CN112437813A - Method for industrially producing NAD (nicotinamide adenine dinucleotide) by enzyme method - Google Patents
Method for industrially producing NAD (nicotinamide adenine dinucleotide) by enzyme method Download PDFInfo
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- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 title description 54
- 229950006238 nadide Drugs 0.000 title description 52
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 title description 52
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
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.
Description
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.
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.
In view of the defects of the prior art mentioned in the background art, the invention aims to solve the technical problems of high production cost, complex operation and long production time of the existing enzymatic industrial production method of NAD, thereby developing a new method for enzymatic production of NAD suitable for large-scale industrialization.
In order to achieve the above object, the present invention provides a method for industrially producing NAD by an enzymatic method, comprising the step of preparing NAD by performing a biocatalytic reaction using nicotinamide riboside and ATP as raw materials in the presence of an NAD synthase comprising 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.
NAD synthase, which is Nicotinamide Adenine Dinucleotide (NAD) synthase, also known as NADR or NADR, is an enzyme that catalyzes the conversion of a substrate into Nicotinamide Adenine Dinucleotide, and has the advantage of catalyzing and synthesizing NAD in one step by using 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 the NAD synthetases presently known to occur in nature, 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. In view of the above, the inventors have conducted extensive experiments and screening on known genes and utilized genetic engineering techniques to screen for known target genesThe fragments are fused and recombined to obtain a series of recombinant NAD synthetases, and finally, fusion products with remarkably improved enzymatic activity are screened out, so that the invention is obtained.
In the method for industrially producing NAD by the enzyme method provided by the invention, Haemophilus influenzae, Saccharomyces cerevisiae, Escherichia coli and Salmonella typhimurium refer to all types of strains under the name of the strain, namely, the corresponding enzyme domains of all types of strains under the Haemophilus influenzae, Saccharomyces cerevisiae, Escherichia coli and Salmonella typhimurium are all applicable to the invention.
Preferably, in the method for industrially producing NAD by the enzymatic method provided by the invention, the nicotinamide ribokinase domain is fused at the C-terminal of the nicotinamide mononucleotide adenyltransferase domain.
More preferably, in the method for industrially producing NAD by the enzymatic method provided by the present invention, the nicotinamide ribokinase domain is fused with the nicotinamide mononucleotide adenylyltransferase domain via a flexible linker, and the sequence of the flexible linker is gsgsgsgsgs. The connecting peptide segment is specially designed according to the structural characteristics of two fused enzyme structural domains and is determined through multiple screening and experimental verification, and compared with other connecting peptide segments, the connecting peptide segment can play a role in enhancing protein expression.
More preferably, in the method for industrially producing NAD by the enzymatic method provided by the present invention, the amino acid sequence of the nicotinamide mononucleotide adenylyltransferase domain derived from Haemophilus influenzae is accession number P44308 in UniProt [52-224]]Is namedhiNMNAT; the amino acid sequences of the nicotinamide ribokinase domain from human, Saccharomyces cerevisiae, Escherichia coli and Salmonella typhimurium are sequentially the UniProt with the accession numbers Q9NWW6, Q9NPI5, P53915, P27278[230-]And P2458 [230-]The corresponding nomenclature is in turnhNRK1、 hNRK2、 yNRK1、 ecNRK andstNRK。
more preferably, in the method for industrially producing NAD by the enzymatic method provided by the present invention, the amino acid sequence of NAD synthase is as shown in SEQ ID NO: 4 to SEQ ID NO: shown in fig. 8.
Preferably, in the method for industrially producing NAD by the enzyme method provided by the invention, the biocatalytic reaction is carried out in Mg2+And in the presence of a buffer.
More preferably, in the method for industrially producing NAD by the enzyme method provided by the invention, nicotinamide riboside, ATP and Mg2+The feeding ratio of (A) to (B) is 1: 2: 0.1-1.
