WO2023004969A1 - 酯酶突变体及其应用 - Google Patents
酯酶突变体及其应用 Download PDFInfo
- Publication number
- WO2023004969A1 WO2023004969A1 PCT/CN2021/119423 CN2021119423W WO2023004969A1 WO 2023004969 A1 WO2023004969 A1 WO 2023004969A1 CN 2021119423 W CN2021119423 W CN 2021119423W WO 2023004969 A1 WO2023004969 A1 WO 2023004969A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- pet
- esterase
- mutant
- esterase mutant
- preparation
- Prior art date
Links
- 108090000371 Esterases Proteins 0.000 title claims abstract description 79
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 24
- 239000006184 cosolvent Substances 0.000 claims abstract description 16
- 102220122845 rs767335346 Human genes 0.000 claims description 208
- 102220515737 Uncharacterized protein IRF1-AS1_H86A_mutation Human genes 0.000 claims description 97
- 102220492673 Integrin alpha-3_H86S_mutation Human genes 0.000 claims description 33
- 210000004027 cell Anatomy 0.000 claims description 26
- 239000013612 plasmid Substances 0.000 claims description 24
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 21
- -1 ester compound Chemical class 0.000 claims description 20
- 230000000694 effects Effects 0.000 claims description 19
- 230000035772 mutation Effects 0.000 claims description 18
- 102220577246 Density-regulated protein_A86Y_mutation Human genes 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- 108020004414 DNA Proteins 0.000 claims description 10
- 238000006555 catalytic reaction Methods 0.000 claims description 9
- 102000053602 DNA Human genes 0.000 claims description 8
- 238000006460 hydrolysis reaction Methods 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 6
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 230000007062 hydrolysis Effects 0.000 claims description 5
- JWUJQDFVADABEY-UHFFFAOYSA-N 2-methyltetrahydrofuran Chemical compound CC1CCCO1 JWUJQDFVADABEY-UHFFFAOYSA-N 0.000 claims description 4
- 102220480224 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1_F88R_mutation Human genes 0.000 claims description 4
- 241000196324 Embryophyta Species 0.000 claims description 4
- 102220621769 Pentraxin-4_H86N_mutation Human genes 0.000 claims description 4
- 102220477300 Ribosome biogenesis protein BOP1_F88Y_mutation Human genes 0.000 claims description 4
- 241000672609 Escherichia coli BL21 Species 0.000 claims description 3
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 claims description 3
- 244000061121 Rauvolfia serpentina Species 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 3
- 210000005253 yeast cell Anatomy 0.000 claims description 3
- GVNVAWHJIKLAGL-UHFFFAOYSA-N 2-(cyclohexen-1-yl)cyclohexan-1-one Chemical compound O=C1CCCCC1C1=CCCCC1 GVNVAWHJIKLAGL-UHFFFAOYSA-N 0.000 claims description 2
- 101150065749 Churc1 gene Proteins 0.000 claims description 2
- 102100038239 Protein Churchill Human genes 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 229910052717 sulfur Inorganic materials 0.000 claims description 2
- 102220324707 rs375086772 Human genes 0.000 claims 2
- 235000001014 amino acid Nutrition 0.000 claims 1
- 230000003197 catalytic effect Effects 0.000 abstract description 26
- 102000004190 Enzymes Human genes 0.000 abstract description 19
- 108090000790 Enzymes Proteins 0.000 abstract description 19
- 108090000623 proteins and genes Proteins 0.000 abstract description 12
- 102000004169 proteins and genes Human genes 0.000 abstract description 9
- 238000009776 industrial production Methods 0.000 abstract description 6
- 238000012216 screening Methods 0.000 abstract description 6
- 238000009510 drug design Methods 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 description 62
- 239000000758 substrate Substances 0.000 description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 238000000034 method Methods 0.000 description 14
- 239000000047 product Substances 0.000 description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 7
- 239000007791 liquid phase Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000002741 site-directed mutagenesis Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 239000012044 organic layer Substances 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003672 processing method Methods 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- NYPJDWWKZLNGGM-UHFFFAOYSA-N fenvalerate Chemical compound C=1C=C(Cl)C=CC=1C(C(C)C)C(=O)OC(C#N)C(C=1)=CC=CC=1OC1=CC=CC=C1 NYPJDWWKZLNGGM-UHFFFAOYSA-N 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 238000013341 scale-up Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 102220604479 Adenosine deaminase_F61A_mutation Human genes 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000005946 Cypermethrin Substances 0.000 description 1
- 239000005892 Deltamethrin Substances 0.000 description 1
