WO2023184822A1 - 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 PDF

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WO2023184822A1
WO2023184822A1 PCT/CN2022/112444 CN2022112444W WO2023184822A1 WO 2023184822 A1 WO2023184822 A1 WO 2023184822A1 CN 2022112444 W CN2022112444 W CN 2022112444W WO 2023184822 A1 WO2023184822 A1 WO 2023184822A1
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nucleotide sequence
enzyme
expression vector
sialic acid
recombinant
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French (fr)
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赵弘
秦国富
于铁妹
潘俊锋
刘建
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深圳瑞德林生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7012—Compounds having a free or esterified carboxyl group attached, directly or through a carbon chain, to a carbon atom of the saccharide radical, e.g. glucuronic acid, neuraminic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/03—Oxo-acid-lyases (4.1.3)
    • C12Y401/03003—N-Acetylneuraminate lyase (4.1.3.3)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use

Definitions

  • the invention relates to the field of biochemistry, and in particular to an enzyme co-expression system and its application in the synthesis of sialic acid.
  • Sialic acid also known as bird's nest acid, is a type of neuraminic acid derivative, usually referring to the compound N-acetylneuraminic acid ⁇ Neu5Ac>. Its main food source is breast milk, but sialic acid is also found in cow's milk, eggs and cheese, but in lower amounts. The content of N-acetylneuraminic acid in bird's nest is high, which is also the main indicator of bird's nest grading standards. Sialic acid is widely used in health foods, biopharmaceuticals, and cosmetics production.
  • the first step is to use N-acetylglucosamine-2-epimerase (AGE) to convert glucosamine (GlcNAc) into N-acetylmannosamine (ManNAc), and the second part is to use N-acetylneuraminic acid Aldolase (NAL) converts ManNAc and pyruvate into N-acetylneuraminic acid (Neu5Ac).
  • AGE N-acetylglucosamine-2-epimerase
  • NAL N-acetylneuraminic acid Aldolase
  • the difficulty of the one-pot continuous reaction is that the isomerase activity is inhibited by the second-step reaction raw material (pyruvic acid).
  • the second-step cleavage reaction is a reversible reaction, and the equilibrium constant of the reaction to generate sialic acid in the forward direction is not high.
  • the present invention provides an enzyme co-expression system and its application in synthesizing sialic acid.
  • the dual-promoter co-expression system used in the present invention also adopts molecular biology means to increase expression, realizing the simultaneous overexpression of two enzymes and realizing the preparation of enzyme liquid through a one-time sterilization process, thereby improving the reaction of the enzyme catalyzed reaction. efficiency.
  • Bacteroides fragilis (Uniprot ID: Q5LEN7) N-acetylglucosamine-2-epimerase (AGE), which is not significantly inhibited by pyruvate, was used.
  • the invention provides N-acetylneuraminic acid aldolase mutants having:
  • the invention also provides recombinant expression vectors including:
  • nucleotide sequence encoding the same protein as the nucleotide sequence shown in (4), but different from the nucleotide sequence shown in (4) due to the degeneracy of the genetic code;
  • nucleotide sequence represented by (4) or (5) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (4) or (5), and being identical to the nucleotide sequence represented by (4) or (5).
  • nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (4), (5) or (6).
  • the above-mentioned recombinant expression vector further includes an AAGTATTAT sequence.
  • the present invention also provides host cells for transformation and/or transfection of the above recombinant expression vector.
  • the present invention also provides recombinase, which is directly or indirectly produced through the above-mentioned recombinant expression vector and/or the above-mentioned host cell.
  • the invention also provides a method for preparing the above-mentioned recombinant enzyme, which includes the following steps:
