CN115044568A - 高稳定型的蔗糖磷酸化酶突变体及其应用 - Google Patents
高稳定型的蔗糖磷酸化酶突变体及其应用 Download PDFInfo
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- CN115044568A CN115044568A CN202210717959.1A CN202210717959A CN115044568A CN 115044568 A CN115044568 A CN 115044568A CN 202210717959 A CN202210717959 A CN 202210717959A CN 115044568 A CN115044568 A CN 115044568A
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- sucrose phosphorylase
- amino acid
- mutant
- sucrose
- asp
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Abstract
本发明提供一种蔗糖磷酸化酶突变体，所述蔗糖磷酸化酶突变体基于参比蔗糖磷酸化酶通过以下位置的突变得到：在参比蔗糖磷酸化酶的自由能低的氨基酸残基处；在参比蔗糖磷酸化酶的不稳定的氨基酸残基处；在参比蔗糖磷酸化酶的与热稳定相关的氨基酸残基处。本申请通过对参比蔗糖磷酸化酶突变得到的蔗糖磷酸化酶突变体与参比蔗糖磷酸化酶相比具有更高的热稳定性，在70℃下，所述蔗糖磷酸化酶突变体的热稳定性比参比蔗糖磷酸化酶的热稳定性提高10％‑98％。
Description
技术领域
本发明属于生物技术领域，具体涉及一种热稳定性提高的蔗糖磷酸化酶突变体及其应用。
背景技术
蔗糖磷酸化酶(EC2.4.1.7，Sucrose phosphorylase，SPase)属于糖基水解酶13(GH13)家族，催化蔗糖与磷酸进行反应，使蔗糖转化为D-果糖和α-D-葡萄糖-1-磷酸(G1P)。该酶是一种催化转移葡萄糖苷键的特异性酶，具有广泛的受体混杂性，可以将蔗糖中的葡萄糖基转移至不同受体，包括磷酸、水、含酚羟基、醇羟基及羧基等物质。该酶在化妆品、药品及食品领域均有广泛应用，可用于多个生物活性物的改性修饰，比如：将氢醌、甘油、抗坏血酸等进行糖基化修饰后获得护肤效果好且性质稳定的化妆品添加剂；以木糖、鼠李糖、半乳糖等为受体，催化合成多一个葡萄糖基的功能性低聚糖；在咖啡酸上加一个葡萄糖基后进行糖基化修饰，能显著提高咖啡酸的稳定性。
蔗糖磷酸化酶首次在肠膜明串珠菌中发现，随后在多种微生物中该酶逐渐被开发。目前报道的该酶大多数为常温菌来源，主要有四类:(1)肠膜明串珠菌(Leuconostocmesenteroides)，其中Leuconostoc mesenteroides ATCC 12291是目前报道产酶量最高的菌株(Kagan BO,Latker SN,Zfasman EM.Phosphoro1ysis of saccharose by culturesof Leuconostoc mesenteroides.1942,Biokhimiya,7:93-108.)；(2)变异链球菌(Streptococcus mutans)，由该菌株产生的葡萄糖基转移酶A可以可逆地催化蔗糖与磷酸生成D-果糖和α-D-葡萄糖-1-磷酸，因此将其鉴定为蔗糖磷酸化酶(Russell R R,MukasaH,Shimamura A,et al.Streptococcus mutans gtfa gene specifies sucrosephosphorylase.1988,Infection and immunity,56(10):2763-2765.)；(3)双歧杆菌(Bifidobacterium)，常见的产蔗糖磷酸化酶的有青春双歧杆菌(Bifidobacteriumadolescentis)(van den Broek L.A.,van Boxtel E.L.,Kievit R.P.,et al.Physico-chemicaland transglucosylation properties of recombinant sucrosephosphorylase from Bifidobacterium adolescentis DSM20083.2004,Appl MicrobiolBiotechnol,65(2):219-227.)和长双歧杆菌(Bifidobacterium longum)(Kullin B,Abratt VR,Reid SJ.A functional analysis of the Bifidobacterium longum cscAand scrP genes in sucrose utilization.Appl Microbiol Biotechnol.2006Oct；72(5):975-81.)；(4)嗜糖假单胞菌(Pseudomonas saccharophila)(DoudoroffMichael.Studies on the phosphorolysis of sucrose.1943,Journal ofBiological Chemistry,151(2):351-361)。
