A carbonyl reductase lsCR mutant and application thereof in catalyzing synthesis of chiral alcohol

Abstract

The application discloses a carbonyl reductase LsCR mutant and application of the carbonyl reductase LsCR mutant in catalyzing synthesis of chiral alcohol. The carbonyl reductase LsCR mutant is obtained by carrying out unit point or multi-point site-directed mutation or site-directed saturation mutation on the 99th and 150th positions of the amino acid sequence shown in SEQ ID NO. 1. The activity of the carbonyl reductase mutant LsCR M6 is increased by 78% compared to the mutant LsCR M4 , the T m value is increased by 17.4 DEG C, and the half-life at 45 DEG C is 103.3 hours. The substrate 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) of the mutant LsCR M6 can reach 600 g / L, the reaction can be completed within 5.0 hours, and the substrate conversion rate is greater than 99%. The carbonyl reductase mutant LsCR M6 has a good industrial application prospect.

Description

(I) Technical Field

[0001] This invention relates to a carbonyl reductase LsCR mutant derived from *Lactobacillus sauerkraut* and its application in chiral biocatalytic synthesis, particularly to the application of the carbonyl reductase LsCR mutant in the synthesis of the chiral aromatic alcohol (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol, a key intermediate in the anticoagulant drug ticagrelor. (II) Background Technology

[0002] Ticagrelor is a major product among small-molecule oral anticoagulants. This drug can reversibly bind to the G protein-coupled receptor P2Y. 12 Ticagrelor inhibits ADP-mediated platelet activation and aggregation. Its antiplatelet action is rapid, significant, and exhibits minimal individual variability, making it a preferred antiplatelet treatment for acute coronary syndrome (ACS) according to domestic and international guidelines. In 2020, ticagrelor ranked 54th in global small molecule drug sales, with global sales of approximately US$1.593 billion, demonstrating significant market potential. Compared to clopidogrel, a similar anticoagulant, ticagrelor has a faster onset of action, stronger inhibition of platelet aggregation, requires no metabolic activation process, significantly reduces the incidence of cardiovascular disease death, myocardial infarction, or stroke, and can overcome clopidogrel resistance symptoms in some patients.

[0003] Ticagrelor, chemically named (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol, consists of three structural parts, the most difficult to synthesize being the chiral (1R,2S)-2-(3,4-difluorophenyl)cyclopropane-1-amine intermediate. In most synthetic routes for chiral cyclopropylamine intermediates, an asymmetric reduction reaction is required to synthesize chiral (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol; this asymmetric reduction reaction introduces a chiral center into the cyclopropylamine structure. Therefore, it is of great significance to study the chiral synthesis technique of optically pure (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol.

[0004] Traditional chemical synthesis methods, in order to achieve stereoselective reduction, introduce hazardous chiral auxiliaries and expensive chiral resolving agents, such as borane methyl sulfide, chiral reagents (S)-α,α-diphenylproline, and trimethyl borate. The addition of these substances increases the difficulty of post-processing. Compared with traditional chemical methods, biocatalysis offers advantages such as milder reaction conditions, better selectivity, simpler post-processing, and environmental friendliness. However, in chemical industrial production, enzymes, as catalysts, need to catalyze many artificially synthesized substances that are very different from natural substrates. The concentrations of substrates and products in the reaction system far exceed the amounts that enzymes can access in the natural environment, thus limiting the efficiency of biocatalysis. Thanks to the rapid development of protein engineering, many enzyme molecules have encountered problems such as low catalytic activity, poor stability, and substrate and / or product inhibition when catalyzing non-natural substrates. By molecularly modifying enzyme molecules and optimizing their various properties, biocatalysis has been widely applied in industrial production.

[0005] Based on patent application (CN115109759 A), this invention constructs a mutant library through rational design and obtains the "optimal mutant" LsCR, which exhibits significantly improved thermal stability and catalytic activity, through further screening. M4 -D150F / V99L(LsCR M6 ). (III) Summary of the Invention

[0006] The purpose of this invention is to address the poor thermal stability of the existing carbonyl reductase LsCR in the asymmetric reduction of 2-chloro-1-(3,4-difluorophenyl)ethyl ketone. This invention provides a stereoselective carbonyl reductase LsCR mutant with improved stability, and utilizes the recombinant bacteria of this LsCR mutant gene and its enzyme solution as a biocatalyst for the asymmetric reduction of carbonyl compounds to prepare chiral alcohols. When the substrate is 2-chloro-1-(3,4-difluorophenyl)ethyl ketone, the mutant LsCR… M4 -D150F / V99L(LsCR M6 It exhibits good thermal stability and has a higher specific enzyme activity than LsCR. M4 Increased by 78%, T m The value increased by 17.4 °C, and the half-life at 45 °C was 103.3 hours, providing a new biocatalyst for the asymmetric synthesis of the chiral intermediate (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol of ticagrelor.

