WO2020258110A1 - Recombinant nad synthetase and gene and use thereof - Google Patents

Recombinant nad synthetase and gene and use thereof Download PDF

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WO2020258110A1
WO2020258110A1 PCT/CN2019/093139 CN2019093139W WO2020258110A1 WO 2020258110 A1 WO2020258110 A1 WO 2020258110A1 CN 2019093139 W CN2019093139 W CN 2019093139W WO 2020258110 A1 WO2020258110 A1 WO 2020258110A1
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recombinant
nad synthetase
nad
nicotinamide
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张章
张琦
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邦泰合盛生物科技（深圳）有限公司
邦泰生物工程(深圳)有限公司
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Priority to PCT/CN2019/093139 priority Critical patent/WO2020258110A1/en
Priority to CN201980044146.7A priority patent/CN112437811B/en
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Abstract

Provided are a recombinant NAD synthetase and a gene and use thereof. Said enzyme comprises a nicotinamide mononucleotide adenosyltransferase domain from Haemophilus influenzae and a nicotinamide ribokinase domain from any one of human, Saccharomyces cerevisiae, Escherichia coli and Salmonella typhimurium, and can be used in industrial production to produce NAD on a large scale by using NR and ATP as raw materials.

Description

一种重组NAD合成酶及其基因和应用A recombinant NAD synthetase and its gene and application 技术领域Technical field
本发明涉及生物酶催化和基因工程技术领域，特别涉及一种通过基因工程技术手段人工获得的重组NAD合成酶、其基因以及用于规模化催化NR和ATP转化生产NAD的工业用途。The invention relates to the technical fields of biological enzyme catalysis and genetic engineering, in particular to a recombinant NAD synthetase artificially obtained through genetic engineering techniques, its gene, and industrial use for catalyzing the conversion of NR and ATP to produce NAD on a large scale.
背景技术Background technique
NAD合成酶，是指烟酰胺腺嘌呤二核苷酸（Nicotinamide Adenine Dinucleotide，缩写NAD）合成酶，也写作NADR或NadR，是一种催化底物转化成烟酰胺腺嘌呤二核苷酸的酶。NAD synthetase refers to nicotinamide adenine dinucleotide (Nicotinamide Adenine Dinucleotide, abbreviated NAD) synthetase, also written as NADR or NadR, which is an enzyme that catalyzes the conversion of a substrate into nicotinamide adenine dinucleotide.
NAD是存在于包括人类细胞在内的几乎所有活细胞中的一种生理物质，对人体无毒副作用，这种物质是很多可催化氧化—还原反应的酶的辅助因子，其参与细胞物质代谢、能量合成、细胞DNA修复等多种生理活动，是线粒体中能量产生链中的控制标志物，被称为辅酶Ⅰ。NAD is a physiological substance that exists in almost all living cells, including human cells, and has no toxic or side effects to the human body. This substance is a cofactor for many enzymes that can catalyze oxidation-reduction reactions, and it participates in cell material metabolism, Energy synthesis, cellular DNA repair and other physiological activities are the control markers in the energy production chain in the mitochondria and are called Coenzyme I.
NAD的用途非常广泛，可以用于化工催化反应、原料药生产、保健品行业、化妆品行业等，市场需求量很大。目前，工业上生产NAD的方法一般包括两种，一种为化学法，另一种为生物催化法，生物催化法因相较于化学法具有反应条件温和、节能环保、无有机溶剂残留等优点而逐渐成为主流。NAD has a wide range of uses, and can be used in chemical catalytic reactions, raw material drug production, health care products, cosmetics, etc., and the market demand is great. At present, there are generally two methods for the industrial production of NAD, one is a chemical method, and the other is a biocatalysis method. Compared with the chemical method, the biocatalysis method has the advantages of mild reaction conditions, energy saving and environmental protection, and no organic solvent residues. And gradually become the mainstream.
