CN115896062A - Nicotinamide riboside kinase mutant and related product and application thereof - Google Patents
Nicotinamide riboside kinase mutant and related product and application thereof Download PDFInfo
- Publication number
- CN115896062A CN115896062A CN202210966503.9A CN202210966503A CN115896062A CN 115896062 A CN115896062 A CN 115896062A CN 202210966503 A CN202210966503 A CN 202210966503A CN 115896062 A CN115896062 A CN 115896062A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- seq
- mutant
- nicotinamide
- nicotinamide riboside
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010021066 nicotinamide riboside kinase Proteins 0.000 title claims abstract description 144
- 150000001413 amino acids Chemical group 0.000 claims abstract description 35
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 8
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229960001230 asparagine Drugs 0.000 claims abstract description 7
- 235000009582 asparagine Nutrition 0.000 claims abstract description 7
- 239000004471 Glycine Substances 0.000 claims abstract description 5
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 36
- 241000894006 Bacteria Species 0.000 claims description 34
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 30
- 235000005152 nicotinamide Nutrition 0.000 claims description 15
- 239000011570 nicotinamide Substances 0.000 claims description 15
- 229960003966 nicotinamide Drugs 0.000 claims description 15
- 125000000539 amino acid group Chemical group 0.000 claims description 14
- 101001076781 Fructilactobacillus sanfranciscensis (strain ATCC 27651 / DSM 20451 / JCM 5668 / CCUG 30143 / KCTC 3205 / NCIMB 702811 / NRRL B-3934 / L-12) Ribose-5-phosphate isomerase A Proteins 0.000 claims description 12
- 102000001253 Protein Kinase Human genes 0.000 claims description 12
- 102000046755 Ribokinases Human genes 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
- FZAQROFXYZPAKI-UHFFFAOYSA-N anthracene-2-sulfonyl chloride Chemical compound C1=CC=CC2=CC3=CC(S(=O)(=O)Cl)=CC=C3C=C21 FZAQROFXYZPAKI-UHFFFAOYSA-N 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
- 239000012620 biological material Substances 0.000 claims description 4
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims description 2
- 238000003259 recombinant expression Methods 0.000 claims description 2
- 102200149807 rs25397 Human genes 0.000 description 46
- 238000006243 chemical reaction Methods 0.000 description 36
- 102000004190 Enzymes Human genes 0.000 description 22
- 108090000790 Enzymes Proteins 0.000 description 22
- 239000013612 plasmid Substances 0.000 description 22
- 239000000243 solution Substances 0.000 description 21
- 239000000758 substrate Substances 0.000 description 13
- 241000588724 Escherichia coli Species 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 102000053602 DNA Human genes 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000010276 construction Methods 0.000 description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- 101150107671 hisB gene Proteins 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- JLEBZPBDRKPWTD-TURQNECASA-O N-ribosylnicotinamide Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)=C1 JLEBZPBDRKPWTD-TURQNECASA-O 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 238000002741 site-directed mutagenesis Methods 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 238000005520 cutting process Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 235000020956 nicotinamide riboside Nutrition 0.000 description 3
- 239000011618 nicotinamide riboside Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- YABIFCKURFRPPO-IVOJBTPCSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyridin-1-ium-3-carboxamide;chloride Chemical compound [Cl-].NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)=C1 YABIFCKURFRPPO-IVOJBTPCSA-N 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 108030003689 Deoxynucleoside kinases Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 244000253911 Saccharomyces fragilis Species 0.000 description 1
- 235000018368 Saccharomyces fragilis Nutrition 0.000 description 1
- IHNHAHWGVLXCCI-FDYHWXHSSA-N [(2r,3r,4r,5s)-3,4,5-triacetyloxyoxolan-2-yl]methyl acetate Chemical compound CC(=O)OC[C@H]1O[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H]1OC(C)=O IHNHAHWGVLXCCI-FDYHWXHSSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
- 238000005915 ammonolysis reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229940031154 kluyveromyces marxianus Drugs 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a nicotinamide riboside kinase mutant and a related product and application thereof, wherein the nicotinamide riboside kinase mutant is obtained by mutating 27 th asparagine and/or 101 th glycine of nicotinamide riboside kinase which is derived from organism and has an amino acid sequence shown as SEQ ID NO. 1.
Description
Technical Field
The invention particularly relates to a nicotinamide riboside kinase mutant and related products and application thereof, belonging to the technical field of genetic engineering.
Background
Nicotinamide Ribokinase (NRK) is a key enzyme in the NAD + salvage synthesis pathway (also called NR salvage pathway), belongs to the deoxynucleoside kinase superfamily and nucleoside monophosphoryl kinase superfamily, plays an important role in NAD + biosynthesis and anticancer therapy, and is widely present in eukaryotes. NRK is capable of phosphorylating Nicotinamide Ribose (NR) to β -Nicotinamide Mononucleotide (NMN), as shown in figure 5.NMN is a naturally-occurring bioactive nucleotide, has two existing forms of alpha and beta, wherein the beta structure is the active form of NMN, has the functions of delaying senility, prolonging life, treating type II diabetes, neurodegenerative diseases, cardiovascular and cerebrovascular diseases and the like, and has wide application prospect in the industries of health care products, foods and cosmetics.