[0007] The technical solution adopted in this invention is:

[0008] The present invention provides a carbonyl reductase LsCR mutant, which is obtained by performing single-site or multi-site site-directed mutation or site-directed saturation mutation on the 99th and 150th amino acid sequences shown in SEQ ID NO.1.

[0009] The preferred carbonyl reductase LsCR mutant of the present invention is made by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following: (1) valine at position 99 is mutated to leucine LsCR. M4 -V99L; (2) Aspartic acid at position 150 is mutated to phenylalanine; (3) Valine at position 99 is mutated to leucine, and aspartic acid at position 150 is mutated to phenylalanine (LsCR) M4 -V99L / D150F, denoted as LsCR M6 The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.5.

[0010] This invention also relates to the coding gene of the carbonyl reductase LsCR mutant, a recombinant vector containing the coding gene, and a genetically engineered bacterium containing the recombinant vector, wherein the recombinant vector uses pET-28a(+) as the expression vector, and the genetically engineered bacterium uses E. coli BL21(DE3) as the host bacterium.

[0011] This invention also provides an application of the LsCR mutant carbonyl reductase in the asymmetric reduction of carbonyl compounds to prepare chiral alcohols. The method of application is as follows: using engineered bacteria containing the LsCR mutant coding gene as a catalyst, wet bacterial cells obtained by induction culture or pure enzyme solution extracted by ultrasonic disruption of wet bacterial cells, carbonyl compounds as substrates, isopropanol as co-substrate, NADPH as coenzyme, and a pH 5-7 buffer solution (preferably pH 6, 100mM sodium citrate buffer) as the reaction medium to form a transformation system. The reaction is carried out at 25-55℃ and 600-1200 rpm. After the reaction is completed, the reaction solution is extracted and purified with ethyl acetate to obtain chiral aromatic alcohols; wherein the carbonyl compound is 2-chloro-1-(3,4-difluorophenyl)ethyl ketone, and the corresponding chiral aromatic alcohol is (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol.

[0012] Furthermore, in the transformation system, the final substrate concentration is 500-1000 g / L, preferably 600 g / L; the final volume concentration of isopropanol is 20%-80%, preferably 40% (v / v); the catalyst dosage is 1-15 g DCW / L (DCW cell dry weight) based on the total dry weight of the bacteria, preferably 1 g DCW / L; the catalyst dosage is 0.01-0.1 mg / mL based on the protein content in the pure enzyme solution, preferably 0.05 mg / mL; and the final NADPH concentration is 0.1 mM.

[0013] Further, the wet bacterial cells are prepared as follows: Engineered bacteria containing the carbonyl reductase LsCR mutant encoding gene are inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 10 h to obtain a seed culture; the seed culture is inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 1.0-5.0% (preferably 4%) and cultured at 37°C and 200 rpm for 2 h (OD). 600 =0.6~0.8), add isopropyl β-D-thiogalactoside (IPTG) to the culture medium to a final concentration of 0.15mM, incubate at 28℃ for 12h, and then centrifuge at 4℃ and 8000rpm for 10min to obtain wet bacterial cells containing carbonyl reductase LsCR mutant.

[0014] Further, the pure enzyme solution was prepared as follows: Wet cells obtained from the fermentation culture of engineered bacteria containing the carbonyl reductase LsCR gene were resuspended at 50 g / L in a pH 6, 100 mM sodium citrate buffer. The mixture was ultrasonically disrupted for 20 min on an ice-water mixture under the following conditions: power 400 W, disruption for 1 s, pause for 3 s. The disrupted mixture was collected to obtain the crude enzyme solution. The crude enzyme solution was centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant was collected. After microfiltration through a 0.45 μm microfiltration membrane, ammonium sulfate was slowly added while stirring in an ice-water bath until the ammonium sulfate saturation in the crude enzyme solution reached 50%. The solution was then allowed to stand at 4°C for 4 h. The precipitate was discarded after centrifugation at 8000 rpm and 4°C for 25 min. Ammonium sulfate was added to the supernatant until 70% ammonium sulfate saturation was achieved. The solution was centrifuged again at 8000 rpm for 25 min, and the supernatant was discarded. The precipitate was dissolved in a 20 mM, pH 7.5 potassium phosphate buffer and then processed using DEAE Sepharose Fast... The mutant protein was purified using a Flow anion exchange column (1.6 × 20 cm) as follows: ① Pre-equilibrate with buffer A (pH 7.5, 20 mM Tris-HCl buffer); ② Wash away contaminating proteins with buffer A at a flow rate of 1.0 mL / min until the conductivity stabilizes; ③ Elute the target protein with buffer B (pH 7.5, 20 mM Tris-HCl buffer containing 1.0 M sodium chloride) at a flow rate of 1 mL / min, starting collection when the conductivity line rises and stopping when it falls back to the baseline, collecting the eluent with the active target peak; Dialyze the collected eluent overnight with 20 mM sodium phosphate buffer (pH 7.5) (dialysis bag molecular weight cutoff of 30 kDa), and collect the cutoff solution, which is the pure enzyme solution of the mutant.

