A mutant of nicotinamide mononucleotide adenylyltransferase

Abstract

The present invention discloses a nicotinamide mononucleotide adenylyltransferase mutant capable of converting NMN into NAD. The enzyme mutant has high catalytic efficiency and can convert a substrate concentration of 100 g / L, greatly improving the production capacity of NAD industrial production and reducing the production cost of NAD, and has practical industrial application value.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and in particular relates to a novel nicotinamide mononucleotide adenylyltransferase mutant. Background Art

[0002] Nicotinamide adenine dinucleotide (abbreviated as: coenzyme I, English name: nicotinamide adeninedinucleotide, NAD) is an indispensable small molecule compound in organisms, participating in redox reactions, energy transfer, material metabolism and signal transduction. NAD is a very common coenzyme of dehydrogenase in organisms. Dehydrogenase plays a decisive role in the metabolism of organisms. Some basic metabolic movements of organisms, such as protein decomposition, carbohydrate decomposition, and fat decomposition, are inseparable from dehydrogenase. Since dehydrogenase must be involved in NAD to promote metabolic operation, it is an indispensable substance in the metabolism of NAD organisms.

[0003] NAD is one of the essential coenzymes for modern biocatalytic reactions. Modern biocatalytic technology simulates the reactions that are completed by enzymes in organisms. The simulation conditions of this reaction are strictly based on the metabolic characteristics of the organism. Therefore, chiral reduction catalyzed by common leucine dehydrogenase, ammonium formate dehydrogenase, glucose dehydrogenase, and some ketoreductases all require the help of NAD to complete the entire reaction. In the materials, chemical, food, and pharmaceutical industries, redox reactions (especially chiral reduction) are the largest, most deeply studied, and most mature type of reactions, so the demand for NAD in the industrial field is huge.

[0004] There are many reports on the preparation methods of NAD, which mainly include chemical synthesis, yeast extraction and enzymatic preparation.

[0005] The chemical synthesis method is subject to certain limitations in industrial production due to its low conversion rate, expensive reagents, and a large number of operations involving silica gel columns and gel chromatography columns.

[0006] Although the process of extracting amino acids from yeast is mature, the equipment is large, the concentration consumes a lot of energy, the production efficiency is low, and the cost is high.

[0007] Enzymatic synthesis mainly uses NMN and ATP as substrates, and NMN is adenylated to synthesize NAD under the action of nicotinamide mononucleotide adenylyltransferase (NMNAT). However, the substrate concentrations of the NMN adenylation reactions reported so far are not very high. In the embodiment described in patent CN103710321B, the NMN concentration is 5mM, which is equivalent to a mass concentration of about 1.6g / L. Considering that the ideal substrate concentration of general chemicals in industrial production is greater than 100g / L, if the substrate concentration catalyzed by the enzyme can be increased, the production capacity can be greatly increased and production costs can be saved.

[0008] Therefore, it is necessary to obtain a nicotinamide mononucleotide adenylyltransferase that can convert high concentrations of NMN into NAD. Summary of the invention

[0009] The purpose of the present invention is to provide a new nicotinamide mononucleotide adenylyltransferase mutant in view of the deficiencies of the prior art.

[0010] On the one hand, the amino acid sequence of the nicotinamide mononucleotide adenylyltransferase mutant provided by the present invention is an amino acid sequence in which the nicotinamide mononucleotide adenylyltransferase shown in SEQ ID NO.2 is mutated as a reference sequence, and the mutation sites are that the histidine at position 18 is mutated to alanine, the proline at position 43 is mutated to glycine, the tyrosine at position 84 is mutated to glycine, and the aspartic acid at position 153 is mutated to proline.

[0011] Furthermore, the amino acid sequence of the nicotinamide mononucleotide adenylyltransferase mutant is shown in SEQ ID NO.4.

[0012] Furthermore, the nucleotide sequence of the nicotinamide mononucleotide adenylyltransferase mutant is shown in SEQ ID NO.3.

[0013] Furthermore, the amino acid sequence SEQ ID NO. 2 used as a reference sequence is mutated based on the wild-type amino acid sequence.

[0014] Furthermore, the wild-type gene sequence of the nicotinamide mononucleotide adenylyltransferase mutant is derived from Staphylococcus aureus, and the accession number of the wild-type template in NCBI is WP_031923054.1.

[0015] Furthermore, the nicotinamide mononucleotide adenylyltransferase mutant is expressed in a genetically engineered bacterium, preferably Escherichia coli or yeast.

