Immobilized ketoreductase mutants and their use in synthesis of phenylbutazone intermediates

Abstract

The application provides a kind of immobilized ketoreductase, immobilization method and its application in preparation of key intermediate of phenylbutazone, the amino acid sequence of the ketoreductase is as SEQ ID NO.2, preferably ketoreductase mutant SEQ ID NO.16, the immobilized ketoreductase includes resin and corresponding ketoreductase.The ketoreductase mutant of the application and the immobilized ketoreductase prepared therefrom have high enzyme activity when carrying out ketone reduction reaction, when used for preparing key intermediate of phenylbutazone, the solvent is pure water, and the environment is friendly.The immobilized transaminase has high conversion rate, good stereoselectivity, good stability, can be reused, and is easier to operate, thereby reducing production cost, and being conducive to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of molecular sieve synthesis, specifically relating to an immobilized ketone reductase mutant and its application in the synthesis of phenanthracene intermediates. Background Technology

[0002] Phenobarbital is a drug developed by Aspire Biopharmaceuticals of South Korea for the treatment of epilepsy. It was approved by the US FDA on November 21, 2019, and by the European EMA on June 16, 2021. Clinical trial results showed that over 20% of patients in the phenobarbital treatment group were seizure-free, demonstrating significant clinical advantages compared to other similar products. According to IMS data, phenobarbital sales reached $450 million in 2024, with a demand for 4 tons of active pharmaceutical ingredient (API), indicating a market expansion rate that has more than doubled. Currently, only Chengdu Aobang has submitted its API application to the CDE (Center for Drug Evaluation) in China on October 10, 2024, which is insufficient to meet the domestic and international API market demand. Its chemical structure is as follows:

[0003]

[0004] Formula C

[0005] (R)-1-(2-chlorophenyl)-2-(2H-tetrazole-2-yl)ethanol is its key chiral intermediate. Its chemical structural formula is as follows:

[0006]

[0007] Formula B

[0008] Korean patent CN 101228138B discloses a method for preparing (R)-1-(2-chlorophenyl)-2-(2H-tetrazole-2-yl)ethanol (5). The reaction pathway is as follows: 2-bromo-2'-chloroacetophenone (compound 2) is used as a raw material, and compound 3 is prepared by ketone reductase catalysis. However, in the synthesis of 5 from 4, substitution reactions can occur at both the 1 and 2 positions of 1H-tetrazole, generating (R)-1-(2-chlorophenyl)-2-(1H-tetrazole-1-yl)ethane-1-ol and compound 5, respectively. Column chromatography separation is required, resulting in high costs and making it unsuitable for large-scale production. Furthermore, the final optical purity is >98%, which does not meet the quality requirements for the active pharmaceutical ingredient.

[0009]

[0010] Subsequently, the Korean company, Acer, improved the process route in its patents CN102803233B and CN02574821B. Their patents redesigned the process route, employing asymmetric metal catalysis and bio-enzyme catalysis for the compounds, respectively. Ruthenium catalysts are expensive, resulting in high raw material costs and increasing the risk of heavy metal contamination in the finished product. Bio-enzyme catalysis uses crude enzyme solutions for the reaction. The main disadvantages of using crude enzyme solutions or wet bacterial cells, which contain a large amount of free enzymes, are: difficulty in enzyme recovery and reuse, leading to high costs; poor stability, sensitivity to the environment, easy inactivation, and difficulty in storage and transportation; and difficulty in separating the reaction product from the enzyme, complicating downstream purification processes, affecting product purity, and hindering continuous industrial production.

[0011]

[0012] In 2024, Yu Zenghui et al. published a new production process document for cinozylate, which improved the original route 2 by using commercially available ketone reductase. However, the catalytic form still used free enzyme solution, and the substrate concentration was only 110 g / L.

[0013] Based on the above situation, and addressing a series of problems arising from the use of metal catalysts or bio-enzymes in the construction of key chiral centers during the synthesis of phenanthracene and its intermediates, this patent specifically screened and verified ketone reduction from three strains from different sources, and further developed corresponding immobilized ketone reductases, with the reaction occurring in a pure aqueous phase. This simplifies the process, effectively reduces production costs, and possesses significant industrialization advantages. Summary of the Invention

