Carbonyl reductase mutants with thermal stability and isopropanol tolerance and uses thereof

Abstract

The present invention discloses a carbonyl reductase mutant with thermal stability and isopropanol tolerance and its use. The carbonyl reductase mutant has an S148L and / or Q169K mutation in the amino acid sequence shown in SEQ ID NO: 1. The present invention further discloses a combination of carbonyl reductases and a method for producing a compound represented by formula II, and the use of the combination of the carbonyl reductases in the production of the compound represented by formula II. Compared with WT, the thermal stability of mutant 35 of the present invention is 2077.7 times, and the isopropanol tolerance is 209.1 times.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical compound production, and in particular, to a carbonyl reductase mutant with thermal stability and isopropanol tolerance and its use.

Background Art

[0002] This application claims the priority of Chinese Patent Application 2022108517951 with an application date of July 19, 2022. This application incorporates the full text of the above Chinese patent application by reference.

[0003] Carbonyl reductase (Ketoredutase, KRED) can directionally reduce prochiral carbonyl compounds to chiral hydroxy compounds and is an important tool for synthesizing chiral drugs. Carbonyl reductase has the advantages of high selectivity and high conversion rate under mild conditions and has been attracting attention in the pharmaceutical industry for a long time. The antiplatelet drug Ticagrelor is an effective drug for the treatment of acute coronary syndrome and was launched in China in November 2012. (S)-2-Chloro-1-(3,4-difluorophenyl)ethanol (Formula II) is an important chiral intermediate in the synthesis of Ticagrelor. The inventor discovered in previous research that LSADH (carbonyl reductase) derived from Leifsonia sp. strain S749 can efficiently reduce 2-chloro-1-(3,4-difluorophenyl)ethanone (Formula I) to synthesize the chiral alcohol intermediate (S)-2-chloro-1-(3,4-difluorophenyl)ethanol (ee>99.9%) of Ticagrelor. However, the inventor found that in the actual production and use of LSADH, its stability is relatively low and it cannot adapt to high-intensity industrial production. LSADH is inactivated early in high concentrations of substrate, product, and isopropanol and cannot exhibit its original performance. Therefore, it is very important to improve the stability of the enzyme so that the enzyme can withstand and adapt to a harsh industrial environment.

Chemical Formula

[0004] The modification of enzyme stability is always an important and difficult point in modification. Existing modification strategies include the introduction of disulfide bonds, the modification of salt bridges, surface charge engineering, and enzyme loop engineering. However, no matter what strategy is used to modify the enzyme, the biggest problem in enzyme stability modification is that while improving the rigidity of the enzyme structure, the original catalytic activity is also reduced. Therefore, in the process of enzyme modification, it is extremely difficult to effectively balance stability and activity. To select a candidate enzyme suitable for modification, the inventors systematically compared carbonyl reductases derived from the chiral intermediate (S)-2-chloro-1-(3,4-difluorophenyl)ethanol of ticagrelor, which have been reported in existing patent documents such as CN110894184A, CN107686447A, CN111763662A, CN109423484A, CN106701840A, CN109112166A, CN109295020A, and Org. Process Res. Dev. 2017, 21, 1595-1601. These naturally derived carbonyl reductases have common problems in industrial use, such as being unable to adapt to industrial production conditions and having low catalytic ability for non-natural substrates. LSADH is an enzyme with great potential because of its high substrate concentration and high catalytic efficiency. Therefore, it has important application value to improve the thermal stability and isopropanol tolerance of the enzyme through reasonable modification.

Summary of the Invention

[0005] The present invention provides a carbonyl reductase mutant with thermal stability and isopropanol tolerance and its use to solve the problems of low thermal stability and isopropanol tolerance of carbonyl reductases in the prior art. The present invention performs molecular evolution of the enzyme through rational design and obtains mutants with significantly improved thermal stability and isopropanol tolerance, which is more useful for large-scale industrial use.

[0006] The first aspect of the present invention provides a carbonyl reductase mutant having an S148L and / or Q169K mutation in the amino acid sequence shown in SEQ ID NO: 1. In some preferred embodiments, the carbonyl reductase mutant further comprises one or more mutations among I145V, A163G, and L207V. Preferably, the carbonyl reductase mutant further comprises one or more mutations among T100P, T111R, and V183N.

