至稳定期。通过离心来收获生物质,并且将其重悬浮在50mM乙酸钠缓冲液(pH 5.5)中。使用Branson型超声波仪(Ultrasonics Corp.Danbury,CT,USA)通过超声法来进行细胞破裂,回收可溶性级分。使用树脂HisPurTM Ni-NTA(Thermo ScientificTM,USA),通过固定化金属离子亲和色谱法(IMAC),将包含多组氨酸尾的重组蛋白质纯化至同质。所述酶对于底物蔗糖的总活性通过测量葡萄糖(其是既在水解反应中也在果糖基化反应中释放的产物)来进行测定。一个单位(U)表示释放1μmol葡萄糖/分钟的酶的量,在以初始速率的反应中,具有处于在100mM乙酸钠缓冲液(pH 5.5)中的1.5M浓度的底物蔗糖和在40℃的温度下。葡萄糖的定量用GOPOD试剂盒(Megazyme,Ireland)来进行。蛋白质的定量通过Bradford方法(Anal.Biochem.72:248-254,1976)来进行,并且作为标准品,采用牛血清白蛋白的级分V。
表1显示了显示出对于果聚糖这种多糖的形成的影响的通过随机诱变获得的LsdA变体的主要特征。所有经修饰的变体都比未突变的酶的活性小。其置换引起菌落的粘液性的完全或部分丧失(Lev 0-2表型)和/或总酶促活性的较小损害的活性位点的氨基酸为R141、H142、F274、N276和R304。氨基酸R141和H142位于朝向LsdA的活性位点的空穴内部的环1(βIB-βIC)中,在那里H142与供者底物的果糖基残基的碳6直接相互作用(亚位点-1)(Martínez-Fleites等人,Biochem.J.390:19-27,2005)。氨基酸F274和N276位于从活性位点的表面向空穴内部推进的环4(βIID-βIIIA)中,在那里可以与果糖基残基的接受者分子的第二个单糖(葡萄糖或果糖)直接地或间接地相互作用(亚位点+2)。氨基酸R304位于定位在活性位点的空穴表面上的环5(IIIB-IIIC)中,在那里可以与果糖基残基的接受者分子的第三个单糖直接地或间接地相互作用(亚位点+3)。
选择氨基酸R141、H142、F274、N276和R304作为用于通过饱和诱变的定向进化实验的靶标。
表1.通过随机诱变获得的LsdA变体的特征
星号(*)表明,该氨基酸不位于活性位点中。
实施例2.属于LsdA的亚位点-1的氨基酸H142的饱和诱变
通过随机诱变获得的经修饰的变体R141C、R141H和H142Y不产生果聚糖这种多糖,但是相对于未突变的酶而言显示出对于蔗糖的总活性的超过50%的降低(参见表1)。氨基酸H142与果糖基残基的供者蔗糖分子的位置Fru-O6'直接相互作用(亚位点-1),并且因此对于诱变研究来说是令人感兴趣的靶标。
为了评价在位置142处的每种可能的氨基酸置换对于果聚糖这种多糖的合成反应的效应,通过饱和诱变来构建LsdA变体文库。在致突变的聚合酶链式反应(PCR)中,使用质粒pALS200作为模板和使用正向引物5'GGATCGTTTCCCATGGCGCATGTAC(SEQ ID NO:1)和反向引物5'GATGCGGGCATGCACGTG(A/C/G/T)(A/C/G/T)(A/C/G/T)GTCGTCG AAACC(SEQ ID NO:2)。后者包含三联体CAC(H142)的简并,并且保持SphI限制位点。PCR的条件为:1)95℃下5分钟,2)95℃下1分钟,3)60℃下1分钟,4)72℃下1分钟,5)72℃下5分钟,30个循环的步骤2至4。将扩增出的产物进行NcoI-SphI消化,并且将片段群体(524bp)用于替代在质粒pALS200中的相应片段。将连接混合物通过电穿孔引入到大肠杆菌DH5α菌株中。进行36个独立转化,并且在具有补充有0.25%(v/v)甘油、5%(w/v)蔗糖和抗生素氨苄青霉素(100μg mL-1)的LB固体培养基(pH 7)的培养皿上进行转化体菌落的选择。在30℃下温育4-5天后,评价转化体的表型。
将总共大约1,000个具有不同粘液性程度的氨苄青霉素抗性菌落独立地接种在具有补充有5%(w/v)蔗糖和0.025%(w/v)溴麝香草酚蓝的LB固体培养基(pH 7.0)的24-孔平板中,并且在30℃下温育4天。从具有Sac+表型的菌落中纯化质粒,并且对整个相应于经突变的和经装配的lsdA基因的区域进行测序。DNA测序结果的分析揭示获得了19个具有氨基酸H142的置换的可能的经修饰的变体。从相应的大肠杆菌克隆的细胞提取物开始通过IMAC纯化每个LsdA变体,并且基于从在100mM乙酸钠缓冲液(pH 5.5)中的浓度为1.5M的底物蔗糖开始和在40℃的温度下葡萄糖释放的初始速率(VG)来定量比活性(U/mg)。蔗果三糖合成的初始速率(VK)与果糖释放到水中的初始速率(VF)之比使得能够建立转移酶活性与水解酶活性的比例(T/H)。
在最终时间(24小时)时的所述反应的产物特性谱通过HPLC来测定,其中使用与折光指数类型的检测器相偶联的Aminex HPX-42柱(Biorad)。作为走样溶剂,使用以0.5mL/分钟的流速的蒸馏水,和操作温度为80℃。注射20μL的经十倍稀释的样品。作为走样标准品,使用葡萄糖(G)、果糖(F)、蔗糖(GF)、1-蔗果三糖(GF2)和1,1-蔗果四糖(GF3)的混合物,其以相同浓度(40mg mL-1)的每种糖来制备。在反应混合物中碳水化合物的最终组成为葡萄糖(G)、果糖(F)、蔗糖(GF)、三糖(GF2)、四糖(GF3)、五糖(GF4)和聚合度为6至11的寡果糖聚糖(GF5-10)。
表2显示了通过氨基酸H142的饱和诱变获得的19个LsdA变体的主要特征。表达经修饰的变体H142A、H142G和H142N的大肠杆菌菌落显示出与包含未经诱变的质粒pALS200的细菌相同的粘液性程度(Lev 5表型)。总共9个经修饰的变体产生非粘液状菌落(Lev0表型),其中H142E和H142P是突出的,因为具有稍微高于未突变的酶的比活性值,尽管果糖向水的转移是占优势的反应。虽然置换H142S不完全抑制果聚糖这种多糖的形成(Lev 3表型),但是有助于最高的总FOS产率,并且保持了GF2的优势。
实施例3.对氨基酸R141进行饱和诱变,同时使置换H142S保持固定
为了获得不产生果聚糖这种多糖、保持高的比活性并且实现增加FOS产率的在亚位点-1处经突变的LsdA变体,构建了双重突变体文库,其中将置换H142S与在位置141处的氨基酸的可能变化中的每一个相组合。LsdA的氨基酸R141的饱和诱变通过PCR来进行,其中使用质粒pALS200作为模板和使用正向引物5'GGATCGTTTCCCATGGCGCATGTAC(SEQ ID NO:1)和反向引物5'GATGCGGGCATGCACGCT(A/C/G/T)(A/C/G/T)(A/C/G/T)GTCGTC GAAACC(SEQ IDNO:3),其保持三联体CAC变化为AGC(H142S),具有三联体CGC(R141)的简并,并且保持SphI限制位点。PCR的条件描述在实施例2中。将593bp的扩增出的片段群体进行NcoI-SphI消化,并且插入到质粒pALS200的相应区域中。将连接混合物通过电穿孔引入到大肠杆菌DH5α菌株中。进行36个独立转化,并且在具有补充有0.25%(v/v)甘油、5%(w/v)蔗糖和抗生素氨苄青霉素(100μg mL-1)的LB固体培养基(pH 7.0)的培养皿上进行转化体菌落的选择。在30℃下温育4-5天后,评价转化体的表型。
将总共大约1,000个氨苄青霉素抗性菌落独立地接种在具有补充有5%(w/v)蔗糖和0.025%(w/v)溴麝香草酚蓝的LB固体培养基(pH 7.0)的24-孔平板中,并且在30℃下温育4天。所有转化体都显示出Sac+表型,并且菌落的粘液性程度在0和3之间波动。对在从独立转化体中回收的质粒中的相应于lsdA基因的区域进行的测序揭示获得了19个与固定突变H142S相组合的氨基酸R141置换的变体。从相应的大肠杆菌克隆的细胞提取物开始通过IMAC纯化每个经修饰的LsdA变体。比活性(U/mg)、转移酶活性与水解酶活性之比(T/H)和反应产物的组成按照在实施例1和2中所描述的来进行测定。
表3显示了包含氨基酸R141的19种可能置换中的每一种并且使置换H142S保持固定的LsdA变体的主要特征。表达具有双重置换(其中氨基酸R141变化为N、G、C、Q、H、E、D、T或M)的变体的大肠杆菌菌落显示出与表达具有单一置换H142S的变体的菌落相似的粘液性影响(Lev 2-3表型)。其余的双重变体产生非粘液状菌落(Lev0表型),其中辨别出K141-S142、Y141-S142、F141-S142、V141-S142、L141-S142和A141-S142这些组合,因为具有与未突变的酶相似的比活性值。这六个不产生果聚糖这种多糖的经修饰的变体对蔗糖进行果糖基化从而生成三糖(GF2)和以较小比例生成四糖(GF3),尽管果糖向水的转移继续是占优势的反应。在这些之中,变体K141-S142具有更大的在转移活性与水解活性之比方面的值。
实施例4.位于LsdA的亚位点+2处的氨基酸F274的定向诱变
表达通过随机诱变获得的经修饰的LsdA变体F274A的大肠杆菌菌落使用蔗糖作为碳源(Sac+表型),并且具有与果聚糖这种多糖的产生相关联的粘液状外观的显著影响(Lev1表型)(参见表1)。氨基酸F274占据与果糖基残基的接受者分子的第二个单糖可能相互作用的活性位点的位置(亚位点+2),并且因此对于定向诱变研究来说是令人感兴趣的靶标。
经修饰的变体F274A的活性比未突变的酶低17%(参见表1)。为了优化在位置274处的突变,进行原始氨基酸F被N、Y和W的定向置换,这通过重叠PCR技术来进行,其中使用质粒pALS200作为模板,和使用下列寡核苷酸作为引物,其中加有下划线的三联体相应于所设计的突变。
BamHI-正向:5’CTGGGGCGGATCCACGCCGACTTC(SEQ ID NO:4)
F274N-正向:5’GCGCAGAACGAAAACTTCAATTTCCGCGATCCG(SEQ ID NO:5)
F274N-反向:5’CGGATCGCGGAAATTGAAGAATTCGTTCTGCGC(SEQ ID NO:6)
F274Y-正向:5’GCGCAGAACGAATACTTCAATTTCCGCGATCCG(SEQ ID NO:7)
F274Y-反向:5’CGGATCGCGGAAATTGAAGTATTCGTTCTGCGC(SEQ ID NO:8)
F274W-正向:5’GCGCAGAACGAATGGTTCAATTTCCGCGATCCG(SEQ ID NO:9)
F274W-反向:5’CGGATCGCGGAAATTGAACCATTCGTTCTGCGC(SEQ ID NO:10)
KpnI-反向:5’GGCGCCAGGGTACCCCCGCGACGG(SEQ ID NO:11)
PCR的条件为:1)95℃所5分钟,2)95℃1分钟,3)60℃1分钟,4)72℃1分钟,5)72℃5分钟,30个循环的步骤2至4。将包含每个所设计的突变的最终扩增产物进行BamHI-KpnI消化,并且将所得的片段(782bp)用于替代在质粒pALS200中的相应片段。将这三个新的构建物独立地引入到大肠杆菌DH5α菌株中。所有对于氨苄青霉素具有抗性的转化体都获得了利用蔗糖作为碳源的能力(Sac+表型)。菌落的粘液性程度依赖于突变的类型而变化。F274被N、Y和W置换的发生通过对从相应的转化体中回收的质粒进行测序来验证。通过固定化金属离子亲和色谱法(IMAC),从大肠杆菌提取物开始,将与多组氨酸尾相融合的变体F274N、F274Y、F274W和F274A纯化至同质。比活性(U/mg)、转移酶活性与水解酶活性之比(T/H)和反应产物的组成按照在实施例1和2中所描述的来进行测定。