[0014] To address the aforementioned technical problems, this invention aims to solve the following issues inherent in existing technologies using free enzyme catalysis: difficulty in enzyme recovery and reuse, high costs, significant wastewater treatment pressure, severe environmental pollution, poor stability and storage, and difficulties in separating enzymes and products after the reaction, leading to complex downstream purification processes, reduced product purity, and challenges in achieving continuous industrial production. To this end, this invention, through sequence alignment analysis, screened three ketone reductases from different bacterial species, all of which can efficiently and stereoselectively catalyze the conversion of compound A. Furthermore, by designing and identifying key active sites, one of the ketone reductases underwent targeted evolution using site-directed saturation mutagenesis, further enhancing its activity to meet the demands of industrial production. Moreover, to overcome the limitations of free enzymes in industrial applications, this invention develops the application of immobilized ketone reductase in the synthesis of phenanthracene and its key intermediates. This catalytic process uses a single aqueous phase system, making it environmentally friendly. The immobilized enzyme exhibits high conversion rate, excellent stereoselectivity, and operational stability in application, simplifying the process flow, effectively reducing production costs, and possessing significant industrial advantages.

[0015] This invention utilizes bioinformatics methods to compare and analyze a large number of known ketone reductase sequences, screening out multiple ketone reductase genes from different sources. Heterologous expression of these genes was achieved in *E. coli* through genetic engineering, and three wild-type ketone reductases with high conversion rates and high chiral selectivity were verified. Furthermore, one of these ketone reductases underwent directed evolution to further enhance its activity. Further immobilization studies using a specific immobilization resin significantly improved the economic efficiency of these enzymes in the catalytic process of compound A.

[0016] The technical solution of this invention is as follows:

[0017] In a first aspect, the present invention provides a microbacterium derived from algae ( xiguobacterium algae) The ketone reductase is obtained through codon optimization, and its amino acid sequence is shown in SEQ ID NO:2. One or more amino acids are replaced, deleted, altered, inserted, or added to the amino acid sequence shown in SEQ ID NO:2 to obtain an amino acid sequence with ≥90% homology, preferably SEQ ID NO:2.

[0018] Secondly, the present invention provides a mutant of ketone reductase, which is obtained by mutating at three sites: positions 190, 193, and 201 of the amino acid sequence SEQ ID NO:2 ketone reductase.

[0019] Preferably, the mutation involves replacing the alanine residue at position 190 with leucine or methionine, the serine residue at position 193 with alanine or cysteine, and the tyrosine residue at position 201 with phenylalanine or tryptophan.

[0020] More preferably, the ketone reductase mutant is obtained by simultaneously mutating positions 190, 193, and 201 of the ketone reductase in SEQ ID NO:2 to obtain an amino acid sequence as shown in SEQ ID NO:16, and the nucleotide sequence encoding the ketone reductase gene is shown in SEQ ID NO:15.

[0021] Thirdly, the present invention provides an isolated nucleotide encoding the above-mentioned ketone reductase or its active fragment or a mutant of ketone reductase. Preferably, the isolated nucleotide has the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:15, or a variant thereof having the same activity or function as the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:15.

[0022] Fourthly, the present invention also provides a recombinant plasmid. The recombinant plasmid carries a nucleotide sequence with a nucleotide sequence number such as SEQ ID NO:1 or SEQ ID NO:15.

[0023] Fifthly, the present invention provides a genetically engineered bacterium. The genetically engineered bacterium contains a mutant of the ketone reductase described in the first aspect or the ketone reductase described in the second aspect, or the nucleotide described in the third aspect, or the recombinant plasmid described in the fourth aspect.

[0024] Sixthly, an immobilized ketone reductase, the immobilized ketone reductase comprising a resin and the free ketone reductase or its active fragment or a mutant of the ketone reductase; preferably, the ketone reductase and the resin are connected by covalent bonds, the resin being an amino or epoxy resin, more preferably, the resin being LXTE-703 (short-chain amino) or LXTE-707 (amino + cyclic amino).

[0025] Seventhly, the use of the ketone reductase, a mutant of the ketone reductase, the nucleotide, the recombinant plasmid, the genetically engineered bacteria, or the immobilized ketone reductase in the synthesis of the phenanthracene intermediate as shown in Formula B:

[0026]

[0027] Formula B.

[0028] Eighthly, the present invention provides a method for synthesizing phenanthracene, wherein the ketone reductase shown in the first aspect is used to synthesize formula B from formula A, catalyzing the reduction of a carbon group to a hydroxyl group, comprising: reacting compound A in a buffer solution in the presence of an enzyme catalytic system to obtain compound B. Then, phenanthracene is prepared based on the compound shown in formula B.