这些微生物来源的蔗糖磷酸化酶普遍热稳定性较差，这大大限制了其应用范围，而且高温下酶易于失活，会增加生产成本，因此亟需获取热稳定性得到改善的蔗糖磷酸化酶变体，用于工业领域相关产品的生产。
发明内容
针对现有技术存在的上述问题，本发明提供一种热稳定性提高的蔗糖磷酸化酶突变体及其应用。
具体来说，本发明涉及如下方面：
1.一种蔗糖磷酸化酶突变体，其特征在于，所述蔗糖磷酸化酶突变体基于参比蔗糖磷酸化酶通过以下位置的突变得到：
在参比蔗糖磷酸化酶的自由能低的氨基酸残基处；
在参比蔗糖磷酸化酶的不稳定的氨基酸残基处；
在参比蔗糖磷酸化酶的与热稳定相关的氨基酸残基处。
2.根据项1所述的蔗糖磷酸化酶突变体，其特征在于，所述蔗糖磷酸化酶突变体相对于参比蔗糖磷酸化酶具有更高的热稳定性，且其酶活不低于参比蔗糖磷酸化酶的85％，其中参比蔗糖磷酸化酶的氨基酸序列如SEQ ID NO.1所示。
3.根据项2所述的蔗糖磷酸化酶突变体，其特征在于，在70℃下，所述蔗糖磷酸化酶突变体的热稳定性比参比蔗糖磷酸化酶的热稳定性提高10％-98％。
4.根据项2所述的蔗糖磷酸化酶突变体，其特征在于，所述突变基于SEQ ID NO.1在以下任意一个或两个以上位置进行：
M22、I25、S155、T165、V233、V241、V310、N320和T439。
5.根据项4所述的蔗糖磷酸化酶突变体，其特征在于，所述突变为基于SEQ IDNO.1进行以下的任意一种或两种以上氨基酸替换：
M22L、M22I、M22V、M22A、I25L、I25E、I25V、I25A、S155E、S155I、S155R、S155T、T165E、T165I、T165V、T165A、V233I、V233A、V233L、V233M、V241I、V241S、V241W、V241M、V310E、V310L、V310M、V310R、N320E、N320P、N320M、N320L、T439E、T439P、T439M和T439L。
6.根据项5所述的蔗糖磷酸化酶突变体，其特征在于，所述氨基酸替换包括以下组合：
S155T+I25T；或
S155T+T165V；或
S155T+T439E；或
S155T+I25T+T165V；或
S155T+165V+T439E。
7.一种核酸分子，其特征在于，包括编码项1-6中任一项所述的蔗糖磷酸化酶突变体的核苷酸序列。
8.一种表达载体，其特征在于，所述表达载体包括编码项1-6中任一项所述的蔗糖磷酸化酶突变体的核苷酸序列。
9.一种宿主细胞，其特征在于，所述宿主细胞包含项8所述的表达载体。
10.一种催化转移葡萄糖苷键的方法，其特征在于，所述方法包括：
使用项1-6中任一项所述的蔗糖磷酸化酶突变体来催化转移葡萄糖苷键。
11.权利要求1-6中任一项所述的蔗糖磷酸化酶突变体、项8所述的表达载体或项9所述的宿主细胞在催化转移葡萄糖苷键中的应用。
12.项8所述的表达载体或项9所述的宿主细胞在表达蔗糖磷酸化酶中的应用。
本申请通过对参比蔗糖磷酸化酶突变得到的蔗糖磷酸化酶突变体与参比蔗糖磷酸化酶相比具有更高的热稳定性，在70℃下，所述蔗糖磷酸化酶突变体的热稳定性比参比蔗糖磷酸化酶的热稳定性提高10％-98％。
具体实施方式
下面结合实施例进一步说明本申请，应当理解，实施例仅用于进一步说明和阐释本申请，并非用于限制本申请。
除非另外定义，本说明书中有关技术的和科学的术语与本领域内的技术人员所通常理解的意思相同。虽然在实验或实际应用中可以应用与此间所述相似或相同的方法和材料，本文还是在下文中对材料和方法做了描述。在相冲突的情况下，以本说明书包括其中定义为准，另外，材料、方法和例子仅供说明，而不具限制性。以下结合具体实施例对本申请作进一步的说明，但不用来限制本申请的范围。
在本文中，20种天然氨基酸中文全称、三字母缩写及其单字母缩写的对应关系为：精氨酸：Arg,R；组氨酸：His,H；赖氨酸，Lys,K；天冬氨酸，Asp,D；谷氨酸：Glu,E；丝氨酸，Ser,S；苏氨酸，Thr,T；天冬酰胺，Asn,N；谷氨酰胺：Gln,Q；半胱氨酸，Cys,C；甘氨酸，Gly,G；脯氨酸，Pro,P；丙氨酸，Ala,A；异亮氨酸，Ile,I；缬氨酸，Vla,V；亮氨酸，Leu,L；甲硫氨酸，Met,M；苯丙氨酸，Phe,F；酪氨酸，Tyr,Y；色氨酸，Trp,W。
在本文中，术语“热稳定性”是蛋白质稳定性的一个层面，用于表征蛋白质对热变性的抵抗程度。
其中，本文中的“突变体”可理解为包含至少一个野生型或天然氨基酸被不同于野生型或天然多肽的氨基酸取代的突变型多肽。突变体可以只包含一个野生型或天然氨基酸取代，称为“点突变”或“单点突变”多肽。另外，突变体可包含两个或更多个野生型或天然氨基酸被不同于野生型或天然多肽的两个或多个氨基酸取代。
取代突变可利用本领域熟知的任何诱变技术制备，包括而不限于定点诱变。其中，定点突变是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组，也可以是质粒)中引入所需变化(通常是表征有利方向的变化)，包括碱基的添加、删除、点突变等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征，是基因研究工作中一种非常有用的手段。