表4显示了所获得的具有氨基酸F274的点置换的四个LsdA变体的催化行为。按照在实施例2中所描述的,通过HPLC来测定与初始浓度为1.5M的蔗糖的反应的产物特性谱。F274通过其他芳香族氨基酸(Y或W)进行的变化不影响果聚糖这种多糖的产生,也不增加总FOS产率,相对于未突变的酶而言。变体F274A和F274N不产生果聚糖这种多糖,并且合成短链FOS,通过与浓度为1.5M的蔗糖进行反应。
实施例5.位于LsdA的亚位点+2处的氨基酸N276的饱和诱变
表达通过随机诱变获得的经修饰的LsdA变体N276H的大肠杆菌菌落使用蔗糖作为碳源(Sac+表型),并且具有与果聚糖这种多糖的产生相关联的粘液状外观的显著影响(Lev2表型)(参见表1)。氨基酸N276占据与果糖基残基的接受者分子的第二个单糖可能相互作用的活性位点的位置(亚位点+2),并且因此对于定向诱变研究来说是令人感兴趣的靶标。
经修饰的变体N276H的活性比未突变的酶低13%(参见表1)。为了评价在位置276处的可能的19种氨基酸置换中的每一种对于果聚糖这种多糖的合成反应的效应,通过饱和诱变构建了LsdA变体文库。在PCR中,使用质粒pALS200以及正向引物5'CGCAGAACGAATTCTTC(A/C/G/T)(A/C/G/T)(A/C/G/T)TTCCGCG(SEQ ID NO:12)(保持EcoRI限制位点并且包含编码N276的三联体AAT的简并)和反向引物5'GGCGCCAGGGTACCCCCGCGACGG(SEQ ID NO:11)(保持KpnI限制位点)。
诱变PCR的条件为:1)95℃5分钟,2)95℃1分钟,3)60℃1分钟,4)72℃1分钟,5)72℃5分钟,30个循环的步骤2至4。将扩增出的产物进行EcoRI-KpnI消化,并且将所得的616bp的片段群体用于替代在质粒pALS200中的相应片段。通过电穿孔将连接混合物引入到大肠杆菌DH5α菌株中。进行20个独立转化,并且在具有补充有0.25%(v/v)甘油、5%(w/v)蔗糖和抗生素氨苄青霉素(100μg mL-1)的LB固体培养基(pH 7)的培养皿上进行转化体菌落的选择。在30℃下温育4-5天后,评价转化体的表型。
将总共大约1,000个具有不同粘液性程度的氨苄青霉素抗性菌落独立地接种在具有补充有5%(w/v)蔗糖和0.025%(w/v)溴麝香草酚蓝的LB固体培养基(pH 7.0)的24-孔平板中,并且在30℃下温育4天。从具有Sac+表型的菌落中纯化质粒,并且对相应于lsdA基因的区域进行测序。DNA测序结果的分析揭示获得了19个氨基酸N276置换的变体。从相应的大肠杆菌克隆的细胞提取物开始通过IMAC纯化每个LsdA变体。比活性(U/mg)、转移酶活性与水解酶活性之比(T/H)和反应产物的组成按照在实施例1和2中所描述的来进行测定。
表5显示了通过氨基酸N276的饱和诱变获得的经修饰的LsdA变体的文库的主要特征。按照在实施例2中所描述的,通过HPLC来测定与初始浓度为1.5M的蔗糖的反应的产物特性谱。大肠杆菌的转化体显示出有明显差别的粘液性程度(Lev 0-5表型)。包含在位置276处的Asn(N)被Pro(P)、Asp(D)、Gly(G)或Ala(A)置换的变体显示出Lev 5表型,因此果聚糖的产生未受影响。经修饰的变体N276H比未突变的酶稍微更有活性,并且产生具有较小粘液性程度的菌落(Lev 2表型)。在产生非粘液状菌落(Lev 0表型)的经修饰的变体中,变体N276R和N276K是突出的,具有与未突变的酶相似的比活性和更高的总FOS产率。
实施例6.位于LsdA的亚位点+3处的氨基酸R304的定向诱变
表达通过随机诱变获得的经修饰的LsdA变体R304C的大肠杆菌菌落使用蔗糖作为碳源(Sac+表型),并且具有与果聚糖这种多糖的产生相关联的粘液状外观的显著影响(Lev2表型)(参见表1)。氨基酸R304占据与果糖基残基的接受者分子的第三个单糖可能相互作用的活性位点的位置(亚位点+3),并且因此对于定向诱变研究来说是令人感兴趣的靶标。
经修饰的变体R304C的活性比未突变的酶低11%(参见表1)。为了优化在位置304处的突变,进行原始氨基酸R被H、K、S和A的定向置换。独立地构建这四个经修饰的变体,这通过重叠PCR技术来进行,其中使用质粒pALS200作为模板,和使用下列寡核苷酸作为引物,其中加有下划线的三联体相应于所设计的突变。
BamHI-正向:5’CTGGGGCGGATCCACGCCGACTTC(SEQ ID NO:4)
R304H-正向:5’GGCAATACCGCGGGCCAGCATGGCGTCGCCAAC(SEQ ID NO:13)
R304H-反向:5’GTTGGCGACGCCATGCTGGCCCGCGGTATTGCC(SEQ ID NO:14)
R304K-正向:5’GGCAATACCGCGGGCCAGAAGGGCGTCGCCAAC(SEQ ID NO:15)
R304K-反向:5’GTTGGCGACGCCCTTCTGGCCCGCGGTATTGCC(SEQ ID NO:16)
R304S-正向:5’GGCAATACCGCGGGCCAGTCGGGCGTCGCCAAC(SEQ ID NO:17)
R304S-反向:5’GTTGGCGACGCCCGACTGGCCCGCGGTATTGCC(SEQ ID NO:18)
R304A-正向:5’GGCAATACCGCGGGCCAGGCCGGCGTCGCCAAC(SEQ ID NO:19)
R304A-反向:5’GTTGGCGACGCCGGCCTGGCCCGCGGTATTGCC(SEQ ID NO:20)
KpnI-反向:5’GGCGCCAGGGTACCCCCGCGACGG(SEQ ID NO:11)
PCR的条件为:1)95℃5分钟,2)95℃1分钟,3)60℃1分钟,4)72℃1分钟,5)72℃5分钟,30个循环的步骤2至4。将包含每个所设计的突变的最终PCR产物进行BamHI-KpnI消化,并且将所得的717bp的片段用于替代在质粒pALS200中的相应片段。将这四个新的构建物独立地引入到大肠杆菌DH5α菌株中。转化体菌落获得了利用蔗糖的能力,并且显示出不同的粘液性程度(Lev 2-4表型)。R304被H、K、S和A置换通过对从氨苄青霉素抗性菌落中回收的质粒进行测序来验证。从大肠杆菌提取物开始,通过IMAC将与多组氨酸尾相融合的经修饰的变体R304H、R304K、R304S和R304A纯化至同质。比活性(U/mg)、转移酶活性与水解酶活性之比(T/H)和反应产物的组成按照在实施例1和2中所描述的来进行测定。
表6显示了所获得的具有氨基酸R304的点置换的五个LsdA变体的催化行为。按照在实施例2中所描述的,通过HPLC来测定与初始浓度为1.5M的蔗糖的反应的产物特性谱。经修饰的变体R304K在与蔗糖(1.5M)的反应中显示出与未突变的酶相似的比活性和高于未突变的酶的总FOS产率。Arg(R)被Lys(K)(另一种碱性氨基酸)置换引起最小的果糖基化与水解之比降低,相比于经修饰的变体R304C、R304H、R304S和R304A而言。这些经修饰的变体合成GF2、GF3和浓度下降的线性FOS(GF4-10)的阶梯,后者由于所述酶的持续机制的影响而不导致高分子量果聚糖的合成。
实施例7.在亚位点+2和+3处具有置换的经修饰的变体的构建
氨基酸N276(亚位点+2)和R304(亚位点+3)位于暴露于活性位点表面的环中,并且专门与果糖基残基的接受者底物相互作用。基于在实施例5和6中所显示的比活性和FOS产率的结果,我们决定构建经双重修饰的变体N276H-R304K、N276R-R304K和N276K-R304K。定向诱变通过PCR技术来进行,其中使用质粒pALS200(LsdA-R304K)作为模板,和使用下列寡核苷酸作为引物,其中加有下划线的三联体相应于所设计的突变。正向引物包含EcoRI限制位点,和反向引物包含KpnI限制位点。
N276H-正向:5’CGCAGAACGAATTCTTCCACTTCCGCG(SEQ ID NO:21)
N276R-正向:5’CGCAGAACGAATTCTTCCGCTTCCGCG(SEQ ID NO:22)
N276K-正向:5’CGCAGAACGAATTCTTCAAGTTCCGCG(SEQ ID NO:23)
KpnI-反向:5’GGCGCCAGGGTACCCCCGCGACGG(SEQ ID NO:11)
PCR的条件为:1)95℃5分钟,2)95℃1分钟,3)60℃1分钟,4)72℃1分钟,5)72℃5分钟,30个循环的步骤2至4。将包含每个所设计的突变的扩增产物进行EcoRI-KpnI消化,并且将所得的片段(616bp)用于替代在质粒pALS200中的相应片段。将这三个构建物独立地引入到大肠杆菌DH5α菌株中。在这三种情况下,对于氨苄青霉素具有抗性的转化体获得了利用蔗糖作为碳源的能力(Sac+表型),并且菌落不显示出粘液性(Lev 0表型)。所设计的突变的正确出现通过DNA测序来验证。从大肠杆菌提取物开始,通过IMAC将具有双重置换并且与多组氨酸尾相融合的这三个变体纯化至同质。用碱性氨基酸(H、R或K)置换N276(亚位点+2)和用另一种碱性的但分子量较低的氨基酸(K)置换R304(亚位点+3)的组合效应保持了所述酶的比活性不变,并且增加了短链FOS(其是通过不聚合而在反应混合物中积累的产物)的产率(表7)。
实施例8.鉴定通过选择不产生果聚糖这种多糖的经修饰的LsdA变体而合成的FOS的类型
通过采用HPAEC-PAD(高效阴离子交换色谱法-脉冲电流检测(High-PerformanceAnion-Exchange Chromatography-Pulsed Amperometric Detection))技术,测定了通过选择不形成果聚糖这种多糖并且保持高的比活性的的九个经修饰的LsdA变体从蔗糖(1.5M)开始合成的短链FOS产物(三糖和四糖)的连接类型。这些经修饰的变体被命名为H142E(SEQ ID NO:26)、R141K-H142S(SEQ ID NO:27)、F274A(SEQ ID NO:28)、N276H(SEQID NO:29)、N276R(SEQ ID NO:30)、R304K(SEQ ID NO:31)、N276H-R304K(SEQ ID NO:32)、N276R-R304K(SEQ ID NO:33)和N276K-R304K(SEQ ID NO:34)。将在不同反应时间时所采的样品在蒸馏水中稀释50倍,并且在保持于30℃下的PA200 Carbopack柱(3x 250mm)上走样。碳水化合物的洗脱用具有100mm NaOH的乙酸钠梯度以0.5mL/分钟的流速来进行。检测通过使用具有金工作电极和Ag/AgCl pH参比电极的Dionex ED40组件来进行。
一旦蔗糖耗尽时的反应产物的HPAEC-PAD分析揭示,未突变的酶和这九个经修饰的变体合成具有β-(2→1)和β-(2→6)果糖基连接的FOS的混合物(表8)。经修饰的变体N276R(亚位点+2)和N276R-R304K对于蔗糖进行果糖基化导致主要形成6-蔗果三糖。相反地,未突变的酶和其余的经修饰的变体产生1-蔗果三糖和1,1-蔗果四糖,分别作为主要的三糖和四糖产物。相关于果糖基化产物的连接类型,经修饰的变体N276R和N276H的有明显差别的行为表明,亚位点+2在决定果糖基残基的接受者分子的定向中发挥重要作用。
表8.通过所选择的变体合成的短链FOS的类型
