CN117535256A - Carbonyl reductase and application thereof in synthesis of vitronectin - Google Patents
Carbonyl reductase and application thereof in synthesis of vitronectin Download PDFInfo
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- CN117535256A CN117535256A CN202311529061.2A CN202311529061A CN117535256A CN 117535256 A CN117535256 A CN 117535256A CN 202311529061 A CN202311529061 A CN 202311529061A CN 117535256 A CN117535256 A CN 117535256A
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- carbonyl reductase
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- 108010031132 Alcohol Oxidoreductases Proteins 0.000 title claims abstract description 128
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- 230000015572 biosynthetic process Effects 0.000 title abstract description 11
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- 108010031318 Vitronectin Proteins 0.000 title abstract description 6
- 102100035140 Vitronectin Human genes 0.000 title abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims abstract description 60
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 5
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 3
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- 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 2
- 108020004705 Codon Proteins 0.000 description 2
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- 101001110310 Lentilactobacillus kefiri NADP-dependent (R)-specific alcohol dehydrogenase Proteins 0.000 description 2
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
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- 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 2
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- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 1
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- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 238000010369 molecular cloning Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- -1 sodium triacetylborohydride Chemical compound 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000000707 stereoselective effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 150000003741 xylose derivatives Chemical class 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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
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- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
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- 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
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- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
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- Physics & Mathematics (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention discloses carbonyl reductase and application thereof in vitronectin synthesis, which takes carbonyl reductase NgADH which is derived from Nakaseomyces glabratus and has an amino acid sequence shown as SEQ ID NO. 2 as a starting sequence and has corresponding mutation sites (such as T221K and the like). The invention also discloses a coding gene of the carbonyl reductase, a vector, a bioengineering bacterium and a method for synthesizing the vitronectin. The invention has the advantages of good stereoselectivity, high optical purity of the product and improved reaction yield.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to carbonyl reductase and application thereof in vitronectin synthesis reaction.
Background
The vitriol (Pro-Xylane), which is a chemical name of hydroxypropyl tetrahydropyran triol, is a xylose derivative with anti-aging active substances, and has wide application in cosmetics. The vitreous color causes the skin to be stronger and more elastic, improves the neck fine lines and prevents aging. The action mechanism is as follows: the vitriol can promote synthesis of glycosaminoglycan and synthesis of collagen type I, type lV and type Vll (Eur J Dermatol.20088 May-Jun;18 (3): 297-302). Studies have shown that S-vitronectin (Ia) is more active than R-vitronectin (Ib) (Bioorg Med Chem Lett,2009,19 (3): 845-849; CN100441588).
The currently marketed products are mainly mixtures in which the S/R isomer ratio is between 5:5 and 7:3. The main reason is that sodium borohydride or sodium triacetylborohydride is adopted for reduction of carbonyl in the prior art, and the reaction stereoselectivity is not ideal. In 2002, the European Leya company reports a method for preparing vitrein by original research chemistry (WO 02/051828A 2), but the method adopts sodium borohydride to reduce carbonyl of beta-acetonylxyloside (II), and has the defects of low reaction stereoselectivity, difficult product separation, environmental pollution and the like.
The prior art also discloses methods employing biocatalytic synthesis. For example, CN111876452a discloses a method for preparing vitronectin by a one-pot method using biological enzymes, but carbonyl reductase information used in the method is not detailed and is difficult to reproduce. For example, CN113416756a discloses a biocatalytic method using Aldehyde Ketone Reductase (AKR) and alcohol dehydrogenase, but the S-configuration diastereomer content prepared is only 94.6%, which is still to be improved. For example CN114507681a discloses a biocatalytic synthesis method using the sorbose reductase OpCR derived from the wild-type structural gene of Ogataea parapolymorpha and mutants thereof, but does not disclose the stereochemistry and chiral purity of the produced product vitrein. Also, for example, CN113717997a discloses a biocatalytic synthesis method using wild-type dehydrogenase RDH derived from soybean rhizobium Bradyrhizobium japonicum USDA and mutants thereof. In general, these carbonyl reductases of natural origin have the general problems of poor adaptability to industrial production conditions, insufficient activity, insufficient catalytic ability on unnatural substrates, and the like in industrial applications.
Thus, there is a need in the art to develop ketoreductase enzymes and/or mutants thereof that are more active, providing more options for the biocatalytic synthesis of S-vitrein.
Disclosure of Invention
Aiming at the problems of low chiral purity, low enzyme catalytic capability, few types of selectable ketoreductase and the like of products in the synthesis of S-vitriol in the prior art, the invention provides carbonyl reductase with high catalytic activity, strong stereoselectivity and high yield from other sources, and also provides a nucleic acid sequence, a recombinant expression plasmid, a genetic engineering strain and a preparation method thereof corresponding to the carbonyl reductase, and application and a reaction system of the carbonyl reductase in the stereospecific synthesis of S-vitriol.
