WO2024017405A1

WO2024017405A1 - Thermostable and isopropanol-tolerant carbonyl reductase mutant and use thereof

Info

Publication number: WO2024017405A1
Application number: PCT/CN2023/114764
Authority: WO
Inventors: 陈少欣; 汤佳伟; 张露文; 吴远杰; 张正玉
Original assignee: 上海医药工业研究院有限公司; 中国医药工业研究总院有限公司
Priority date: 2022-07-19
Filing date: 2023-08-24
Publication date: 2024-01-25
Also published as: CN117417909A

Abstract

Disclosed are a thermostable and isopropanol-tolerant carbonyl reductase mutant and a use thereof. The carbonyl reductase mutant is subjected to S148L and/or Q169K mutations on an amino acid sequence as shown in SEQ ID NO: 1. Further disclosed are a carbonyl reductase combination and a method for preparing a compound as shown in formula II, and a use of the carbonyl reductase combination in the preparation of the compound as shown in formula II. The thermal stability of mutant 35 of the present invention is 2077.7 times that of a WT enzyme, and the isopropanol tolerance of the mutant 35 is 209.1 times that of the WT enzyme.

Description

A thermostable and isopropanol-tolerant carbonyl reductase mutant and its application

This application claims the priority of Chinese patent application 2022108517951 with a filing date of 2022/7/19. This application cites the full text of the above-mentioned Chinese patent application.

Technical field

The present invention relates to the field of pharmaceutical compound preparation, and in particular to a thermostable and isopropyl alcohol-tolerant carbonyl reductase mutant and its application.

Background technique

Carbonyl reductase (Ketoredutase, KRED) can directionally reduce prochiral carbonyl compounds into chiral hydroxy compounds and is an important tool for the synthesis of chiral drugs. Carbonyl reductase, which has the advantages of high selectivity and high conversion rate under mild conditions, has long been valued by the pharmaceutical industry. The antiplatelet therapeutic drug Ticagrelor is an effective drug for the treatment of acute coronary syndrome and was launched in my country in November 2012. (S)-2-Chloro-1-(3,4-difluorophenyl)ethanol (Formula II) is an important chiral intermediate in the synthesis of ticagrelor. In previous research, the inventor found that LSADH (carbonyl reductase) derived from Leifsonia sp.strain S749 can efficiently reduce 2-chloro-1-(3,4-difluorophenyl)ethanone (formula I) to synthesize alternative Graylo chiral alcohol intermediate (S)-2-chloro-1-(3,4-difluorophenyl)ethanol (ee>99.9%). However, in the actual production and application of LSADH, the inventor found that its stability was relatively low and it was unable to adapt to high-intensity industrial production. LSADH is deactivated prematurely in high concentrations of substrates, products and isopropanol, and cannot perform as it should. Therefore, it is of great significance to improve the stability of enzymes so that they can withstand and adapt to harsh industrial environments.

Enzyme stability modification has always been a hot and difficult point in modification. Existing modification strategies include the introduction of disulfide bonds, modification of salt bridges, surface charge engineering, and enzyme cyclization engineering. But no matter which strategy is used to modify the enzyme, the biggest problem in enzyme stability modification is that while improving the rigidity of the enzyme structure, it will also reduce its original catalytic activity. Therefore, in the process of enzyme modification, it is extremely challenging to effectively balance stability and activity. In order to select suitable candidate enzymes for transformation, the inventors combined CN110894184A, CN107686447A, CN111763662A, CN109423484A, CN106701840A, CN109112166A, CN109295020A and Org.Process Res.Dev.2017,21,1595-1601 can prepare alternative products reported in existing patent documents. The carbonyl reductase derived from Greenough chiral intermediate (S)-2-chloro-1-(3,4-difluorophenyl)ethanol was systematically compared. These naturally derived carbonyl reductases have common problems in industrial applications such as their inability to adapt to industrial production conditions and their low catalytic ability for non-natural substrates. LSADH, with its higher substrate concentration and higher catalytic efficiency, is an enzyme with great potential. Therefore, improving the thermal stability and isopropyl alcohol tolerance of the enzyme through rational modification has important practical application value.

Contents of the invention

In order to solve the problems of poor thermal stability and poor isopropyl alcohol tolerance of carbonyl reductase in the prior art, the present invention provides a thermally stable and isopropyl alcohol tolerant carbonyl reductase mutant and its application. The present invention carries out molecular evolution of the enzyme through rational design to obtain a mutant with greatly improved thermal stability and isopropyl alcohol tolerance, which is more conducive to large-scale industrial application.

A first aspect of the present invention provides a carbonyl reductase mutant, which has S148L and/or Q169K mutations in the amino acid sequence shown in SEQ ID NO:1.