- 102220587930 Deoxyhypusine hydroxylase_M86A_mutation Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108091007476 Microbial Esterases Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 101000693619 Starmerella bombicola Lactone esterase Proteins 0.000 description 1
- 102220503482 Superoxide dismutase [Cu-Zn]_H81A_mutation Human genes 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- FPQFYIAXQDXNOR-QDKLYSGJSA-N alpha-Zearalenol Chemical compound O=C1O[C@@H](C)CCC[C@H](O)CCC\C=C\C2=CC(O)=CC(O)=C21 FPQFYIAXQDXNOR-QDKLYSGJSA-N 0.000 description 1
- KEOYKWIOAINZSQ-UHFFFAOYSA-N alpha-Zearalenol Natural products CC1CCCC(O)CCC=CCc2cc(O)cc(O)c2C(=O)O1 KEOYKWIOAINZSQ-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229960005424 cypermethrin Drugs 0.000 description 1
- KAATUXNTWXVJKI-UHFFFAOYSA-N cypermethrin Chemical compound CC1(C)C(C=C(Cl)Cl)C1C(=O)OC(C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 KAATUXNTWXVJKI-UHFFFAOYSA-N 0.000 description 1
- 229960002483 decamethrin Drugs 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- OWZREIFADZCYQD-NSHGMRRFSA-N deltamethrin Chemical compound CC1(C)[C@@H](C=C(Br)Br)[C@H]1C(=O)O[C@H](C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 OWZREIFADZCYQD-NSHGMRRFSA-N 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 239000002728 pyrethroid Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01017—Gluconolactonase (3.1.1.17)
-
- 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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
Definitions
- the invention relates to the field of industrial enzyme preparation, in particular to an esterase mutant and its application.
- Esterase is a kind of hydrolase, which is a general term for enzymes that catalyze the hydrolysis of esters and widely exists in plants, animals and microorganisms.
- Animal pancreas esterase and microbial esterase are the main sources, mainly fungi, followed by bacteria, and esterases from different sources have different catalytic characteristics and catalytic activities. Esterase has been commercialized and plays an important role in food, medicine, chemical industry and other fields.
- CN 107058362 A Disclosed in CN 107058362 A is the esterase gene est816 and its recombinant esterase, which has high-efficiency soluble expression in the expression system of E. , cypermethrin, fenvalerate and deltamethrin) have a strong degradation effect, and the degradation rate is as high as more than 90%. It has broad application prospects in pyrethroid pesticide residues.
- a kind of lactonase is disclosed in CN 106929493 A, and its amino acid sequence is that the 167th amino acid of the sequence numbered 5 has valine mutated into histidine, which improves the degradation efficiency of ⁇ -zearalenol .
- biocatalysts When biocatalysts are applied in organic synthesis, they are unnatural substrates in most cases, and for unnatural substrates, their reactivity, stability and selectivity are often not very good, so esterases that can be really widely used are not not much.
- the main purpose of the present invention is to provide an esterase mutant and its application to solve the problems of low esterase activity and/or low stereoselectivity in the prior art.
- an esterase mutant is provided, the amino acid sequence of the esterase mutant has the following amino acid mutations on the basis of SEQ ID NO: 1: G19S, G19S+H86S, G19S+ H86A, G19S+H86Q, G19S+H86M, G19S+H86T, G19S+H86C, G19S+H86N, G19S+F88N, G19S+F88R, G19S+F88Y, G19S+F88K, G19S+S111T, G19S+S111V, G19S, M113I G19S+M113L, G19S+M113V, G19S+F125Y, G19S+F125S, G19S+Y128F, G19S+L157V, G19S+M166L, G19S+L187V, G19S+L187I, G19S+S218Y, G19S+S218;
- amino acid sequence of the esterase mutant has more than 90%, preferably more than 95%, more preferably more than 99% homology with the mutated amino acid sequence.
- esterase mutant is derived from Rauvolfia serpentina.
- a DNA molecule encoding the above-mentioned esterase mutant is provided.
- the recombinant plasmid is selected from any one of the following: pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a (+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET -29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET- 40b(+),
- a non-plant host cell containing any one of the aforementioned recombinant plasmids.
- the host cell is a prokaryotic cell or a eukaryotic cell
- the eukaryotic cell is a yeast cell.
- the host cells are competent cells.
- the competent cells are Escherichia coli BL21 cells or Escherichia coli W3110 cells.
- a preparation method of a chiral compound comprising: using the aforementioned esterase mutant to catalyze the hydrolysis of the ester compound represented by formula I into the acid represented by formula II Alcohol compound shown in compound and formula III;
- n 1, 2, 3 or 4;
- X C, O or S
- R 1 CH 3 , CH 2 CH 3 , CH 2 -CH 2 CH 3 or CHCH 3 CH 3 ,
- R 2 H, F, Cl, Br, CH 3 or CH 2 CH 3 .
