CN116590271A - Glucose isomerase with enhanced heat resistance and application thereof - Google Patents
Glucose isomerase with enhanced heat resistance and application thereof Download PDFInfo
- Publication number
- CN116590271A CN116590271A CN202310795231.5A CN202310795231A CN116590271A CN 116590271 A CN116590271 A CN 116590271A CN 202310795231 A CN202310795231 A CN 202310795231A CN 116590271 A CN116590271 A CN 116590271A
- Authority
- CN
- China
- Prior art keywords
- glucose
- fructose
- glucose isomerase
- 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
- 108700040099 Xylose isomerases Proteins 0.000 title claims abstract description 39
- 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 abstract description 37
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 claims abstract description 24
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 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
- 239000008103 glucose Substances 0.000 claims description 19
- 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
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 241000588724 Escherichia coli Species 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 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 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 8
- 230000014509 gene expression Effects 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 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
- 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
- 238000002360 preparation method Methods 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
- 239000007788 liquid Substances 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 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
- 238000005273 aeration Methods 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 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
- 238000011081 inoculation 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
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- 239000010413 mother solution Substances 0.000 claims description 2
- 230000009465 prokaryotic expression Effects 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 29
- 238000006555 catalytic reaction Methods 0.000 abstract description 2
- 239000012452 mother liquor Substances 0.000 abstract 1
- 229930091371 Fructose Natural products 0.000 description 12
- 239000005715 Fructose Substances 0.000 description 12
- 235000021433 fructose syrup Nutrition 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 235000013305 food Nutrition 0.000 description 6
- 238000006317 isomerization reaction Methods 0.000 description 6
- 239000006188 syrup Substances 0.000 description 6
- 235000020357 syrup Nutrition 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 4
- PJVXUVWGSCCGHT-ZPYZYFCMSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;(3s,4r,5r)-1,3,4,5,6-pentahydroxyhexan-2-one Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO PJVXUVWGSCCGHT-ZPYZYFCMSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000000758 substrate 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
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 1
- ZAQJHHRNXZUBTE-WUJLRWPWSA-N D-xylulose Chemical compound OC[C@@H](O)[C@H](O)C(=O)CO ZAQJHHRNXZUBTE-WUJLRWPWSA-N 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 238000010960 commercial process Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229960004903 invert sugar Drugs 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 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
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 102220145534 rs886058950 Human genes 0.000 description 1
- 235000014214 soft drink Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000004127 xylose metabolism 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
- C12N9/90—Isomerases (5.)
- C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
-
- 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
- C12P19/02—Monosaccharides
-
- 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
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
- C12Y503/01005—Xylose isomerase (5.3.1.5)
-
- 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
-
- 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
Abstract
The invention relates to the technical field of enzyme catalysis, in particular to a glucose isomerase with enhanced heat resistance and application thereof, wherein the amino acid sequence of the glucose isomerase is shown as SEQ ID NO:1. When in use, the D-glucose mother liquor is taken, PB and pure water are added, and crude enzyme solution of glucose isomerase is added for reaction at 80 ℃. The glucose isomerase with enhanced heat resistance can catalyze D-glucose to generate D-fructose at high temperature; compared with wild glucose isomerase, the method can be applied to high-temperature conversion of D-glucose to generate D-fructose, and has stronger thermal stability.
Description
Technical Field
The invention relates to the technical field of enzyme catalysis, in particular to a glucose isomerase with enhanced heat resistance.
Background
Glucose isomerase, also known as D-xylose isomerase, is an enzyme with great industrial application value that catalyzes the isomerization of D-xylose to xylulose in the first step of xylose metabolism in many microorganisms. Glucose isomerase is involved in sugar metabolism and is widely used in the industrial production of high fructose syrup (HFCS) and bioethanol. Such isomerization reactions are important in industrial processes, such as the production of fructose syrups in the food industry. Fructose syrup itself is popular in the united states and japan as a sweetener and is an alternative source of sucrose or invert sugar in the pharmaceutical, food and beverage industries. Under the push of the growing market demand for fructose and glucose syrup, glucose isomerase has been increasingly used for producing fructose and glucose syrup from corn starch hydrolysates since 1970 s, and has been juxtaposed with amylase and protease as three of the most consumed industrial enzymes. Because fructose syrups containing 55% or more fructose have a higher sweetness than sucrose, there is a greater demand for such syrups in the food and beverage industries, and as the fructose syrup market grows, so does the demand for more efficient glucose isomerase.
In the existing high fructose syrup production process, thermolabile glucose isomerase from a mesophilic microorganism is used in an immobilized enzyme reactor to produce 40% to 42% high fructose syrup at an operating temperature of 50 ℃ and 58 ℃ with a residence time of less than 1 hour. If this temperature is exceeded, undesirable browning products may be formed due to non-enzymatic reactions (e.g., maillard reactions) between the reducing sugars and the proteins. However, this temperature limitation is detrimental to high glucose conversion. In fact, the isomerisation of glucose will reach a reaction equilibrium, which is shifted towards fructose at higher temperatures. In this process, an additional expensive chromatographic step is required to obtain the desired 55% fructose syrup concentration. The fructose-glucose syrup-55 is a more desirable variety of fructose-glucose syrup for food and soft drink applications. In the process of producing the fructose-glucose syrup, the final conversion rate of D-glucose to D-fructose is highly dependent on the temperature, and the higher the temperature is, the more favorable the yield of D-fructose is. To avoid a complex concentration step downstream, higher temperatures are required to obtain fructose syrup-55 directly by promoting the equilibrium of the isomerization reaction toward D-fructose formation. Theoretically, due to the equilibrium of the isomerization reaction, isomerization at high temperatures (about 95 ℃) and lower pH (about 4.5-5.5) is required to achieve the desired fructose levels in the syrup. If this can be achieved, no additional concentration step is required.
Briefly, compared to the enzymes currently in use, industrially valuable glucose isomerase must have a lower pH, a higher temperature, and a higher pH for Ca 2+ Resistance to inhibition and higher affinity to glucose. Accordingly, there is currently a great deal of research effort directed to the identification of thermostable and acid-stable glucose isomerase that has a higher affinity for glucose. In addition, advances in recombinant DNA technology and protein engineering open up new possibilities for developing economically viable commercial processes. For this reason, researchers have attempted to isolate microorganisms capable of producing glucose isomerase with high activity and stability at high temperature.
U.S. patent WO2004/044129 recognizes that there is a need to discover glucose isomerase that retains a high level of activity at high temperatures and low pH. However, the peptides disclosed in this document have optimal activity only above pH 5 and are unstable above 90 ℃. More importantly, to maintain high levels of activity, these enzymes require cobalt (Co 2+ ) As cofactor. And Co 2+ And is not suitable for producing high fructose syrup for human consumption. It is not only associated with a number of possible health problems, but also the disposal of waste media may lead to environmental pollution. The most suitable ions are those commonly used in food-related applications including Mn 2+ And Mg (magnesium) 2+ Etc. However, these ions are generally only effective when combined with enzymes from non-thermophilic organisms or only microthermophilic organisms.
Thus, the optimal glucose isomerase for use in the food industry should meet the following conditions: it can still function efficiently under acidic conditions (i.e., a pH of about 6 or less) to avoid browning reactions; it can tolerate higher substrate and product inhibition (e.g., greater than 200g/L glucose) in order to increase production efficiency per unit volume; it should remain stable at high reaction temperatures (i.e., 80 ℃ or higher) to promote high fructose conversion; the catalyst has higher catalytic activity, can reach a reaction balance point in a shorter time, and reduces energy consumption and manpower consumption; it can be free of harmful metal ions, such as Co 2+ The method comprises the steps of carrying out a first treatment on the surface of the It can be used in common microorganisms such as large scaleRecombinant preparation is efficiently carried out in large quantities in enterobacteria or bacillus, so that the production is convenient and the cost is reduced.
Therefore, a new glucose isomerase or mutant is developed, the high temperature tolerance and the catalytic efficiency under the high temperature reaction condition are improved, and the novel glucose isomerase or mutant has great practical value.
Disclosure of Invention
The invention aims to provide glucose isomerase 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 glucose isomerase having an amino acid sequence as set forth in SEQ ID NO:1.
The glucose isomerase with enhanced heat resistance can be used for catalyzing D-glucose to generate D-fructose, and the using method comprises the following steps: adding PB and pure water into the D-glucose mother solution, adding a crude enzyme solution of glucose isomerase, and reacting at 80 ℃.