  • the present invention also provides a method for synthesizing sialic acid, which uses glucosamine and pyruvate as raw materials and is catalyzed by the above-mentioned recombinant enzyme and/or the recombinant enzyme prepared by the above-mentioned preparation method to prepare the above-mentioned sialic acid.
  • the molar ratio of glucosamine and pyruvate is (270-500): (450-600).
  • the recombinant enzyme in the above method includes: isomerase (AGE), and the added amount is 7430 to 22290 U.
  • AGE isomerase
  • the recombinant enzyme in the above method also includes: aldolase (NAL).
  • NAL aldolase
  • the aldolase is an N-acetylneuraminic acid aldolase mutant provided by the present invention. , which has:
  • the added amount of aldolase (NAL) in the above method is 7790 to 23370 U.
  • the present invention also provides the application of the above mutant, the above recombinant expression vector, the above host cell, the above recombinant enzyme and/or the recombinant enzyme prepared by the above preparation method in directly or indirectly preparing sialic acid and/or products containing sialic acid.
  • the above-mentioned products include: one or more of food, cosmetics, and drugs.
  • the invention provides N-acetylneuraminic acid aldolase mutants having:
  • the present invention uses molecular biology methods to effectively express the two enzymes required for the two-step reaction at the same time, and increases the expression level of the two co-expressed enzymes to achieve a more efficient reaction.
  • the specific embodiment is to use the more advantageous expression vector pRSFDuet-1, with dual promoters to express AGE and NAL respectively, still on the same vector.
  • the sequence AAGTATTAT which enhances recombinant expression, was used to increase the expression level of the second-step enzyme NAL. This is beneficial to improve the efficiency of a cascade reaction in which the first step is fast and the second step is slow. Over-co-expression enables a single sterilization process to meet the reaction process requirements, shortens the process route, and saves costs.
  • the enzyme used in the present invention is expressed as a protease for the first time and used in this synthesis reaction (the sequence is published in NCBI, but there is no literature report on the actual catalytic activity).
  • isomerase AGE is an excellent enzyme that is not significantly inhibited by pyruvate, as shown in SEQ ID NO: 12.
  • the enzyme in the present invention is a modified enzyme with three mutation points.
  • the forward catalytic activity of the modified enzyme has been significantly improved, by approximately 67% to 88%. . It is beneficial to improve the balance of the forward condensation reaction in the second step, making it easier for the reaction to proceed in the direction of synthesizing the target product, thereby increasing the yield.
  • Figure 1 shows the agarose gel identification diagram of the PCR amplified fragments; the left picture shows the PCR amplified DNA fragments [pRSFDuet-1-MCS1 - ], [age] and [nal(with AAGTATTAT)]; the right picture shows PCR amplified DNA fragment [pRSFDuet-1-age-MCS2 - ];
  • Figure 2 shows a schematic structural diagram of the vector connecting the target protein; specifically, it is a schematic structural diagram of the dual expression vector pRSFDuet-1-age-nal;
  • Figure 3 shows protein electrophoresis pattern: overexpression of AGE and NAL
  • Figure 4 shows the reaction liquid phase diagram of Example 5; wherein: in Figure 4A, the lower picture is the liquid phase when the reaction is 0 h, and the upper picture is the liquid phase when the reaction is 3h; Figure 4B is the data of the corresponding spectrum;
  • Figure 5A shows the nuclear magnetic spectrum (1HNMR) of the product, 1HNMR (400MHz) ⁇ 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);
  • Figure 5B shows a partial enlarged view;
  • Figure 6A shows the product nuclear magnetic spectrum (13C NMR), 13CNMR (101MHz,) ⁇ 174.80, 173.19, 95.22, 70.37, 70.09, 68.18, 66.65, 63.12, 52.01, 38.77, 22.03;
  • Figure 6B shows a partial enlargement;
  • Figure 7 shows the product mass spectrum
  • Figure 8 shows the first step of the enzymatic reaction; wherein: GlcNAc glucosamine, ManNAcN-acetylmannosamine, GlcNAc 2-epimeraseN-acetylglucosamine-2-epimerase (AGE);
  • Figure 9 shows the second step of the enzyme reaction; wherein: ManNAc N-acetylmannosamine, Pyruvate pyruvate, Neu5Ac N-acetylneuraminic acid, N-acetylneuraminic acid lyase N-acetylneuraminic acid aldolase (NAL);
  • Figure 10 shows the reaction liquid phase diagram of Example 6
  • Figure 11 shows the reaction liquid phase diagram of Example 7.
  • the invention discloses an enzyme co-expression system and its application in synthesizing sialic acid.
  • the raw materials and reagents used in the construction of the vector, the culture and expression of sialic acid synthase, the preparation of crude enzyme solution, the determination of enzyme activity, and the enzyme-catalyzed synthesis of sialic acid according to the present invention can all be purchased from the market.
  • the modified LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.
  • the target genes AGE and NAL were synthesized through gene synthesis.
  • the gene sequences are shown in Table 1.
  • the synthesized gene was ligated and inserted into the pET28a expression vector using the two sites of NdeI and XhoI restriction endonucleases to form the original plasmids pET28a-age and pET28a-nal.
  • the four PCR reactions are as follows: pRSFDuet-1 is used as the template and primers P01 and P02 are used to amplify to obtain the pRSFDuet-1-MCS1 - fragment.
  • primers P01 and P02 are used to amplify to obtain the pRSFDuet-1-MCS1 - fragment.
  • the age fragment was obtained.
  • pET28a-nal as a template and using primers P05 and P06 to amplify
  • the fragment of nal (with AAGTATTAT) was obtained.
  • the complete plasmid pRSFDuet-1-age-nal obtained in Example 1 was transformed into competent intestinal cells B121 (DE3) by heat shock. Carry out plate culture, and finally select single clones for modified LB liquid culture. When the cell OD reaches about 0.6, add about 1 mL of the remaining cell solution to preserve the seeds. Add 0.5 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) to the remaining cell solution to induce protein expression at 25°C for 10 hours. Finally, collect the cells by high-speed centrifugation. (6000rpm, 15min) to obtain wet cells. Take a small amount of cells and mix them evenly with Tris-HCl buffer (50mM, pH 8.0).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • culture the seed cells take out the strain cryopreservation tube prepared in Example 2(1) from the -80 degree refrigerator, and while keeping it frozen, take a small amount and apply it on a resistant plate, and culture it at 37°C overnight. On the morning of the next day, pick a single colony and insert it into 5 mL of modified LB culture medium (37°C) containing 50 ⁇ g/mL kanamycin for culture. When the cells grow to the logarithmic phase, inoculate into 250 mL containing the same antibiotic.
  • One unit of enzyme activity is defined as the amount of enzyme required to convert 1 ⁇ mol of substrate in one minute at pH 7.5 and a certain temperature.
  • the isomerase AGE activity measurement system includes: 100mM N-acetylglucosamine, 10mM MgCl 2 , 0.5mM ATP, 100mM Tris-HCl solution, quenched with 0.05% H 2 SO 4 after 15 minutes of reaction, calculated based on HPLC results Enzyme activity.
  • the activity measurement system for the aldolase NAL cleavage direction includes: 50mM N-acetylneuraminic acid, 100mM Tris-HCl solution, quenched with 0.05% H 2 SO 4 after 15 minutes of reaction, and the enzyme activity is calculated based on the HPLC results.
  • Thermo Fisher ultimate3000 high performance liquid chromatography was used to detect the substrate and product (0.05% H 2 SO 4 was used as the mobile phase, the flow rate was 0.5 mL/min, the column temperature was 65°C, and the absorption wavelength was 205 nm).
  • N-acetylneuraminic acid can reach up to 425mM/L (i.e. 131.4g/L), and the synthesis rate is 43.8g*L -1 *h -1 .
  • the conversion calculated from N-acetylglucosamine was 85%.
  • the reaction solution was centrifuged to remove the protein and purified, and N-acetylneuraminic acid was successfully isolated. And its structure was characterized, as shown in Figures 5 to 7.
  • N-acetylneuraminic acid can reach up to 178mM/L (i.e. 55g/L), and the synthesis rate is 18.3g*L -1 *h -1 , from N- The calculated conversion to acetylglucosamine was 59%.
  • the yield of N-acetylneuraminic acid can reach up to 139mM/L (i.e. 43g/L), and the synthesis rate is 14.3g*L -1 *h -1 , from N- The calculated conversion to acetylglucosamine was 52%.