- ester compound is any one of the following:
- esterase mutant catalyzes the hydrolysis reaction of the ester compound represented by formula I at a temperature of 20°C to 40°C. Further, ester compounds and esterase are dissolved in potassium phosphate buffer to form a catalytic reaction system, wherein the potassium phosphate buffer
- the concentration is 0.1M ⁇ 1M, and the pH value is 6.0 ⁇ 7.5.
- the mass ratio of the esterase mutant to the ester compound is 0.2mg-2mg:20mg.
- the mass ratio of the esterase mutant to the ester compound is 0.1g-0.5g:10g.
- the catalytic reaction system also contains a co-solvent, and the co-solvent is selected from any one of the following: DMSO, DCM and 2-MeTHF.
- volume percentage of the co-solvent in the reaction system is ⁇ 20%.
- the esterase mutant obtained through rational design and several rounds of enzyme evolution screening on the basis of the amino acid sequence shown in SEQ ID NO: 1, compared with the wild-type esterase, the protein structure and function have changed.
- the catalytic activity and/or stereoselectivity of esterase mutants are greatly improved, and when the system contains some organic co-solvents, they still have relatively stable catalytic activity and/or stereoselectivity selective.
- the improved catalytic activity and/or stereoselectivity of the lipase mutant reduces the amount of enzyme to a certain extent, reduces the difficulty of post-treatment, and is suitable for industrial production.
- an esterase mutant is provided, the amino acid sequence of the esterase mutant is obtained by the following mutations in the amino acid sequence shown in SEQ ID NO: 1: G19S, G19S+H86S, G19S +H86A, G19S+H86Q, G19S+H86M, G19S+H86T, G19S+H86C, G19S+H86N, G19S+F88N, G19S+F88R, G19S+F88Y, G19S+F88K, G19S+S111T, G19S+S111V, G19S+S111V, G19S+M113 , G19S+M113L, G19S+M113V, G19S+F125Y, G19S+F125S, G19S+Y128F, G19S+L157V, G19S+M166L, G19S+L187V, G19S+L187I, G19S+S218Y, G19S+S+S218H,
- the amino acid sequence of the above-mentioned esterase mutant is obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, and the important sites involved in the mutation include but are not limited to one or more of the following sites: G19S, H86S ,H86A,H86Q,H86M,H86T,H86C,H86N,F88N,F88R,F88Y,F88K,S111T,S111V,M113A,M113G,M113I,M113L,M113V,F125Y,F125S,Y128F,M137F,M1377YM,M1371M,M1371 .
- the above-mentioned esterase mutants are obtained through rational design and several rounds of enzyme evolution screening on the basis of the amino acid sequence shown in SEQ ID NO: 1. Compared with the parent esterase, the protein structure and function have changed. In practical application, the catalytic activity and/or stereoselectivity of the esterase mutant has been greatly improved, and when the system contains some organic co-solvents, it still has relatively stable catalytic activity and/or stereoselectivity . In addition, the improved catalytic activity and/or stereoselectivity of the esterase mutant reduces the amount of enzyme used to a certain extent, reduces the difficulty of post-treatment, and is suitable for industrial production. In addition, the improved catalytic activity and/or stereoselectivity of the esterase mutant reduces the amount of enzyme used to a certain extent, reduces the difficulty of post-treatment, and is suitable for industrial production.
- amino acid sequence of SEQ ID NO: 1 (derived from Rauvolfia serpentina) is as follows:
- a mutation site is introduced into SEQ ID NO: 1 by whole plasmid PCR, the activity and selectivity of the mutants are detected, and the mutants with improved activity and selectivity are selected.
- sequence SEQ ID NO: 1 design primers for site-directed mutagenesis, and use pET-22b(+) as an expression vector to obtain a mutant plasmid with the target gene by means of site-directed mutagenesis.
- site-directed mutagenesis refers to the introduction of desired changes (usually changes that characterize a favorable direction) into the target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR), including bases Additions, deletions, point mutations, etc.
- PCR polymerase chain reaction
- Site-directed mutagenesis can quickly and efficiently improve the properties and characterization of the target protein expressed in DNA, and is a very useful method in gene research.
- the method of introducing site-directed mutagenesis by whole-plasmid PCR is simple and effective, and is currently used more frequently.
- the principle is that a pair of primers (forward and reverse) containing the mutation site are annealed to the template plasmid and then "circularly extended" with a polymerase.