[0015] The carbonyl reductase LsCR and its mutant in this invention both have a full-length base sequence of 759 bp, from the first base to the 759th base, with the start codon being ATG and the stop codon being TAA.

[0016] The LsCR mutant carbonyl reductase described in this invention M6 LsCR was obtained using site-directed saturation mutagenesis. M4 The carbonyl reductase gene (SEQ ID NO.1) was mutated, and the resulting mutant plasmid was transformed into E. coli BL21(DE3) competent cells by heat shock. The obtained strain was inoculated, transformed, induced, and the cells were recovered. Optically pure (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol was prepared by catalyzing the asymmetric reduction of 2-chloro-1-(3,4-difluorophenyl)ethanol using resuspended bacterial solution. The specific method is as follows: Step 1: Activate the starting strain E. coli BL21(DE3) / pET28a(+)-lscr M4 And extract plasmid pET28a(+)-lscr M4 Stored at -20℃. The second step involved site-directed and combinatorial mutagenesis at V99 and D150 sites to construct a mutant library. Gas chromatography was used to detect the product configuration, yielding the highly stable, strictly S-selective carbonyl reductase mutant LsCR. M4 -V99L / D150F(LsCR M6 Carbonyl reductase mutant LsCR M6 Asymmetric reduction of 2-chloro-1-(3,4-difluorophenyl)ethyl ketone increased specific enzyme activity by 78% compared to LsCR-M4, T m The value increased by 17.4°C, and the half-life at 45°C was 103.3 h.

[0017] This invention relates to the inoculation, transfer, induction, and cell recovery of genetically engineered bacteria containing the LsCR mutant carbonyl reductase. The culture medium can be any medium in the art that can promote bacterial growth and produce the present invention, with LB medium being preferred: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in distilled water, and adjusted to pH 7.5. There are no particular limitations on the culture method and conditions; these can be optimized based on factors such as host type and culture method.

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following: the stability of the carbonyl reductase LsCR mutant constructed by the present invention is significantly improved, wherein the mutant LsCR M6 The enzyme activity was compared with that of the control group LsCR carbonyl reductase. M4 Increased by 78%, T mThe value increased by 17.4°C, and the half-life at 45°C was 103.3 h. When the LsCR mutant carbonyl reductase of this invention catalyzes 2-chloro-1-(3,4-difluorophenyl)ethyl ketone, the LsCR mutant in the reaction system... M6 When the concentration of the substrate 2-chloro-1-(3,4-difluorophenyl)ethyl ketone is 1 g DCW / L, and the feed concentration can reach 600 g / L, the concentration of the product (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol gradually increases over time. The reaction can be completed within 5 hours, with a substrate conversion rate greater than 99%, and the product ee P The value has consistently remained above 99.7%. (iv) Description of the attached drawings

[0019] Figure 1 This is a schematic diagram of the reaction catalyzed by carbonyl reductase LsCR to prepare (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol through the asymmetric reduction of 2-chloro-1-(3,4-difluorophenyl)ethyl ketone.

[0020] Figure 2 The image shows the gas chromatograms of the substrate 2-chloro-1-(3,4-difluorophenyl) ethyl ketone (CFPO) and the products (1R)-2-chloro-1-(3,4-difluorophenyl)ethanol ((R)-CFPL) and (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL).

[0021] Figure 3 It is a carbonyl reductase mutant LsCR M6 SDS-PAGE electrophoresis image of purified enzyme. Lane 1: Control LsCR M4 Purified enzyme; Lane 2: LsCR M6 Purified enzyme; M: standard protein molecule.

[0022] Figure 4 For LsCR M6 Half-life at 45°C.

[0023] Figure 5 It is LsCR M6 T m Value (defined as the temperature at which the unfolded protein fraction f equals 0.5).

[0024] Figure 6 It is LsCR M6 T 50 30 Value (defined as the temperature at which half of the enzyme activity is lost after 30 minutes of incubation).

[0025] Figure 7 It is LsCR M6 Optimize the optimal reaction pH.

[0026] Figure 8 It utilizes the carbonyl reductase mutant LsCR M6 Time-process graph of the asymmetric reduction of 2-chloro-1-(3,4-difluorophenyl)ethyl ketone by coupling with isopropanol to prepare (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol.

[0027] Figure 9 It is LsCR M6 Circular dichroism spectra at different temperatures.

[0028] Figure 10 It is LsCR M6 Tolerance to different organic solvents.

[0029] Figure 11 Different volumes of isopropanol affect LsCR M6 The effect of catalytic reactions.