NAD的生物催化法生产具体是指，以烟酰胺单核苷酸（NMN）和三磷酸腺苷（ATP）为原料，在烟酰胺单核苷酸腺苷转移酶（NMNAT）的催化作用下，生产NAD。此种方法存在一个弊端，就是NMN的价格特别昂贵，从而导致NAD的生产成本极高，在市场上没有竞争优势。因此，工业上多采用以NMN的前体物质烟酰胺核糖（NR）替代NMN，同时加入用于催化NR转化成NMN的生物催化剂烟酰胺核糖激酶（NRK）的生物催化方法。此种方法的缺点在于，需要进行两步酶催化反应，导致反应时间延长，生产操作步骤增多。NAD合成酶的优势就在于能够以NR和ATP为原料，经一步催化合成NAD。The biocatalytic production of NAD specifically refers to the production of NAD using nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) as raw materials, under the catalysis of nicotinamide mononucleotide adenosine transferase (NMNAT). One disadvantage of this method is that the price of NMN is extremely expensive, which leads to extremely high production costs of NAD and no competitive advantage in the market. Therefore, the industry mostly adopts a biocatalytic method of replacing NMN with nicotinamide ribose (NR), the precursor material of NMN, and adding the biocatalyst nicotinamide ribokinase (NRK) for catalyzing the conversion of NR to NMN. The disadvantage of this method is that it requires two steps of enzyme-catalyzed reaction, which results in prolonged reaction time and increased production operation steps. The advantage of NAD synthetase is that it can use NR and ATP as raw materials to catalyze the synthesis of NAD in one step.
目前已在多种生物体内发现了自然存在的NAD合成酶，如，来源于鼠伤寒沙门氏菌（ Salmonella typhimurium）中的 stNadR，来源于流感嗜血杆菌（ Haemophilus influenzae）中的 hiNadR,，来源于大肠杆菌（ Escherichia coli）中的 ecNadR等。但是这些自然存在的NAD合成酶的活性存在明显的偏向性，如， hiNadR虽具有较高的将NMN转化为NAD的活性，但将NR转化为NMN的活性则相对弱一些，而 stNadR、 ecNadR的活性则刚好与之相反。因此，在采用这些自然存在的NAD合成酶催化NR和ATP转化生产NAD时存在转化率偏低的问题，导致生产NAD的产量低、成本高，从而无法满足在工业上应用的条件，限制了NAD合成酶在规模化工业生产中的应用。 At present, naturally occurring NAD synthase has been found in a variety of organisms, such as st NadR derived from Salmonella typhimurium and hi NadR derived from Haemophilus influenzae . Ec NadR in Escherichia coli and so on. However, the activities of these naturally-occurring NAD synthetases have obvious biases. For example, although hi NadR has a high activity of converting NMN to NAD, the activity of converting NR to NMN is relatively weak, while st NadR, The activity of ec NadR is just the opposite. Therefore, when these naturally occurring NAD synthetases are used to catalyze the conversion of NR and ATP to produce NAD, there is a problem of low conversion rate, resulting in low yield and high cost of producing NAD, which cannot meet the conditions for industrial application and restricts NAD. The application of synthetase in large-scale industrial production.
技术问题technical problem
鉴于上述背景技术中提到的现有技术的不足，本发明的目的在于克服自然存在的NAD合成酶催化NR和ATP转化成NAD的转化率偏低的技术问题，开发一种适于规模化工业应用的重组NAD合成酶，从而解决现有NAD的工业化生产方法存在的生产成本高、操作繁琐的技术难题。In view of the shortcomings of the prior art mentioned in the above-mentioned background art, the purpose of the present invention is to overcome the technical problem of the low conversion rate of NR and ATP into NAD that naturally existed NAD synthetase catalyzes, and develops a suitable for large-scale industry The applied recombinant NAD synthetase can solve the technical problems of high production cost and complicated operation in the existing industrial production method of NAD.
技术解决方案Technical solutions
为实现上述目的，发明人对目前已知基因进行了大量的试验和筛选，并利用基因工程学技术手段对筛选到的已知目的基因片段进行融合从而获得一系列重组NAD合成酶，最终筛选出其中酶活性得到显著提高的融合产物。基于此，本发明提供了一种重组NAD合成酶 ，所述重组NAD合成酶包含来源于流感嗜血杆菌的烟酰胺单核苷酸腺苷转移酶结构域以及来源于人类、酿酒酵母、大肠杆菌和鼠伤寒沙门氏菌中的任意一种的烟酰胺核糖激酶结构域。 In order to achieve the above-mentioned purpose, the inventor conducted a large number of experiments and screenings on the currently known genes, and used genetic engineering techniques to fuse the selected known target gene fragments to obtain a series of recombinant NAD synthetase, and finally screened out The fusion product in which the enzyme activity is significantly improved. Based on this, the present invention provides a recombinant NAD synthetase comprising a nicotinamide mononucleotide adenosyl transferase domain derived from Haemophilus influenzae and derived from humans, Saccharomyces cerevisiae, and Escherichia coli And the nicotinamide ribokinase domain of any of Salmonella typhimurium.
本发明提供的上述重组NAD合成酶中，流感嗜血杆菌、酿酒酵母、大肠杆菌和鼠伤寒沙门氏菌是指该名称菌种下的所有类型的菌株，即，来源于流感嗜血杆菌、酿酒酵母、大肠杆菌和鼠伤寒沙门氏菌菌种下的所有类型的菌株的对应酶结构域都适用本发明。In the above-mentioned recombinant NAD synthetase provided by the present invention, Haemophilus influenzae, Saccharomyces cerevisiae, Escherichia coli and Salmonella typhimurium refer to all types of strains under this name, namely, derived from Haemophilus influenzae, Saccharomyces cerevisiae, The corresponding enzyme domains of all types of strains of Escherichia coli and Salmonella typhimurium are applicable to the present invention.
优选地，本发明提供的上述重组NAD合成酶中，烟酰胺核糖激酶结构域融合在烟酰胺单核苷酸腺苷转移酶结构域的C端。Preferably, in the aforementioned recombinant NAD synthetase provided by the present invention, the nicotinamide ribokinase domain is fused to the C-terminus of the nicotinamide mononucleotide adenosyl transferase domain.
更优选地，本发明提供的上述重组NAD合成酶中，烟酰胺核糖激酶结构域通过柔性连接肽段与烟酰胺单核苷酸腺苷转移酶结构域进行融合，前述柔性连接肽段的序列为GSGSGSGS。该连接肽段是发明人针对融合的两个酶结构域的结构特征特别设计并经多次筛选和试验验证而确定的，相较于其他连接肽段，该肽段可起到增强蛋白表达的作用。More preferably, in the aforementioned recombinant NAD synthetase provided by the present invention, the nicotinamide ribokinase domain is fused with the nicotinamide mononucleotide adenosyl transferase domain through a flexible connecting peptide, and the sequence of the aforementioned flexible connecting peptide is GSGSGSGS. This connecting peptide is specially designed by the inventor for the structural characteristics of the two enzyme domains fused and confirmed by multiple screenings and experiments. Compared with other connecting peptides, this peptide can enhance protein expression. effect.
更优选地，本发明提供的上述重组NAD合成酶中，来源于流感嗜血杆菌的烟酰胺单核苷酸腺苷转移酶结构域的氨基酸序列为UniProt中登录号为P44308[52-224]的氨基酸序列，命名为 hiNMNAT；来源于人类、酿酒酵母、大肠杆菌和鼠伤寒沙门氏菌的烟酰胺核糖激酶结构域的氨基酸序列依次为UniProt中登录号为Q9NWW6、Q9NPI5、P53915、P27278[230-410]和P24518[230-410]的氨基酸序列，对应的命名依次为 hNRK1、 hNRK2、 yNRK1、 ecNRK和 stNRK。 More preferably, in the above-mentioned recombinant NAD synthetase provided by the present invention, the amino acid sequence of the nicotinamide mononucleotide adenosyl transferase domain derived from Haemophilus influenzae is that of UniProt with the accession number P44308[52-224] The amino acid sequence is named hi NMNAT; the amino acid sequence of the nicotinamide ribokinase domain derived from human, Saccharomyces cerevisiae, Escherichia coli and Salmonella typhimurium is the accession number of UniProt as Q9NWW6, Q9NPI5, P53915, P27278 [230-410] And the amino acid sequence of P24518[230-410], the corresponding names are h NRK1, h NRK2, y NRK1, ec NRK and st NRK.
更优选地，本发明提供的上述重组NAD合成酶的氨基酸序列如SEQ ID NO：4至SEQ ID NO：8所示。More preferably, the amino acid sequence of the aforementioned recombinant NAD synthetase provided by the present invention is shown in SEQ ID NO: 4 to SEQ ID NO: 8.
本发明同时还提供了一种基因序列，该基因序列编码本发明提供的上述重组NAD合成酶。The present invention also provides a gene sequence, which encodes the above-mentioned recombinant NAD synthetase provided by the present invention.
本发明同时还提供了一种生物材料，包括重组载体、重组细胞或者重组微生物，该生物材料含有本发明提供的上述基因序列。即，本发明提供的生物材料是含有本发明提供的上述基因序列的重组载体，或者是含有本发明提供的上述基因序列的重组细胞，又或者是含有本发明提供的上述基因序列的重组微生物。The invention also provides a biological material, including a recombinant vector, a recombinant cell or a recombinant microorganism, the biological material containing the above-mentioned gene sequence provided by the invention. That is, the biological material provided by the present invention is a recombinant vector containing the aforementioned gene sequence provided by the present invention, or a recombinant cell containing the aforementioned gene sequence provided by the present invention, or a recombinant microorganism containing the aforementioned gene sequence provided by the present invention.
另外，本发明还提供了上述重组NAD合成酶的用途，即应用于工业生产中以NR和ATP为原料规模化生产NAD。In addition, the present invention also provides the use of the above-mentioned recombinant NAD synthetase, that is, it is applied to large-scale production of NAD using NR and ATP as raw materials in industrial production.
有益效果Beneficial effect
与现有技术相比，本发明具有以下优点：Compared with the prior art, the present invention has the following advantages:
1、与自然界存在的NAD合成酶相比，本发明提供的重组NAD合成酶的酶活力显著提高，经试验检测，其酶活力是自然界存在的常用NAD合成酶的3.5-65倍，其催化NR和ATP转化生成NAD的产率增加了400%以上，从而能够适用于NAD的规模化工业大生产中。1. Compared with the NAD synthetase that exists in nature, the enzyme activity of the recombinant NAD synthetase provided by the present invention is significantly improved. The enzyme activity of the recombinant NAD synthetase provided by the present invention is 3.5-65 times that of the commonly used NAD synthetase in nature. It catalyzes NR The yield of NAD from the conversion of ATP and ATP has increased by more than 400%, which can be applied to large-scale industrial production of NAD.
2、与现有的生物催化生产NAD的方法相比，应用本发明提供的重组NAD合成酶生产NAD时，可实现以NR和ATP为原料一步催化生成NAD，其投料成本降低的同时，又能缩短反应时间，减少工业操作步骤，从而大大降低了生产成本。2. Compared with the existing methods of biocatalytic production of NAD, when the recombinant NAD synthetase provided by the present invention is used to produce NAD, it can realize the one-step catalytic production of NAD with NR and ATP as raw materials, which reduces the feed cost and at the same time. Shorten the reaction time, reduce the industrial operation steps, thereby greatly reducing the production cost.