[0007] In some preferred embodiments, the mutant is selected from the group consisting of the following. (1) S148L, S148L / Q169K, I145V / S148L / A163G / L207V, I145V / S148L / A163G / Q169K / L207V, T100P / T111R / I145V / S148L / A163G / Q169K / V183N / L207V or G78R / A81E / T100P / S105P / T111R / Q125R / I145V / S148L / A163G / V183N / L207V; (2) Q169K, S148V / Q169K, I145V / A163G / Q169K / L207V, T100P / I145V / A163G / Q169K / L207V or T100P / T111R / I145V / A163G / Q169K / V183N / L207V.

[0008] In some preferred embodiments, the amino acid sequence of the carbonyl reductase mutant is shown in SEQ ID NO: 4.

[0009] The second aspect of the present invention provides another carbonyl reductase mutant having a mutation selected from the following group in the amino acid sequence shown in SEQ ID NO: 1. (1) I145V / A163G / V183N / L207V; (2) T100P / I145V / A163G / L207V; (3) S148V / Q169T.

[0010] The third aspect of the present invention provides an isolated nucleic acid encoding the carbonyl reductase mutant according to the first aspect of the present invention. In some embodiments, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 3 or 5.

[0011] The fourth aspect of the present invention provides a recombinant expression vector containing the isolated nucleic acid according to the third aspect of the present invention.

[0012] The fifth aspect of the present invention provides a genetically engineered bacterium containing the isolated nucleic acid according to the third aspect of the present invention or containing the recombinant expression vector according to the fourth aspect of the present invention.

[0013] The sixth aspect of the present invention provides a combination of carbonyl reductases, including at least one of the carbonyl reductase mutants according to the first aspect of the present invention and wild-type carbonyl reductase, or including at least two of the carbonyl reductase mutants according to the first aspect of the present invention.

[0014] The seventh aspect of the present invention provides a method for producing a compound represented by formula II, which includes catalyzing the compound represented by formula I with the carbonyl reductase mutant according to the first aspect of the present invention, or the genetically engineered bacterium according to the fifth aspect of the present invention, or the combination of carbonyl reductases according to the sixth aspect of the present invention.

Chemical formula

[0015] Preferably, the carbonyl reductase mutant has the T100P / T111R / I145V / S148L / A163G / Q169K / V183N / L207V mutation in the amino acid sequence shown in SEQ ID NO: 1.

[0016] In some preferred embodiments, the method includes the following steps. In a buffer solution, the carbonyl reductase mutant, NAD +, isopropanol, and a compound represented by formula I are added, and a compound represented by formula II is obtained by reaction. Preferably, the buffer solution is a phosphate buffer solution with a concentration of 0.1 M, a pH of 7.0, a reaction temperature of 40 to 50 °C, for example, 45 °C.

[0017] More preferably, the method further includes extraction of the compound represented by formula II, and the extraction includes the following steps. (1) Adding methyl tert-butyl ether for extraction, inactivating proteins, filtering, and re-extracting the aqueous layer with methyl tert-butyl ether. (2) Combining the extracted products, washing the organic layer with water and / or brine, and drying. (3) Filtering and concentrating by rotary evaporation to obtain a purified compound represented by formula II.

[0018] In some more preferred embodiments, the reaction conditions of the reaction are shown below. In step (1), the protein inactivation temperature is 55 to 65 °C, preferably 60 °C, the time is 0.5 to 1.5 hours, for example, 1 hour, diatomaceous earth is used for filtration, and the amount of diatomaceous earth is preferably 8% to 12%, for example, 10%. In step (2), it is dried with anhydrous sodium sulfate, and the organic layer is washed with water and 5% brine.

[0019] The eighth aspect of the present invention provides the use of the carbonyl reductase mutant described in the first aspect of the present invention, or the genetically engineered bacterium described in the fifth aspect of the present invention, or the combination of carbonyl reductase described in the sixth aspect of the present invention in the production of the compound represented by formula II.