在本文中，术语“氨基酸替换”或“氨基酸取代”可以互换使用。
本申请提供一种蔗糖磷酸化酶突变体，所述蔗糖磷酸化酶突变体基于参比蔗糖磷酸化酶通过以下位置的突变得到：
在参比蔗糖磷酸化酶的自由能低的氨基酸残基处；
在参比蔗糖磷酸化酶的不稳定的氨基酸残基处；
在参比蔗糖磷酸化酶的与热稳定相关的氨基酸残基处。
其中，参比蔗糖磷酸化酶来源于青春双歧杆菌，由504个氨基酸组成，氨基酸序列SEQ ID NO:1所示。
参比蔗糖磷酸化酶的自由能低的氨基酸残基可以通过以下方式获得：PoPMuSiC软件预测每个氨基酸残基的解折叠自由能，找到自由能低的位点。
参比蔗糖磷酸化酶的不稳定的氨基酸残基可以通过以下方式获得：B-factor的在线分析软件计算蛋白质各个残基的B-factor值，从而分析出该蛋白质中不稳定的区域。
参比蔗糖磷酸化酶的与热稳定相关的氨基酸残基可以通过以下方式获得：基于不同来源的蔗糖磷酸化酶的同源多序列比对，找到与热稳定性相关的氨基酸位点。
本申请所述蔗糖磷酸化酶突变体相对于参比蔗糖磷酸化酶具有更高的热稳定性，且其酶活不低于参比蔗糖磷酸化酶的85％。
进一步地，在70℃下，所述蔗糖磷酸化酶突变体的热稳定性比参比蔗糖磷酸化酶的热稳定性提高10％-98％。其中，蔗糖磷酸化酶突变体的热稳定性比参比蔗糖磷酸化酶的热稳定性提高值通过以下方式计算：
(突变体酶活残留比例-原始酶活残留比例)/原始酶活残留比例。
在一个具体的实施方式中，所述突变为基于SEQ ID NO.1在以下任意一个或两个以上位置进行的氨基酸替换：
M22、I25、S155、T165、V233、V241、V310、N320和T439。
例如，所述氨基酸替换可以发生在M22、I25、S155、T165、V233、V241、V310、N320和T439中的任意一个位置、任意两个位置、任意三个位置、任意四个位置、任意五个位置、任意六个位置、任意七个位置、任意八个位置或在九个位置均发生氨基酸替换。
在一个具体的实施方式中，所述氨基酸替换为基于SEQ ID NO.1进行以下的任意一种或两种以上氨基酸替换：
M22L、M22I、M22V、M22A、I25L、I25E、I25V、I25A、S155E、S155I、S155R、S155T、T165E、T165I、T165V、T165A、V233I、V233A、V233L、V233M、V241I、V241S、V241W、V241M、V310E、V310L、V310M、V310R、N320E、N320P、N320M、N320L、T439E、T439P、T439M和T439L。
例如，所述氨基酸替换为基于SEQ ID NO.1进行以下的任意一种、任意两种或任意三种氨基酸替换：
M22L、M22I、M22V、M22A、I25L、I25E、I25V、I25A、S155E、S155I、S155R、S155T、T165E、T165I、T165V、T165A、V233I、V233A、V233L、V233M、V241I、V241S、V241W、V241M、V310E、V310L、V310M、V310R、N320E、N320P、N320M、N320L、T439E、T439P、T439M和T439L。
在一个具体的实施方式中，所述氨基酸替换包括以下的任意一种：M22L、M22I、M22V、M22A、I25E、I25V、I25A、S155R、T165E、T165V、V233I、V233A、V233L、V233M、V241I、V241W、V310E、V310L、V310M、V310R、N320E、N320P、T439P、T439M和T439L。
在一个具体的实施方式中，所述氨基酸替换包括以下的任意一种：I25L、S155E、S155I、S155T、T165E、T165V、V241S、V241W、N320M、N320L和T439E。
在一个具体的实施方式中，所述氨基酸替换包括以下组合：S155T+I25T。
在一个具体的实施方式中，所述氨基酸替换包括以下组合：S155T+T165V。
在一个具体的实施方式中，所述氨基酸替换包括以下组合：S155T+T439E。
在一个具体的实施方式中，所述氨基酸替换包括以下组合：S155T+I25T+T165V。
在一个具体的实施方式中，所述氨基酸替换包括以下组合：S155T+T165V+T439E。
本申请还提供一种核酸分子，其包括编码上述的蔗糖磷酸化酶突变体的核苷酸序列。
本申请还提供一种表达载体，其包括编码上述的蔗糖磷酸化酶突变体的核苷酸序列。
如本文所使用的，术语“载体”用于描述可以被工程化以含有可以在宿主细胞中扩增的一种多核苷酸或多种多核苷酸的核酸分子。载体包括但不限于：单链，双链或部分双链的核酸分子；包含一个或多个游离末端，没有游离末端(例如环状)的核酸分子；包含DNA，RNA或两者的核酸分子；以及本领域已知的其他多核苷酸种类。一种类型的载体是“质粒”，其是指可以插入额外DNA片段的环状双链DNA环，例如通过标准分子克隆技术。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和游离型哺乳动物载体)。其他载体(例如，非游离型哺乳动物载体)在引入宿主细胞后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。此外，某些载体能够指导外源插入基因的表达。此类载体在本文中称为“表达载体”。重组表达载体包含适于在宿主细胞中表达核酸的形式的本申请的核酸，这意味着重组表达载体包括一种或多种调节元件，其可以基于用于表达的、可以与待表达的核酸序列可操作地连接。
