阅读说明：本技术 一种生物催化的高纯度nmn生产工艺 (Biocatalytic high-purity NMN production process ) 是由 丰明乾 朱元奎 刘路 于 2021-09-09 设计创作，主要内容包括：本发明涉及一种生物催化的高纯度NMN生产工艺,S1、构建重组表达载体,S2、利用重组表达载体转化宿主细胞,获得遗传工程化的宿主细胞,S3、培养遗传工程化的宿主细胞,持续性表达烟酰胺核苷激酶,S4、添加底物烟酰胺核苷,继续培养细胞,利用摇瓶法培养或发酵罐培养,S5、离心收取上清,进行HPLC反应。本发明的优点：成本低,生产工艺简单等优点。(The invention relates to a biocatalytic high-purity NMN production process, S1, constructing a recombinant expression vector, S2, transforming a host cell by using the recombinant expression vector to obtain a genetically engineered host cell, S3, culturing the genetically engineered host cell, continuously expressing nicotinamide riboside kinase, S4, adding substrate nicotinamide riboside, continuously culturing the cell, culturing by using a shake flask method or a fermentation tank, S5, centrifuging, collecting supernatant, and carrying out HPLC reaction. The invention has the advantages that: low cost, simple production process and the like.)

1. A biocatalytic high purity NMN production process is characterized in that: comprises using nicotinamide riboside as substrate, using full 293F cell containing nicotinamide riboside kinase to perform one-step method to efficiently prepare beta-nicotinamide mononucleotide, the expression is as follows:

2. a biocatalytic high purity NMN production process according to claim 1, characterized by: the amino acid sequence of the enzyme is shown as protein shown in SEQ ID No. 1.

3. A biocatalytic high purity NMN production process according to claim 1, characterized by: the nucleotide sequence of the gene for coding the protein shown by SEQ ID No.1 is shown as SEQ ID No. 2.

4. A recombinant expression vector characterized by: the recombinant expression vector contains a gene for nicotinamide riboside kinase for NMN preparation.

5. A genetically engineered host cell characterized by: the host cell comprises a recombinant expression vector for the gene for nicotinamide riboside kinase for NMN production.

6. A genetically engineered host cell according to claim 5, wherein: the host cell is 293F cell.

7. A method of preparing high purity NMN, characterized by: the method comprises the following steps:

s1, constructing a recombinant expression vector,

s2, transforming the host cell by the recombinant expression vector to obtain genetically engineered host cell,

s3, culturing the genetically engineered host cell to continuously express nicotinamide riboside kinase,

s4, adding substrate nicotinamide riboside, continuously culturing the cells, culturing by using a shake flask method or a fermentation tank,

s5, centrifuging to collect the supernatant, and carrying out HPLC reaction.

8. The method of preparing high purity NMN according to claim 7, wherein: and step S3, culturing the constructed stable transgenic nicotinamide riboside kinase 293F cells in a shake flask by using a serum-free culture medium, and adjusting the density to 5-6 milliion/ml.

9. The method of preparing high purity NMN according to claim 7, wherein: the step S4 is to add 50mM nicotinamide riboside to the culture medium and shake the reaction for 4h at 37 ℃.

Technical Field

The invention relates to the technical field of enzyme engineering, in particular to a biocatalytic high-purity NMN production process.

Background

NMN is known as beta-nicotinamide mononucleotide and is one of the precursors of the cofactor NAD + of the human longevity protein. Can quickly become NAD + in human body, exerts fundamental influence on human health, leads hundreds of life activities in human body, and controls the rhythm of diseases and aging.

NAD + has too large molecular weight, cannot be directly absorbed and supplemented by oral administration, and mainly depends on cell synthesis in vivo, but the synthesis amount is very low, and the level of NAD + self-composed in the elderly human body is far from reaching the required level, and a large number of advanced scientific researches show that: NMN can directly promote the self-generation of NAD + in vivo, and is the most ideal pipeline for supplementing NAD +. The NMN is supplemented, so that the content of NAD < + > in a body can be effectively improved, the physiological decline of the organism is remarkably slowed down, the energy metabolism is enhanced, and the service life is prolonged. Recent research shows that the NMN in organisms is regulated to have better treatment and repair effects on cardiovascular and cerebrovascular diseases, neurodegenerative diseases, aging degenerative diseases and the like.

However, most of the NMN is derived from chemical synthesis at present, the production rate is low, the product purity is low, the product contains about 50% of chiral molecular NMN, and the NMN is toxic and difficult to separate, so the yield is very low, and a large amount of organic solvents are used, and the damage to the environment is serious.

In recent years, the biocatalysis technology is used for the production of NMN, is green biological conversion, has almost zero environmental pollution, does not generate chiral molecules NMN, has high yield and low cost, and is a great revolution of the NMN production technology. However, the designed synthetic methods are relatively complex, and the conversion rate and the yield are generally not high.

Disclosure of Invention

The technical problem to be solved by the present invention is to solve the above problems and to provide a biocatalytic high purity NMN production process that provides conversion and yield.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a biocatalytic high-purity NMN production process comprises the steps of taking nicotinamide riboside as a substrate, and carrying out one-step method high-efficiency preparation of beta-nicotinamide mononucleotide by using a cell containing nicotinamide riboside kinase 293F, wherein the expression is as follows:

the amino acid sequence of the enzyme is shown as protein shown in SEQ ID No. 1.

The nucleotide sequence of the gene for coding the protein shown by SEQ ID No.1 is shown as SEQ ID No. 2.

A recombinant expression vector comprising a gene for nicotinamide riboside kinase for NMN production.

A genetically engineered host cell comprising a recombinant expression vector for the gene of nicotinamide riboside kinase for NMN production.

The host cell is 293F cell.

A method of preparing high purity NMN, comprising the steps of:

s1, constructing a recombinant expression vector,

s2, transforming the host cell by the recombinant expression vector to obtain genetically engineered host cell,

s3, culturing the genetically engineered host cell to continuously express nicotinamide riboside kinase,

s4, adding substrate nicotinamide riboside, continuously culturing the cells, culturing by using a shake flask method or a fermentation tank,

s5, centrifuging to collect the supernatant, and carrying out HPLC reaction.

And step S3, culturing the constructed stable transgenic nicotinamide riboside kinase 293F cells in a shake flask by using a serum-free culture medium, and adjusting the density to 5-6 milliion/ml.

The step S4 is to add 50mM nicotinamide riboside to the culture medium and shake the reaction for 4h at 37 ℃.

After adopting the structure, the invention has the following advantages: the whole-cell catalyst has the advantages of low cost, simple production process and the like. The NRK1 gene is cloned into a cell by means of synthetic biology, and NMN can be prepared in multiple batches under the catalysis of the whole cell of the genetic engineering, so that the yield of the NMN is improved, and the production cost is reduced; overcomes the defects of the synthesis of the current chemical synthesis method and the complexity of the catalysis of the existing biological enzyme method, provides the nicotinamide riboside kinase 294F cell and the process for synthesizing NMN by biological catalysis, effectively reduces the complexity of the reaction, can be safely and efficiently used in actual production, can repeatedly use production raw materials for many times, and further effectively reduces the investment of actual cost.

Drawings

FIG. 1 is a map of an expression plasmid of the present invention.

FIG. 2 is a graph showing the analysis of the result of the catalytic synthesis of NMN by 293F-whole cells according to the present invention.

FIG. 3 is an analytical graph of a 10g/L NMN standard of the present invention.

Detailed Description

The present invention is described in further detail below.

Combining all the figures, a biocatalytic high-purity NMN production process comprises the steps of taking nicotinamide riboside as a substrate, and carrying out one-step method high-efficiency preparation of beta-nicotinamide mononucleotide by using a cell containing nicotinamide riboside kinase 293F, wherein the expression is as follows:

the amino acid sequence of the enzyme is shown as protein shown in SEQ ID No. 1.

The nucleotide sequence of the gene for coding the protein shown by SEQ ID No.1 is shown as SEQ ID No. 2.

A recombinant expression vector comprising a gene for nicotinamide riboside kinase for NMN production.

A genetically engineered host cell comprising a recombinant expression vector for the gene of nicotinamide riboside kinase for NMN production.

The host cell is 293F cell.

A method of preparing high purity NMN, comprising the steps of:

s1, constructing a recombinant expression vector,

s2, transforming the host cell by the recombinant expression vector to obtain genetically engineered host cell,

s3, culturing the genetically engineered host cell to continuously express nicotinamide riboside kinase,

s4, adding substrate nicotinamide riboside, continuously culturing the cells, culturing by using a shake flask method or a fermentation tank,

s5, centrifuging to collect the supernatant, and carrying out HPLC reaction.

And step S3, culturing the constructed stable transgenic nicotinamide riboside kinase 293F cells in a shake flask by using a serum-free culture medium, and adjusting the density to 5-6 milliion/ml.

The step S4 is to add 50mM nicotinamide riboside to the culture medium and shake the reaction for 4h at 37 ℃.

In the practice of the present invention, the construction of an enzyme gene expression system is described

The NRK1 gene is constructed on the PBSPS plasmid vector by using restriction enzyme cutting sites EcoRV and BspEI to form a recombinant plasmid PBSPS-NRK 1-His. The constructed recombinant plasmid was linearized with HindIII enzyme, the linearized recombinant plasmid was transfected into 293F cells using PEI, 3 days after transfection, the transfected 293F cells were aliquoted into 96-well plates and screened using 2ug/ml puro. After 2 weeks of screening in a 96-well plate, supernatant of the surviving monoclonal cell line was subjected to ELISA detection using anti-His secondary antibody, and a monoclonal 293F cell line highly expressing NKR1 was screened. Carrying out mass propagation, and finally carrying out suspension culture in a shake flask.

EXAMPLE two catalytic Synthesis of NMN in 293F-Whole cells

The constructed whole 293F cells of stable nicotinamide riboside kinase (NRK1) were cultured in a shake flask using serum-free medium at a density of 5-6 milliion/ml. Nicotinamide Nucleoside (NR) 50mM was added to the medium and reacted at 37 ℃ with shaking for 4 h. The supernatant was collected by centrifugation and subjected to HPLC reaction. By comparison with the standard, the production amount of NMN was 13.21g/L, and the conversion efficiency was 87.4%. After the cells were collected again and cultured in a serum-free medium, catalytic synthesis of NMN was repeated, and by comparison with the standard, NMN production of 12.54g/L was obtained, with a conversion efficiency of 82.7%. Indicating that the cells can be reused.

The experimental results further show that the invention can be safely and efficiently used in actual production, and effectively reduce the investment of actual cost.

The amino acid sequence (SEQ ID NO: 1) of nicotinamide riboside kinase (NRK1) contained in the 293F-intact stable cells of the invention is as follows:

MKTFIIGISGVTNSGKTTLAKNLQKHLPNCSVISQDDFFKPESEIETDKNGFLQYDVLEALNMEKMMSAISCWMESARHSVVSTDQESAEEIPILIIEGFLLFNYKPLDTIWNRSYFLTIPYEECKRRRSTRVYQPPDSPGYFDGHVWPMYLKYRQEMQDITWEVVYLDGTKSEEDLFLQVYEDLIQELAKQKCLQVTA。

the nucleotide sequence (SEQ ID NO: 2) of nicotinamide riboside kinase (NRK1) contained in the 293F-intact stable cells of the invention is as follows: ATGAAAACATTTATCATTGGAATCAGTGGTGTGACAAACAGTGGCAAAACAACACTGGCTAAGAATTTGCAGAAACACCTCCCAAATTGCAGTGTCATATCTCAGGATGATTTCTTCAAGCCAGAGTCTGAGATAGAGACAGATAAAAATGGATTTTTGCAGTACGATGTGCTTGAAGCACTTAACATGGAAAAAATGATGTCAGCCATTTCCTGCTGGATGGAAAGCGCAAGACACTCTGTGGTATCAACAGACCAGGAAAGTGCTGAGGAAATTCCCATTTTAATCATCGAAGGTTTTCTTCTTTTTAATTATAAGCCCCTTGACACTATATGGAATAGAAGCTATTTCCTGACTATTCCATATGAAGAATGTAAAAGGAGGAGGAGTACAAGGGTCTATCAGCCTCCAGACTCTCCGGGATACTTTGATGGCCATGTGTGGCCCATGTATCTAAAGTACAGACAAGAAATGCAGGACATCACATGGGAAGTTGTGTACCTGGATGGAACAAAATCTGAAGAGGACCTCTTTTTGCAAGTATATGAAGATCTAATACAAGAACTAGCAAAGCAAAAGTGTTTGCAAGTGACAGCATAA are provided.

The working principle of the invention is as follows: the whole-cell catalyst has the advantages of low cost, simple production process and the like. And the NRK1 gene is cloned into a cell by means of synthetic biology, and the NMN can be prepared in multiple batches under the catalysis of the whole cell of the genetic engineering, so that the yield of the NMN is improved, and the production cost is reduced.

The present invention and its embodiments have been described above, but the description is not limitative, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.

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