CN116904421A - NADH pyrophosphatase mutant with enhanced heat resistance and application thereof - Google Patents
NADH pyrophosphatase mutant with enhanced heat resistance and application thereof Download PDFInfo
- Publication number
- CN116904421A CN116904421A CN202310875031.0A CN202310875031A CN116904421A CN 116904421 A CN116904421 A CN 116904421A CN 202310875031 A CN202310875031 A CN 202310875031A CN 116904421 A CN116904421 A CN 116904421A
- Authority
- CN
- China
- Prior art keywords
- nadh
- mutant
- pyrophosphatase
- fermentation
- thermostable
- 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
- 108010067588 nucleotide pyrophosphatase Proteins 0.000 title claims abstract description 26
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 28
- 102000004190 Enzymes Human genes 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 19
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims abstract description 18
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 27
- 238000000855 fermentation Methods 0.000 claims description 22
- 230000004151 fermentation Effects 0.000 claims description 22
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
- 229940041514 candida albicans extract Drugs 0.000 claims description 12
- 239000012138 yeast extract Substances 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 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 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 8
- 230000014509 gene expression Effects 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
- 235000019270 ammonium chloride Nutrition 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 6
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 6
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 6
- 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 claims description 6
- 229930027917 kanamycin Natural products 0.000 claims description 6
- 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 claims description 6
- 229960000318 kanamycin Drugs 0.000 claims description 6
- 229930182823 kanamycin A Natural products 0.000 claims description 6
- 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 claims description 6
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 6
- 235000011152 sodium sulphate Nutrition 0.000 claims description 6
- 239000012137 tryptone Substances 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 4
- 108091033319 polynucleotide Proteins 0.000 claims description 4
- 239000002157 polynucleotide Substances 0.000 claims description 4
- 102000040430 polynucleotide Human genes 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 3
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 3
- 238000005273 aeration Methods 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 235000011007 phosphoric acid Nutrition 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000009465 prokaryotic expression Effects 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 26
- XQHMUSRSLNRVGA-TURQNECASA-N NMNH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1 XQHMUSRSLNRVGA-TURQNECASA-N 0.000 description 12
- 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 description 11
- 229950006238 nadide Drugs 0.000 description 10
- DAYLJWODMCOQEW-TURQNECASA-O NMN(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(O)=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-O 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 235000020956 nicotinamide riboside Nutrition 0.000 description 4
- 239000011618 nicotinamide riboside Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 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 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 108700010070 Codon Usage Proteins 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000006722 reduction reaction Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 description 1
- OOXNYFKPOPJIOT-UHFFFAOYSA-N 5-(3-bromophenyl)-7-(6-morpholin-4-ylpyridin-3-yl)pyrido[2,3-d]pyrimidin-4-amine;dihydrochloride Chemical compound Cl.Cl.C=12C(N)=NC=NC2=NC(C=2C=NC(=CC=2)N2CCOCC2)=CC=1C1=CC=CC(Br)=C1 OOXNYFKPOPJIOT-UHFFFAOYSA-N 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 108010076278 Adenosine kinase Proteins 0.000 description 1
- 102100032534 Adenosine kinase Human genes 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 101150109562 Nmrk1 gene Proteins 0.000 description 1
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical class C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 231100000446 genotoxin Toxicity 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 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
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 102220121910 rs114986640 Human genes 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 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
- C12N9/14—Hydrolases (3)
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
- C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
- C12Y306/01022—NAD+ diphosphatase (3.6.1.22)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the technical field of enzyme catalysis, in particular to a heat-resistant enhanced NADH pyrophosphatase mutant and application thereof, wherein the amino acid sequence of the NADH pyrophosphatase mutant is shown as SEQ ID NO:1. When in use, NADH dry powder is taken and added with MgCl 2 、MnCl 2 And Tris-HCl, adding crude enzyme solution of NADH pyrophosphatase mutant, and reacting at 30-60 ℃. Compared with wild NADH pyrophosphatase, the NADH pyrophosphatase mutant can be applied to catalyzing NADH at high temperature and shows stronger heat stability.