- the so-called circular extension means that the polymerase extends the primer according to the template and returns to The 5' end of the primer is terminated, and then undergoes repeated heating and annealing extension cycles. This reaction is different from rolling circle amplification and will not form multiple tandem copies.
- the extension products of the forward and reverse primers are annealed and paired to form a nicked open-circle plasmid.
- Dpn I digestion extension product because the original template plasmid is derived from conventional Escherichia coli, it is modified by dam methylation, and it is sensitive to Dpn I and is cut up, while the plasmid with mutated sequence synthesized in vitro has no methylation Without being cut, it can be successfully transformed in the subsequent transformation, and the clone of the mutant plasmid can be obtained.
- beneficial amino acid positions can be combined to obtain mutants with better traits.
- Saturation mutation is a method to obtain mutants in which amino acids at target sites are replaced by other 19 amino acids in a short period of time by modifying the coding gene of the target protein. This method is not only a powerful tool for protein directional modification, but also an important means for the study of protein structure-function relationship. Saturation mutations can often obtain more ideal evolutionary bodies than single point mutations. For these problems that the site-directed mutation method cannot solve, it is precisely the unique feature that the saturation mutation method is good at.
- the aforementioned mutant plasmids were transformed into E. coli cells, and overexpressed in E. coli. Crude enzymes were then obtained by sonicating cells. Optimum conditions for esterase-induced expression: 25°C, 0.06mM IPTG for overnight induction.
- the mutants screened by the present application have undergone a large number of experimental verifications. Under the condition that the substrate is not over-converted in the form of a racemate, it is finally proved that the catalytic activity and/or stereoselectivity of the enzyme-catalyzed reaction are significantly improved (such as e.e. from the initial ⁇ 10% to at least 80% and above), largely meeting the needs of industrial production.
- a DNA molecule encoding any of the above-mentioned esterase mutants is also provided.
- the encoded esterase mutant has the advantages of high catalytic activity and/or high stereoselectivity.
- a recombinant plasmid is also provided, and the recombinant plasmid is connected with the above-mentioned DNA molecule.
- the DNA molecule can encode any of the above-mentioned esterase mutants with high catalytic activity and/or high stereoselectivity.
- any recombinant plasmid that can be used to express the DNA molecule of the above-mentioned esterase is suitable for the present invention.
- the recombinant plasmid is selected from one of the following: pET-22b(+), pET-21b(+), pET-3a(+), pET-3d(+), pET-11a(+ ), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET- 21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a (+), pET-29a
- a non-plant host cell containing any one of the above recombinant plasmids is also provided.
- Specific host cells may be prokaryotic cells or eukaryotic cells, preferably eukaryotic cells are yeast cells. More preferably, the above-mentioned host cells are competent cells, and it is further preferred that the competent cells are Escherichia coli BL21 cells or Escherichia coli W3110 cells.
- a preparation method of a chiral compound comprising: using any one of the aforementioned esterase mutants to catalyze the hydrolysis of the ester compound shown in formula I into formula Acid compounds shown in II and alcohol compounds shown in formula III;
- ester compound is any one of the following:
- the esterase mutant has higher catalytic activity or higher stereoselectivity at a temperature of 20°C to 40°C, and thus can catalyze the hydrolysis of the ester compound represented by formula I reaction.
- potassium phosphate buffer, ester compound and esterase form a catalytic reaction system, wherein the concentration of potassium phosphate buffer is preferably 0.1M-1M, and the pH value is preferably 6.0-7.5. Under this condition, they can catalyze different substrates, and have higher activity and/or higher stereoselectivity.
- the mass ratio of the esterase mutant to the ester compound is 0.2 mg to 2 mg: 20 mg, which is suitable for a small reaction volume (for example, 0.6 mL), and the corresponding substrate
- the mass range of the enzyme is 2mg-0.2mg.
- the mass ratio of the esterase mutant to the ester compound is 0.1g-0.5g:10g.
- This dosage ratio is especially suitable for carrying out in a large reaction system (for example, 100mL), and when the corresponding substrate dosage is 10g, the mass range of the enzyme is 0.1g-0.5g.
- the mutants obtained by the evolutionary screening of this application can not only catalyze the hydrolysis reaction of the substrate under suitable buffer conditions, but also have high stability, catalytic activity and/or stereogenicity in the presence of certain co-solvents. selective.
- the reaction system further contains a co-solvent, and the co-solvent is selected from any one of the following: DMSO, DCM and 2-MeTHF.
- the co-solvent is selected from any one of the following: DMSO, DCM and 2-MeTHF.
- the volume percentage of the co-solvent in the reaction system is ⁇ 20%.