[0030] Figure 12 It is LsCR M6 Optimization of the optimal reaction temperature. (V) Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0032] Example 1: Construction of a stereoselective mutant library of carbonyl reductase LsCR

[0033] 1. Starting strain

[0034] The engineered strain E. coli BL21(DE3) / pET28a(+)-lscr, constructed by mutating the carbonyl reductase LsCR (amino acid sequence shown in SEQ ID NO.2, encoding gene nucleotide sequence shown in SEQ ID NO.3) derived from *Levilactobacillus suantsaii*, as described in patent application CN115109759 A, is described in this paper. M4 The originating strain is denoted as strain LsCR. M4 The plasmid pET-28a(+)-lscr-M4 was activated and extracted, containing the carbonyl reductase LsCR. M4 The amino acid sequence is shown in SEQ ID NO.1. The preparation of the carbonyl reductase LsCR mutant library was achieved through site-directed saturation mutagenesis and combinatorial mutagenesis.

[0035] SEQ ID NO.1

[0036] MSHRLDGKVAIVTGGTLGIGLAIADKFVAEGAKVMITGRHADVGEKAAKSIGGPDVIQFFHHDATDEQGWVDLFDATEKAFGPVTTVVNNAGMAVNKSVEDTTTEEWHRQLAVNLDGVFFGTRLGIQRMKNKNLGASIINMSSIAGLVGDPNLGAYNATKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ。

[0037] SEQ ID NO.2

[0038] MSHRLDGKVAIVTGGTLGIGLAIADKFVAEGAKVMITGRHADVGEKAAKSIGGPDVIQFFHHDATDEQGWVDLFDATEKAFGPVTTVVNNAGMAVNKSVENTTTEEWHRQLAVNLDAVFFGTRLGIQRMKNKNLGASIINMSSIEGFVGDPNLGAYNATKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ。

[0039] SEQ ID NO.3

[0040] ATGAGCCATCGCCTGGATGGTAAAGTTGCCATTGTTACGGGTGGTACCCTGGGTATTGGTCTGGCCATTGCAGATAAATTTGTGGCAGAAGGTGCCAAAGTGATGATTACCGGTCGCCATGCAGATGTTGGTGAAAAAGCAGCAAAAAGCATTGGTGGTCCGGATGTTATTCAGTTTTTTCATCATGATGCAACAGATGAACAGGGTTGGGTTGATCTGTTTGATGCAACAGAAAAAGCATTTGGTCCGGTTACAACCGTTGTGAATAATGCAGGTATGGCAGTTAACAAAAGCGTCGAAAATACAACCACCGAAGAATGGCATCGTCAGCTGGCAGTGAATCTGGATGCAGTGTTTTTTGGTACACGTCTGGGCATTCAGCGTATGAAAAATAAAAACCTGGGCGCAAGCATTATTAATATGAGCAGCATTGAAGGTTTTGTTGGTGATCCGAATCTGGGTGCATATAATGCCACTAAAGGCGCAGTTCGTATTATGTCAAAAAGCGCAGCACTGGATTGTGCACTGAAAGATTATGATGTTCGCGTGAATACAGTTCATCCGGGTTATATTAAAACACCTCTGGTTGATGATCTGCCGGGTGCGGAAGAAGCCATGAGCCAGCGTACCAAAACCCCGATGGGTCATATTGGTGAACCGAATGATATCGCATATATTTGTGTTTATCTGGCAAGCAATGAAAGTAAATTTGCAACCGGTAGTGAATTTGTAGTTGATGGTGGTTATACCGCACAGTAA。

[0041] 2. Site-directed mutation

[0042] (1) Construction of mutation library

[0043] Using the vector pET28a(+)-lscr in the strain LsCR M4 M4 as a template, the polymerase chain reaction (PCR) is carried out using the primers in Table 1 according to the following procedure.

[0044] ​PCR reaction system (25 μL): 1 μL upstream primer (100 μM), 1 μL downstream primer (100 μM), 12.5 μL 2×Phanta buffer, 0.5 μL dNTP mixture (10 mM each), 1 μL plasmid template, 0.5 μL DNA polymerase and 8.5 μL ultrapure water.

[0045] The PCR amplification procedure is as follows:

[0046] Step 1: 98℃ for 3 minutes;

[0047] Step 2: 98℃ for 15 seconds;

[0048] Step 3: 60℃ for 15 seconds;

[0049] Step 4: 72℃2000bp / 1min (skip to step 2, 30cycles);

[0050] Step 5: 72℃ for 10 minutes;

[0051] Step 6: 16℃ for 15 minutes;

[0052] Step 7: Store at 4℃ or take samples immediately for agarose gel electrophoresis.

[0053] Table 1 Primer Design for Carbonyl Reductase Mutation

[0054]

[0055]

[0056] The recombinant plasmid purified using the Clean-up purification kit (Axygen Scientific, Int, catalog number AP-PCR-250) was transferred to E. coli BL21(DE3) competent cells, and the clones were seeded into 10 mL LB agar plates and cultured at 37°C for 12 h.

[0057] (2) Screening of mutants

[0058] Positive clones were randomly selected from the plates and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C for 12 h to obtain a seed culture. 2 mL of the seed culture was sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing. After confirming the sequencing results were correct, the cells were inoculated at a rate of 1% (v / v) into 50 mL of LB liquid medium containing 50 μg / mL kanamycin. The cells were incubated at 37°C with shaking at 200 rpm for 2 h. Then, a final concentration of 0.15 mM IPTG was added, and the cells were incubated at 28°C for 12 h. The resulting cells were centrifuged at 8000 rpm at 4°C for 10 min to obtain the corresponding wet bacterial cells.