本发明的实施方式Embodiments of the invention
下面结合具体实施例对本发明做进一步的详细说明，以下实施例是对本发明的解释，本发明并不局限于以下实施例。若无特别说明，本发明实施例中所使用的原料及试剂皆为市售商品，实施例中未注明具体条件者，均按常规条件或制造商建议的条件进行。The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are an explanation of the present invention, and the present invention is not limited to the following embodiments. Unless otherwise specified, the raw materials and reagents used in the examples of the present invention are all commercially available products. If specific conditions are not specified in the examples, the procedures are carried out under conventional conditions or conditions recommended by the manufacturer.
1、NAD合成酶质粒的构建1. Construction of NAD synthetase plasmid
（1）自然界存在的NAD合成酶质粒(1) NAD synthetase plasmids that exist in nature
设计如下引物对（SEQ ID NO：9至SEQ ID NO：14）Design the following primer pairs (SEQ ID NO: 9 to SEQ ID NO: 14)
ecNadR-1-NdeⅠ-up：CCCATATGTCGTCATTTGATTACCTG ec NadR-1-NdeⅠ-up: CCCATATGTCGTCATTTGATTACCTG
ecNadR-end-XhoⅠ-dn：CCCTCGAGTTATCTCTGCTCCCCCATCATCT ec NadR-end-XhoⅠ-dn: CCCTCGAGTTATCTCTGCTCCCCCATCATCT
stNadR-1-NdeⅠ-up：CCCATATGTCATCGTTCGACTATCTCAA st NadR-1-NdeⅠ-up: CCCATATGTCATCGTTCGACTATCTCAA
stNadR-end-XhoⅠ-dn：CCCTCGAGTTATCCCTGCTCGCCCATCATC st NadR-end-XhoⅠ-dn: CCCTCGAGTTATCCCTGCTCGCCCATCATC
hiNadR-52-NdeⅠ-up：CCCATATGTCAAAAACAAAAGAGAAAAA hi NadR-52-NdeⅠ-up: CCCATATGTCAAAAACAAAAGAGAAAAA
hiNadR-end-NdeⅠ-up：CCCTCGAGTCATTGAGATGTCCCTTTTAT hi NadR-end-NdeⅠ-up: CCCTCGAGTCATTGAGATGTCCCTTTTAT
利用PCR扩增技术分别对来源于大肠杆菌（ Escherichia coli）、鼠伤寒沙门氏菌（ Salmonella typhimurium）和流感嗜血杆菌（ Haemophilus influenzae）中的NAD合成酶（ ecNadR、 stNadR和 hiNadR）的基因序列进行扩增，然后利用限制性内切酶NdeⅠ和XhoⅠ将扩增产物连接到载体pET-28a上，分别得到质粒pET28a- ecNadR、pET28a- stNadR和pET28a- hiNadR，经测序确认其氨基酸序列分别如SEQ ID NO：1至SEQ ID NO：3所示。 The gene sequences of NAD synthetases ( ec NadR, st NadR and hi NadR) from Escherichia coli , Salmonella typhimurium and Haemophilus influenzae were analyzed by PCR amplification technology. After amplification, the amplified products were ligated to the vector pET-28a using restriction enzymes NdeI and XhoI to obtain plasmids pET28a- ec NadR, pET28a- st NadR and pET28a- hi NadR, and their amino acid sequences were confirmed by sequencing They are shown in SEQ ID NO:1 to SEQ ID NO:3, respectively.