图例:1K,1-蔗果三糖;6K,6-蔗果三糖;NK,新蔗果三糖;1,1K,1,1-蔗果四糖;1,6K,1,6-蔗果四糖;6,6K,6,6-蔗果四糖。
实施例9.在巴斯德毕赤酵母中产生经修饰的LsdA变体
选择在实施例8中所描述的九个经修饰的LsdA变体用于评价其在巴斯德毕赤酵母这种酵母中经由重组体的产生。在每种情况下,通过PCR来扩增编码LsdA蛋白的成熟区域(消除了构成在前体蛋白中存在的信号肽的前30个氨基酸)的1.7kb的DNA片段,其中使用携带具有相应于研究中的氨基酸置换(表9)的三联体变化的lsdA基因的质粒作为模板。正向引物5’AGTGCGCTATCGATAGGGAATTTCAGC(SEQ ID NO:24)和反向引物5’TTACTGGTCTAGAAATTGGCGAACCTG(SEQ ID NO:25)在其序列中包含碱基变化,以分别产生ClaI和XbaI限制位点。PCR的条件描述在实施例2中。将每个扩增出的产物(没有终止密码子的基因)进行ClaI-XbaI消化,并且插入到pGAPZαC载体(Invitrogen,San Diego,CA,USA)的相应位点中。以这种方式,每个经修饰的LsdA变体的成熟区域通过N-末端与酿酒酵母(Saccharomyces cerevisiae)的α因子的信号肽相融合和通过C-末端键与myc表位和六组氨酸尾相融合。将该杂合基因在组成性的巴斯德毕赤酵母甘油醛-3-磷酸脱氢酶(GAP)启动子的控制之下进行翻译。通过电穿孔将每个连接混合物引入到大肠杆菌DH5α菌株中,并且在没有氯化钠且补充有抗生素zeocin(25μg mL-1)的LB培养基中选择转化体。按照Invitrogen手册的指导(Invitrogen San Diego,CA,USA),将每个质粒在其位于GAP启动子中的唯一AvrII位点处进行消化,并且通过电穿孔引入到巴斯德毕赤酵母X-33菌株中。在补充有zeocin(100μg mL-1)的YPG培养基(1%(w/v)酵母提取物,2%(w/v)蛋白胨,2%(w/v)甘油,1.5%(w/v)琼脂)上选择转化体,然后在SYP固体培养基(1%(w/v)酵母提取物,2%(w/v)蛋白胨,5%(w/v)蔗糖,0.025%(w/v)溴麝香草酚蓝,pH 6.5)上评价菌落的表型。表达经修饰的LsdA变体或未突变的酶的zeocin抗性菌落(Zeor)获得了水解蔗糖的能力(Sac+表型)并且从消耗通过该重组酶的作用而释放出的葡萄糖开始来产生酸,根据通过SYP培养基的颜色从绿色(pH 6.5)至黄色(pH<5.5)的变化所证明的。源自用没有插入片段的pGAPZαC载体进行电穿孔的菌落不能利用蔗糖并且将培养基的颜色变为蓝色(pH>7.5)。挑选在20个在筛选实验中显示出较高蔗糖酶活性的菌落(Zeor,Suc+)之中选择出的每个经修饰的LsdA变体的克隆,以评价其在增加的培养条件下在发酵罐水平上的行为。
在每种情况下,在筛上用200mL的生长培养基接种有效容积为5升的发酵罐(Marubishi,Japan)直至稳定期。采用相同的培养基用于在筛上和在发酵罐中重组酵母的生长,其包含:5%(v/v)甘油;0.5%(w/v)酵母提取物;2.2%(w/v)(NH4)2SO4;1.82%(w/v)K2HPO4;0.75%(w/v)MgSO4·7H2O;0.05%(w/v)CaCl2·2H2O;维生素;和痕量矿物质。在发酵的第一阶段,为了将溶解氧的浓度保持在超过20%的值,自动增加搅拌500至900rpm,并且将通气保持在1vvm(空气体积/培养基体积/分钟)。一旦溶解氧的值增加(其表明碳源被耗尽),就转入第二个供料阶段。在该时刻,增加空气流至2vvm,并且开始以通过溶解氧的变化所控制的流速来供应1.5L的50%(v/v)甘油溶液。发酵罐在受控的温度(28-30℃)和pH(5.0-6.0)条件下进行操作。通过与用没有插入片段的pGAPZαC载体进行转化的X-33菌株的生长进行比较,在72小时的培养期间在表达LsdA变体的重组克隆中的任一个之中均没有观察到细胞生长的抑制(表9)。
通过离心将每个最终培养物分开为两个级分。将沉降物(细胞生物质)和培养物上清液与100mM蔗糖一起进行温育,并且使用“Glucose-Trinder”试剂盒(Sigma,USA)来定量产物葡萄糖的浓度。表9显示了表达所选择的LsdA变体的重组克隆的行为。所有LsdA变体(包括未突变的酶)都在酵母的周质间隙中积累,并且在较小程度上分泌至培养基。相对于在大肠杆菌中表达的等价的经修饰的变体而言,N-糖基化的出现增加了所述酶的热稳定性并且不引起产物特性谱的变化。
表9.表达经修饰的LsdA变体的巴斯德毕赤酵母克隆的生长和糖解活性
所述值表示三次实验的平均值±标准偏差。*周质活性是指在完整细胞中的糖解酶单位/升培养基。底物蔗糖通过扩散进入酵母的周质间隙。
实施例10.获得在巴斯德毕赤酵母中产生的LsdA变体的稳定的酶制备物
在5L规模的发酵罐中使表达在表9中提到的LsdA变体的重组克隆生长72小时,其中采用在实施例9中所描述的培养基和操作条件。通过离心来沉降细胞生物质,并且按照制造商的说明书,使培养物上清液在使用Hydrosart膜(孔隙率0.2μm)的Sartocon Slice 200设备(Sartorius,Germany)中经历过滤过程。然后,采用相同的Sartocon Slice 200设备,但是用Hydrosart超滤膜(孔隙率30kDa),逆pH 6.0的0.1M乙酸钠缓冲液,将过滤物通过渗滤浓缩10倍。在冷冻干燥过程后,酶制备物的活性在9,000至12,000U/克产物的范围内波动。一个单位(U)表示释放1μmol葡萄糖/分钟的酶的量,在以初始速率的反应中,具有处于在100mM乙酸钠缓冲液(pH 5.5)中的1.5M浓度的底物蔗糖和在40℃的温度下。酶制备物的活性在冷藏条件(4-10℃)下在至少1年的储存期间保持稳定。
实施例11.建立具有蔗糖的完全转变的用于合成宽FOS谱的级联反应双酶促方法
在所选择的九个经修饰的LsdA变体(H142E、R141K-H142S、F274A、N276H、N276R、R304K、N276H-R304K、N276R-R304K、N276K-R304K)中,LsdA不水解具有β-(2→1)果糖基连接的FOS的特性保持不变。该特性使得下述方面成为可能:在先前通过真菌或植物来源的果糖基转移酶反应而获得的具有高1-蔗果三糖、1,1-蔗果四糖和1,1,1-蔗果五糖含量的糖浆中的剩余蔗糖的转变之中使用这些经修饰的LsdA变体或未突变的酶。
在本实施例中,用来自高羊茅这种植物的蔗糖:蔗糖-1-果糖基转移酶(1-SST,EC2.4.1.99)(其在巴斯德毕赤酵母这种酵母中通过重组途径获得并且被命名为Sa1-SSTrec)来起始具有两个步骤的级联反应双酶促方法。在pH 5.5、45℃的温度和100rpm的推动搅拌下将该酶与蔗糖溶液(700g/L)一起进行温育。当1-蔗果三糖的含量达到总碳水化合物组成的50%(w/w)时,通过在30分钟的时间间隔内将温度逐渐增加至80℃来使该酶失活。在第二个步骤中,将在实施例10中产生的经修饰的LsdA或未突变的酶的稳定制备物添加至反应罐,并且在45℃下进行温育直至取得蔗糖的完全转变。
通过HPAEC-PAD技术,在该方法的每个步骤结束时测定在反应混合物中存在的产物短链FOS(GF2和GF3)的连接类型,如在实施例8中所描述的。表10显示了在Sa1-SSTrec与蔗糖的反应(步骤1)中取得的1-蔗果三糖的高产率,并且证实了该酶不能形成β-(2→6)连接的FOS。在使Sa1-SSTrec失活后向反应混合物中掺入未突变的LsdA或经修饰的变体使得能够消耗剩余蔗糖并且扩大FOS的谱。三糖(GF2)和四糖(GF3)的产率和最终组成依赖于在所述双酶促方法的第二个步骤中所采用的LsdA变体而变化。
在亚位点-1处具有氨基酸置换的两个变体(H142E和R141K-H142S)将蔗糖水解为葡萄糖和果糖,并且不改变1-蔗果三糖和1,1-蔗果四糖的总和。其他七个经修饰的LsdA变体以四糖(GF3)、五糖(GF4)和任选地己糖(GF5)的合成(这是其中蔗糖作为果糖基残基的供者起作用的反应)为代价降低了1-蔗果三糖的含量,并且因此增加了总FOS产率。特别令人感兴趣的是通过变体N276R和N276R-R304来合成1,6-蔗果四糖,作为果糖基残基从蔗糖转移至1-蔗果三糖的末端果糖的碳6的OH基团的结果。令人惊讶地,在步骤2的反应条件下,未突变的LsdA酶不形成果聚糖这种多糖。
实施例12.建立用于产生没有葡萄糖的FOS糖浆的双酶促方法
为了消除在从蔗糖开始产生的FOS糖浆中存在的副产物葡萄糖,改变在实施例11中所描述的双酶促方法的第二个步骤。在新的方法中,Sa1-SSTrec反应(步骤1)的产物的转化不用经修饰的LsdA的可溶性制备物来进行,而是用巴斯德毕赤酵母克隆的整个细胞,其在周质中积累经修饰的LsdA酶。为了使该生物催化剂可重复使用,将重组酵母的细胞固定在藻酸钙基质上,并且在4℃下活地储存在蔗糖溶液(1.75M)中。
图1显示了所实施的两个类型的双酶促方法的中间和最后步骤的HPLC分析的色谱图。在pH 5.5和45℃下Sa1-SSTrec与蔗糖(700g/L)的反应(步骤1)产生比例为9:1的1-蔗果三糖(GF2)和1,1-蔗果四糖(GF3),并且其总和占总碳水化合物组成的56%(w/w)(图1A和图1C)。在该方法的第二个步骤中使用经修饰的LsdA(变体R141K-H142S)的可溶性制备物使得能够完全水解剩余蔗糖,而不改变1-蔗果三糖和1,1-蔗果四糖的总和(图1B)。在备选的方案中,表达经修饰的LsdA(变体R141K-H142S)的固定化酵母消耗蔗糖和产物单糖,而不改变FOS的含量和组成(图1D)。通过表达经修饰的LsdA的固定化酵母来消除在FOS糖浆中的葡萄糖使得能够将其应用扩大到旨在糖尿病患者的益生元食品制剂。
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Gln Gly Asn Phe Ser Arg Gln Glu Ala Ala Arg Met Ala His Arg Pro
1 5 10 15
Gly Val Met Pro Arg Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu Ala
20 25 30
Gly Val Pro Gly Phe Pro Leu Pro Ser Ile His Thr Gln Gln Ala Tyr
35 40 45
Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu
50 55 60
Gln Ile Lys Ala His Ser Asp Ala Thr Val Ala Ala Gly Gln Asn Ser
65 70 75 80
Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val
85 90 95
Ile Asn Pro Asp Val Trp Val Trp Asp Thr Trp Thr Leu Ile Asp Lys
100 105 110
His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu
115 120 125
Thr Ala Asp Pro Asn Ala Gly Tyr Gly Phe Asp Asp Arg Glu Val His
130 135 140
Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg
145 150 155 160
Arg Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp
165 170 175
Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln Ala
180 185 190
Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His Gly Asn Thr Val
195 200 205
Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn
210 215 220
Asn Ile Thr Pro Pro Gln Ala Ile Ile Thr Gln Thr Leu Gly Arg Ile
225 230 235 240
His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr
245 250 255
Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn Gly Ala Gln Asn
260 265 270
Glu Phe Phe Asn Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His
275 280 285
Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Arg
290 295 300
Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp
305 310 315 320
Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr
325 330 335
Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser
340 345 350
Lys Trp Lys Phe Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp
355 360 365
Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile
370 375 380
Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro
385 390 395 400
Asp Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln
405 410 415
Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu
420 425 430
Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln Asn Pro
435 440 445
Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val
450 455 460
Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala
465 470 475 480
Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu
485 490 495
Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn
500 505 510
Arg Ala Asp Val Asn Ile Ala Gly Phe Ile Gln Asp Leu Phe Gly Gln
515 520 525
Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val
530 535 540
Leu Ala Gln Val Arg Gln Phe Leu Asn Gln
545 550
<210> 27
<211> 554
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:经修饰的果聚糖蔗糖酶R141K-H142S
<400> 27
Gln Gly Asn Phe Ser Arg Gln Glu Ala Ala Arg Met Ala His Arg Pro
1 5 10 15
Gly Val Met Pro Arg Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu Ala
20 25 30
Gly Val Pro Gly Phe Pro Leu Pro Ser Ile His Thr Gln Gln Ala Tyr
35 40 45
Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu
50 55 60
Gln Ile Lys Ala His Ser Asp Ala Thr Val Ala Ala Gly Gln Asn Ser
65 70 75 80
Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val
85 90 95
Ile Asn Pro Asp Val Trp Val Trp Asp Thr Trp Thr Leu Ile Asp Lys
100 105 110
His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu
115 120 125
Thr Ala Asp Pro Asn Ala Gly Tyr Gly Phe Asp Asp Lys Ser Val His
130 135 140
Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg
145 150 155 160
Arg Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp
165 170 175
Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln Ala
180 185 190
Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His Gly Asn Thr Val
195 200 205
Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn
210 215 220
Asn Ile Thr Pro Pro Gln Ala Ile Ile Thr Gln Thr Leu Gly Arg Ile
225 230 235 240
His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr
245 250 255
Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn Gly Ala Gln Asn
260 265 270
Glu Phe Phe Asn Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His
275 280 285
Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Arg
290 295 300
Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp
305 310 315 320
Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr
325 330 335
Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser
340 345 350
Lys Trp Lys Phe Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp
355 360 365
Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile
370 375 380
Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro
385 390 395 400
Asp Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln
405 410 415
Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu
420 425 430
Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln Asn Pro
435 440 445
Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val
450 455 460
Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala
465 470 475 480
Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu
485 490 495
Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn
500 505 510
Arg Ala Asp Val Asn Ile Ala Gly Phe Ile Gln Asp Leu Phe Gly Gln
515 520 525
Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val
530 535 540
Leu Ala Gln Val Arg Gln Phe Leu Asn Gln
545 550
<210> 28
<211> 554
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:经修饰的果聚糖蔗糖酶F274A
<400> 28
Gln Gly Asn Phe Ser Arg Gln Glu Ala Ala Arg Met Ala His Arg Pro
1 5 10 15
Gly Val Met Pro Arg Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu Ala
20 25 30
Gly Val Pro Gly Phe Pro Leu Pro Ser Ile His Thr Gln Gln Ala Tyr
35 40 45
Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu
50 55 60
Gln Ile Lys Ala His Ser Asp Ala Thr Val Ala Ala Gly Gln Asn Ser
65 70 75 80
Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val
85 90 95
Ile Asn Pro Asp Val Trp Val Trp Asp Thr Trp Thr Leu Ile Asp Lys
100 105 110
His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu
115 120 125
Thr Ala Asp Pro Asn Ala Gly Tyr Gly Phe Asp Asp Arg His Val His
130 135 140
Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg
145 150 155 160
Arg Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp
165 170 175
Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln Ala
180 185 190
Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His Gly Asn Thr Val
195 200 205
Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn
210 215 220
Asn Ile Thr Pro Pro Gln Ala Ile Ile Thr Gln Thr Leu Gly Arg Ile
225 230 235 240
His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr
245 250 255
Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn Gly Ala Gln Asn
260 265 270
Glu Ala Phe Asn Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His
275 280 285
Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Arg
290 295 300
Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp
305 310 315 320
Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr
325 330 335
Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser
340 345 350
Lys Trp Lys Phe Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp
355 360 365
Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile
370 375 380
Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro
385 390 395 400
Asp Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln
405 410 415
Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu
420 425 430
Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln Asn Pro
435 440 445
Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val
450 455 460
Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala
465 470 475 480
Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu
485 490 495
Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn
500 505 510
Arg Ala Asp Val Asn Ile Ala Gly Phe Ile Gln Asp Leu Phe Gly Gln
515 520 525
Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val
530 535 540
Leu Ala Gln Val Arg Gln Phe Leu Asn Gln
545 550
<210> 29
<211> 554
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:经修饰的果聚糖蔗糖酶N276H
<400> 29
Gln Gly Asn Phe Ser Arg Gln Glu Ala Ala Arg Met Ala His Arg Pro
1 5 10 15
Gly Val Met Pro Arg Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu Ala
20 25 30
Gly Val Pro Gly Phe Pro Leu Pro Ser Ile His Thr Gln Gln Ala Tyr
35 40 45
Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu
50 55 60
Gln Ile Lys Ala His Ser Asp Ala Thr Val Ala Ala Gly Gln Asn Ser
65 70 75 80
Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val
85 90 95
Ile Asn Pro Asp Val Trp Val Trp Asp Thr Trp Thr Leu Ile Asp Lys
100 105 110
His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu
115 120 125
Thr Ala Asp Pro Asn Ala Gly Tyr Gly Phe Asp Asp Arg His Val His
130 135 140
Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg
145 150 155 160
Arg Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp
165 170 175
Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln Ala
180 185 190
Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His Gly Asn Thr Val
195 200 205
Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn
210 215 220
Asn Ile Thr Pro Pro Gln Ala Ile Ile Thr Gln Thr Leu Gly Arg Ile
225 230 235 240
His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr
245 250 255
Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn Gly Ala Gln Asn
260 265 270
Glu Phe Phe His Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His
275 280 285
Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Arg
290 295 300
Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp
305 310 315 320
Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr
325 330 335
Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser
340 345 350
Lys Trp Lys Phe Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp
355 360 365
Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile
370 375 380
Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro
385 390 395 400
Asp Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln
405 410 415
Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu
420 425 430
Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln Asn Pro
435 440 445
Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val
450 455 460
Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala
465 470 475 480
Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu
485 490 495
Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn
500 505 510
Arg Ala Asp Val Asn Ile Ala Gly Phe Ile Gln Asp Leu Phe Gly Gln
515 520 525
Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val
530 535 540
Leu Ala Gln Val Arg Gln Phe Leu Asn Gln
545 550
<210> 30
<211> 554
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:经修饰的果聚糖蔗糖酶N276R
<400> 30
Gln Gly Asn Phe Ser Arg Gln Glu Ala Ala Arg Met Ala His Arg Pro
1 5 10 15
