WO2021082352A1 - 一种合成乳-n-二糖的方法 - Google Patents

一种合成乳-n-二糖的方法 Download PDF

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WO2021082352A1
WO2021082352A1 PCT/CN2020/083230 CN2020083230W WO2021082352A1 WO 2021082352 A1 WO2021082352 A1 WO 2021082352A1 CN 2020083230 W CN2020083230 W CN 2020083230W WO 2021082352 A1 WO2021082352 A1 WO 2021082352A1
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lacto
disaccharide
concentration
synthesizing
atp
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French (fr)
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方诩
杜志强
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烟台华康荣赞生物科技有限公司
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Priority to AU2020327339A priority Critical patent/AU2020327339B2/en
Priority to US17/271,054 priority patent/US20220243239A1/en
Priority to EP20828248.3A priority patent/EP3842539A4/en
Publication of WO2021082352A1 publication Critical patent/WO2021082352A1/zh

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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01211—1,3-Beta-galactosyl-N-acetylhexosamine phosphorylase (2.4.1.211)
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01006—Galactokinase (2.7.1.6)
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    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02001—Acetate kinase (2.7.2.1)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0114—Lacto-N-biosidase (3.2.1.140)

Definitions

  • the invention belongs to the technical field of bioengineering and oligosaccharide synthesis, and specifically relates to a method for synthesizing lacto-N-disaccharide.
  • breast milk as the only material and energy link between the newborn and the mother after birth, is considered the gold standard of infant nutrition.
  • breast milk In addition to providing nutrients such as protein, fat and carbohydrates necessary for infant growth, breast milk also provides active components such as enzymes, antibodies, growth factors and oligosaccharides that can promote infant health and development.
  • active components such as enzymes, antibodies, growth factors and oligosaccharides that can promote infant health and development.
  • HMOs Human Milk Oligosaccharides
  • HMOs Human Milk Oligosaccharides
  • HMOs can not only act as prebiotics for intestinal probiotics, regulate and promote the maturation of the intestinal immune barrier of infants, but also act as bait molecules for pathogens, have antibacterial and antifungal effects, and protect infants and young children from pathogens. infection. Therefore, research on HMOs is of great significance to the health of infants and young children, and by adding HMOs to the milk powder to simulate breast milk components, it is particularly important for some infants who cannot breastfeed and the nutrition and health protection of infants after lactation.
  • Patent name "A fucosyltransferase and its genetically engineered bacteria and application” (CN201611147477.8)
  • Patent name "Production of human milk oligosaccharides in a microbial host with modified input/output” (CN201680052611.8)
  • Patent name "A fucosyltransferase and its application” (CN201611147478.2)
  • Patent name "Method for preparing N-acetyllactosamine-containing tetrasaccharide lactose-N-neotetraose (LNNT)" (CN201510751641.5).
  • the present invention utilizes a multi-enzyme catalytic system with good biological safety and wide application, and by introducing an ATP regeneration cycle system in the multi-enzyme reaction system, the synthesis of lacto-N-disaccharide and the utilization of substrate .
  • the invention provides a new way for the synthesis of lacto-N-disaccharide, which lays a foundation for the large-scale industrial production of lacto-N-disaccharide, and has important economic value and social benefits.
  • Gal galactose
  • GlcNAc acetylglucosamine
  • Lac lactose
  • GalK galactose kinase
  • LNBP lacto-N-disaccharide phospholytic enzyme
  • acetyl phosphate and acetate kinase are also added to regenerate ATP in situ.
  • the method also includes: separating the product milk-N-disaccharide and the ATP and ADP present in the system.
  • the synthesis principle of the present invention is as follows: the present invention constructs a multi-enzyme catalytic system using galactose as a substrate to synthesize lacto-N-disaccharide.
  • the reaction substrate, galactose is catalyzed by galactose kinase to produce galactose-1-phosphate, and ATP is degraded into ADP; further galactose-1-phosphate is produced under the action of lacto-N-disaccharide phospholytic enzyme (LNBP) Lacto-N-disaccharide.