[0020] Unless it violates the common general knowledge in this technical field, the above-mentioned preferred conditions can be arbitrarily combined to obtain each preferred embodiment of the present invention. The reagents and raw materials used in the present invention are commercially available.

[0021] The positive progressive effects of the present invention are: The carbonyl reductase mutant of the present invention has improved catalytic efficiency compared to the wild type and has good thermal stability and isopropanol tolerance. In one preferred example of the present invention, the catalytic efficiency of mutant 35 is increased by 41 times compared to the wild type LSADH, the Tm is increased by 23.3 °C, the half-life at 55 °C is increased from 3 minutes to 60 hours, the isopropanol tolerance is increased by 80%, and compared with the WT, the thermal stability of mutant 35 is 2077.7 times and the isopropanol tolerance is 209.1 times.

Brief Description of the Drawings

[0022]

Figure 1

Modes for Carrying Out the Invention

[0023] Hereinafter, the present invention will be further described by way of embodiments, but the present invention is not limited to the scope of the above embodiments. In the following examples, experimental methods for which specific conditions are not described are selected according to ordinary methods and conditions, or the product description.

[0024] Example 1: Construction of carbonyl reductase LSADH recombinant bacteria and its mutant library The carbonyl reductase LSADH (SEQ ID NO: 1) gene derived from Leifsonia sp. strain S749 was synthesized into the pET28a(+) vector by GenScript Nanjing after codon optimization (SEQ ID NO: 3). Then, the plasmid containing the LSADH gene was introduced into the host Escherichia coli BL21(DE3) competent cells to obtain recombinant gene recombinant bacteria of carbonyl reductase (LSADH).

[0025] Based on the structural analysis of carbonyl reductase LSADH and the prediction by HotSpot Wizard, the inventors selected residues such as E53, A63, G78, A81, A98, T100, S105, T111, G123, Q125, A132, A143, S148, S154, T159, A163, Q169, Y175, K179, V180, V183, G186, V195, S200, L204, L207, A212, and S224 within the active pocket of LSADH, and constructed a site-directed mutagenesis library and a combinatorial mutagenesis library. The specific operation was to introduce the corresponding mutations by overlap PCR, transfer the constructed plasmid library into Escherichia coli BL21(DE3), and then spread it on LB solid medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C. The next day, single colonies were collected into 96-well plates containing 400 μL of LB medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C. Next, 10 μL of the seed solution was aspirated from the 96-well plate cultured overnight and transferred into a new 96-well plate (containing 400 μL of fermentation medium containing 50 μg / mL kanamycin), and cultured with shaking at 37°C until the OD 600 value > 0.8, then IPTG with a final concentration of 1 mM was added, and cultured at 28°C for 20 hours to induce the expression of LSADH mutants. Finally, the 96-well plate was transferred to a centrifuge and centrifuged at 4000 g for 30 minutes to collect the bacteria, which were stored at -20°C for later use.

[0026] The composition of the fermentation medium is as follows: yeast extract (2.4%), soy peptone (1.2%), sodium chloride (0.3%), glycerol (0.5%), dipotassium hydrogen phosphate (0.2%), magnesium sulfate heptahydrate (0.05%).

[0027] Example 2: Preparation and Screening of Crude Enzyme Solution of Carbonyl Reductase LSADH Mutants Using 200 μL of lysis buffer (0.1 M phosphate buffer, pH 7.0 containing 1000 U of lysozyme), the cells stored above were resuspended, then lysed at 30 °C for 1 hour, placed in a centrifuge at 4 °C and 4000 g for centrifugation for 30 minutes, and the clear supernatant was aspirated to measure the activity of the mutants. The crude enzyme solutions of LSADH and the mutants were immersed in isopropanol at concentrations of 40%, 60%, and 80% at 55 °C respectively to perform a stability test. The test system (total volume: 200 μL) consisted of 4 mM of 2-chloro-1-(3,4-difluorophenyl)ethanone substrate, 1 mM of NADH, 20% of DMSO (v / v), K2HPO4-KH2PO4 phosphate buffer (100 mM, pH 7.0), and 10 μL of LSADH or mutant enzyme solution. The protein concentrations of the crude enzyme solutions of LSADH and the mutants were diluted and adjusted according to the actual activity measurement. The activities of LSADH and its mutants were calculated and characterized based on the change value of NADH absorbance at 340 nm.

