CN114507658B - 酶共表达系统及其在合成唾液酸的应用 - Google Patents
酶共表达系统及其在合成唾液酸的应用 Download PDFInfo
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- CN114507658B CN114507658B CN202210340323.XA CN202210340323A CN114507658B CN 114507658 B CN114507658 B CN 114507658B CN 202210340323 A CN202210340323 A CN 202210340323A CN 114507658 B CN114507658 B CN 114507658B
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- recombinase
- sialic acid
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Abstract
本发明涉及生物化学领域，尤其涉及酶共表达系统及其在合成唾液酸的应用。本发明提供了N‑乙酰神经氨酸醛缩酶（NAL）突变体具有如SEQ ID NO:1所示的氨基酸序列；或在上述氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或与上述氨基酸序列同源性90%以上的序列。本发明采用的双启动子的共表达系统，同时采用了增加表达的分子生物学手段，实现了两个酶同时过度表达，实现一次破菌工艺制得酶液，从而提高酶催化反应的反应效率。同时，对镰孢梭菌(Clostridium scindens)NAL进行了改造，提高了正向反应生产唾液酸的合成效率。本发明中酶催化过程的产率，符合工业化生产的要求。
Description
技术领域
本发明涉及生物化学领域，尤其涉及酶共表达系统及其在合成唾液酸的应用。
背景技术
唾液酸，又称燕窝酸，是一类神经氨酸的衍生物，通常情况下指化合物N-乙酰神经氨酸（N-acetylneuraminic acid <Neu5Ac>）。其主要食物来源是母乳，在牛奶、鸡蛋和奶酪中也存在唾液酸，但含量较低。燕窝中的N-乙酰神经氨酸含量较高，也是燕窝分级标准的主要指标。唾液酸在健康食品，生物制药，以及化妆品生产方面都有广泛的应用。
由于唾液酸的天然产量比较低，开发研究出有效的合成路线一直是工业生产中的热点。其中根据N-乙酰神经氨酸在体内的合成路径，使用价格比较合理的原料，可以用两步酶法进行生物合成。第一步为用N-乙酰葡萄糖胺-2-差向异构酶（AGE）将葡萄糖胺（GlcNAc）转化成N-乙酰甘露糖胺（ManNAc），第二部分为用N-乙酰神经氨酸醛缩酶（NAL）将ManNAc和丙酮酸（Pyruvate）连接转化成N-乙酰神经氨酸（Neu5Ac）。一锅法连续反应的难点是异构酶活性受第二步反应原料（丙酮酸）的抑制，第二步裂解反应是个可逆反应，正向生成唾液酸的反应平衡常数不高。此类方法在一些文章中有公开发表，多数过程是采用首先制备两个酶液（AGE和NAL），然后通过液体酶或者固定酶的方法进行反应。然而目前此类报道中唾液酸的反应效率没有达到符合工业生产的要求。
另外，从酶催化效率方面分析，首先目前文献中使用的第一步反应酶（AGE）受丙酮酸抑制严重。另外一个关键问题是第二步反应的产率低。第二步反应中天然的N-乙酰神经氨酸醛缩酶（NAL）都存在催化平衡（可逆）反应的问题，正向反应生产唾液酸产率低，也大大限制了这一过程的工业化。
发明内容
有鉴于此，本发明提供了酶共表达系统及其在合成唾液酸的应用。本发明采用的双启动子的共表达系统，同时采用了增加表达的分子生物学手段，实现了两个酶的同时过度表达，实现一次破菌工艺制得酶液，从而提高酶催化反应的反应效率。经筛选后采用了受丙酮酸抑制不明显的脆弱拟杆菌（Bacteroides fragilis）(Uniprot ID: Q5LEN7) N-乙酰葡萄糖胺-2-差向异构酶（AGE）。同时，对第二步反应的镰孢梭菌(Clostridium scindens)（Uniprot ID:B0NDA1）NAL进行了改造，提高了正向反应生产唾液酸的合成效率。综合三个技术特点后，本专利中的酶催化过程的产率（85%），符合工业化生产的要求。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了N-乙酰神经氨酸醛缩酶突变体具有：
