CN115927409A - Halogen alcohol dehalogenase mutant with improved stereoselectivity and activity and application thereof - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a halohydrin dehalogenase mutant with improved stereoselectivity and activity and application thereof, wherein the halohydrin dehalogenase mutant comprises mutants M1 (I104H) and M2 (I104F), the nucleotide sequence of M1 is shown as SEQ ID NO.1, and the amino acid sequence of M1 is shown as SEQ ID NO. 2; the M2 nucleotide sequence is shown as SEQ ID NO.3, and the M2 amino acid sequence is shown as SEQ ID NO. 4. The mutant is obtained by a fixed-point (iterative) saturation mutation method on the basis of HheG recombinant Escherichia coli. Compared with the original HheG, the mutants M1 and M2 catalyze the production of S-4-phenyl oxazolidinone from styrene oxide by 40.6 percent, and the yields of S-4-phenyl oxazolidinone by the styrene oxide are respectively increased to 47.7 percent and 42.4 percent, and the ee values are respectively increased to 97.5 percent and 98.3 percent from 55.6 percent. The mutant can be used for synthesizing chiral 4-substituted oxazolidinone.
Description
Technical Field
The invention relates to the technical field of bioengineering, and particularly relates to a halohydrin dehalogenase mutant with improved stereoselectivity and activity and application thereof.
Background
Chiral oxazolidinone is a five-membered heterocyclic compound containing oxygen and nitrogen, and the molecular skeleton of the compound is widely applied in the fields of chemistry and medicine. In 1981, evans et al first reported that chiral 4-substituted and 4,5-disubstituted-2-oxazolidinones can be used as chiral auxiliary agents; thereafter, a series of chiral auxiliaries containing an oxazolidinone structure were reported in succession. Meanwhile, chiral oxazolidinone as an important structural unit is widely concerned for synthesizing an antibacterial drug with a brand new structure. Chiral oxazolidinones are found in bioactive molecules and natural products, in addition to their use as antibacterial agents. In view of the importance of chiral oxazolidinones in the fields of chemistry and medicine, there is a continuing effort to study the synthesis of chiral oxazolidinones and their derivatives. At present, the synthesis methods of chiral oxazolidinone and derivatives thereof mainly comprise chemical methods and biological methods. The chemical process consisting essentially of CO 2 The involved asymmetric cyclization, kinetic resolution, epoxide ring opening, asymmetric ammoxidation of olefin, asymmetric hydrogenation of unsaturated heterocycle and the like; the biological method mainly comprises the steps of catalyzing epoxide ring opening by halohydrin dehalogenase, catalyzing C-H bond amination of azidoformate by P450 enzyme, catalyzing amino alcohol kinetic resolution by lipase, and the like. Although the chiral oxazolidinone compound synthesized by a biological method has certain advantages, the method still has some defects: (1) the highest theoretical yield of chemical resolution is only 50%; (2) In the dynamic kinetic resolution of epoxides, the substrate requires an epoxide substrate with a halogen atom; (3) Although novel regioselective ring opening of epoxides has been discoveredForm HheG of chiral 4-substituted oxazolidinone, but have poor stereoselectivity. In order to solve the existing problems, the applicant takes original halohydrin dehalogenase (HheG) as original enzyme and obtains excellent mutants M1 and M2 by a method of site-directed (iterative) saturation mutation molecular modification, thereby solving the problems of low reaction activity, low stereoselectivity and the like at present.
Disclosure of Invention
The invention aims to provide a halohydrin dehalogenase mutant with improved stereoselectivity and activity, and a chiral 4-phenyl oxazolidinone compound is prepared.
The wild type gene of the mutant gene of the halohydrin dehalogenase is derived from pET-28b (+) of an expression vector of HheG recombinant Escherichia coli (Ilumato bacillus coccus YM16-304, NCBI ID.
A mutant of halohydrin dehalogenase, which is characterized in that the amino acid sequence is shown in SEQ ID NO.2 and SEQ ID NO. 4.
The mutant achieves substantially complete resolution of 10mM ethylene oxide.
The invention also claims a vector and an engineering bacterium for expressing the mutant, and application of the mutant in the aspects of food, environment, pharmacy and the like.
The invention has the beneficial effects that:
according to the invention, hheG is used as a template, and site-directed (iterative) saturation mutation is carried out on a site I104, and the result shows that most of strains mainly generate S-4-phenyloxazolidinone, the activity and the stereoselectivity are good, the mutants M1 and M2 are similar in activity and selectivity, compared with WT, the yield of S-4-phenyloxazolidinone is respectively improved from 40.6% to 47.7% and 42.4%, and the ee value is respectively improved from 55.6% to 97.5% and 98.3%.
Drawings
FIG. 1 scheme of catalytic Process for mutants M1 and M2;
FIG. 2 liquid phase diagram of catalytic reaction of mutants M1 and M2
FIG. 3 scheme of the M1 gram scale reaction of the mutants;
FIG. 4 (S) -4-phenyloxazolidinone standard curve plotting;
Detailed Description
LB medium (g/L): tryptone 10, yeast powder 5 and NaCl 10.