[0028] The mutants with significantly improved stability were retested in a 2 mL activity measurement system. Specifically, it included 20 g / L of 2-chloro-1-(3,4-difluorophenyl)ethanone substrate, 20% of isopropanol (v / v), 0.1 g / L of NAD + , K2HPO4-KH2PO4 phosphate buffer (100 mM, pH 7.0), and 100 μl of the crude enzyme solution before or after treatment. After reacting at 28 °C for 15 minutes, the conversion rate was detected by HPLC. The relative stability of each mutant is shown in Table 1. * represents the improvement multiples of the thermal stability and isopropanol tolerance of the mutant compared to wild-type LSADH.

[0029] Table 1 Some mutants and their relative activities

Table 1

[0030] Example 3: Measurement of the enzyme properties of carbonyl reductase LSADH and mutant 35 Considering that the stability and isopropanol tolerance of mutant 35 (the amino acid sequence is shown in SEQ ID NO: 4 and the nucleotide sequence is shown in SEQ ID NO: 5) were significantly improved, the inventors further investigated the kinetic parameters of mutant 35 compared with wild-type LSADH, the changes in the half-life at 55 °C, Tm and T1550 (Table 2). K m and k cat / K m From the values of, it was found that the affinity of mutant 35 for 2-chloro-1-(3,4-difluorophenyl)ethanone increased 8-fold and the catalytic efficiency increased 40.5-fold compared with wild-type LSADH. In terms of thermal stability, the half-life (t1 / 2) of mutant 35 at 55 °C was 60 hours, while the half-life (t1 / 2) of wild-type LSADH was only 3 minutes. As shown in Figure 1, mutant 35 could maintain 80% of its catalytic activity even after culturing at 55 °C for 24 hours, and 20% of its catalytic activity remained even after culturing for 144 hours. However, wild-type LSADH had no activity after culturing at 55 °C for 15 minutes. At the same time, we also measured the melting temperature of mutant 35 and found that the Tm value of mutant 35 increased from 39.5 °C of wild-type LSADH to 62.8 °C. This indicates that mutant 35 unfolds at a higher temperature than wild-type LSADH and gradually destroys its secondary structure. To further verify the change in the activity of LSADH after structural destruction due to temperature increase, we measured the residual enzyme activities of LSADH and mutant 35 after culturing at various temperatures such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 °C for 15 minutes. The results showed that the T1550 value of mutant 35 increased from 40.0 °C of wild-type LSADH to 57.2 °C. Therefore, the activity and thermal stability of mutant 35 are significantly improved compared with LSADH.

[0031] Table 2 Kinetic parameters, Tm and T1550 of LSADH and mutants

Table 2

[0032] Example 4: Biocatalytic production of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol on a kilogram scale Take 0.1 M phosphate buffer (12 L), add NAD + (20 g), add isopropanol (8 L), add the mutant 35 (0.4 kg) obtained from the above fermentation, stir vigorously, and add a solution of compound I (2-chloro-1-(3,4-difluorophenyl)ethanone) (10 kg) in batches. At 45 °C, monitor by HPLC when the reaction conversion rate reached >98%, and stop the reaction. Add methyl tert-butyl ether (40 L) for extraction, heat to 60 °C and keep warm for 1 hour to inactivate the protein. Next, add 10% diatomaceous earth to filter the cells, and extract the aqueous layer again with methyl tert-butyl ether (20 L). Combine the organic layers, wash with water and 5% brine, and dry over anhydrous sodium sulfate. Finally, filter and concentrate by rotary evaporation to obtain compound II ((S)-2-chloro-1-(3,4-difluorophenyl)ethanol) (9.3 kg), and the ee value was 99%.