（1）、如SEQ ID NO:1所示的氨基酸序列；或
（2）、在如（1）所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或
（3）、与如（1）所示的氨基酸序列同源性90%以上的序列。
本发明还提供了重组表达载体包括：
（4）、如SEQ ID NO:2和SEQ ID NO:3所示的核苷酸序列；或
（5）、与（4）所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与（4）所示的核苷酸序列不同的核苷酸序列；或
（6）、与（4）或（5）所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与（4）或（5）所示的核苷酸序列功能相同或相似的核苷酸序列；或
（7）、与（4）、（5）或（6）所述核苷酸序列具有至少90%序列同源性的核苷酸序列。
在本发明的一些实施方案中，上述重组表达载体还包括AAGTATTAT序列。
本发明还提供了宿主细胞，转化和/或转染上述重组表达载体。
本发明还提供了重组酶，经上述重组表达载体和/或上述宿主细胞直接或间接制得。
本发明还提供了上述重组酶制备方法，包括以下步骤：
S1：获得上述重组表达载体；
S2：取上述重组表达载体转化、表达，获得上述重组酶。
本发明还提供了合成唾液酸的方法，以葡萄糖胺和丙酮酸为原料，经如上述重组酶和/或上述制备方法制得的重组酶催化，制得上述唾液酸。
在本发明的一些实施方案中，上述方法中，所述葡萄糖胺和所述丙酮酸的摩尔比为（270~500）：（450~600）。
在本发明的一些实施方案中，上述方法中所述重组酶包括：异构酶（AGE），其添加量为7430~22290 U。
在本发明的一些实施方案中，上述方法中所述重组酶还包括：醛缩酶（NAL），作为优选，所述醛缩酶为本发明提供的N-乙酰神经氨酸醛缩酶突变体，其具有：
（1）、如SEQ ID NO:1所示的氨基酸序列；或
（2）、在如（1）所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或
（3）、与如（1）所示的氨基酸序列同源性90%以上的序列。
在本发明的一些实施方案中，上述方法中所述醛缩酶（NAL）的添加量为7790~23370 U。
本发明还提供了上述突变体、上述重组表达载体、上述宿主细胞、上述重组酶和/或上述制备方法制得的重组酶在直接或间接制备唾液酸和/或含有唾液酸的产品中的应用，上述产品包括：食品、化妆品、药物中的一种或多种。
本发明提供了N-乙酰神经氨酸醛缩酶突变体具有：
（1）、如SEQ ID NO:1所示的氨基酸序列；或
（2）、在如（1）所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或
（3）、与如（1）所示的氨基酸序列同源性90%以上的序列。
本发明的有益效果包括：
（1）本发明运用分子生物学的手段，有效同时表达了两步反应需要的两个酶，并提高了两个共表达酶的表达量，实现了更高效的反应。具体体现为采用更具优势的表达载体RSFDuet-1，双启动子分别表达AGE和NAL，仍在同一载体上。另外，运用了增强重组表达的序列AAGTATTAT，提高了第二步酶NAL的表达水平。这有利于提高第一步快速，第二步慢速的级联反应的效率。过度共表达实现了一次破菌过程就能满足反应工艺要求，缩短了工艺路线，节约了成本。
（2）本发明中采用的酶，是首次被表达成蛋白酶，应用于这个合成反应中的（序列是NCBI中发表的，但是没有文献报道过实际催化活性）。其中异构酶（AGE）是受丙酮酸抑制作用不明显的优异酶，如SEQ ID NO:12所示。