More preferably, in the method for industrially producing NAD by the enzyme method provided by the invention, Mg2+From MgCl2Or MgSO 24Provided is a method.
More preferably, in the method for industrially producing NAD by the enzymatic method provided by the invention, the buffer solution is a phosphate buffer solution with pH of 6.5-8.0.
More preferably, the buffer is phosphate buffer of pH7.2, which is the optimum pH for the reaction and maximizes conversion.
More preferably, in the method for industrially producing NAD by the enzymatic method provided by the invention, the concentration of the phosphate buffer is 20-100 mM.
More preferably, the phosphate buffer is present at a concentration of 100mM, which is more advantageous for stabilizing the pH in the reaction system.
Preferably, the NAD synthase used in the method for industrially producing NAD by the enzymatic method provided by the present invention is an immobilized enzyme immobilized on an epoxy-based carrier.
More preferably, the epoxy-based carrier is LX-1000EP, Sepabeads-EP or Eupergit C.
Preferably, the NAD synthase used in the method for industrially producing NAD by the enzymatic method provided by the present invention is immobilized by the following method:
(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.
More preferably, in the above immobilization method, the concentration of the alkali metal inorganic salt solution in the step (1) is 0.5 to 1M.
More preferably, in the above immobilization method, the amount of the carrier material added in step (1) is 1g/50mg NAD synthase.
In the immobilization method, the secondary immobilization operation in the step (2) is helpful for enhancing the stability of the immobilized enzyme, and the pH value of 9-10 is the key point for improving the stability of the immobilized enzyme and is helpful for forming new covalent linkage between enzyme molecules and a carrier material.
More preferably, in the above immobilization method, the concentration of the alkali metal inorganic salt solution in the step (2) is 50 to 200 mM.
More preferably, the concentration of the alkali metal inorganic salt solution in step (2) is 100 mM.
More preferably, in the above immobilization method, the alkali metal inorganic salt solution in step (1) and step (2) is at least one of sodium chloride, sodium phosphate and potassium phosphate.
More preferably, in the immobilization method, the concentration of the hydrophilic amino acid solution in step (3) is 1 to 3M.
More preferably, in the immobilization method, the hydrophilic amino acid in step (3) is glycine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline or aspartic acid.
Compared with the prior art, the method for industrially producing NAD by the enzyme method has the following advantages:
1. the method uses the NAD synthetase artificially synthesized based on the gene recombination technology, compared with the NAD synthetase existing in the nature, the enzyme activity of the NAD synthetase artificially synthesized is obviously improved, through test detection, the enzyme activity is 3.5-65 times of that of the common NAD synthetase existing in the nature, and the yield of the NAD generated by catalyzing the conversion of NR and ATP is increased by more than 400 percent, so that the method can be suitable for the large-scale chemical industry production of the NAD.
2. Compared with the existing method for producing NAD by biocatalysis, the method can realize one-step catalytic production of NAD by taking NR and ATP as raw materials, and can shorten the reaction time and reduce the industrial operation steps while reducing the feeding cost, thereby greatly reducing the production cost.
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%.
Claims (10)
- 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.
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| CN107955791A (en) * | 2017-11-20 | 2018-04-24 | 苏州东和盛昌生物科技有限公司 | It is a kind of can a step be catalyzed nicotinamide adenine dinucleotide strain and its screening technique and application |
| WO2018211028A1 (en) * | 2017-05-18 | 2018-11-22 | Dsm Ip Assets B.V. | Microbial production of nicotinamide riboside |
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| WO2018211028A1 (en) * | 2017-05-18 | 2018-11-22 | Dsm Ip Assets B.V. | Microbial production of nicotinamide riboside |
| CN107955791A (en) * | 2017-11-20 | 2018-04-24 | 苏州东和盛昌生物科技有限公司 | It is a kind of can a step be catalyzed nicotinamide adenine dinucleotide strain and its screening technique and application |
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