Description
Technical Field
The invention relates to the technical field of enzyme catalysis, in particular to a heat-resistant NADH pyrophosphatase mutant and application thereof.
Background
Nicotinamide Adenine Dinucleotide (NAD) is critical to organisms because it participates in hundreds of biological reactions and regulates key biological processes such as metabolism and DNA repair. Research shows that up-regulating NAD biosynthesis through gene operation can raise stress resistance and prolong life of yeast and drosophila; increasing NAD levels proved to delay progestin and other degenerative diseases in mice. Moreover, there is sufficient evidence that Nicotinamide Riboside (NR) and Nicotinamide Mononucleotide (NMN) are potent NAD enhancers because they increase cellular NAD levels and confer a variety of health benefits; supplementation with NR or NMN may protect mice from age-related health deterioration.
Currently, these studies have focused mainly on the oxidized form of NAD precursors, since most NAD-consuming enzymes use NAD as a substrate. Little is known about the effect of the reduced form of NAD precursor. Recent studies have shown that reduced forms of NR (denoted NRH) can enhance NAD better in cells and tissues than NR or NMN. NRH increases resistance to genotoxin-induced cell death, and NRH conversion to NMNH is independent of Nrk1 or Nrk2.NRH is converted to NMNH by adenosine kinase to synthesize NAD and demonstrates that oral NRH can prevent acute kidney injury in mice.
The university of Qinghua developed a chemical reduction method to synthesize reduced NMN, called NMNH, and studied the biological impact of NMNH on cellular processes, found that NMNH was a better NAD enhancer than NMN both in vitro and in vivo, and found that NMNH increased cellular NADH levels, induced reduction stress, and inhibited cell growth, glycolysis, and TCA cycle.
Although the absorption effect of the reduced Nicotinamide Mononucleotide (NMNH) is better than that of NMN, the reduced nicotinamide mononucleotide has poor stability of aqueous solution, is extremely easy to degrade at normal temperature and neutral pH, is further aggravated with the extension of time, and causes great obstacle to mass production. To solve this problem, there are two schemes worth trying: one is to promote the enzyme activity and shorten the total reaction time so as to reduce the degradation occurrence time of NMNH, and the reaction is finished as soon as possible to carry out the extraction process; and the other is to raise the pH value of the total reaction system, so that the stability of NMNH is enhanced and the degradation degree in the reaction process is slowed down. The first scheme can be realized by screening high-enzyme activity mutants and improving the heat resistance of the enzyme, and the collision frequency of enzyme molecules at high temperature is increased, so that the enzymatic reaction process can be accelerated to a certain extent, and the screening of the enzyme mutants with enhanced heat resistance has high practical significance.
Disclosure of Invention
The invention aims to provide an NADH pyrophosphatase mutant with enhanced heat resistance and application thereof.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a thermostable enhanced mutant NADH pyrophosphatase having an amino acid sequence as set forth in SEQ ID NO:1.
The thermostable enhanced NADH pyrophosphatase mutant of the present invention can be used for catalyzing NADH, and the use method thereof is as follows: taking NADH dry powder, adding MgCl 2 、MnCl 2 And Tris-HCl, adding crude enzyme solution of NADH pyrophosphatase mutant, and reacting at 30-60 ℃.