（2）本发明提供的重组NAD合成酶质粒(2) The recombinant NAD synthetase plasmid provided by the present invention
参考蛋白数据库公开的 hiNMNAT、 hNRK1、 hNRK2、 yNRK1、 ecNRK和 stNRK的氨基酸序列（UniProt登录号分别为：P44308[52-224]、Q9NWW6、Q9NPI5、P53915、P27278[230-410]和P24518[230-410]），结合序列比对和结构功能分析，设计带有柔性连接肽段GSGSGSGS序列的引物将 hiNMNAT和 hNRK1、 hNRK2、 yNRK1、 ecNRK、 stNRK的基因序列分别扩增出来，然后以扩增产物为模板，利用引物将 hiNMNAT分别与 hNRK1、 hNRK2、 yNRK1、 ecNRK、 stNRK进行融合PCR，即得到本发明提供的重组NAD合成酶 hihNadR1、 hihNadR2、 hiyNadR、 hiecNadR和 histNadR的融合基因片段，其对应的氨基酸序列分别如SEQ ID NO：4至SEQ ID NO：8所示。再利用限制性内切酶NdeⅠ和XhoⅠ将融合基因片段连接到载体pET-22b上，分别得到质粒pET22b- hihNadR1、pET22b- hihNadR2、pET22b- hiyNadR、pET22b- hiecNadR和pET22b- histNadR。 Refer to the amino acid sequences of hi NMNAT, h NRK1, h NRK2, y NRK1, ec NRK and st NRK published in the protein database (UniProt accession numbers are: P44308[52-224], Q9NWW6, Q9NPI5, P53915, P27278[230-410 ] And P24518[230-410]), combined with sequence alignment and structure and function analysis, designed primers with flexible connecting peptide GSGSGSGS sequence to combine hi NMNAT and h NRK1, h NRK2, y NRK1, ec NRK, st NRK genes sequences was amplified, and then the amplified product as a template, primers, respectively, and hi NMNAT h NRK1, h NRK2, y NRK1 , ec NRK, st NRK the PCR fusion, i.e. recombinant NAD synthetase of the present invention provides hih The corresponding amino acid sequences of the fusion gene fragments of NadR1, hih NadR2, hiy NadR, hiec NadR and hist NadR are shown in SEQ ID NO: 4 to SEQ ID NO: 8, respectively. Restriction enzymes NdeI and XhoI were used to connect the fusion gene fragments to the vector pET-22b to obtain plasmids pET22b- hih NadR1, pET22b- hih NadR2, pET22b- hiy NadR, pET22b- hiec NadR and pET22b- hist NadR.
2、NAD合成酶酶液的制备2. Preparation of NAD synthase enzyme solution
将第1部分构建好的NAD合成酶质粒分别转化50μL BL21 (DE3)感受态细胞，加入900μL Luria Broth (LB)培养基37℃活化1h，接入10-20mL LB培养基（含100mg/L氨苄青霉素或50mg/L卡那霉素）中37℃培养6h-16h，然后接入1-4L LB培养基（含100mg/L氨苄青霉素或50mg/L卡那霉素）中37℃培养至OD 600=0.8-1，调节温度至16-37℃，加入0.2-1mM IPTG诱导蛋白表达。4-20h后离心收集菌体，用20mL破菌液（20mM Tris-HCl pH7.5, 100mM NaCl, 10mM咪唑）重悬。然后用均质机破碎细胞，离心（4℃, 12000g, 25min）收集上清液。 The NAD synthetase plasmid constructed in Part 1 was transformed into 50μL BL21 (DE3) competent cells respectively, and 900μL Luria Broth (LB) medium was added to activate at 37℃ for 1h, and then 10-20mL LB medium (containing 100mg/L ampicillin) Cultured in penicillin or 50mg/L kanamycin) at 37°C for 6h-16h, then connected to 1-4L LB medium (containing 100mg/L ampicillin or 50mg/L kanamycin) at 37°C to OD 600 =0.8-1, adjust the temperature to 16-37℃, add 0.2-1mM IPTG to induce protein expression. After 4-20 hours, the cells were collected by centrifugation, and resuspended in 20 mL of bactericidal solution (20mM Tris-HCl pH7.5, 100mM NaCl, 10mM imidazole). Then crush the cells with a homogenizer, and collect the supernatant by centrifugation (4°C, 12000g, 25min).
