CN115478057A - Ketoreductase mutant for preparing (S) -3, 4-dihydroxy-2' -chlorophenethanol - Google Patents
Ketoreductase mutant for preparing (S) -3, 4-dihydroxy-2' -chlorophenethanol Download PDFInfo
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Abstract
The invention discloses a ketoreductase mutant, which can convert 3, 4-dihydroxy-2 '-chloroacetophenone into (S) -3, 4-dihydroxy-2' -chlorophenethanol ethanol so as to prepare S-noradrenaline and S-adrenaline. The ketoreductase mutant has the advantages of high substrate conversion concentration of 200g/L, conversion rate of more than 95%, ee value of more than 99%, simple reaction process, low cost and little pollution, and can selectively obtain S-adrenaline and S-noradrenaline.
Description
The technical field is as follows:
the invention belongs to the technical field of biocatalysis, and particularly relates to a ketoreductase mutant which can be used for preparing an intermediate (S) -3, 4-dihydroxy-2' -chlorophenethanol for removing S-noradrenaline and S-adrenaline.
Background art:
norepinephrine, chemical name (R) -4- (2-amino-1-hydroxyethyl) -1, 2-benzenediol, is a substance formed by removing N-methyl group from epinephrine, and also belongs to catecholamine in chemical structure. Norepinephrine is an anti-shock vasoactive drug, and is mainly used for rescuing shock caused by acute hypotension and peripheral vasodilation, and the like.
The noradrenaline has optical activity, and the levorotatory curative effect is 27 times that of the dextrorotatory body, so the R-type noradrenaline is widely applied to clinically rescuing shock caused by acute hypotension and peripheral vasodilatation and the like. In production preparation, quality analysis and related organism experiments of R-type noradrenaline, S-type noradrenaline molecules play an important role in standard samples or control experiments. However, the prior art does not specially use a synthesis process of S-type noradrenaline, but prepares the S-type noradrenaline optical isomer by resolving a racemic noradrenaline molecule by means of the original R-type noradrenaline synthesis route. And with the continuous promotion of the S-shaped noradrenaline in clinical research, the market demand of the S-shaped noradrenaline is further expanded. However, with the improvement of the noradrenaline synthesis process, more and more manufacturers adopt asymmetric synthesis methods to directly prepare R-type noradrenaline, and S-type noradrenaline cannot be obtained by a splitting method, so that the output of the S-type noradrenaline is reduced, and the supply price is increased.
According to the existing report of the process for preparing noradrenaline, the method for preparing S-type noradrenaline by utilizing the synthetic route shown by Scheme1 is the simplest, but the premise is that the problem of the preparation process of (S) -3, 4-dihydroxy-2' -chlorophenethanol is required to be solved.
In addition, (S) -3, 4-dihydroxy-2' -chlorophenethanol is also an important intermediate for preparing (S) -epinephrine.
In view of the above, there is a need for a process for efficiently preparing chiral compound (S) -3, 4-dihydroxy-2' -chlorophenethanol, which satisfies the growing market demand, wherein the key point is the selection of ketoreductase.
The invention content is as follows:
the present invention aims to provide a novel ketoreductase mutant against the disadvantages of the prior art.
On one hand, the invention utilizes protein engineering technology to carry out semi-rational and rational design on ketoreductase derived from Lactobacillus kefiri, and obtains 2 ketoreductase mutants Mut1 and Mut2 with high catalytic efficiency under the action of high-flux screening.
Further, the amino acid sequences of the ketoreductase mutants Mut1 and Mut2 are shown in SEQ ID NO.3 and 5, respectively.
Furthermore, the ketoreductase mutant Mut1 is obtained by mutating partial sites on the basis of a wild type sequence, wherein the mutation is specifically as follows: the 14 th site is Ala, the 72 th site is Phe, the 94 th site is Thr, the 116 th site is Ile, the 143 th site is Ser, the 147 th site is Leu, the 156 th site is Tyr, the 160 th site is Lys, the 199 th site is His, the 202 th site is Leu, and the 237 th site is Pro.
Furthermore, the ketoreductase mutant Mut2 is obtained by carrying out substitution and deletion on the basis of the sequence of the ketoreductase mutant Mut 1.