- the mutant of the present application still has higher catalytic activity and/or Stereoselectivity.
- the activity represents the conversion rate
- the activity value 20 of the substrate 1 is used as an example for illustration here: in 0.3M potassium phosphate Buffer pH 7.0, 20°C, 2mg of wild-type enzyme, 3h, the conversion rate of substrate 1 was 20%.
- the activity is reduced and increased multiples, --- reduced by less than 5 times, -- reduced by 2-5 times, - reduced by 1-2 times, + increased by 1-2 times, ++ increased by 2-5 times, + ++ increased by 5-10 times, ++++ increased by more than 10 times.
- the ee value is less than 0% (refers to the ee value of the product, because the substrate is racemized, that is, both the S configuration and the R configuration, for example, each accounting for about 50%, the enzyme will have a choice during the catalytic process Catalytic S and R configurations, for example, the conversion of substrates that selectively catalyze the R configuration is more, and there are more corresponding R configurations in the product.
- the activity is reduced and increased multiples, --- reduced by less than 5 times, -- reduced by 2-5 times, - reduced by 1-2 times, + increased by 1-2 times, ++ increased by 2-5 times, + ++ increased by 5-10 times, ++++ increased by more than 10 times.
- ee value less than 0%# ee value 0-50%*, ee value 50-60%**, ee value 60-70%***, ee value 70%-80%* ***, ee value 80%-95%*****, ee value greater than 95%******.
- the activity is reduced and increased multiples, --- reduced by less than 5 times, -- reduced by 2-5 times, - reduced by 1-2 times, + increased by 1-2 times, ++ increased by 2-5 times, + ++ increased by 5-10 times, ++++ increased by more than 10 times.
- ee value less than 0%# ee value 0-50%*, ee value 50-60%**, ee value 60-70%***, ee value 70%-80%* ***, ee value 80%-95%*****, ee value greater than 95%******.
- the activity is reduced and increased multiples, --- reduced by less than 5 times, -- reduced by 2-5 times, - reduced by 1-2 times, + increased by 1-2 times, ++ increased by 2-5 times, + ++ increased by 5-10 times, ++++ increased by more than 10 times.
- ee value less than 0%# ee value 0-50%*, ee value 50-60%**, ee value 60-70%***, ee value 70%-80%* ***, ee value 80%-95%*****, ee value greater than 95%******.
- the activity is reduced and increased multiples, --- reduced by less than 5 times, -- reduced by 2-5 times, - reduced by 1-2 times, + increased by 1-2 times, ++ increased by 2-5 times, + ++ increased by 5-10 times, ++++ increased by more than 10 times.
- ee value less than 0%# ee value 0-50%*, ee value 50-60%**, ee value 60-70%***, ee value 70%-80%* ***, ee value 80%-95%*****, ee value greater than 95%******.
- the activity is reduced and increased multiples, --- reduced by less than 5 times, -- reduced by 2-5 times, - reduced by 1-2 times, + increased by 1-2 times, ++ increased by 2-5 times, + ++ increased by 5-10 times, ++++ increased by more than 10 times.
- ee value less than 0%# ee value 0-50%*, ee value 50-60%**, ee value 60-70%***, ee value 70%-80%* ***, ee value 80%-95%*****, ee value greater than 95%******.
- reaction conditions in Table 7 are: containing different co-solvents, the buffer is 0.3M potassium phosphate buffer pH 6.5, and the reaction temperature is 20°C.
- reaction conditions in Table 8 are: no co-solvent, and the reaction temperature is 20°C.
- reaction conditions in Table 9 are: no co-solvent, the buffer is 0.3M potassium phosphate buffer, and the reaction temperature is 20°C.
- reaction conditions in Table 10 are: no co-solvent, the buffer is 0.3M potassium phosphate buffer, pH 6.5.
- the activity is reduced and increased multiples, --- reduced by less than 5 times, -- reduced by 2-5 times, - reduced by 1-2 times, + increased by 1-2 times, ++ increased by 2-5 times, + ++ increased by 5-10 times, ++++ increased by more than 10 times.
- ee value less than 0%# ee value 0-50%*, ee value 50-60%**, ee value 60-70%***, ee value 70%-80%* ***, ee value 80%-95%*****, ee value greater than 95%******.
- Substrate 1 was added to 10g, 100mL reaction system, esterase (G19S+H86A+M113I+S218F+F219Y+M137L) 100mg, 0.3M potassium phosphate buffer pH 6.5. React at 20°C, track the reaction time and take samples for detection, and adjust the pH at about 6.4. When reacting for 3 hours, the conversion rate is 49%, and the e.e. value is 99%.