本申请还提供一种宿主细胞，其包含权利要求上述表达载体。
如本文所使用的，术语“宿主细胞”意指任何细胞类型，其易受包含本申请的多核苷酸的核酸构建体或表达载体的转化、转染、转导等。术语“宿主细胞”涵盖亲本细胞的任何后代，其由于复制过程发生的突变与亲本细胞不完全相同。
本申请还提供一种催化转移葡萄糖苷键的方法，所述方法包括：
使用上述的蔗糖磷酸化酶突变体来催化转移葡萄糖苷键。
本申请还包括上述蔗糖磷酸化酶突变体、表达载体或宿主细胞在催化转移葡萄糖苷键中的应用。
对于本申请中的蔗糖磷酸化酶突变体和参比蔗糖磷酸化酶的酶活，可以通过以下方法来确定：
蔗糖和磷酸盐在蔗糖磷酸化酶的催化下生成葡萄糖-1-磷酸和果糖。测定生成产物果糖的量从而计算出蔗糖磷酸化酶的酶活。总反应式如下：
蔗糖+磷酸盐＝(SP)>葡萄糖1-磷酸+果糖
酶活定义为：在特定条件下(在本申请中为50℃)，将每分钟水解蔗糖生成1μmo1的果糖所需酶量定义为蔗糖磷酸化酶的一个酶活力单位。
对于本申请中的蔗糖磷酸化酶突变体和参比蔗糖磷酸化酶的比酶活，可以通过以下方法来确定：
利用Brandford法测定所制备酶液中蛋白质的含量，根据SDS-PAGE中灰度计算目标蛋白的含量，比酶活计算公式如下:
比酶活＝酶活/酶液中目标蛋白浓度。
比酶活定义:每毫克蛋白所具有的酶活力。
实施例
实施例1蔗糖磷酸化酶野生酶Basp的表达
将核苷酸序列为SEQ ID NO.2的聚合酶链式反应(Polymerase Chain Reaction，PCR)产物纯化后用NcoI和XhoI核酸内切酶进行双酶切消化处理，与经相同酶消化的pET-20b载体用T4连接酶16℃过夜连接后，转化大肠杆菌DH5α感受态细胞。涂布于含有1.5％琼脂的LB(含100μg/μL的氨苄青霉素)平板，重组载体命名为pET-20b-basp。
实施例2构建蔗糖磷酸化酶Basp的突变体
首先通过SWISS-MODEL在线软件对该酶的氨基酸序列进行同源建模，获得其三维空间结构。通过以下三种方式找到突变位点：①通过PoPMuSiC软件预测每个氨基酸残基的解折叠自由能，找到自由能低的位点进行突变设计；②通过B-factor的在线分析软件计算蛋白质各个残基的B-factor值，从而分析出该蛋白质中不稳定的区域，并通过合理的设计进行改造；③基于不同来源的蔗糖磷酸化酶的同源多序列比对，找到与热稳定性相关的氨基酸位点进行定点突变改造。之后利用Pymol软件对于突变位点进行模拟突变，计算氢键、离子键等分子内作用力及其构型的改变，得到每个氨基酸位点拟突变的氨基酸。
最终针对三种方式获得的突变如表1和表2所示。表1所示的单点突变为：M22L、M22I、M22V、M22A、I25L、I25E、I25V、I25A、S155E、S155I、S155R、S155T、T165E、T165I、T165V、T165A、V233I、V233A、V233L、V233M、V241I、V241S、V241W、V241M、V310E、V310L、V310M、V310R、N320E、N320P、N320M、N320L、T439E、T439P、T439M和T439L。表2中的两点和三点突变为：S155T+I25T、S155T+165V、S155T+T439E、S155T+I25T+T165V、S155T+165V+T439E。
表1
表2
对上述突变设计定点突变引物，对蔗糖磷酸化酶基因basp进行定点突变。
PCR的反应条件如下：
2ng/μl pET20b-basp(携带有来自青春双歧杆菌的蔗糖磷酸化酶基因basp)1μl,2.5mM dNTPs 2μl，10μM引物(F以及R)1.25μl，以及Ta Q5高保真DNA聚合酶0.25μl。PCR扩增条件：98℃30s；98℃10s，60℃30s，72℃3min，20个循环；72℃5min；4℃保温。
PCR产物经DpnI酶37℃处理2h去除原质粒后，利用DNA纯化试剂盒进行纯化后转化大肠杆菌DH5α感受态细胞。涂布于含有1.5％琼脂的LB(含100μg/μL的氨苄青霉素)平板，获取含蔗糖磷酸化酶的突变体质粒的重组菌株。
实施例3蔗糖磷酸化酶的表达和纯化
将实施例1和2中得到的重组载体测序正确后(测序服务有北京睿博兴科生物科技有限公司提供)，用热激法转化大肠杆菌BL21(DE3)获得蔗糖磷酸化酶野生型及其突变体的重组大肠杆菌BL21菌株。挑取单克隆至液体培养基过夜培养活化，转接至新鲜的液体培养基中，于OD600＝0.6-0.8时加入终浓度为10μM的IPTG进行诱导表达目标蛋白。继续培养4h后，收集菌体细胞，用裂解缓冲液(20mM Tris-HCl，0.5M NaCl，20mM咪唑)重悬菌体后再次离心洗涤菌体，用裂解缓冲液再次重悬菌体调节OD600＝10，用超声破碎仪间歇破碎10min，离心去除沉淀，留取上清，即为粗酶液。