[0016] On the other hand, the nicotinamide mononucleotide adenylyltransferase mutant provided by the present invention can convert NMN into NAD.

[0017] Furthermore, the NMN concentration is 100 g / L.

[0018] Furthermore, the nicotinamide mononucleotide adenylyltransferase mutant is nicotinamide mononucleotide adenylyltransferase enzyme powder or whole cells or cell disrupted liquid containing the nicotinamide mononucleotide adenylyltransferase.

[0019] Furthermore, the concentration of the nicotinamide mononucleotide adenylyltransferase powder is 1 to 20 g / L.

[0020] Furthermore, the nicotinamide mononucleotide adenylyltransferase cell concentration is 5 to 60 g / L.

[0021] Furthermore, the reaction is carried out in a buffer solution, wherein the buffer solution is a phosphate buffer or a triethanolamine buffer, preferably a phosphate buffer, and the concentration of the buffer solution is 50 to 100 mmol / L.

[0022] Furthermore, the reaction is carried out at a pH of 5 to 8 and a temperature of 20 to 45°C.

[0023] The beneficial effect of the present invention is that the nicotinamide mononucleotide adenylyltransferase mutant disclosed in the present invention can convert 100 g / L NMN into NAD, which is more than 5 times higher than the conversion capacity of the mutant template. At the same time, the thermal stability of the mutant is also improved. After being prepared into immobilized cells, it can be repeatedly applied more than 20 times, which greatly improves the production capacity of NAD industrial production, reduces production costs, is conducive to improving the market competitiveness of products, and has practical industrial application value. DETAILED DESCRIPTION

[0024] The technical content of the present invention is further described below in conjunction with specific embodiments, the purpose of which is to provide a better understanding of the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0025] Example 1 Construction and expression of nicotinamide mononucleotide adenylyltransferase mutants

[0026] The structure of nicotinamide mononucleotide adenylyltransferase of SEQ ID NO.2 (the corresponding nucleotide sequence is SEQ ID NO.1, which is used as a mutant template and control group and named CT) was simulated by computer and docked with the substrate. It was speculated that the amino acids at positions 18, 43, 84, and 153 are located at the exit of the active center or substrate pocket, which may be closely related to the catalytic activity of the enzyme. After saturation mutation and screening of these sites, and combined mutation of positive mutants, H18A / P43G / Y84G / D153P was screened using a 96-well plate, and the catalytic efficiency was significantly improved compared with the mutant template, which was named Mu (i.e., SEQ ID No.4, the corresponding nucleotide sequence is SEQ ID No.2). The cells of mutant Mu and mutant template CT were transferred from glycerol tubes to 5 mL of LB test tube culture medium containing kanamycin, cultured at 37°C for 12 h, and the activated culture was transferred to 400 mL of 2YT liquid culture medium containing kanamycin at a 1% inoculum amount, cultured at 37°C until OD reached 0.6-0.8, and IPTG (final concentration 0.1 mM) was added to induce culture at 25°C for 16 h, and the bacteria were collected by centrifugation.

[0027] Example 2 Comparison of the responses of nicotinamide mononucleotide adenylyltransferase mutants and control cells

[0028] The reaction system was prepared according to the system in Table 1, and the reaction volume was 10 mL. First, other materials except cells were added, and the pH was adjusted to 7.0 with NaOH, and then mutant Mu or CT cells were added, blown evenly with a pipette, and reacted for 18 hours in a 37°C water bath with magnetic stirring, and then samples were taken, and an equal volume of 0.1M HCl was added, and the samples were centrifuged and sent to HPLC for detection. The results showed that the conversion rate of mutant Mu reached 96.3% after 18 hours, while the conversion rate of the CT control group was only 42.4%.

[0029] Table 1 Nicotinamide mononucleotide adenylyltransferase reaction system

[0030] raw material concentration NMN 100g / L ATP 190g / L <![CDATA[MgCl 2 ]]> 1.2g / L cell 50g / L pH 7.0 Potassium Phosphate Buffer 100mM

[0031] Example 3 Preparation of immobilized cells of nicotinamide mononucleotide adenylyltransferase mutants

[0032] Add Mu bacterial cells to 100mM phosphate buffer to prepare a bacterial suspension with a concentration of 20% (v / v). Add an amino resin carrier to the bacterial suspension, stir evenly, and then add 25% glutaraldehyde for cross-linking. After stirring at 25°C for 24 hours, filter and drain, then rinse the immobilized cells twice with 4°C distilled water, and vacuum dry to obtain immobilized Mu cells.

[0033] Example 4 Preparation of NAD using CT cells

[0034] Add 1.7 g NMN, 3.0 g ATP, 0.12 g MgCl to a 200 mL round-bottom flask. 2 , 50 mL of 100 mM phosphate buffer at pH 7.0, add water to 100 mL, add 5 g of CT cells, stir in a 37°C water bath for 18 h. Samples were taken for HPLC testing, and the results showed that the conversion rate was 99.2%.

[0035] Example 5 Preparation of NAD using CT cells

[0036] Add 10 g NMN, 19 g ATP, and 0.12 g MgCl to a 200 mL round bottom flask. 2 , 50 mL of 100 mM phosphate buffer at pH 7.0, adjust the pH to 7.0, make up to 100 mL with buffer, add 5 g of CT cells, stir in a 37°C water bath for 18 h. Samples were taken for HPLC detection, and the results showed that the conversion rate was 42.4%.

[0037] Example 6 Preparation of NAD using mutant Mu cells

[0038] Add 10 g NMN, 19 g ATP, and 0.12 g MgCl to a 200 mL round bottom flask. 2 , 50 mL of 100 mM phosphate buffer at pH 7.0, adjust the pH to 7.0, make up to 100 mL with buffer, add 5 g of Mu cells, stir in a 37°C water bath for 18 h. Samples were taken for HPLC testing, and the results showed that the conversion rate was 95.7%.

[0039] Example 7 Preparation of NAD Using Immobilized Mu Cells

[0040] Add 10 g NMN, 19 g ATP, and 0.12 g MgCl to a 200 mL round bottom flask. 2 , 50 mL of 100 mM phosphate buffer at pH 7.0, adjust the pH to 7.0, make up to 100 mL with buffer, add 8.5 g of Mu immobilized cells, stir and react in a 37°C water bath for 18 hours. Samples were taken for HPLC detection, and the results showed that the conversion rate was 95.3%.

[0041] The fixed cells were collected by centrifugation and the above system was repeatedly applied. At the 20th application, the conversion rate still reached 91.2%. Sequence Listing <110> Shangke Biopharmaceuticals (Shanghai) Co., Ltd. <120> A mutant of nicotinamide mononucleotide adenylyltransferase <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 570 <212> DNA / RNA <213> Artificial Sequence <400> 1 atgaaaaaga taatacttta cggcggtcag tttaacccta tccatactgc acatatgata 60 gtagctagcg aagtatttca tgaattacag ccaactgaat tttatttttt acctagtttt 120 atgtctccat tgaaaaagca ccatgatttt atagacgttc agcacagatt aacaatgata 180 cagatgatta tcgacgagct tggttttgga gatatttgtg acgatgaaat taaacgtggt 240 ggtcaaagtt atacctatga cacgatcaag gcattcaagg agcaacacaa agacagtgag 300 ttgtactttg ttattgggac ggatcagtat aaccaactag agaaatggta tcaaattgaa 360 tacttaaaag aaatggttac ttttgtagtt gtaaatggtg acaaaaatag tcaaaatgtt 420 gaaaatgcta tgattgcaat tcagatacct agggtagata taagttcgac aatgattcga 480 caaagagtta gtgaaccgaa atctatccaa gttcttgttc ctaaatccgt tgaaaactat 540 attaaggggg aaggattata tgaacattga 570 <210> 2 <211> 189 <212> PRT <213> Artificial Sequence <400> 2 Met Lys Lys Ile Ile Leu Tyr Gly Gly Gln Phe Asn Pro Ile His Thr 1 5 10 15 Ala His Met Ile Val Ala Ser Glu Val Phe His Glu Leu Gln Pro Thr 20 25 30 Glu Phe Tyr Phe Leu Pro Ser Phe Met Ser Pro Leu Lys Lys His His 35 40 45 Asp Phe Ile Asp Val Gln His Arg Leu Thr Met Ile Gln Met Ile Ile 50 55 60 Asp Glu Leu Gly Phe Gly Asp Ile Cys Asp Asp Glu Ile Lys Arg Gly 65 70 75 80 Gly Gln Ser Tyr Thr Tyr Asp Thr Ile Lys Ala Phe Lys Glu Gln His 85 90 95 Lys Asp Ser Glu Leu Tyr Phe Val Ile Gly Thr Asp Gln Tyr Asn Gln 100 105 110 Leu Glu Lys Trp Tyr Gln Ile Glu Tyr Leu Lys Glu Met Val Thr Phe 115 120 125 Val Val Val Asn Gly Asp Lys Asn Ser Gln Asn Val Glu Asn Ala Met 130 135 140 Ile Ala Ile Gln Ile Pro Arg Val Asp Ile Ser Ser Thr Met Ile Arg 145 150 155 160 Gln Arg Val Ser Glu Pro Lys Ser Ile Gln Val Leu Val Pro Lys Ser 165 170 175 Val Glu Asn Tyr Ile Lys Gly Glu Gly Leu Tyr Glu His 180 185 <210> 3 <211> 570 <212> DNA / RNA <213> Artificial Sequence <400> 3 atgaaaaaga taatacttta cggcggtcag tttaacccta tccatactgc agctatgata 60 gtagctagcg aagtatttca tgaattacag ccaactgaat tttatttttt acctagtttt 120 atgtctggat tgaaaaagca ccatgatttt atagacgttc agcacagatt aacaatgata 180 cagatgatta tcgacgagct tggttttgga gatatttgtg acgatgaaat taaacgtggt 240 ggtcaaagtg gtacctatga cacgatcaag gcattcaagg agcaacacaa agacagtgag 300 ttgtactttg ttattgggac ggatcagtat aaccaactag agaaatggta tcaaattgaa 360 tacttaaaag aaatggttac ttttgtagtt gtaaatggtg acaaaaatag tcaaaatgtt 420 gaaaatgcta tgattgcaat tcagatacct agggtaccta taagttcgac aatgattcga 480 caaagagtta gtgaaccgaa atctatccaa gttcttgttc ctaaatccgt tgaaaactat 540 attaaggggg aaggattata tgaacattga 570 <210> 4 <211> 189 <212> PRT <213> Artificial Sequence <400> 4 Met Lys Lys Ile Ile Leu Tyr Gly Gly Gln Phe Asn Pro Ile His Thr 1 5 10 15 Ala Ala Met Ile Val Ala Ser Glu Val Phe His Glu Leu Gln Pro Thr 20 25 30 Glu Phe Tyr Phe Leu Pro Ser Phe Met Ser Gly Leu Lys Lys His His 35 40 45 Asp Phe Ile Asp Val Gln His Arg Leu Thr Met Ile Gln Met Ile Ile 50 55 60 Asp Glu Leu Gly Phe Gly Asp Ile Cys Asp Asp Glu Ile Lys Arg Gly 65 70 75 80 Gly Gln Ser Gly Thr Tyr Asp Thr Ile Lys Ala Phe Lys Glu Gln His 85 90 95 Lys Asp Ser Glu Leu Tyr Phe Val Ile Gly Thr Asp Gln Tyr Asn Gln 100 105 110 Leu Glu Lys Trp Tyr Gln Ile Glu Tyr Leu Lys Glu Met Val Thr Phe 115 120 125 Val Val Val Asn Gly Asp Lys Asn Ser Gln Asn Val Glu Asn Ala Met 130 135 140 Ile Ala Ile Gln Ile Pro Arg Val Pro Ile Ser Ser Thr Met Ile Arg 145 150 155 160 Gln Arg Val Ser Glu Pro Lys Ser Ile Gln Val Leu Val Pro Lys Ser 165 170 175 Val Glu Asn Tyr Ile Lys Gly Glu Gly Leu Tyr Glu His 180 185

Claims

1. A nicotinamide mononucleotide adenylyltransferase mutant, characterized in that: The amino acid sequence of the nicotinamide mononucleotide adenylyltransferase mutant is shown in SEQ ID NO.

4.

2. The nicotinamide mononucleotide adenylyltransferase mutant according to claim 1, characterized in that The gene nucleotide sequence of the nicotinamide mononucleotide adenylyltransferase mutant is shown in SEQ ID NO.

3.

3. The nicotinamide mononucleotide adenylyltransferase mutant according to claim 1, characterized in that The nicotinamide mononucleotide adenylyltransferase mutant is expressed in genetically engineered bacteria.

4. The nicotinamide mononucleotide adenylyltransferase mutant according to claim 1, characterized in that The nicotinamide mononucleotide adenylyltransferase mutant is capable of converting NMN into NAD.

5. The nicotinamide mononucleotide adenylyltransferase mutant according to claim 4, characterized in that: The NMN concentration is 100g / L.