The preparation method of the crude enzyme liquid comprises the following steps:
(1) By a primer splicing method, SEQ ID NO:1 and cloning the corresponding coding polynucleotide sequence of the protein shown in the formula 1 into a prokaryotic expression vector to realize high expression in escherichia coli;
(2) By shake flask fermentation or fed-batch fermentation
(1) Shaking flask fermentation
E.coli single colony containing the expression vector is selected and inoculated in 10mL of culture medium A after autoclaving, and is cultured at 30 ℃ and 250rpm overnight;
taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1:100 was inoculated into 100mL of the autoclaved medium B, cultured at 30℃until the cell OD 5-6 was reached, and the flask was immediately placed in a 25℃shaker at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and incubation was continued at 25℃and 250rpm for 16 hours;
after the culture, the culture solution was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells; then washing the bacterial precipitate twice with distilled water, collecting bacterial precipitate, and preserving at-70 ℃; simultaneously taking a small amount of thalli for SDS-PAGE detection;
(2) fed-batch fermentation
Fed-batch fermentation was performed in a computer controlled bioreactor, a 200ml seed shake flask was prepared from a single colony of E.coli harboring the expression vector, and the bioreactor was accessed when the culture of the seed shake flask was OD 2.0; the temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration during the fermentation was automatically controlled at 30% by the stirring rate and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% v/v orthophosphoric acid and 30% v/v aqueous ammonia; during the fermentation, when the dissolved oxygen is greatly raised, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when the OD600 is 50.0, the temperature is controlled to be 25 ℃, 0.1mM IPTG is used for inducing expression for 16 hours, and the collected thalli are centrifugally preserved at the temperature of minus 25 ℃, and when in use, 2 kg of pure water is added for each kg of wet thalli.
Wherein, the culture medium A is: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and kanamycin is added to 50mg/L.
Wherein, the culture medium B is: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and kanamycin is added to 50mg/L.
Wherein, the culture medium used for fed-batch fermentation is: 24g/L of yeast extract, 12g/L of peptone, 0.4% w/v glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, pH 7.0.
Compared with the prior art, the invention has the beneficial effects that:
the thermostable NADH pyrophosphatase mutant can catalyze NADH at high temperature, and compared with wild NADH pyrophosphatase, the thermostable NADH pyrophosphatase mutant has stronger thermostability and can obtain better social benefit and economic value.
Drawings
FIG. 1 shows the results of a high performance liquid chromatography with 0.5g/L NADH; 19.2 minutes are the substrate NADH.
FIG. 2 shows the results of a 0.4g/L NMNH HPLC; 8.2 minutes was the product NMNH peak.
FIG. 3 shows the results of the 15-minute reaction in example 5.
FIG. 4 shows the results of the 15-minute reaction in comparative example 1.
FIG. 5 shows the results of the 23 hour reaction in example 6.
FIG. 6 shows the results of the 15-minute reaction in example 7.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The instruments and reagents used in this example are commercially available products unless otherwise specified.
The liquid phase detection conditions referred to in the following examples are as follows:
mobile phase: phase A: 15.6g of sodium dihydrogen phosphate dihydrate is dissolved in 900mL, and after complete dissolution, water is added to fix the volume of 1000mL. Then 20mL of methanol is added for complete dissolution, and then ultrasonic filtration is performed. And B phase: methanol. A, B=95% and 5%.
Retention time: isocratic elution was performed for 22 minutes.
Column type: shimadzu C18.6 x 250mm x 5 μm.
Ultraviolet wavelength: 340nm.
Column temperature: 30 ℃.
Sample injection volume: 10 mu L.
EXAMPLE 1 obtaining the wild-type NADH pyrophosphatase Gene sequence
The secondary structure and codon preference of the gene are adjusted by a total gene synthesis method so as to realize high expression in escherichia coli. The Primer Premier (http:// Primer3.Ut. Ee /) and OPTIMIZER (http:// genome. Uro. Es/OPTIMIZER /) were used for design, and the difference in annealing temperature (Tm) was controlled within 3 ℃, the Primer length was controlled within 60base, the Primer sequences were as shown in Table 2, and the obtained primers were dissolved in double distilled water and then added to the following reaction system so that the final concentration of each Primer was 30nM and the final concentration of the head-to-tail primers was 0.6. Mu.M.