粗酶液经镍离子亲和层析柱进行纯化，纯化过程如下：
①20mL清水冲洗柱子；
②20mL binding buffer平衡柱子；
③破碎后的菌液离心后，上清过柱；
④20mL binding buffer冲洗，收集洗脱液；
⑤含50mM咪唑的Tris-HCl缓冲液冲洗，清洗杂蛋白；
⑥含200mM咪唑Tris-HCl冲洗柱子，收集洗脱液，即为纯化的蔗糖磷酸化酶；
⑦含500mM咪唑Tris-HCl冲洗柱子，去除柱体所有蛋白；
⑧清水冲洗柱子，保存在20％乙醇中。
将上述方法纯化得到的纯化的蔗糖磷酸化酶利用变性的聚丙烯酰胺凝胶电泳(SDS-PAGE)检测蛋白的纯化效果，将纯化好的蔗糖磷酸化酶在4℃保存以备后续测定使用。
实施例4测定蔗糖磷酸化酶及其变体在70℃下的热稳定性
(1)蔗糖磷酸化酶酶活测定：
蔗糖和磷酸盐在蔗糖磷酸化酶的催化下生成葡萄糖-1-磷酸和果糖。测定生成产物果糖的量从而计算出蔗糖磷酸化酶的酶活。总反应式如下：
蔗糖+磷酸盐＝(SP)>葡萄糖1-磷酸+果糖
酶活定义为：将每分钟水解蔗糖生成1μmo1的果糖所需酶量定义为蔗糖磷酸化酶的一个酶活力单位。
果糖标准曲线的制定
取7个5.0mL离心管将果糖标准液按表1配制不同果糖浓度的溶液，体系为1.0mL，然后加入DNS 1.5mL，混匀后放入沸水浴中煮沸15min，放入冰水中冷却后用酶标仪测定540nm下的吸光值，以果糖浓度为横坐标，以540nm下的吸光值为纵坐标，绘制果糖标准曲线，如表3所示。
表3
反应体系如下：将纯化的蔗糖磷酸化酶酶液稀释一定倍数后，按照表4配置反应体系，体系为1.0mL，50℃准确反应10min，然后加入DNS 1.5mL，混匀后放入沸水浴中煮沸15min，放入冰水中冷却后用酶标仪测定540nm下的吸光值，计算其酶活。
表4
酶活计算方法如下：
式中A：吸光值；b：截距；n：稀释倍数；M：果糖分子量；k：斜率；t：反应时间(min)
蔗糖磷酸化酶比酶活的测定：
利用Brandford法测定所制备酶液中蛋白质的含量，根据SDS-PAGE中灰度计算目标蛋白的含量，比酶活计算公式如下:
比酶活＝酶活/酶液中目标蛋白浓度。
(2)蔗糖磷酸化酶热稳定性测定：
将上述纯化的蔗糖磷酸化酶酶液稀释一定倍数后，在70℃下温育45min取出，放于冰上10min，测定50℃下的酶的残余活性，与未经70℃处理的酶液进行酶活力比较。发现在前面实施例2中描述过的变体比野生型在70℃下的稳定性有所变化，酶活结果如表1和表2所示。
从以上结果可以确定，本申请的蔗糖磷酸化酶突变体相比于野生型酶具有更高的稳定性，并且具体来说，含有S155T的酶突变体经过分析发现在70℃的热稳定性提高78.26％，且S155T、T165V和T439E组合突变得到最高的热稳定性，酶活残留较野生型提高97.87％。因此可以确定，本申请的蔗糖磷酸化酶突变体热稳定性相比于野生型酶来说显著增加。
上述参照实施例对该种蔗糖磷酸化酶变体及其构建方法与应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本申请总体构思下的变化和修改，应属本申请的保护范围。
序列表
SEQ ID NO.1
MKNKVQLITYADRLGDGTIKSMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLAKGEESEYYPMFLTMSSVFPNGATEEDLAGIYRPRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTDSDKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDFALPPLLLHALSTGHVEPVAHWTDIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESEAATGAAASNLDLYQVNSTYYSALGCNDQHYIAARAVQFFLPGVPQVYYVGALAGKNDMELLNKTNNGRDINRHYYSTAEIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTPTDTSISFTWRGETSEATLTFEPKRGLGVDNTTPVAMLEWHDSAGDHRSDDLIANPPVVA
SEQ ID NO.2
ATGAAAAACAAGGTGCAGCTCATCACTTACGCCGACCGCCTTGGCGACGGCACCATCAAGTCGATGACCGACATTCTGCGCACCCGCTTCGACGGCGTGTACGACGGCGTTCACATCCTGCCGTTCTTCACCCCGTTCGACGGCGCCGACGCAGGCTTCGACCCGATCGACCACACCAAGGTCGACGAACGTCTCGGCAGCTGGGACGACGTCGCCGAACTCTCCAAGACCCACAACATCATGGTCGACGCCATCGTCAACCACATGAGTTGGGAATCCAAGCAGTTCCAGGACGTGCTGGCCAAGGGCGAGGAGTCCGAATACTATCCGATGTTCCTCACCATGAGCTCCGTGTTCCCGAACGGCGCCACCGAAGAGGACCTGGCCGGCATCTACCGTCCGCGTCCGGGCCTGCCGTTCACCCACTACAAGTTCGCCGGCAAGACCCGCCTCGTGTGGGTCAGCTTCACCCCGCAGCAGGTGGACATCGACACCGATTCCGACAAGGGTTGGGAATACCTCATGTCGATTTTCGACCAGATGGCCGCCTCTCACGTCAGCTACATCCGCCTCGACGCCGTCGGCTATGGCGCCAAGGAAGCCGGCACCAGCTGCTTCATGACCCCGAAGACCTTCAAGCTGATCTCCCGTCTGCGTGAGGAAGGCGTCAAGCGCGGTCTGGAAATCCTCATCGAAGTGCACTCCTACTACAAGAAGCAGGTCGAAATCGCATCCAAGGTGGACCGCGTCTACGACTTCGCCCTGCCTCCGCTGCTGCTGCACGCGCTGAGCACCGGCCACGTCGAGCCCGTCGCCCACTGGACCGACATACGCCCGAACAACGCCGTCACCGTGCTCGATACGCACGACGGCATCGGCGTGATCGACATCGGCTCCGACCAGCTCGACCGCTCGCTCAAGGGTCTCGTGCCGGATGAGGACGTGGACAACCTCGTCAACACCATCCACGCCAACACCCACGGCGAATCCGAGGCAGCCACTGGCGCCGCCGCATCCAATCTCGACCTCTACCAGGTCAACAGCACCTACTATTCGGCGCTCGGGTGCAACGACCAGCACTACATCGCCGCCCGCGCGGTGCAGTTCTTCCTGCCGGGCGTGCCGCAAGTCTACTACGTCGGCGCGCTCGCCGGCAAGAACGACATGGAGCTGCTGAACAAGACGAATAACGGCCGCGACATCAATCGCCATTACTACTCCACCGCGGAAATCGACGAGAACCTCAAGCGTCCGGTCGTCAAGGCCCTGAACGCGCTCGCCAAGTTCCGCAACGAGCTCGACGCGTTCGACGGCACGTTCTCGTACACCACCCCGACCGACACGTCCATCAGCTTCACCTGGCGCGGCGAAACCAGCGAGGCCACGCTGACGTTCGAGCCGAAGCGCGGTCTCGGTGTGGACAACACTACGCCGGTCGCCATGTTGGAATGGCACGATTCCGCGGGAGACCACCGTTCGGATGATCTGATCGCCAATCCGCCTGTCGTCGCCTGA
序列表
<110> 华熙生物科技股份有限公司
华熙生物科技（天津）有限公司
<120> 高稳定型的蔗糖磷酸化酶突变体及其应用
<130> TPF02082
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 504
<212> PRT
<213> 人工序列
<220>
<223> 人工序列描述：人工合成的序列
<400> 1
Met Lys Asn Lys Val Gln Leu Ile Thr Tyr Ala Asp Arg Leu Gly Asp
1 5 10 15
Gly Thr Ile Lys Ser Met Thr Asp Ile Leu Arg Thr Arg Phe Asp Gly
20 25 30
Val Tyr Asp Gly Val His Ile Leu Pro Phe Phe Thr Pro Phe Asp Gly
35 40 45
Ala Asp Ala Gly Phe Asp Pro Ile Asp His Thr Lys Val Asp Glu Arg
50 55 60
Leu Gly Ser Trp Asp Asp Val Ala Glu Leu Ser Lys Thr His Asn Ile
65 70 75 80
Met Val Asp Ala Ile Val Asn His Met Ser Trp Glu Ser Lys Gln Phe
85 90 95
Gln Asp Val Leu Ala Lys Gly Glu Glu Ser Glu Tyr Tyr Pro Met Phe
100 105 110
Leu Thr Met Ser Ser Val Phe Pro Asn Gly Ala Thr Glu Glu Asp Leu
115 120 125
Ala Gly Ile Tyr Arg Pro Arg Pro Gly Leu Pro Phe Thr His Tyr Lys
130 135 140
Phe Ala Gly Lys Thr Arg Leu Val Trp Val Ser Phe Thr Pro Gln Gln
145 150 155 160
Val Asp Ile Asp Thr Asp Ser Asp Lys Gly Trp Glu Tyr Leu Met Ser
165 170 175
Ile Phe Asp Gln Met Ala Ala Ser His Val Ser Tyr Ile Arg Leu Asp
180 185 190