[0029] ,

[0030] The ketone reductase is the ketone reductase or its active fragment provided in the first aspect, or a mutant of the ketone reductase provided in the second aspect, or a ketone reductase or its active fragment encoded by the nucleotides of the third aspect, or an immobilized ketone reductase of the sixth aspect.

[0031] The ketone reductase or its active fragment or mutant ketone reductase is in the form of whole Escherichia coli cells, Escherichia coli cell lysate, lyophilized or spray-dried enzyme powder.

[0032] The enzyme catalytic system may also include a coenzyme cycle system, wherein the coenzyme cycle system is at least one of the following:

[0033] Lactate dehydrogenase-coenzyme cycle system: includes lactate dehydrogenase, lactate, and coenzyme;

[0034] The glucose dehydrogenase-coenzyme cycle system includes glucose dehydrogenase, glucose, and coenzyme.

[0035] The coenzyme may include reduced coenzyme I or reduced coenzyme II.

[0036] In some preferred embodiments, the coenzyme is reduced coenzyme I.

[0037] The reaction temperature can be 15-45°C. In some embodiments, the reaction temperature is 20-40°C. In some embodiments, the reaction temperature is 35-37°C, and in some preferred embodiments, the reaction temperature is 37°C.

[0038] The pH of the buffer solution can be 6.0-10.0. In some embodiments, the pH of the buffer solution is 6.0-9.0. In some embodiments, the pH of the buffer solution is 7.0-8.0. In some preferred embodiments, the pH of the buffer solution is 7.0.

[0039] The buffer solution may include or be a phosphate buffer solution.

[0040] The concentration of compound A is 10 g / L to 200 g / L, preferably 100 g / L.

[0041] The beneficial effects of this invention are as follows:

[0042] The ketone reductase of this invention exhibits high conversion rate, excellent stereoselectivity, and operational stability in applications. The catalytic process utilizes a single aqueous phase system, making it environmentally friendly. Immobilized enzymes simplify the process flow, effectively reduce production costs, and possess significant industrialization advantages, specifically:

[0043] (1) Through directed evolution technology, the wild-type enzyme activity (conversion rate in 1 hour) is increased by 8-40 times, the substrate concentration is increased from 20 g / L in the wild type to 200 g / L (superior to 110 g / L in the existing technology), and the conversion rate and stereoselectivity are high (optical purity is increased to >99%).

[0044] (2) Pure aqueous phase reaction reduces the use of organic solvents, simplifies downstream purification, and reduces wastewater treatment pressure and production costs.

[0045] (3) Compared with the original route, column chromatography separation is avoided; compared with the metal catalyst route, expensive costs and heavy metal risks are avoided; in the free enzyme base, this patent has studied the immobilized enzyme carrier and immobilization method. Compared with the free enzyme route, while maintaining the same substrate concentration and chiral selectivity as the free enzyme, the stability of the enzyme is improved, the generation of solid waste such as diatomaceous earth is avoided, and the post-processing operation is improved.

[0046] The ee value, or enantiomeric composition of a compound sample, can be described using the terms "enantiomeric excess" or "ee%". It indicates the excess of one enantiomer over another, usually expressed as a percentage. Attached Figure Description

[0047] Figure 1 Equation A is transformed into Equation B;

[0048] Figure 2 Typical conversion graph of ketone reductase catalyzing the conversion of formula A to formula B;

[0049] Figure 3 Comparison of two configurations, A and B;

[0050] Figure 4 Typical spectrum of Bee value detection;

[0051] Figure 5 LC-MS spectra of Formula A;

[0052] Figure 6 LC-MS spectra of formula B. Detailed Implementation

[0053] The technical solution of the present invention will be further explained and described below with reference to specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention should be determined by the contents of the claims. Modifications and substitutions made by those skilled in the art to the technical solution of the present invention without creative effort all fall within the scope of protection of the present invention.

[0054] Example 1: Source of ketoreductase and construction of expression vector

[0055] Based on the reported amino acid sequences of ketone reductases and a codon optimization algorithm, a ketone reductase (KRED) encoding gene for expression in *E. coli* was designed. The codon-optimized gene and its encoded polypeptide are listed in Table 1. The complete gene sequence was synthesized, and restriction enzyme sites were designed at both ends. Bam HI and Hin dIII, subcloned into the corresponding expression vector pET-28a(+) Bam HI and Hin dIII, placing the expression of the ketone reductase gene in the T7 promoter and lacI Recombinant expression plasmids were obtained under the control of repressor genes.