In a first aspect, the present invention provides a carbonyl reductase, which takes carbonyl reductase NgADH derived from Nakaseomyces glabratus and having an amino acid sequence shown in SEQ ID NO. 2 as a starting sequence, and has the following mutation sites:
mutant 7: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site T221K;
mutant 13: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site T41A/C139T/T221K;
mutant 15: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/F92T/I99Y/C139T/T221K;
mutant 16: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/F92C/F94W/C139T/T221K;
mutant 17: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site N49R/H78Y/I99Y/C139T/T221K;
mutant 18: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/F92V/V108L/C139T/Y208I/T221K;
mutant 19: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of D29E/H73E/F94V/C139T/Y208I/T221K/K279N;
mutant 20: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/F92V/C139T/Y208I/T221K/A272V/I302V;
mutant 21: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T22I/D29E/H78Y/C139T/T221K/A272V/N309D;
mutant 22: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/H78Y/F92L/F94V/C139T/Y208I/T221K/A272V;
mutant 23: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T22I/D29E/H78Y/C139T/T221K/A272V/K279N/N309D;
mutant 24: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T22I/D29E/H78Y/V108I/T221K/A272V/K279N/L296I; or (b)
Mutant 25: the amino acid sequence corresponding to SEQ ID NO. 2 and having mutation sites N49R/F94L/C139T/Y208T/T221K/D240T/A272V/I302V.
In some embodiments, the carbonyl reductase is mutant 13.
In some embodiments, the carbonyl reductase is mutant 15.
In some embodiments, the carbonyl reductase is mutant 16.
In some embodiments, the carbonyl reductase is mutant 17.
In some embodiments, the carbonyl reductase is mutant 18.
In some embodiments, the carbonyl reductase is mutant 19.
In some embodiments, the carbonyl reductase is mutant 20.
In some embodiments, the carbonyl reductase is mutant 21.
In some embodiments, the carbonyl reductase is mutant 22.
In some embodiments, the carbonyl reductase is mutant 23.
In some embodiments, the carbonyl reductase is mutant 24.
In some embodiments, the carbonyl reductase is mutant 25.
The carbonyl reductase provided by the invention is used for carrying out molecular modification on carbonyl reductase NgADH (the amino acid sequence of which is shown as SEQ ID NO: 2) from Nakaseomyces glabratus, so as to obtain a carbonyl reductase mutant with improved enzyme activity. The invention takes wild carbonyl reductase NgADH as a template and utilizes a random point mutation kitII Site-Directed Mutagenesis Kit) or mutation of the wild-type carbonyl reductase gene NgADH by semi-rational design to obtain a plasmid library containing the evolved carbonyl reductase gene. Mutant sites with improved enzymatic activity are obtained by high-throughput screening, and mutant libraries are built for the mutant sites, and mutants with improved activity and preferred mutant sites are screened.
Compared with wild carbonyl reductase NgADH, the carbonyl reductase provided by the invention has higher enzyme activity and better product stereoselectivity.
In a second aspect, the present invention also provides a gene encoding the carbonyl reductase according to the first aspect. The nucleotide sequence of the coding gene takes the sequence shown in SEQ ID NO. 1 as a starting sequence, and the corresponding mutation (such as base substitution) is carried out on codons according to the difference between the amino acid sequence of the carbonyl reductase and SEQ ID NO. 2.
The present invention also provides an isolated nucleic acid encoding the carbonyl reductase of the first aspect.
Wherein the preparation method of the nucleic acid can be a preparation method conventional in the art, and preferably comprises: the nucleic acid molecule encoding the carbonyl reductase is obtained by a gene cloning technique or by an artificial full sequence synthesis method.
In a third aspect, the present invention also provides an expression vector carrying the carbonyl-reduced coding gene according to the second aspect.
In a preferred embodiment, the expression vector is a recombinant plasmid.
In a preferred embodiment, the expression vector is a pET22b (+) vector.
Wherein the recombinant expression vector can be obtained by a method conventional in the art, and is generally constructed by ligating the nucleic acid to various expression vectors.
The invention also provides a recombinant expression transformant containing the expression vector. The recombinant expression transformant comprises a host cell and a target gene transferred into the host cell, wherein the target gene comprises the gene encoding the carbonyl reductase in the second aspect.