（3）本发明中的酶与天然酶对比，醛缩酶（NAL）是带有三个突变点的改造酶，改造后的酶正向催化活性得到了显著提高，约提高了67%~88%。有利于提高第二步正向缩合反应的平衡，使反应更易于向合成目标产物的方向进行，从而提高了产率。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示PCR扩增后片段的琼脂糖凝胶鉴定图；其中，左图为PCR扩增DNA片段[pRSFDuet-1-MCS1-]、[age]和[nal (with AAGTATTAT)]；右图为PCR扩增DNA片段[pRSFDuet-1-age-MCS2-]；
图2示载体连接目标蛋白结构示意图；具体为双表达载体pRSFDuet-1-age-nal结构示意图；
图3示蛋白电泳图：AGE和NAL过度表达；
图4A中，下图为实施例5反应0 h时的液相，上图为实施例5反应3 h时的液相；图4B为实施例5相应谱图的数据；
图5A示产物核磁图谱（1H NMR），1H NMR (400 MHz) δ 4.11 – 3.98 (m,2H)3.97–3.86(m,1H),3.81(m,1H),3.76–3.67(m,1H),3.64–3.47(m,2H),2.29m,1H),2.02(s,3H),1.93–1.79(m,1H)；图5B示局部放大图；
图6A示产物核磁图谱（13C NMR）,13C NMR(101 MHz,) δ 174.80，173.19，95.22,70.37,70.09,68.18,66.65,63.12,52.01,38.77,22.03；图6B示局部放大图；
图7示产物质谱图；
图8示第一步酶反应；其中：GlcNAc葡萄糖胺，ManNAc N-乙酰甘露糖胺, GlcNAc2-epimerase N-乙酰葡萄糖胺-2-差向异构酶（AGE）；
图9示第二步酶反应；其中：ManNAc N-乙酰甘露糖胺, Pyruvate丙酮酸， Neu5AcN-乙酰神经氨酸, N-acetylneuraminic acid lyase N-乙酰神经氨酸醛缩酶（NAL）；
图10示实施例6的反应液相图；
图11示实施例7的反应液相图。
具体实施方式
本发明公开了酶共表达系统及其在合成唾液酸的应用。
应该理解，表述“……中的一种或多种”单独地包括每个在所述表述后叙述的物体以及所述叙述的物体中的两者或更多者的各种不同组合，除非从上下文和用法中另有理解。与三个或更多个叙述的物体相结合的表述“和/或”应该被理解为具有相同的含义，除非从上下文另有理解。
术语“包括”、“具有”或“含有”，包括其语法同义语的使用，通常应该被理解为开放性和非限制性的，例如不排除其他未叙述的要素或步骤，除非另有具体陈述或从上下文另有理解。
应该理解，只要本发明仍可操作，步骤的顺序或执行某些行动的顺序并不重要。此外，两个或更多个步骤或行动可以同时进行。
本文中的任何和所有实例或示例性语言如“例如”或“包括”的使用，仅仅打算更好地说明本发明，并且除非提出权利要求，否则不对本发明的范围构成限制。本说明书中的任何语言都不应解释为指示任何未要求保护的要素对于本发明的实践是必不可少的。
此外，用以界定本发明的数值范围与参数皆是约略的数值，此处已尽可能精确地呈现具体实施例中的相关数值。然而，任何数值本质上不可避免地含有因个别测试方法所致的标准偏差。因此，除非另有明确的说明，应当理解本公开所用的所有范围、数量、数值与百分比均经过“约”的修饰。在此处，“约”通常是指实际数值在一特定数值或范围的正负10%、5%、1%或0.5%之内。
本发明载体构建、唾液酸合成酶的培养表达、粗酶液的制备、酶活测定、酶催化合成唾液酸中，所用原料及试剂均可由市场购得。
改良LB培养基构成为：1% 胰蛋白胨、0.5% 酵母粉，1% NaCl，1% 磷酸氢二钾、1%磷酸氢二钾以及5%的甘油。
下面结合实施例，进一步阐述本发明：
实施例1 利用同源重组法构建质粒载体pRSFDuet-1-age-nal
（1）构建AGE，NAL目标基因
通过基因合成目标基因AGE和NAL，基因序列如表1。合成后的基因连接插入到pET28a表达载体，使用NdeI和XhoI限制性内切酶的两个位点，形成pET28a-age和pET28a-nal原始质粒。
表1 基因序列
其中，三个突变点，下划线和粗体表示突变后的基因序列。
（2）设计引物
通过PCR扩增，得到同源重组需要的4个质粒片段。引物名称和序列请见表2：
表2 引物名称和序列