- the reaction sample was processed, 100mL of dichloromethane was added for extraction, and after sufficient shaking, the organic layer was separated, and an appropriate amount of anhydrous sodium sulfate was added, further filtered, and the organic layer was subjected to rotary evaporation, and finally obtained
- the 4.8g sample had a purity of 98% and an e.e. value of 98%. Further nuclear magnetic detection showed a yield of 45%.
- the amplified reaction was carried out.
- Substrate 4 was added to 10g, 100mL reaction system, esterase (G19S+H86A+M113I+S218F+F219Y+M137L) 100mg, 0.3M potassium phosphate buffer pH 6.5. React at 20°C, track the reaction time and take samples for detection, and adjust the pH at about 6.4. When reacting for 6 hours, the conversion rate is 49%, and the e.e. value is 99%.
- the reaction sample was processed, 100mL of dichloromethane was added for extraction, and after sufficient shaking, the organic layer was separated, and an appropriate amount of anhydrous sodium sulfate was added, further filtered, and the organic layer was subjected to rotary evaporation, and finally obtained
- the 4.6g sample had a purity of 98% and an e.e. value of 98%. Further nuclear magnetic detection showed a yield of 44%.
- the above-mentioned embodiments of the present invention have achieved the following technical effects: the esterase obtained through rational design and several rounds of enzyme evolution screening on the basis of the amino acid sequence shown in SEQ ID NO: 1 Mutants, compared with the parent esterase, have undergone changes in protein structure and function.
- the catalytic activity and/or stereoselectivity of the esterase mutants have been greatly improved, and when the system contains certain When using an organic co-solvent, it still has relatively stable catalytic activity and/or stereoselectivity.
- the improved catalytic activity and/or stereoselectivity of the lipase mutant reduces the amount of enzyme to a certain extent, reduces the difficulty of post-treatment, and is suitable for industrial production.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
Claims (18)
- 一种酯酶突变体,其特征在于,(1)所述酯酶突变体的氨基酸序列在SEQ ID NO:1基础上发生如下氨基酸突变:G19S、G19S+H86S、G19S+H86A、G19S+H86Q、G19S+H86M、G19S+H86T、G19S+H86C、G19S+H86N、G19S+F88N、G19S+F88R、G19S+F88Y、G19S+F88K、G19S+S111T、G19S+S111V、G19S+M113I、G19S+M113L、G19S+M113V、G19S+F125Y、G19S+F125S、G19S+Y128F、G19S+L157V、G19S+M166L、G19S+L187V、G19S+L187I、G19S+S218Y、G19S+S218H、G19S+S218F、G19S+S218N、G19S+H86S+S111T、G19S+H86S+S111V、G19S+H86S+M113A、G19S+H86S+M113G、G19S+H86S+M113I、G19S+H86S+M113L、G19S+H86S+M113V、G19S+H86S+M113I+L157A、G19S+H86S+M113I+L157G、G19S+H86S+M113I+L157V、G19S+H86A+S111T、G19S+H86A+S111V、G19S+H86A+M113A、G19S+H86A+M113G、G19S+H86A+M113I、G19S+H86A+M113L、G19S+H86A+M113V、G19S+H86A+M113I+S218E、G19S+H86A+M113I+S218F、G19S+H86A+M113I+S218I、G19S+H86A+M113I+S218L、G19S+H86A+M113I+S218M、G19S+H86A+M113I+S218T、G19S+H86A+M113I+S218V、G19S+H86A+M113I+S218Y、G19S+H86A+M113I+S218F+A86C、G19S+H86A+M113I+S218F+A86M、G19S+H86A+M113I+S218F+A86N、G19S+H86A+M113I+S218F+A86Q、G19S+H86A+M113