Further, the ketoreductase mutant is derived from wild-type Lactobacillus kefir, and the accession number of the wild-type template NCBI is 4RF2_A.
Further, the ketoreductase mutant is constructed in vitro on a recombinant expression vector, preferably a PET series vector.
Further, the host cell of the ketoreductase mutant is Escherichia coli, and the cell is BL21 (DE 3), PLysS or Origami B (DE 3).
Further, after expression of the host cells under IPTG induction, proteins of the two mutants can be obtained. Catalysis of high concentrations of substrate is also achieved with whole cells in the presence of high concentrations of isopropanol.
On the other hand, the ketoreductase mutant provided by the invention can be used for converting 3, 4-dihydroxy-2 '-chloroacetophenone into (S) -3, 4-dihydroxy-2' -chlorophenethanol, and adopts the following technical scheme:
furthermore, the concentration of the 3, 4-dihydroxy-2' -chloroacetophenone can reach 200g/L.
Further, the ketoreductase mutant is ketoreductase enzyme powder or a whole cell or cell disruption solution containing the ketoreductase, preferably a ketoreductase whole cell.
Further, the reaction requires the addition of a coenzyme system, preferably NADPH, at a concentration of less than 0.1mM.
Further, the reaction utilizes the reverse reaction of the ketoreductase mutant, and takes isopropanol as a substrate to realize the regeneration of coenzyme NADPH.
Further, the concentration of isopropanol in the reaction is controlled between 20% and 80%, preferably 80%.
Further, the reaction temperature of the reaction is controlled to be 25 to 45 ℃, preferably 37 ℃.
Further, the pH of the reaction is controlled to be between 5.0 and 7.5, preferably 6.0.
The invention has the beneficial effects that the invention provides a novel ketoreductase mutant, the concentration of a conversion substrate is up to 200g/L, the conversion rate is more than 95.5%, and the chiral ee value is more than 99%. The reaction process is simple, the cost is low, the pollution is small, and S-adrenaline and S-noradrenaline can be selectively obtained.
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Detailed Description
The technical content of the present invention is further described below with reference to specific examples for better understanding of the content of the present invention, but the scope of the present invention is not limited thereto.
Example 1 construction of ketoreductase KRED mutation library
Taking the structure of the wild-type ketoreductase of SEQ ID No.1 (the corresponding nucleotide sequence is SEQ ID No. 2) as reference, and respectively butting the structure with a substrate and an S-shaped product through computer simulation to obtain the optimal structure and energy. In order to allow the S-type product to better exist in the active pocket of ketoreductase KRED, saturation mutation screening is carried out on the amino acid generating steric hindrance, and 11000 monoclonal library volumes are constructed. Through the saturated primer design of the sites, the whole plasmid amplification reaction of the recombinant plasmid pET-21a-kred is realized under the participation of high-fidelity polymerase, then FD DpnI restriction enzyme is utilized to digest the template of a PCR product, the PCR product is purified and transformed into escherichia coli BL21 (DE 3) competence, then 1mL of LB culture medium is added for incubation at 37 ℃, and then the LB culture medium is coated on the plasmid containing 50ug/L Amp + The LB solid plates of (1) were subjected to inverted culture in an incubator at 37 ℃ for 18 hours to grow full plate monoclonals, and these plates were obtainedAnd constructing a mutation library for later screening.
EXAMPLE 2 screening of ketoreductase KRED mutants
188 single clones were randomly picked from each plate for 96-well shake culture, and two wild-type strains were inoculated into each 96-well plate for later analysis as a control, totaling 20 96-well plates. The specific operation is as follows: adding 400uL of LB culture medium into a sterile 96-well plate, culturing at 37 ℃ for about 12h, transferring the plate into a second 96-well plate according to the inoculation amount of 10%, culturing for about 3h, adding IPTG with the final concentration of 0.1mM to induce expression for 16h, and inducing the temperature to be 25 ℃. After the culture, the supernatant was centrifuged and discarded, and the supernatant was frozen in a freezer at-20 ℃ for further use.
The transformation system was prepared according to Table 1, and 300uL of the resulting solution was transferred to the above centrifuged 96-well plate by means of a line gun, reacted at 37 ℃ for 4 hours (which is a reaction time for the control group to convert about half of the substrate) in a homothermal shaker at 300rpm, and inactivated at 80 ℃. And finally, performing absorptiometry detection and chiral HPLC analysis on all conversion reaction liquid under 340nm, improving the catalytic efficiency and the chiral value by more than 10% as positive values, sequencing and analyzing mutant information, performing next iteration mutation on the basis of the mutation, and so on, gradually completing iteration saturation mutation of all sites, and taking the final mutant with high catalytic activity and chiral selectivity as the optimal result of the evolution.
TABLE 1 reaction solution System for screening saturated mutant library
| Raw materials | Concentration of |
| Substrate | 20g/L |
| Isopropanol (I-propanol) | 20％ |
| NADH | 2g/L |
| pH 7.0 Potassium phosphate Buffer | 100mM |
EXAMPLE 4 recombinant expression of ketoreductase
The pre-screened positive mutant strain is prepared by performing amplification fermentation, and BL21 (DE 3) cells containing target gene are inoculated to Amp + The mixture was cultured overnight at 37 ℃ in a resistant LB tube to obtain a primary seed culture. Transferring the seed culture solution into a 2YT liquid culture medium containing resistance according to the inoculation ratio of 1%, placing the culture medium in a shaking table, culturing for 3-5h at 37 ℃ and 200rpm, cooling to 20 ℃ when OD reaches 0.6-0.8, adding IPTG (isopropyl-beta-thiogalactoside) with the concentration controlled at 0.1mM, and performing overnight induction expression. The fermentation broth was centrifuged to collect cells, the collected cells were lysed with 20mM Tris-HCl buffer (pH 6.5), and the cells were disrupted by sonication. Then, the mixture is centrifuged at 12000rpm for 10min, and the obtained supernatant is the transaminase protein, and the expression condition of the protein can be seen through electrophoresis.
Example 4 catalytic reaction of wild-type ketoreductase
To a 50mL reaction flask was added 0.2g of 3, 4-dihydroxy-2' -chloroacetophenone, dissolved in 4mL of isopropanol, 6mg of coenzyme NADPH was added, 16mL of water was added, pH was adjusted to 7.0 with 0.1M NaOH, and finally 1g of cells (containing wild-type ketoreductase) were added. Stirring the mixture in a water bath at 37 ℃ for reaction, and controlling the pH value to be between 6.0 and 7.5 in the reaction process by using 0.1M NaOH. After the reaction was completed for 24 hours, the reaction mixture was dissolved in 2-fold acetonitrile and analyzed by HPLC, and the conversion was 65%. The target product is obtained after rotary evaporation at 40 ℃, and the ee value is only 45 percent.
Example 5 catalytic reaction of ketoreductase mutant Mut1
A50 mL reaction flask was charged with 4g of 3, 4-dihydroxy-2' -chloroacetophenone, dissolved in 16mL of isopropanol, 6mg of coenzyme NADPH was added, 4mL of water was added, the pH was adjusted to 6.5 with 0.1M sodium hydroxide solution, and finally 1g of ketoreductase enzyme powder (mutant Mut 1) was added. Stirring the mixture in a water bath at 37 ℃ for reaction, and controlling the pH value to be between 6.0 and 7.5 in the reaction process by using 0.1M sodium hydroxide solution. After the reaction was completed for 24 hours, the reaction mixture was dissolved in 2-fold acetonitrile and analyzed by HPLC, and the conversion was 95%. The target product is obtained after rotary evaporation at 40 ℃, and the ee value is 99%.
Example 6 catalytic reaction of ketoreductase mutant Mut2
To a 50mL reaction flask was added 4g of 3, 4-dihydroxy-2' -chloroacetophenone, dissolved in 16mL of isopropanol, 6mg of coenzyme NADPH was added, 4mL of water was added, the pH was adjusted to 6.5 with 0.1M sodium hydroxide solution, and finally 1g of cells (ketoreductase-containing mutant Mut 2) were added. Stirring the mixture in a water bath at 37 ℃ for reaction, and controlling the pH value to be between 6.0 and 7.5 in the reaction process by using 0.1M sodium hydroxide solution. After the reaction was completed for 24 hours, the reaction mixture was dissolved in 2-fold acetonitrile and analyzed by HPLC, whereby the conversion was 99%. The target product is obtained after rotary evaporation at 40 ℃, and the ee value is 99%.