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 in the inoculation ratio of example, 100mL of the autoclaved medium B was inoculated, 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% of glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, and the pH value is 7.0.
Compared with the prior art, the invention has the beneficial effects that:
the glucose isomerase with enhanced heat resistance of the invention can catalyze D-glucose to generate D-fructose at high temperature. Compared with wild glucose isomerase, the method can be applied to high-temperature conversion of D-glucose to generate D-fructose, has stronger thermal stability, and can obtain better social benefit and economic value.
Drawings
FIG. 1 shows the results of high performance liquid chromatography of 20g/L fructose. 8.4 minutes was the product fructose peak.
FIG. 2 shows the results of high performance liquid chromatography of 20g/L glucose. The 10 minutes is the glucose peak.
FIG. 3 shows the results of the reaction of comparative example 1 for 20 hours.
FIG. 4 shows the results of the reaction of example 6 for 20 hours.
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: acetonitrile: water = 70:30
A detector: differential detector
Flow rate: 1mL/min
Column temperature: 40 DEG C
Differential detector cell temperature: 40 DEG C
An amino column 250mm x 4.6 μm was used.
Example 1 obtaining the wild-type glucose isomerase 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 CSGIwt, having the corresponding amino acid sequence of SEQ ID NO:3.
TABLE 2
EXAMPLE 2 acquisition of the Gene sequence of glucose isomerase mutant
The thermostable enhanced glucose isomerase mutant of the present invention, which is derived from the nucleotide sequence of SEQ ID NO:3, a wild-type glucose isomerase. Glucose isomerase 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
| 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:2, designated CSGI1, the corresponding amino acid sequence of which is SEQ ID NO:1. compared with the sequence of CSGIwt, there are three mutations A44S, K380R, K390R.
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 carried out in a computer-controlled bioreactor (Shanghai state of China) with a capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L dihydrogenphosphate 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 Heat treatment and residual enzyme Activity detection
Each enzyme was incubated at 85℃for 0, 0.5, 1, 2 hours in a 10ml tube (2 ml system added with 0.5ml crude enzyme solution). Then, 500. Mu.l of the heat-treated enzyme extract and 500. Mu.l of a substrate buffer (100 mM PB buffer pH6.8, 20mM MgSO 4 ，1mM MnCl 2 360g/L D-glucose, pH 6.8) was mixed and incubated at 55℃for 4 hours to evaluate residual enzyme activity. Finally, the substrate residue and the formation of the product were detected by using a high-performance liquid chromatography, and a 0-hour CSGIwt sample was used as a standard 100%, which had a fructose concentration of 97.51g/L, a glucose concentration of 91.13g/L and a conversion of 54.17% at 4 hours. The reaction results after the heat treatment for 2 hours and for 4 hours are shown below.
Therefore, the mutant can retain more enzyme activity after high-temperature treatment, and has larger promotion than wild type protein.
Comparative example 1 high temperature reaction control example
2ml of the system, 50mM PB buffer pH6.8, 10mM MgSO, was prepared in a 10ml reaction tube 4 ，0.5mM MnCl 2 400g/L D-glucose, 50. Mu.l/ml CSGIwt crude enzyme solution. The reaction was carried out in a water bath at 80℃for 4 hours and samples were taken overnight. As shown in FIG. 3, the results of the high performance liquid phase detection after spotting and diluting 5 times show that the fructose concentration is 100.56g/L in 20 hours, and the enzyme activity loss is large in the high temperature reaction.
Example 6 high temperature reaction example
2ml of the system, 50mM PB buffer pH6.8, 10mM MgSO, was prepared in a 10ml reaction tube 4 ，0.5mM MnCl 2 400g/L D-glucose, 50. Mu.l/ml CSGI1 crude enzyme solution. The reaction was carried out in a water bath at 80℃for 4 hours and samples were taken overnight. The plate was spotted and diluted 5-fold for high performance liquid detection, as shown in FIG. 4, and the result showed that the fructose concentration was 230.77g/L for 20 hours.
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 (7)
1. A glucose isomerase with enhanced thermostability, characterized in that: the amino acid sequence is shown in SEQ ID NO:1.
2. Use of the thermostable enhanced glucose isomerase according to claim 1 for catalyzing the production of D-fructose from D-glucose.
3. Use of a thermostable enhanced glucose isomerase according to claim 2 for catalyzing the production of D-fructose from D-glucose, characterized in that: adding PB and pure water into the D-glucose mother solution, adding the crude enzyme solution of the glucose isomerase in the claim 1, and reacting at 80 ℃.
4. Use of a thermostable enhanced glucose isomerase for catalyzing the production of D-fructose from D-glucose according to claim 3, characterized in that the preparation method of the crude enzyme solution 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;
taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1:100 in the inoculation ratio of example, 100mL of the autoclaved medium B was inoculated, 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 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. Use of a thermostable enhanced glucose isomerase according to claim 4 for catalyzing the production of D-fructose from D-glucose, characterized in that: in the preparation method of the crude enzyme liquid, 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 kanamycin is added to 50mg/L.
6. Use of a thermostable enhanced glucose isomerase according to claim 4 for catalyzing the production of D-fructose from D-glucose, characterized in that: in the preparation method of the crude enzyme liquid, 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 kanamycin is added to 50mg/L.
7. Use of a thermostable enhanced glucose isomerase according to claim 4 for catalyzing the production of D-fructose from D-glucose, characterized in that: 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% of glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, and the pH value is 7.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310795231.5A CN116590271A (en) | 2023-06-30 | 2023-06-30 | Glucose isomerase with enhanced heat resistance and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310795231.5A CN116590271A (en) | 2023-06-30 | 2023-06-30 | Glucose isomerase with enhanced heat resistance and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116590271A true CN116590271A (en) | 2023-08-15 |
Family
ID=87595897
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310795231.5A Pending CN116590271A (en) | 2023-06-30 | 2023-06-30 | Glucose isomerase with enhanced heat resistance and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116590271A (en) |
-
2023
- 2023-06-30 CN CN202310795231.5A patent/CN116590271A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1097632C (en) | A mutant E. coli strain with increased succinic acid production | |
| US11781118B2 (en) | Preparation of L-amino acid deaminase mutant and application thereof | |
| CN1247790C (en) | Method for producing ascorbic acid intermediates | |
| CN111518806B (en) | Acetobacter pasteurianus promoter and application thereof | |
| JP2022544085A (en) | Psicose 3-epimerase mutant, genetically engineered fungus for expressing same, immobilized enzyme and immobilization method thereof | |
| CN110396507B (en) | L-pantolactone dehydrogenase from Cneubacter physcomitrella | |
| CN111763662A (en) | Ketoreductase and application thereof in synthesis of ticagrelor intermediate | |
| CN117535256A (en) | Carbonyl reductase and application thereof in synthesis of vitronectin | |
| CN114350630B (en) | L-pantolactone dehydrogenase, mutant and application thereof | |
| CN107400667B (en) | Immobilization method of cell containing recombinant high-temperature-resistant glucose isomerase | |
| CN112921025B (en) | Mutant of epimerase, coding gene, amino acid sequence and application thereof | |
| CN116590271A (en) | Glucose isomerase with enhanced heat resistance and application thereof | |
| CN115747183A (en) | Ketoreductase mutant and application thereof | |
| CN116694610A (en) | Glucose isomerase mutant and application thereof | |
| CN116622691A (en) | Glucose isomerase suitable for low pH condition and application thereof | |
| CN110904088B (en) | High-temperature-resistant D-psicose3-epimerase, mutant and application thereof | |
| CN116606845A (en) | Glucose isomerase suitable for high-temperature reaction and application | |
| CN116891845A (en) | High-activity glucose isomerase and application thereof in catalyzing high-concentration glucose | |
| CN101892228B (en) | Engineering bacteria with high tolerance to acrylamide and acrylonitrile for producing nitrile hydratase and application thereof | |
| CN113957065B (en) | Sucrose isomerase with high conversion rate and application thereof | |
| CN114134136B (en) | Rapid preparation method of isomaltulose under high reaction concentration and sucrose isomerase thereof | |
| CN114058610B (en) | High-activity sucrose isomerase and application thereof | |
| CN117757764A (en) | Formate dehydrogenase mutant and application thereof in preparation of reduced nicotinamide adenine dinucleotide | |
| CN116904421A (en) | NADH pyrophosphatase mutant with enhanced heat resistance and application thereof | |
| CN117327671A (en) | Novel ketoreductase mutant and preparation method 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 |