Gly Val Met Pro Arg Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu Ala
20 25 30
Gly Val Pro Gly Phe Pro Leu Pro Ser Ile His Thr Gln Gln Ala Tyr
35 40 45
Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu
50 55 60
Gln Ile Lys Ala His Ser Asp Ala Thr Val Ala Ala Gly Gln Asn Ser
65 70 75 80
Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val
85 90 95
Ile Asn Pro Asp Val Trp Val Trp Asp Thr Trp Thr Leu Ile Asp Lys
100 105 110
His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu
115 120 125
Thr Ala Asp Pro Asn Ala Gly Tyr Gly Phe Asp Asp Arg His Val His
130 135 140
Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg
145 150 155 160
Arg Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp
165 170 175
Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln Ala
180 185 190
Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His Gly Asn Thr Val
195 200 205
Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn
210 215 220
Asn Ile Thr Pro Pro Gln Ala Ile Ile Thr Gln Thr Leu Gly Arg Ile
225 230 235 240
His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr
245 250 255
Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn Gly Ala Gln Asn
260 265 270
Glu Phe Phe Arg Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His
275 280 285
Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Arg
290 295 300
Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp
305 310 315 320
Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr
325 330 335
Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser
340 345 350
Lys Trp Lys Phe Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp
355 360 365
Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile
370 375 380
Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro
385 390 395 400
Asp Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln
405 410 415
Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu
420 425 430
Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln Asn Pro
435 440 445
Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val
450 455 460
Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala
465 470 475 480
Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu
485 490 495
Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn
500 505 510
Arg Ala Asp Val Asn Ile Ala Gly Phe Ile Gln Asp Leu Phe Gly Gln
515 520 525
Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val
530 535 540
Leu Ala Gln Val Arg Gln Phe Leu Asn Gln
545 550
<210> 31
<211> 554
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:经修饰的果聚糖蔗糖酶R304K
<400> 31
Gln Gly Asn Phe Ser Arg Gln Glu Ala Ala Arg Met Ala His Arg Pro
1 5 10 15
Gly Val Met Pro Arg Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu Ala
20 25 30
Gly Val Pro Gly Phe Pro Leu Pro Ser Ile His Thr Gln Gln Ala Tyr
35 40 45
Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu
50 55 60
Gln Ile Lys Ala His Ser Asp Ala Thr Val Ala Ala Gly Gln Asn Ser
65 70 75 80
Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val
85 90 95
Ile Asn Pro Asp Val Trp Val Trp Asp Thr Trp Thr Leu Ile Asp Lys
100 105 110
His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu
115 120 125
Thr Ala Asp Pro Asn Ala Gly Tyr Gly Phe Asp Asp Arg His Val His
130 135 140
Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg
145 150 155 160
Arg Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp
165 170 175
Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln Ala
180 185 190
Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His Gly Asn Thr Val
195 200 205
Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn
210 215 220
Asn Ile Thr Pro Pro Gln Ala Ile Ile Thr Gln Thr Leu Gly Arg Ile
225 230 235 240
His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr
245 250 255
Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn Gly Ala Gln Asn
260 265 270
Glu Phe Phe Asn Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His
275 280 285
Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Lys
290 295 300
Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp
305 310 315 320
Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr
325 330 335
Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser
340 345 350
Lys Trp Lys Phe Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp
355 360 365
Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile
370 375 380
Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro
385 390 395 400
Asp Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln
405 410 415
Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu
420 425 430
Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln Asn Pro
435 440 445
Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val
450 455 460
Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala
465 470 475 480
Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu
485 490 495
Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn
500 505 510
Arg Ala Asp Val Asn Ile Ala Gly Phe Ile Gln Asp Leu Phe Gly Gln