TABLE 1
| 2mMdNTP mix(2mM eachdNTP) | 5μl |
| 10×Pfubuffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH 2 O | So that the total volume of the reaction system was 50. Mu.l |
The prepared PCR reaction system is placed in a Bo-Japanese patent application (XP) cycler gene amplification instrument for amplification according to the following procedures: 98℃30s,55℃45s,72℃120s,35x. The DNA fragment obtained by PCR was cut and purified, and cloned into NdeI/XhoI site of pET30a by homologous recombination. The monoclonal was picked for sequencing. The DNA sequence which is sequenced successfully is SEQ ID NO:4, designated PKNPYwt, the corresponding amino acid sequence of which is SEQ ID NO:3.
TABLE 2
| 1 | ATGCGTACCGGTCGTTGGCAGTCTGCTCTGCTGGACCCGGCTGCTGCTGGTGGTTG |
| 2 | GTTAGCGTCACCCAGGAACTGCTGTTTGTAGTGAGCCAGAGCCCAACCACCAGCAGCAGC |
| 3 | GTTCCTGGGTGACGCTAACGGTGTTCTGTTCCCGCGTGAATGGCTGAAACGTCAGGACCT |
| 4 | TCACCGTCGAAGTGACCAACACCGTGTTCAGACAGAACACGCAGGTCCTGACGTTTCAGC |
| 5 | TGGTCACTTCGACGGTGACGCTATCTACCTGCTGGAAGTTGACGCTCCGGAACGTCTGGA |
| 6 | AGCTTCCAGCATGAAGTGACGCAGACCGATCCAGTCGCAACCTTCCAGACGTTCCGGAGC |
| 7 | GTCACTTCATGCTGGAAGCTGACGAAGACCTGTTCGCTATGCTGGGTTTCGCTTCTCAGA |
| 8 | GCAAGAACCGCAGAAACGGTTTTCACGAGCCCAGGTACCGATCTGAGAAGCGAAACCCAG |
| 9 | CGTTTCTGCGGTTCTTGCGGTGCTCCGATGCAGCGTATGCCGCGTGACCGTGCTATGCGT |
| 10 | CGGAGACAGCAGCGGGTAACGCTGGATGTCGCAGGTTTCGCAACGCATAGCACGGTCACG |
| 11 | CCCGCTGCTGTCTCCGTCTATGATCGTTCTGGTTACCCGTGGTGACGAACTGCTGCTGGC |
| 12 | AGCCAGGGTAGAGTACATACCCGGAACGAAACGCGGAGAACGAGCCAGCAGCAGTTCGTC |
| 13 | GGTATGTACTCTACCCTGGCTGGTTTCTGCGAACCGGGTGAATCTGTTGAACACTGCGTT |
| 14 | ACCGATTTCCAGACCAACTTCTTCACGAACTTCACGAGCAACGCAGTGTTCAACAGATTC |
| 15 | GAAGTTGGTCTGGAAATCGGTAACATCCGTTACCTGGGTTCTCAGTCTTGGCCGTTCCCG |
| 16 | CACCAGAAACGTAGTCAGCGTGGAAACCCAGCATCAGAGAGTGCGGGAACGGCCAAGACT |
| 17 | CGCTGACTACGTTTCTGGTGAAATCGTTATGCAGCCGGACGAAATCGAAGACGCTCGTTG |
| 18 | ACGACCAGCCGGCAGACGCGGCAGTTCGTCGATACGGAACCAACGAGCGTCTTCGATTTC |
| 19 | CTGCCGGCTGGTCGTTCTATCGCTCGTTACCTGATCGACGTTTTCCTGGCTCGTCGTGCT |
| 20 | TTAGTGACCACCACCCGGCAGAACCGGGTCCGGCAGACCAGCACGACGAGCCAGG |
EXAMPLE 2 acquisition of Gene sequence of NADH pyrophosphatase mutant
The NADH pyrophosphatase suitable for high temperature reaction of the present invention is derived from the nucleotide sequence of SEQ ID NO:3, wild-type NADH pyrophosphatase. NADH pyrophosphatase mutants and polynucleotides encoding such mutants may be prepared using methods commonly used by those skilled in the art. Mutants can be obtained by subjecting the enzyme-encoding enzyme to in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, directed evolution methods, and the like.