[0056] Table 1

[0057]

[0058] Example 2 Construction and Induced Expression of Recombinant Engineered Bacteria

[0059] The constructed recombinant expression plasmid was transformed into E. coli. E. coli BL21(DE3) competent cells were plated on LB agar plates with kanamycin resistance (final concentration of 50 μg / mL) and cultured overnight at 37°C. Single colonies were picked from the plates and transferred to LB liquid medium for 8 h of culture. The cells were collected and plasmids were extracted for sequencing verification.

[0060] The correctly sequenced engineered strain was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 200 rpm for 8 h to obtain a seed culture. Subsequently, the seed culture was transferred at a 2% (v / v) inoculation rate to fresh LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm to obtain an OD concentration. 600 To a final concentration of 0.2 mmol / L, IPTG was added to induce ketoreductase protein expression. The cells were cultured at 25°C and 200 rpm for 15 h, then centrifuged at 4°C and 12000 rpm for 10 min to collect the bacterial cells. The cells were then resuspended in 100 mM pH 7.0 sodium phosphate buffer at a ratio of buffer:cells = 10:1, and sonicated to obtain the crude enzyme solution, which was stored at -20°C for later use.

[0061] Example 3 Screening for the synthesis of (R)-1-(2-chlorophenyl)-2-(2H-tetrazole-2-yl)ethanol (Formula B) catalyzed by recombinant engineered bacteria

[0062] The crude enzyme solution obtained in Example 2 was used to catalyze the synthesis of (R)-1-(2-chlorophenyl)-2-(2H-tetrazole-2-yl)ethanol. The screening reaction system was as follows: 4.5 ml of 100 mM sodium phosphate buffer (adjusted to pH 7.0) and 0.1 g of substrate were added to a 10 ml EP tube and stirred until homogeneous. Then, 0.125 g of glucose, 10 mg of NADP, and 10 mg of glucose dehydrogenase were added. Finally, 0.5 ml of the crude ketoreductase solution prepared in Example 2 was added, with a substrate concentration of 20 g / L. The reaction conditions were 37 °C and 180 rpm. Samples were taken at 1 h and 24 h for analysis. The preferred sequence SEQ ID NO:2 was determined to have better conversion rate and ee value, as shown in Table 2.

[0063] Conversion rate: The percentage of product calculated using the area normalization method.

[0064] The detection method is as follows: Thermo Fisher Vanquish; column: Waters XBridge C18, 4.6*150mm 3.5um; flow rate: 1ml / min; detection wavelength: 210nm; column temperature: 35℃; mobile phase A: 10mM KHPO4 solution (pH 9.2): acetonitrile = 95:5; mobile phase B: acetonitrile; gradient table: 0-22min, 5%B-70%B, 22-27min, 70%B-70%B, 27-27.1min, 70%B-5%B, 27.1-32min, 5%B-5%B. Figure 2 Typical conversion graphs of formula A to formula B catalyzed by ketone reductase; Figure 3 Comparison chart of the two configurations of formula B; Figure 4 This is a typical spectrum for Bee value detection. Figure 5 The LC-MS spectrum of Equation A; Figure 6 The LC-MS spectrum of Equation B is shown.

[0065] ee value: S represents configuration enantiomer;

[0066] The ee value detection method is as follows: Agilent 1260, column: Daicel CHIRALPAK AD-H, 4.6*250mm, 5um; flow rate: 1mL / min; detection wavelength: 210nm; column temperature: 25℃; mobile phase: ethanol: n-hexane = 30:70 (v:v). The screening results are shown in Table 2.

[0067] Table 2

[0068]

[0069] Based on the conversion rate and ee value results shown in the table above, the preferred sequence is SEQ ID NO:2.

[0070] Example 4 Construction and screening of SEQ ID NO:2 ketoreductase mutant

[0071] To further improve the enzyme activity and substrate concentration of SEQ ID NO:2, the sequence was subjected to targeted mutation through computer simulation and experimental verification.

[0072] (1) First round of mutant screening

[0073] Based on the enzyme gene sequence and three-dimensional structure analysis, key amino acid sites in SEQ ID NO:2 were identified, and combined saturation mutations were performed on alanine at position 190 and serine at position 193. Based on the codon bias of *E. coli*, targeted mutation primers were designed (see Table 3), and whole-plasmid PCR amplification was performed.

[0074] Table 3. Primers for full-plasmid PCR amplification in the first round of site-directed saturation mutagenesis

[0075]

[0076] Using the recombinant expression plasmid pET28a-KRED as a template, site-directed mutagenesis was performed using whole plasmid PCR.

