CN114438052B - Nicotinamide mononucleotide adenyltransferase mutant and application thereof - Google Patents
Nicotinamide mononucleotide adenyltransferase mutant and application thereof Download PDFInfo
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- CN114438052B CN114438052B CN202011191122.5A CN202011191122A CN114438052B CN 114438052 B CN114438052 B CN 114438052B CN 202011191122 A CN202011191122 A CN 202011191122A CN 114438052 B CN114438052 B CN 114438052B
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- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
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- C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
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Abstract
The invention discloses a nicotinamide mononucleotide adenyltransferase mutant which can convert NMN into NAD. The enzyme has good thermal stability, can be repeatedly used for more than 20 times after being prepared into immobilized cells, greatly reduces the production cost of NAD, is beneficial to improving the market competitiveness of products, and has practical industrial application value.
Description
Technical field:
the invention belongs to the technical field of protein engineering, and particularly relates to a nicotinamide mononucleotide adenyltransferase mutant and an application thereof in converting NMN into NAD.
The background technology is as follows:
nicotinamide adenine dinucleotide (coenzyme I, english name: nicotinamide adenine dinucleotide, NAD) is an indispensable small molecular compound in organisms, and participates in oxidation-reduction reaction, energy transfer, substance metabolism, signal transduction and other processes. NAD is a very common coenzyme for dehydrogenases in organisms, which play a decisive role in the metabolism of organisms, and some organisms' essential metabolic movements, such as proteolysis, carbohydrate lysis, lipolysis, are independent of dehydrogenases. Since dehydrogenases must be involved in NAD in order to promote metabolic operations, they are indispensable substances in NAD organism metabolism.
NAD is one of the essential coenzymes for modern biocatalytic reactions. Modern biocatalytic techniques mimic reactions in vivo that are performed using enzymes, the conditions of which are strictly based on metabolic characteristics in vivo, so chiral reduction as catalyzed by common leucine dehydrogenase, ammonium formate dehydrogenase, glucose dehydrogenase, and partial ketoreductase enzymes all require the assistance of NAD to complete the entire reaction. In the material, chemical, food and pharmaceutical industries, the oxidation-reduction reaction (in particular chiral reduction) is the most numerous, intensive and mature reaction, so the demand of NAD in the industrial field is enormous.
There are many reports on the preparation method of NAD, and there are mainly three types of chemical synthesis, yeast extraction and enzymatic methods.
Chemical synthesis is limited in industrial production due to low conversion, expensive reagents, and a large number of operations of silica gel column and gel chromatographic column.
The method for extracting yeast has the advantages of mature process, huge equipment, high concentration energy consumption, low production efficiency and high cost.
The enzymatic synthesis mainly uses beta-Nicotinamide Mononucleotide (NMN) and ATP as substrates, and under the action of nicotinamide mononucleotide adenyltransferase (nicotinamide mononucleotide adenylyltransferase, NMNAT), NMN is adenylized to synthesize NAD. Since a large amount of enzymes are required to be used in production, a large amount of production costs are occupied. In order to reduce the production cost, the enzyme can be prepared into immobilized enzyme to increase the repeated application times of the enzyme, but the high and low thermal stability of the enzyme limits the immobilization technology to be successfully implemented. If the enzyme itself is gradually deactivated at the reaction temperature, the effect of subsequent batches may be rapidly deteriorated, although the effect on the conversion in the first reaction may not be very pronounced.
Thus, obtaining nicotinamide mononucleotide adenylyltransferase with higher thermostability has a direct positive impact on the subsequent preparation of immobilized enzyme and repeated use.
The invention comprises the following steps:
the invention aims at overcoming the defects of the prior art and providing a novel nicotinamide mononucleotide adenyltransferase mutant.
In one aspect, the amino acid sequence of the nicotinamide mononucleotide adenyltransferase mutant provided by the invention is an amino acid sequence which takes nicotinamide mononucleotide adenyltransferase shown in SEQ ID NO.2 as a reference sequence to be mutated, wherein aspartic acid at a 32 rd position is mutated into threonine, arginine at a 133 rd position is mutated into glycine, and glycine at a 166 th position is mutated into proline.
Further, the amino acid sequence of the nicotinamide mononucleotide adenyltransferase mutant is shown as SEQ ID NO. 4.
Further, the nucleotide sequence of the nicotinamide mononucleotide adenyltransferase mutant is shown as SEQ ID NO. 3.
Further, the wild-type gene sequence of the nicotinamide mononucleotide adenyltransferase mutant is derived from staphylococcus aureus (Staphylococcus aureus), and the accession number of the wild-type template NCBI is WP_031923054.1.
Furthermore, nicotinamide mononucleotide adenylyltransferase mutants are expressed in genetically engineered bacteria, preferably E.coli or yeast.
On the other hand, the nicotinamide mononucleotide adenyltransferase mutant provided by the invention can convert NMN into NAD.
Further, the nicotinamide mononucleotide adenylate transferase mutant is nicotinamide mononucleotide adenylate transferase enzyme powder or whole cells or cell disruption solution containing the nicotinamide mononucleotide adenylate transferase.
Further, the concentration of the nicotinamide mononucleotide adenylate transferase enzyme powder is 1-10 g/L.
Further, the nicotinamide mononucleotide adenylyltransferase cell concentration is 5-50 g/L.
Further, the NMN concentration is 2-20 g/L.
Further, 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 is 50-100 mmol/L.
Further, the reaction is carried out at a ph=5 to 8 and a temperature of 20 to 45 ℃.
The nicotinamide mononucleotide adenylyltransferase mutant disclosed by the invention can convert NMN into NAD. The enzyme has good thermal stability, can be repeatedly used for more than 20 times after being prepared into immobilized cells, greatly reduces the production cost of NAD, is beneficial to improving the market competitiveness of products, and has practical industrial application value.
Detailed Description
The technical content of the present invention will be further described with reference to specific embodiments, for better understanding of the content of the present invention, but the scope of the present invention is not limited thereto.
EXAMPLE 1 construction of site-directed saturation mutant library of nicotinamide mononucleotide adenyltransferase mutants
The nicotinamide mononucleotide adenylyltransferase mutant of SEQ ID No.2 (corresponding nucleotide sequence is SEQ ID No. 1) is butted with a substrate through a computer simulation structure, and the 32 site, 133 site and 166 site which are positioned in a loop region are presumed to be possibly related to the stability effect of the enzyme, and a saturated mutant library is constructed for the sites, wherein the specific sequence information is shown in Table 1.
TABLE 1 primer sequences for saturation mutagenesis
The underlined sequences in Table 1 are mutation sites, and the vectors with mutated genes were amplified using a full plasmid PCR amplification reaction. Then, the PCR product was subjected to recombinant plasmid template digestion with DpnI restriction enzyme, purified, transformed into E.coli BL21 (DE 3) for competence, then spread on LB plate containing 50ug/L Kan, and cultured upside down in an incubator at 37℃for 18 hours to give a monoclonal.
EXAMPLE 2 high throughput screening of Positive clones
188 monoclonals are randomly selected for each mutant to carry out 96-well plate shake culture, and each 96-well plate is inoculated with two unmutated strains as a control group, and 6 96-well plates are used in total. The specific operation is as follows: 400uL of LB medium was added to a sterile 96-well plate, incubated at 37℃for 16 hours, transferred to a second 96-well plate containing 2YT medium at 10% of the inoculum size, and the medium was added with antibiotic and IPTG at a final concentration of 0.1mM, and induced to express at 25℃for 24 hours. Centrifuging after the culture is finished, discarding supernatant, and freezing in a refrigerator at-20 ℃ for standby.
For screening, 96-well plates were removed from the refrigerator, 300uL of 10mM PBS (pH 7.0) was added to each well, and oven heat inactivated at 50℃for 6 hours. A living system was prepared according to Table 2, and 300uL of the living system was sucked by a discharge gun and transferred to the above heat-inactivated 96-well plate, and reacted at a constant temperature oscillator of 30℃and a rotation speed of 300rpm for 18 hours. And then carrying out HPLC detection on all the conversion reaction solutions, selecting the conversion rate higher than that of a control group as a candidate, and carrying out repeated detection and confirmation.
TABLE 2 reaction liquid System for screening saturated mutant library
| Raw materials | Concentration of |
| NMN | 100mM |
| ATP | 100mM |
| MgCl 2 | 20mM |
| pH7.0 Potassium phosphate Buffer | 200mM |
EXAMPLE 3 sequencing of Positive candidate mutants
In each saturated mutant library, mutants with highest conversion rate (higher than that of the control group WT) after heat inactivation are selected for sequencing, and the mutants are respectively found to be D32T, R133G and G166P according to sequence alignment. Table 3 shows the sequence mutation information obtained by sequencing, which was designated Mu01-Mu03.
TABLE 3 codon and amino acid mutation information after sequencing
| Name | Site | Sample | Codon | Mutation |
| Mu01 | D32 | 1F9 | GAT->ACT | D32T |
| Mu02 | R133 | 1D6 | CGA->GGT | R133G |
| Mu03 | G166 | 2H3 | GGG->CCG | G166P |
EXAMPLE 4 construction of combinatorial mutants
On the basis of the D32T mutant (Mu 01), a site-directed mutation primer is designed for R133G and G166P, and a combined mutant of three sites, namely an amino acid sequence shown in Seq ID No.4, is constructed and named Mu04. Mu04 and unmutated control WT were simultaneously inoculated with 5mL of LB tube medium containing kanamycin, cultured at 37℃for 12 hours, the activated culture was transferred to 100mL of 2YT liquid medium containing kanamycin in an inoculum size of 1%, OD was cultured at 37℃to 0.6-0.8, IPTG (final concentration 0.1 mM) was added, induced culture was performed at 25℃for 16 hours, and cells were collected by centrifugation.
Example 5 Heat stability test of combination mutants
The combined mutant and unmutated wild-type cells were each prepared as 300uL of 100g/L cell suspension and placed in an oven at 50℃for heat inactivation for 6 hours. A living system was prepared according to Table 2, and 300uL of the living system was sucked by a discharge gun and transferred to the above heat-inactivated 96-well plate, and reacted at a constant temperature oscillator of 30℃and a rotation speed of 300rpm for 18 hours. The reaction was then subjected to HPLC detection, and the result showed that Mu04 after heat inactivation for 6 hours had a substrate conversion of 97.1% after 18 hours of reaction, whereas the WT control group had a conversion of 30.7%.
EXAMPLE 6 preparation of nicotinamide mononucleotide adenylyltransferase mutant Mu04 cells/enzyme powder
Inoculating Mu04 mutant into 5mL LB test tube culture medium containing kanamycin for activation culture (culturing for 12 hours at 37 ℃), transferring the activation culture into 400mL 2YT liquid culture medium containing kanamycin according to the inoculum size of 1%, culturing OD to 0.6-0.8 at 37 ℃, adding IPTG (final concentration of 0.1 mM), performing induction culture at 25 ℃ for 16 hours, and centrifugally collecting thalli to obtain the nicotinamide mononucleotide adenyltransferase mutant Mu04 thalli cell.
After 20g of cells were resuspended in 40mL of phosphate buffer (10 mM, pH 7.5), the cells were homogenized and broken in a homogenizer, and the supernatant was collected by centrifugation, pre-frozen at-20℃and lyophilized in vacuo for 48 hours, followed by grinding to obtain an enzyme powder of nicotinamide mononucleotide adenyltransferase mutant Mu04.
EXAMPLE 7 preparation of nicotinamide mononucleotide adenylyltransferase immobilized cells
Mu04 cells were added to 100mM phosphate buffer to prepare a bacterial suspension having a concentration of 20% (v/v). Adding an amino resin carrier into the bacterial suspension, uniformly stirring, adding glutaraldehyde for crosslinking, stirring at 25 ℃ for 6 hours, filtering, draining, leaching the immobilized cells twice by using distilled water at 4 ℃, and draining in vacuum to obtain the immobilized Mu04 cells.
Example 8 preparation of NAD with Mu04 cells
Into a 200mL round bottom flask was added 50mL of a pre-formulated 200mM phosphate buffer pH7.0, 1mL of a pre-formulated 1M MgCl 2 1.7g NMN,3.0g ATP, pH7.0 was adjusted, 100mL of water was made up, mu04 cells (3 g) were added to the mixture, and the mixture was stirred in a water bath at 30℃for 18 hours. The results of the sampling HPLC detection showed the conversion rate to be 99.2%.
Example 9 preparation of NAD with immobilized Mu04 cells
Into a 200mL round bottom flask was added 50mL of a pre-formulated 200mM phosphate buffer pH7.0, 1mL of a pre-formulated 1M MgCl 2 1.7g NMN,3.0g ATP, pH7.0 was adjusted, 100mL of water was made up, mu04 immobilized cells (6.5 g) were added to a water bath at 30℃and reacted for 18 hours with stirring. The results of the sampling HPLC detection showed the conversion rate of 99.0%.
The immobilized cells are collected by centrifugation and repeatedly used according to the system, and the conversion rate is 95.2% when the immobilized cells are used for 20 th time.
Sequence listing
<120> nicotinamide mononucleotide adenyltransferase mutant and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 570
<212> DNA/RNA
<213> Staphylococcus aureus
<400> 1
atgaaaaaga taatacttta cggcggtcag tttaacccta tccatactgc acatatgata 60
gtagctagcg aagtatttca tgaattacag ccagatgaat 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 gtaaatcgag acaaaaatag tcaaaatgtt 420
gaaaatgcta tgattgcaat tcagatacct agggtagata taagttcgac aatgattcga 480
caaagagtta gtgaagggaa atctatccaa gttcttgttc ctaaatccgt tgaaaactat 540
attaaggggg aaggattata tgaacattga 570
<210> 2
<211> 189
<212> PRT
<213> Staphylococcus aureus
<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 Asp
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 Arg 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 Gly 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 (Artificial Sequence)
<400> 3
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> 4
<211> 189
<212> PRT
<213> Artificial sequence (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 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
Claims (4)
1. A nicotinamide mononucleotide adenyltransferase mutant is characterized in that the amino acid sequence of the nicotinamide mononucleotide adenyltransferase mutant is shown as SEQ ID NO. 4.
2. The nicotinamide mononucleotide adenyltransferase mutant of claim 1, which has a nucleotide sequence of a gene encoding the nicotinamide mononucleotide adenyltransferase mutant as shown in SEQ ID No. 3.
3. The nicotinamide mononucleotide adenyltransferase mutant of claim 1, which is expressed in genetically engineered bacteria.
4. The nicotinamide mononucleotide adenyltransferase mutant of claim 1, which is capable of converting NMN to NAD.
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| CN115873820B (en) * | 2021-09-29 | 2025-04-04 | 尚科生物医药（上海）有限公司 | A mutant of nicotinamide mononucleotide adenylyltransferase |
| CN118048332B (en) * | 2024-04-16 | 2024-06-21 | 邦泰生物工程(深圳)有限公司 | High-catalytic-performance glucose dehydrogenase mutant and application thereof in NMNH synthesis |
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| WO2014019108A1 (en) * | 2012-07-28 | 2014-02-06 | 深圳华大基因研究院 | Nmnat1 mutant gene, primers, kit, and method for detecting same and use thereof |
| CN105755019A (en) * | 2016-03-07 | 2016-07-13 | 华南师范大学 | Nicotinamide mononucleotide adenylyl transferase gene and application thereof |
| CN106884029A (en) * | 2015-12-16 | 2017-06-23 | 中国科学院大连化学物理研究所 | The method of enzyme' s catalysis niacinamide cytimidine dinucleotides |
| CN108884483A (en) * | 2016-03-16 | 2018-11-23 | 帝斯曼知识产权资产管理有限公司 | The micro-organisms of niacin riboside |
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| US7608276B2 (en) * | 2001-03-27 | 2009-10-27 | Novartis Vaccines And Diagnostics Srl | Staphylococcus aureus proteins and nucleic acids |
| WO2014019108A1 (en) * | 2012-07-28 | 2014-02-06 | 深圳华大基因研究院 | Nmnat1 mutant gene, primers, kit, and method for detecting same and use thereof |
| CN106884029A (en) * | 2015-12-16 | 2017-06-23 | 中国科学院大连化学物理研究所 | The method of enzyme' s catalysis niacinamide cytimidine dinucleotides |
| CN105755019A (en) * | 2016-03-07 | 2016-07-13 | 华南师范大学 | Nicotinamide mononucleotide adenylyl transferase gene and application thereof |
| CN108884483A (en) * | 2016-03-16 | 2018-11-23 | 帝斯曼知识产权资产管理有限公司 | The micro-organisms of niacin riboside |
Non-Patent Citations (2)
| Title |
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| Characterization and mutational analysis of a nicotinamide mononucleotide deamidase from agrobacterium tumefaciens showing high thermal stability and catalytic efficiency;Ana Belen Marinez-Monino等;PLOS ONE;第12卷(第4期);e0174759第1-20页 * |
| WP_031923054.1;GenPept;GenPept;序列 * |
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