[0033] The sequences used in this specification are shown below. Original amino acid sequence of carbonyl reductase (SEQ ID NO: 1): MAQYDVADRSAIVTGGGSGIGRAVALTLAASGAAVLVTDLNEEHAQAVVAEIEAAGGKAAALAGDVTDPAFGEASVAGANALAPLKIAVNNAGIGGEAATVGDYSLDSWRTVIEVNLNAVFYGMQPQLKAMAANGGGAIVNMASILGSVGFANSSAYVTAKHALLGLTQNAALEYAADKVRVVAVGPGFIRTPLVEANLSADALAFLEGKHALGRLGEPEEVASLVAFLASDAASFITGSYHLVDGGYTAQ*

[0034] Original nucleotide sequence of carbonyl reductase (SEQ ID NO: 3): atggcgcagtatgatgtggcggatcgcagcgcgattgtgaccggcggcggcagcggcattggccgcgcggtggcgctgaccctggcggcgagcggcgcggcggtgctggtgaccgatctgaacgaagaacatgcgcaggcggtggtggcggaaattgaagcggcgggcggcaaagcggcggcgctggcgggcgatgtgaccgatccggcgtttggcgaagcgagcgtggcgggcgcgaacgcgctggcgccgctgaaaattgcggtgaacaacgcgggcattggcggcgaagcggcgaccgtgggcgattatagcctggatagctggcgcaccgtgattgaagtgaacctgaacgcggtgttttatggtatgcagccgcagctgaaagcgatggcggcgaacggcggcggcgcgattgtgaacatggcgagcattctgggcagcgtgggctttgcgaacagcagcgcgtatgtgaccgcgaaacatgcgctgctgggcctgacccagaacgcggcgctggaatatgcggcggataaagtgcgcgtggtggcggtgggcccgggctttattcgcaccccgctggtggaagcgaacctgagcgcggatgcgctggcgtttctggaaggcaaacatgcgctgggccgcctgggcgaaccggaagaagtggcgagcctggtggcgtttctggcgagcgatgcggcgagctttattaccggcagctatcatctggtggatggcggctataccgcgcagtaa

[0035] Amino acid sequence of mutant 35 (SEQ ID NO: 4): MAQYDVADRSAIVTGGGSGIGRAVALTLAASGAAVLVTDLNEEHAQAVVAEIEAAGGKAAALAGDVTDPAFGEASVAGANALAPLKIAVNNAGIGGEAAPVGDYSLDSWRRVIEVNLNAVFYGMQPQLKAMAANGGGAIVNMASVLGLVGFANSSAYVTAKHGLLGLTKNAALEYAADKVRVNAVGPGFIRTPLVEANLSADALAFVEGKHALGRLGEPEEVASLVAFLASDAASFITGSYHLVDGGYTAQ*

[0036] Nucleotide sequence of mutant 35 (SEQ ID NO: 4): ATGGCGCAGTATGATGTGGCGGATCGCAGCGCGATTGTGACCGGCGGCGGCAGCGGCATTGGCCGCGCGGTGGCGCTGACCCTGGCGGCGAGCGGCGCGGCGGTGCTGGTGACCGATCTGAACGAAGAACATGCGCAGGCGGTGGTGGCGGAAATTGAAGCGGCGGGCGGCAAAGCGGCGGCGCTGGCGGGCGATGTGACCGATCCGGCGTTTGGCGAAGCGAGCGTGGCGGGCGCGAACGCGCTGGCGCCGCTGAAAATTGCGGTGAACAACGCGGGCATTGGCGGCGAAGCGGCGCCGGTGGGCGATTATAGCCTGGATAGCTGGCGCCGCGTGATTGAAGTGAACCTGAACGCGGTGTTTTATGGTATGCAGCCGCAGCTGAAAGCGATGGCGGCGAACGGCGGCGGCGCGATTGTGAACATGGCGAGCGTGCTGGGCCTGGTGGGCTTTGCGAACAGCAGCGCGTATGTGACCGCGAAACATGGCCTGCTGGGCCTGACCAAAAACGCGGCGCTGGAATATGCGGCGGATAAAGTGCGCGTGAACGCGGTGGGCCCGGGCTTTATTCGCACCCCGCTGGTGGAAGCGAACCTGAGCGCGGATGCGCTGGCGTTTGTGGAAGGCAAACATGCGCTGGGCCGCCTGGGCGAACCGGAAGAAGTGGCGAGCCTGGTGGCGTTTCTGGCGAGCGATGCGGCGAGCTTTATTACCGGCAGCTATCATCTGGTGGATGGCGGCTATACCGCGCAGTAA

Claims

1. A carbonyl reductase mutant having an S148L and / or Q169K mutation in the amino acid sequence shown in SEQ ID NO:

1.

2. The carbonyl reductase mutant further comprises one or more mutations among I145V, A163G, and L207V, Preferably, the carbonyl reductase mutant further comprises one or more mutations among T100P, T111R, and V183N, and is the carbonyl reductase mutant according to claim 1.

3. (1) S148L, S148L / Q169K, I145V / S148L / A163G / L207V, I145V / S148L / A163G / Q169K / L207V, T100P / T111R / I145V / S148L / A163G / Q169K / V183N / L207V, or G78R / A81E / T100P / S105P / T111R / Q125R / I145V / S148L / A163G / V183N / L207V; (2) Q169K, S148V / Q169K, I145V / A163G / Q169K / L207V, T100P / I145V / A163G / Q169K / L207V, or T100P / T111R / I145V / A163G / Q169K / V183N / L207V; Selected from the group consisting of Preferably, the amino acid sequence of the carbonyl reductase mutant is shown in SEQ ID NO: 4, and is the carbonyl reductase mutant according to claim 1.

4. The carbonyl reductase has, in the amino acid sequence shown in SEQ ID NO: 1, (1) I145V / A163G / V183N / L207V; (2) T100P / I145V / A163G / L207V; (3) S148V / Q169T And has a mutation selected from the group consisting of, and is a carbonyl reductase mutant.

5. Encoding the carbonyl reductase mutant according to any one of claims 1 to 4, Preferably, an isolated nucleic acid characterized in that the nucleotide sequence is shown in SEQ ID NO: 3 or 5.

6. A recombinant expression vector comprising the isolated nucleic acid according to claim 5.

7. A genetically engineered bacterium comprising the isolated nucleic acid according to claim 5, or comprising the recombinant expression vector according to claim 6.

8. At least one of the carbonyl reductase mutants according to any one of claims 1 to 4, and a wild-type carbonyl reductase, or a combination of carbonyl reductase, characterized by comprising at least two of the carbonyl reductase mutants according to any one of claims 1 to 4.

9. A method for producing a compound represented by formula II, characterized by comprising catalyzing the compound represented by formula I with the carbonyl reductase mutant according to any one of claims 1 to 4, or the recombinant bacterium according to claim 7, or the combination of carbonyl reductase according to claim 8. 【Chemical 1】 (Preferably, the carbonyl reductase mutant has T100P / T111R / I145V / S148L / A163G / Q169K / V183N / L207V mutations in the amino acid sequence shown in SEQ ID NO: 1.)

10. The method comprises Add the carbonyl reductase mutant, NAD ＋ , isopropanol, and the compound represented by formula I to a buffer solution, and obtain the compound represented by formula II by reaction, Preferably, the buffer solution is a phosphate buffer solution with a concentration of 0.1 M, pH 7.0, and a reaction temperature of 40 to 50 °C including More preferably, the method further comprises extracting the compound represented by formula II, and the extraction comprises (1) adding methyl tert-butyl ether for extraction, inactivating proteins, filtering, and re-extracting the aqueous layer with methyl tert-butyl ether; (2) combining the extracted products, washing the organic layer with water and / or brine, and drying; (3) filtering and concentrating by rotary evaporation to obtain the purified compound represented by formula II The method according to claim 9, characterized by comprising the above steps.

11. The reaction conditions of the above reaction are In step (1), the protein inactivation temperature is 55 to 65 °C, preferably 60 °C, the time is 0.5 to 1.5 hours, kieselguhr is used for filtration, and the amount of kieselguhr is preferably 8% to 12%; In step (2), drying with anhydrous sodium sulfate and washing the organic layer with water and 5% brine. The method according to claim 10, characterized by the above steps.

12. Use of the carbonyl reductase mutant according to any one of claims 1 to 4, or the recombinant bacterium according to claim 7, or the combination of carbonyl reductase according to claim 8, in the production of the compound represented by formula II.

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