The main synthesis method of NMN at present is a chemical method and a biological enzyme method, wherein the chemical method takes nicotinic acid tetraethoxy and tetraacetyl ribose as raw materials, and NMN is generated through a plurality of reactions such as carboxyl neutralization, phosphorylation, ammonolysis and the like. The main biological enzyme synthesis method is to use nicotinamide riboside and ATP as substrates, and nicotinamide riboside kinase to catalyze and generate NMN in one step, so that the yield is high, the product purity is high, but the reaction time is too long, and the degradation of NMN into nicotinamide under high pH (such as neutral condition and alkaline condition) is still a limiting factor.
In the prior art, escherichia coli is used as an expression host to express nicotinamide ribokinase derived from saccharomyces cerevisiae, human or kluyveromyces marxianus. Most of nicotinamide riboside kinases have the problem of high Km value, so that the dosage of the enzyme needs to be increased in the actual production, the utilization rate of a substrate is not high, and the cost of subsequent purification is increased due to the residue of the substrate.
Disclosure of Invention
Compared with wild kinase, the nicotinamide riboside kinase mutant provided by the invention has the characteristics of low Km value and low optimal PH value, and has a good application prospect in preparation of beta-nicotinamide mononucleotide.
The technical scheme of the invention is as follows:
the invention aims to provide a nicotinamide riboside kinase mutant, wherein the nicotinamide riboside kinase mutant is obtained by mutating asparagine at position 27 and/or glycine at position 101 of nicotinamide riboside kinase which is derived from organisms and has an amino acid sequence shown as SEQ ID NO. 1.
Further, the amino acid sequence of the nicotinamide riboside kinase mutant is any one of b1-b 6:
a1, generating a nicotinamide riboside kinase mutant of an amino acid sequence shown as SEQ ID NO. 2 when asparagine at position 27 in the amino acid sequence shown as SEQ ID NO. 1 is mutated;
a2, generating a nicotinamide riboside kinase mutant of an amino acid sequence shown as SEQ ID NO. 3 when 101 th nucleotide in the amino acid sequence shown as SEQ ID NO. 1 is mutated;
a3, generating the nicotinamide riboside kinase mutant of the amino acid sequence shown in SEQ ID NO. 4 when the 27 th asparagine and the 101 th glycine in the amino acid sequence shown in SEQ ID NO. 1 are mutated;
a4, when the amino acid sequence shown in SEQ ID NO. 2 is substituted and/or deleted and/or added by one or more amino acid residues except the 27 th amino acid residue, and the mutant is the nicotinamide riboside kinase which has the same function and is derived from the amino acid sequence shown in SEQ ID NO. 2;
a5, when the amino acid sequence shown in SEQ ID NO. 3 is substituted and/or deleted and/or added by one or more amino acid residues except the 101 th amino acid residue, and the nicotinamide ribokinase mutant which has the same function and is derived from the amino acid sequence shown in SEQ ID NO. 3;
a6, when the amino acid sequence shown in SEQ ID NO. 4 is substituted and/or deleted and/or added by one or more amino acid residues except the 27 th amino acid residue and the 101 th amino acid residue, and the mutant is the nicotinamide riboside kinase which has the same function and is derived from the amino acid sequence shown in SEQ ID NO. 4.
The second purpose of the invention is to provide an application of the nicotinamide riboside kinase mutant as nicotinamide riboside kinase in preparing beta-nicotinamide mononucleotide
The present invention also provides a gene encoding the nicotinamide riboside kinase mutant.
Further, the nucleotide sequence of the coding gene of the nicotinamide riboside kinase mutant is any one of c1-c 3:
c1, the nucleotide sequence of the coding gene of the nicotinamide riboside kinase mutant with the amino acid sequence shown as SEQ ID NO. 2 is shown as SEQ ID NO. 8;
c2, the nucleotide sequence of the coding gene of the nicotinamide riboside kinase mutant with the amino acid sequence shown as SEQ ID NO. 3 is shown as SEQ ID NO. 9;
c3, the nucleotide sequence of the coding gene of the nicotinamide riboside kinase mutant with the amino acid sequence shown as SEQ ID NO. 4 is shown as SEQ ID NO. 10.
The fourth object of the present invention is to provide a related biomaterial containing the gene, wherein the related biomaterial is any one of the following c1 to c 3:
c1, a recombinant expression vector containing the gene;
c2, an expression cassette containing the gene;
c3, recombinant bacteria containing the genes are obtained by introducing the genes into host bacteria.
The fifth purpose of the invention is to provide a preparation method of nicotinamide riboside kinase, which comprises culturing the bacteria and obtaining nicotinamide riboside kinase from recombinant bacteria.
Compared with the prior art, the invention has the beneficial effects that:
1. compared with wild nicotinamide riboside kinase, the nicotinamide riboside kinase mutant provided by the invention has greatly improved substrate specific activity, and has huge application potential in NMN production.
2. The reaction kinetics Km value of the nicotinamide riboside kinase mutant provided by the invention on a substrate is reduced by different amplitudes compared with that of wild-type nicotinamide riboside kinase, so that the nicotinamide riboside kinase mutant has obvious advantages on affinity on the substrate compared with wild-type nicotinamide riboside kinase, and can greatly improve the conversion rate and the utilization rate of the substrate.
Reference numerals
FIG. 1 is SDS-PAGE electrophoresis of supernatant obtained by cell disruption of recombinant strain of nicotinamide riboside kinase mutant in example 1 of the present invention;
wherein, the Lane M is a protein Marker with the molecular weight of 15-150 KDa;
FIG. 2 is a standard curve for β -nicotinamide mononucleotide in example 2 of the invention;
FIG. 3 shows pBAD-NRK/K12, and NRK in example 4 of the present invention A27T The strain/K12, NRK A101D K12 StrainAnd NRK A27T/A101D Time profile of NMN production by K12 strain;
FIG. 4 shows NRK and NRK in example 2 of the present invention A27T 、NRK A101D 、NRK A27T/A101D K of cat a/Km ratio diagram;
FIG. 5 is a schematic representation of the conversion of nicotinamide riboside to beta-nicotinamide mononucleotide according to the prior art.
Detailed Description
The invention will be further described with reference to the accompanying drawings and preferred embodiments, which are given for illustration only and are not intended to limit the scope of the invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified;
the quantitative tests in the following examples, all set up three replicates and the results averaged.
The experimental methods in the following examples are all conventional methods unless otherwise specified;
coli K12 (Tomoya Baba, takeshi Ara, miki Hasegawa, yuki Takai, yoshiko Okumura, miki Baba, kirilla Datsenko, masaru Tomita, barry L Wanner and Hirotada Mori1.Construction of Escherichia coli K-12in-frame, single-gene knock out variants: the Keio molecular Systems biology (2006): 1-11), publicly available from the university of engineering, which was used only for repeating the experiments related to the present invention, but not for other uses;
the pBADhisB vector in the following examples is product of Invitrogen corporation, catalog number V430-01; the promoter positioned at the upstream of a Multiple Cloning Site (MCS) in the pBAD/hisB vector is an ara promoter, the upstream of the MCS at the downstream of the ara promoter is provided with a histidine tag, and the histidine tag is provided with an initiation codon ATG (codon usage index), so that an exogenous gene inserted into the MCS can have no initiation codon as long as a triplet codon conforms to the initiation codon and no frame shift exists;
the following examples were prepared with the following formulation of self-induction medium ZYM: 100mL A +2mL B +2mL C +200 μ L D +100 μ L E; wherein the compositions of components A, B, C, D and E are as follows:
A-ZY: the nutrient solution consists of a solute and a solvent, wherein the solvent is water, and the solute comprises tryptone with the mass concentration of 1% and yeast powder with the mass concentration of 0.5%;
b-50 XM: the solute consists of a solute and a solvent, wherein the solvent is water, and the solute is: 1.25M Na 2 HPO 4 、1.25M KH 2 PO 4 、2.5M NH 4 Cl and 0.25M Na 2 SO 4 ；
C-50X 5052: the solvent is water, and the solute comprises 25% by mass of glycerol, 2.5% by mass of glucose and 10% by mass of L-arabinose;
d: consists of solute and solvent, the solvent is water, the solute is 1M MgSO 4 ；
E-1000X trace elements: the solute consists of a solute and a solvent, wherein the solvent is water, and the solute is: 50mM FeCl 3 ，20mM CaCl 2 ，10mM MnCl 2 ，10mM ZnSO 4 ，2mM CoCl 2 、2mM NiCl 2 、2mM Na 2 Mo 4 、2mM Na 2 SeO 3 And 2mM H 3 BO 3 。
Example 1 construction of wild type Nicotinamide riboside kinase Gene recombinant bacteria
1.Construction of recombinant plasmid of nicotinamide riboside kinase gene
The fragment between Bgl II and EcoRI enzyme cutting sites of a pBAD/hisB carrier is replaced by a double-stranded DNA molecule (coding gene NRK of nicotinamide riboside kinase derived from human (Homo sapiens)) shown as SEQ ID NO. 1 to obtain a wild type nicotinamide riboside kinase gene recombinant plasmid pBAD/hisB-NRK (sequencing verification is carried out);
wherein, the DNA molecule shown in SEQ ID NO.5 codes the protein (nicotinamide riboside kinase, named NRK) shown in the sequence SEQ ID NO. 1;
2. construction of wild type nicotinamide riboside kinase gene recombinant bacteria and control bacteria
1. Introducing the recombinant plasmid pBAD/hisB-NRK prepared by the steps into a K12 strain of Escherichia coli (Escherichia coli) to obtain a wild type nicotinamide riboside kinase gene recombinant strain NRK/K12; the supernatant obtained by the centrifugation after the disruption of the recombinant bacteria NRK/K12 is verified to have the expression of Nicotinamide Riboside Kinase (NRK) (28.3 KD) through SDS-PAGE electrophoresis (see lanes 8 and 9 in figure 1);
2. introducing the pBAD/hisB vector into a K12 strain of Escherichia coli (Escherichia coli) to obtain a control strain pBAD/K12; the supernatant obtained by centrifugation after disruption of the pBAD/K12 strain was examined by SDS-PAGE in lanes 9 and 10 of FIG. 1.
Example 2 acquisition of Nicotinamide riboside kinase mutant Gene and construction of recombinant bacterium
1. Nicotinamide riboside kinase NRK A27T Construction of coding gene and recombinant bacterium thereof
1. The recombinant plasmid pBAD/hisB-NRK obtained in example 1 was used as a template with a primer NRK A27T -F and primer NRK A27T The primer pair consisting of the-R is subjected to site-directed mutagenesis by utilizing a site-directed mutagenesis kit (TransGen, cat # FM 111-01), and a recombinant plasmid with successful mutagenesis is obtained by screening and is named as a recombinant plasmid pBAD/hisB-NRK A27T ；
NRK A27T -F：5’-CTGCGTGCGCTGCCGACTTGCTGCGT-3' (positions 67-92 of the sequence shown in SEQ ID No.6, the underlined part is the site of mutation compared to the sequence shown in SEQ ID No. 5);
NRK A27T -R：5’-GTCGGCAGCGCACGCAGCAGGCTAT-3' (the reverse complement of positions 59-83 of the sequence shown in SEQ ID No.6, the underlined part is the site of mutation compared to the sequence shown in SEQ ID No. 5);
through sequencing, the recombinant plasmid pBAD/hisB-NRK A27T Is a recombinant plasmid obtained by replacing a fragment between Bgl II and EcoRI enzyme cutting sites of a pBAD/hisB carrier by a double-stranded DNA molecule with a sequence shown as SEQ ID No. 6; the double-stranded DNA molecule with the sequence shown as SEQ ID No.6 replaces the fragment between BglII and EcoRI enzyme cutting sites of the pBAD/hisB carrier to obtain recombinant plasmid;
wherein, the DNA molecule shown in SEQ ID No.6 codes the protein (nicotinamide riboside kinase, named NRK) shown in sequence SEQ ID No. 2 A27T )；
2. The recombinant plasmid pBAD/hisB-NRK obtained by the steps A27T Introducing into Escherichia coli (Escherichia coli) K12 strain to obtain recombinant strain NRK A27T The ratio of the component A to the component B is/K12; recombinant bacterium NRK A27T The supernatant obtained by centrifugation after the/K12 crushing is verified to be visible Nicotinamide Riboside Kinase (NRK) through SDS-PAGE electrophoresis A27T ) (28.3 kD) was expressed (see lanes 5, 6 in FIG. 1).
2. Nicotinamide riboside kinase NRK A101D Construction of coding gene and recombinant bacterium thereof
1. The recombinant plasmid pBAD/hisB-NRK obtained in example 1 was used as a template with a primer NRK A101D -F and primer NRK A101D The primer pair consisting of the-R is subjected to site-directed mutagenesis by utilizing a site-directed mutagenesis kit (TransGen, cargo number: FM 111-01), and a recombinant plasmid with successful mutagenesis is obtained by screening and is named as a recombinant plasmid pBAD/hisB-NRK 101D ；
NRK 101D -F：5’-TGCTGCTGGAAGACTTTCTGCTGTAT-3' (positions 67-92 of the sequence shown in SEQ ID No.7, the underlined portion is the site of mutation compared to the sequence shown in SEQ ID No. 5);
NRK 101D -R：5’-TCTTCCAGCAGCAGAATATGGGTAT-3' (the reverse complement of position 59-83 of the sequence shown in SEQ ID No.7, the underlined part is the site of mutation compared to the sequence shown in SEQ ID No. 5);
through sequencing, the recombinant plasmid pBAD/hisB-NRK A101D Is a recombinant plasmid obtained by replacing a fragment between BglII and EcoRI enzyme cutting sites of a pBAD/hisB carrier by a double-stranded DNA molecule with a sequence shown as SEQ ID No. 7;
wherein, the DNA molecule shown in SEQ ID No.7 codes the protein (nicotinamide riboside kinase, named NRK) shown in the sequence SEQ ID No. 3 A101D )；
2. The recombinant plasmid pBAD/hisB-NRK obtained in the step 1 A101D Introducing into Escherichia coli (Escherichia coli) K12 strain to obtain recombinant strain NRK A101D K12; recombinant bacterium NRK A101D The supernatant obtained by centrifugation after the/K12 crushing is verified to be visible nicotinamide riboside by SDS-PAGE electrophoresisKinase (NRK) A101D ) (28.3 KD) is expressed (see lanes 3 and 4 in FIG. 1);
3. nicotinamide riboside kinase NRK A27T/A101D Construction of coding gene and recombinant bacterium thereof
1. The recombinant plasmid pBAD/hisB-NRK obtained in example 1 was used A27T As template, use primer NRK A101D -F and primer NRK A101D The primer pair consisting of the-R is subjected to site-directed mutagenesis by utilizing a site-directed mutagenesis kit (TransGen, cargo number: FM 111-01), and a recombinant plasmid with successful mutagenesis is obtained by screening and is named as a recombinant plasmid pBAD/hisB-NRK A27T/A101D ；
Through sequencing, the recombinant plasmid pBAD/hisB-NRK A27T/A101D Is a recombinant plasmid obtained by replacing a fragment between Bgl II and EcoRI enzyme cutting sites of a pBAD/hisB carrier by a double-stranded DNA molecule with a sequence shown as SEQ ID No. 8;