Ala Val Gly Tyr Gly Ala Lys Glu Ala Gly Thr Ser Cys Phe Met Thr
195 200 205
Pro Lys Thr Phe Lys Leu Ile Ser Arg Leu Arg Glu Glu Gly Val Lys
210 215 220
Arg Gly Leu Glu Ile Leu Ile Glu Val His Ser Tyr Tyr Lys Lys Gln
225 230 235 240
Val Glu Ile Ala Ser Lys Val Asp Arg Val Tyr Asp Phe Ala Leu Pro
245 250 255
Pro Leu Leu Leu His Ala Leu Ser Thr Gly His Val Glu Pro Val Ala
260 265 270
His Trp Thr Asp Ile Arg Pro Asn Asn Ala Val Thr Val Leu Asp Thr
275 280 285
His Asp Gly Ile Gly Val Ile Asp Ile Gly Ser Asp Gln Leu Asp Arg
290 295 300
Ser Leu Lys Gly Leu Val Pro Asp Glu Asp Val Asp Asn Leu Val Asn
305 310 315 320
Thr Ile His Ala Asn Thr His Gly Glu Ser Glu Ala Ala Thr Gly Ala
325 330 335
Ala Ala Ser Asn Leu Asp Leu Tyr Gln Val Asn Ser Thr Tyr Tyr Ser
340 345 350
Ala Leu Gly Cys Asn Asp Gln His Tyr Ile Ala Ala Arg Ala Val Gln
355 360 365
Phe Phe Leu Pro Gly Val Pro Gln Val Tyr Tyr Val Gly Ala Leu Ala
370 375 380
Gly Lys Asn Asp Met Glu Leu Leu Asn Lys Thr Asn Asn Gly Arg Asp
385 390 395 400
Ile Asn Arg His Tyr Tyr Ser Thr Ala Glu Ile Asp Glu Asn Leu Lys
405 410 415
Arg Pro Val Val Lys Ala Leu Asn Ala Leu Ala Lys Phe Arg Asn Glu
420 425 430
Leu Asp Ala Phe Asp Gly Thr Phe Ser Tyr Thr Thr Pro Thr Asp Thr
435 440 445
Ser Ile Ser Phe Thr Trp Arg Gly Glu Thr Ser Glu Ala Thr Leu Thr
450 455 460
Phe Glu Pro Lys Arg Gly Leu Gly Val Asp Asn Thr Thr Pro Val Ala
465 470 475 480
Met Leu Glu Trp His Asp Ser Ala Gly Asp His Arg Ser Asp Asp Leu
485 490 495
Ile Ala Asn Pro Pro Val Val Ala
500
<210> 2
<211> 1515
<212> DNA
<213> 人工序列
<220>
<223> 人工序列描述：人工合成的序列
<400> 2
atgaaaaaca aggtgcagct catcacttac gccgaccgcc ttggcgacgg caccatcaag 60
tcgatgaccg acattctgcg cacccgcttc gacggcgtgt acgacggcgt tcacatcctg 120
ccgttcttca ccccgttcga cggcgccgac gcaggcttcg acccgatcga ccacaccaag 180
gtcgacgaac gtctcggcag ctgggacgac gtcgccgaac tctccaagac ccacaacatc 240
atggtcgacg ccatcgtcaa ccacatgagt tgggaatcca agcagttcca ggacgtgctg 300
gccaagggcg aggagtccga atactatccg atgttcctca ccatgagctc cgtgttcccg 360
aacggcgcca ccgaagagga cctggccggc atctaccgtc cgcgtccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgc ctcgtgtggg tcagcttcac cccgcagcag 480
gtggacatcg acaccgattc cgacaagggt tgggaatacc tcatgtcgat tttcgaccag 540
atggccgcct ctcacgtcag ctacatccgc ctcgacgccg tcggctatgg cgccaaggaa 600
