CN115109764A - NAD kinase mutant and application thereof - Google Patents

NAD kinase mutant and application thereof Download PDF

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CN115109764A
CN115109764A CN202110302988.7A CN202110302988A CN115109764A CN 115109764 A CN115109764 A CN 115109764A CN 202110302988 A CN202110302988 A CN 202110302988A CN 115109764 A CN115109764 A CN 115109764A
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张小飞
倪旎
竺伟
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SYNCOZYMES (SHANGHAI) CO Ltd
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Abstract

The invention discloses an NAD kinase mutant which can convert NAD into NADP. Compared with wild type NAD kinase, the NAD kinase mutant has the advantages of improving the catalytic concentration of a substrate, reducing the reaction time and having great industrial application value.

Description

NAD kinase mutant and application thereof
The technical field is as follows:
the invention belongs to the technical field of protein engineering, and particularly relates to an NAD kinase mutant capable of converting NAD into NADP.
Background art:
oxidized beta-Nicotinamide adenine dinucleotide phosphate (coenzyme II, NADP) is an extremely important nucleotide coenzyme. Oxidized coenzyme II transfers protons, electrons, and energy in redox reactions and is involved in many cellular metabolic reactions. Coenzyme II has wide application in the fields of life science, enzyme catalysis asymmetric synthesis and medical care.
Coenzyme II is widely present in organisms, but is present in very low amounts. The current methods for the synthesis of NADP are mainly divided into chemical and biological methods. The chemical method takes nicotinamide as a raw material, and synthesizes NADP through multi-step reaction, but the chemical method has the problems of long reaction route, harsh reaction conditions, poor selectivity, easy generation of byproducts, low yield and the like, and has the problems of high production cost, need of using an organic solvent and environmental pollution.
Biological methods are further classified into conventional fermentation methods and enzyme methods, wherein the fermentation method is to obtain NADP by separating and extracting yeast or other microorganisms using fermentation or other microorganism culture techniques, such as the fermentation method used by Roche corporation. However, the route has the disadvantages of high raw material consumption and energy consumption, low atom utilization rate, limited yield and high production cost, and limits the wide application of NADP. The enzymatic synthesis of NADP is a more efficient reaction, and has the advantages of mild reaction conditions, strong stereoselectivity, high conversion rate compared with a fermentation method and the like.
NADP is synthesized in vivo mainly by catalyzing NAD phosphorylation with ATP or a polyphosphoric acid (poly (p)) derivative as a phosphate donor under the action of NAD kinase. In organisms with sequenced genomes, most of the organisms can search homologous genes of NAD kinase, the catalytic reaction of the NAD kinase is the last step of an NADP biosynthesis pathway, and the overexpression of the NAD kinase can effectively improve the in-vivo NADP content.
Document Journal of Biological Chemistry 1950,182(7) 805-813 first purified NAD kinase from Saccharomyces cerevisiae cells in 1950, after which researchers purified NAD kinase also from the gut of pigeons. The Journal of Bioscience and Bioengineering,2004,98(5):391-393 reported the crystal structure of NAD kinase from Mycobacterium tuberculosis H37Rv, providing a powerful tool for the later studies of protein engineering. In 2005, oganesian reported thermophilic NAD kinase from Thermotoga maritima, providing information reference for structural modification of NAD kinase. In 2019, Kimura reports the NAD kinase derived from Myxococcus xanthus, and provides more resources for later researches. The patent CN103409442A modifies NAD kinase, and the final product NADP only obtains 14.5g/L, which also severely restricts the application of industrialization.
At present, the reported NAD kinase generally has the problem of low conversion rate in the conversion of preparing NADP, and because NAD and NADP have similar structures, obvious substrate residues exist after the reaction is finished, and the NAD which is not completely converted can be removed by post-treatment, so that the post-treatment process becomes complicated, and the industrial production cost is high.
In order to improve the catalytic efficiency of the NAD kinase, reduce the cost of the biological catalytic synthesis of the NADP and improve the industrial application value of the NAD kinase, the invention modifies the amino acid to obtain the NAD kinase mutant with high catalytic activity.
The invention content is as follows:
the invention aims to provide a novel NAD kinase mutant aiming at the defects of the prior art.
On one hand, the amino acid sequence of the NAD kinase mutant provided by the invention is an amino acid sequence which takes mutation by taking the NAD kinase shown in SEQ ID NO.1 as a reference sequence, and single-point or multi-point mutation is taken at sites 94, 113, 143, 198 and 283. Wherein, the Tyr at the 94 th position is mutated into Arg, the Ser at the 113 th position is mutated into Arg or Thr, the Met at the 143 th position is mutated into Ile, the Ala at the 198 th position is mutated into Ser, and the Val at the 283 th position is mutated into Ala or Gly.
Furthermore, the amino acid sequence of the NAD kinase mutant is shown in SEQ ID NO.3, 5, 7, 9, 11 and 13.
Further, the gene nucleotide sequence of the NAD kinase mutant is shown in SEQ ID No.4, 6, 8, 10, 12 and 14.
Further, the NAD kinase mutant is derived from Myxococcus xanthus and accession number of the wild-type template NCBI is AAK 82999.1.
Further, the NAD kinase mutant was expressed in e.coli BL (21) DE 3.
Further, the expression vector of the NAD kinase mutant is pET-24 a.
In another aspect, the invention provides the use of NAD kinase mutants that can be used in a process for the preparation of NADP to catalyze the conversion of NAD to NADP.
Further, the concentration of the substrate NAD is 10-42 g/L.
Further, the NAD kinase mutant is NAD kinase enzyme powder or a whole cell or a cell disruption solution containing the NAD kinase, preferably an NAD kinase cell.
Further, the concentration of the NAD kinase enzyme powder is 1-10 g/L.
Furthermore, the cell concentration of the NAD kinase is 5-50 g/L.
Further, the reaction is carried out in a buffer solution, wherein the buffer solution is phosphate buffer solution or Tris-hydroxymethyl aminomethane hydrochloride (Tris-HCl) buffer solution, preferably phosphate buffer solution, and the concentration of the buffer solution is 50-100 mmol/L.
Further, the reactive phosphorus donor is provided by polyphosphoric acid or ATP at a concentration greater than or equal to 20 g/L.
Further, the reaction is carried out at a pH of 6.0 to 8.0 and a temperature of 30 to 60 ℃.
Furthermore, the reaction time required by the reaction is 7-24 h.
The NAD kinase mutant disclosed by the invention has the beneficial effects that NAD can be converted into NADP by the NAD kinase mutant, the catalytic activity is improved, the catalysis of the substrate with the highest concentration of 42g/L is realized, the catalysis time is reduced from 24h to 7h, the generation of byproducts is reduced, the production cost is reduced, and the NAD kinase mutant has great industrial application value.
Drawings
FIG. 1 Gene electrophoresis of NAD kinase in example 1
FIG. 2 expression electropherogram of NAD kinase in example 1
FIG. 3 Gene amplification electropherogram of NAD kinase mutant in example 2
FIG. 4 mapping of NAD kinase mutant transformation products in example 10
Detailed Description
The technical content of the present invention is further described below with reference to specific examples for better understanding of the content of the present invention, but the scope of the present invention is not limited thereto.
Example 1 inducible expression of wild-type NAD kinase
The gene NAD of NAD kinase is directly designed on pET-24a to construct recombinant plasmid pET-24 a-NAD. NdeI and EcoRI restriction enzyme sites are respectively introduced into both ends of the nad gene and directly synthesized. The synthesized recombinant plasmid is transferred into BL21(DE3) to be competent to obtain a recombinant strain. The universal primer T7/T7-ter is used as the upstream and downstream fragments on the combined vector to amplify the target gene and identify the size of the gene, the result is shown in figure 1, all monoclonals have the gene with normal size, and the downstream protein expression can be further carried out.
Transfer of monoclonals from transformation plates into the presence of Kan + Resistant LB tubes were incubated overnight at 37 ℃. Inoculating the seed liquid to the seed containing kan according to the inoculation amount of 1.5% + Culturing the bacteria in resistant 2YT medium at 37 deg.C until the organism OD 600 When the value reaches about 0.6, IPTG induction is carried out, the temperature is reduced to 25 ℃, thalli are collected after expression for 16h, cells are crushed for electrophoretic analysis, and the result is shown in figure 2, the size of protein is consistent with the expected size, and the protein has good soluble expression.
Example 2 construction of NAD kinase mutants
With the knowledge of bioinformatics and structural biology, hot spot amino acids Y94, S113, M143, A198 and V283 which can promote catalytic activity in NAD kinase are analyzed, and a series of mutants are designed through simulated docking: Y94R/S113R/M143I/A198S, Y94R/S113T/M143I/A198S, Y94R/S113R/M143I/A198S/V283A, Y94R/S113T/M143I/A198S/V283A, Y94R/S113R/M143I/A198S/V283G and Y94R/S113T/M143I/A198S/V283G. According to the rationally designed mutant, the primers for mutation are constructed by using software, the specific information is shown in table 1, and the underlined part in the table is the amino acid after mutation.
TABLE 1 nucleic acid sequences of site-directed mutagenesis primers
Figure BDA0002987002580000051
Amplification of full-length plasmids was achieved using conventional techniques of PCR with high fidelity polymerase Primer STAR max DNA, degenerate and extend using different temperatures. The result of the amplification was checked by electrophoresis (see FIG. 3), and each pair of primers was able to amplify the whole plasmid fragment perfectly.
The PCR amplification product was digested with FD DpnI restriction enzyme, the wild-type template DNA sequence was removed, transferred to BL21(DE3) competent cells, and plated on Kan cells containing 100ug/mL + On a resistant LB plate, a first plate was used,culturing at 37 deg.c overnight, and sequencing the single clone to determine whether the site is mutated. And (5) sequentially superposing mutations at the later stage to obtain the initially designed mutant.
Example 3 catalytic Activity assay of NAD kinase mutants
And (4) carrying out induced expression on the mutants with correct sequencing to respectively obtain respective recombinant cells. Freezing and crushing in a low-temperature refrigerator at-40 deg.C. 30mg of each mutant cell was weighed in a 1mL reaction system, and the substrate was dissolved in 100mM phosphate buffer pH 7.0 to raise the concentration from 10g/L to 42g/L, MgCl 2 ATP was added at 8mg, controlled at 0.1 mM. The reaction was carried out in a shaker at 40 ℃ and samples were taken for the 7h and 24h reaction periods, respectively, for analysis of the conversion effect. The data are shown in Table 2, the catalytic efficiency of the mutant is gradually improved, and finally the Y94R/S113T/M143I/A198S/V283G mutant can catalyze 42g/L of substrate in 7h, so that the catalytic activity is obviously improved.
TABLE 2 catalytic Activity of different mutants
Figure BDA0002987002580000061
Example 4 conversion of wild-type NAD kinase
In a 100mL reaction system, 1g of substrate NAD, 3g of wild type NAD kinase cells and anhydrous MgCl are weighed 2 9.5g and 2g of sodium polyphosphate, wherein the raw materials are dissolved in 100mM phosphate buffer solution, the reaction temperature is controlled in a water bath at 40 ℃, and the pH value in the reaction process is controlled to be stabilized at 7.0 by using 0.1M NaOH. The conversion rate after 7h of reaction is 60%, and after low-temperature rotary evaporation, 0.64g of white powder solid product is obtained, and the yield reaches 95.5%.
Example 5 transformation of NAD kinase mutants
In a 100mL reaction system, weighing 2g of substrate NAD, 3g of mutant (SEQ ID NO.3) cells and anhydrous MgCl 2 9.5g and 2g of sodium polyphosphate, wherein the raw materials are dissolved in 100mM tris hydrochloride buffer solution, the reaction temperature is controlled in a water bath at 40 ℃, and the pH value in the reaction process is controlled to be stable at 7.0 by using 0.1M NaOH. After 7h reaction, mainly the product NADP is obtainedAfter low-temperature rotary evaporation, 1.56g of white powder solid product is obtained, and the yield reaches 95.3%.
Example 6 transformation of NAD kinase mutants
In a 100mL reaction system, weighing substrate NAD 2g, mutant (SEQ ID NO.5) cell 3g, anhydrous MgCl 2 9.5g and 2g of sodium polyphosphate, wherein the raw materials are dissolved in 100mM phosphate buffer solution, the reaction temperature is controlled in a water bath at 40 ℃, and the pH value in the reaction process is controlled to be stabilized at 7.0 by using 0.1M NaOH. After 7h of reaction, the product NADP is mainly obtained, and after low-temperature rotary evaporation, 1.84g of white powder solid product is obtained, and the yield reaches 95.6%.
Example 7 transformation of NAD kinase mutants
In a 100mL reaction system, 3g of substrate NAD, 3g of mutant (SEQ ID NO.7) cells and anhydrous MgCl are weighed 2 9.5g of sodium polyphosphate and 2g of sodium polyphosphate, wherein the raw materials are dissolved in 100mM phosphate buffer solution, the reaction temperature is controlled in a water bath at 40 ℃, and the pH value in the reaction process is controlled to be stable at 7.0 by using 0.1M NaOH. After 7h of reaction, the product NADP is mainly obtained, and 2.53g of white powder solid product is obtained after low-temperature rotary evaporation, and the yield reaches 95.4%.
Example 8 transformation of NAD kinase mutants
In a 100mL reaction system, 3g of substrate NAD, 3g of mutant (SEQ ID NO.9) cells and anhydrous MgCl are weighed 2 9.5g and 2g of sodium polyphosphate, wherein the raw materials are dissolved in 100mM phosphate buffer solution, the reaction temperature is controlled in a water bath at 40 ℃, and the pH value in the reaction process is controlled to be stabilized at 7.0 by using 0.1M NaOH. After 7h of reaction, the product NADP is mainly obtained, and 2.6g of white powder solid product is obtained after low-temperature rotary evaporation, and the yield reaches 95%.
Example 9 transformation of NAD kinase mutants
In a 100mL reaction system, 4.2g of substrate NAD, 3g of mutant (SEQ ID NO.11) cells and anhydrous MgCl are weighed 2 9.5g and 2g of sodium polyphosphate, wherein the raw materials are dissolved in 100mM phosphate buffer solution, the reaction temperature is controlled in a water bath at 40 ℃, and the pH value in the reaction process is controlled to be stabilized at 7.0 by using 0.1M NaOH. Reacting for 7h to obtain mainly NADP product, and low-temperature rotary steaming to obtain3.7g of white powder solid product is obtained, and the yield reaches 95.3 percent.
Example 10 transformation of NAD kinase mutants
In a 100mL reaction system, 4.2g of substrate NAD, 3g of mutant (SEQ ID NO.13) cells and anhydrous MgCl are weighed 2 9.5g of sodium polyphosphate and 2g of sodium polyphosphate, wherein the raw materials are dissolved in 100mM phosphate buffer solution, the reaction temperature is controlled in a water bath at 40 ℃, and the pH value in the reaction process is controlled to be stable at 7.0 by using 0.1M NaOH. After 7 hours of reaction, the product NADP is mainly obtained, the conversion rate is 100%, 4.5g of white powder solid product is obtained after low-temperature rotary evaporation, and the yield reaches 95.7%.
Sequence listing
<110> Shang Ke biomedical (Shanghai) Co., Ltd
<120> NAD kinase mutant and application thereof
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<211> 921
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atggtttctg ctggtggtca cccgggtatc tctcacgctc gtccgggtcc gtcttggacc 60
acccgtcgtg gttgcgttca gaccctggct atcgttgcta aacgtgacaa accggaagct 120
gttgctctgg ctgctcagat ccgtgaacgt tacccgcacc tgtctgttct ggctgaccgt 180
accctggctc acgaactggg ttggccgcgt gttgacgacc gtgaactggt tacccgtgct 240
gacctgatgg ttgttctggg tggtgacggt accctgatcc gtgctgctcg tctgctgggt 300
ggtcgtggtg ttccgatcct gggtgttaac ctgggtaccc tgggtttcat gaccgaagtt 360
ccggttgaag aactgtaccc gatgctggaa caggttctgg ctggtcgttt ccaggttgac 420
tctcgtatca aactgtcttg ccgtctgctg cgtggtggtc gtgttctgat cgaagacgaa 480
gttctgaacg acgttgttat caacaaaggt gctctggctc gtatcgctga ccacgaaacc 540
tctatcgacg gtgttccgat caccacctac aaatctgacg gtgttatcct gtctaccccg 600
accggttcta ccgcttactc tctgtctgct ggtggtccga tcgttcaccc gtctgttgac 660
tgcaccgttc tgtctccgat ctgctctcac gctctgaccc agcgttctat cgttgttccg 720
gctgaccgta ccatccgtgt taccctgcgt tctgaaaccg ctgacaccta cctgaccatc 780
gacggtcaga ccggtcacgg tctgcagggt ggtgactgca tcgaagttgt tcgttctcac 840
aaccgtgtta acctggttcg taacccgaaa gttgcttact tctctatcct gcgtcagaaa 900
ctgcactggg gtgaacgtta a 921
<210> 7
<211> 306
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met Val Ser Ala Gly Gly His Pro Gly Ile Ser His Ala Ala Pro Gly
1 5 10 15
Pro Ser Thr Thr Thr Ala Ala Gly Cys Val Gly Thr Leu Ala Ile Val
20 25 30
Ala Leu Ala Ala Leu Pro Gly Ala Val Ala Leu Ala Ala Gly Ile Ala
35 40 45
Gly Ala Thr Pro His Leu Ser Val Leu Ala Ala Ala Thr Leu Ala His
50 55 60
Gly Leu Gly Thr Pro Ala Val Ala Ala Ala Gly Leu Val Thr Ala Ala
65 70 75 80
Ala Leu Met Val Val Leu Gly Gly Ala Gly Thr Leu Ile Ala Ala Ala
85 90 95
Ala Leu Leu Gly Gly Ala Gly Val Pro Ile Leu Gly Val Ala Leu Gly
100 105 110
Ala Leu Gly Pro Met Thr Gly Val Pro Val Gly Gly Leu Thr Pro Met
115 120 125
Leu Gly Gly Val Leu Ala Gly Ala Pro Gly Val Ala Ser Ala Ile Leu
130 135 140
Leu Ser Cys Ala Leu Leu Ala Gly Gly Ala Val Leu Ile Gly Ala Gly
145 150 155 160
Val Leu Ala Ala Val Val Ile Ala Leu Gly Ala Leu Ala Ala Ile Ala
165 170 175
Ala His Gly Thr Ser Ile Ala Gly Val Pro Ile Thr Thr Thr Leu Ser
180 185 190
Ala Gly Val Ile Leu Ser Thr Pro Thr Gly Ser Thr Ala Thr Ser Leu
195 200 205
Ser Ala Gly Gly Pro Ile Val His Pro Ser Val Ala Cys Thr Val Leu
210 215 220
Ser Pro Ile Cys Ser His Ala Leu Thr Gly Ala Ser Ile Val Val Pro
225 230 235 240
Ala Ala Ala Thr Ile Ala Val Thr Leu Ala Ser Gly Thr Ala Ala Thr
245 250 255
Thr Leu Thr Ile Ala Gly Gly Thr Gly His Gly Leu Gly Gly Gly Ala
260 265 270
Cys Ile Gly Val Val Ala Ser His Ala Ala Ala Ala Leu Val Ala Ala
275 280 285
Pro Leu Val Ala Thr Pro Ser Ile Leu Ala Gly Leu Leu His Thr Gly
290 295 300
Gly Ala
305
<210> 8
<211> 921
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atggtttctg ctggtggtca cccgggtatc tctcacgctc gtccgggtcc gtcttggacc 60
acccgtcgtg gttgcgttca gaccctggct atcgttgcta aacgtgacaa accggaagct 120
gttgctctgg ctgctcagat ccgtgaacgt tacccgcacc tgtctgttct ggctgaccgt 180
accctggctc acgaactggg ttggccgcgt gttgacgacc gtgaactggt tacccgtgct 240
gacctgatgg ttgttctggg tggtgacggt accctgatcc gtgctgctcg tctgctgggt 300
ggtcgtggtg ttccgatcct gggtgttaac ctgggtcgtc tgggtttcat gaccgaagtt 360
ccggttgaag aactgtaccc gatgctggaa caggttctgg ctggtcgttt ccaggttgac 420
tctcgtatca aactgtcttg ccgtctgctg cgtggtggtc gtgttctgat cgaagacgaa 480
gttctgaacg acgttgttat caacaaaggt gctctggctc gtatcgctga ccacgaaacc 540
tctatcgacg gtgttccgat caccacctac aaatctgacg gtgttatcct gtctaccccg 600
accggttcta ccgcttactc tctgtctgct ggtggtccga tcgttcaccc gtctgttgac 660
tgcaccgttc tgtctccgat ctgctctcac gctctgaccc agcgttctat cgttgttccg 720
gctgaccgta ccatccgtgt taccctgcgt tctgaaaccg ctgacaccta cctgaccatc 780
gacggtcaga ccggtcacgg tctgcagggt ggtgactgca tcgaagttgt tcgttctcac 840
aaccgtgcta acctggttcg taacccgaaa gttgcttact tctctatcct gcgtcagaaa 900
ctgcactggg gtgaacgtta a 921
<210> 9
<211> 306
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Val Ser Ala Gly Gly His Pro Gly Ile Ser His Ala Ala Pro Gly
1 5 10 15
Pro Ser Thr Thr Thr Ala Ala Gly Cys Val Gly Thr Leu Ala Ile Val
20 25 30
Ala Leu Ala Ala Leu Pro Gly Ala Val Ala Leu Ala Ala Gly Ile Ala
35 40 45
Gly Ala Thr Pro His Leu Ser Val Leu Ala Ala Ala Thr Leu Ala His
50 55 60
Gly Leu Gly Thr Pro Ala Val Ala Ala Ala Gly Leu Val Thr Ala Ala
65 70 75 80
Ala Leu Met Val Val Leu Gly Gly Ala Gly Thr Leu Ile Ala Ala Ala
85 90 95
Ala Leu Leu Gly Gly Ala Gly Val Pro Ile Leu Gly Val Ala Leu Gly
100 105 110
Thr Leu Gly Pro Met Thr Gly Val Pro Val Gly Gly Leu Thr Pro Met
115 120 125
Leu Gly Gly Val Leu Ala Gly Ala Pro Gly Val Ala Ser Ala Ile Leu
130 135 140
Leu Ser Cys Ala Leu Leu Ala Gly Gly Ala Val Leu Ile Gly Ala Gly
145 150 155 160
Val Leu Ala Ala Val Val Ile Ala Leu Gly Ala Leu Ala Ala Ile Ala
165 170 175
Ala His Gly Thr Ser Ile Ala Gly Val Pro Ile Thr Thr Thr Leu Ser
180 185 190
Ala Gly Val Ile Leu Ser Thr Pro Thr Gly Ser Thr Ala Thr Ser Leu
195 200 205
Ser Ala Gly Gly Pro Ile Val His Pro Ser Val Ala Cys Thr Val Leu
210 215 220
Ser Pro Ile Cys Ser His Ala Leu Thr Gly Ala Ser Ile Val Val Pro
225 230 235 240
Ala Ala Ala Thr Ile Ala Val Thr Leu Ala Ser Gly Thr Ala Ala Thr
245 250 255
Thr Leu Thr Ile Ala Gly Gly Thr Gly His Gly Leu Gly Gly Gly Ala
260 265 270
Cys Ile Gly Val Val Ala Ser His Ala Ala Ala Ala Leu Val Ala Ala
275 280 285
Pro Leu Val Ala Thr Pro Ser Ile Leu Ala Gly Leu Leu His Thr Gly
290 295 300
Gly Ala
305
<210> 10
<211> 921
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atggtttctg ctggtggtca cccgggtatc tctcacgctc gtccgggtcc gtcttggacc 60
acccgtcgtg gttgcgttca gaccctggct atcgttgcta aacgtgacaa accggaagct 120
gttgctctgg ctgctcagat ccgtgaacgt tacccgcacc tgtctgttct ggctgaccgt 180
accctggctc acgaactggg ttggccgcgt gttgacgacc gtgaactggt tacccgtgct 240
gacctgatgg ttgttctggg tggtgacggt accctgatcc gtgctgctcg tctgctgggt 300
ggtcgtggtg ttccgatcct gggtgttaac ctgggtaccc tgggtttcat gaccgaagtt 360
ccggttgaag aactgtaccc gatgctggaa caggttctgg ctggtcgttt ccaggttgac 420
tctcgtatca aactgtcttg ccgtctgctg cgtggtggtc gtgttctgat cgaagacgaa 480
gttctgaacg acgttgttat caacaaaggt gctctggctc gtatcgctga ccacgaaacc 540
tctatcgacg gtgttccgat caccacctac aaatctgacg gtgttatcct gtctaccccg 600
accggttcta ccgcttactc tctgtctgct ggtggtccga tcgttcaccc gtctgttgac 660
tgcaccgttc tgtctccgat ctgctctcac gctctgaccc agcgttctat cgttgttccg 720
gctgaccgta ccatccgtgt taccctgcgt tctgaaaccg ctgacaccta cctgaccatc 780
gacggtcaga ccggtcacgg tctgcagggt ggtgactgca tcgaagttgt tcgttctcac 840
aaccgtgcta acctggttcg taacccgaaa gttgcttact tctctatcct gcgtcagaaa 900
ctgcactggg gtgaacgtta a 921
<210> 11
<211> 306
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Val Ser Ala Gly Gly His Pro Gly Ile Ser His Ala Ala Pro Gly
1 5 10 15
Pro Ser Thr Thr Thr Ala Ala Gly Cys Val Gly Thr Leu Ala Ile Val
20 25 30
Ala Leu Ala Ala Leu Pro Gly Ala Val Ala Leu Ala Ala Gly Ile Ala
35 40 45
Gly Ala Thr Pro His Leu Ser Val Leu Ala Ala Ala Thr Leu Ala His
50 55 60
Gly Leu Gly Thr Pro Ala Val Ala Ala Ala Gly Leu Val Thr Ala Ala
65 70 75 80
Ala Leu Met Val Val Leu Gly Gly Ala Gly Thr Leu Ile Ala Ala Ala
85 90 95
Ala Leu Leu Gly Gly Ala Gly Val Pro Ile Leu Gly Val Ala Leu Gly
100 105 110
Ala Leu Gly Pro Met Thr Gly Val Pro Val Gly Gly Leu Thr Pro Met
115 120 125
Leu Gly Gly Val Leu Ala Gly Ala Pro Gly Val Ala Ser Ala Ile Leu
130 135 140
Leu Ser Cys Ala Leu Leu Ala Gly Gly Ala Val Leu Ile Gly Ala Gly
145 150 155 160
Val Leu Ala Ala Val Val Ile Ala Leu Gly Ala Leu Ala Ala Ile Ala
165 170 175
Ala His Gly Thr Ser Ile Ala Gly Val Pro Ile Thr Thr Thr Leu Ser
180 185 190
Ala Gly Val Ile Leu Ser Thr Pro Thr Gly Ser Thr Ala Thr Ser Leu