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Abstract

提供了N-乙酰神经氨酸醛缩酶(NAL)突变体，将其和N-乙酰葡萄糖胺-2-差向异构酶(AGE)共表达的系统及其在合成唾液酸的应用。采用双启动子的共表达系统实现了两个酶同时过度表达，提高酶催化反应的反应效率。同时，对NAL的突变改造提高了正向反应生产唾液酸的合成效率。

Description

酶共表达系统及其在合成唾液酸的应用
本申请要求于2022年04月02日提交中国专利局、申请号为202210340323.X、发明名称为“酶共表达系统及其在合成唾液酸的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
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7430～22290U。
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7790～23370U。
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pRSFDuet-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4示实施例5反应液相图；其中：图4A中，下图为反应0h时的液相，上图为反应3h时的液相；图4B为相应谱图的数据；
图5A示产物核磁图谱(1HNMR)，1HNMR(400MHz)δ4.11–3.98(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),13CNMR(101MHz,)δ174.80，173.19，95.22,70.37,70.09,68.18,66.65,63.12,52.01,38.77,22.03；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，ManNAcN-乙酰甘露糖胺,GlcNAc 2-epimeraseN-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，Neu5Ac N-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
Figure PCTCN2022112444-appb-000001
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
Figure PCTCN2022112444-appb-000002
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(with AAGTATTAT)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1mL保种，剩余细胞液加入0.5mM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(6000rpm，15min)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(50mM，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5mL含50μg/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250mL含同样抗菌素的LB培养液中，最后再转入5L培养发酵罐里进行培养；当细胞OD～15加入0.5mM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(6000rpm，15min)获得湿细胞40～60g。取少量细胞与三羟甲基氨基甲烷盐酸(Tris-HCl)缓冲液(50mM，pH8.0)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(50mM Tris-HCl，pH＝7.5)，利用高压均质破碎细胞壁，高速离心后(12000rpm，10min)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pH7.5，一定温度下，一分钟转化1μmol的底物所需酶量。
其中异构酶AGE测活体系包括：100mM N-乙酰葡萄糖胺，10mM MgCl 2，0.5mM ATP，100mM Tris-HCl溶液，反应15min后用0.05％的H 2SO 4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：50mM N-乙酰神经氨酸，100mM  Tris-HCl溶液，反应15min后用0.05％的H 2SO 4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H 2SO 4为流动相，流速0.5mL/min，柱温为65℃，吸收波长为205nm)。
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779U/mL(35℃)，以及395U/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
Figure PCTCN2022112444-appb-000003
实施例5酶催化合成唾液酸
取实施例3获得30mL粗酶液加入到反应液中(N-乙酰葡萄糖胺500mM、MgCl 210mM、ATP 2.5mM、丙酮酸钠600mM，50mM的Tris-HCl缓冲液溶解)，于pH7.5、37℃下反应。反应3h后，取1.5mL反应液于12000rpm下离心2min后，将上清用0.22μm的滤膜过滤后，用HPLC检测N-乙酰神经氨酸的产量。反应0h及3h的液相图谱详见图4，其中N-乙酰神经氨酸的产量最高可达425mM/L(即131.4g/L)，合成速率为43.8g*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5～7所示。
实施例6酶催化合成唾液酸
取实施例3获得20mL粗酶液加入到反应液中(N-乙酰葡萄糖胺300mM、MgCl 210mM、ATP 2.5mM、丙酮酸钠500mM，50mM的Tris-HCl缓冲液溶解)，于pH 7.5、37℃下反应。反应3h后，取1.5mL反应液于12000rpm下离心2min后，将上清用0.22μm的滤膜过滤后，用HPLC检测N-乙酰神经氨酸的产量。反应3h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178mM/L(即55g/L)，合成速率为18.3g*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10mL粗酶液加入到反应液中(N-乙酰葡萄糖胺270mM、MgCl 2 10mM、ATP 2.5mM、丙酮酸钠450mM，50mM的Tris-HCl缓冲液溶解)，于pH 7.5、37℃下反应。反应3h后，取1.5mL反应液于12000rpm下离心2min后，将上清用0.22μm的滤膜过滤后，用HPLC检测N-乙酰神经氨酸的产量。反应3h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139mM/L(即43g/L)，合成速率为14.3g*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。
以上对本发明所提供的酶共表达系统及其在合成唾液酸的应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  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：
    (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。
  2. 重组表达载体，其特征在于，所述重组表达载体包括：
    (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。
  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所述的宿主细胞直接或间接制得。
  6. 如权利要求5所述重组酶的制备方法，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7或权利要求8所述的方法，其特征在于，所述重组酶的添加量为7430～23370U。
  10. 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、药物中的一种或多种。
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