四个PCR反应分别为，以pRSFDuet-1为模板，用引物P01，P02扩增，得到pRSFDuet-1-MCS1-片段。以pET28a-age为模板，用引物P03，P04扩增，得到age片段。以pET28a-nal为模板，用引物P05，P06扩增，得到nal (with AAGTATTAT)的片段。最后，通过反向PCR，以pRSFDuet-1-age为模板，用引物P07，P08扩增，得到pRSFDuet-1-age-MCS2-的片段。扩增后片段的琼脂糖凝胶鉴定图，请见图1。
（3）同源重组
使用同源重组酶，将片段pRSFDuet-1-MCS1-和age按摩尔比2：1的比例构建pRSFDuet-1-age。然后，以pRSFDuet-1-age为模板，扩增删除MCS2的Linearized vector，获得pRSFDuet-1-age-MCS2-后，使用同源重组酶，将片段pRSFDuet-1-age-MCS2-和nal (withAAGTATTAT)按DNA摩尔比2:1的比例构建pRSFDuet-1-age-nal，载体连接目标蛋白结构示意图，请见图2。
实施例2 唾液酸合成酶的培养表达
（1）质粒转化
将实施例1获得的完整质粒pRSFDuet-1-age-nal，经热击转化进感受态细胞大肠Bl21(DE3)。进行平板培养，最后挑单克隆进行改良LB液体培养。当细胞OD达到0.6左右，提出约1 mL保种，剩余细胞液加入0.5 mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG) 25 ℃诱导蛋白表达10小时，最后高速离心收集细胞(6000 rpm，15 min)获得湿细胞。取少量细胞与三羟甲基氨基甲烷盐酸(Tris-HCl)缓冲液(50 mM，pH 8.0)混合均匀，然后利用冻融法破碎细胞，高速离心后上清液利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定蛋白表达，蛋白表达结果请见图3。
（2）扩大培养
首先培养种子细胞：从-80度冰箱取出实施例2（1）制备的菌种冻存管，在保持冻结的状态下，取少量进行涂抗性平板，37度过夜培养。第二日早晨，挑取一个单克隆菌落，接入5 mL含50 μg/mL卡那霉素的改良LB培养液中(37 ℃)进行培养，当细胞生长至对数期后接种到250 mL含同样抗菌素的LB培养液中，最后再转入5 L培养发酵罐里进行培养；当细胞OD~15加入0.5 mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG) 25℃诱导蛋白表达12小时，最后高速离心收集细胞(6000 rpm，15 min)获得湿细胞40~60 g。取少量细胞与三羟甲基氨基甲烷盐酸(Tris-HCl)缓冲液(50 mM，pH 8.0)混合均匀，然后利用冻融法破碎细胞，高速离心后上清液利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定蛋白表达。
实施例3 粗酶液的制备
取实施例2中，经确认正确蛋白表达后的大肠杆菌细胞，重悬于适当体积的缓冲液后（50 mM Tris-HCl，pH=7.5），利用高压均质破碎细胞壁，高速离心后(12000 rpm，10 min)获得含两种酶的上清液，即为粗酶液。
实施例4 测定酶活
一个酶活力单位定义为：在pH 7.5，一定温度下，一分钟转化1 μmol的底物所需酶量。
其中异构酶AGE测活体系包括：100 mM N-乙酰葡萄糖胺，10 mM MgCl2，0.5 mMATP，100 mM Tris-HCl溶液，反应15 min后用0.05 %的H2SO4进行淬灭，根据HPLC结果计算酶活。
醛缩酶NAL裂解方向的测活体系包括：50 mM N-乙酰神经氨酸，100 mM Tris-HCl溶液，反应15 min后用0.05 %的H2SO4进行淬灭，根据HPLC结果计算酶活。采用赛默飞ultimate3000高效液相色谱进行底物及产物的检测（以0.05 %的H2SO4为流动相，流速0.5mL/min，柱温为65 ℃，吸收波长为205 nm）。
经过计算，由实施例3重组大肠杆菌破碎得到的粗酶液中，其异构酶AGE活力为743U/mL（37 ℃），醛缩酶NAL活力为779 U/mL（35 ℃），以及395 U/mL（25 ℃）。具体酶活计算过程中的原始数据见下表3：
表3 酶活计算过程