gccggcacca gctgcttcat gaccccgaag accttcaagc tgatctcccg tctgcgtgag 660
gaaggcgtca agcgcggtct ggaaatcctc atcgaagtgc actcctacta caagaagcag 720
gtcgaaatcg catccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctgctg 780
cacgcgctga gcaccggcca cgtcgagccc gtcgcccact ggaccgacat acgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgac 900
cagctcgacc gctcgctcaa gggtctcgtg ccggatgagg acgtggacaa cctcgtcaac 960
accatccacg ccaacaccca cggcgaatcc gaggcagcca ctggcgccgc cgcatccaat 1020
ctcgacctct accaggtcaa cagcacctac tattcggcgc tcgggtgcaa cgaccagcac 1080
tacatcgccg cccgcgcggt gcagttcttc ctgccgggcg tgccgcaagt ctactacgtc 1140
ggcgcgctcg ccggcaagaa cgacatggag ctgctgaaca agacgaataa cggccgcgac 1200
atcaatcgcc attactactc caccgcggaa atcgacgaga acctcaagcg tccggtcgtc 1260
aaggccctga acgcgctcgc caagttccgc aacgagctcg acgcgttcga cggcacgttc 1320
tcgtacacca ccccgaccga cacgtccatc agcttcacct ggcgcggcga aaccagcgag 1380
gccacgctga cgttcgagcc gaagcgcggt ctcggtgtgg acaacactac gccggtcgcc 1440
atgttggaat ggcacgattc cgcgggagac caccgttcgg atgatctgat cgccaatccg 1500
cctgtcgtcg cctga 1515
Claims (10)
1.一种蔗糖磷酸化酶突变体，其特征在于，所述蔗糖磷酸化酶突变体基于参比蔗糖磷酸化酶通过以下位置的突变得到：
在参比蔗糖磷酸化酶的自由能低的氨基酸残基处；
在参比蔗糖磷酸化酶的不稳定的氨基酸残基处；
在参比蔗糖磷酸化酶的与热稳定相关的氨基酸残基处。
2.根据权利要求1所述的蔗糖磷酸化酶突变体，其特征在于，所述蔗糖磷酸化酶突变体相对于参比蔗糖磷酸化酶具有更高的热稳定性，且其酶活不低于参比蔗糖磷酸化酶的85％，其中参比蔗糖磷酸化酶的氨基酸序列如SEQ ID NO.1所示。
3.根据权利要求2所述的蔗糖磷酸化酶突变体，其特征在于，在70℃下，所述蔗糖磷酸化酶突变体的热稳定性比参比蔗糖磷酸化酶的热稳定性提高10％-98％。
4.根据权利要求2所述的蔗糖磷酸化酶突变体，其特征在于，所述突变基于SEQ IDNO.1在以下任意一个或两个以上位置进行：
M22、I25、S155、T165、V233、V241、V310、N320和T439。
5.根据权利要求4所述的蔗糖磷酸化酶突变体，其特征在于，所述突变为基于SEQ IDNO.1进行以下的任意一种或两种以上氨基酸替换：
M22L、M22I、M22V、M22A、I25L、I25E、I25V、I25A、S155E、S155I、S155R、S155T、T165E、T165I、T165V、T165A、V233I、V233A、V233L、V233M、V241I、V241S、V241W、V241M、V310E、V310L、V310M、V310R、N320E、N320P、N320M、N320L、T439E、T439P、T439M和T439L。
优选的，所述氨基酸替换包括以下组合：
S155T+I25T；或
S155T+T165V；或
S155T+T439E；或
S155T+I25T+T165V；或
S155T+165V+T439E。
6.一种核酸分子，其特征在于，包括编码权利要求1-5中任一项所述的蔗糖磷酸化酶突变体的核苷酸序列。
7.一种表达载体，其特征在于，所述表达载体包括编码权利要求1-5中任一项所述的蔗糖磷酸化酶突变体的核苷酸序列。
8.一种宿主细胞，其特征在于，所述宿主细胞包含权利要求7所述的表达载体。
9.一种催化转移葡萄糖苷键的方法，其特征在于，所述方法包括：
使用权利要求1-5中任一项所述的蔗糖磷酸化酶突变体来催化转移葡萄糖苷键。
10.权利要求1-5中任一项所述的蔗糖磷酸化酶突变体、权利要求7所述的表达载体或权利要求8所述的宿主细胞在催化转移葡萄糖苷键中的应用。
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