上清液中加入30mL Buffer A (20mM Tris-HCl pH7.5, 100mM NaCl)平衡后的重力柱（30mL柱体积含4mL Ni-NTA凝胶），吸附半小时后收集含未结合蛋白流穿液，30mL Buffer B (20mM Tris-HCl pH7.5, 100mM NaCl, 20mM咪唑)冲洗杂蛋白两遍后，10mL Buffer C (20mM Tris-HCl pH7.5, 100mM NaCl, 500mM咪唑)孵育10min后收集含结合目的蛋白的洗脱液，SDS-PAGE蛋白电泳结果显示洗脱液为高纯度目的蛋白，即得NAD合成酶酶液。Add 30mL Buffer A (20mM Tris-HCl pH7.5, 100mM NaCl) to the supernatant and equilibrate the gravity column (30mL column volume contains 4mL Ni-NTA gel), and collect the flow-through liquid containing unbound protein after adsorption for half an hour , 30mL Buffer B (20mM Tris-HCl pH7.5, After washing the contaminated protein twice with 100mM NaCl, 20mM imidazole, 10mL Buffer C (20mM Tris-HCl pH7.5, 100mM NaCl, 500mM imidazole) incubate for 10min and collect the eluate containing the bound target protein. SDS-PAGE protein electrophoresis results It shows that the eluate is high-purity target protein, that is, NAD synthase enzyme solution is obtained.
3、NAD合成酶酶活力测定3. Determination of NAD synthase enzyme activity
第2部分制备得到的酶液经NanoDrop 2000测定蛋白浓度后稀释至1g/L，取100μL加入到400μL反应液（100mM pH7.2磷酸盐缓冲液、烟酰胺核糖10mM、ATP 20mM、MgCl 2 10mM）中，37℃反应15min。反应结束后，通过高效液相色谱（HPLC）测定反应液中烟酰胺腺嘌呤二核苷酸的含量，测定结果如表1所示。一个酶活力单位（U）定义为上述条件下，每分钟转化一微摩尔烟酰胺核糖为烟酰胺腺嘌呤二核苷酸所需酶量。 The enzyme solution prepared in Part 2 was diluted to 1g/L by NanoDrop 2000 after measuring the protein concentration, and 100μL was added to 400μL reaction solution (100mM pH7.2 phosphate buffer, nicotinamide ribose 10mM, ATP 20mM, MgCl 2 10mM) In the middle, react at 37°C for 15 min. After the reaction, the content of nicotinamide adenine dinucleotide in the reaction solution was determined by high performance liquid chromatography (HPLC). The results are shown in Table 1. An enzyme activity unit (U) is defined as the amount of enzyme required to convert one micromole nicotinamide ribose into nicotinamide adenine dinucleotide per minute under the above conditions.
表1Table 1
酶液Enzyme Solution 序列来源Sequence source NAD生成量NAD generation 酶活U/mgEnzyme activity U/mg
ecNadR ec NadR 大肠杆菌Escherichia coli 0.2mM0.2mM 0.050.05
stNadR st NadR 鼠伤寒沙门氏菌Salmonella typhimurium 0.2mM0.2mM 0.060.06
hiNadR hi NadR 流感嗜血杆菌Haemophilus influenzae 0.1mM0.1mM 0.030.03
hihNadR1 hih NadR1 本发明this invention 0.8 mM0.8 mM 0.210.21
hihNadR2 hih NadR2 本发明this invention 7.0 mM7.0 mM 1.871.87
hiyNadR hiy NadR 本发明this invention 1.6 mM1.6 mM 0.430.43
hiecNadR hiec NadR 本发明this invention 5.9 mM5.9 mM 1.571.57
histNadR hist NadR 本发明this invention 7.4 mM7.4 mM 1.971.97
 To

Claims (8)

  1. 一种重组NAD合成酶，其特征在于：所述重组NAD合成酶包含来源于流感嗜血杆菌的烟酰胺单核苷酸腺苷转移酶结构域以及来源于人类、酿酒酵母、大肠杆菌和鼠伤寒沙门氏菌中的任意一种的烟酰胺核糖激酶结构域。A recombinant NAD synthetase, characterized in that: the recombinant NAD synthetase comprises a nicotinamide mononucleotide adenosyl transferase domain derived from Haemophilus influenzae and derived from humans, Saccharomyces cerevisiae, Escherichia coli and Typhimurium The nicotinamide ribokinase domain of any of Salmonella species.