Wherein, the recombinant expression transformant is generally prepared by transforming the expression vector into a host microorganism. The host microorganism is preferably E.coli (E.coli), more preferably E.coli DH 5. Alpha. The recombinant expression vector (such as plasmid) is transformed into escherichia coli DH5 alpha, and the optimized genetic engineering strain is obtained. The transformation method may be selected from those conventional in the art, such as an electrotransformation method, a heat shock method, etc.
In a fourth aspect, the present invention also provides the use of a carbonyl reductase as described in the first aspect above for catalyzing carbonyl compounds.
Specifically, the invention provides an application of the carbonyl reductase as a catalyst in preparing S-vitronectin by biocatalysis and reduction of carbonyl by using beta-acetonylxyloside as a substrate.
The specific method is that the beta-acetonylxyloside is taken as a substrate to prepare the S-vitronectin through biocatalysis reduction of carbonyl, and the method comprises the following steps:
(a) In a liquid reaction system, using a compound beta-acetonylxyloside shown in a formula II as a substrate, and carrying out asymmetric reduction reaction under the catalysis of carbonyl reductase to obtain a compound S-vitronectin shown in a formula Ia;
(b) Optionally separating the compound S-vitronectin of formula Ia from the reaction system after the reaction of step (a);
wherein the carbonyl reductase is the carbonyl reductase of the first aspect.
In some embodiments, the concentration of the compound of formula II in the reaction system is 1-1000g/L.
In some specific embodiments, the concentration of the compound of formula II in the reaction system is 10g/L or more, 30g/L or more, 50g/L or more, 70g/L or more, 90g/L or more, 100g/L or more, 120g/L or more, 140g/L or more, 160g/L or more, 180g/L or more, 200g/L or more, 220g/L or more, 240g/L or more, 260g/L or more, 280g/L or more, 300g/L or more, 320g/L or more, 350g/L or more, 400g/L or more, or 450g/L or more.
In some embodiments, the concentration of the compound of formula II in the reaction system is 50-500g/L; preferably, the concentration of the compound of formula II is 100-400g/L; further preferably 150-300g/L.
In some embodiments, a co-substrate is also present in the reaction system.
In some specific embodiments, the co-substrate is selected from the group consisting of: isopropanol, glucose, ammonium formate, sodium formate, or a combination thereof.
In some specific embodiments, the co-substrate is isopropanol.
In some embodiments, the concentration of the co-substrate mass volume in the reaction system is from 5% to 50% (w/v).
In some specific embodiments, the coenzyme is selected from the group consisting of: reduced coenzyme, oxidized coenzyme, or a combination thereof.
In some specific embodiments, the reduced coenzyme is selected from the group consisting of: NADH, NADPH, or combinations thereof.
In some specific embodiments, the oxidative coenzyme is selected from the group consisting of: nad+, nadp+, or a combination thereof.
In some embodiments, the nad+ to substrate dosage ratio is 0.01% to 2.0% (w/w); preferably, the ratio is 0.1% to 1.0% (w/w).
In some specific embodiments, the ratio of the amount of nadp+ to the amount of substrate is 0.01% to 2.0% (w/w); preferably, the ratio is 0.1% to 1.0% (w/w).
In some embodiments, enzymes for coenzyme regeneration are also present in the reaction system. Wherein the enzyme for coenzyme regeneration is selected from the group consisting of: alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, or a combination thereof.
In some specific embodiments, the enzyme for coenzyme regeneration is glucose dehydrogenase.
In some specific embodiments, in step (a), the reaction temperature is from 10 ℃ to 45 ℃. The reaction temperature may also be selected from 20℃to 40℃or from 25℃to 35 ℃.
In some embodiments, in step (a), the reaction time is from 0.1 to 240 hours. The appropriate reaction endpoint is determined by monitoring the conversion of the compound of formula I.
In some embodiments, in step (a), the pH of the reaction system is from 6.0 to 9.0; preferably, the pH is 6.0 to 8.0; further preferably, the pH is 6.5 to 7.5.
In some embodiments, the carbonyl reductase is present in the reaction system in the form of: an enzyme in free form, an immobilized enzyme, or an enzyme in bacterial form.
In some specific embodiments, the reaction system is an aqueous solvent system.
In some specific embodiments, the reaction system is a phosphate buffer salt system.
In some embodiments, the reaction system contains a solvent selected from the group consisting of: water, alcohol, or a combination thereof.
In some embodiments, the co-solvent is present in a volume percent concentration of 5-50% (v/v); preferably, the co-solvent is present in a volume percentage concentration of 5-30% (v/v).
In some specific embodiments, in step (b), the ee value of the compound of formula II in the reaction system after the reaction is greater than or equal to 99%; preferably, the ee value of the compounds of the formula II is greater than or equal to 99.9%.