实施例5 酶催化合成唾液酸
取实施例3获得30 mL粗酶液加入到反应液中（N-乙酰葡萄糖胺500 mM、MgCl2 10mM、ATP 2.5 mM、丙酮酸钠600 mM，50 mM的Tris-HCl缓冲液溶解），于pH 7.5、37 ℃下反应。反应3 h后，取1.5 mL反应液于12000 rpm下离心2 min后，将上清用0.22 μm的滤膜过滤后，用HPLC检测N-乙酰神经氨酸的产量。反应0 h及3 h的液相图谱详见图4A、图4B，其中N-乙酰神经氨酸的产量最高可达425 mM/L（即131.4 g/L），合成速率为43.8 g*L-1*h-1，从N-乙酰葡萄糖胺计算的转化率为85 %。反应结束后，将反应液离心除去蛋白并进行纯化操作，成功分离了N-乙酰神经氨酸。并对其进行了结构表征，如图5A、图5B、图6A、图6B、图7所示。
实施例6 酶催化合成唾液酸
取实施例3获得20 mL粗酶液加入到反应液中（N-乙酰葡萄糖胺300 mM、MgCl2 10mM、ATP 2.5 mM、丙酮酸钠500 mM，50 mM的Tris-HCl缓冲液溶解），于pH 7.5、37 ℃下反应。反应3 h后，取1.5 mL反应液于12000 rpm下离心2 min后，将上清用0.22 μm的滤膜过滤后，用HPLC检测N-乙酰神经氨酸的产量。反应3 h的液相图谱详见图10，其中N-乙酰神经氨酸的产量最高可达178 mM/L（即55 g/L），合成速率为18.3 g*L-1*h-1，从N-乙酰葡萄糖胺计算的转化率为59 %。
实施例7 酶催化合成唾液酸
取实施例3获得10 mL粗酶液加入到反应液中（N-乙酰葡萄糖胺270 mM、MgCl2 10mM、ATP 2.5 mM、丙酮酸钠450 mM，50 mM的Tris-HCl缓冲液溶解），于pH 7.5、37 ℃下反应。反应3 h后，取1.5 mL反应液于12000 rpm下离心2 min后，将上清用0.22 μm的滤膜过滤后，用HPLC检测N-乙酰神经氨酸的产量。反应3 h的液相图谱详见图11，其中N-乙酰神经氨酸的产量最高可达139 mM/L（即43 g/L），合成速率为14.3 g*L-1*h-1，从N-乙酰葡萄糖胺计算的转化率为52 %。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 深圳瑞德林生物技术有限公司
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<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Asn Thr Thr Glu Tyr Leu Gln Thr Trp Ser Asp Ser Tyr Lys Asn
1 5 10 15
Asp Met Ile Ser Asn Ile Met Pro Phe Trp Met Lys Tyr Gly Trp Asp
20 25 30
Arg Lys Asn Gly Gly Val Tyr Thr Cys Val Asp Arg Asp Gly Gln Leu
35 40 45
Met Asp Thr Thr Lys Ser Val Trp Phe Gln Gly Arg Phe Ala Phe Thr
50 55 60
Cys Ser Tyr Ala Tyr Asn His Ile Glu Arg Asn Thr Glu Trp Leu Ala
65 70 75 80
Ala Ala Lys Ser Thr Leu Asp Phe Ile Glu Ala His Cys Phe Asp Thr
85 90 95
Asp Gly Arg Met Phe Phe Glu Val Thr Glu Thr Gly Leu Pro Ile Arg
100 105 110
Lys Arg Arg Tyr Val Phe Ser Glu Thr Phe Ala Ala Ile Ala Met Ser
115 120 125
Glu Tyr Ala Ile Ala Ser Gly Asp His Ser Tyr Ala Val Lys Ala Leu
130 135 140
Lys Leu Phe Asn Asp Arg His Phe Leu Ser Thr Pro Gly Ile Leu Glu
145 150 155 160
Pro Lys Tyr Cys Glu Arg Val Gln Met Lys Gly His Ser Ile Ile Met
165 170 175
Ile Leu Ile Asn Val Ala Ser Arg Ile Arg Ala Ala Ile Asn Asp Pro
180 185 190
Val Leu Asp Arg Gln Ile Glu Glu Ser Ile Ala Ile Leu His Lys Asp
195 200 205
Phe Met His Pro Glu Phe Lys Ala Leu Leu Glu Thr Val Gly Pro Asn
210 215 220
Gly Glu Phe Ile Asp Thr Asn Ala Thr Arg Thr Ile Asn Pro Gly His
225 230 235 240
Cys Ile Glu Thr Ser Trp Phe Ile Leu Glu Glu Ala Lys Asn Arg Asn
245 250 255
Trp Asp Lys Glu Met Val Asp Thr Ala Leu Thr Ile Leu Asp Trp Ser
260 265 270
Trp Glu Trp Gly Trp Asp Lys Glu Tyr Gly Gly Ile Ile Asn Phe Arg
275 280 285
Asp Cys Arg Asn Leu Pro Ser Gln Asp Tyr Ala His Asp Met Lys Phe
290 295 300
Trp Trp Pro Gln Thr Glu Ala Ile Ile Ala Thr Leu Tyr Ala Tyr Gln
305 310 315 320
Ala Thr Lys Asn Glu Lys Tyr Leu Ala Met His Lys Gln Ile Ser Asp
325 330 335
Trp Thr Tyr Ala His Phe Pro Asp Ala Glu Phe Gly Trp Tyr Gly Tyr
340 345 350
Leu His Arg Asp Gly Thr Ile Ser Gln Pro Ala Lys Gly Asn Leu Phe
355 360 365
Lys Gly Pro Phe His Ile Pro Arg Met Met Thr Lys Gly Tyr Ala Leu
370 375 380
Cys Gln Glu Leu Leu Ser Glu Lys
385 390
Claims (9)
1.N-乙酰神经氨酸醛缩酶突变体，其特征在于，所述突变体具有如SEQ ID NO:1所示的氨基酸序列。
2.重组表达载体，其特征在于，所述重组表达载体包括：
（1）、如SEQ ID NO:2和SEQ ID NO:3所示的核苷酸序列；或
（2）、与（1）所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与（1）所示的核苷酸序列不同的核苷酸序列。
3.如权利要求2所述的重组表达载体，其特征在于，所述重组表达载体还包括AAGTATTAT序列。
4.宿主细胞，其特征在于，转化和/或转染如权利要求2或权利要求3所述的重组表达载体。
5.重组酶，其特征在于，经如权利要求2或权利要求3所述的重组表达载体和/或如权利要求4所述的宿主细胞制得；
所述重组酶同时包含如SEQ ID NO:2所示核苷酸序列编码的异构酶和如SEQ ID NO:3所示核苷酸序列编码的醛缩酶。
6.如权利要求5所述的重组酶的制备方法，其特征在于，包括以下步骤：
S1：获得如权利要求2或权利要求3所述的重组表达载体；
S2：取所述重组表达载体转化、表达，获得所述重组酶。
7.合成唾液酸的方法，其特征在于，以葡萄糖胺和丙酮酸为原料，经如权利要求5所述的重组酶和/或如权利要求6所述制备方法制得的重组酶催化，制得所述唾液酸。
8.如权利要求7所述的方法，其特征在于，所述葡萄糖胺与丙酮酸的摩尔比为（270~500）：（450~600）。
