阅读说明：本技术 一种重组nad合成酶及其基因和应用 (Recombinant NAD synthetase, gene and application thereof ) 是由 张章 张琦 于 2019-06-27 设计创作，主要内容包括：提供一种重组NAD合成酶及其基因和应用。该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,能够应用于工业生产中以NR和ATP为原料规模化生产NAD。(Provides a recombinant NAD synthetase, a gene and an application thereof. The enzyme comprises a nicotinamide mononucleotide adenylyltransferase structural domain derived from haemophilus influenzae and a nicotinamide ribokinase structural domain derived from any one of human beings, saccharomyces cerevisiae, escherichia coli and salmonella typhimurium, and can be applied to large-scale production of NAD by taking NR and ATP as raw materials in industrial production.)

A recombinant NAD synthase characterized by: the recombinant NAD synthase comprises a nicotinamide mononucleotide adenylyltransferase domain derived from Haemophilus influenzae and a nicotinamide ribokinase domain derived from any one of human, Saccharomyces cerevisiae, Escherichia coli and Salmonella typhimurium.

The recombinant NAD synthase of claim 1, wherein: the nicotinamide ribokinase domain is fused at the C-terminus of the nicotinamide mononucleotide adenylyltransferase domain.

The recombinant NAD synthase according to claim 1 or 2, characterized in that: the nicotinamide ribokinase structural domain is fused with the nicotinamide mononucleotide adenyl transferase structural domain through a flexible connecting peptide segment, and the sequence of the flexible connecting peptide segment is GSGSGSGS.

The recombinant NAD synthase according to claim 1 or 2, characterized in that: the amino acid sequence of the nicotinamide mononucleotide adenyltransferase structural domain derived from haemophilus influenzae is the amino acid sequence with the accession number P44308[52-224] in UniProt; the amino acid sequence of the nicotinamide ribokinase domain derived from human, Saccharomyces cerevisiae, Escherichia coli and Salmonella typhimurium is the amino acid sequence with the accession numbers Q9NWW6, Q9NPI5, P53915, P27278[ 230-.

The recombinant NAD synthase of claim 4, wherein: the amino acid sequence of the recombinant NAD synthetase is shown as SEQ ID NO: 4 to SEQ ID NO: shown in fig. 8.

A gene sequence characterized by: the gene sequence encodes the recombinant NAD synthase of any one of claims 1-5.

A biomaterial comprising a recombinant vector, a recombinant cell or a recombinant microorganism, wherein: the biological material contains the gene sequence of claim 6.

Use of the recombinant NAD synthase of any one of claims 1 to 5, characterized in that: the application is that the recombinant NAD synthetase is applied to industrial production to produce NAD in a large scale by taking NR and ATP as raw materials.

Technical Field

The invention relates to the technical field of biological enzyme catalysis and genetic engineering, in particular to a recombinant NAD synthetase artificially obtained by a genetic engineering technical means, a gene thereof and industrial application of the recombinant NAD synthetase in large-scale catalytic NR and ATP conversion production.

Background

NAD synthase, which is a Nicotinamide Adenine Dinucleotide (NAD) synthase, also known as NADR or NADR, is an enzyme that catalyzes the conversion of a substrate to Nicotinamide Adenine Dinucleotide.

NAD is a physiological substance present in almost all living cells including human cells, has no toxic side effects on human bodies, is a cofactor of many enzymes that catalyze oxidation-reduction reactions, is involved in various physiological activities such as cellular substance metabolism, energy synthesis, cellular DNA repair, etc., is a control marker in energy-generating chains in mitochondria, and is called coenzyme i.

The NAD has wide application, can be used for chemical catalytic reaction, raw material medicine production, health product industry, cosmetic industry and the like, and has large market demand. At present, two methods for industrially producing NAD generally comprise a chemical method and a biological catalysis method, and the biological catalysis method gradually becomes a mainstream because of the advantages of mild reaction conditions, energy conservation, environmental protection, no organic solvent residue and the like compared with the chemical method.