[0059] The wet bacterial cells were screened for dominant mutants under the following conditions: 50 g / L substrate 2-chloro-1-(3,4-difluorophenyl) ethyl ketone, 40% (v / v) isopropanol, 100 g / L wet bacterial cells, and the volume was brought up to 1 mL with 100 mM citrate buffer at pH 6. The reaction was carried out at 45 °C, pH 6, and 1000 rpm for 3 min.

[0060] Add 1 mL of ethyl acetate to each reaction system, shake thoroughly, and centrifuge at 12000 rpm for 2 min. After removing residual moisture by adding a small amount of anhydrous sodium sulfate to the organic phase, perform gas chromatography to determine the configuration and content of the product. Using relative enzyme activity as the screening index, the results are shown in Table 2. The obtained dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored at -80℃. The mutant strain E. coli BL21(DE3) / pET-28a(+)-lscr was obtained. M4 -V99L, E.coli BL21(DE3) / pET-28a(+)-lscr M4 -D150F.

[0061] The formula for calculating residual enzyme activity is:

[0062]

[0063] Relative enzyme activity is measured by carbonyl reductase LsCR M4 The enzyme activity was obtained at 100%.

[0064] The gas chromatography detection conditions were as follows: BGB-174 column, detector 250℃, injection port 250℃, column temperature 160℃, carrier gas nitrogen, and split ratio 30:1 (v / v).

[0065] Table 2. Relative enzyme activities of LsCR mutants

[0066]

[0067] 3. Combinatorial mutation

[0068] Use pET28a(+)-lscr M4 Using -D150F as a template, polymerase chain reaction (PCR) was performed using the following upstream and downstream primers. The recombinant plasmid purified using a Clean-up purification kit (Axygen Scientific, Int, catalog number AP-PCR-250) was transferred to E. coli BL21(DE3) competent cells, and clones were inoculated into 10 mL LB agar plates and cultured at 37°C for 12 h. Screening was performed using the method in step 2, and the results are shown in Table 3. The mutant strain E. coli BL21(DE3) / pET-28a(+)-lscr was obtained. M4 -V99L / D150F, denoted as strain LsCR M6 The highly thermally stable carbonyl reductase mutant LsCR M4 -V99L / D150F (amino acid sequence shown in SEQ ID NO.4, encoding gene nucleotide sequence shown in SEQ ID NO.5), gas chromatogram shown in [image missing]. Figure 2 .

[0069] Upstream primer: AGCctgGAAGATACAACCACCGAAGAATGGCA;

[0070] Downstream primer: GTTGTATCTTCcagGCTTTTGTTAACTGCCATACCTGC.

[0071] Table 3. Relative enzyme activities of LsCR mutants

[0072]

[0073] SEQ ID NO.4

[0074] MSHRLDGKVAIVTGGTLGIGLAIADKFVAEGAKVMITGRHADVGEKAAKSIGGPDVIQFFHHDATDEQGWVDLFDATEKAFGPVTTVVNNAGMAVNKSLEDTTTEEWHRQLAVNLDGVFFGTRLGI QRMKNKNLGASIINMSSIAGLVGFPNLGAYNATKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ.

[0075] SEQ ID NO.5

[0076] .

[0077] Example 2: Induced expression of carbonyl reductase LsCR and mutant wet cells

[0078] The starting strain LsCR was respectively M4 The strain LsCR constructed using the method in Example 2 M6The seed culture was obtained by inoculating 10 mL of LB liquid medium containing 50 μg / mL kanamycin and culturing at 37°C for 12 h. 2 mL of the seed culture was then inoculated at a 4% (v / v) inoculation rate into 50 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm for 2 h. Then, 0.15 mM IPTG inducer was added, and the culture was incubated at 28°C for 12 h. The resulting cells were centrifuged at 8000 rpm at 4°C for 10 min to obtain the corresponding wet cells.

[0079] Example 3: Purification and Enzyme Activity Determination of the LsCR Mutant Carbonyl Reductase

[0080] (1) Purification of carbonyl reductase

[0081] Starting strain LsCR M4 The carbonyl reductase mutant strain LsCR obtained in Example 1 M6 The wet bacterial cells obtained after induction of expression according to the method described in Example 2 were resuspended in 20mM sodium phosphate buffer (pH 7.5) at a final concentration of 100g / L. The mixture was then sonicated for 6 min on an ice-water bath under the following sonication conditions: 400W power, 1s sonication followed by a 1s pause, to obtain a crude enzyme solution. The solution was centrifuged at 8000 rpm for 10 min at 4°C, and 20 mL of the supernatant was collected. After microfiltration through a 0.45 μm microfiltration membrane, ammonium sulfate was slowly added while stirring in an ice-water bath until the ammonium sulfate saturation in the crude enzyme solution reached 50%. The solution was then incubated at 4°C for 4 h. The precipitate was discarded after centrifugation at 8000 rpm for 25 min at 4°C. Ammonium sulfate was added to the supernatant until 70% ammonium sulfate saturation was achieved. The solution was centrifuged again at 8000 rpm for 25 min, the supernatant was discarded, and the precipitate was dissolved in 5 mL of 20mM potassium phosphate buffer (pH 7.5). The protein was then purified using a DEAE Sepharose Fast Flow anion exchange column.