In some preferred embodiments, the carbonyl reductase mutant further includes one or more mutations of I145V, A163G and L207V.

Preferably, the carbonyl reductase mutant further includes one or more mutations of T100P, T111R and V183N.

In some preferred embodiments, the mutant is selected from the group consisting of:

(1)S148L, S148L/Q169K, I145V/S148L/A163G/L207V, I145V/S148L/A163G/Q169K/L207V, T100P/T111R/I145V/S148L/A163G/Q169K/V183N/L207V or G 78R/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.

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

A second aspect of the invention provides another carbonyl reductase mutant, which has a mutation selected from the following group on the amino acid sequence shown in SEQ ID NO: 1:

(1)I145V/A163G/V183N/L207V;

(2)T100P/I145V/A163G/L207V;

(3)S148V/Q169T.

A third aspect of the invention provides an isolated nucleic acid encoding a carbonyl reductase mutant as described in the first aspect of the invention.

In some embodiments, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO: 3 or 5.

The fourth aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the third aspect of the present invention.

The fifth aspect of the present invention provides a genetically engineered bacterium, which contains the isolated nucleic acid as described in the third aspect of the present invention, or the recombinant expression vector as described in the fourth aspect of the present invention.

A sixth aspect of the present invention provides a carbonyl reductase combination, which includes at least one carbonyl reductase mutant according to the first aspect of the present invention and a wild-type carbonyl reductase, or includes at least two of the first aspect of the present invention. The carbonyl reductase mutant.

The seventh aspect of the present invention provides a method for preparing a compound represented by formula II, which includes using 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 present invention. The carbonyl reductase combination according to the sixth aspect of the invention catalyzes the compound represented by Formula I.

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

In some preferred embodiments, the method includes the following steps:

In the buffer, add the carbonyl reductase mutant, NAD ⁺ , isopropanol and the compound represented by formula I; the compound represented by formula II is obtained by the reaction.

Preferably, the buffer is a phosphate buffer with a concentration of 0.1M, a pH of 7.0, and a reaction temperature of 40-50°C, such as 45°C.

More preferably, the method also includes extracting the compound represented by Formula II, and the extraction includes the following steps:

(1) Add methyl tertiary ether for extraction; inactivate the protein, filter, and extract the water layer again with methyl tertiary ether;

(2) Combine the extracted products, wash the organic layer with water and/or brine, and dry;

(3) Filtration, rotary evaporation and concentration to obtain the purified compound represented by Formula II.

In some more preferred embodiments, the reaction conditions of the reaction are as follows:

In step (1), the protein inactivation temperature is 55-65°C; preferably 60°C; the time is 0.5-1.5h, for example 1h; diatomite is used for filtration, and the amount of diatomite is preferably 8%-12%. For example 10%;

In step (2), dry with anhydrous sodium sulfate, and wash the organic layer with water and 5% brine.

The eighth aspect of the present invention provides 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 carbonyl reductase combination described in the sixth aspect of the present invention in the preparation of formula II Applications of the compounds shown.

On the basis of common sense in the field, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progressive effects of the present invention are:

The carbonyl reductase mutant of the present invention has improved catalytic efficiency compared with the wild type, and has good thermal stability and isopropyl alcohol tolerance. In a preferred example of the present invention, the catalytic efficiency of mutant 35 is increased by 41 times compared with wild-type LSADH; Tm is increased by 23.3°C, and the half-life at 55°C is increased from 3 minutes to 60 hours; the tolerance to isopropyl alcohol is increased to 80%; Relative to WT, mutant 35 has a thermal stability of 2077.7 times and an isopropyl alcohol tolerance of 209.1 times.

Description of drawings

Figure 1 shows the half-life of wild-type carbonyl reductase LSADH and its mutants at 55°C.

Detailed ways

The present invention is further described below by means of examples, but the present invention is not limited to the scope of the described examples. Experimental methods without specifying specific conditions in the following examples were carried out in accordance with ordinary Specific methods and conditions, or choose according to product instructions.

Example 1: Construction of carbonyl reductase LSADH engineering bacteria and its mutant library

The carbonyl reductase LSADH (SEQ ID NO:1) gene derived from Leifsonia sp.strain S749 was codon optimized (SEQ ID NO:3) and synthesized by Nanjing GenScript into the pET28a(+) vector. Subsequently, the plasmid containing the LSADH gene was introduced into the host E. coli BL21 (DE3) competent cells to obtain a recombinant genetically engineered strain of carbonyl reductase (LSADH).