Sequence listing
<110> Shang Ke biomedical (Shanghai) Co., ltd
<120> a ketoreductase mutant for preparing (S) -3, 4-dihydroxy-2' -chlorophenethanol
<160> 6
<170> SIPOSequenceListing 1.0
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<213> Lactobacillus kefiri
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Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
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Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
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Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
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Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
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Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
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Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
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Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
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Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
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Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
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Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
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Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
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Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
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caacacgatg cttctgatga agccggctgg actaagttgt ttgatacgac tgaagaagca 240
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gataccacaa ctgaagaatg gcgcaagctg ctctcagtta acttggatgg tgtcttcttc 360
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ggggcagaag aaatgatgtc acagcggacc aagacaccaa tgggtcatat cggtgaacct 660
aacgatatcg cttggatctg tgtttacctg gcatctgacg aatctaaatt tgccactggt 720
gcagaattcg ttgtcgatgg tggatacact gctcaataa 759
<210> 3
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<213> Artificial Sequence (Artificial Sequence)
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Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Ala Gly Thr
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Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
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Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
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Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
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Ser Asp Glu Ala Gly Trp Thr Phe Leu Phe Asp Thr Thr Glu Glu Ala
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Val Asn Leu Ile Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
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<213> Artificial Sequence (Artificial Sequence)
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Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Ala Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
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Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
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Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
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Ser Asp Glu Ala Gly Trp Thr Phe Leu Phe Asp Thr Thr Glu Glu Ala
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Val Asn Leu Ile Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
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Glu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
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Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
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ttggcaatcg ctgataagtt tgttgaagaa ggcgcaaagg ttgttattac cggccgtcac 120
gctgatgtag gtgaaaaagc tgccaaatca atcggcggca cagacgttat ccgttttgtc 180
caacacgatg cttctgatga agccggctgg actttcttgt ttgatacgac tgaagaagca 240
tttggcccag ttaccacggt tgtcaacaat gccggaatta cggtcagcaa gagtgttgaa 300
gataccacaa ctgaagaatg gcgcaagctg ctctcagtta acttgattgg tgtcttcttc 360
ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420
atgtcatcta tcgaaggtct tgttggtgat ccaactctgg gtgcatacaa cgcttcaaaa 480
ggtgctgtca gaattatgtc taaatcagct gccttggatt gcgctttgaa ggactacgat 540
gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcatgaa 600
gggctagaag aaatgatgtc acagcggacc catatcggtg aacctaacga tatcgcttgg 660
atctgtgttt acctggcatc tgacgaatct aaacctgcca ctggtgcaga attcgttgtc 720
gatggtggat acactgctca ataa 744
Claims (4)
1. A ketoreductase mutant for preparing (S) -3, 4-dihydroxy-2' -chlorophenethanol, which is characterized in that the amino acid sequence of the ketoreductase mutant is shown in SEQ ID NO.3 and SEQ ID NO. 5.
2. The ketoreductase mutant of claim 1, in which the ketoreductase mutant has the gene nucleotide sequence shown in SEQ ID No.4 and 6.
3. The ketoreductase mutant of claim 1, wherein the ketoreductase mutant is expressed in BL21 (DE 3), PLysS or Origami B (DE 3).
4. The ketoreductase mutant of claim 1, which can convert 3, 4-dihydroxy-2 '-chloroacetophenone to (S) -3, 4-dihydroxy-2' -chlorophenethanol.
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|---|---|---|---|
| CN202110600537.1A CN115478057A (en) | 2021-05-31 | 2021-05-31 | Ketoreductase mutant for preparing (S) -3, 4-dihydroxy-2' -chlorophenethanol |
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| CN115478057A true CN115478057A (en) | 2022-12-16 |
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| CN202110600537.1A Pending CN115478057A (en) | 2021-05-31 | 2021-05-31 | Ketoreductase mutant for preparing (S) -3, 4-dihydroxy-2' -chlorophenethanol |
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- 2021-05-31 CN CN202110600537.1A patent/CN115478057A/en active Pending
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