[0082] The mutant protein was purified using a DEAE Sepharose Fast Flow anion exchange column. The specific procedures were as follows: ① Pre-equilibrate with buffer A (20 mM Tris-HCl buffer, pH 7.5). ② Wash away unbound impurities with buffer A at a flow rate of 1.0 mL / min until conductivity stabilizes. ③ Elute the target protein with buffer B (pH 7.5, 20 mM Tris-HCl buffer containing 1 M sodium chloride) for 2 column volumes at a flow rate of 1.0 mL / min. Collect the eluent from the peak showing activity until it reaches baseline. Dialyze the collected eluent to 20 mM phosphate buffer (pH 7.5) overnight (dialysis bag molecular weight cutoff 30 kDa). Collect the cutoff solution, which is the purified carbonyl reductase enzyme solution. 5 mL of LsCR was obtained.M4 Pure enzyme solution, protein concentration 1.2 g / L; 5 mL LsCR M6 The purified enzyme solution had a protein concentration of 1.4 g / L. All purification steps were performed at 4°C. Protein concentration was determined using a quinoline carboxylic acid protein assay kit (Nanjing Kaiji Biotechnology Development Co., Ltd., Nanjing).

[0083] Protein size was identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the electrophoresis results are shown below. Figure 3 The expression level of the target enzyme in the mutant strain did not change significantly compared to that in the original strain. Therefore, the increased enzyme activity in the mutant strain was not due to an increase in enzyme expression, but rather to an increase in the enzyme's specific activity.

[0084] (2) Determination of the specific enzyme activity of carbonyl reductase

[0085] Enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 micromole of (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol per minute at 45°C and pH 6. Specific enzyme activity is defined as the number of activity units per milligram of enzyme protein, U / mg.

[0086] Enzyme activity assay standard conditions: 10 mM substrate 2-chloro-1-(3,4-difluorophenyl)ethyl ketone, 1 mM NADPH, protein concentration 0.05 μg / μL pure enzyme solution, and brought to 500 μL with 100 mM citrate buffer at pH 6. The reaction was carried out at 45℃, pH 6, and 1000 rpm for 3 min. Gas chromatography detection and analysis were performed using the method in Example 1. The results are shown in Table 4.

[0087] Table 4. Enzyme activity of LsCR mutants

[0088]

[0089] Example 4: Determination of the stability of the carbonyl reductase LsCR mutant

[0090] (1) Determination of the half-life of carbonyl reductase

[0091] The LsCR prepared in Example 3 M6 The pure enzyme solution was diluted to 1 μg / μL with 100mM, pH 7.5 potassium phosphate buffer, dispensed into EP tubes, and incubated in a 45℃ water bath. Samples were taken every 10 hours. After the temperature returned to room temperature, the remaining enzyme activity after incubation at different temperatures was determined according to the enzyme activity detection method in Example 1.

[0092] Half-life (t) 1 / 2 Calculated based on the thermal deactivation equation:

[0093] At =A0exp(-k d t) Formula (2)

[0094] In the thermal deactivation equation (2), k d For the non-dynamic constants, A0 and A t These represent the initial enzyme activity and the enzyme activity at time t, respectively. Figure 4 Take ln(A t Plot a graph with A0 as the ordinate and incubation time t as the abscissa, and fit the experimental data to obtain k. d Using the formula ln2 / k d The half-life t is calculated. 1 / 2 .

[0095] (2) Carbonyl reductase T m Measurement

[0096] The LsCR mutant was analyzed by circular dichroism spectroscopy (CD): The LsCR obtained in Example 3 was analyzed by circular dichroism spectroscopy. M6 The pure enzyme solution was diluted with deionized water to a protein concentration of 0.1 μg / μL. 200 μL was added to a 5 mm quartz cuvette, and the cuvette was agitated until no air bubbles were present. The cuvette was then placed in a heating bath with a temperature range of 25–95 °C and a temperature gradient of 5 °C. The UV wavelength range was set to 160–280 nm. Based on the formula, a nonlinear curve was fitted to the α value at a wavelength of 222 nm. Figure 5 .

[0097] T m The calculation formula is

[0098]

[0099] In formula (3), θ t For the ellipticity at any temperature, θ F The ellipticity corresponding to the fully folded form, θ U The corresponding ellipticity in the non-folded form.