TB medium (g/L): tryptone 12, yeast powder 24 and glycerol 5.
EXAMPLE 1 acquisition of a mutant Gene of a halohydrin dehalogenase
In the present study, a site-directed (iterative) saturation mutagenesis method was used to perform protein engineering modification of the halohydrin dehalogenase, and the mutations were introduced as detailed in table 1 below.
TABLE 1 site I104 saturation mutation primer design
The 50 μ L PCR reaction system was: primer
0.5 μ L of HS, 0.2 μ L of each primer, 2.0 μ L of template, 10 μ L of 5 XPS buffer, 4 μ L of dNTP mix, and 50 μ L of double distilled water. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min, denaturation at 98 ℃ for 30s, annealing at Tm-5 ℃ for 5s, performing 30 cycles, continuously extending at 72 ℃ for 10min, cooling to 4 ℃, and finishing the reaction. After the PCR was completed, dpnI buffer (5.2. Mu.L) and DpnI enzyme (1.8. Mu.L) were added to the PCR tube, mixed well, and cultured in a 37 ℃ incubator for 3-5 hours. And purifying, recovering and converting the PCR product obtained by digestion according to the steps of the gel recovery kit. Transformants were sequenced by Shanghai worker.
Example 2 culture expression of halohydrin dehalogenase recombinant E.coli
Picking single colony of halohydrin dehalogenase or sucking 2% of glycerol strain liquid in volume ratio, inoculating the single colony or the glycerol strain liquid into a test tube containing 5mL of TB liquid culture medium, adding Kan to the final concentration of 50 mu g/mL, and culturing for 8-10h in a shaking table at 37 ℃ and 250 rpm. Taking the first-level bacterial liquid in the test tube according to the volume ratio of 2 percent, adding the first-level bacterial liquid into a 250mL shake flask containing 50mL TB liquid culture medium, adding Kan to the final concentration of 50 mu g/mL, and shaking the mixture in a shaking table at 37 ℃ and 250rpmCultured to OD 600 Is 0.6-0.8. The inducer IPTG was added to a final concentration of 0.2mM and induced at 25 ℃ for 12h in a shaker at 250 rpm.
EXAMPLE 3 mutant catalytic reaction System
The strains were collected according to example 2, and the cells were suspended by shaking with the addition of a corresponding volume of PB buffer (pH =7.5, 50mM) to give a cell concentration of 10g cdw/L, and 5mL of the suspension was put into a 50mL centrifuge tube, and 30. Mu.L of NaOCN (final concentration: 6 mM) and 50. Mu.L of ethylene oxide (final concentration: 10 mM) were added and reacted for 6 hours in a 30 ℃ 250rpm constant temperature shaker (see FIG. 1). After the reaction, 5mL of ethyl acetate (containing 3mM 3-phenylpropanenitrile internal standard) was aspirated into the centrifuge tube, followed by shaking for sufficient extraction, standing for stratification or centrifugation (conditions: 4 ℃, 9000rpm, 3 min), and then the supernatant was removed by anhydrous Na 2 SO 4 Drying, filtering with 0.22 μm filter membrane, placing into liquid vial, and analyzing by normal phase-high performance liquid chromatograph and gas chromatograph. As shown in Table 2, the mutants M1 and M2 are similar in both activity and selectivity, and compared with WT, the yield of S-4-phenyloxazolidinone is increased from 40.6% to 47.7% and 42.4%, respectively, and the ee value is increased from 55.6% to 97.5% and 98.3%, respectively, to substantially complete resolution of 10mM substrate (see FIG. 2).
TABLE 2 site I104 saturation mutation screening results
Example 4 gram order reaction
To prove the utility of the method, according to example 2, the strain was cultured and collected, PB buffer suspension (M1 cell concentration 10g cdw/L) was calculated and added to a corresponding volume of pH 7.5, 272mL of the cell suspension was put in a 1000mL three-necked flask, 1g (30 mM) of racemic ethylene oxide and 18mM NaOCN were added, and the mixture was put in a 30 ℃ water bath and reacted for 3 hours with stirring (see FIG. 3). After the reaction was completed, the reaction mixture was extracted with ethyl acetate(3X 300 mL) and the organic phase was separated by centrifugation and washed with anhydrous Na 2 SO 4 Drying and concentration under reduced pressure, and purification by column chromatography to give enantiomerically pure 4-substituted oxazolidinone (petroleum ether: ethyl acetate =3:1, dichloromethane: ethyl acetate = 20. And performing gram-level reaction on the template substrate by using the mutant M1 with better stereoselectivity as a catalyst. 1g of styrene oxide is put into 272mL of aqueous phase reaction system, and the mixture is separated and purified by column chromatography to obtain 534.4mg of (S) -4-phenyl oxazolidinone, wherein the resolution yield is 38.9 percent, and the ee is 94.6 percent.