Based on the structural analysis of carbonyl reductase LSADH and HotSpot Wizard prediction, the inventors selected E53, A63, G78, A81, A98, T100, S105, T111, G123, Q125, A132, A143, S148, S154, and T159 in the active pocket of LSADH. , A163, Q169, Y175, K179, V180, V183, G186, V195, S200, L204, L207, A212 and S224 and other residues, a site-directed mutation library and a combinatorial mutation library were established. The specific operation is as follows: introduce the corresponding mutations through overlapping PCR, transfer the constructed plasmid library into E. coli BL21 (DE3), and then spread it onto LB solid medium (containing 50 μg/mL kanamycin), and incubate at 37 Incubate overnight at ℃. The next day, single colonies were picked into a 96-well plate containing 400 μL LB medium (containing 50 μg/mL kanamycin) and cultured at 37°C overnight. Then transfer 10 μL of seed solution from the 96-well plate cultured overnight to a new 96-well plate (containing 400 μL of fermentation medium containing 50 μg/mL kanamycin), and culture with shaking at 37°C until the OD ₆₀₀ value is >0.8. IPTG with a final concentration of 1mM was added and cultured at 28°C for 20 hours to induce expression of the LSADH mutant. Finally, the 96-well plate was moved to a centrifuge, centrifuged at 4000g for 30 minutes to collect the bacteria, and stored at -20°C for later use.

The fermentation medium formula 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 %).

Example 2: Preparation and screening of crude enzyme solution of carbonyl reductase LSADH mutant

Use 200 μL lysis buffer (0.1 M phosphate buffer containing 1000 U lysozyme, pH 7.0) to resuspend the above-preserved bacterial cells, then lyse it at 30°C for 1 hour, put it into a centrifuge at 4°C, 4000g, centrifuge for 30 minutes, and remove the clarification The supernatants were used to determine mutant activity. LSADH and mutant crude After the enzyme solution was soaked at 55°C and isopropyl alcohol with concentrations of 40%, 60%, and 80%, the stability test was performed. The test system (total volume 200 μL) is as follows: 4mM 2-chloro-1-(3,4-difluorophenyl)ethanone substrate, 1mM NADH, 20% DMSO (v/v), K ₂ HPO ₄ -KH ₂ PO ₄ phosphate buffer (100mM, pH 7.0) and 10μL LSADH or mutant enzyme solution. The protein concentration of crude enzyme solution of LSADH and mutants was diluted and adjusted according to the actual activity measurement. The activity of LSADH and its mutants was calculated and characterized based on the change value of NADH absorbance at 340 nm.

Mutants with significantly improved stability were retested in a 2mL activity measurement system, specifically: 20g/L 2-chloro-1-(3,4-difluorophenyl)ethanone substrate, 20% isopropyl alcohol (v /v), 0.1g/L NAD ⁺ , K ₂ HPO ₄ -KH ₂ PO ₄ phosphate buffer (100mM, pH 7.0) and 100μl 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. *Compared to wild-type LSADH, the mutant's thermal stability and isopropyl alcohol tolerance are improved fold.

Table 1 Some mutants and their relative activities

Example 3: Determination of enzymatic properties of carbonyl reductase LSADH and mutant 35

In view of the significantly improved stability and isopropanol tolerance of mutant 35 (the amino acid sequence is shown in SEQ ID NO: 4, the nucleotide sequence is shown in SEQ ID NO: 5), the inventor further investigated the comparison of mutant 35 Kinetic parameters, half-life at 55°C, Tm and changes (Table 2). From the K _m and k _cat /K _m values, it can be seen that the affinity of mutant 35 for 2-chloro-1-(3,4-difluorophenyl)ethanone is increased by 8 times compared with wild-type LSADH. Catalytic efficiency increased by 40.5 times. In terms of thermal stability, the half-life (t1/2) of mutant 35 at 55°C is 60 hours, while the half-life (t1/2) of wild-type LSADH is only 3 minutes. As shown in Figure 1, mutant 35 can still maintain 80% of the catalytic activity after incubation at 55°C for 24 hours, and 20% of the catalytic activity remains after 144 hours of incubation. while wild type LSADH is no longer active after incubation 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 shows that mutant 35 unfolds and gradually destroys the secondary structure at a higher temperature than wild-type LSADH. In order to further verify the changes in the activity of LSADH after its structure is destroyed as the temperature increases, we measured the activity of LSADH and mutant 35 at different temperatures such as 25, 30, 35, 40, 45, 50, 55, 60, 65, and 70°C. Residual enzyme activity after 15 min of incubation. The results showed that mutant 35 The value increased from 40.0°C for wild-type LSADH to 57.2°C. Therefore, the activity and thermal stability of mutant 35 are significantly improved compared to LSADH.