515 520 525
Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val
530 535 540
Leu Ala Gln Val Arg Gln Phe Leu Asn Gln
545 550
<210> 32
<211> 554
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:经修饰的果聚糖蔗糖酶N276H-R304K
<400> 32
Gln Gly Asn Phe Ser Arg Gln Glu Ala Ala Arg Met Ala His Arg Pro
1 5 10 15
Gly Val Met Pro Arg Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu Ala
20 25 30
Gly Val Pro Gly Phe Pro Leu Pro Ser Ile His Thr Gln Gln Ala Tyr
35 40 45
Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu
50 55 60
Gln Ile Lys Ala His Ser Asp Ala Thr Val Ala Ala Gly Gln Asn Ser
65 70 75 80
Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val
85 90 95
Ile Asn Pro Asp Val Trp Val Trp Asp Thr Trp Thr Leu Ile Asp Lys
100 105 110
His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu
115 120 125
Thr Ala Asp Pro Asn Ala Gly Tyr Gly Phe Asp Asp Arg His Val His
130 135 140
Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg
145 150 155 160
Arg Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp
165 170 175
Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln Ala
180 185 190
Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His Gly Asn Thr Val
195 200 205
Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn
210 215 220
Asn Ile Thr Pro Pro Gln Ala Ile Ile Thr Gln Thr Leu Gly Arg Ile
225 230 235 240
His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr
245 250 255
Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn Gly Ala Gln Asn
260 265 270
Glu Phe Phe His Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His
275 280 285
Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Lys
290 295 300
Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp
305 310 315 320
Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr
325 330 335
Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser
340 345 350
Lys Trp Lys Phe Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp
355 360 365
Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile
370 375 380
Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro
385 390 395 400
Asp Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln
405 410 415
Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu
420 425 430
Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln Asn Pro
435 440 445
Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val
450 455 460
Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala
465 470 475 480
Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu
485 490 495
Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn
500 505 510
Arg Ala Asp Val Asn Ile Ala Gly Phe Ile Gln Asp Leu Phe Gly Gln
515 520 525
Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val
530 535 540
Leu Ala Gln Val Arg Gln Phe Leu Asn Gln
545 550
<210> 33
<211> 554
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:经修饰的果聚糖蔗糖酶N276R-R304K
<400> 33
Gln Gly Asn Phe Ser Arg Gln Glu Ala Ala Arg Met Ala His Arg Pro
1 5 10 15
Gly Val Met Pro Arg Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu Ala
20 25 30
Gly Val Pro Gly Phe Pro Leu Pro Ser Ile His Thr Gln Gln Ala Tyr
35 40 45
Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu
50 55 60
Gln Ile Lys Ala His Ser Asp Ala Thr Val Ala Ala Gly Gln Asn Ser
65 70 75 80
Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val
85 90 95
Ile Asn Pro Asp Val Trp Val Trp Asp Thr Trp Thr Leu Ile Asp Lys
100 105 110
His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu
115 120 125
Thr Ala Asp Pro Asn Ala Gly Tyr Gly Phe Asp Asp Arg His Val His
130 135 140
Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg
145 150 155 160
Arg Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp
165 170 175
Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln Ala
180 185 190
Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His Gly Asn Thr Val
195 200 205
Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn
210 215 220
Asn Ile Thr Pro Pro Gln Ala Ile Ile Thr Gln Thr Leu Gly Arg Ile
225 230 235 240
His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr
245 250 255
Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn Gly Ala Gln Asn
260 265 270
Glu Phe Phe Arg Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His
275 280 285
Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Lys
290 295 300
Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp
305 310 315 320
Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr
325 330 335
Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser
340 345 350
Lys Trp Lys Phe Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp
355 360 365
Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile
370 375 380
Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro
385 390 395 400
Asp Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln
405 410 415
Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu
420 425 430
Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln Asn Pro
435 440 445
Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val
450 455 460
Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala
465 470 475 480
Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu
485 490 495
Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn
500 505 510
Arg Ala Asp Val Asn Ile Ala Gly Phe Ile Gln Asp Leu Phe Gly Gln
515 520 525
Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val
530 535 540
Leu Ala Gln Val Arg Gln Phe Leu Asn Gln
545 550
<210> 34
<211> 554
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述:经修饰的果聚糖蔗糖酶N276K-R304K
<400> 34
Gln Gly Asn Phe Ser Arg Gln Glu Ala Ala Arg Met Ala His Arg Pro
1 5 10 15
Gly Val Met Pro Arg Gly Gly Pro Leu Phe Pro Gly Arg Ser Leu Ala
20 25 30
Gly Val Pro Gly Phe Pro Leu Pro Ser Ile His Thr Gln Gln Ala Tyr
35 40 45
Asp Pro Gln Ser Asp Phe Thr Ala Arg Trp Thr Arg Ala Asp Ala Leu
50 55 60
Gln Ile Lys Ala His Ser Asp Ala Thr Val Ala Ala Gly Gln Asn Ser
65 70 75 80
Leu Pro Ala Gln Leu Thr Met Pro Asn Ile Pro Ala Asp Phe Pro Val
85 90 95
Ile Asn Pro Asp Val Trp Val Trp Asp Thr Trp Thr Leu Ile Asp Lys
100 105 110
His Ala Asp Gln Phe Ser Tyr Asn Gly Trp Glu Val Ile Phe Cys Leu
115 120 125
Thr Ala Asp Pro Asn Ala Gly Tyr Gly Phe Asp Asp Arg His Val His
130 135 140
Ala Arg Ile Gly Phe Phe Tyr Arg Arg Ala Gly Ile Pro Ala Ser Arg
145 150 155 160
Arg Pro Val Asn Gly Gly Trp Thr Tyr Gly Gly His Leu Phe Pro Asp
165 170 175
Gly Ala Ser Ala Gln Val Tyr Ala Gly Gln Thr Tyr Thr Asn Gln Ala
180 185 190
Glu Trp Ser Gly Ser Ser Arg Leu Met Gln Ile His Gly Asn Thr Val
195 200 205
Ser Val Phe Tyr Thr Asp Val Ala Phe Asn Arg Asp Ala Asn Ala Asn
210 215 220
Asn Ile Thr Pro Pro Gln Ala Ile Ile Thr Gln Thr Leu Gly Arg Ile
225 230 235 240
His Ala Asp Phe Asn His Val Trp Phe Thr Gly Phe Thr Ala His Thr
245 250 255
Pro Leu Leu Gln Pro Asp Gly Val Leu Tyr Gln Asn Gly Ala Gln Asn
260 265 270
Glu Phe Phe Lys Phe Arg Asp Pro Phe Thr Phe Glu Asp Pro Lys His
275 280 285
Pro Gly Val Asn Tyr Met Val Phe Glu Gly Asn Thr Ala Gly Gln Lys
290 295 300
Gly Val Ala Asn Cys Thr Glu Ala Asp Leu Gly Phe Arg Pro Asn Asp
305 310 315 320
Pro Asn Ala Glu Thr Leu Gln Glu Val Leu Asp Ser Gly Ala Tyr Tyr
325 330 335
Gln Lys Ala Asn Ile Gly Leu Ala Ile Ala Thr Asp Ser Thr Leu Ser
340 345 350
Lys Trp Lys Phe Leu Ser Pro Leu Ile Ser Ala Asn Cys Val Asn Asp
355 360 365
Gln Thr Glu Arg Pro Gln Val Tyr Leu His Asn Gly Lys Tyr Tyr Ile
370 375 380
Phe Thr Ile Ser His Arg Thr Thr Phe Ala Ala Gly Val Asp Gly Pro
385 390 395 400
Asp Gly Val Tyr Gly Phe Val Gly Asp Gly Ile Arg Ser Asp Phe Gln
405 410 415
Pro Met Asn Tyr Gly Ser Gly Leu Thr Met Gly Asn Pro Thr Asp Leu
420 425 430
Asn Thr Ala Ala Gly Thr Asp Phe Asp Pro Ser Pro Asp Gln Asn Pro
435 440 445
Arg Ala Phe Gln Ser Tyr Ser His Tyr Val Met Pro Gly Gly Leu Val
450 455 460
Glu Ser Phe Ile Asp Thr Val Glu Asn Arg Arg Gly Gly Thr Leu Ala
465 470 475 480
Pro Thr Val Arg Val Arg Ile Ala Gln Asn Ala Ser Ala Val Asp Leu
485 490 495
Arg Tyr Gly Asn Gly Gly Leu Gly Gly Tyr Gly Asp Ile Pro Ala Asn
500 505 510
Arg Ala Asp Val Asn Ile Ala Gly Phe Ile Gln Asp Leu Phe Gly Gln
515 520 525
Pro Thr Ser Gly Leu Ala Ala Gln Ala Ser Thr Asn Asn Ala Gln Val
530 535 540
Leu Ala Gln Val Arg Gln Phe Leu Asn Gln
545 550
Claims (13)
1.经修饰的重氮营养葡糖酸醋酸杆菌(Gluconacetobacter diazotrophicus)果聚糖蔗糖酶(LsdA),其特征在于,其包含从由SEQ ID NO:26-SEQ ID NO:34组成的序列的组中选择的氨基酸序列。
2.权利要求1的果聚糖蔗糖酶,其在经遗传修饰的宿主微生物中产生。
3.权利要求2的果聚糖蔗糖酶,其中所述经遗传修饰的宿主微生物为巴斯德毕赤酵母(Pichia pastoris)这种酵母或大肠杆菌(Escherichia coli)这种细菌。
4.经遗传修饰的微生物,其特征在于,其产生权利要求1至3中任一项的经修饰的果聚糖蔗糖酶。
5.权利要求4的经遗传修饰的微生物,其为巴斯德毕赤酵母这种酵母或大肠杆菌这种细菌。
6.酶制备物,其特征在于,其包含权利要求1至3中任一项的经修饰的果聚糖蔗糖酶。
7.权利要求6的酶制备物,其特征在于,其为液体或冻干物。
8.用于将蔗糖转变为果寡糖(FOS)的酶促方法,其特征在于,所述方法包括使用权利要求1至3中任一项的经修饰的果聚糖蔗糖酶或者权利要求4至5的经遗传修饰的微生物。
9.用于将蔗糖转变为果寡糖(FOS)的双酶促方法,其特征在于,在该方法的第一步中使用具有果糖基转移酶活性的酶,和在第二步中使用权利要求1至3中任一项的经修饰的果聚糖蔗糖酶、未突变的果聚糖蔗糖酶或权利要求4至5的经遗传修饰的微生物。
10.权利要求9的方法,其中在第一步中所使用的酶是蔗糖:蔗糖-1-果糖基转移酶(1-SST)类型的。
11.权利要求1至3中任一项的经修饰的果聚糖蔗糖酶或者权利要求4至5的经遗传修饰的微生物在用于将蔗糖转变为果寡糖(FOS)的酶促方法中的用途。
12.权利要求11的用途,其中所述方法为双酶级联反应,其中在第一步中使用具有果糖基转移酶活性的酶,和在第二步中使用所述经修饰的果聚糖蔗糖酶或所述经遗传修饰的微生物。
13.权利要求12的用途,其中在第一步中所使用的酶是蔗糖:蔗糖-1-果糖基转移酶(1-SST)类型的。
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CU2021000020A CU20210020A7 (es) | 2021-03-30 | 2021-03-30 | Levanasacarasa modificada para la producción optimizada de fructooligosacáridos |
PCT/CU2022/050003 WO2022207017A1 (es) | 2021-03-30 | 2022-03-23 | Levanasacarasa modificada para la produccion optimizada de fructooligosacaridos |
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CN (1) | CN117413056A (zh) |
AR (1) | AR125233A1 (zh) |
BR (1) | BR112023020286A2 (zh) |
CA (1) | CA3215929A1 (zh) |
CO (1) | CO2023014456A2 (zh) |
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CO2023014456A2 (es) | 2024-03-07 |
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CA3215929A1 (en) | 2022-10-06 |
CU20210020A7 (es) | 2022-11-07 |
BR112023020286A2 (pt) | 2024-01-23 |
WO2022207017A1 (es) | 2022-10-06 |
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