[0100] (3) Carbonyl reductase T 50 30 Determination of value

[0101] The LsCR prepared in Example 3 M6 The pure enzyme solution was diluted to 1 μg / μL with 100 mM phosphate buffer (pH 7.5), aliquoted into EP tubes, and incubated in a water bath at 45–70 °C for 30 min. After incubation on ice, the remaining enzyme activity was measured at different temperatures according to the enzyme activity detection method described in Example 1. Figure 6 As shown, the T value of this enzyme was calculated through curve fitting. 50 30The temperature was 64.6℃.

[0102] Example 5: Carbonyl reductase mutant LsCR M6 The optimal catalytic pH for pure enzyme solution

[0103] LsCR prepared according to the method in Example 3 with a protein concentration of 0.05 mg / mL was added to 10 mL of PBS buffer (pH 3.0-6.0 citrate buffer; pH 6.0-8.0 phosphate buffer; pH 8.0-10.0 Tris-HCl buffer) at different pH values ​​(3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.0-8.0) and 100 mM. M6 Pure enzyme solution, 10 mM substrate 2-chloro-1-(3,4-difluorophenyl)ethyl ketone (CFPO), 2 mM NADPH, reacted at 45°C and 800 rpm for 5 min. Enzyme activity at different pH values ​​was detected according to the method in Example 3 to optimize LsCR. M6 The pH value of the catalytic reaction is shown in the results. Figure 7 The pH optimization range was 3.0-10.0, and the optimal catalytic pH value was determined to be 5.5 through optimization.

[0104] Example 6: Carbonyl reductase mutant LsCR M6 Asymmetric synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol

[0105] According to the description in Example 2, the carbonyl reductase mutant LsCR was obtained by fermentation. M6 Wet bacteria. First, LsCR M6 The wet bacterial cells were resuspended in pH 6, 100 mM citrate buffer. LsCR in the reaction system... M6 The addition amount of wet bacterial cells was 1 g DCW / L based on a bacterial cell dry weight ratio of 4:1 (w / w). The dosage of the substrate 2-chloro-1-(3,4-difluorophenyl)ethyl ketone (CFPO) was 600 g / L, and NADP... + The conversion system consisted of 50 mL of 0.1 mM citrate buffer (100 mM, pH 6) as the reaction medium. 40% (v / v) isopropanol was added to the system. The reaction was carried out at 45°C and 800 rpm for 510 min. The reaction results are as follows: Figure 8 As shown. Mutant LsCR M6 The substrate of 600 g / L can be completely converted to the product (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) within 270 min, and the product ee p >99.7%. Under the same conditions, the carbonyl reductase mutant LsCR M4The asymmetric synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol requires 13 hours for complete conversion.

[0106] Example 7: Carbonyl reductase mutant LsCR M6 Determination of secondary structure

[0107] The mutant was analyzed for circular dichroism using a CD spectrometer. The carbonyl reductase LsCR prepared in Example 3 was used... M6 The pure enzyme solution was diluted to 0.1 μg / μL with 100 mM phosphate buffer (pH 7.5), and 200 μL of the diluted solution was transferred to a 0.5 mm optical path quartz cuvette and placed in the sample chamber. The temperature range was set to 25-95℃ with a temperature gradient of 5℃, and the UV wavelength was 180-260 nm. The measurement results are as follows: Figure 9 As shown, the mutant LsCR M6 The secondary structure of the protein fluctuates with increasing temperature, indicating that protein structural stability decreases with increasing temperature. When the temperature increases from 75℃ to 95℃, the mutant LsCR... M6 The fluctuations are most pronounced at this point, indicating that the changes in its secondary structure are the greatest, suggesting that the mutant has strong thermal stability.

[0108] Example 8: Organic solvents on the carbonyl reductase mutant LsCR M6 Effect of catalytic reactions

[0109] In Example 6, isopropanol was replaced with a hydrophilic organic solvent (5% v / v) and a hydrophobic organic solvent (10% v / v), respectively. The selected hydrophilic organic solvents were: N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), isopropanol (IPA), ethanol (EtOH), and methanol (MeOH); the hydrophobic organic solvent was toluene. Other procedures were the same as in Example 6. Figure 10 As shown, isopropanol has a good promoting effect on enzyme activity. Since isopropanol can be used as a co-substrate in the coenzyme regeneration system, choosing isopropanol as an organic solvent is the optimal choice.

[0110] Example 9: Isopropanol-induced carbonyl reductase mutant LsCR M6 Effect of catalytic reaction

[0111] The isopropanol addition amounts in Example 6 were changed to 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%, respectively, with the remaining operations the same as in Example 6. Figure 11As shown, the conversion rate was highest when the isopropanol addition (isopropanol / total system, v / v) was 40%, and the conversion efficiency increased with increasing isopropanol addition. However, there was no significant difference in conversion rate between 20% and 60% isopropanol addition. Furthermore, when the isopropanol addition exceeded 60%, the conversion rate showed a decreasing trend. Therefore, considering both the production cost and substrate conversion rate of the whole-cell catalytic reaction, the optimal isopropanol addition was selected as 40% (v / v).