In some specific embodiments, in step (b), greater than or equal to 95% of the compound of formula I is converted to the compound of formula II in the reacted reaction system; preferably, > 98% of the compound of formula I is converted into the compound of formula II; more preferably, 99% or more of the compound of formula I is converted to the compound of formula II.
In a fifth aspect, the present invention provides a reaction system comprising:
(1) An aqueous solvent;
(2) A substrate, wherein the substrate is beta-acetonylxyloside;
(3) A coenzyme;
(4) Carbonyl reductase; the carbonyl reductase is the carbonyl reductase of the first aspect;
(5) A co-substrate.
In some specific embodiments, the coenzyme is selected from the group consisting of: reduced coenzyme, oxidized coenzyme, or a combination thereof.
In some specific embodiments, the reduced coenzyme is selected from the group consisting of: NADH, NADPH, or combinations thereof.
In some specific embodiments, the oxidative coenzyme is selected from the group consisting of: nad+, nadp+, or a combination thereof.
In some embodiments, the nad+ to substrate dosage ratio is 0.01% to 2.0% (w/w); preferably, the ratio is 0.1% to 1.0% (w/w).
In some specific embodiments, the ratio of the amount of nadp+ to the amount of substrate is 0.01% to 2.0% (w/w); preferably, the ratio is 0.1% to 1.0% (w/w).
In some specific embodiments, the co-substrate is selected from the group consisting of: isopropanol, glucose, ammonium formate, or a combination thereof.
In some specific embodiments, the co-substrate is isopropanol.
In some embodiments, the concentration of the co-substrate mass volume in the reaction system is from 5% to 50% (w/v).
In some embodiments, the substrate concentration in the reaction system is in the range of 1 to 1000g/L.
The reaction system provided by the invention can perform enzymatic reaction, and can prepare the S-vitriol with high optical purity at high conversion rate. Preferably, the optical purity is not less than 98% of ee value; more preferably, the optical purity is not less than 99% of ee value.
Technical terminology
Carbonyl reductase
In the present invention, a "carbonyl reductase" is an enzyme capable of stereoselectively catalyzing the asymmetric reduction of a potentially chiral ketone to a chiral alcohol.
In the present invention, the carbonyl reductase includes wild-type and mutant types. Furthermore, it may be isolated or recombinant. The wild-type carbonyl reductase mentioned in the present invention is carbonyl reductase NgADH gene (accession number KAI 8399263) from Nakaseomyces glabratus, its amino acid sequence is shown in SEQ ID No. 2, and its coding gene is shown in SEQ ID No. 1.
The carbonyl reductase provided by the invention is a series of mutants obtained by screening on the basis of wild carbonyl reductase, has higher activity than the wild carbonyl reductase, and has 100% stereoselectivity to S-vitrein.
As the reaction system, wet cells, crude enzyme solution, crude enzyme powder, pure enzyme, etc. of the above carbonyl reductase can be used. To obtain higher conversion efficiency, it is preferable to use a crude enzyme solution. The ratio of the amount of carbonyl reductase to the amount of substrate is preferably 1% to 6% (w/w) or the ratio of the mass of resting cell cells to the mass of substrate is 10% to 100%.
Coenzyme A
In the present invention, "coenzyme" means a coenzyme capable of realizing electron transfer in a redox reaction.
Typically, the coenzyme of the invention is the reducing coenzyme NADH, NADPH or the oxidizing coenzyme NAD+, NADP+. The oxidative coenzymes NAD+, NADP+ are preferred because of the relatively high price and cost of the reduced coenzymes.
When selecting an oxidative coenzyme, a method for achieving coenzyme regeneration is required, which mainly comprises three of (1) glucose dehydrogenase and co-substrate glucose; (2) Alcohol dehydrogenase and co-substrate isopropanol, (3) formate dehydrogenase and co-substrate ammonium formate.
In a preferred embodiment, the coenzyme is NAD+, and the coenzyme regeneration system is an alcohol dehydrogenase, preferably the alcohol dehydrogenase is combined with a co-substrate isopropanol. The ratio of NAD+ usage to substrate usage is 0.01% -2.0% (w/w). The buffer system is phosphate buffer salt with the concentration of 0.1mol/L. The pH of the buffer is 6-10.