9.如权利要求1所述的突变体、如权利要求2或权利要求3所述的重组表达载体、如权利要求4所述的宿主细胞、如权利要求5所述的重组酶和/或如权利要求6所述制备方法制得的重组酶在直接或间接制备唾液酸和/或含有唾液酸的产品中的应用；所述产品包括：食品、化妆品或药物。
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| PCT/CN2022/112444 WO2023184822A1 (zh) | 2022-04-02 | 2022-08-15 | 酶共表达系统及其在合成唾液酸的应用 |
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| EP1737969B2 (en) * | 2004-04-15 | 2018-09-12 | GlycoFi, Inc. | Production of galactosylated glycoproteins in lower eukaryotes |
| AU2012261528B2 (en) * | 2006-02-09 | 2014-04-24 | Centre National De La Recherche Scientifique | Synthesis of sialic acid in plants |
| CN101165190B (zh) * | 2006-10-17 | 2011-08-24 | 中国科学院上海生命科学研究院 | 固定化双酶法制备n-乙酰神经氨酸 |
| KR100888513B1 (ko) * | 2006-12-15 | 2009-03-12 | 주식회사 진켐 | 신규 ｎ―아세틸글루코사민―２―에피머라아제 및 이를이용한 ｃｍｐ―ｎ―아세틸뉴라민산의 제조방법 |
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| CN101979644A (zh) * | 2010-09-10 | 2011-02-23 | 扬州博生源生物科技有限公司 | 融合蛋白一步催化制备n-乙酰神经氨酸的方法 |
| CN102649965A (zh) * | 2011-02-23 | 2012-08-29 | 王革 | 用基因工程技术制备唾液酸 |
| CN103060300A (zh) * | 2011-10-20 | 2013-04-24 | 中国科学院微生物研究所 | N-乙酰神经氨酸醛缩酶及其编码基因与应用 |
| CN103060302A (zh) * | 2011-10-20 | 2013-04-24 | 中国科学院微生物研究所 | 痢疾志贺氏菌的n-乙酰神经氨酸醛缩酶及编码基因与应用 |
| CN103602627B (zh) * | 2013-11-25 | 2015-03-18 | 武汉中科光谷绿色生物技术有限公司 | 一种产n-乙酰神经氨酸大肠杆菌工程菌及其构建方法和应用 |
| CN104878035A (zh) * | 2015-04-20 | 2015-09-02 | 江南大学 | 一种产n-乙酰神经氨酸重组微生物的构建方法及应用 |
| CN108285886A (zh) * | 2018-01-30 | 2018-07-17 | 江南大学 | 重组枯草芽孢杆菌全细胞转化生产n-乙酰神经氨酸的方法 |
| CN109971696A (zh) * | 2019-03-20 | 2019-07-05 | 江南大学 | 一种全细胞转化法高产n-乙酰神经氨酸的重组菌及应用 |
| CN110129391A (zh) * | 2019-04-24 | 2019-08-16 | 湖州硕邦生物科技有限公司 | 一种采用固定双酶制备n-乙酰神经氨酸的方法 |
| CN113337495B (zh) * | 2021-06-03 | 2022-10-11 | 江南大学 | 一种提高唾液酸产量的方法与应用 |
| CN114507658B (zh) * | 2022-04-02 | 2022-07-05 | 深圳瑞德林生物技术有限公司 | 酶共表达系统及其在合成唾液酸的应用 |
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