阅读说明：本技术 酶法工业化生产nad的方法 (Method for industrially producing NAD (nicotinamide adenine dinucleotide) by enzyme method ) 是由 张章 张琦 于 2019-06-27 设计创作，主要内容包括：酶法工业化生产NAD的方法,该方法以烟酰胺核糖和ATP为原料,在NAD合成酶存在的条件下进行生物催化反应制备NAD,其中,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。该方法可实现以NR和ATP为原料一步催化生产NAD。(The method for industrially producing NAD by enzyme method takes nicotinamide riboside and ATP as raw materials, and prepares NAD by biocatalytic reaction in the presence of NAD synthetase, wherein the NAD synthetase comprises nicotinamide mononucleotide adenyl transferase structural domain derived from haemophilus influenzae and nicotinamide ribokinase structural domain derived from any one of human, saccharomyces cerevisiae, escherichia coli and salmonella typhimurium. The method can realize one-step catalytic production of NAD by using NR and ATP as raw materials.)

The method for industrially producing NAD by an enzyme method is characterized by comprising the following steps: the NAD is prepared by taking nicotinamide riboside and ATP as raw materials and carrying out a biocatalytic reaction in the presence of NAD synthetase, wherein the NAD synthetase comprises a nicotinamide mononucleotide adenyl transferase structural domain derived from haemophilus influenzae and a nicotinamide ribokinase structural domain derived from any one of human, saccharomyces cerevisiae, escherichia coli and salmonella typhimurium.

The enzymatic industrial production method of NAD according to claim 1, characterized in that: the nicotinamide ribokinase domain is fused at the C-terminus of the nicotinamide mononucleotide adenylyltransferase domain.

The method for industrially producing NAD by an enzymatic method 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 method for industrially producing NAD by an enzymatic method 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 method for industrially producing NAD by an enzymatic method according to claim 4, wherein: the amino acid sequence of the NAD synthetase is shown as SEQ ID NO: 4 to SEQ ID NO: shown in fig. 8.

The enzymatic industrial production method of NAD according to claim 1, characterized in that: the biocatalytic reaction is in Mg²⁺And in the presence of a buffer.

The method for industrially producing NAD by an enzymatic method according to claim 6, wherein: the nicotinamide riboside, ATP and Mg ²⁺The feeding ratio of (A) to (B) is 1: 2: 0.1-1.

The method for industrially producing NAD by an enzymatic method according to claim 6, wherein: the buffer solution is phosphate buffer solution with pH value of 6.5-8.0.

The enzymatic industrial production method of NAD according to claim 1, characterized in that: the NAD synthetase is immobilized enzyme fixed on epoxy carrier.

The method for industrially producing NAD by an enzymatic method according to claim 9, wherein the NAD synthase is immobilized by:

(1) primary fixation: adding an alkali metal inorganic salt solution with the pH of 7.0-8.3 and an immobilized carrier material into the NAD synthetase, stirring to fully combine the NAD synthetase with the carrier material, and collecting primary immobilized enzyme;

(2) secondary fixation: adding an alkali metal inorganic salt solution with the pH of 9-10 into the primary immobilized enzyme in the step (1), stirring for 1-4 days, and collecting secondary immobilized enzyme;

(3) hydrophilic sealing: and (3) adding a hydrophilic amino acid solution with the pH value of 8.0-8.5 into the secondary immobilized enzyme obtained in the step (2), stirring for 8-30 hours, collecting the immobilized enzyme subjected to hydrophilic sealing, and washing with water to obtain the immobilized enzyme.

Technical Field

The invention relates to the technical field of enzyme method preparation and gene engineering of coenzyme products, in particular to a method for industrially producing NAD (nicotinamide adenine dinucleotide) by a biocatalysis technology.

Background

NAD, an acronym for Nicotinamide Adenine Dinucleotide (Nicotinamide Adenine Dinucleotide), is a physiological substance present in almost all living cells including human cells, has no toxic or side effects on human bodies, is a cofactor of many enzymes that catalyze oxidation-reduction reactions, participates in various physiological activities such as cellular substance metabolism, energy synthesis, and cellular DNA repair, 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 include a chemical method and a biocatalytic method (i.e., an enzymatic method), and the biocatalytic method is gradually the mainstream because of the advantages of mild reaction conditions, energy saving, 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. However, this method has some disadvantages, for example, it needs two biological enzymes and two-step enzyme catalysis reaction, and in order to avoid affecting the conversion rate, it is often to feed twice, which results in increasing the number of production operation steps and prolonging the reaction time.

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 the reaction solution (100 mM phosphate buffer, pH7.2, nicotinamide)Ribose 10mM, ATP 20mM, MgCl₂10 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 measurement results are 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

4. Immobilization of NAD synthetase

Adding sodium phosphate solution with the concentration of 1M, pH being 7 into the eluent collected from the 2 nd part, adding the immobilized carrier LX-1000EP according to the protein concentration of 50mg/g in the eluent, stirring for 24h at normal temperature, collecting the immobilized enzyme, transferring into 10mL of sodium phosphate solution with the concentration of 100mM and the pH value of 9, stirring for 72h at normal temperature to enhance the stability of the immobilized enzyme, then collecting the immobilized enzyme, transferring into 10mL of glycine solution with the concentration of 3M, pH being 8.5, and stirring for 24h at normal temperature to seal the immobilized enzyme carrier. Finally, the immobilized enzyme is collected and washed by pure water, and is stored at 4 ℃.

5. With immobilised enzymeshihNadR2 preparation of NAD

50mL of the reaction mixture (100 mM sodium phosphate solution pH7.2, nicotinamide ribose 10mM, ATP 20mM, MgCl₂10 mM) 1.36g was addedhihNadR2 immobilized enzyme (enzyme amount 1 g/L), at 37 ℃ 150rpm reaction for 30 min. After the reaction, the immobilized enzyme was collected on a filter cloth, and the NAD content in the reaction solution was measured by High Performance Liquid Chromatography (HPLC). The conversion of nicotinamide riboside to NAD was determined to be 95%.

6. With immobilised enzymeshiecNadR preparation of NAD

50mL of the reaction mixture (100 mM sodium phosphate solution pH7.2, nicotinamide ribose 10mM, ATP 20mM, MgCl₂10 mM) 1.44g was addedhiecNadR immobilized enzyme (enzyme amount 1 g/L), 37 ℃ 150rpm reaction for 30 min. After the reaction, the immobilized enzyme was collected on a filter cloth, and the NAD content in the reaction solution was measured by High Performance Liquid Chromatography (HPLC). Conversion of nicotinamide riboside to NAD was determined to be 80%.

7. With immobilised enzymeshistNadR preparation of NAD

50mL of the reaction mixture (100 mM sodium phosphate solution pH7.2, nicotinamide ribose 10mM, ATP 20mM, MgCl₂10 mM) 1.21g was addedhistNadR immobilized enzyme (enzyme amount 1 g/L), 37 ℃ 150rpm reaction for 30 min. After the reaction, the immobilized enzyme was collected on a filter cloth, and the NAD content in the reaction solution was measured by High Performance Liquid Chromatography (HPLC). Conversion of nicotinamide riboside to NAD was determined to be 98%.