Table 2 Kinetic parameters, Tm and values of LSADH and mutants

Example 4: Biocatalytic preparation of kilogram-scale (S)-2-chloro-1-(3,4-difluorophenyl)ethanol

Take 0.1M phosphate buffer (12L), add NAD ⁺ (20g), add isopropyl alcohol (8L), add mutant 35 (0.4kg) obtained from the above fermentation, stir vigorously and add compound I (2-chloro -1-(3,4-difluorophenyl)ethanone) (10kg) solution, 45°C, HPLC monitoring when the reaction conversion rate is >98%, terminate the reaction. Add methyl tertiary ether (40L) for extraction, heat to 60°C and incubate for 1 hour to inactivate the protein. Then 10% diatomaceous earth was added to filter the bacterial cells, and the aqueous layer was extracted again with methyl tertiary ether (20L). The organic layers were combined, washed with water and 5% saline, and dried over anhydrous sodium sulfate. Finally, compound II ((S)-2-chloro-1-(3,4-difluorophenyl)ethanol) (9.3kg) was obtained by filtration and rotary evaporation, with an ee value of 99%.

The sequence used in this article is as follows:

Original amino acid sequence of carbonyl reductase (SEQ ID NO:1):

Original nucleotide sequence of carbonyl reductase (SEQ ID NO:3):

Mutant 35 amino acid sequence (SEQ ID NO:4):

Mutant 35 nucleotide sequence (SEQ ID NO:4):

Claims

A carbonyl reductase mutant, characterized in that the carbonyl reductase mutant has S148L and/or Q169K mutations on the amino acid sequence shown in SEQ ID NO:1.
The carbonyl reductase mutant according to claim 1, wherein the carbonyl reductase mutant further includes one or more mutations in I145V, A163G and L207V;

Preferably, the carbonyl reductase mutant further includes one or more mutations of T100P, T111R and V183N.
The carbonyl reductase mutant according to claim 1, characterized in that it is selected from the following group:

(1)S148L, S148L/Q169K, I145V/S148L/A163G/L207V, I145V/S148L/A163G/Q169K/L207V, T100P/T111R/I145V/S148L/A163G/Q169K/V183N/L207V or G 78R/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;

Preferably, the amino acid sequence of the carbonyl reductase mutant is shown in SEQ ID NO: 4.
A carbonyl reductase mutant, characterized in that the carbonyl reductase has a mutation selected from the following group on the amino acid sequence shown in SEQ ID NO:1:

(1)I145V/A163G/V183N/L207V;

(2)T100P/I145V/A163G/L207V;

(3)S148V/Q169T.
An isolated nucleic acid, characterized in that the nucleic acid encodes the carbonyl reductase mutant according to any one of claims 1-4;

Preferably, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 3 or 5.
A recombinant expression vector, characterized in that the recombinant expression vector contains as claimed in The isolated nucleic acid described in 5.
A genetically engineered bacterium, characterized in that the genetically engineered bacterium contains the isolated nucleic acid as claimed in claim 5, or the recombinant expression vector as claimed in claim 6.
A carbonyl reductase combination, characterized in that the carbonyl reductase combination includes at least one carbonyl reductase mutant according to any one of claims 1 to 4, and a wild-type carbonyl reductase, or includes at least two The carbonyl reductase mutant according to any one of claims 1-4.
A method for preparing a compound represented by Formula II, characterized in that it includes using a carbonyl reductase mutant as claimed in any one of claims 1-4 or a genetically engineered bacterium as claimed in claim 7 or as The carbonyl reductase combination of claim 8 catalyzes the compound shown in formula I;

Preferably, the carbonyl reductase mutant has a T100P/T111R/I145V/S148L/A163G/Q169K/V183N/L207V mutation on the amino acid sequence shown in SEQ ID NO:1.
The method according to claim 9, characterized in that the method includes the following steps:

In the buffer, add the carbonyl reductase mutant, NAD + , isopropanol and the compound represented by formula I; the reaction obtains the compound represented by formula II;

Preferably, the buffer is phosphate buffer, the concentration is 0.1M, the pH is 7.0, and the reaction temperature is 40-50°C;

More preferably, the method also includes extracting the compound represented by formula II, and the extraction includes the following steps: Steps:

(1) Add methyl tertiary ether for extraction; inactivate the protein, filter, and extract the water layer again with methyl tertiary ether;

(2) Combine the extracted products, wash the organic layer with water and/or brine, and dry;

(3) Filtration, rotary evaporation and concentration to obtain the purified compound represented by formula II.
The method of claim 10, wherein the reaction conditions of the reaction are as follows:

In step (1), the protein inactivation temperature is 55-65°C; preferably 60°C; the time is 0.5-1.5h; diatomite is used for filtration, and the amount of diatomite is preferably 8%-12%;

In step (2), dry with anhydrous sodium sulfate, and wash the organic layer with water and 5% brine.
The carbonyl reductase mutant according to any one of claims 1 to 4 or the genetically engineered bacterium according to claim 7 or the carbonyl reductase according to claim 8 is combined in the preparation of a compound represented by formula II Applications.