195 200 205
Ser Ala Gly Gly Pro Ile Val His Pro Ser Val Ala Cys Thr Val Leu
210 215 220
Ser Pro Ile Cys Ser His Ala Leu Thr Gly Ala Ser Ile Val Val Pro
225 230 235 240
Ala Ala Ala Thr Ile Ala Val Thr Leu Ala Ser Gly Thr Ala Ala Thr
245 250 255
Thr Leu Thr Ile Ala Gly Gly Thr Gly His Gly Leu Gly Gly Gly Ala
260 265 270
Cys Ile Gly Val Val Ala Ser His Ala Ala Gly Ala Leu Val Ala Ala
275 280 285
Pro Leu Val Ala Thr Pro Ser Ile Leu Ala Gly Leu Leu His Thr Gly
290 295 300
Gly Ala
305
<210> 12
<211> 921
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atggtttctg ctggtggtca cccgggtatc tctcacgctc gtccgggtcc gtcttggacc 60
acccgtcgtg gttgcgttca gaccctggct atcgttgcta aacgtgacaa accggaagct 120
gttgctctgg ctgctcagat ccgtgaacgt tacccgcacc tgtctgttct ggctgaccgt 180
accctggctc acgaactggg ttggccgcgt gttgacgacc gtgaactggt tacccgtgct 240
gacctgatgg ttgttctggg tggtgacggt accctgatcc gtgctgctcg tctgctgggt 300
ggtcgtggtg ttccgatcct gggtgttaac ctgggtcgtc tgggtttcat gaccgaagtt 360
ccggttgaag aactgtaccc gatgctggaa caggttctgg ctggtcgttt ccaggttgac 420
tctcgtatca aactgtcttg ccgtctgctg cgtggtggtc gtgttctgat cgaagacgaa 480
gttctgaacg acgttgttat caacaaaggt gctctggctc gtatcgctga ccacgaaacc 540
tctatcgacg gtgttccgat caccacctac aaatctgacg gtgttatcct gtctaccccg 600
accggttcta ccgcttactc tctgtctgct ggtggtccga tcgttcaccc gtctgttgac 660
tgcaccgttc tgtctccgat ctgctctcac gctctgaccc agcgttctat cgttgttccg 720
gctgaccgta ccatccgtgt taccctgcgt tctgaaaccg ctgacaccta cctgaccatc 780
gacggtcaga ccggtcacgg tctgcagggt ggtgactgca tcgaagttgt tcgttctcac 840
aaccgtggta acctggttcg taacccgaaa gttgcttact tctctatcct gcgtcagaaa 900
ctgcactggg gtgaacgtta a 921
<210> 13
<211> 306
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Met Val Ser Ala Gly Gly His Pro Gly Ile Ser His Ala Ala Pro Gly
1 5 10 15
Pro Ser Thr Thr Thr Ala Ala Gly Cys Val Gly Thr Leu Ala Ile Val
20 25 30
Ala Leu Ala Ala Leu Pro Gly Ala Val Ala Leu Ala Ala Gly Ile Ala
35 40 45
Gly Ala Thr Pro His Leu Ser Val Leu Ala Ala Ala Thr Leu Ala His
50 55 60
Gly Leu Gly Thr Pro Ala Val Ala Ala Ala Gly Leu Val Thr Ala Ala
65 70 75 80
Ala Leu Met Val Val Leu Gly Gly Ala Gly Thr Leu Ile Ala Ala Ala
85 90 95
Ala Leu Leu Gly Gly Ala Gly Val Pro Ile Leu Gly Val Ala Leu Gly
100 105 110
Thr Leu Gly Pro Met Thr Gly Val Pro Val Gly Gly Leu Thr Pro Met
115 120 125
Leu Gly Gly Val Leu Ala Gly Ala Pro Gly Val Ala Ser Ala Ile Leu
130 135 140
Leu Ser Cys Ala Leu Leu Ala Gly Gly Ala Val Leu Ile Gly Ala Gly
145 150 155 160
Val Leu Ala Ala Val Val Ile Ala Leu Gly Ala Leu Ala Ala Ile Ala
165 170 175
Ala His Gly Thr Ser Ile Ala Gly Val Pro Ile Thr Thr Thr Leu Ser
180 185 190
Ala Gly Val Ile Leu Ser Thr Pro Thr Gly Ser Thr Ala Thr Ser Leu
195 200 205
Ser Ala Gly Gly Pro Ile Val His Pro Ser Val Ala Cys Thr Val Leu
210 215 220
Ser Pro Ile Cys Ser His Ala Leu Thr Gly Ala Ser Ile Val Val Pro
225 230 235 240
Ala Ala Ala Thr Ile Ala Val Thr Leu Ala Ser Gly Thr Ala Ala Thr
245 250 255
Thr Leu Thr Ile Ala Gly Gly Thr Gly His Gly Leu Gly Gly Gly Ala
260 265 270
Cys Ile Gly Val Val Ala Ser His Ala Ala Gly Ala Leu Val Ala Ala
275 280 285
Pro Leu Val Ala Thr Pro Ser Ile Leu Ala Gly Leu Leu His Thr Gly
290 295 300
Gly Ala
305
<210> 14
<211> 921
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atggtttctg ctggtggtca cccgggtatc tctcacgctc gtccgggtcc gtcttggacc 60
acccgtcgtg gttgcgttca gaccctggct atcgttgcta aacgtgacaa accggaagct 120
gttgctctgg ctgctcagat ccgtgaacgt tacccgcacc tgtctgttct ggctgaccgt 180
accctggctc acgaactggg ttggccgcgt gttgacgacc gtgaactggt tacccgtgct 240
gacctgatgg ttgttctggg tggtgacggt accctgatcc gtgctgctcg tctgctgggt 300
ggtcgtggtg ttccgatcct gggtgttaac ctgggtaccc tgggtttcat gaccgaagtt 360
ccggttgaag aactgtaccc gatgctggaa caggttctgg ctggtcgttt ccaggttgac 420
tctcgtatca aactgtcttg ccgtctgctg cgtggtggtc gtgttctgat cgaagacgaa 480
gttctgaacg acgttgttat caacaaaggt gctctggctc gtatcgctga ccacgaaacc 540
tctatcgacg gtgttccgat caccacctac aaatctgacg gtgttatcct gtctaccccg 600
accggttcta ccgcttactc tctgtctgct ggtggtccga tcgttcaccc gtctgttgac 660
tgcaccgttc tgtctccgat ctgctctcac gctctgaccc agcgttctat cgttgttccg 720
gctgaccgta ccatccgtgt taccctgcgt tctgaaaccg ctgacaccta cctgaccatc 780
gacggtcaga ccggtcacgg tctgcagggt ggtgactgca tcgaagttgt tcgttctcac 840
aaccgtggta acctggttcg taacccgaaa gttgcttact tctctatcct gcgtcagaaa 900
ctgcactggg gtgaacgtta a 921

Claims (5)

1. A mutant NAD kinase is characterized in that single-point or multi-point mutation is generated at sites 94, 113, 143, 198 and 283 by taking an amino acid sequence of a wild type NAD kinase shown in SEQ ID NO.1 as a reference sequence. Wherein, the Tyr at the 94 th position is mutated into Arg, the Ser at the 113 th position is mutated into Arg or Thr, the Met at the 143 th position is mutated into Ile, the Ala at the 198 th position is mutated into Ser, and the Val at the 283 th position is mutated into Ala or Gly.
2. The NAD kinase mutant according to claim 1, wherein the amino acid sequence of the NAD kinase mutant is shown in SEQ ID No.3, 5, 7, 9, 11, 13.
3. The NAD kinase mutant according to claim 1, characterized in that the gene nucleotide sequence of the NAD kinase mutant is shown in SEQ ID Nos. 4, 6, 8, 10, 12 and 14.
4. The NAD kinase mutant according to claim 1, which is expressed in E.coli BL (21) DE 3.
5. The NAD kinase mutant according to claim 1, wherein the NAD kinase mutant is capable of converting NAD to NADP.
CN202110302988.7A 2021-03-22 2021-03-22 NAD kinase mutant and application thereof Pending CN115109764A (en)

Priority Applications (1)

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Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
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Publications (1)

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Country Status (1)

Country Link
CN (1) CN115109764A (en)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
YOSHIO KIMURA 等: "Catalytic activity profile of polyP: AMP phosphotransferase from Myxococcus xanthus", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 131, no. 2, XP086476242, DOI: 10.1016/j.jbiosc.2020.09.016 *
YOSHIO KIMURA 等: "Enzymatic characteristics of a polyphosphate/ATP-NAD kinase, Pank, from Myxococcus xanthus", CURRENT MICROBIOLOGY, vol. 77 *
石廷玉;王怀林;谢建平;: "多聚磷酸盐及其代谢酶的研究进展", 生理科学进展, no. 03 *

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Application publication date: 20220927