The biological catalysis method for producing the NAD specifically comprises the step of producing the NAD by taking Nicotinamide Mononucleotide (NMN) and Adenosine Triphosphate (ATP) as raw materials under the catalysis action of nicotinamide mononucleotide adenyl transferase (NMNAT). This method has a drawback that NMN is extremely expensive, resulting in extremely high production cost of NAD and no competitive advantage in the market. Therefore, the industry has adopted a biocatalytic method in which Nicotinamide Riboside (NR), a precursor of NMN, is substituted for NMN, while a biocatalyst, Nicotinamide Ribokinase (NRK), for catalyzing the conversion of NR into NMN is added. This method has disadvantages in that two enzymatic reactions are required, resulting in a prolonged reaction time and an increase in the number of production operations. The NAD synthetase has the advantage that NAD can be synthesized by one-step catalysis by taking NR and ATP as raw materials.

Naturally occurring NAD synthetases have now been found in a variety of organisms, e.g., from Salmonella typhimurium (A)Salmonella typhimurium) In (1)stNadR derived from Haemophilus influenzae (b) ((b))Haemophilus influenzae) In (1)hiNadR, derived from Escherichia coli (E. coli)Escherichia coli) In (1)ecNadR, and the like. There is a significant bias in the activity of these naturally occurring NAD synthetases, however, e.g.,hiNadR has higher activity of converting NMN into NAD, but has weaker activity of converting NR into NMN, andstNadR、 ecthe NadR activity is exactly opposite. Therefore, when the naturally-existing NAD synthetase is used for catalyzing NR and ATP conversion to produce NAD, the conversion rate is low, the yield of NAD production is low, the cost is high, and the conditions for industrial application cannot be met, so that the application of NAD synthetase in large-scale industrial production is limited.

Modes for carrying out the invention

The present invention will be described in further detail with reference to specific examples, which are illustrative of the present invention and are not to be construed as being limited thereto. Unless otherwise specified, the starting materials and reagents used in the examples of the present invention are commercially available, and those not specifically mentioned in the examples are carried out under conventional conditions or conditions recommended by the manufacturer.

1. Construction of NAD synthetase plasmid

(1) Naturally occurring NAD synthase plasmids

The following primer pairs (SEQ ID NO: 9 to SEQ ID NO: 14) were designed

ecNadR-1-NdeⅠ-up：CCCATATGTCGTCATTTGATTACCTG

ecNadR-end-XhoⅠ-dn：CCCTCGAGTTATCTCTGCTCCCCCATCATCT

stNadR-1-NdeⅠ-up：CCCATATGTCATCGTTCGACTATCTCAA

stNadR-end-XhoⅠ-dn：CCCTCGAGTTATCCCTGCTCGCCCATCATC

hiNadR-52-NdeⅠ-up：CCCATATGTCAAAAACAAAAGAGAAAAA

hiNadR-end-NdeⅠ-up：CCCTCGAGTCATTGAGATGTCCCTTTTAT

Respectively using PCR amplification technology to make the DNA fragment be respectively used for the DNA fragment derived from Escherichia coli (E. coli)Escherichia coli) Salmonella typhimurium (Salmonella typhimurium) And Haemophilus influenzae: (Haemophilus influenzae) NAD synthase (a) of (b)ecNadR、 stNadR andhiNadR), then utilizing restriction enzymes Nde I and Xho I to connect the amplification product to the carrier pET-28a so as to respectively obtain the plasmid pET28a-ecNadR、pET28a- stNadR and pET28a-hiAnd NadR, the amino acid sequences of which are confirmed by sequencing to be respectively shown as SEQ ID NO: 1 to SEQ ID NO: 3, respectively.