EXAMPLE 5 analysis of the product
(1) The 4-phenyl oxazolidinone product is analyzed by high performance liquid chromatography, and the method comprises the following steps: chiralcel AY-H, hexane/i-PrOH =90/10, flow rate =1.0mL/min,214nm,60min; the epoxy substrate is analyzed in gas phase by the following method: BGB-175,90 ℃, flow rate =2.0mL/min,28min.
(2) Calculation of ee value: the ee value of the product was calculated from the peak area based on the analysis result of high performance liquid chromatography. The calculation formula is as follows:
[ R ] represents the peak area of the R-configuration product, and [ S ] represents the peak area of the S-configuration product
EXAMPLE 6 establishment of the product 4-phenyl oxazolidinone Standard Curve
Weighing 0.0666g (98%) of solid white powder 4-phenyl oxazolidinone, placing the solid white powder into a dry and clean 10mL volumetric flask, dissolving the solid white powder in ethyl acetate (an internal standard solution containing 3mM 3-phenyl propionitrile), fixing the volume to a scale mark, and shaking up to obtain mother liquor A with the concentration of 40 mM. Respectively sucking the mother liquor A and the internal standard solution, and uniformly mixing to obtain solutions with the concentrations of 16mM, 12mM, 10mM, 8mM, 6mM, 5mM, 4mM, 2mM, 1mM and 0 mM. Respectively extracting 1mL of the solution with 1mL of PB buffer solution with pH of 7.5, standing for layering, collecting the upper layer of ethyl acetate, and extracting with anhydrous Na 2 SO 4 Drying and filtering with 0.22 μm organic filter membrane, loading the filtrate into a clean sample bottle, and analyzing with normal phase-high performance liquid chromatograph. Taking the peak area (Ai) of the product and the internal standard solution of 3-benzeneThe ratio of the peak area (As) of the propionitrile is ordinate, the concentration of the product is abscissa, and the standard curve of the 4-phenyl oxazolidinone is obtained As follows: y =0.223x +0.0926 2 =0.9993 (fig. 4).
The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A mutant halohydrin dehalogenase gene having improved stereoselectivity and activity, comprising: the nucleotide sequence of the mutant gene is shown in SEQ ID NO.1 and SEQ ID NO.3.
2. A mutant halohydrin dehalogenase having improved stereoselectivity and activity, comprising: the amino acid sequence of the mutant is shown as SEQ ID NO.2 and SEQ ID NO. 4.
3. A recombinant vector comprising the mutant gene of the halohydrin dehalogenase having improved stereoselectivity and activity of claim 1.
4. A genetically engineered bacterium producing the halohydrin dehalogenase mutant having improved stereoselectivity and activity of claim 2, wherein: the genetically engineered bacterial species comprising a mutant gene of a halohydrin dehalogenase according to claim 1 or a recombinant vector according to claim 3.
5. The genetically engineered bacterium of claim 4, wherein: the host cell of the genetic engineering bacteria is Escherichia coli BL21 (DE 3).
6. A mutant halohydrin dehalogenase having improved stereoselectivity and activity in accordance with claim 2 wherein: the mutant is obtained by mutating isoleucine at position 104 into histidine M1 (I104H) and phenylalanine M2 (I104F).
7. The use of a mutant halohydrin dehalogenase having improved stereoselectivity and activity in accordance with claim 2 in food, environmental, pharmaceutical applications, etc.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745556A (en) * | 2015-03-05 | 2015-07-01 | 浙江工业大学 | Recombinant halohydrin dehalogenase, and mutant and engineering strain and applications thereof |
| CN109593069A (en) * | 2019-01-24 | 2019-04-09 | 遵义医学院 | A kind of method of biocatalysis synthesis 4- substituted oxazolidine ketone compound |
| CN111647588A (en) * | 2020-05-22 | 2020-09-11 | 浙江工业大学 | Halogen alcohol dehalogenase mutant and application thereof in synthesis of chiral epichlorohydrin |
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Patent Citations (3)
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| CN104745556A (en) * | 2015-03-05 | 2015-07-01 | 浙江工业大学 | Recombinant halohydrin dehalogenase, and mutant and engineering strain and applications thereof |
| CN109593069A (en) * | 2019-01-24 | 2019-04-09 | 遵义医学院 | A kind of method of biocatalysis synthesis 4- substituted oxazolidine ketone compound |
| CN111647588A (en) * | 2020-05-22 | 2020-09-11 | 浙江工业大学 | Halogen alcohol dehalogenase mutant and application thereof in synthesis of chiral epichlorohydrin |
Non-Patent Citations (1)
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| "SDR family oxidoreductase[llumatobacter coccineus] wp_015443096.1", pages 1, Retrieved from the Internet <URL:httpswww.ncbi.nlm.nih.govprotein505255994sat=5&satkey=689171306> * |
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