Stereoisomers of
In the present invention, stereoisomers refer to isomers produced by different spatial arrangements of atoms in molecules, and can be classified into cis-trans isomers and enantiomers, and also into enantiomers and diastereomers. In chemical or enzymatic reactions, one stereoisomer preferentially forms over the other, known as stereoselectivity. The stereoselectivity may be partial, where formation of one stereoisomer is favored over another, or the stereoselectivity may be complete, where only one stereoisomer is formed. When a stereoisomer is an enantiomer, stereoselectivity refers to enantioselectivity, i.e., the fraction of one enantiomer in the sum of the two enantiomers (usually reported as a percentage). Which (typically is a percentage) is generally reported in the art alternatively as the enantiomeric excess (ee) calculated therefrom according to the formula: [ major enantiomer-minor enantiomer ]/[ major enantiomer + minor enantiomer ]. When stereoisomers are diastereomers, stereoselectivity refers to diastereoselectivity, i.e. the fraction of one diastereomer in a mixture of two diastereomers (usually reported as a percentage), usually optionally reported as diastereomeric excess (d.e.). Enantiomeric excess and diastereomeric excess are types of stereoisomer excess. In the present invention, the substantially stereoisomers are pure and the enzyme is capable of converting the substrate to a corresponding product having at least about 95%, 96%, 97%, 98%, or 99% stereoisomer excess; preferably, at least about 98% stereoisomer excess; more preferably, at least about 99% stereoisomer is present in excess.
Biocatalytic preparation method
The invention provides a method for preparing a compound S-vitronectin (Ia) by using carbonyl reductase to catalyze and reduce beta-acetonylxyloside (II). Wherein the carbonyl reductase is a mutant of carbonyl reductase NgADH from Nakaseomyces glabratus, and the reaction formula is as follows:
according to the preferred system, the preparation method is carried out as follows: mixing and stirring the substrate and the phosphate buffer, adding wet thalli, crude enzyme solution, crude enzyme powder or pure enzyme, adding coenzyme NAD+ and co-substrate, adjusting pH between 6.5 and 7.5 with sodium bicarbonate, maintaining at 20-40deg.C, and monitoring by TLC or HPLC, preferably until the reaction conversion rate reaches more than 98% (e.g. 98%,99% or 100%). After the reaction is finished, centrifuging or filtering by diatomite, removing thalli, taking supernatant, nano-filtering to remove salt, concentrating, crystallizing and purifying to obtain the product.
The invention has the main technical effects that:
1) Compared with the prior art, the carbonyl reductase obtained by screening and the preparation method provided by the invention are used for preparing the S-vitriol, so that the yield is obviously improved, and the cost is reduced.
2) The carbonyl reductase obtained by screening has high stereoselectivity in the reaction of preparing S-vitrein by catalytic reduction of beta-acetonylxyloside, and the optical purity of S-configuration products is high, and the ee value reaches 100%.
3) The invention provides a new source of carbonyl reductase for the enzymatic synthesis of S-vitrein, has the advantages of high catalytic activity, strong stereoselectivity and higher reaction yield, and provides more and better choices for industrial production.
Drawings
FIG. 1 is a chiral HPLC plot of the racemate of the glassy gene, wherein the retention time of the S-glassy gene configuration is about 10.42min and the retention time of the R-glassy gene configuration is 11.45min.
FIG. 2 is a chiral HPLC plot of the compound of formula Ia obtained by carbonyl reductase catalysis of the present application, showing an ee value of 100% for the S configuration.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. 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 experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor LaboratoryPress, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials referred to in the present invention are available from commercial sources unless otherwise specified.
Example 1: preliminary screening of carbonyl reductase
In this example, a plurality of carbonyl reductases were initially screened, wherein,
RDH is derived from Bradyrhizobium diazoefficiens, and Genbank number thereof is WP_011089402;
LSADH is derived from Leiffsonia sp.strain S749, genbank accession number AB213459;
ARQR is derived from Agrobacterium radiobacter ECU2556, genbank accession number wp_015918093.1;
AKR is derived from Thermotoga maritima MSB, genbank accession number wp_004082270.1;
NgADH is Nakaseomyces glabratus, genbank accession number KAI8399263.
The screening results are shown in Table 1, wherein specific reaction conditions are noted correspondingly.
TABLE 1 screening results for partial carbonyl reductase
| Numbering device | Carbonyl reductase | Conversion (%) | ee value (%) | Configuration of |
| 1 | RDH b | 1 | Undetected | Undetected |
| 2 | LSADH a | 70 | 95 | R |
| 3 | ARQR b | 31 | 98 | R |
| 4 | AKR b | 7 | 91 | S |
| 5 | NgADH b | 85 | 100 | S |
a represents the reaction conditions: 2mL of reaction system (1) 10g/L of substrate, 20g/L of glucose, 1mM of NAD (P), 10g/L of GDH, phosphate buffer (0.1M, pH 7.0), 28℃at 220rpm,20h.
b represents the reaction conditions: 2mL of reaction system (2) 10g/L of substrate, 20% isopropyl alcohol, 1mM NAD (P), phosphate buffer (0.1M, pH 7.0), 28℃at 220rpm,20h.
As shown in Table 1, the carbonyl reductases from different sources have significant differences in the reduction effect on the substrate beta-acetonylxyloside of the present invention, and the three-dimensional configuration of the product is also different. Carbonyl reductase NgADH has advantages over other carbonyl reductases in both conversion and stereoselectivity.
Example 2: construction of carbonyl reductase engineering bacteria
The carbonyl reductase NgADH gene (accession No. KAI 8399263) from Nakaseomyces glabratus was synthesized by codon optimization, cloned into pET22b (+) vector, transferred into E.coli DH 5. Alpha. Competent cells, and cultured on ampicillin-resistant plates. And (3) selecting single colony of the positive transformant for culture, extracting plasmid for sequencing, extracting recombinant plasmid after determining, then introducing competent cells of host escherichia coli BL21 (DE 3) into an ampicillin resistance flat plate for culture, and selecting single colony for culture in LB culture medium to obtain recombinant genetically engineered bacteria of carbonyl reductase.
Example 3: construction and screening of carbonyl reductase mutant library
Wild carbonyl reductase NgADH is used as a template, and a random point mutation kit is usedII Site-Directed Mutagenesis Kit) to obtain a plasmid library containing the evolved carbonyl reductase gene by error-prone PCR or by semi-rational design of mutation of the wild-type carbonyl reductase gene NgADH. The constructed plasmid library was transferred into E.coli BL21 (DE 3) and then spread onto LB solid medium containing 50. Mu.g/mL ampicillin and cultured overnight in an incubator at 37 ℃. Single colonies were picked into 96-well plates containing 400. Mu.L of LB medium (50. Mu.g/mL of ampicillin) and cultured overnight at 37℃and 200rpm to give a seed solution for carbonyl reductase mutation. mu.L of the carbonyl reductase mutant seed solution was pipetted into a 96-well plate containing 400. Mu.L of fermentation medium (50. Mu.g/mL ampicillin) and incubated at 37℃and 800rpm to OD 600 Value of>0.8. The mutants were induced to express using isopropyl thiogalactoside (IPTG) at a final concentration of 0.5mM at 28 ℃ and then cultured for a further 20h. The cells were collected by centrifugation at 3000g for 30min in a 96-well plate into a centrifuge, then resuspended in 200. Mu.L of lysis buffer (0.1M phosphate buffer containing 1000U of lysozyme, pH 7.0), lysed at 30℃for 1h, and then centrifuged at 4℃for 4000g for 30min in the centrifuge, and the clarified supernatant was aspirated to determine mutant activity. mu.L of the reaction solution (containing 0.4mM substrate, 1mM NADPH) was added to a new 96-well ELISA plate, and after 10. Mu.L of the supernatant was added, the change in NADPH was detected at absorbance of 340 nm. The amount of NADPH consumed was used to calculate the level of enzyme activity of the reaction mutants, and the relative activities of the respective mutants are shown in Table 1.
The fermentation medium formulation is as follows: yeast extract (2.0%), tryptone (1.2%), sodium chloride (0.3%), glycerol (1%), dipotassium hydrogen phosphate (0.2%), magnesium sulfate heptahydrate (0.05%).
Table 2 partial mutants and their relative activities
* The activity of the wild-type carbonyl reductase NgADH (SEQ ID NO: 2) was set to 100%.
As shown in Table 2, the carbonyl reductase NgADH activity-enhancing sensitivity type mutation residue position is L296I, V108L, T41 37139S, D E, F92V, T221 98279 5294W, T141P and the like, and the relative activity is more than 130%, particularly the activity of V108L, F92V, T221K is better. At the same time, it was also unexpectedly found that the relative activity of mutants 13, 15-25 reached more than 500%.
Example 4: application of mutant in preparation of S-vitronectin by reduction of beta-acetonylxyloside
Reaction conditions: taking 0.05M phosphate buffer (2 mL), adding NADP + (0.001 g), glucose dehydrogenase GDH (0.03 g), glucose (0.2 g) were added and dissolved sufficiently, and the above-mentioned fermented mutant carbonyl reductase or wild-type carbonyl reductase NgADH (0.06 g) was added. The substrate β -acetonylxyloside (0.1 g) was added in portions with vigorous stirring and reacted at 30℃and 220rpm for 3 hours. 2mL of acetonitrile was added for quenching. The conversion and the stereoselectivity (%) ee value of the product were measured using a differential assay, as shown in Table 3.
TABLE 3 catalytic Activity and stereoselectivity of wild-type and mutant carbonyl reductases
EXAMPLE 5 preparation of the Graded Compound S-Botrytis by biocatalysis
Taking 0.05M phosphate buffer (100 mL), adding NADP + (0.01 g), glucose dehydrogenase GDH (1.5 g) and glucose (20 g) were added to the mixture to dissolve the mixture sufficiently, and the above-mentioned fermented mutant carbonyl was addedPrimordial enzyme mutant 15 (4 g). The substrate beta-acetonylxyloside (10 g) was added and reacted at 30℃and 200rpm, and the pH was adjusted to between 6.5 and 7.5 with sodium bicarbonate. Monitoring reaction conversion>At 98%, the reaction was terminated. Removing thalli by centrifugation, filtering supernatant to remove salt, concentrating under reduced pressure, crystallizing and purifying to obtain 9.1g of product, wherein the purity is 99.9%, the yield is 91%, and the ee value is 100%.
EXAMPLE 6 preparation of the Graded Compound S-Botrytis by biocatalysis
Taking 0.05M phosphate buffer (100 mL), adding NADP + (0.01 g) glucose dehydrogenase GDH (1.5 g) was added, glucose (20 g) was added for sufficient dissolution, and the above-mentioned fermentation mutant carbonyl reductase mutant 17 (3 g) was added. The substrate beta-acetonylxyloside (10 g) was added and reacted at 30℃and 200rpm, and the pH was adjusted to between 6.5 and 7.5 with sodium bicarbonate. Monitoring reaction conversion>At 98%, the reaction was terminated. Removing thalli by centrifugation, filtering supernatant to remove salt, concentrating under reduced pressure, crystallizing and purifying to obtain 9.2g of product, wherein the purity is 99.9%, the yield is 92%, and the ee value is 100%.
EXAMPLE 7 preparation of the Graded Compound S-Botrytis by biocatalysis
Taking 0.05M phosphate buffer (100 mL), adding NADP + (0.01 g), glucose dehydrogenase GDH (1.5 g), glucose (20 g) were added and dissolved sufficiently, and the above-mentioned fermentation mutant carbonyl reductase mutant 19 (3 g) was added. The substrate beta-acetonylxyloside (10 g) was added and reacted at 30℃and 220rpm, and the pH was adjusted to between 6.5 and 7.5 with sodium bicarbonate. Monitoring reaction conversion>At 98%, the reaction was terminated. Removing thalli by centrifugation, filtering supernatant to remove salt, concentrating under reduced pressure, crystallizing and purifying to obtain 9.5g of product, wherein the purity is 99.9%, the yield is 95%, and the ee value is 100%.
EXAMPLE 7 preparation of the Graded Compound S-Botrytis by biocatalysis
Taking 0.05M phosphate buffer (100 mL), adding NADP + (0.01 g) was added formate dehydrogenase FDH (1.5 g), sodium formate (20 g) was added for sufficient dissolution, and the above-mentioned fermented mutant carbonyl reductase mutant 16 (3 g) was added. The substrate beta-acetonylxyloside (20 g) was added, reacted at 30℃and 220rpm, and the pH was adjusted to between 6.5 and 7.5 with sodium bicarbonate. Monitoring reaction conversion>At 98%, the reaction was terminated. Removing thalli by centrifugation, removing salt by nanofiltration of supernatant,concentrating under reduced pressure, crystallizing and purifying to obtain 18.7g of product with purity of 99.9%, yield of 93.5% and ee value of 100%.
EXAMPLE 8 preparation of the Graded Compound S-Botrytis by biocatalysis
Taking 0.05M phosphate buffer (100 mL), adding NADP + (0.01 g), formate dehydrogenase FDH (1.5 g), sodium formate (20 g) were added and dissolved sufficiently, and the above-mentioned fermented mutant carbonyl reductase mutant 20 (3 g) was added. The substrate beta-acetonylxyloside (20 g) was added, reacted at 30℃and 220rpm, and the pH was adjusted to between 6.5 and 7.5 with sodium bicarbonate. Monitoring reaction conversion>At 98%, the reaction was terminated. The thalli is removed by centrifugation, the supernatant is subjected to nanofiltration for desalting, reduced pressure concentration and crystallization purification, 18.9g of product is obtained, the purity is 99.9%, the yield is 94.5%, and the ee value is 100%.
EXAMPLE 9 preparation of the Graded Compound S-Botrytis by biocatalysis
Taking 0.05M phosphate buffer (160 mL), adding NADP + (0.02 g), isopropyl alcohol dehydrogenase (3 g) was added, isopropyl alcohol (40 mL) was added for sufficient dissolution, and the above-mentioned fermentation mutant carbonyl reductase mutant 22 (3 g) was added. The substrate beta-acetonylxyloside (20 g) was added, reacted at 30℃and 220rpm, and the pH was adjusted to between 6.5 and 7.5 with sodium bicarbonate. Monitoring reaction conversion>At 98%, the reaction was terminated. Removing thalli by centrifugation, filtering supernatant to remove salt, concentrating under reduced pressure, crystallizing and purifying to obtain 19.4g of product, wherein the purity is 99.9%, the yield is 97%, and the ee value is 100%.
EXAMPLE 10 preparation of the Graded Compound S-Botrytis by biocatalysis
Taking 0.05M phosphate buffer (160 mL), adding NADP + (0.015 g) was added with isopropyl alcohol dehydrogenase (3 g), added with isopropyl alcohol (40 mL) to be sufficiently dissolved, and added with the above-mentioned fermented mutant carbonyl reductase mutant 24 (3 g). The substrate beta-acetonylxyloside (20 g) was added and reacted at 30℃and 200rpm, and the pH was adjusted to between 6.0 and 8.0 with sodium bicarbonate. Monitoring reaction conversion>At 98%, the reaction was terminated. The thalli is removed by centrifugation, the supernatant is subjected to nanofiltration for desalting, reduced pressure concentration and crystallization purification, 19.2g of product is obtained, the purity is 99.9%, the yield is 96%, and the ee value is 100%.
In summary, the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (18)
1. The carbonyl reductase is characterized in that the carbonyl reductase takes carbonyl reductase NgADH which is derived from Nakaseomyces glabratus and has an amino acid sequence shown as SEQ ID NO. 2 as a starting sequence, and has the following mutation sites:
mutant 7: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site T221K;
mutant 13: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site T41A/C139T/T221K;
mutant 15: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/F92T/I99Y/C139T/T221K;
mutant 16: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/F92C/F94W/C139T/T221K;
mutant 17: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site N49R/H78Y/I99Y/C139T/T221K;
mutant 18: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/F92V/V108L/C139T/Y208I/T221K;
mutant 19: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of D29E/H73E/F94V/C139T/Y208I/T221K/K279N;
mutant 20: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/F92V/C139T/Y208I/T221K/A272V/I302V;
mutant 21: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T22I/D29E/H78Y/C139T/T221K/A272V/N309D;
mutant 22: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T41A/H78Y/F92L/F94V/C139T/Y208I/T221K/A272V;
mutant 23: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T22I/D29E/H78Y/C139T/T221K/A272V/K279N/N309D;
mutant 24: an amino acid sequence corresponding to SEQ ID NO. 2 and having a mutation site of T22I/D29E/H78Y/V108I/T221K/A272V/K279N/L296I; or (b)
Mutant 25: the amino acid sequence corresponding to SEQ ID NO. 2 and having mutation sites N49R/F94L/C139T/Y208T/T221K/D240T/A272V/I302V.
2. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 13.
3. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 15.
4. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 16.
5. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 17.
6. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 18.
7. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 19.
8. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 20.
9. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 21.
10. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 22.
11. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 23.
12. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 24.
13. The carbonyl reductase of claim 1, wherein the carbonyl reductase is mutant 25.
14. A coding gene encoding the carbonyl reductase according to any one of claims 1-13.
15. An expression vector, wherein the expression vector is loaded with a gene encoding the carbonyl reductase of claim 14; preferably, the expression vector is a recombinant plasmid; preferably, the expression vector is a pET22b (+) vector.
16. A genetically engineered bacterium comprising a host cell and a gene of interest transferred into the host cell, the gene of interest comprising a gene encoding the carbonyl reductase of claim 14; preferably, the host cell is E.coli.
17. The method for preparing the S-vitronectin by taking the beta-acetonylxyloside as a substrate through biocatalysis and reduction of carbonyl is characterized by comprising the following steps:
(a) In a liquid reaction system, using a compound beta-acetonylxyloside shown in a formula II as a substrate, and carrying out asymmetric reduction reaction under the catalysis of carbonyl reductase to obtain a compound S-vitronectin shown in a formula Ia;
(b) Optionally separating the compound S-vitronectin of formula Ia from the reaction system after the reaction of step (a);
wherein the carbonyl reductase is a carbonyl reductase according to any one of claims 1 to 13.
18. A reaction system, characterized in that the reaction system comprises:
(1) An aqueous solvent;
(2) A substrate, wherein the substrate is beta-acetonylxyloside;
(3) A coenzyme;
(4) Carbonyl reductase; the carbonyl reductase being a carbonyl reductase according to any one of claims 1 to 13;
(5) A co-substrate.
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| CN117737149B (en) * | 2024-02-20 | 2024-05-07 | 山东君泰药业有限公司 | Method for synthesizing high-purity S-vitronectin by enzyme catalysis |
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