CN115141815A - 蔗糖磷酸化酶突变体及其应用 - Google Patents
蔗糖磷酸化酶突变体及其应用 Download PDFInfo
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- CN115141815A CN115141815A CN202210699240.XA CN202210699240A CN115141815A CN 115141815 A CN115141815 A CN 115141815A CN 202210699240 A CN202210699240 A CN 202210699240A CN 115141815 A CN115141815 A CN 115141815A
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- sucrose phosphorylase
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Abstract
本申请公开了一种蔗糖磷酸化酶突变体及其应用,所述蔗糖磷酸化酶突变体包含一种或两种以上的基于参考序列的突变体。本申请所述的突变体具有较高的催化活性,其有利于扩大蔗糖磷酸化酶转糖苷作用于工业化的应用前景,实现大规模工业化应用。
Description
技术领域
本申请涉及工业生物技术领域,尤其涉及一种蔗糖磷酸化酶突变体及其应用。
背景技术
糖基化作用是自然界中最重要的生化反应之一,能赋予天然产物结构多样性,改善化合物水溶性、稳定性、生物利用度等性质,所得产物能广泛应用于食品、医药及个人护理等领域。碳水化合物活性酶(Carbohydrate-Active enZymes,CAZy)是一类对糖苷键有活性的酶,可以降解、修饰以及生成糖苷键。这些酶可以分为4类:糖苷水解酶(GHs)、糖苷磷酸化酶(GPs)、转糖苷酶(TGs)以及糖基转移酶(GTs)。GHs包含糖苷酶和转糖苷酶,能够参与糖苷键的水解和合成,GHs分布广泛,糖基供体廉价,在生物技术和生物医学中有广泛的应用。蔗糖磷酸化酶(Sucrose phosphorylase,SPase,EC 2.4.1.7) 属于GH13家族,能够可逆地催化蔗糖与无机磷酸盐反应生成D-果糖(D-Fru) 和α-D-葡萄糖-1-磷酸(α-D-Glc-1-P)。SPase具有广泛的受体底物特异性,能将蔗糖的葡萄糖基转移至多种受体化合物,根据参与反应的类型可将其作用受体分为3类:水;无机磷酸;含醇羟基、酚羟基或羧基的糖基受体化合物。 SPase在糖基化应用中具有突出表现,尤其是对非碳水化合物的糖基化活性使其逐渐成为酶工程研究的热点。
虽然SPase的糖基受体底物特异性广泛,具有较好的工业应用前景,但其对一些受体底物的亲和力不佳,导致产率低。因此,需要通过理性突变、定向进化等策略进行天然SPase的基因修饰,实现其催化特性的定向改造。
发明内容
为了解决现有技术中已知的蔗糖磷酸化酶部分受体特异性低、酶活低的问题,本申请提供了一种蔗糖磷酸化酶突变体,所提供的变体酶活有所提高,从而可以增强底物特异性,提高产率,降低生产成本,具有广阔的工业应用前景。
本申请具体技术方案如下:
1.一种蔗糖磷酸化酶突变体,其中,包含一种或两种以上的基于参考序列的突变体,所述参考序列的氨基酸序列如SEQ ID NO:1所示。
2.根据项1所述的蔗糖磷酸化酶突变体,其中,所述突变体的氨基酸序列包含对应于SEQ ID NO:1的I25、Y34、E63、S155、T165、L214、N280、 V310、V374和K428中的至少一个位点的氨基酸突变。
3.根据项2所述的蔗糖磷酸化酶突变体,其中,所述突变体的氨基酸序列包含对应于SEQ ID NO:1的I25、Y34、E63、S155、T165、L214、N280、 V310、V374和K428中的一个或两个位点的氨基酸突变。
4.根据项2或3所述的蔗糖磷酸化酶突变体,其中,所述突变体的氨基酸序列包含对应于SEQ ID NO:1的以下任意一种或两种以上氨基酸替换:
I25L、I25A、I25V、Y34F、Y34W、E63P、E63H、E63W、S155T、T165V、 T165L、T165I、T165G、T165A、L214F、L214W、L214Y、N280Y、N280T、 N280F、V310L、V310I、V310F、V374I、V374L、K428R、K428H。
5.根据项2-4中任一项所述的蔗糖磷酸化酶突变体,其中,所述蔗糖磷酸化酶突变体选自下述任一的突变体:S155T、S155T/T165V、S155T/T165L、 S155T/T165I、S155T/T165G、S155T/T165A、V310L、V310I、V310F、 L214F/V310L、L214F/V310I、L214F/V310F、I25L/V310L、E63P/V310L、 S155T/V310L、T165V/V310L;优选选自下述中的任一突变体:V310L、V310I、 V310F、L214F/V310L、L214F/V310I、L214F/V310F、I25L/V310L、E63P/V310L、S155T/V310L、T165V/V310L。
6.一种核酸,其编码项1-5中任一项所述的蔗糖磷酸化酶突变体。
7.一种表达载体,其包含权利要求1所述的核酸。
8.一种基因工程菌,其包含项7所述的表达载体。
9.根据项8所述的基因工程菌,其中,所述基因工程菌的宿主菌为大肠杆菌。
10.项7中所述的表达载体、项8-9中任一项所述的基因工程菌在制备蔗糖磷酸化酶突变体中的应用。
11.一种催化转移葡萄糖苷键的方法,其中,所述方法包括:
使用项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。
发明的效果
本申请所提供的多种酶活提高的蔗糖磷酸化酶突变体,其是在青春双歧杆菌来源的蔗糖磷酸化酶的基础上进行定点突变实现蔗糖磷酸化酶酶活提高的,酶活的提高有利于扩大蔗糖磷酸化酶转糖苷作用于工业化的应用前景,实现大规模工业化应用。
附图说明
图1是果糖的标准曲线示意图。
具体实施方式
下面结合附图所描述的实施方式对本申请做以详细说明,其中所有附图中相同的数字表示相同的特征。虽然附图中显示了本申请的具体实施例,然而应当理解,可以以各种形式实现本申请而不应被这里阐述的实施例所限制。相反,提供这些实施例是为了能够更透彻地理解本申请,并且能够将本申请的范围完整的传达给本领域的技术人员。
需要说明的是,在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解,技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异作为区分组件的方式,而是以组件在功能上的差异作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为开放式用语,故应解释成“包含但不限定于”。说明书后续描述为实施本申请的较佳实施方式,然而所述描述乃以说明书的一般原则为目的,并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。
本申请提供了一种蔗糖磷酸化酶突变体,其中,其包含一种或两种以上的基于参考序列的突变体。
所述参考序列指的是野生型的序列。
在一些实施方式中,所述参考序列的氨基酸序列如SEQ ID NO:1所示。在一些实施方式中,编码SEQ ID NO:1的核苷酸序列如SEQ ID NO:2所示。
SEQ ID NO:1的氨基酸序列如下:
MKNKVQLITYADRLGDGTIKSMTDILRTRFDGVYDGVHILPFFTPFD GADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQ FQDVLAKGEESEYYPMFLTMSSVFPNGATEEDLAGIYRPRPGLPFTHYKF AGKTRLVWVTFTPQQVDIDTDSDKGWEYLMSIFDQMAASHVSYIRLDA VGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIA SKVDRVYDFALPPLLLHALSTGHVEPVAHWTDIRPNNAVTVLDTHDGIG VIDIGSDQLDRSLKGLIPDEDVDNLVNTIHANTHGESEAATGAAASNLDL YQVNSTYYSALGCNDQHYIAARAVQFFLPGVPQVYYVGALAGKNDMEL LNKTNNGRDINRHYYSTAEIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTPTDTSISFTWRGETSEATLTFEPKRGLGVDNTTPVAMLEWHDSAGD HRSDDLIANPPVVA
SEQ ID NO:2的核苷酸序列如下:
atgaaaaacaaggtgcagctcatcacttacgccgaccgccttggcgacggcaccatcaagtcgatgaccga cattctgcgcacccgcttcgacggcgtgtacgacggcgttcacatcctgccgttcttcaccccgttcgacggcgccg acgcaggcttcgacccgatcgaccacaccaaggtcgacgaacgtctcggcagctgggacgacgtcgccgaactc tccaagacccacaacatcatggtcgacgccatcgtcaaccacatgagttgggaatccaagcagttccaggacgtgc tggccaagggcgaggagtccgaatactatccgatgttcctcaccatgagctccgtgttcccgaacggcgccaccga agaggacctggccggcatctaccgtccgcgtccgggcctgccgttcacccactacaagttcgccggcaagacccg cctcgtgtgggtcagcttcaccccgcagcaggtggacatcgacaccgattccgacaagggttgggaatacctcatgt cgattttcgaccagatggccgcctctcacgtcagctacatccgcctcgacgccgtcggctatggcgccaaggaagc cggcaccagctgcttcatgaccccgaagaccttcaagctgatctcccgtctgcgtgaggaaggcgtcaagcgcggt ctggaaatcctcatcgaagtgcactcctactacaagaagcaggtcgaaatcgcatccaaggtggaccgcgtctacg acttcgccctgcctccgctgctgctgcacgcgctgagcaccggccacgtcgagcccgtcgcccactggaccgaca tacgcccgaacaacgccgtcaccgtgctcgatacgcacgacggcatcggcgtgatcgacatcggctccgaccagc tcgaccgctcgctcaagggtctcgtgccggatgaggacgtggacaacctcgtcaacaccatccacgccaacaccc acggcgaatccgaggcagccactggcgccgccgcatccaatctcgacctctaccaggtcaacagcacctactattc ggcgctcgggtgcaacgaccagcactacatcgccgcccgcgcggtgcagttcttcctgccgggcgtgccgcaagt ctactacgtcggcgcgctcgccggcaagaacgacatggagctgctgaacaagacgaataacggccgcgacatca atcgccattactactccaccgcggaaatcgacgagaacctcaagcgtccggtcgtcaaggccctgaacgcgctcgc caagttccgcaacgagctcgacgcgttcgacggcacgttctcgtacaccaccccgaccgacacgtccatcagcttc acctggcgcggcgaaaccagcgaggccacgctgacgttcgagccgaagcgcggtctcggtgtggacaacactac gccggtcgccatgttggaatggcacgattccgcgggagaccaccgttcggatgatctgatcgccaatccgcctgtc gtcgcc
所述蔗糖磷酸化酶(Sucrose phosphorylase,SPase,EC 2.4.1.7)能够可逆地催化蔗糖与无机磷酸盐反应生成D-果糖(D-Fru)和α-D-葡萄糖-1-磷酸 (α-D-Glc-1-P)。SPase具有广泛的受体底物特异性,能将蔗糖的葡萄糖基转移至多种受体化合物,根据参与反应的类型可将其作用受体分为3类:水;无机磷酸;含醇羟基、酚羟基或羧基的糖基受体化合物。SPase在糖基化应用中具有突出表现,尤其是对非碳水化合物的糖基化活性使其逐渐成为酶工程研究的热点。
在一些实施方式中,所述突变体的氨基酸序列包含对应于SEQ ID NO:1 的I25、Y34、E63、S155、T165、L214、N280、V310、V374和K428中的至少一个位点的氨基酸突变。
所述对应于具有本领域普通技术人员通常理解的含义。具体地说,“对应于”表示两条序列经同源性或序列相同性比对后,一条序列与另一条序列中的指定位置相对应的位置。
所述突变体是指相对于野生型蛋白的氨基酸,例如野生型的序列SEQ ID NO:1的序列来源于青春双歧杆菌的蔗糖磷酸化酶,或来源于此类酶的基础上,包含一个或更多个位置处的改变,即取代、插入和/或缺失,并仍然保留其活性。
本申请所述的蔗糖磷酸化酶突变体是在源自青春双歧杆菌的蔗糖磷酸化酶的基础上进行定点突变得到的,本发明通过使用不同来源的蔗糖磷酸化酶进行突变,得到源自青春双歧杆菌的蔗糖磷酸化酶进行定点突变后,所得到的突变体酶活性大大提高。
对于定点突变的方法,本发明不作任何限制,其可以根据本领域常规的方法进行定点突变,例如可以采用定向诱变、随机诱变或合成寡核苷酸的构建,进而将所突变得到的DNA序列在宿主菌中进行表达,得到氨基酸序列发生取代、插入和/或缺失的突变体。
在一些实施方式中,所述突变体的氨基酸序列具包含对应于SEQ ID NO:1的I25、Y34、E63、S155、T165、L214、N280、V310、V374和K428 中的一个位点的氨基酸突变。
在一些实施方式中,所述突变体的氨基酸序列具包含对应于SEQ ID NO:1的I25、Y34、E63、S155、T165、L214、N280、V310、V374和K428 中的两个位点的氨基酸突变。
例如,所述突变体的氨基酸序列为I25/Y34、I25/E63、I25/S155、I25/T165、 I25/L214、I25/N280、I25/V310、I25/V374、I25/K428、Y34/E63、Y34/S155、 Y34/T165、Y34/L214、Y34/N280、Y34/V310、Y34/V374、Y34/K428、E63/S155、 E63/T165、E63/L214、E63/N280、E63/V310、E63/V374、E63/K428、S155/T165、 S155/L214、S155/N280、S155/V310、S155/V374、S155/K428、T165/L214、 T165/N280、T165/V310、T165/V374、T165/K428、L214/N280、L214/V310、 L214/V374、L214/K428、N280/V310、N280/V374、N280/K428、V310/V374、 V310/K428或V374/K428。
上述所述的突变体在SEQ ID NO:1的相应位置发生两个氨基酸突变的突变体,其中,“/”表示突变位点的组合,例如,“I25/E63”表示第25位的异亮氨酸和第63位的谷氨酸均发生突变,即双突变体。
本申请的氨基酸由单字母或三字母代码表示,具有如下含义:A:Ala(丙氨酸);R:Arg(精氨酸);N:Asn(天冬酰胺);I:Ile(异亮氨酸);L:Leu (亮氨酸);V:Val(缬氨酸);Y:Tyr(酪氨酸);F:Phe(苯丙氨酸); W:Trp(色氨酸);E:Glu(谷氨酸);P:Pro(脯氨酸);H:His(组氨酸); S:Ser(丝氨酸);T:Thr(苏氨酸);G:Gly(甘氨酸);K:Lys(赖氨酸)
对于I25L\A\V,指的是SEQ ID NO:1中的第25位的I突变为L或A或 V,对于其他位置的突变,其和上述类似。
在一些实施方式中,所述蔗糖磷酸化酶突变体选自下述任一的突变体: S155T、S155T/T165V、S155T/T165L、S155T/T165I、S155T/T165G、 S155T/T165A、V310L、V310I、V310F、L214F/V310L、L214F/V310I、 L214F/V310F、I25L/V310L、E63P/V310L、S155T/V310L、T165V/V310L。
对于“I25L/E63P”,其表示第25位的异亮氨酸突变为亮氨酸,且第63 位的谷氨酸突变为脯氨酸。
在一些实施方式中,所述蔗糖磷酸化酶突变体选自下述任一的突变体: V310L、V310I、V310F、L214F/V310L、L214F/V310I、L214F/V310F、 I25L/V310L、E63P/V310L、S155T/V310L、T165V/V310L。
在一些实施方式中,所述蔗糖磷酸化酶突变体选自下述任一的突变体: S155T、S155T/T165V、S155T/T165L、S155T/T165I、S155T/T165G、S155T/T165A。本申请提供的上述突变体,其酶活较突变前均有提高,突变体酶活提高14-351%,优选为100-351%,最优选提高351%,所得到的突变体可以为该酶的催化机理研究提供更多的基础,并提高了该酶的工业应用潜力。
其中,突变体酶活提高通过以下方式计算:(突变体的相对酶活-野生型的相对酶活)×100%。
本发明所述的突变体与参考序列的同源性为90%以上。
本申请提供了一种核酸,其编码上述所述的蔗糖磷酸化酶突变体。
本申请提供了一种表达载体,其包含上述所述的核酸。在一些实施方式中,所述表达载体为质粒,进一步优选为pET-20b。
本申请提供了一种基因工程菌,其包含上述所述的表达载体,优选的,所述基因工程菌是将所述核酸连接载体得到表达载体,再导入宿主菌中得到基因工程菌。
对于将核酸连接载体得到表达载体的方法以及将表达载体导入宿主菌中的方法,本申请不作任何限制,其是本领域常用的方法。
在一些实施方式中,所述宿主菌为大肠杆菌。
本申请提供了上述所述的表达载体或基因工程菌在制备蔗糖磷酸化酶突变体中的应用。
本申请提供了一种催化转移葡萄糖苷键的方法,其所述方法包括:使用上述所述的蔗糖磷酸化酶突变体来催化转移葡萄糖苷键。
本申请提供了一种蔗糖磷酸化酶突变体的制备方法,其包括培养上述所述的基因工程菌,使其表达表达蔗糖磷酸化酶突变体。
对于培养方法,本发明不作任何限制,其是本领域常规的方法,例如按 1-5%接种量将基因工程菌接入到LB液体培养基中,于37℃,200rpm/min 培养至OD600达到0.6-0.8后,加入IPTG诱导剂于25-30℃,200rpm/min诱导8-10h后收集菌液,离心收集菌体,重悬后破碎菌体,离心收集上清得到突变体。
本申请使用上述所述的突变体进行催化,能够大大提高催化效率,所述突变体的活性较野生型的活性显著增加。
本申请对参考序列进行突变,所得到的突变体的催化性能得到显著提高,其在催化底物时,能够大大提高催化效率,这可以为蔗糖磷酸化酶的催化机理研究提供了更多的基础,并提高了该酶的工业应用潜力。
实施例
本申请对试验中所用到的材料以及试验方法进行一般性和/或具体的描述,在下面的实施例中,如果无其他特别的说明,%表示wt%,即重量百分数。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