  2. 根据权利要求1所述的重组NAD合成酶，其特征在于：所述烟酰胺核糖激酶结构域融合在所述烟酰胺单核苷酸腺苷转移酶结构域的C端。The recombinant NAD synthetase of claim 1, wherein the nicotinamide ribokinase domain is fused to the C-terminus of the nicotinamide mononucleotide adenosyl transferase domain.
  3. 根据权利要求1或2所述的重组NAD合成酶，其特征在于：所述烟酰胺核糖激酶结构域通过柔性连接肽段与所述烟酰胺单核苷酸腺苷转移酶结构域进行融合，所述柔性连接肽段的序列为GSGSGSGS。The recombinant NAD synthetase according to claim 1 or 2, wherein the nicotinamide ribokinase domain is fused with the nicotinamide mononucleotide adenosyl transferase domain through a flexible connecting peptide, so The sequence of the flexible connecting peptide is GSGSGSGS.
  4. 根据权利要求1或2所述的重组NAD合成酶，其特征在于：所述来源于流感嗜血杆菌的烟酰胺单核苷酸腺苷转移酶结构域的氨基酸序列为UniProt中登录号为P44308[52-224]的氨基酸序列；所述来源于人类、酿酒酵母、大肠杆菌和鼠伤寒沙门氏菌的烟酰胺核糖激酶结构域的氨基酸序列依次为UniProt中登录号为Q9NWW6、Q9NPI5、P53915、P27278[230-410]和P24518[230-410]的氨基酸序列。The recombinant NAD synthetase according to claim 1 or 2, wherein the amino acid sequence of the nicotinamide mononucleotide adenosyl transferase domain derived from Haemophilus influenzae is UniProt with accession number P44308[ 52-224]; the amino acid sequence of the nicotinamide ribokinase domain derived from human, Saccharomyces cerevisiae, Escherichia coli and Salmonella typhimurium is the accession number of UniProt as Q9NWW6, Q9NPI5, P53915, P27278 [230- 410] and P24518 [230-410] amino acid sequence.
  5. 根据权利要求4所述的重组NAD合成酶，其特征在于：所述重组NAD合成酶的氨基酸序列如SEQ ID NO：4至SEQ ID NO：8所示。The recombinant NAD synthetase according to claim 4, wherein the amino acid sequence of the recombinant NAD synthetase is as shown in SEQ ID NO: 4 to SEQ ID NO: 8.
  6. 一种基因序列，其特征在于：所述基因序列编码权利要求1-5任一项所述的重组NAD合成酶。A gene sequence, characterized in that: the gene sequence encodes the recombinant NAD synthetase according to any one of claims 1-5.
  7. 一种生物材料，包括重组载体、重组细胞或者重组微生物，其特征在于：所述生物材料含有权利要求6所述的基因序列。A biological material, comprising a recombinant vector, a recombinant cell, or a recombinant microorganism, characterized in that the biological material contains the gene sequence of claim 6.
  8. 权利要求1至5任一项所述的重组NAD合成酶的用途，其特征在于：所述用途为将所述重组NAD合成酶应用于工业生产中以NR和ATP为原料规模化生产NAD。The use of the recombinant NAD synthetase according to any one of claims 1 to 5, characterized in that: the use is to apply the recombinant NAD synthetase to industrial production to produce NAD on a large scale using NR and ATP as raw materials.
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