(2) The recombinant NAD synthetase plasmid provided by the invention

Published by reference to protein databaseshiNMNAT、 hNRK1、 hNRK2、 yNRK1、 ecNRK andstthe amino acid sequence of NRK (UniProt accession numbers: P44308[52-224, respectively)]、Q9NWW6、Q9NPI5、P53915、P27278[230-410]And P2458 [230-]) Combining sequence comparison and structural function analysis to design a primer with a flexible connecting peptide segment GSGSGSGS sequencehiNMNAT andhNRK1、 hNRK2、 yNRK1、 ecNRK、 stNRK gene sequences are respectively amplified, and then amplification products are used as templates, and primers are used for amplifying the NRK gene sequenceshiNMNAT respectively withhNRK1、 hNRK2、 yNRK1、 ecNRK、 stNRK is fused with PCR to obtain the recombinant NAD synthetase provided by the inventionhihNadR1、 hihNadR2、 hiyNadR、 hiecNadR andhistthe corresponding amino acid sequences of the fusion gene segments of NadR are respectively shown in SEQ ID NO: 4 to SEQ ID NO:shown in fig. 8. Then the fusion gene fragment is connected to a vector pET-22b by utilizing restriction enzymes Nde I and Xho I to respectively obtain plasmids pET22b-hihNadR1、pET22b- hihNadR2、pET22b- hiyNadR、pET22b- hiecNadR and pET22b-histNadR。

2. Preparation of NAD synthetase enzyme solution

The NAD synthase plasmids constructed in part 1 were transformed into 50. mu.L of BL21 (DE3) competent cells, respectively, added to 900. mu.L of Luria Broth (LB) medium at 37 ℃ for activation for 1 hour, inoculated into 10-20mL of LB medium (containing 100mg/L ampicillin or 50mg/L kanamycin) for culture at 37 ℃ for 6 hours-16 hours, and then inoculated into 1-4L of LB medium (containing 100mg/L ampicillin or 50mg/L kanamycin) for culture at 37 ℃ to OD₆₀₀And (4) =0.8-1, adjusting the temperature to 16-37 ℃, and adding 0.2-1mM IPTG to induce protein expression. After 4-20h, the cells were collected by centrifugation and resuspended in 20mL of a lysate (20mM Tris-HCl pH7.5, 100mM NaCl, 10mM imidazole). Then, the cells were disrupted by a homogenizer and centrifuged (4 ℃, 12000g, 25 min) to collect the supernatant.

Adding 30mL Buffer A (20mM Tris-HCl pH7.5, 100mM NaCl) balanced gravity column (30 mL column volume contains 4mL Ni-NTA gel), adsorbing for half an hour, collecting flow-through liquid containing unbound protein, washing the hybrid protein twice with 30mL Buffer B (20mM Tris-HCl pH7.5, 100mM NaCl, 20mM imidazole), incubating for 10min with 10mL Buffer C (20mM Tris-HCl pH7.5, 100mM NaCl, 500mM imidazole), collecting eluent containing bound target protein, and performing SDS-PAGE protein electrophoresis to show that the eluent is high-purity target protein, thus obtaining the NAD synthetase enzyme liquid.

3. Determination of enzymatic Activity of NAD synthetase

The enzyme solution prepared in part 2 was diluted to 1g/L after protein concentration measurement by NanoDrop 2000, and 100. mu.L of the enzyme solution was added to 400. mu.L of a reaction solution (100 mM phosphate buffer solution, pH7.2, nicotinamide ribose 10mM, ATP 20mM, MgCl, etc.)₂10 mM), and reacted at 37 ℃ for 15 min. After the reaction, the content of nicotinamide adenine dinucleotide in the reaction solution was measured by High Performance Liquid Chromatography (HPLC), and the results were measuredAs shown in table 1. One enzyme activity unit (U) is defined as the amount of enzyme required to convert one micromole of nicotinamide riboside per minute to nicotinamide adenine dinucleotide under the conditions described above.

TABLE 1

Enzyme solution	Sequence origin	Amount of NAD produced	Enzyme activity U/mg
ecNadR	Escherichia coli	0.2mM	0.05
stNadR	Salmonella typhimurium	0.2mM	0.06
hiNadR	Haemophilus influenzae	0.1mM	0.03
hihNadR1	The invention	0.8 mM	0.21
hihNadR2	The invention	7.0 mM	1.87
hiyNadR	The invention	1.6 mM	0.43
hiecNadR	The invention	5.9 mM	1.57
histNadR	The invention	7.4 mM	1.97