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-SPase。
实施例2单个定点突变体的制备与表达
(1)靶位点设计和分析
a.同源建模
通过SWISS-MODEL在线软件对蔗糖磷酸化酶的序列进行同源建模,获得其三维空间结构。使用MEGA7.0进行多序列比对,使用PyMol对其模型进行可视化分析和处理。
b.模型验证
利用PROCHECK和Verify-3D程序对模型进行评估。Ramachandran plot 用于阐述肽平面内两个二面角(φ与ψ)的比值,以表明氨基酸残基的允许和不允许的构想,通过PROCHECK计算的Psi/Phi Ramachandran图来评估模型的立体化学可靠性。蛋白质侧链氨基酸残基的兼容性通过Verify-3D软件进行评价,至少80%氨基酸残基得分≥0.2则通过验证。
c.酶与配体的分子对接
采用Discoverystudio软件中的分子对接程序进行分子对接,根据其相互作用热区匹配的原理对接到酶的结合口袋中,观察其间的相互作用。
d.拟突变氨基酸及其替换对象的选择
基于不同来源的蔗糖磷酸化酶的同源多序列比对,排除保守氨基酸位点,选择非保守位点上的氨基酸作为拟突变氨基酸,将它们替换为性质相似或其他蔗糖磷酸化酶中出现频率高的氨基酸,形成一系列拟突变酶。
e.运用程序分别将各种拟突变酶与配体进行分子对接模拟,分别计算各种对接复合物的结合自由能,其值越低,表明受体与配体之间的亲和力越高,从而以提高酶活力,得到突变位点。
(2)制备和表达
利用SDM PCR技术,以原始的pET-20b-SPase为模板进行定点突变,获得携带编码蔗糖磷酸化酶突变体I25L\A\V、Y34F\W、E63P\H\W、S155T、 T165V\L\I\G\A、L214F\W\Y、N280Y\W\F、V310L\I\F、V374I\L、K428R\H 的重组质粒pET-20b-SPase1~pET-20b-SPase27,其中,定点突变方法如下:
PCR程序:98℃预变性30s;98℃变性10s,64℃退火30s,72℃延伸3min,循环20次;72℃延伸10min.
Dpn I消化:PCR产物45μL,Dpn I 0.5μL,37℃1h。
然后将得到的重组载体pET-20b-SPase1~pET-20b-SPase27分别转化 DH5α感受态细胞,挑取单克隆送交青岛睿博兴科测序公司进行DBA测序分析确定正确的转化子。
将测序正确的质粒转入BL21感受态细胞,挑取单菌落接种于含有 100g/L氨苄青霉素的试管里(10mL LB)过夜培养,转接至50ml LB培养基中,37℃,200rpm培养至OD600=0.6-0.8,添加IPTG至终浓度10μM,继续培养4h,发酵液8000rpm离心10min后去除上清,留取菌体沉淀,用水重悬至OD600约50,超声破碎15min,60℃30min热处理,离心去除沉淀,留取上清进行酶活测定同时进行SDS-PAGE凝胶电泳分析,结果表明这些突变体蛋白均正常表达,其中,酶活测定的方法如下:
1)果糖标准曲线测定
取7个5.0mL离心管将果糖标准液配制不同果糖浓度的溶液(表1),体系为1.0mL,然后加入DNS试剂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,其如图1所示。
表1不同果糖的浓度
2)酶活测定方法:
蔗糖和磷酸盐在SPase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SPase的酶活。将所得到的酶液稀释一定倍数后,按照表2配置反应体系,体系为1.0mL,50℃准确反应10min,然后加入DNS 试剂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,计算酶活,将每分钟水解蔗糖生成1μmol的果糖所需酶量定义为SPase的一个酶活单位(U),所得到的酶活如表3所示,其中,野生型的的相对活性为100。
表2催化体系
组分 | 体积 |
50mM磷酸缓冲液(pH 6.5) | 450μL |
5%蔗糖 | 500μL |
酶液 | 50μL |
DNS | 1.5mL |
表3单突变体酶活表
从表3可以看出,本申请所得到的突变体酶活相较于野生型的活性显著增加。
实施例3双突变体的制备与表达
首先选择与野生型的相对活性相比具有增加活性的突变位点中的20个变异体,即表3中的M2、M3、M4、M6、M7、M8、M9、M10、M11、M12、 M13、M14、M15、M16、M17、M18、M20、M21、M22和M23,分析相应基因的碱基序列并分析氨基酸突变信息,进行组合突变并表达,方法和实施例1相同,得到双突变体,如表4中的M28-M44所示,并按照实施例1 相同的进行酶活测定,其结果如表4所示。
表4双突变体酶活表
名称 | 双突变体 | 变异的数量 | 相对活性 |
WT | - | 100 | |
M28 | I25L和E63P | 2 | 152 |
M29 | I25L和E63H | 2 | 167 |
M30 | I25L和E63W | 2 | 125 |
M31 | S155T和T165V | 2 | 195 |
M32 | S155T和T165L | 2 | 163 |
M33 | S155T和T165I | 2 | 133 |
M34 | S155T和T165G | 2 | 126 |
M35 | S155T和T165A | 2 | 125 |
M36 | L214F和V310L | 2 | 235 |
M37 | L214F和V310I | 2 | 278 |
M38 | L214F和V310F | 2 | 254 |
M39 | I25L和V310L | 2 | 208 |
M40 | E63P和V310L | 2 | 370 |
M41 | S155T和V310L | 2 | 451 |
M42 | T165V和V310L | 2 | 326 |
M43 | V374I和K428R | 2 | 135 |
M44 | V374I和K428H | 2 | 98 |
从表4可以看出,本申请所得到的双突变体具有较高的催化活性,其相对野生型,催化活性提高14-351%,优选为100-351%,最优选提高351%。
以上所述,仅是本申请的较佳实施例而已,并非是对本申请作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本申请技术方案内容,依据本申请的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本申请技术方案的保护范围。
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> 蔗糖磷酸化酶突变体及其应用
<130> TPF02083
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 504
<212> PRT
<213> 人工序列
<220>
<223> 人工序列说明:人工合成的序列
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Claims (10)
1.一种蔗糖磷酸化酶突变体,其中,包含一种或两种以上的基于参考序列的突变体,所述参考序列的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的蔗糖磷酸化酶突变体,其中,所述突变体的氨基酸序列包含对应于SEQ ID NO:1的I25、Y34、E63、S155、T165、L214、N280、V310、V374和K428中的至少一个位点的氨基酸突变;优选的,所述突变体的氨基酸序列包含对应于SEQ ID NO:1的I25、Y34、E63、S155、T165、L214、N280、V310、V374和K428中的一个或两个位点的氨基酸突变。
3.根据权利要求2所述的蔗糖磷酸化酶突变体,其中,所述突变体的氨基酸序列包含对应于SEQ ID NO:1的以下任意一种或两种以上氨基酸替换:
I25L、I25A、I25V、Y34F、Y34W、E63P、E63H、E63W、S155T、T165V、T165L、T165I、T165G、T165A、L214F、L214W、L214Y、N280Y、N280W、N280F、V310L、V310I、V310F、V374I、V374L、K428R、K428H。
4.根据权利要求3中任一项所述的蔗糖磷酸化酶突变体,其中,所述蔗糖磷酸化酶突变体选自下述任一的突变体:S155T、S155T/T165V、S155T/T165L、S155T/T165I、S155T/T165G、S155T/T165A、V310L、V310I、V310F、L214F/V310L、L214F/V310I、L214F/V310F、I25L/V310L、E63P/V310L、S155T/V310L、T165V/V310L;优选选自下述中的任一突变体:V310L、V310I、E63P/V310L、S155T/V310L、T165V/V310L。
5.一种核酸,其编码权利要求1-4中任一项所述的蔗糖磷酸化酶突变体。
6.一种表达载体,其包含权利要求5所述的核酸。
7.一种基因工程菌,其包含权利要求6所述的表达载体。
8.根据权利要求7所述的基因工程菌,其中,所述基因工程菌的宿主菌为大肠杆菌。
9.权利要求6中所述的表达载体、权利要求7或8中所述的基因工程菌在表达蔗糖磷酸化酶中的应用。
10.一种催化转移葡萄糖苷键的方法,其中,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。
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