I+S218F+A86S、G19S+H86A+M113I+S218F+M137F、G19S+H86A+M113I+S218F+M137L、G19S+H86A+M113I+S218F+M137Y、G19S+H86A+M113I+S218F+M137E、G19S+H86A+M113I+S218F+M137W、G19S+H86A+M113I+S218F+F219Y、G19S+H86A+M113I+S218F+F219L、G19S+H86A+M113I+S218F+F219T、G19S+H86A+M113I+S218F+F219Q、G19S+H86A+M113I+S218F+F219Y+M137F、G19S+H86A+M113I+S218F+F219Y+M137L、G19S+H86A+M113I+S218F+F219Y+M137W、G19S+H86A+M113I+S218F+F219Y+M137Y或G19S+H86A+M113I+S218F+F219Y+M137S;或者所述酯酶突变体的氨基酸序列与发生突变的氨基酸序列具有80%以上同源性且具有酯酶活性。
- 根据权利要求1所述的酯酶突变体,其特征在于,所述酯酶突变体的氨基酸序列与发生突变的氨基酸序列具有90%以上,优选为95%以上,更优选为99%以上同源性且具有酯酶活性。
- 根据权利要求1或2所述的酯酶突变体,其特征在于,所述酯酶突变体来源于Rauvolfia serpentina。
- 一种DNA分子,其特征在于,编码权利要求1至3中任一项所述的酯酶突变体。
- 一种重组质粒,其特征在于,所述重组质粒连接有权利要求4所述的DNA分子。
- 根据权利要求5所述的重组质粒,其特征在于,所述重组质粒选自如下任意一种:pET-21b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-8、pUC-18以及pUC-19。
- 一种非植物的宿主细胞,其特征在于,所述宿主细胞含有权利要求5或6所述的重组质粒。
- 根据权利要求7所述的宿主细胞,其特征在于,所述宿主细胞为原核细胞或真核细胞,所述真核细胞为酵母细胞。
- 根据权利要求8所述的宿主细胞,其特征在于,所述宿主细胞为感受态细胞。
- 根据权利要求9所述的宿主细胞,其特征在于,所述感受态细胞为大肠杆菌BL21细胞或大肠杆菌W3110。
- 根据权利要求11所述的制备方法,其特征在于,所述酯酶突变体在20℃~40℃的温度下催化式I所示的酯类化合物发生水解反应。
- 根据权利要求11所述的制备方法,其特征在于,所述酯类化合物及所述酯酶溶于磷酸钾缓冲液中形成催化反应体系,其中磷酸钾缓冲液的浓度为0.1M~1M,pH值为6.0~7.5。
- 根据权利要求11所述的制备方法,其特征在于,所述酯酶突变体与所述酯类化合物的质量比为0.2mg~2mg:20mg。
- 根据权利要求11所述的制备方法,其特征在于,所述酯酶突变体与所述酯类化合物的质量比为0.1g~0.5g:10g。
- 根据权利要求14所述的制备方法,其特征在于,所述催化反应体系中还含有助溶剂,所述助溶剂选自如下任意一种:DMSO、DCM及2-MeTHF。
- 根据权利要求17所述的制备方法,其特征在于,所述助溶剂在所述催化反应体系中的体积百分含量≤20%。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020247006955A KR20240039032A (ko) | 2021-07-28 | 2021-09-18 | 에스테라아제 돌연변이체 및 이의 응용 |
EP21951543.4A EP4365286A1 (en) | 2021-07-28 | 2021-09-18 | Esterase mutant and use thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110853890.0A CN113308450B (zh) | 2021-07-28 | 2021-07-28 | 酯酶突变体及其应用 |
CN202110853890.0 | 2021-07-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023004969A1 true WO2023004969A1 (zh) | 2023-02-02 |
Family
ID=77381572
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2021/119423 WO2023004969A1 (zh) | 2021-07-28 | 2021-09-18 | 酯酶突变体及其应用 |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP4365286A1 (zh) |
KR (1) | KR20240039032A (zh) |
CN (1) | CN113308450B (zh) |
WO (1) | WO2023004969A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117070494A (zh) * | 2023-10-13 | 2023-11-17 | 天津凯莱英生物科技有限公司 | 酯酶突变体及其应用 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113308450B (zh) * | 2021-07-28 | 2021-11-12 | 凯莱英医药集团(天津)股份有限公司 | 酯酶突变体及其应用 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101001954A (zh) * | 2004-05-27 | 2007-07-18 | 西尔斯公司 | 用于改变植物特征的核苷酸序列及其编码的多肽 |
CN102573451A (zh) * | 2009-07-20 | 2012-07-11 | 希尔雷斯股份有限公司 | 具有增加的生物质的转基因植物 |
CN106929493A (zh) | 2015-12-30 | 2017-07-07 | 中国科学院天津工业生物技术研究所 | 内酯酶及利用内酯酶降解α-玉米赤霉烯醇的方法 |
CN107058362A (zh) | 2017-01-11 | 2017-08-18 | 中山大学 | 酯酶基因est816及其重组酯酶在降解拟除虫菊酯类农药方面的应用 |
CN112375751A (zh) * | 2021-01-18 | 2021-02-19 | 凯莱英生命科学技术(天津)有限公司 | 脂肪酶突变体及其应用 |
CN112430585A (zh) * | 2021-01-27 | 2021-03-02 | 凯莱英生命科学技术(天津)有限公司 | 酯酶突变体及其应用 |