  • the ADP obtained above combines with acetyl phosphate to be regenerated into ATP to realize the regeneration of ATP. Only low concentration of ATP needs to be put into the reactant of the present invention, which greatly reduces the reaction cost and improves the substrate conversion efficiency, avoids the effect of high concentration of ATP to inhibit galactoskinase, so that the substrate can be completely converted and the reaction time is shortened.
  • the second aspect of the present invention provides the synthetic lacto-N-disaccharide prepared by the above-mentioned synthesis method.
  • the synthesis of all oligosaccharides or polysaccharides containing and/or using lacto-N-disaccharide as the bone structure is also within the protection scope of the present invention.
  • galactose is used as a raw material substrate, and the oligosaccharide structure synthesized by two-step catalysis includes lacto-N-disaccharide.
  • all oligosaccharides or polysaccharides with lacto-N-disaccharide as bone structure can be synthesized.
  • the preparation cost of the product is low and the practical value is high.
  • the invention utilizes a multi-enzyme catalytic system with good biological safety and wide application, and by introducing an ATP regeneration cycle system into the multi-enzyme reaction system, a simplified and economical coupling of enzymatic synthesis of lacto-N-disaccharide and ATP regeneration is established.
  • the system improves the synthesis of lacto-N-disaccharide and the utilization rate of the substrate.
  • the present invention provides a new way for the synthesis of lacto-N-disaccharide, which lays a foundation for the large-scale industrial production of lacto-N-disaccharide and its analogs, and has important economic value and social benefits.
  • the raw materials used in the present invention are easy to obtain, the synthesis method is efficient and gentle, simple and convenient, low cost, environmentally friendly, suitable for industrial production, and has good practical application value.
  • Figure 1 is a schematic diagram of the present invention using a galactose substrate to catalyze the production of lacto-N-disaccharide in two steps.
  • Fig. 2 is a comparison diagram of lacto-N-disaccharide synthesis at an initial amount of 5mM ATP after the introduction of ATP regeneration cycle in Example 2 of the present invention.
  • Fig. 3 is a comparison diagram of lacto-N-disaccharide synthesis at an initial amount of 7.5 mM ATP after the introduction of ATP regeneration cycle in Example 3 of the present invention.
  • a method for synthesizing lacto-N-disaccharide comprising:
  • galactoskinase and lacto-N-biose phospholytic enzyme are added to prepare lacto-N-biose;
  • acetyl phosphate and acetate kinase are also added to regenerate ATP in situ.
  • the invention aims at consuming one molecule of ATP energy in the galactoskinase catalysis step, and is constructed but not limited to realize ATP energy regeneration in acetyl phosphate with acetate kinase.
  • the experimental results of the present invention prove that by introducing the energy regeneration cycle, the ATP regeneration cycle and the full conversion of the substrate can be realized, thereby effectively improving the synthesis efficiency of lacto-N-disaccharide.
  • the method further includes: separating the product lacto-N-disaccharide and ATP and ADP present in the reaction system.
  • the source of the enzymes used is not particularly limited. It can be extracted from natural bacteria, yeast or fungi and other microorganisms; it can be produced by genetically engineered bacteria through genetic recombination; It can be extracted from natural plant tissues or animal tissues.
  • the product form of the enzyme is also not particularly limited. It can be solid, powder, liquid, or an immobilized enzyme fixed on a carrier by physical or chemical methods; the enzyme can be a commercially available product or a self-made product by an enterprise or individual. .
  • the galactoskinase, lacto-N-biose phospholytic enzyme (LNBP) and acetate kinase are all produced by genetically engineered bacteria by genetic recombination.
  • the production method includes: separately cloning expression vectors derived from galactoskinase, lacto-N-biose phospholytic enzyme and acetate kinase; obtaining the target enzyme protein by culturing and inducing the host of the corresponding expression vector.
  • the expression vector is any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid, an F cosmid, a phage, or an artificial chromosome;
  • the viral vector may include Adenovirus vectors, retrovirus vectors or adeno-associated virus vectors
  • artificial chromosomes include bacterial artificial chromosomes (BAC), phage P1-derived vectors (PAC), yeast artificial chromosomes (YAC) or mammalian artificial chromosomes (MAC); further preferred Is a plasmid; more preferably a pET-28a plasmid;
  • the host includes but is not limited to bacteria, fungi and eukaryotic cells, and is further selected from Escherichia coli, Bacillus, Bacillus subtilis, Saccharomyces cerevisiae, Trichoderma reesei, and oxalic acid Penicillium; more preferably Escherichia coli BL21 (DE3).
  • the production method includes: respectively cloning the expression plasmids pET28a-galk, pET28a-Lnbp and pET28a-ack derived from galactoskinase, LNBP and acetate kinase; by culturing and inducing the corresponding expression plasmids
  • the BL21(DE3) strain is purified (preferably purified by a nickel column) to obtain the target enzyme protein.
  • the galactoskinase source strains include but are not limited to Escherichia coli; the LNBP source strains include but are not limited to Bifidobacterium; the ACK source strains include but are not limited to Escherichia coli.
  • the amino acid sequence of the galactoskinase is shown in SEQ ID No. 1;
  • amino acid sequence of the LNBP is shown in SEQ ID No. 2;
  • amino acid sequence of the acetate kinase is shown in SEQ ID No. 3.
  • the reaction system further contains ATP, and further, the ATP concentration is 5-15 mM (preferably 7.5 mM).
  • the reaction system also contains magnesium ions (MgCl 2 is preferably used) and Tris-HCl buffer.
  • the MgCl 2 concentration is 1-10 mM (preferably 3 mM); the concentration of the Tris-HCl buffer is 10-200 mM (preferably 100 mM).
  • the reaction temperature of the reaction system is 25-45°C
  • the reaction pH is 5.8-7.5.
  • the concentration of the galactose and acetylglucosamine substrates are both 10-20 mM.
  • the concentration of the galactoskinase is 1-10 U/mL.
  • the LNBP enzyme concentration is 100-300 U/mL.
  • the concentration of acetate kinase required for in-situ regeneration of ATP is 1-10 U/mL, and the concentration of acetyl phosphate is 2.5-5 mM.
  • lacto-N-disaccharide synthesized by the above-mentioned synthesis method is provided.
  • Example 1 Using galactose as a substrate to synthesize lacto-N-disaccharide
  • reaction detection profile (HPLC) comparison results verify the synthesis of lacto-N-disaccharide.
  • Example 3 Add 10 mM galactose, 5 mM ATP, 5 mM acetyl phosphate, 10 mM GlcNAc, 3 mM MgCl 2 , 100 mM Tris-HCl, 5 U Galk, 3 U ACK, 168 U LNBP to a 1 mL reaction system.
  • the initial concentration of ATP in the Control control is 10mM, and the ATP cyclic reaction is not introduced into the reaction system.
  • the reaction system is boiled for 5min, centrifuged to take the supernatant, after filtering the membrane, Biorad-HPX column, RID Detect the corresponding substrate and product.
  • LNB synthesis increased by 1.49 times compared to the control group (no ATP added).

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Abstract

提供一种合成乳-N-二糖的方法，包括在含有半乳糖、乙酰葡萄糖胺、乳糖为底物的反应体系中，加入半乳糖激酶、乳-N-二糖磷解酶制备乳-N-二糖，同时还在反应体系中加入乙酰磷酸、乙酸激酶进行ATP的原位再生，提高乳-N-二糖合成及底物利用率。

Description

一种合成乳-N-二糖的方法 技术领域
本发明属于生物工程及寡糖合成技术领域，具体涉及一种合成乳-N-二糖的方法。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
母乳作为新生儿出生后唯一与母体联系的物质和能量纽带，被认为是婴儿营养的黄金标准。除了提供婴儿生长所必需的蛋白质、脂肪和碳水化合物等营养物质之外，母乳还提供了能够促进婴儿健康和发育的酶、抗体、生长因子和寡糖等活性组分。进一步研究发现，与牛羊乳相比，最大的不同在于母乳中含有丰富的功能性人乳寡糖及其衍生物(Human Milk Oligosaccharides，HMOs)，其含量高达12-24g/L，仅次于乳糖和脂质成分，为母乳中第三大营养物质。HMOs对于人体，特别是针对婴幼儿的消化系统、肠道健康及免疫系统完善有特殊的功效。近期研究发现，HMOs不仅可以作为肠道益生菌的益生元，调节和促进婴儿肠道免疫屏障的成熟，而且可以作为病原体的诱饵分子、具有抑制细菌和抗真菌作用，保护婴幼儿免受病原体的感染。因此针对HMOs的研究对于婴幼儿健康有着重要意义，并且通过在奶粉中添加HMOs模拟母乳成分，对于一些无法进行母乳喂养的婴儿以及哺乳期后婴儿的营养健康保障尤为重要。
然而，有限的来源及难以利用化学方法规模化合成，制约了其生物学功能的研究和广泛应用。为了突破此瓶颈，因其来源途径有限，亟需建立简洁、高效、经济地大量获得核心结构乳-N-二糖(LNB，Galβ1-3GlcNAc，化学结构式如下)的合成途径。因此，生物法合成乳-N-二糖(LNB)的技术成本低，且合成过程简单，分离方法简便。
Figure PCTCN2020083230-appb-000001
目前已有多项专利集中在利用大肠杆菌中生产人乳寡糖生物合成技术，如：
专利名：“一种岩藻糖基转移酶及其基因工程菌和应用”(CN201611147477.8)
专利名：“在具有经改造的输入/输出的微生物宿主中人乳寡糖的产生”(CN201680052611.8)
专利名：“一种岩藻糖基转移酶及其应用”(CN201611147478.2)
专利名：“用于制备含N-乙酰乳糖胺的四糖乳糖-N-新四糖(LNNT)的方法”(CN201510751641.5)。
上述专利中均采用以大肠杆菌作为宿主，异源生产人乳寡糖的技术。但是大肠杆菌内毒素的去除是大规模工业生产中的一个重大挑战。内毒素是革兰氏阴性细菌细胞壁中的一种成分，也称为脂多糖，是一种对人体能够产生毒性物质。然而，对于尤其是作为婴儿奶粉的添加剂人乳寡糖来说，食品安全性显然是非常重要。因此寻找安全性好、产量稳定、生产效率高从而使之更适合规模工业化生产的人乳寡糖的合成方法尤为重要。
发明内容
针对上述现有技术的不足，本发明利用生物安全性好且应用广泛的多酶催化体系，通过在多酶反应体系中引入ATP再生循环系统，提高乳-N-二糖合成及底物利用率。本发明提供了一个新的乳-N-二糖合成途径，为乳-N-二糖的大规模工业生产奠定了基础，具有重要的经济价值和社会效益。
本发明的第一个方面，提供一种合成乳-N-二糖的方法，所述方法包括：
在含有半乳糖(Gal)、乙酰葡萄糖胺(GlcNAc)、乳糖(Lac)为底物的反应体系中，加入半乳糖激酶(GalK)、乳-N-二糖磷解酶(LNBP)制备乳-N-二糖；
在上述反应体系中，还加入乙酰磷酸、乙酸激酶(ACK)进行ATP的原位再生。
进一步的，所述方法还包括：分离产物乳-N-二糖及体系中存在的ATP、ADP。
本发明的合成原理如下：本发明构建以半乳糖为底物的多酶催化体系合成乳-N-二糖。反应底物半乳糖在半乳糖激酶催化下生成半乳糖-1-磷酸，同时ATP降解为ADP；进一步的半乳糖-1-磷酸在乳-N-二糖磷解酶(LNBP)作用下，生成乳-N-二糖。在乙酸激酶(ACK)作用下，上述得到的ADP结合乙酰磷酸再生为ATP，实现ATP的再生。本发明反应物中只需要投入低浓度的ATP，大大降低反应成本及提高了底物转化效率，避免了高浓度ATP抑制半乳糖激酶的作用，从而使得底物实现完全转化，且反应时间缩短。
本发明的第二个方面，提供上述合成方法制备合成的乳-N-二糖。另外，基于本发明的合成方法，合成所有含有和/或以乳-N-二糖为骨骼构造的寡糖或多糖亦在本发明的保护范围之内。
本发明有益技术效果：
本发明以半乳糖为原料底物，通过两步催化所合成的寡糖结构包括乳-N-二糖，同时，还可以合成所有以乳-N-二糖为骨骼构造的寡糖或多糖，产品制备成本低、实用价值高。本发明利用生物安全性好且应用广泛的多酶催化体系，通过在多酶反应体系中引入ATP再生循环系统，建立了简化和经济的酶法合成乳-N-二糖与ATP再生的偶联体系，提高乳-N-二糖合成及底物利用率。
综上，本发明提供了一个新的乳-N-二糖合成途径，为乳-N-二糖及其类似物的大规模工业生产奠定了基础，具有重要的经济价值和社会效益。同时本发明所用原料易得，合成方法高效温和、简便易行，成本低、绿色环保、适合产业化生产，具有良好的实际应用之价值。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示 意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为本发明以半乳糖底物两步催化生成乳-N-二糖的示意图。
图2为本发明实施例2中引入ATP再生循环后，5mM ATP起始量下乳-N-二糖合成比较图。
图3为本发明实施例3中引入ATP再生循环后，7.5mM ATP起始量下乳-N-二糖合成比较图。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列具体实施方式中如果未注明具体条件的实验方法，通常按照本领域技术内的分子生物学的常规方法和条件，这种技术和条件在文献中有完整解释。参见例如Sambrook等人，《分子克隆：实验手册》中所述的技术和条件，或按照制造厂商所建议的条件。
如前所述，目前人乳寡糖难以使用化学方法规模化合成，而采用生物技术异源生产人乳寡糖，除普遍生产效率不高之外，又易产生食品安全性问题。
有鉴于此，本发明的一个具体实施方式中，提供一种合成乳-N-二糖的方法，所述方法包括：
在含有半乳糖、乙酰葡萄糖胺、乳糖为底物的反应体系中，加入半乳糖激酶、 乳-N-二糖磷解酶制备乳-N-二糖；
在上述反应体系中，还加入乙酰磷酸、乙酸激酶进行ATP的原位再生。本发明针对半乳糖激酶催化步骤中消耗一分子ATP能量，构建以但不局限与乙酸激酶在乙酰磷酸实现ATP能量再生。本发明实验结果证明，通过引入能量再生循环，能够实现ATP再生循环以及底物的全转化，从而有效提高乳-N-二糖的合成效率。
本发明的又一具体实施方式中，所述方法还包括：分离产物乳-N-二糖及反应体系中存在的ATP、ADP。
本发明中，所用到的酶类的来源没有特别的限制，可以是自然界的细菌、也可以是酵母或真菌等微生物菌体中提取而得；可以是通过基因重组由基因工程菌生产而得；可以是由自然界的植物组织或动物组织中提取而得。酶的产品形态也无特别限制，可以是固体、粉末、液体、或通过物理或化学的方法固定在载体上的固定化酶；酶可以是市售的商品，也可以是企业或个人自制的产品。
本发明的又一具体实施方式中，所述半乳糖激酶、乳-N-二糖磷解酶(LNBP)和乙酸激酶均采用基因重组由基因工程菌生产获得。
具体的，所述生产方法包括：分别克隆来源于半乳糖激酶、乳-N-二糖磷解酶及乙酸激酶表达载体；通过培养、诱导相应表达载体的宿主，获得目标酶蛋白。
本发明的又一具体实施方式中，所述表达载体为病毒载体、质粒、噬菌体、噬菌粒、黏粒、F黏粒、噬菌体或人工染色体中的任意一种或多种；病毒载体可包括腺病毒载体、逆转录病毒载体或腺伴随病毒载体，人工染色体包括细菌人工染色体(BAC)、噬菌体P1衍生的载体(PAC)、酵母人工染色体(YAC)或哺乳动物人工染色体(MAC)；进一步优选为质粒；更进一步优选为pET-28a质粒；
本发明的又一具体实施方式中，所述宿主包括但不限于细菌、真菌和真核细胞，进一步选自埃希氏大肠杆菌、芽孢杆菌、枯草芽孢杆菌、酿酒酵母、里氏木霉和草酸青霉；更进一步优选为大肠杆菌BL21(DE3)。
本发明的又一具体实施方式中，所述生产方法包括：分别克隆来源于半乳糖 激酶、LNBP及乙酸激酶表达质粒pET28a-galk，pET28a-Lnbp和pET28a-ack；通过培养、诱导相应表达质粒的BL21(DE3)菌株，通过纯化(优选采用镍柱纯化)，获得目标酶蛋白。
其中，所述半乳糖激酶来源菌种包括但不限于大肠杆菌；LNBP来源菌种包括但不限于双歧杆菌；ACK来源菌种包括但不限于大肠杆菌。
本发明的又一具体实施方式中，
所述半乳糖激酶的氨基酸序列如SEQ ID No.1所示；
所述LNBP的氨基酸序列如SEQ ID No.2所示；
所述乙酸激酶的氨基酸序列如SEQ ID No.3所示。
本发明的又一具体实施方式中，反应体系中还含有ATP，进一步所述ATP浓度为5-15mM(优选为7.5mM)。
反应体系中还含有镁离子(优选使用MgCl 2)和Tris-HCl缓冲液。
本发明的又一具体实施方式中，所述MgCl 2浓度为1-10mM(优选为3mM)；；所述Tris-HCl缓冲液的浓度为10-200mM(优选为100mM)。
本发明的又一具体实施方式中，反应体系的反应温度为25-45℃，反应pH值为5.8-7.5。
本发明的又一具体实施方式中，所述半乳糖、乙酰葡萄糖胺底物浓度均为10-20mM。
本发明的又一具体实施方式中，所述半乳糖激酶浓度为1-10U/mL。
本发明的又一具体实施方式中，所述LNBP酶浓度为100-300U/mL。
本发明的又一具体实施方式中，ATP原位再生所需的乙酸激酶浓度为1-10U/mL，乙酰磷酸浓度为2.5-5mM。
本发明的又一具体实施方式中，提供上述合成方法合成的乳-N-二糖。另外，基于本发明的合成方法，合成所有含有以乳-N-二糖为骨骼构造的寡糖或多糖亦在本发明的保护范围之内。
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件进行。
实施例1：以半乳糖为底物合成乳-N-二糖
在1mL反应体系中加入10mM半乳糖，10mM ATP，10mM GlcNAc，3mM MgCl 2，100mM Tris-HCl，5U Galk，168U乳-N-二糖磷解酶。如表1所示，其中Control为不添加乳-N-二糖磷解酶(LNBP)的对照组，37℃反应12h后，反应体系煮沸5min，离心取上清，过滤膜后，Biorad-HPX柱，RID检测相应底物及产物，反应检测图谱(HPLC)比较结果验证合成了乳-N-二糖。
表1 以半乳糖底物两步催化生成乳-N-二糖的反应表
Figure PCTCN2020083230-appb-000002
实施例2：引入ATP再生循环合成乳-N-二糖
在1mL反应体系中加入10mM半乳糖，7.5mM ATP，2.5mM乙酰磷酸，10mM GlcNAc，3mM MgCl 2，100mM Tris-HCl，5U Galk，3U ACK，168U LNBP。如表2所示，Control对照中ATP初始浓度为10mM，反应体系中不引入ATP循环反应，37℃反应12h后，反应体系煮沸5min，离心取上清，过滤膜后，Biorad-HPX柱，RID检测相应底物及产物。如图2所示，ATP-1反应条件下，LNB合成相比于对照组(未加入ATP)提高1.60倍。
实施例3：在1mL反应体系中加入10mM半乳糖，5mM ATP，5mM乙酰磷酸，10mM GlcNAc，3mM MgCl 2，100mM Tris-HCl，5U Galk，3U ACK，168U LNBP。如表2所示，Control对照中ATP初始浓度为10mM，反应体系中不引入ATP循环反应，37℃反应12h后，反应体系煮沸5min，离心取上清，过 滤膜后，Biorad-HPX柱，RID检测相应底物及产物。如图3所示，ATP-2反应条件下，LNB合成相比于对照组(未加入ATP)提高1.49倍。
表2 引入ATP再生循环后以半乳糖底物两步催化生成乳-N-二糖的反应表
Figure PCTCN2020083230-appb-000003
应注意的是，以上实例仅用于说明本发明的技术方案而非对其进行限制。尽管参照所给出的实例对本发明进行了详细说明，但是本领域的普通技术人员可根据需要对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。
Figure PCTCN2020083230-appb-000004
Figure PCTCN2020083230-appb-000005
Figure PCTCN2020083230-appb-000006
Figure PCTCN2020083230-appb-000007
Figure PCTCN2020083230-appb-000008
Figure PCTCN2020083230-appb-000009
Figure PCTCN2020083230-appb-000010
Figure PCTCN2020083230-appb-000011
Figure PCTCN2020083230-appb-000012
Figure PCTCN2020083230-appb-000013
Figure PCTCN2020083230-appb-000014

Claims (12)

  1. 一种合成乳-N-二糖的方法，其特征在于，所述方法包括：
    在含有半乳糖、乙酰葡萄糖胺、乳糖为底物的反应体系中，加入半乳糖激酶、乳-N-二糖磷解酶制备乳-N-二糖；
    在上述反应体系中，还加入乙酰磷酸、乙酸激酶进行ATP的原位再生。
  2. 如权利要求1所述的合成乳-N-二糖的方法，其特征在于，所述方法还包括：分离产物乳-N-二糖及反应体系中存在的ATP、ADP。
  3. 如权利要求1所述的合成乳-N-二糖的方法，其特征在于，所述半乳糖激酶、乳-N-二糖磷解酶和乙酸激酶均采用基因重组由基因工程菌生产获得。
  4. 如权利要求3所述的合成乳-N-二糖的方法，其特征在于，生产方法包括：分别克隆来源于半乳糖激酶、乳-N-二糖磷解酶及乙酸激酶表达载体；通过培养、诱导相应表达载体的宿主，获得目标酶蛋白。
  5. 如权利要求1所述的合成乳-N-二糖的方法，其特征在于，
    所述半乳糖激酶的氨基酸序列如SEQ ID No.1所示；
    所述乳-N-二糖磷解酶的氨基酸序列如SEQ ID No.2所示；
    所述乙酸激酶的氨基酸序列如SEQ ID No.3所示。
  6. 如权利要求1所述的合成乳-N-二糖的方法，其特征在于，反应体系中还含有ATP；所述ATP浓度为5-15mM。
  7. 如权利要求6所述的合成乳-N-二糖的方法，其特征在于，所述ATP浓度为7.5mM。
  8. 如权利要求1所述的合成乳-N-二糖的方法，其特征在于，反应体系中还含有MgCl 2和Tris-HCl缓冲液；
    所述MgCl 2浓度为1-10mM；所述Tris-HCl缓冲液的浓度为10-200mM。
  9. 如权利要求8所述的合成乳-N-二糖的方法，其特征在于，所述MgCl 2浓度为3mM；所述Tris-HCl缓冲液的浓度为100mM。
  10. 如权利要求1所述的合成乳-N-二糖的方法，其特征在于，反应体系的 反应温度为25-45℃，反应pH值为5.8-7.5。
  11. 如权利要求1所述的合成乳-N-二糖的方法，其特征在于，所述半乳糖、乙酰葡萄糖胺底物浓度均为10-20mM；
    所述半乳糖激酶浓度为1-10U/mL；
    所述LNBP酶浓度为100-300U/mL；
    乙酸激酶浓度为1-10U/mL；
    乙酰磷酸浓度为2.5-5mM。
  12. 权利要求1-11任一项所述合成方法合成的乳-N-二糖。
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