The secondary structure and codon preference of the gene are adjusted by a total gene synthesis method so as to realize high expression in escherichia coli. The Primer Premier (http:// Primer3.Ut. Ee /) and OPTIMIZER (http:// genome. Uro. Es/OPTIMIZER /) were used for design, and the difference in annealing temperature (Tm) was controlled within 3 ℃, the Primer length was controlled within 60base, the Primer sequences were as shown in Table 4, and the obtained primers were dissolved in double distilled water and then added to the following reaction system so that the final concentration of each Primer was 30nM and the final concentration of the head-to-tail primers was 0.6. Mu.M.
TABLE 3 Table 3
| 2mM dNTP mix(2mM eachdNTP) | 5μl |
| 10×Pfubuffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH 2 O | So that the total volume of the reaction system was 50. Mu.l |
The prepared PCR reaction system is placed in a Bo-Japanese patent application (XP) cycler gene amplification instrument for amplification according to the following procedures: 98℃30s,55℃45s,72℃120s,35x. The DNA fragment obtained by PCR was cut and purified, and cloned into NdeI/XhoI site of pET30a by homologous recombination. The monoclonal was picked for sequencing. The DNA sequence which is sequenced successfully is SEQ ID NO:2, designated PKNPY1, having the corresponding amino acid sequence of SEQ ID NO:1. compared to the sequence of PKNPYwt, there are three mutations V68L, E109H, R242S.
TABLE 4 Table 4
Example 3 shake flask expression test
E.coli single colonies containing the expression vector were picked and inoculated into 10ml of autoclaved medium: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and kanamycin is added to 50mg/L. Culturing at 30℃and 250rpm overnight.
Taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1: an inoculation ratio of 100 was inoculated into 100ml of autoclaved medium: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and kanamycin is added to 50mg/L. The cells were cultured at 30℃until the cell OD 5-6 was reached, and the flask was immediately placed in a 25℃shaker at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and the incubation was continued at 25℃and 250rpm for 16 hours.
After the completion of the culture, the culture was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells. Then the bacterial cell precipitate is washed twice with distilled water, and the bacterial cells are collected and stored at-70 ℃. And simultaneously taking a small amount of thalli for SDS-PAGE detection.
Example 4 fed-batch fermentation
Fed-batch fermentation was performed in a computer controlled bioreactor (Shanghai state of China) with 15L capacity and 8L working volume using 24g/L yeast extract, 12g/L peptone, 0.4% w/v glucose, 2.31g/L phosphatase and 12.54g/L dipotassium hydrogen phosphate, pH 7.0.
E.coli single colonies containing the expression vector were prepared into 200ml seed shake flasks and were accessed into the bioreactor when the culture of the seed shake flasks was OD 2.0. The temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration was automatically controlled at 30% by stirring rate (rpm) and aeration supply cascade, and the pH of the medium was maintained at 7.0 by 50% (v/v) orthophosphoric acid and 30% (v/v) aqueous ammonia. During the fermentation process, when the dissolved oxygen is greatly raised, the feeding is started. The feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol. When OD600 was about 50.0 (wet weight: about 100 g/L), the temperature was controlled at 25℃and expression was induced with 0.1mM IPTG for 16 hours, and the cells were collected by centrifugation and stored at-25℃and used by adding 2 kg of pure water per kg of wet cells.
EXAMPLE 5 mutant high temperature reaction
Water bath reaction at 60 deg.c and 0.5mM MgCl 2 ，0.5mM MnCl 2 20mM Tris-HCl, pH=8.0; 60mM ADH dry powder; 10g/L of crude enzyme PKNPY 1. Samples were taken 15 minutes to detect product formation. The results in FIG. 3 show that the PKNPY1 system has reacted more than 95% of the substrate at 15 minutes. Since the byproduct adenylate absorbs weakly at 340nm, it is not shownObvious absorption peaks are shown, but do not affect the judgment of the overall reaction.
Comparative example 1 wild-type protein high temperature reaction
Water bath reaction at 60 deg.c and 0.5mM MgCl 2 ，0.5mM MnCl 2 20mM Tris-HCl, pH=8.0; 60mM NADH dry powder; 10g/L of crude enzyme PKNPYwt. Samples were taken 15 minutes to detect product formation. The results of FIG. 4 show that only about 1/5 of NADH was reacted, a large amount of substrate remained in the reaction system, and that PKNPYwt was low in the enzyme activity under the above conditions.
EXAMPLE 6 prolonged reaction time production of product
Water bath reaction at 60 deg.c and 0.5mM MgCl 2 ，0.5mM MnCl 2 20mM Tris-HCl, pH=8.0; 60mM NADH dry powder; 10g/L of crude enzyme PKNPY 1. Samples were taken after 23 hours to detect product formation. The results in fig. 5 show that NMNH is mostly degraded, showing the instability of the product solution.
EXAMPLE 7 mutant Normal temperature reaction
30 ℃ water bath reaction, 0.5mM MgCl 2 ，0.5mM MnCl 2 20mM Tris-HCl, pH=8.0; 60mM NADH dry powder; 10g/L of crude enzyme PKNPY 1. Samples were taken 15 minutes to detect product formation. The results in FIG. 6 show that only about 3/4 of NADH has reacted, and that significant substrate remains in the reaction system, which can cause significant interference with subsequent separation and extraction.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A thermostable enhanced NADH pyrophosphatase mutant characterized in that: the amino acid sequence is shown in SEQ ID NO:1.
2. Use of the thermostable enhanced NADH pyrophosphatase mutant of claim 1 for catalyzing NADH.
3. The use of a thermostable enhanced mutant NADH pyrophosphatase according to claim 2 for catalyzing NADH, characterized in that: taking NADH dry powder, adding MgCl 2 、MnCl 2 And Tris-HCl, adding crude enzyme solution of NADH pyrophosphatase mutant, and reacting at 30-60 ℃.
4. The use of a thermostable enhanced mutant NADH pyrophosphatase according to claim 3, for catalyzing NADH, wherein said crude enzyme solution preparation method comprises the steps of:
(1) By a primer splicing method, SEQ ID NO:1 and cloning the corresponding coding polynucleotide sequence of the protein shown in the formula 1 into a prokaryotic expression vector to realize high expression in escherichia coli;
(2) By shake flask fermentation or fed-batch fermentation
(1) Shaking flask fermentation
E.coli single colony containing the expression vector is selected and inoculated in 10mL of culture medium A after autoclaving, and is cultured at 30 ℃ and 250rpm overnight; the culture medium A is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and adding kanamycin to 50mg/L;
taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1: inoculating 100 to 100mL of autoclaved culture medium B, culturing at 30deg.C until the cell OD 5-6 is reached, immediately placing the triangular flask in 25 deg.C shaking table, and culturing at 250rpm for 1 hr; IPTG was added to a final concentration of 0.1mM and incubation was continued at 25℃and 250rpm for 16 hours; the culture medium B is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and adding kanamycin to 50mg/L;
after the culture, the culture solution was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells; then washing the bacterial precipitate twice with distilled water, collecting bacterial precipitate, and preserving at-70 ℃; simultaneously taking a small amount of thalli for SDS-PAGE detection;
(2) fed-batch fermentation
Fed-batch fermentation was performed in a computer controlled bioreactor, a 200ml seed shake flask was prepared from a single colony of E.coli harboring the expression vector, and the bioreactor was accessed when the culture of the seed shake flask was OD 2.0; the temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration during the fermentation was automatically controlled at 30% by the stirring rate and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% v/v orthophosphoric acid and 30% v/v aqueous ammonia; during fermentation, when dissolved oxygen rise occurs, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when the OD600 was 50.0, the temperature was controlled at 25℃and expression was induced with 0.1mM IPTG for 16 hours, and the cells were harvested by centrifugation and stored at-25 ℃.
5. The use of a thermostable enhanced mutant NADH pyrophosphatase according to claim 4, for catalyzing NADH, wherein: in the preparation method of the crude enzyme liquid, the culture medium used for fed-batch fermentation is as follows: 24g/L of yeast extract, 12g/L of peptone, 0.4% w/v glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, pH 7.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310875031.0A CN116904421A (en) | 2023-07-17 | 2023-07-17 | NADH pyrophosphatase mutant with enhanced heat resistance and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310875031.0A CN116904421A (en) | 2023-07-17 | 2023-07-17 | NADH pyrophosphatase mutant with enhanced heat resistance and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116904421A true CN116904421A (en) | 2023-10-20 |
Family
ID=88359828
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310875031.0A Pending CN116904421A (en) | 2023-07-17 | 2023-07-17 | NADH pyrophosphatase mutant with enhanced heat resistance and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116904421A (en) |
-
2023
- 2023-07-17 CN CN202310875031.0A patent/CN116904421A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109735553B (en) | Preparation method of anti-AIDS drug atazanavir intermediate | |
| CN1247790C (en) | Method for producing ascorbic acid intermediates | |
| CN108220276B (en) | Cephalosporin C acylase mutant and application thereof in 7-aminocephalosporanic acid production | |
| WO2018023207A1 (en) | Method for preparing nicotinamide mononucleotide | |
| TWI601825B (en) | Process for the enantioselective enzymatic reduction of intermediates | |
| CN109055324B (en) | Improved ketoreductase and application thereof | |
| CN117535256A (en) | Carbonyl reductase and application thereof in synthesis of vitronectin | |
| CN109943577B (en) | Biotransformation method of anti-AIDS drug atazanavir intermediate | |
| CN1191369C (en) | Process for producing adenosylmethionine by metabolic engineering bacteria | |
| CN108220218B (en) | Microbial strain modified by kinase gene with phosphorylation function and application thereof | |
| CN115747183A (en) | Ketoreductase mutant and application thereof | |
| CN116904421A (en) | NADH pyrophosphatase mutant with enhanced heat resistance and application thereof | |
| CN117143844A (en) | NADH pyrophosphatase suitable for alkaline pH and application thereof | |
| CN116855561A (en) | Process for preparing reduced nicotinamide mononucleotide under high-temperature and high-pH conditions | |
| JPWO2016060171A1 (en) | Yeast producing ethanol from xylose | |
| CN114752574B (en) | Enzyme catalysis system, catalase, preparation method and application | |
| CN117904067A (en) | Transaminase mutant for producing sitagliptin intermediate | |
| CN114517174B (en) | Engineering bacteria for synthesizing notoginseng element and application | |
| CN116790569B (en) | Pyruvic acid decarboxylase mutant and application thereof in preparation of alpha-hydroxyketone compound | |
| CN114875011B (en) | AMP phosphotransferase mutant, coding gene thereof and application thereof in ATP synthesis | |
| CN116925993B (en) | Genetically engineered strains and methods for enzyme-catalyzed production of cytidine acids | |
| CN117757764A (en) | Formate dehydrogenase mutant and application thereof in preparation of reduced nicotinamide adenine dinucleotide | |
| JP7375052B2 (en) | Ketoreductase mutant and method for producing chiral alcohol | |
| CN116622691A (en) | Glucose isomerase suitable for low pH condition and application thereof | |
| CN116606845A (en) | Glucose isomerase suitable for high-temperature reaction and application |
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 |