CN113308450A (zh) * | 2021-07-28 | 2021-08-27 | 凯莱英医药集团(天津)股份有限公司 | 酯酶突变体及其应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE202019102623U1 (de) * | 2019-05-10 | 2019-05-27 | Ursula Pirzer | Wasserlösliches Getränkepulver |
CN111068013A (zh) * | 2020-01-07 | 2020-04-28 | 深圳简体美健康信息咨询有限公司 | 一种治疗痤疮的中药面膜及其制备方法 |
-
2021
- 2021-07-28 CN CN202110853890.0A patent/CN113308450B/zh active Active
- 2021-09-18 WO PCT/CN2021/119423 patent/WO2023004969A1/zh active Application Filing
- 2021-09-18 EP EP21951543.4A patent/EP4365286A1/en active Pending
- 2021-09-18 KR KR1020247006955A patent/KR20240039032A/ko unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101001954A (zh) * | 2004-05-27 | 2007-07-18 | 西尔斯公司 | 用于改变植物特征的核苷酸序列及其编码的多肽 |
CN102573451A (zh) * | 2009-07-20 | 2012-07-11 | 希尔雷斯股份有限公司 | 具有增加的生物质的转基因植物 |
CN106929493A (zh) | 2015-12-30 | 2017-07-07 | 中国科学院天津工业生物技术研究所 | 内酯酶及利用内酯酶降解α-玉米赤霉烯醇的方法 |
CN107058362A (zh) | 2017-01-11 | 2017-08-18 | 中山大学 | 酯酶基因est816及其重组酯酶在降解拟除虫菊酯类农药方面的应用 |
CN112375751A (zh) * | 2021-01-18 | 2021-02-19 | 凯莱英生命科学技术(天津)有限公司 | 脂肪酶突变体及其应用 |
CN112430585A (zh) * | 2021-01-27 | 2021-03-02 | 凯莱英生命科学技术(天津)有限公司 | 酯酶突变体及其应用 |
CN113308450A (zh) * | 2021-07-28 | 2021-08-27 | 凯莱英医药集团(天津)股份有限公司 | 酯酶突变体及其应用 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117070494A (zh) * | 2023-10-13 | 2023-11-17 | 天津凯莱英生物科技有限公司 | 酯酶突变体及其应用 |
CN117070494B (zh) * | 2023-10-13 | 2024-01-19 | 天津凯莱英生物科技有限公司 | 酯酶突变体及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN113308450A (zh) | 2021-08-27 |
EP4365286A1 (en) | 2024-05-08 |
KR20240039032A (ko) | 2024-03-26 |
CN113308450B (zh) | 2021-11-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11702640B2 (en) | Monooxygenase mutant, preparation method and application thereof | |
WO2023004969A1 (zh) | 酯酶突变体及其应用 | |
CN111187759A (zh) | 用于制备烟酰胺单核苷酸的酶组合物及酶法制备烟酰胺单核苷酸的方法 | |
CN111094557A (zh) | 醇脱氢酶突变体及其在双芳基手性醇合成中的应用 | |
CN109402074B (zh) | 单加氧酶突变体及其应用 | |
CN113774036B (zh) | 一种亚胺还原酶突变体及其应用 | |
WO2022160408A1 (zh) | 酯酶突变体及其应用 | |
WO2022151568A1 (zh) | 脂肪酶突变体及其应用 | |
CN110628742A (zh) | 转氨酶突变体及其应用 | |
US11162081B2 (en) | Ketoreductase mutant and application thereof | |
Hülsmeyer et al. | Crystal structure of cis‐biphenyl‐2, 3‐dihydrodiol‐2, 3‐dehydrogenase from a PCB degrader at 2.0 Å resolution | |
US11603521B2 (en) | Amino acid dehydrogenase mutant and use thereof | |
EP3974523A1 (en) | Transaminase mutant and use thereof | |
CN113502281B (zh) | 脂肪酶突变体及其应用 | |
JP2023012497A (ja) | モノオキシゲナーゼ突然変異体及びその使用 | |
WO2020093191A1 (zh) | 单加氧酶突变体及其应用 | |
CN117070494B (zh) | 酯酶突变体及其应用 | |
WO2023123589A1 (zh) | 转氨酶突变体及其应用 | |
CN114525265B (zh) | 转氨酶突变体及其应用 | |
JP4513967B2 (ja) | D−アミノアシラーゼの活性向上方法 | |
CN117230034A (zh) | 一种高稳定性哺乳动物尿酸氧化酶突变体 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21951543 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2024505054 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2021951543 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2021951543 Country of ref document: EP Effective date: 20240131 |
|
ENP | Entry into the national phase |
Ref document number: 20247006955 Country of ref document: KR Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |