CN116410944A - Dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate - Google Patents

Dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate Download PDF

Info

Publication number
CN116410944A
CN116410944A CN202111641233.6A CN202111641233A CN116410944A CN 116410944 A CN116410944 A CN 116410944A CN 202111641233 A CN202111641233 A CN 202111641233A CN 116410944 A CN116410944 A CN 116410944A
Authority
CN
China
Prior art keywords
dehydrogenase
hydroxybutyrate
methyl
acetoacetate
ethyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111641233.6A
Other languages
Chinese (zh)
Inventor
程占冰
孙传民
田振华
王舒
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ecolab Biotechnology Shanghai Co ltd
Original Assignee
Ecolab Biotechnology Shanghai Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ecolab Biotechnology Shanghai Co ltd filed Critical Ecolab Biotechnology Shanghai Co ltd
Priority to CN202111641233.6A priority Critical patent/CN116410944A/en
Publication of CN116410944A publication Critical patent/CN116410944A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—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
    • 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
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate. The dehydrogenase comprises one or more of the following amino acid residue differences compared to SEQ ID NO. 1: Q97V, H145L and Y188F; and has the activity not lower than that of the wild dehydrogenase with the amino acid sequence shown as SEQ ID NO. 1. The dehydrogenase prepared by the invention has high conversion rate and yield which can be up to 99.8% and 95% respectively in the application of preparing (R) -3-hydroxybutyrate methyl ester or ethyl ester, and the purity of the prepared product is more than 99%, and the ee value can be up to 99.8%.

Description

Dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate
Technical Field
The invention belongs to the technical field of enzyme engineering biology, and particularly relates to dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate.
Background
(R) -3-hydroxybutyric acid is also called beta-hydroxybutyric acid, is a ketone body with chiral activity and has antibacterial, insecticidal and antiviral activities; the chiral module can be used for synthesizing fine chemicals such as antibiotics, vitamins, aromatic hydrocarbons, pheromones and the like, and can also be used as a precursor substance of polyester to produce biodegradable plastics; because the half-life of the (R) -3-hydroxybutyric acid in the human body is short and the human body has good tolerance to the (R) -3-hydroxybutyric acid, the (R) -3-hydroxybutyric acid can be directly used as an oral medicine; under the condition of sugar deficiency, the (R) -3-hydroxybutyric acid can partially protect and stabilize nerve cells; there is also some evidence that (R) -3-hydroxybutyric acid has great potential in enhancing cardiac efficiency, preventing brain damage, and in pharmaceutical value.
The (R) -3-hydroxybutyric acid may be obtained by hydrolysis of (R) -3-hydroxybutyrate such as methyl (R) -3-hydroxybutyrate or ethyl (R) -3-hydroxybutyrate.
Figure BDA0003443462680000011
The (R) -3-hydroxybutyrate methyl ester or (R) -3-hydroxybutyrate ethyl ester contains two functional groups in the molecule, can be used for preparing various natural products and pharmaceutical preparations, such as synthesizing imipenem, meropenem, panipenem, faropenem, ertapenem, doripenem, biapenem and other antibiotics, beta-polypeptides and the like, and is also an important chiral intermediate for synthesizing L-carnitine.
At present, the preparation methods of (R) -3-hydroxybutyric acid methyl ester or (R) -3-hydroxybutyric acid ethyl ester reported and disclosed at home and abroad mainly comprise two methods: resolution of racemates and asymmetric reductive synthesis.
The method for resolving the racemized methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate by utilizing the chromatography to obtain a product with a single configuration has the defects of high investment cost on equipment and chiral stationary phase, high consumption of mobile phase and difficult solvent recovery, so the method is limited to laboratory-scale preparation.
The single-configuration 3-hydroxybutyrate methyl ester or 3-hydroxybutyrate ethyl ester is obtained by utilizing an enzymatic chiral resolution technology, and has high production cost, low raw material utilization rate and certain limitation on industrial application.
The main defects of the (R) -3-hydroxybutyric acid methyl ester or (R) -3-hydroxybutyric acid ethyl ester prepared by adopting a chemical asymmetric synthesis method are that the reaction conditions are harsh, the reaction steps are complicated, the yield is low, most of the organic solvents used in the reaction are toxic, and the environmental pollution is easy to cause; and the chiral catalyst is difficult to prepare, expensive, easy to remain in the product and difficult to remove, and is difficult to recycle, so that the cost is high, the benefit is low, and the industrial application process of the chiral catalyst is limited.
The asymmetric synthesis catalyzed by biological cells or enzymes has the advantages of high stereoselectivity, green and safe reaction conditions, few byproducts and the like.
It is reported that strain Pichia membranaefaciens Hansen ZJPH07 capable of catalyzing the asymmetric reduction of ethyl acetoacetate (EAA) into ethyl (R) -3-hydroxybutyrate ((R) -EHB) is obtained by screening Hou Jing and the like, the catalytic efficiency is improved by utilizing ultraviolet mutagenesis and introducing a system containing ionic liquid medium, after Pichia membranaefaciens-218 obtained by ultraviolet mutagenesis is added with ionic liquid [ BMIM ] [ BF4], 550mmol/L of substrate EAA reduction can be catalyzed, the yield is 73%, the ee value of the product is 77.8%, and compared with the reaction in an aqueous phase (the substrate concentration is 350mmol/L, the yield is 65.1%, the ee value is 68.5%), the catalytic efficiency and the stereoselectivity are improved. But neither the yields nor the ee values are very high.
Patent CN109852593B discloses a method for asymmetrically synthesizing R-3-ethylhydroxybutyrate by an enzymatic method under the action of recombinant ketoreductase, formate dehydrogenase and coenzyme by using ethyl acetoacetate as a substrate and ammonium formate as a hydrogen donor, wherein the ketoreductase is derived from candida magnolia (Candida magnoliae), and when an enzyme mutant CmCR (S176C, Y191A) with GenBank accession No. AB036927 is used as an enzyme catalyst, the conversion rate of ethyl acetoacetate can reach 99%, and the ee value of the product reaches 99.9%. However, ammonium formate and formate dehydrogenase are used as coenzyme circulation systems in the reaction, and the reaction cost of the enzyme is increased.
Patent CN111705068B discloses a stereoselective ketoreductase derived from Leifsonia sp.strain S749 and an asymmetric synthesis of ethyl (R) -3-hydroxybutyrate using such ketoreductase. According to the method, ethyl acetoacetate is used as a substrate, isopropanol is used as a coenzyme circulation system, enzymes with additional coenzyme circulation are not needed, the (R) -3-hydroxy ethyl butyrate is generated under the catalysis of ketoreductase, the conversion rate reaches 99.5%, and the ee value reaches 99.5%.
Disclosure of Invention
The invention aims to overcome the defect that in the prior art, dehydrogenase capable of effectively catalyzing the formation of (R) -3-hydroxybutyrate is rare, and provides the dehydrogenase and application thereof in the preparation of (R) -3-hydroxybutyrate.
The invention mainly solves the technical problems through the following technical scheme.
The present invention provides a dehydrogenase comprising one or more of the following amino acid residue differences compared to SEQ ID NO: 1: Q97V, H145L and Y188F;
and has the activity not lower than that of the wild dehydrogenase with the amino acid sequence shown as SEQ ID NO. 1. For example, at least the function of the wild-type dehydrogenase is retained.
Preferably, the dehydrogenase has the following amino acid differences compared to the wild-type dehydrogenase:
(1) Q97V, H145L or Y188F;
(2) Any two of Q97V, H145L and Y188F; preferably H145L and Y188F;
(3) Q97V, H145L and Y188F.
The invention also provides an isolated nucleic acid encoding a dehydrogenase as described above.
The invention also provides a recombinant expression vector comprising an isolated nucleic acid as described above.
The invention also provides a transformant comprising an isolated nucleic acid as described above or a recombinant expression vector as described above.
The host cell used for preparing the transformant in the present invention is preferably Escherichia coli (Escherichia coli) such as E.coli BL21 (DE 3).
The present invention also provides a method for preparing the dehydrogenase as described above, comprising: the transformant as described above is cultured under conditions suitable for expression of the dehydrogenase.
The invention also provides a preparation method of the (R) -3-hydroxybutyrate, which comprises the following steps:
under the catalytic action of dehydrogenase and coenzyme, the substrate methyl acetoacetate or ethyl acetoacetate is used to obtain the product methyl (R) -3-hydroxybutyrate or ethyl; the dehydrogenase is the dehydrogenase prepared by the method.
In the preparation method of the present invention, the coenzyme may be any coenzyme conventional in the art, for example NADH, NAD + NADPH or NADP + Preferably NAD + Or NADH.
In a preferred embodiment of the present invention, isopropanol is also included in the reaction system.
The volume concentration of the substrate methyl acetoacetate or ethyl acetoacetate in the reaction system accounting for the total volume of the reaction system is preferably 30-55%, preferably 44-48%.
In the reaction system, the molar ratio of isopropyl alcohol to methyl acetoacetate or ethyl acetoacetate as a substrate is preferably (1 to 5): 1, more preferably 1.3:1.
In the reaction system, the mass ratio of the coenzyme to the substrate methyl acetoacetate or ethyl acetoacetate is preferably 1 (100-2000), more preferably 1:1000.
In the reaction system, the mass ratio of the dosage of the dehydrogenase thallus to the substrate methyl acetoacetate or ethyl acetoacetate can be 1 (10-100), and is preferably 1:50.
The form of the dehydrogenase used may be conventional in the art, and may be, for example, a dehydrogenase cell, a crude enzyme solution, a pure enzyme solution, or an immobilized enzyme.
The preparation of the dehydrogenase cell may comprise the following operations:
the transformant as described above was inoculated into LB medium and shake-cultured at 200rpm at 37℃until OD 600 To 0.8-1.0, IPTG was added to a final concentration of 0.05mM and the temperature was lowered to 30℃for overnight induction. After the induction culture was completed, the mixture was centrifuged at 5000rpm for 20 minutes, and the supernatant was discarded to collect dehydrogenase cells.
The LB medium may contain 30. Mu.g/mL kanamycin antibiotic.
The composition of the LB liquid medium is, for example: peptone 10g/L, yeast powder 5g/L and NaCl 10g/L.
In the reaction system, the catalysis is preferably performed in PBS buffer with pH of 7.0.
The invention also provides the use of a dehydrogenase as described above for the preparation of (R) -3-hydroxybutyrate, for example (R) -3-hydroxybutyrate methyl or ethyl.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
the dehydrogenase prepared by the invention has high conversion rate and yield which can be up to 99.8% and 95% respectively in the application of preparing (R) -3-hydroxybutyrate methyl ester or ethyl ester, and the purity of the prepared product is more than 99%, and the ee value can be up to 99.8%. Has good application prospect.
Drawings
FIG. 1 is a reaction scheme of the present invention.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The experimental methods in the invention are all conventional methods unless otherwise specified, and specific reference is made to the "molecular cloning Experimental guidelines" by J.Sam Broker et al for gene cloning operations.
pET28a plasmid was purchased from Novagen; ndeI enzyme, hindIII enzyme, are purchased from Thermo Fisher; e.coli BL21 (DE 3) competent cells were purchased from the company of biotechnology limited, prosperous in the ancient cooking vessel, beijing.
The reaction route of the invention is shown in figure 1.
EXAMPLE 1 construction of dehydrogenase Gene mutants
The gene sequence SEQ ID NO. 2 of dehydrogenase (SEQ ID NO. 1) with PDB number of 5X8H is fully synthesized, and is connected into a pET28a expression vector by enzyme cutting sites NdeI and HindIII to construct a recombinant plasmid pET28a-ChADH. The gene synthesis company is Suzhou Jin Weizhi biotechnology limited company (Suzhou industrial park star lake street 218 biological nano-technology park C3).
The recombinant plasmid pET28a-ChADH is transformed into E.coli BL21 (DE 3) competent cells, coated on LB medium containing 30 mug/mL of kanamycin, cultured overnight at 37 ℃, and harvested to obtain recombinant escherichia coli containing pET28a-ChADH plasmid.
The recombinant plasmid pET28a-ChADH is used as a template, a primer pair is designed aiming at the mutation of the Q97 site, the H145 site and the Y188 site of SEQ ID NO. 1, and a PCR technology is adopted to introduce nucleotide mutation into the ChADH gene (SEQ ID NO. 2) in vitro, so as to construct a single-point mutation library.
Combination mutation: the single-point mutation is combined with a plurality of different mutations in an overlap PCR mode to form a novel mutant.
TABLE 1 primer sequences
Figure BDA0003443462680000061
TABLE 2 PCR amplification System
Reagent(s) Dosage of
Prime STAR Max Premix(2X) 10μL
Forward primer (10. Mu.M) 0.5μL
Reverse primer (10. Mu.M) 0.5μL
DNA template 0.1μL
Deionized water 8.9μL
TABLE 3 PCR amplification procedure
Figure BDA0003443462680000062
The PCR product was digested by adding DpnI enzyme at 37℃for 2 hours. The reaction was completed and transformed into E.coli BL21 (DE 3) competent cells, which were plated on LB medium containing 30. Mu.g/mL of calicheamicin, cultured overnight at 37℃and harvested to obtain recombinant E.coli containing the mutant.
TABLE 4 mutant library
Numbering device Mutation site
1 Wild Type (WT)
2 Q97V
3 H145L
4 Y188F
5 H145L-Y188F
6 Q97V-H145L-Y188F
Example 2 enzymatic Activity on different substrates
1. Preparation of crude enzyme solution
The recombinant plasmid-containing E.coli of example 1 was inoculated into LB medium containing 30. Mu.g/mL kanamycin antibiotic, shake cultured at 37℃at 200rpm, and the mixture was allowed to stand for OD 600 To 0.8-1.0, IPTG was added to a final concentration of 0.05mM and the temperature was lowered to 30℃for overnight induction. After the induction culture is finished, centrifuging at 5000rpm for 20min, discarding supernatant, collecting thalli, and storing in a refrigerator at-20 ℃ for later use.
LB liquid medium composition: peptone 10g/L, yeast powder 5g/L, naCl 10g/L, dissolved in deionized water, and sterilized at 121 deg.C for 20 min.
Taking 5g of thalli, adding 50mL of phosphate buffer (pH 7.0,25 mM) to resuspend the thalli, homogenizing and crushing for 3min at 4 ℃ and 800mbar, centrifuging for 30min at 15 ℃ at 5000rpm, and leaving supernatant to prepare crude enzyme solution, and refrigerating at 4 ℃ for later use.
2. Isopropyl alcohol is used as a substrate, and the enzyme activity detection method comprises the following steps:
at 30℃900. Mu.L of pH 7.0 50mM phosphate buffer, 50. Mu.L of isopropanol, 10. Mu.L of 25mM NAD were added to 1mL of the reaction system + The rate of NADH production was measured at 340nm with 40. Mu.L of the diluted enzyme solution. The enzyme activity is defined as the amount of enzyme required to produce 1. Mu. Mol NADH per minute at 30℃being 1U.
3. Methyl acetoacetate (or ethyl acetoacetate) is used as a substrate, and the enzyme activity detection method comprises the following steps:
to 1mL of the reaction system, 900. Mu.L of a pH 7.0 50mM phosphate buffer, 50. Mu.L of methyl acetoacetate (or ethyl acetoacetate), 10. Mu.L of 25mM NADH, and 40. Mu.L of the diluted enzyme solution were added at 30℃to determine the consumption rate of NADH at 340 nm. The enzyme activity is defined as the amount of enzyme required to consume 1. Mu. Mol NADH per minute at 30℃being 1U.
The results of the enzyme activity assay under different substrate conditions are shown in Table 5.
TABLE 5 detection results of enzyme Activity under different substrate conditions
Figure BDA0003443462680000081
Example 3 asymmetric Synthesis of methyl (R) -3-hydroxybutyrate
5mL 100mM PBS (pH 7.0) was used as a buffer, 50g (46 mL) of methyl acetoacetate, 34g (43 mL) of isopropyl alcohol, 10mL of Enz.6 (Q97V-H145L-Y188F) enzyme solution, 50mg of coenzyme NAD, the reaction temperature was 35 ℃, the reaction time was 12H, and the conversion rate reached 99.6%.
The reaction solution was concentrated under reduced pressure to remove the solvent, and then 48.13g of methyl hydroxybutyrate was collected by distillation under reduced pressure, the yield was 95%, the purity was over 99%, and the ee value was 99.8%.
Example 4 asymmetric Synthesis of ethyl (R) -3-hydroxybutyrate
5mL 100mM PBS (pH 7.0) was used as a buffer, 50g (49 mL) of ethyl acetoacetate, 30g (38 mL) of isopropyl alcohol, 10mL of Enz.6 (Q97V-H145L-Y188F) enzyme solution, 50mg of coenzyme NAD, a reaction temperature of 35℃and a reaction time of 12H were added, and the conversion rate reached 99.8%.
The reaction solution was concentrated under reduced pressure to remove the solvent, and then 47.12g of methyl hydroxybutyrate was collected by distillation under reduced pressure, whereby the yield was 93%, the purity was over 99%, and the ee value was 99.8%.
SEQUENCE LISTING
<110> chess Ke Lai Biotechnology (Shanghai) stock Co., ltd
<120> a dehydrogenase and its use in the preparation of (R) -3-hydroxybutyrate
<130> P210110422C
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 249
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid sequence of dehydrogenase
<400> 1
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp His Gly Asn Lys Ala Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Ile Ala Cys Asn Asn Ala Gly Ile Gly Gly Glu
85 90 95
Gln Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Ile Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Ile
130 135 140
His Gly Ile Val Ala Ala Pro Leu Ser Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Glu Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Gly Pro Ala Tyr Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 2
<211> 750
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleic acid sequence of dehydrogenase
<400> 2
atgggtattc tggataataa agttgcactg gttaccggtg caggtagcgg tattggtctg 60
gcagttgcac atagctatgc gaaagaaggt gcaaaagtga ttgtgagcga tattaatgaa 120
gatcatggta ataaagcagt tgaagatatt aaagcacagg gtggtgaagc cagctttgtg 180
aaagcggata ccagcaatcc ggaagaagtt gaagccctgg tgaaacgtac cgttgaaatt 240
tatggtcgtc tggatattgc atgtaataat gcaggtattg gtggtgaaca ggcactggcc 300
ggtgattatg gtctggatag ctggcgtaaa gtgctgagca ttaatctgga tggtgttttc 360
tatggttgta aatatgaact ggaacagatg gagaaaaatg gtggtggtgt gattgttaat 420
atggccagca ttcatggtat tgtggcagca ccgctgagca gcgcctatac cagcgcaaaa 480
catgccgttg ttggtctgac caaaaatatt ggtgcggaat atggtcagaa aaatattcgt 540
tgtaatgcag ttggtccggc gtatattgaa accccgctgc tggaaagcct gaccaaagaa 600
atgaaagaag cactgattag caaacatccg atgggtcgtc tgggtaaacc ggaagaagtt 660
gcagaactgg ttctgtttct gagcagcgaa aaaagtagct ttatgaccgg tggttattat 720
ctggttgatg gtggttatac cgccgtttaa 750
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Forward primer Q97V
<400> 3
gcaggtattg gtggtgaagt ggcactggcc ggtgattatg 40
<210> 4
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> reverse primer Q97V
<400> 4
cataatcacc ggccagtgcc acttcaccac caatacctgc 40
<210> 5
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> Forward primer H145L
<400> 5
gttaatatgg ccagcattct gggtattgtg gcagcaccg 39
<210> 6
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> reverse primer H145L
<400> 6
cggtgctgcc acaataccca gaatgctggc catattaac 39
<210> 7
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Forward primer Y188F
<400> 7
gtaatgcagt tggtccggcg ttcattgaaa ccccgctgct g 41
<210> 8
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> reverse primer Y188F
<400> 8
cagcagcggg gtttcaatga acgccggacc aactgcatta c 41

Claims (10)

1. A dehydrogenase, characterized in that it comprises one or more of the following amino acid residue differences compared to SEQ ID No. 1: Q97V, H145L and Y188F;
and has the activity not lower than that of the wild dehydrogenase with the amino acid sequence shown as SEQ ID NO. 1.
2. The dehydrogenase of claim 1, wherein said dehydrogenase has the following amino acid differences compared to said wild-type dehydrogenase:
(1) Q97V, H145L or Y188F;
(2) Any two of Q97V, H145L and Y188F; preferably H145L and Y188F;
(3) Q97V, H145L and Y188F.
3. An isolated nucleic acid encoding the dehydrogenase of claim 1 or 2.
4. A recombinant expression vector comprising the isolated nucleic acid of claim 3.
5. A transformant comprising the isolated nucleic acid of claim 3 or the recombinant expression vector of claim 4;
the host cell used for the preparation of the transformant is preferably Escherichia coli (Escherichia coli) such as E.coli BL21 (DE 3).
6. A method of preparing the dehydrogenase of claim 1 or 2, comprising: culturing the transformant according to claim 5 under conditions suitable for expression of the dehydrogenase.
7. A process for preparing (R) -3-hydroxybutyrate comprising:
the methyl or ethyl acetoacetate substrate is catalyzed by a coenzyme, a dehydrogenase according to claim 1 or 2, to yield the product methyl or ethyl (R) -3-hydroxybutyrate.
8. The method according to claim 7, wherein the coenzyme is NADH or NAD + NADPH or NADP + Preferably NAD + Or NADH;
preferably, the reaction system further comprises isopropanol.
9. The process according to claim 7, wherein the substrate methyl acetoacetate or ethyl acetoacetate is present in a volume concentration of 30% to 55%, preferably 44% to 48%, based on the total volume of the reaction system;
and/or the molar ratio of isopropanol to the substrate methyl or ethyl acetoacetate is (1-5): 1, preferably 1.3:1;
and/or the mass ratio of the coenzyme to the substrate methyl acetoacetate or ethyl acetoacetate is 1 (100-2000), preferably 1:1000;
and/or the mass ratio of the dehydrogenase thallus dosage to the substrate methyl acetoacetate or ethyl acetoacetate is 1 (10-100), preferably 1:50;
and/or, the catalysis is performed in a PBS buffer at pH 7.0.
10. Use of a dehydrogenase according to claim 1 or 2 for the preparation of methyl (R) -3-hydroxybutyrate or ethyl ester.
CN202111641233.6A 2021-12-29 2021-12-29 Dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate Pending CN116410944A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111641233.6A CN116410944A (en) 2021-12-29 2021-12-29 Dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111641233.6A CN116410944A (en) 2021-12-29 2021-12-29 Dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate

Publications (1)

Publication Number Publication Date
CN116410944A true CN116410944A (en) 2023-07-11

Family

ID=87058148

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111641233.6A Pending CN116410944A (en) 2021-12-29 2021-12-29 Dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate

Country Status (1)

Country Link
CN (1) CN116410944A (en)

Similar Documents

Publication Publication Date Title
CN113717997B (en) Enzyme composition and method for synthesizing vitronectin by chemical enzyme method
CN111094557B (en) Alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohol
EP3739043A1 (en) Glutamate dehydrogenase mutant and application thereof
CN108048416B (en) Improved ketoreductase mutant and preparation method and application thereof
EP4086343A1 (en) Use of biological enzyme for preparing orlistat intermediate, and preparation method
CN110791493A (en) Aspartate ammonia lyase mutant and application thereof
CN114507681B (en) Sorbose reductase OpCR gene, mutant and encoded protein and application thereof in preparation of vitronectin
CN103898177B (en) Prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof
US6387668B1 (en) Enantioselective epoxide hydrolases and genes encoding these
CN116926028A (en) Dehydrogenase mutant and application thereof in synthesis of S-vitronectin
CN113355299B (en) Ketoacid reductase, gene, engineering bacterium and application in synthesis of chiral aromatic 2-hydroxy acid
CN107828752B (en) Saccharopolyase, preparation method and application in production of alpha-arbutin
CN103898178B (en) Enzyme process prepares high chiral pure (S)-3-piperidine alcohols and the method for derivant thereof
CN110592035B (en) Carbonyl reductase mutant, recombinant expression vector and application of carbonyl reductase mutant in production of chiral alcohol
CN115433721B (en) Carbonyl reductase mutant and application thereof
CN111718883A (en) Recombinant corynebacterium crenatum capable of producing agmatine and application thereof
CN116814572A (en) Carbonyl reductase and mutant thereof and application of carbonyl reductase and mutant in preparation of chiral (R) -8-chloro-6-hydroxy ethyl octanoate
CN108048423B (en) Kidney bean epoxide hydrolase mutant with improved catalytic activity and application thereof
CN116410944A (en) Dehydrogenase and application thereof in preparation of (R) -3-hydroxybutyrate
CN114908129B (en) Dehydrogenase for the preparation of (R) -4-chloro-3-hydroxybutyric acid ethyl ester
CN114525291A (en) Carbonyl reductase and method for preparing (R) -4-chloro-3-hydroxy ethyl butyrate by using same
CN109943618B (en) Application of recombinant lipase in resolution of (R, S) -alpha-ethyl-2-oxo-1-pyrrolidine methyl acetate
CN112410380A (en) Preparation method of 2, 4-difluorobenzylamine
CN109576312B (en) Method for preparing (R) -2-chloro-1- (3-hydroxyphenyl) ethanol
CN112813040B (en) Old yellow enzyme OYE1 mutant protein and application thereof in enzyme-catalyzed asymmetric reduction of 2-aryl acrylic acid derivatives

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
CB02 Change of applicant information

Address after: Room 3114, Building B, 555 Dongchuan Road, Minhang District, Shanghai, 200241

Applicant after: Yikelai Biotechnology (Group) Co.,Ltd.

Address before: Room 3114, Building B, 555 Dongchuan Road, Minhang District, Shanghai, 200241

Applicant before: Ecolab Biotechnology (Shanghai) Co.,Ltd.

CB02 Change of applicant information
SE01 Entry into force of request for substantive examination