wherein, the DNA molecule shown in SEQ ID No.8 codes the protein (nicotinamide riboside kinase, named NRK) shown in sequence SEQ ID No. 4 A27T/A101D )；
2. The recombinant plasmid pBAD/hisB-NRK obtained in the step 1 A27T/A101D Introducing into Escherichia coli (Escherichia coli) K12 strain to obtain recombinant strain NRK A27T/A101D K12; recombinant bacterium NRK A27T/A101D SDS-PAGE electrophoresis of the supernatant obtained by centrifugation after the/K12 disruption verifies that the Nicotinamide Riboside Kinase (NRK) can be seen A27T/A101D ) (28.3 KD) is expressed (see lanes 1 and 2in FIG. 1); example 3 specific Activity and acid dependence assay of Nicotinamide riboside kinase
Bacteria to be detected: reference bacterium pBAD/K12, recombinant bacterium NRK/K12 and recombinant bacterium NRK A27T K12 and recombinant bacterium NRK A101D K12 and recombinant bacterium NRK A27T/A101D /K12；
1. Inoculating the bacteria to be detected into LB solid culture medium containing 100 mug/mL streptomycin, and culturing for 12h at 37 ℃;
2. after completing the step 1, picking a single colony, inoculating the single colony in an LB liquid culture medium containing 100 mu g/mL streptomycin, and culturing the single colony overnight at 37 ℃ and 220rpm by shaking;
3. after the step 2 is completed, inoculating the culture into a self-induction culture medium ZYM according to the inoculation amount of 1 percent (volume percentage), and oscillating overnight for 16h at the temperature of 30 ℃ and at the speed of 200 rpm;
4. after the step 3 is finished, centrifuging the culture system for 10min at the temperature of 4 ℃ and the pressure of 8000g, collecting thalli sediment, carrying out ultrasonic crushing (ice bath ultrasound, the power of 35%, the work time of 5s, the pause time of 3s and the total time of 5 min) after the thalli sediment is re-suspended by adopting a buffer solution (50 mM sodium phosphate, 20mM imidazole, 300mM sodium chloride and the pH value of 7.4), centrifuging for 5min at the temperature of 10000g after the ultrasound is finished, and taking a supernatant (crude enzyme solution);
5. filtering the crude enzyme solution obtained in step 4 with 0.22 μm water system membrane, separating and purifying with HisTrap FF affinity chromatography column (GE Healthcare, cat # 17-5255-01), eluting with eluent (50 mM sodium phosphate, 250mM imidazole, 300mM sodium chloride, pH 7.4), and collecting the eluent (enzyme solution);
6. desalting the eluate (enzyme solution) obtained in step 5 with HiTrap desalting column (GE Healthcare, cat. No. 17-1408-01) with 200mM acetic acid-sodium acetate buffer solution (pH 4.6), and collecting eluate at protein peak to obtain pure enzyme solution; the protein concentration of the pure enzyme solution is measured by adopting an improved Bradford method kit (Shanghai Biotech, the product number is SK 3041); specific results are shown in table 1;
the NMN concentration is measured by a High Performance Liquid Chromatography (HPLC) method, and the specific measurement steps are as follows:
(1) Drawing of NMN standard curve
Accurately weighing 0.03342gNMN standard (Sigma, product number: N3501) and placing in a 100mL volumetric flask, adding 50mL high-purity water, stirring to completely dissolve, and then adding high-purity water to fix the volume to 100mL; after shaking up, sequentially diluting the mixture into NMN standard solutions with different concentrations (0.3342 g/L, 0.2674g/L, 0.2005g/L, 0.1337g/L and 0.0668 g/L) by using high-purity water;
filtering prepared NMN standard solutions with different concentrations by using a water system filter head with the diameter of 0.22 mu m, collecting filtrate, performing quantitative determination by using HPLC, and drawing a NMN standard curve by using the peak area of an absorption peak with the wavelength of 260nm as a horizontal coordinate and the concentration of an NMN standard substance as a vertical coordinate (the result is shown in figure 2);
the HPLC measurement conditions were as follows:
a chromatographic column: welch Ultimate AQC18 reverse phase column (5 μm, 250X 4.6 mm);
column temperature: 35 ℃;
detection wavelength: 360nm;
mobile phase: 5mM ammonium dihydrogen phosphate solution;
flow rate: 1mL/min;
(2) Determination of NMN concentration in reaction solution
Diluting the solution to be detected by 100 times, filtering by using a 0.22 mu m water system filter head, collecting the filtrate, and carrying out quantitative determination by using HPLC; and converting the mass concentration of NMN in the reaction solution according to the peak area of the absorption peak of the conversion solution at 260nm and the standard curve of NMN.
Calculation of the NMN content: y is NMN ＝39.5162X-0.102；
Definition of 1 enzyme activity (U): under the above reaction conditions, the amount of enzyme required to catalyze the formation of 1. Mu. Mol of beta-nicotinamide mononucleotide from 1. Mu. Mol of nicotinamide ribochloride per minute
Calculation of enzyme Activity (U):
wherein: c NMN The concentration (g/L) of NMN in the reaction solution, V the volume (L) of the reaction solution, M the molecular weight (334.22 g/mol) of NMN, t the enzymatic time (min), and 1000 the conversion coefficient of mg and g;
calculation of specific activity:
wherein: u is enzyme activity, C Protein Is the concentration of the enzyme in the reaction solution;
TABLE 2 specific activity of different nicotinamide riboside kinases (unit: U/mg)
| NRK | NRK A27T | NRK A101D | NRK A27T/A101D |
| 1.56±0.568 | 2.84±0.453 | 5.66±0.536 | 7.83±0.749 |
The results are shown in table 2, and the specific activities of the three nicotinamide riboside kinase mutants to the substrate are greatly improved compared with the wild type. Wherein the mutant NRK A27T/A101D The specific activity of the mutant is improved by 5.02 times compared with the wild type, and the mutant NRK A27T The specific activity of the mutant is improved by 1.82 times compared with the wild type, and the mutant NRK A101D The specific activity of the polypeptide is improved by 3.63 times compared with that of the wild type; the results show that three nicotinamide riboside kinase mutants, especially double mutant NRKs A27T/A101D Compared with wild nicotinamide riboside kinase, the nicotinamide riboside kinase has obvious advantages in specific activity and has huge application potential in NMN production.
The concentration was 300mM NRCL, 350mM ATP, 25mM MgCl 2 Diluting the aqueous solution of the proportion to a gradient concentration of 0.01-8mmol/L, and carrying out enzymatic reaction on the aqueous solution and purified nicotinamide riboside kinase enzyme liquid under the optimal condition; fitting by a Lineweaver-Burk model, drawing a reaction kinetics curve, and calculating the reaction kinetics Km;
TABLE 3 kinetics of the reaction of the different nicotinamide riboside kinases (Unit: mM)
| NRK | NRK A27T | NRK A101D | NRK A27T/A101D |
| 0.267±0.023 | 0.153±0.007 | 0.068±0.036 | 0.043±0.009 |
The results are shown in table 3, the Km values of the reaction kinetics of the three nicotinamide riboside kinase mutants to the substrate are reduced in different ranges compared with the wild type; wherein the mutant NRK A27T/A101D The Km value of the mutant is reduced by 6.21 times compared with the wild type, and the mutant NRK A27T The Km value of the mutant is reduced by 1.75 times compared with the wild type, and the mutant NRK A101D The Km value of the gene is reduced by 3.93 times compared with the wild type; the results show that three nicotinamide riboside kinase mutants, especially double mutant NRKs A27T/A101D The Km value is lower, so the protein has obvious advantages in affinity to a substrate compared with wild nicotinamide riboside kinase, and can greatly improve the conversion rate and the utilization rate of the substrate, and simultaneously, the protein is shown in 4,K cat The improvement of the Km ratio can effectively improve the utilization efficiency of the enzyme to the substrate.
Example 4 analysis of Whole-cell catalysis ability of recombinant bacteria to Nicotinamide riboside chloride
And (3) bacteria to be detected: reference bacterium pBAD/K12, recombinant bacterium NRK/K12 and recombinant bacterium NRK A27T /K12, recombinant strain NRK A101D K12 and recombinant strain NRK A27T/A101D /K12；
1. Inoculating the bacteria to be detected into an LB solid culture medium containing 100 mu g/mL streptomycin, and culturing for 12h at 37 ℃;
2. after completing the step 1, picking single colony, inoculating the single colony in LB liquid culture medium containing 100 ug/mL streptomycin, and culturing overnight at 37 ℃ and 220rpm with shaking;
3. after the step 2 is completed, inoculating the culture into a self-induction culture medium ZYM according to the inoculation amount of 1 percent (volume percentage), and oscillating overnight for 16h at the temperature of 30 ℃ and at the speed of 200 rpm;
4. after the step 3 is finished, collecting the culture system, centrifuging for 10min at the temperature of 4 ℃ under the condition of 8000g, and collecting thalli; washing the thallus with 10mM sodium chloride water solution for 1 time, and collecting the thallus again under the same centrifugation condition;
5. after the step 4 is finished, resuspending the thallus precipitate by adopting 300mM NRCL, 350mM ATP and 25mM MgCl2 aqueous solution to obtain initial transformation liquid; the thallus content in the initial conversion solution is 10OD/L in terms of wet weight; carrying out conversion of nicotinamide ribochloride to beta-nicotinamide mononucleotide on the initial conversion solution at 37 ℃,100 rpm and pH =5.5, and collecting the conversion solutions for 1, 2, 3, 4, 5 and 6h respectively;
the experiment is repeated for three times, and 4 transformation systems are arranged at each time point of each repetition;
the NMN yield in the conversion solution was measured by High Performance Liquid Chromatography (HPLC) in the same manner as in example 3;
the results are shown in FIG. 3, recombinant strain NRK A27T/A101D Conversion is carried out for 5h, the NMN yield reaches 97.81g/L, the conversion rate reaches 97.61 percent, and the conversion rate is 24.45 g/L.h; recombinant bacterium NRK A27T The conversion is 5h, the NMN yield is 72.87g/L, the conversion rate is 72.68 percent, and the conversion rate is 18.22 g/L.h; recombinant bacterium NRK A101D The conversion is 5h, the NMN yield is 80.42g/L, the conversion rate is 80.21 percent, and the conversion rate is 20.11 g/L.h; the conversion of the reference strain NRK/K12 is 5h, the NMN yield is only 63.37g/L, the conversion rate is 63.20 percent, and the conversion rate is 15.84 g/L.h; the recombinant strain pBAD-NRK/K12 is converted for 5h, the NMN yield is only 4.06g/L, the conversion rate is only 4.05%, and the conversion rate is 1.02 g/L.h.