[0077] The PCR amplification reaction system (50 μL) for saturation mutation was as follows: 1 ng of recombinant plasmid template, 10 μM of each of the two mutant primers, 10 mM dNTP Mix, 25 μL of 2×Max Buffer, 1 μL of Phanta Max Super-Fidelity DNA Polymerase (2 U / 50 μL), and ddH2O added to 50 μL. The PCR reaction procedure was as follows: (1) 95℃ pre-denaturation for 30 s; (2) 95℃ denaturation for 15 s; (3) 72℃ annealing for 15 s; (4) 72℃ extension for 7 min. Steps (2) to (4) were performed for a total of 30 cycles. Finally, the product was extended at 72℃ for 5 min and stored at 4℃. The amplified PCR product was digested with Dpn I at 37°C for 2 hours. The digested product was then efficiently recombined under the catalysis of Exnase II. The recombinant product was transformed into E. coli BL21(DE3) competent cells, plated on kanamycin-resistant (final concentration 50 μg / mL) LB agar plates, and incubated overnight at 37°C. Single colonies were then picked from the plates.

[0078] The crude enzyme solution for mutant expression was prepared according to the method described in Example 2. The conversion rate and ee value of the mutant were then determined according to the method described in Example 3, with the screening pressure increased to 50 g / L while keeping other conditions unchanged. The results are shown in Table 4. Replacing the alanine residue at position 190 of SEQ ID NO:2 ketoreductase with leucine and methionine, and replacing the serine residue at position 193 with alanine and cysteine, significantly improved the substrate conversion rate of the mutant, especially for E. coli BL21(DE3) / pET28a-KRED. A190M / S193C It has greater application potential in the catalytic preparation of (R)-1-(2-chlorophenyl)-2-(2H-tetrazole-2-yl)ethanol compounds.

[0079] Table 4. Effects of the first round of mutation on conversion rate and ee value (partial)

[0080]

[0081] (2) Second round of mutant screening

[0082] The KRED mutant, which exhibits improved enzyme activity and stereoselectivity in the first round of mutations. A190M / S193CBased on the primers in Table 5, the recombinant expression plasmid pET28a-KRED was used. A190M / S193C Using it as a template, site-directed mutagenesis was performed using whole plasmid PCR.

[0083] Table 5. Primers for full-plasmid PCR amplification of the second round of site-directed saturation mutagenesis

[0084]

[0085] The site-directed mutants were subjected to activity assays according to the method in Example 3, with the screening pressure further increased to 100 g / L while keeping other conditions unchanged. The results are shown in Table 6. The results indicate that after saturation superposition mutagenesis at site 201, the optimal mutant for the second round of mutagenesis was KRED. A190M / S193C / Y201W The conversion rate was 99.8%, and the ee value was 99.9%, significantly better than the wild type. Specific data is shown in the table below. KRED A190M / S193C / Y201W The nucleotide and amino acid sequences are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively.

[0086] Table 6. Effects of the second round of mutation on conversion rate and ee value (partial)

[0087]

[0088] Example 5: Preparation of (R)-1-(2-chlorophenyl)-2-(2H-tetrazole-2-yl)ethanol (Formula B) catalyzed by crude ketoreductase solution. SEQ ID NO:16

[0089] 100g of substrate was added to 450mL of 100mM sodium phosphate buffer (adjusted to pH 7.0) and stirred until homogeneous. Then, 125g of glucose, 1g of NADP, and 1g of glucose dehydrogenase were added. Finally, 50mL of crude SEQ ID NO:16 ketoreductase solution prepared in Example 2 was added, with a substrate concentration of 200g / L. The reaction conditions were 37℃, and the pH was controlled to 7.0 by adding 1M sodium hydroxide solution. After 4 hours, a sample was taken for testing, and the conversion rate was 99.72%. 1L of dichloromethane was added to the reaction solution, and hydrochloric acid was added dropwise to adjust the pH to 1.0. Then, 100g of diatomaceous earth was added, stirred, and filtered. The filtrate was allowed to stand and separate into layers. The solution was washed twice with 400mL of water and desolventized under reduced pressure to obtain an oily substance. Add 100 mL of isopropanol, stir to dissolve, cool to 0 °C, add 1 L of n-heptane dropwise, precipitate, keep warm and stir for 2 h, filter to obtain wet product, dry under vacuum at 50 °C to obtain 79.5 g of dry product with purity ≥99% and ee value ≥99.9%.

[0090] Example 6: Screening and preparation of immobilized enzyme vectors.

[0091] Take 10g of SEQ ID NO:16 ketoreductase wet cells, add 100M 0.1M pH7.0 phosphate buffer, mix well, high-pressure homogenize and disrupt the cells, centrifuge to collect the supernatant enzyme solution, then add 0.02g PLP (pyridoxal phosphate), stir to dissolve, add 10g resin (resin activated according to the corresponding instructions, specific models are shown in Table 7, all purchased from Lanxiao Technology Co., Ltd.) for fixation, fix at 25℃, 180rpm for 18h, filter, wash with buffer, and filter to obtain the corresponding immobilized enzyme. The conversion rate detection method is as follows:

[0092] In 20 ml of 0.1 M sodium phosphate buffer (adjusted to pH 7.0), 2 g of substrate was added and stirred until homogeneous. Then, 125 g of glucose, 100 mg of NADP (coenzyme II-amyl adenine dinucleotide phosphate), and 100 mg of glucose dehydrogenase were added, and the mixture was preheated to 37 °C. 2 g of immobilized enzyme was weighed and added to the preheated reaction solution, and the reaction was carried out at 37 °C for 1 h. A sample was taken, diluted with acetonitrile, and sent for HPLC analysis to determine the conversion rate. The results are shown in Table 7.

[0093] Table 7

[0094]

[0095] Based on the screening results of immobilized enzyme carriers and the immobilization cost, the immobilized enzyme prepared with LXTE-707 resin was optimized for catalytic reaction.

[0096] Example 7: Preparation of (R)-1-(2-chlorophenyl)-2-(2H-tetrazole-2-yl)ethanol (Formula B) by catalytic reaction of 2-ketoreductase immobilized enzyme in aqueous phase.

[0097] In 1 L of 0.1 M sodium phosphate buffer (adjusted to pH 7.0), 100 g of substrate was added and stirred until homogeneous. Then, 1250 g of glucose, 1 g of NADP, and 1 g of glucose dehydrogenase were added, and the mixture was preheated to 37 °C. 100 g of immobilized enzyme KRED51-707 was weighed and added to the preheated reaction solution. The reaction was carried out at 37 °C, with the pH controlled at 7.0 using 1 M sodium hydroxide solution. After 24 h, a sample was taken for analysis, and the conversion rate was 97.8%. 500 mL of ethyl acetate was added to the reaction solution, stirred, and filtered. The filtrate was allowed to stand and separate into layers to remove the immobilized enzyme. The sample was washed twice with 200 mL of water and desolvated under reduced pressure to obtain an oily substance. The resulting oily substance was added to 50 mL of isopropanol, stirred and dissolved, cooled to 0 °C, and 500 mL of n-heptane was added dropwise to precipitate. The mixture was kept warm and stirred for 2 h, filtered, and the wet product was obtained. The wet product was dried under vacuum at 50 °C to obtain 75.5 g of dry product with a purity ≥99% and an ee value ≥99.9%.

[0098] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A mutant of a ketone reductase, characterized in that: The mutant of the ketone reductase was obtained by mutating at three sites: 190, 193 and 201 of the ketone reductase in SEQ ID NO:

2. Specifically, the mutation was to replace the alanine residue at position 190 with methionine, the serine residue at position 193 with cysteine, and the tyrosine residue at position 201 with tryptophan. The mutated amino acid sequence is SEQ ID NO:

16. The mutant of the ketone reductase was used to synthesize the phenanthate intermediate as shown in Formula B: Formula B; The synthesis of phenanthracene involves the following steps: A ketone reductase reacts with the substrate of formula A in a buffer solution in the presence of an enzyme catalyst, with water as the solvent, to obtain compound B. Then, phenanthracene is prepared based on compound B. 。 2. An isolated nucleotide, characterized in that: The nucleotide encodes a mutant of the ketone reductase of claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid carries the nucleotides described in claim 2.

4. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain a mutant of the ketone reductase of claim 1, or the nucleotide of claim 2, or the recombinant plasmid of claim 3.

5. An immobilized ketone reductase, characterized in that, The immobilized ketone reductase is prepared from a resin and a mutant of the ketone reductase of claim 1; the ketone reductase is covalently linked to the resin.

6. The immobilized ketone reductase according to claim 5, characterized in that, The resin is an amino or epoxy resin.

7. The mutant of the ketone reductase according to claim 1, characterized in that, The concentration of compound A is 10 g / L to 200 g / L.

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