[0112] Example 10: Temperature-dependent carbonyl reductase mutant LsCR M6 Effect of catalytic reaction

[0113] The reaction temperature of Example 6 was changed to 35, 40, 45, 50, 55, 60, 65, and 70°C, respectively, while the remaining operations were the same as in Example 6. Figure 12 As shown, the optimal temperature for enzyme catalytic activity is 45-50℃. When the temperature is below 45℃, the enzyme activity shows a slow upward trend as the reaction temperature increases. When the temperature is above 50℃, the enzyme activity gradually begins to decrease, especially when the temperature rises above 60℃, the enzyme activity shows a sharp downward trend, and above 75℃, the enzyme activity is almost completely lost.

Claims

1. A carbonyl reductase LsCR mutant, characterized in that, The carbonyl reductase LsCR mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following: (1) mutating aspartic acid at position 150 to phenylalanine; (2) mutating valine at position 99 to leucine and mutating aspartic acid at position 150 to phenylalanine.

2. A recombinant vector containing the gene encoding the carbonyl reductase LsCR mutant as described in claim 1.

3. A recombinant genetically engineered bacterium constructed from the recombinant vector of claim 2.

4. The application of the LsCR mutant of carbonyl reductase according to claim 1 in the asymmetric reduction of carbonyl compounds to prepare chiral alcohols, characterized in that, The carbonyl compound is 2-chloro-1-(3,4-difluorophenyl)ethyl ketone, and the corresponding chiral aromatic alcohol is (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol.

5. The application as described in claim 4, characterized in that, The method of application is as follows: using engineered bacteria containing the LsCR mutant gene encoding carbonyl reductase as a catalyst, wet bacterial cells obtained by induction culture or pure enzyme solution extracted by ultrasonic disruption of wet bacterial cells are used as catalysts. Carbonyl compounds are used as substrates, isopropanol as co-substrate, NADPH as coenzyme, and a buffer solution with pH 5-7 as the reaction medium to form a transformation system. The reaction is carried out at 25-55℃ and 600-1200 rpm. After the reaction is completed, the reaction solution is extracted and purified with ethyl acetate to obtain chiral alcohol.

6. The application as described in claim 5, characterized in that, In the conversion system, the final substrate concentration is 500-1000 g / L; the final volume concentration of isopropanol is 20%-80%; the catalyst dosage is 1-15 g DCW / L based on the total dry weight of the bacteria; the catalyst dosage is 0.01-0.1 mg / mL based on the protein content in the pure enzyme solution; and the final NADPH concentration is 0.1 mM.

7. The application as described in claim 5, characterized in that, The wet bacterial cells were prepared as follows: engineered bacteria containing the carbonyl reductase LsCR mutant encoding gene were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37℃ for 10 h to obtain seed culture; The seed culture was inoculated at a volume concentration of 1.0-5.0% into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm until OD500. 600 When the value is 0.6~0.8, add 0.15 mM isopropyl thiogalactoside to the culture medium and incubate at 28℃ for 12 h. Then, centrifuge at 4℃ and 8000 rpm for 10 min to obtain wet bacterial cells containing carbonyl reductase LsCR mutant.

8. The application as described in claim 5, characterized in that, The pure enzyme solution was prepared as follows: Wet cells obtained from fermentation culture of engineered bacteria containing the LsCR mutant gene encoding carbonyl reductase were resuspended at 50 g / L in a pH 6, 100 mM sodium citrate buffer. The mixture was ultrasonically disrupted for 20 min on an ice-water mixture under the following conditions: 400 W power, 1 s disruption, 3 s pause. The disrupted mixture was collected to obtain the crude enzyme solution. The crude enzyme solution was centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant was collected. After microfiltration through a 0.45 μm microfiltration membrane, ammonium sulfate was slowly added while stirring in an ice-water bath until the ammonium sulfate saturation in the crude enzyme solution reached 50%. The solution was then allowed to stand at 4°C for 4 h. The precipitate was discarded after centrifugation at 8000 rpm and 4°C for 25 min. Ammonium sulfate was added to the supernatant until 70% ammonium sulfate saturation was achieved. The solution was centrifuged again at 8000 rpm for 25 min, and the supernatant was discarded. The precipitate was dissolved in a 20 mM, pH 7.5 potassium phosphate buffer and then processed using DEAE Sepharose. Fast Flow anion exchange column purification is performed as follows: ① Pre-equilibrate with buffer A; buffer A is a pH 7.5, 20 mM Tris-HCl buffer; ② Wash away contaminating proteins with buffer A at a flow rate of 1.0 mL / min until the conductivity stabilizes; ③ Elute the target protein with buffer B at a flow rate of 1 mL / min, starting collection when the conductivity increases and stopping when it decreases to a constant value, collecting the eluent containing the active target peak; Dialyze the collected eluent overnight with a pH 7.5, 20 mM sodium phosphate buffer, and collect the retentate, which is the pure enzyme solution; buffer B is a pH 7.5, 20 mM Tris-HCl buffer containing 1 M sodium chloride.

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