TABLE 4 NMN production (units: g/L) of nicotinamide riboside kinase at different pHs
| NRK | NRK A27T | NRK A101D | NRK A27T/A101D | |
| PH＝5.5 | 4.06 | 72.87 | 80.42 | 97.81 |
| PH＝6.0 | 3.18 | 67.34 | 60.59 | 87.63 |
| PH＝6.5 | 2.74 | 59.91 | 48.78 | 80.56 |
The results in Table 4 show that three nicotinamide riboside kinase mutants, especially double mutant NRKs A27T/A101D Lower Km, lower NMN production with decreasing pHThe increase is most obvious, the utilization efficiency of the enzyme to the substrate is effectively improved, the optimal PH is reduced, and the problem of degradation of NMN at higher PH can be avoided.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (8)
1. A nicotinamide riboside kinase mutant, characterized in that the nicotinamide riboside kinase mutant is obtained by mutating asparagine at position 27 and/or glycine at position 101 of nicotinamide riboside kinase derived from an organism having an amino acid sequence shown in SEQ ID NO. 1.
2. The nicotinamide riboside kinase mutant according to claim 1, wherein the amino acid sequence of the nicotinamide riboside kinase mutant is any one of a1 to a 6:
a1, when 27 th asparagine in an amino acid sequence shown in SEQ ID NO. 1 is mutated, generating a nicotinamide riboside kinase mutant of the amino acid sequence shown in SEQ ID NO. 2;
a2, generating the nicotinamide riboside kinase mutant of the amino acid sequence shown in SEQ ID NO. 3 when the 101 th nucleotide in the amino acid sequence shown in SEQ ID NO. 1 is mutated;
a3, generating the nicotinamide riboside kinase mutant of the amino acid sequence shown in SEQ ID NO. 4 when the 27 th asparagine and the 101 th glycine in the amino acid sequence shown in SEQ ID NO. 1 are mutated;
a4, when the amino acid sequence shown in SEQ ID NO. 2 is substituted and/or deleted and/or added by one or more amino acid residues except the 27 th amino acid residue, and the mutant is the nicotinamide riboside kinase which has the same function and is derived from the amino acid sequence shown in SEQ ID NO. 2;
a5, when the amino acid sequence shown in SEQ ID NO. 3 is substituted and/or deleted and/or added by one or more amino acid residues except the 101 th amino acid residue, the mutant is the nicotinamide ribokinase mutant which has the same function and is derived from the amino acid sequence shown in SEQ ID NO. 3;
a6, when the amino acid sequence shown in SEQ ID NO. 4 is substituted and/or deleted and/or added by one or more amino acid residues except the 27 th amino acid residue and the 101 th amino acid residue, and the mutant is the nicotinamide riboside kinase which has the same function and is derived from the amino acid sequence shown in SEQ ID NO. 4.
3. Use of a nicotinamide ribokinase mutant as defined in claim 1 or 2 for the preparation of beta-nicotinamide mononucleotide.
4. Use of the nicotinamide ribokinase mutant of claim 1 or 2 for preparing a nicotinamide riboside kinase related product.
5. A gene encoding the nicotinamide ribokinase mutant of claim 1.
6. The gene of the nicotinamide ribokinase mutant, which is characterized in that the nucleotide sequence of the gene encoding the nicotinamide ribokinase mutant is any one of b1 to b 3:
b1, the nucleotide sequence of the coding gene of the nicotinamide ribokinase mutant with the amino acid sequence shown as SEQ ID NO. 2 is shown as SEQ ID NO. 8;
b2, the nucleotide sequence of the coding gene of the nicotinamide ribokinase mutant with the amino acid sequence shown as SEQ ID NO. 3 is shown as SEQ ID NO. 9;
b3, the nucleotide sequence of the coding gene of the nicotinamide ribokinase mutant with the amino acid sequence shown as SEQ ID NO. 4 is shown as SEQ ID NO. 10.
7. A related biomaterial containing the gene according to claim 5 or 6, said related biomaterial being any one of the following c1 to c 3:
c1, a recombinant expression vector containing the gene of claim 3 or 4;
c2, an expression cassette comprising the gene of claim 3 or 4;
c3, a recombinant bacterium containing the gene of claim 3 or 4, and a host bacterium into which the gene is introduced.
8. A method for producing nicotinamide riboside kinase, characterized by culturing the recombinant bacterium according to claim 5 and obtaining nicotinamide riboside kinase from the recombinant bacterium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210966503.9A CN115896062A (en) | 2022-08-10 | 2022-08-10 | Nicotinamide riboside kinase mutant and related product and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210966503.9A CN115896062A (en) | 2022-08-10 | 2022-08-10 | Nicotinamide riboside kinase mutant and related product and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115896062A true CN115896062A (en) | 2023-04-04 |
Family
ID=86488572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210966503.9A Pending CN115896062A (en) | 2022-08-10 | 2022-08-10 | Nicotinamide riboside kinase mutant and related product and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115896062A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070027095A1 (en) * | 2004-02-10 | 2007-02-01 | Brenner Charles M | Nicotinamide riboside kinase compositions and methods for using the same |
| CN110373398A (en) * | 2019-08-06 | 2019-10-25 | 江苏诚信药业有限公司 | A kind of niacinamide ribokinase mutant and its application |
| CN112553178A (en) * | 2020-12-25 | 2021-03-26 | 中山俊凯生物技术开发有限公司 | Nicotinamide ribokinase mutant with enhanced thermal stability and activity and coding gene and application thereof |
| CN112608910A (en) * | 2020-12-15 | 2021-04-06 | 深圳希吉亚生物技术有限公司 | Nicotinamide ribokinase and application thereof |
-
2022
- 2022-08-10 CN CN202210966503.9A patent/CN115896062A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070027095A1 (en) * | 2004-02-10 | 2007-02-01 | Brenner Charles M | Nicotinamide riboside kinase compositions and methods for using the same |
| CN110373398A (en) * | 2019-08-06 | 2019-10-25 | 江苏诚信药业有限公司 | A kind of niacinamide ribokinase mutant and its application |
| CN112608910A (en) * | 2020-12-15 | 2021-04-06 | 深圳希吉亚生物技术有限公司 | Nicotinamide ribokinase and application thereof |
| CN112553178A (en) * | 2020-12-25 | 2021-03-26 | 中山俊凯生物技术开发有限公司 | Nicotinamide ribokinase mutant with enhanced thermal stability and activity and coding gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| KHAN JA等: "Crystal structure of human nicotinamide riboside kinase", 《STRUCTURE》, vol. 15, no. 08, 14 August 2007 (2007-08-14), pages 1005 - 1013, XP022192479, DOI: 10.1016/j.str.2007.06.017 * |
| 任丽梅等: "β-烟酰胺单核苷酸功能与合成研究进展", 《生物资源》, vol. 43, no. 02, 9 April 2021 (2021-04-09), pages 127 - 132 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111718915B (en) | Nicotinamide phosphoribosyl transferase mutant, recombinant expression vector and recombinant bacterium containing mutant and application | |
| CN112795606B (en) | Enzymatic synthesis method of beta-nicotinamide mononucleotide | |
| CN111471668B (en) | Nitrilase mutant and application thereof in preparation of 1-cyanocyclohexylacetic acid | |
| CN108342378B (en) | Glutamic acid decarboxylase mutant and coding gene and application thereof | |
| CN112877307B (en) | Amino acid dehydrogenase mutant and application thereof | |
| CN109251881A (en) | The Escherichia coli recombinant strain and its application of one plant of heterogenous expression nitrile hydratase | |
| CN115960875A (en) | Alginate lyase mutant enzyme with improved thermal stability | |
| CN113337495B (en) | Method for improving sialic acid yield and application | |
| CN112980906B (en) | Enzyme composition for preparing beta-nicotinamide mononucleotide and application thereof | |
| CN115896062A (en) | Nicotinamide riboside kinase mutant and related product and application thereof | |
| CN108865913B (en) | Method for constructing recombinant bacterium capable of efficiently secreting and expressing chondroitin sulfate hydrolase | |
| CN113755473B (en) | Glucoamylase mutant M5 with improved secretion expression level as well as gene and application thereof | |
| CN114277022B (en) | Nitrile hydratase mutant with high activity and high thermal stability | |
| CN107653257A (en) | A kind of encoding gene, recombinant expression carrier and the application of nicotinamide mononucleotide adenylyl transferase | |
| CN108588056B (en) | Low-temperature α -amylase Tcamy and gene and application thereof | |
| CN109234251B (en) | Protein and application of nucleic acid molecule for coding protein in preparation of phosphohydrolase | |
| CN116949017B (en) | High-activity lactase mutant and application thereof | |
| CN111057698B (en) | L-arabinose isomerase, mutant and application thereof | |
| CN110904087B (en) | L-arabinose epimerase mutant and application thereof | |
| CN117230049B (en) | Algin lyase mutant with improved thermal stability and application thereof | |
| CN114107246B (en) | Uridine-cytidine kinase mutant and application thereof in production of cytidine acid | |
| CN113774045B (en) | Glucoamylase mutant M3 with improved secretion expression level as well as gene and application thereof | |
| CN115058402B (en) | Nicotinamide ribokinase mutant and coding gene and application thereof | |
| CN112481245B (en) | Recombinant D-tagatose 3 epimerase DTE-CM and construction method and application thereof | |
| CN115094075A (en) | Alginate lyase high-yield strain and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |