CN117511907A - Nicotinamide adenine dinucleotide kinase mutant and application thereof - Google Patents
Nicotinamide adenine dinucleotide kinase mutant and application thereof Download PDFInfo
- Publication number
- CN117511907A CN117511907A CN202311316999.6A CN202311316999A CN117511907A CN 117511907 A CN117511907 A CN 117511907A CN 202311316999 A CN202311316999 A CN 202311316999A CN 117511907 A CN117511907 A CN 117511907A
- Authority
- CN
- China
- Prior art keywords
- mutant
- nadk
- adenine dinucleotide
- nicotinamide adenine
- mutation
- 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
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 title claims abstract description 23
- 229950006238 nadide Drugs 0.000 title claims abstract description 23
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 title claims abstract description 22
- 108091000080 Phosphotransferase Proteins 0.000 title claims abstract description 16
- 102000020233 phosphotransferase Human genes 0.000 title claims abstract description 16
- 230000035772 mutation Effects 0.000 claims abstract description 26
- 230000003197 catalytic effect Effects 0.000 claims abstract description 13
- 239000004475 Arginine Substances 0.000 claims description 12
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 12
- 230000014509 gene expression Effects 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 6
- 108091033319 polynucleotide Proteins 0.000 claims description 5
- 239000002157 polynucleotide Substances 0.000 claims description 5
- 102000040430 polynucleotide Human genes 0.000 claims description 5
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 4
- 239000004472 Lysine Substances 0.000 claims description 4
- 235000013922 glutamic acid Nutrition 0.000 claims description 4
- 239000004220 glutamic acid Substances 0.000 claims description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 102000001253 Protein Kinase Human genes 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 108060006633 protein kinase Proteins 0.000 claims description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 2
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims description 2
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 2
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 claims 1
- 241001138501 Salmonella enterica Species 0.000 claims 1
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 abstract description 19
- 102000004190 Enzymes Human genes 0.000 abstract description 18
- 108090000790 Enzymes Proteins 0.000 abstract description 18
- 108090000623 proteins and genes Proteins 0.000 abstract description 11
- 239000000758 substrate Substances 0.000 abstract description 10
- 238000006555 catalytic reaction Methods 0.000 abstract description 7
- 238000009776 industrial production Methods 0.000 abstract description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 101001112714 Homo sapiens NAD kinase Proteins 0.000 abstract 2
- 102100023515 NAD kinase Human genes 0.000 abstract 2
- 238000006243 chemical reaction Methods 0.000 description 13
- 238000000034 method Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000012064 sodium phosphate buffer Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 101150084853 nadK gene Proteins 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 2
- 229960002064 kanamycin sulfate Drugs 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003032 molecular docking Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 101100409047 Chlorobaculum tepidum (strain ATCC 49652 / DSM 12025 / NBRC 103806 / TLS) ppk2 gene Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 241000405383 Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 Species 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000001195 anabolic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 108020000161 polyphosphate kinase Proteins 0.000 description 1
- 101150040316 ppk2 gene Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical compound [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 description 1
- 235000019982 sodium hexametaphosphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07001—Nicotinamide-nucleotide adenylyltransferase (2.7.7.1)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the technical fields of genetic engineering and enzyme catalysis, and particularly relates to a nicotinamide adenine dinucleotide kinase mutant and application thereof in catalyzing and generating NADP+ by taking nicotinamide adenine dinucleotide as a substrate. The mutation is one or more of R120H, R236E and R237K. The mutant enzyme expressed by the NADK variant gene obtained by the invention has higher catalytic activity on NAD+, and compared with wild NADK, the catalytic efficiency is increased by 1.9 times. The variant gene provides a wide application prospect for the industrial production of NADP+.
Description
Technical Field
The invention belongs to the technical fields of genetic engineering and enzyme catalysis, and particularly relates to a nicotinamide adenine dinucleotide kinase mutant and application thereof in catalyzing and generating NADP+ by taking nicotinamide adenine dinucleotide as a substrate.
Background
NADP is an abbreviation for nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate), abbreviated as coenzyme II, an oxidative coenzyme which is widely involved in the redox metabolism of living organisms and in a range of other biochemical reactions. It acts as a hydrogen transporter during biological oxidation and participates in various anabolic reactions, such as the synthesis of lipids, fatty acids and nucleotides, in the form of reduced NADPH. Coenzyme II has wide application in the fields of life science, enzyme catalysis asymmetric synthesis, medical care and the like.
Coenzyme II is widely present in organisms but in very low levels. At present, various methods for preparing nicotinamide adenine dinucleotide phosphate are available, including chemical synthesis, biological enzyme catalysis, yeast fermentation extraction and the like. 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 higher production cost, and the problem of environmental pollution is caused by the need of using an organic solvent. For the method of yeast fermentation extraction, since the content of NADP+ in yeast is too low, the method has a great limitation on an industrial production scale. Biological enzyme catalysis is gradually the mainstream method for industrially preparing NADP+ because of the inherent advantages of high efficiency, environmental protection, mild reaction conditions, strong stereoselectivity, high conversion rate compared with a fermentation method and the like.
In order to improve the catalytic efficiency of nicotinamide adenine dinucleotide kinase (nicotinamide adenine dinucleotide kinase, NADK), reduce the biocatalysis synthesis cost of NADP and improve the industrial application value of NADK, the invention utilizes a saturation mutation technology to randomly mutate NADK genes, and obtains NADK variant genes with improved enzyme activity through a large number of screening.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is how to improve the catalytic efficiency of nicotinamide adenine dinucleotide kinase, reduce the biocatalytic synthesis cost of NADP, and improve the industrial application value of NADK.
To achieve the above object, the present invention provides a Nicotinamide Adenine Dinucleotide Kinase (NADK) mutant having higher catalytic activity compared to a wild type.
Preferably, the mutation site of the mutation is arginine on the wild type of nicotinamide adenine dinucleotide kinase.
Preferably, the mutation site of the mutation is arginine at position 120, 236 or 237 on the wild type nicotinamide adenine dinucleotide kinase.
Preferably, the mutation is from arginine to histidine, glutamic acid or lysine.
Preferably, the mutant is one or more of R120H, R236E and R237K.
Preferably, the mutant is R120H; R236E; R237K; R236E, R237K; or R120H, R236E, R237K.
Preferably, the wild type nicotinamide adenine dinucleotide kinase is derived from salmonella typhimurium (Salmonella enterica subsp. Enterica serovar Typhimurium str. LT2), the coding nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of which is shown in SEQ ID NO. 2.
Preferably, the amino acid sequences of the mutants are shown in SEQ ID NO. 4, 6, 8, 10 and 12.
In a preferred embodiment of the present invention, there is provided a polynucleotide encoding the nicotinamide adenine dinucleotide kinase mutant described above.
Preferably, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO. 3, 5, 7, 9 and 11.
In another preferred embodiment of the present invention, there is provided a vector comprising the polynucleotide described above.
In a further preferred embodiment of the present invention, there is provided an expression system comprising the vector described above.
According to the prior public knowledge, any gene is connected into various expression vectors after being operated or transformed, is transformed into a proper host cell, and can over-express target protein through induction under proper conditions. Therefore, the expression vector of the NADK enzyme and the mutant thereof can be pET or pCW or pUC or pPIC9k and the like, and the expression host can be escherichia coli, pichia pastoris, streptomycete, bacillus subtilis and the like.
The NADK gene sequence is synthesized by Changzhou-radix Yuan biotechnology Co Ltd, and is codon optimized for the codon preference of colibacillus, and NdeI and HindIII restriction endonuclease sites are respectively added at two ends of the coding region. The target gene fragment is subjected to restriction enzyme NdeI and HindIII digestion, then is connected with a pET28a (+) vector (Novagen company) subjected to double digestion, is transformed and screened to obtain a positive plasmid, and the positive plasmid NADK002-pET28a (+) is transferred into BL21 (DE 3) host bacteria, so that an in vitro heterologous expression system of NADK is constructed.
In a further preferred embodiment of the present invention there is provided the use of a mutant of nicotinamide adenine dinucleotide kinase as described above for the production of nicotinamide adenine dinucleotide phosphate.
The invention aims to provide a NADK mutant gene and application thereof, and overcomes the defect of poor catalytic activity of the traditional NADK in the process of catalyzing and synthesizing NADP+. The mutant enzyme expressed by the NADK variant gene obtained by the invention has higher catalytic activity on NAD+, and compared with wild NADK, the catalytic efficiency is increased by 1.9 times. The variant gene provides a wide application prospect for the industrial production of NADP+.
According to the invention, through analyzing the three-dimensional protein structure of NADK, one or more possible sites related to catalysis are predicted by utilizing the energy minimum principle and a molecular docking technology, then the NADK gene is mutated by utilizing a point saturation mutation technology, and the mutant with improved catalytic activity is obtained through screening. More specifically, when arginine (R) at position 120 is mutated to histidine (H), the catalytic activity of the mutant is increased relative to NADK. When arginine (R) at position 236 is mutated to glutamic acid (E), mutant enzyme activity is increased. When arginine (R) at position 237 is mutated to lysine (K), mutant enzyme activity is increased relative to NADK. When the above-mentioned 3-site mutations are combined in pairs or three, the catalytic activity of some mutants is improved more than that of a single mutant.
The conception, specific structure, and technical effects of the present invention will be further described below to fully understand the objects, features, and effects of the present invention.
Detailed Description
The following description will be given to various preferred embodiments of the present invention to make the technical contents thereof more clear and easy to understand. The present invention may be embodied in many different forms of embodiments and the scope of the present invention is not limited to only the embodiments described herein.
In the examples, the experimental procedures, which are not specified in particular conditions, were carried out according to conventional molecular biology experimental procedures, as described in the guidelines for molecular cloning experiments (J. Sambrook, D.W. Lassel, huang Peitang, wang Jiaxi, zhu Houchu, et al, third edition, beijing: science Press, 2002), or according to the methods recommended by the manufacturers of the kits.
EXAMPLE construction of prokaryotic expression System
The NADK gene fragment was synthesized by Changzhou-radix Yuan biotechnology Co., ltd and recombined onto the PUC57 vector. After double digestion with restriction enzymes NdeI and HindIII (available from New England Biolabs, NEB) for 4h at 37℃the digested products were separated by 1% agarose gel electrophoresis and recovered by gel digestion (gel recovery kit was available from Tiangen Biochemical Co., ltd.). Followed by ligation overnight at 16℃with the expression vector pET28a (+) (Novagen) subjected to the same double cleavage under the action of T4 DNA ligase (available from Takara). DH5a competent cells (purchased from Tiangen Biochemical technology (Beijing)) were transformed with the ligation solution, and colony PCR screening and sequencing verification were performed to obtain positive recombinant plasmid NADK-pET28a (+).
The positive recombinant plasmid NADK-pET28a (+) is transformed into expression host bacterium BL21 (DE 3) (purchased from Tiangen Biochemical technology (Beijing) limited company) to obtain prokaryotic expression strain NADK-pET28a (+)/BL 21 (DE 3) which is used as a primary strain for subsequent random mutation and fermentation.
The polyphosphate kinase (PPK 2, from E.coli) for ATP regeneration is synthesized by Changzhou-ary biotechnology Co., ltd, and the subsequent construction of recombinant expression plasmid is the same as that of NADK-pET28a (+) plasmid, and the expression strain is obtained after transfer into BL21 (DE 3).
Example two enzyme shake flask fermentation to prepare enzyme lyophilized powder
The expression strain NADK-pET28a (+)/BL 21 (DE 3) constructed above was cultured in 5ml LB liquid medium [ 10g/l tryptone (OXIO), 5g/l yeast powder (OXIO), 10g/l sodium chloride (national reagent) ] containing 30. Mu.g/ml kanamycin sulfate. The culture conditions were 37℃and 200rpm shaking, and the culture time was overnight. The overnight cultured broth was inoculated at a ratio of 1% (V/V) into 500ml LB liquid medium containing 30. Mu.g/ml kanamycin sulfate, and the culture was continued under shaking conditions of 200rpm at 37 ℃. When the OD600 value of the bacterial liquid reaches between 0.8 and 1.0, an inducer IPTG (isopropyl-. Beta. -D-thiogalactoside, IPTG) with a final concentration of 0.1mM is added, and the induction is carried out at 30 ℃ overnight. After induction, the cells were collected by centrifugation at 8000rpm at 4℃and suspended in 50mM sodium phosphate buffer pH 8.5. Followed by ultrasonication (ultrasonic power of 200W, crushing time of 3 seconds/dwell time of 5 seconds, total duration of 20 minutes). Then, the mixture was centrifuged at 12000rpm at 4℃for 20 minutes, and the supernatant was lyophilized to obtain a crude enzyme powder.
EXAMPLE construction of triple mutant library
Construction of the mutant: three-dimensional structural simulation of NADK was performed using homology modeling and the potential sites involved in catalysis and substrate binding were predicted using molecular docking and energy minimization principles, initially identified as three sites 120, 236, 237. Then, the four sites were subjected to saturation mutation respectively using NADK-pET28a (+) recombinant plasmid as a template (for specific mutation operations, reference is made to Stratagene Corp.)Site-Directed Mutagenesis Kit description of operation). Wherein the 120-site mutation forward primer: GGAAGGCNNKTATATTAGTGAAAAACGTTTTCTG, reverse primer: ATATAMNNGCCTTCCAGCACATCACT;236 mutation forward primer: TAGCCATNNKCGCAGCGATCTGGAAATTAGTTGC, reverse primer: CTGCGMNNATGGCTAAAGCGCAGGCG;237 site mutation forward primer: CCATCGTNNKAGCGATCTGGAAATTAGTTGCG, reverse primer: TCGCTMNNACGATGGCTAAAGCGCAGG.
Example four culture of mutants
Mutant culture: after transforming BL21 (DE 3) host bacteria with the above-obtained plasmid, the plasmid was spread on LB solid medium containing 30. Mu.g/ml kanamycin, and cultured overnight at 37℃in an inverted manner, and then the monoclonal antibody was picked up from the plate and cultured in a 96-well plate. The overnight cultured bacterial liquid was transferred to a 96-well plate containing fresh LB medium, and after shaking culture at 37℃and 220rpm for 4 hours, IPTG was added to the culture to give a final concentration of 0.1mM for induction, and the culture was continued overnight at 30 ℃. The cells were collected by centrifugation at 4000rpm for 10min at 4℃and suspended in 50mM sodium phosphate buffer pH8.5, followed by centrifugation to obtain a supernatant.
EXAMPLE five screening of mutants
Screening of mutants: using a substrate NAD concentration of 10g/L, ATP concentration of 50g/L and 10mM magnesium sulfate heptahydrate as reaction conditions, an appropriate amount of the supernatant prepared above was mixed with 10mM sodium phosphate buffer pH7.0, and the volume was made up to 2ml. Next, the mixture was placed under a constant temperature magnetic stirrer at 37℃to carry out a stirring reaction. After 20 minutes of reaction, samples were taken for HPLC detection.
The mutation sites contained in the clones with significantly improved mutant enzyme activity were as follows:
arginine (R) at position 120 is mutated to histidine (H);
arginine (R) at position 236 is mutated to glutamic acid (E);
arginine (R) at position 237 is mutated to lysine (K).
And then carrying out pairwise combined mutation and three combined mutations on the sites, wherein the activity detection shows that the catalytic activity of the combined mutation of the individual sites is obviously improved compared with that of single-point mutation, and the specific enzyme activity values are shown in the following table:
1U is defined as the amount of enzyme required to produce 1. Mu. Mol of product per unit of time (1 min).
Example six biocatalysis of mutants
400mg of substrate NAD was dissolved in 10mL of 50mM sodium phosphate buffer pH6.5, after the substrate was completely dissolved, 25mM sodium hexametaphosphate, 5mM ATP, 25mM magnesium sulfate heptahydrate, NADK mutant and PPK2 enzyme were added in amounts of 2g/L and 1g/L, respectively, and the reaction was stirred under a magnetic stirrer at a constant temperature of 50℃and carried out for 2 hours, followed by HPLC detection. The substrate conversion rate is more than 99%, and the generation rate of NADP can reach 95%.
The substrate conversion and product formation rates for the different mutants are shown in Table 2 below.
TABLE 2 substrate conversion and product formation of NADK with different mutants
| Amino acid sequence numbering | Mutant name | Substrate conversion (%) | Product yield (%) |
| SEQ ID NO:2 | WildtypeNADK | 82％ | 78％ |
| SEQ ID NO:4 | R236E | 84％ | 80％ |
| SEQ ID NO:6 | R120H | 85％ | 81％ |
| SEQ ID NO:8 | R237K | 85％ | 81％ |
| SEQ ID NO:10 | R236E-R237K | 90％ | 85％ |
| SEQ ID NO:12 | R236E-R237K-R120H | 99％ | 95％ |
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, the technical solutions obtained by logic analysis, reasoning or limited experiments based on the prior art by those skilled in the art according to the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. A mutant of Nicotinamide Adenine Dinucleotide Kinase (NADK) having a mutation site of arginine on a wild type of nicotinamide adenine dinucleotide kinase, the mutant having a higher catalytic activity compared to the wild type.
2. The NADK mutant of claim 1, wherein the mutation site of the mutation is arginine at position 120, 236 or 237 on a wild-type nicotinamide adenine dinucleotide kinase.
3. The NADK mutant according to claim 2, wherein the mutation is a mutation from arginine to histidine, glutamic acid or lysine.
4. The NADK mutant of claim 3, wherein the mutation is one or more of R120H, R E and R237K.
5. The NADK mutant of claim 4, wherein the mutant is R120H; R236E; R237K; R236E, R237K; or R120H, R236E, R237K.
6. The NADK mutant according to any one of claims 1-5, wherein the wild type nicotinamide adenine dinucleotide kinase is derived from salmonella typhimurium (Salmonella enterica subsp.
7. A polynucleotide encoding the nicotinamide adenine dinucleotide kinase mutant of claims 1-6.
8. A vector comprising the polynucleotide of claim 7.
9. An expression system comprising the nucleotide of claim 7 and/or the vector of claim 8.
10. Use of the nicotinamide adenine dinucleotide kinase mutant according to claims 1-6 for the production of nicotinamide adenine dinucleotide phosphate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311316999.6A CN117511907A (en) | 2023-10-12 | 2023-10-12 | Nicotinamide adenine dinucleotide kinase mutant and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311316999.6A CN117511907A (en) | 2023-10-12 | 2023-10-12 | Nicotinamide adenine dinucleotide kinase mutant and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117511907A true CN117511907A (en) | 2024-02-06 |
Family
ID=89740785
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311316999.6A Pending CN117511907A (en) | 2023-10-12 | 2023-10-12 | Nicotinamide adenine dinucleotide kinase mutant and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117511907A (en) |
-
2023
- 2023-10-12 CN CN202311316999.6A patent/CN117511907A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108424900B (en) | Nitrilase mutant and construction method and application thereof | |
| CN110373397B (en) | Nicotinamide phosphoribosyl transferase mutant and application thereof | |
| CN111254129B (en) | Polyphosphate kinase mutant and application thereof | |
| CN112159831B (en) | Method for preparing nicotinamide mononucleotide | |
| CN105331642B (en) | Method for catalytically producing α -ketoglutaric acid by using L-glutamic acid oxidase | |
| CN108060114B (en) | A kind of Escherichia coli of fermenting and producing l-Alanine and its application | |
| CN109266664B (en) | Method for improving stability of glutamate oxidase by using fusion truncated expression strategy | |
| Zhang et al. | Exploring a highly D-galactose specific L-arabinose isomerase from Bifidobacterium adolescentis for D-tagatose production | |
| CN112574970B (en) | Nicotinamide mononucleotide adenyltransferase mutant and application thereof | |
| CN114107252A (en) | CL7 protein, high-activity recombinant TET enzyme CL7-NgTET1, prokaryotic expression vector and application thereof | |
| CN114277022B (en) | Nitrile hydratase mutant with high activity and high thermal stability | |
| CN114774379B (en) | Carbonyl reductase mutant with improved heat stability | |
| CN117511907A (en) | Nicotinamide adenine dinucleotide kinase mutant and application thereof | |
| CN115896081A (en) | Aspartase mutant and application thereof | |
| US20210238576A1 (en) | L-aspartate alpha-decarboxylase Mutant and Application thereof | |
| CN112852764B (en) | Mutant protein of ketonizing enzyme and application | |
| CN111690675B (en) | Recombinant bacterium for expressing nitrile hydratase mutant and preparation method and application thereof | |
| CN107760736B (en) | Method for promoting indigo biosynthesis conversion yield | |
| CN111172143A (en) | D-xylonic acid dehydratase and application thereof | |
| CN114277013B (en) | NAD kinase mutant and application thereof | |
| CN114196658B (en) | Nitrilase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid | |
| CN104805108B (en) | A kind of fast construction method of high copy gene multiple repeats | |
| Yuan et al. | Production of 5′-Inosinic acid by whole-cell biocatalyst expressing a mutated acid phosphatase/phosphotransferase | |
| CN110872595B (en) | Acid-resistant expression cassette and application thereof in fermentation production of organic acid | |
| CN114438052B (en) | Nicotinamide mononucleotide adenyltransferase mutant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20240226 Address after: No. 28, Jiuzhou Avenue, Torch Development Zone, Zhongshan City, Guangdong Province Applicant after: Zhongshan bailing Biotechnology Co.,Ltd. Country or region after: China Address before: 528437 room 901, 9 / F, building A8, No.12 Biovalley Avenue, Torch Development Zone, Zhongshan City, Guangdong Province Applicant before: Zhongshan Junkai Biotechnology Development Co.,Ltd. Country or region before: China |
|
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |