A method for synthesizing chiral amino alcohol compounds

Abstract

The application discloses a synthesis method of chiral amino alcohol compounds. The synthesis method of the chiral amino alcohol compounds comprises the following steps: in the presence of a carbonyl reductase and a coenzyme, a carbonyl compound shown as formula II is subjected to a reduction reaction shown as formula I in an organic solvent and a buffer solution to obtain a conversion solution containing the chiral amino alcohol compound shown as formula I. 1 The application uses a racemic aziridine chiral compound as a starting material, and through enzyme reaction of recombined carbonyl reductase, a single configuration chiral amino alcohol is efficiently synthesized, an important intermediate with a high de value is obtained, and production cost is reduced.

Description

Technical Field

[0001] This invention relates to a method for synthesizing chiral amino alcohol compounds. Background Technology

[0002] Florfenicol is a broad-spectrum chloramphenicol antibiotic for animal use, developed by Nagab-hushan et al. of Schering-Plough in the late 1970s. Given its superior efficacy compared to chloramphenicol and thiamphenicol in the prevention and treatment of animal diseases, florfenicol has a broader application prospect, and its synthesis has received considerable attention.

[0003] In recent years, research on the synthesis of chiral compounds using enzymatic reduction has been extensive. Our company has invented two Chinese patents, CN102827042A and CN103936638A, during the development of a new process for florfenicol. Among them, patent CN103936638A contains a very important intermediate, a single-configuration chiral amino alcohol, which is synthesized via chemical methods requiring chemical resolution and chiral reduction, resulting in low efficiency.

[0004] The synthetic route in patent CN103936638A is as follows:

[0005]

[0006] The synthetic route disclosed in patent CN106316898B is as follows:

[0007]

[0008] The above method obtains chiral amino alcohols with different configurations by using different reducing reagents. The chiral amino carbonyl groups are obtained by chemical resolution and require repeated recrystallization to obtain chiral amino carbonyl compounds with a single configuration. Summary of the Invention

[0009] The problem this invention aims to solve is to overcome the deficiency of existing methods for preparing chiral amino alcohols, the key intermediate of florfenicol, by using a single method. This method provides a biosynthetic method for chiral amino alcohol compounds. It utilizes a microbial / enzyme catalysis technique, employing an achiral precursor and a biocatalytic synthesis system, to prepare single chiral amino alcohol compounds through biocatalysis and transformation. The biocatalytic synthesis system used in this method is characterized by high efficiency, low cost, and no pollution; it exhibits high efficiency, high yield, and low cost, and has significant commercial application value.

[0010] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0011] This invention provides a method for synthesizing chiral amino alcohols, comprising the following steps: in an organic solvent and a buffer solution, in the presence of a carbonyl reductase and a coenzyme system, a carbonyl compound as shown in Formula II is subjected to a reduction reaction as shown below to obtain a conversion solution containing a chiral amino alcohol compound as shown in Formula I; wherein the carbonyl reductase is derived from Rhodotorula toruloides.

[0012]

[0013] Among them, R 1 It is (S)-1-phenylethyl and / or (R)-1-phenylethyl;

[0014] * indicates a chiral carbon, which is in the S configuration or a mixture of S and R configurations.

[0015] In one embodiment of the present invention, the carbonyl reductase (accession number CGMCC 2.1389) from Rhodotorula toruloides is selected from the following group:

[0016] (i) Its amino acid sequence is shown in SEQ ID NO.1;

[0017] And / or, (ii) to replace, delete, alter, insert or add one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 within the range of maintaining enzyme activity, to obtain an amino acid sequence, for example, that is more than 95% homologous to the amino acid sequence shown in SEQ ID NO.1.

[0018] In one embodiment of the present invention, the gene sequence encoding the carbonyl reductase is selected from the following group:

[0019] (a) The sequence shown in SEQ ID NO.2;

[0020] (b) a polynucleotide complementary to the sequence defined in (a); or

[0021] (c) and (a) define any polynucleotide or complementary sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99% or more of sequence identity.

[0022] In one embodiment of the present invention, the carbonyl reductase may be in a conventional form for this type of reaction in the art, such as a free enzyme, lyophilized powder, lysed enzyme solution, immobilized enzyme, or enzyme in cell form (carbonyl reductase genetically engineered bacteria whole cell); preferably in the present invention, it is a genetically engineered bacteria whole cell lysed enzyme solution containing the carbonyl reductase.

[0023] In one aspect of the present invention, when the carbonyl reductase is a whole-cell lysis enzyme solution of genetically engineered bacteria containing the carbonyl reductase, the mass ratio of the carbonyl compound as shown in Formula II to the enzyme solution can be 2:1.

[0024] In one aspect of the present invention, in the reduction reaction, when the carbonyl reductase is a whole-cell lysis enzyme solution of genetically engineered bacteria containing the carbonyl reductase, the concentration of the whole-cell lysis enzyme solution of genetically engineered bacteria can be 50 g / L-100 g / L (for example, 50 g / L); the enzyme activity of the carbonyl reductase can be 100 μ / mg.

[0025] In one embodiment of the present invention, the carbonyl reductase is preferably (R,R)-carbonyl reductase.

[0026] The organic solvent can be a conventional organic solvent used in this type of reaction in the art. In this invention, it is preferably one or more of DMSO, DMF, 3,3',5,5'-tetramethylbiphenyl dihydroxyethyl ether (TMBE), and methanol. The amount of the organic solvent used can be conventional for this type of reaction, as long as it does not affect the reaction, for example, enough to dissolve the substrate. In one aspect of this invention, in the reduction reaction, the volume percentage concentration of the organic solvent in the reaction system is % to 10% (e.g., 5%).

[0027] In this invention, the buffer solution can be a conventional buffer solution used in this type of reaction in the art, such as phosphate buffer solution, carbonate buffer solution, Tri-HCl buffer solution, borate buffer solution, glycine buffer solution, citrate buffer solution or MOPS buffer solution; in one embodiment of this invention, Tris-hydrochloric acid buffer solution (Tris: tris(hydroxymethyl)aminomethane, for example, concentration of 0.05 mol / L, 25°C) is preferred.

[0028] In one aspect of the present invention, the pH value of the buffer solution may be a conventional pH value for this type of reaction in the art, such as 7.0-9.0; preferably pH=8.0.

[0029] In one embodiment of the present invention, the molar percentage of the coenzyme in the coenzyme system to the carbonyl compound as shown in Formula II may be 0.01%-1.0%, preferably 0.1%-0.6%.

[0030] In one aspect of the present invention, in the reduction reaction, the concentration of the coenzyme in the coenzyme system in the reaction system can be 1 mM-10 mM (e.g., 5 mM).

[0031] In one aspect of the present invention, the coenzyme in the coenzyme system is selected from the group consisting of: reducing coenzymes, oxidizing coenzymes, or combinations thereof; for example, NADH, NADPH, NAD. + NADP + or combinations thereof; preferably NADH and NAD. + or combinations thereof. When the coenzyme in the coenzyme system contains NAD... + The device may also include an enzyme (dehydrogenase) for the regeneration of the coenzyme and a corresponding cosubstrate (hydrogen donor); the regenerated enzyme may be selected from the group consisting of alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, or combinations thereof; the cosubstrate may be selected from the group consisting of isopropanol, glucose, ammonium formate, or combinations thereof; preferably, the regenerated enzyme and its corresponding cosubstrate are glucose dehydrogenase (EC1.1.1.47) and glucose.

[0032] In one aspect of the present invention, when the coenzyme contains NAD + When the coenzyme comprises a regenerated enzyme and its corresponding cosubstrate, the molar ratio of the cosubstrate to the carbonyl compound as shown in Formula II may be a conventional molar ratio in this type of reaction in the art, for example, 1.5:1.

[0033] In one embodiment of the present invention, the concentration of the co-substrate in the reduction reaction may be 90 g / L.

[0034] In one aspect of the present invention, during the reduction reaction, when the coenzyme in the coenzyme system contains NAD... + When using the regenerated enzyme and its corresponding co-substrate, the concentration of the regenerated enzyme in the reaction system can be 25 mg / L; the enzyme activity of the regenerated enzyme can be 500 μ / mg.

[0035] In one aspect of the present invention, the temperature of the reduction reaction can be a conventional temperature for this type of reaction in the art, such as 10°C-50°C, preferably 20°C-40°C, and more preferably 25°C-35°C.

[0036] In one aspect of the present invention, the reduction reaction time can be 1-120 hours, preferably 5-72 hours, and more preferably 15-24 hours.

[0037] In one embodiment of the present invention, the method for synthesizing the chiral amino alcohol compound includes the following steps: in an organic solvent and a Tris-hydrochloric acid buffer solution, controlling the pH to 8.0, in the presence of a carbonyl reductase and a coenzyme system, a carbonyl compound as shown in Formula II is subjected to the reduction reaction shown, to obtain a conversion solution containing a chiral amino alcohol compound as shown in Formula I; the carbonyl reductase is derived from Rhodotorula glutinis; the coenzyme system includes NAD+ as an oxidizing coenzyme.+ The enzyme used for coenzyme regeneration is glucose dehydrogenase, and its corresponding co-substrate is glucose. Preferably, the amino acid sequence of the carbonyl reductase is as shown in SEQ ID NO.1, "and / or" has more than 95% homology with the amino acid sequence shown in SEQ ID NO.1.

[0038] In one embodiment of the present invention, the synthesis method further includes: after the reduction reaction is completed, separating the chiral amino alcohol compound as shown in Formula I from the conversion solution. The separation step can be a conventional separation step in this type of reaction in the art, which may include: centrifuging the bacterial cells, adding 5% NaOH to the resulting supernatant, filtering to remove the precipitated solid, extracting the remaining conversion solution with an organic solvent (e.g., dichloromethane), washing (e.g., water, saturated brine), drying (e.g., conventional anhydrous sodium sulfate), and concentrating to obtain the chiral amino alcohol compound as shown in Formula I. The organic solvent can be a conventional organic solvent in this type of extraction in the art, such as dichloromethane.

[0039] In one aspect of the present invention, the de value of the chiral amino alcohol compound as shown in Formula I is ≥90%, preferably ≥95%, and more preferably ≥99%.

[0040] In one aspect of the invention, the conversion of the carbonyl compound of Formula II to the chiral amino alcohol compound of Formula I is ≥80%, for example ≥85%, preferably ≥95%, and more preferably ≥99%.

[0041] The present invention also provides a reaction system comprising:

[0042] Organic solvents, buffer solutions, carbonyl reductases, coenzymes, enzymes used for coenzyme regeneration and their corresponding cosubstrates, and carbonyl compounds as shown in Formula II;

[0043] Organic solvents, buffer solutions, carbonyl reductases, coenzymes, enzymes used for coenzyme regeneration and their corresponding cosubstrates, and carbonyl compounds as shown in Formula II are defined as described above.

[0044] In one embodiment of the present invention, the reaction system comprises the following:

[0045] Organic solvents, buffer solutions, carbonyl reductases, coenzymes, enzymes used for coenzyme regeneration and their corresponding cosubstrates, and carbonyl compounds as shown in Formula II.

[0046] the term

[0047] Enantiomeric excess (ee): This is usually used to characterize the excess of one enantiomer relative to another in a chiral molecule.

[0048] Diastereomeric excess (de): This is usually used to characterize the excess of one diastereomer relative to another in a molecule with two or more chiral centers.

[0049] In this invention, "carbonyl reductase" refers to an enzyme capable of stereoselectively and asymmetrically catalyzing the reduction of prochiral ketones to chiral alcohols. Stereoselectivity is defined as enantiomeric excess (ee) ≥ 80% and diastereomeric excess (de) ≥ 80%.

[0050] In this invention, the definition of configuration is based on chiral amino alcohols as shown in Formula I, wherein the configuration of the hydroxyl group is R and the configuration of the chiral carbon on the aziridine is R. Any carbonyl reductase that can stereoselectively recognize the chiral carbon on the aziridine with the R-configuration in the carbonyl compound shown in Formula II and reduce the carbonyl group in the carbonyl compound shown in Formula II to the R-configuration hydroxyl group is defined as (R,R)-carbonyl reductase in the technical solution of this invention.

[0051] In this invention, the carbonyl reductase can be wild-type or mutant. Furthermore, it can be isolated or recombinant.

[0052] The carbonyl reductases used in this invention can be derived from different species. The amino acid sequence of a typical carbonyl reductase is shown in SEQ ID No. 1, and its encoding gene is shown in SEQ ID No. 2.

[0053] SEQ ID No.1

[0054]

[0055] SEQ ID No.2

[0056]

[0057] The carbonyl reductase of the present invention also includes an amino acid sequence obtained by substituting, deleting, altering, inserting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 while maintaining enzyme activity.

[0058] In this invention, the carbonyl reductase can be used in various forms. For example, resting cells or wet bacterial cells expressing the carbonyl reductase of this invention can be used, as well as various other forms such as crude enzyme solution, pure enzyme, or crude enzyme powder, or immobilized enzyme. Preferably, to obtain higher conversion efficiency and reduce costs, crude enzyme solution is preferred, such as whole-cell lysis enzyme solution of genetically engineered bacteria containing the carbonyl reductase.

[0059] In this invention, "coenzyme" refers to a coenzyme capable of electron transfer in redox reactions. The coenzyme in this invention is a reducing coenzyme NADH, NADPH, or an oxidizing coenzyme NAD. + NADP + Due to the high cost of reducing coenzymes, oxidized coenzyme NAD+ is preferred. + NADP + When selecting an oxidative coenzyme, it is necessary to choose a method to achieve coenzyme regeneration, which mainly includes three types: (1) glucose dehydrogenase and glucose as a cosubstrate; (2) alcohol dehydrogenase and isopropanol as a cosubstrate; and (3) formate dehydrogenase and ammonium formate as a cosubstrate. In a preferred embodiment, the coenzyme contains NAD. + The coenzyme regeneration system consists of glucose dehydrogenase and oxidative coenzyme NAD. + The molar percentage of the dosage to the substrate is 0.01%–1.0%, and the buffer system is a 0.05 mol / L Tris-hydrochloric acid buffer solution. The pH of the buffer solution is 7.0–9.0.

[0060] In this invention, a co-solvent may or may not be added to the reaction system. The term "co-solvent" refers to a substance that, when a poorly soluble substance reacts with a third substance added to the solvent, forms a soluble intermolecular complex, association, or double salt, thereby increasing the solubility of the poorly soluble substance in the solvent. This third substance is called a co-solvent.

[0061] In this invention, unless otherwise specified, the concentration refers to the final concentration of the compound in the entire reaction system before the reaction.

[0062] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0063] The carbonyl reductase and 1-phenylethyl-2-[4-(methylsulfonyl)phenyl]formylaziridine of the present invention are both self-made, and other reagents and raw materials used are commercially available.

[0064] The significant advantages of this invention are as follows: It utilizes an enzymatic asymmetric reduction reaction to prepare chiral amino alcohols, a crucial intermediate in the preparation of florfenicol, avoiding chemical resolution and chiral reduction. The desired chiral amino alcohol compound is obtained directly through the specificity and particularity of the enzyme, thus shortening the process steps and facilitating industrial production. The method of this invention exhibits strong stereoselectivity, mild reaction conditions, simple operation, low cost, minimal pollution, low catalyst usage, high efficiency, yield >90%, and high product optical purity (chiral de value >99.5%). Furthermore, it constructs two chiral centers in a single reaction, thereby greatly improving production efficiency and reducing production costs. Detailed Implementation

[0065] The present invention is further illustrated below by way of examples, but the invention is not limited to the scope of the examples described herein. Experimental methods not specifically described in the following examples were performed according to conventional methods and conditions (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or as selected according to the product instructions.

[0066] The carbonyl reductase and 1-phenylethyl-2-[4-(methylsulfonyl)phenyl]formylaziridine in the following examples were prepared in-house and are only one example. Other sources are also applicable to this invention as long as they meet the requirements for carbonyl reductase, coenzyme and 1-phenylethyl-2-[4-(methylsulfonyl)phenyl]formylaziridine.

[0067] Unless otherwise specified, the experimental methods used in this invention are conventional methods.

[0068] The HPLC analysis method was as follows: Column: Yuexu Ultimate LP-C18 (4.6*250mm, 5μm); Column temperature: 35℃; Flow rate: 0.8mL / min; Detection wavelength: 224nm; Mobile phase: Acetonitrile: 50mM potassium dihydrogen phosphate buffer (pH=5.5) = 4:6. Isocratic elution was used.

[0069] In this invention, the construction of the whole cell of the genetically engineered bacterium containing the carbonyl reductase can be carried out using conventional methods in the art, for example, the steps are as follows:

[0070] 1. Optimization of carbonyl reductase genes

[0071] The basic method involves optimizing the codons of the carbonyl reductase gene based on the complete genome sequence of *Rhodotorula toruloides*, eliminating some infrequently used codons, and simultaneously optimizing the primary structure of the mRNA through synonymous conversion, enabling the ribosome to translate smoothly along the start codon. Synonymous conversion is also used to eliminate Nde I and Xho I restriction sites. The carbonyl reductase gene is then synthesized through complete genome synthesis.

[0072] 2. Construction of carbonyl reductase gene expression vector

[0073] The basic method is to synthesize an optimized carbonyl reductase gene through whole-genome synthesis, and then, according to the designed restriction sites, link the carbonyl reductase gene fragment to the pET-28a plasmid to construct a recombinant plasmid.

[0074] 3. Construction of recombinant carbonyl reductase expression strains

[0075] The basic method involves transforming the constructed recombinant plasmid into Escherichia coli to obtain recombinant E. coli that overexpress carbonyl reductase, i.e., whole cells of engineered bacteria containing carbonyl reductase.

[0076] Example 1

[0077] Step (1) uses a genetically engineered strain derived from the carbonyl reductase of Rhodotorula toruloides. The specific preparation method is as follows:

[0078] (1) Construct an expression vector containing a carbonyl reductase gene:

[0079] Rhodotorula purpureus Toruloides were purchased from the China Culture Collection Center (accession number CGMCC 2.1389). Carbonyl reductase from Rhodotorula glutinis was randomly mutated, and strains with high enzyme activity were screened. The GC content and rare codons were optimized, and the entire genome was synthesized to obtain the carbonyl reductase, resulting in the pET-28a recombinant plasmid. The optimized amino acid sequence is shown in SEQ ID No. 1.

[0080] (2) Transformation of recombinant plasmids into host cells

[0081] The recombinant plasmid was transformed into competent Escherichia coli BL21(DE3) (Tiangen Biotech Co., Ltd.), plated on LB solid medium containing 50 μg / mL kanamycin, and cultured at 37°C for 20-24 h to obtain preliminary positive clones.

[0082] (3) Positive clones were obtained by screening with an resistant culture medium.

[0083] Preliminary positive clones were picked and cultured overnight at 37°C and 200 rpm in 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The plasmids were extracted, digested with restriction endonucleases NdeI and XhoI, verified by electrophoresis, and sequenced. After verification, colonies containing the plasmid were considered positive clones, which were the carbonyl reductase genetically engineered bacteria derived from Rhodotorula glutinis.

[0084] (4) Expression method of carbonyl reductase: The engineered carbonyl reductase bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C in a shaker; then, at an inoculation rate of 2% (v / v), they were transferred into LB liquid medium containing 50 μg / mL kanamycin and cultured until OD. 600 After the concentration was reduced to 0.8, IPTG was added for induction. The final concentration of IPTG was 0.2 mmol / L. The temperature was lowered to 25℃ and induced for 16 h. The cells were then collected by centrifugation to obtain whole cells of the carbonyl reductase genetically engineered bacteria derived from Rhodotorula glutinis.

[0085] The whole cells of genetically engineered bacteria with carbonyl reductase activity were ultrasonically disrupted to obtain a whole-cell disruption enzyme solution of carbonyl reductase genetically engineered bacteria.

[0086] The glucose dehydrogenase used was a commercial enzyme purchased from Sigma-Aldrich (CAS: 9028-53-9).

[0087] Step (2) Enzymatic conversion reaction

[0088]

[0089] The reaction was carried out in a 1 L shake flask with a reaction volume of 300 mL. 1-Phenethyl-2-[4-(methylsulfonyl)phenyl]formylaziridine (33.0 g, 0.1 mol) was dissolved in 15 mL DMSO, and glucose (27.0 g, 0.15 mol) was added. Tris-hydrochloric acid buffer solution (pH 8.0) was used as the solvent. Whole cells of genetically engineered Rhodotorula glutinis derived from carbonyl reductase and glucose dehydrogenase were used as catalysts (500 μg / mg, 25 mg / L). Coenzyme NAD+ was also added. +The concentration of carbonyl reductase (100 μ / mg) from Rhodotorula glutinis was controlled at 50 g / L. The enzyme activity of the purified reductase protein was determined using a 0.25 ml reaction system containing Tris-HCl, pH 8.0, 2 mmol / L NADH, 0.1 mmol / L 1-phenylethyl-2-[4-(methylsulfonyl)phenyl]formylaziridine compound, and an appropriate amount of enzyme. The decrease in absorbance at 340 nm was measured. Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the oxidation of 1 μmol NADH per minute under the above conditions. + The concentration was 5 mM. The pH of the conversion system was controlled at 8.0, the conversion temperature at 30℃, the shaking speed at 160 r / min (monitored by HPLC until the starting material disappeared), and the conversion time was 18 h. After conversion, a conversion solution containing (R)-[4-(methylsulfonyl)phenyl][(R)-1-phenylethyl-aziridin-2-yl]methanol was obtained.

[0090] Step (3) Product preparation

[0091] The conversion solution containing compound (R)-[4-(methylsulfonyl)phenyl][(R)-1-phenylethyl-aziridin-2-yl]methanol obtained in step (2) was purified by the following steps: Cells were collected by centrifugation, and 5% NaOH was added to the supernatant. After stirring for half an hour, a large amount of white solid was obtained. After filtration, the reaction solution was extracted twice with dichloromethane. The organic layers were combined, washed once with water, and then washed once with saturated brine. After drying with anhydrous sodium sulfate, the product was evaporated to dryness to obtain 29.7 g. The conversion rate was 99.3%, yielding (R)-[4-(methylsulfonyl)phenyl][(R)-1-R1-aziridin-2-yl]methanol with an ee value of 99.8%, a DE value of 99.5%, and a yield of 90%. The retention time of the target product [4-(methylsulfonyl)phenyl][(R)-1-phenylethyl-aziridin-2-yl]methanol was 6.90, and the retention time of (R)-[4-(methylsulfonyl)phenyl][(S-1-phenylethyl-aziridin-2-yl]methanol was 4.76. SEQUENCE LISTING <110> Hubei Meitian Biotechnology Co., Ltd. <120> A method for synthesizing chiral amino alcohols <130> P19015436C <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> carbonyl reduction <400> 1 Met Ser Ser Pro Thr Pro Asn Val Tyr Val Ile Ser Gly Ala Ser Arg 1 5 10 15 Gly Ile Gly Phe Ala Ile Thr Ser Ile Leu Ala Gln Arg Asp Asn Val 20 25 30 Leu Ile Phe Ala Gly Ala Arg Asp Leu Lys Ser Thr Gln Leu Asn Glu 35 40 45 Leu Ala Leu Lys Ser Gly Gly Lys Val Val Pro Val Lys Leu Glu Ser 50 55 60 Thr Ser Val Glu Asp Ala Ala Ala Leu Ala Lys Val Val Glu Glu Lys 65 70 75 80 Ala Gly Lys Val Asp Tyr Val Leu Ala Val Ala Gly Ile Ser Gln Ser 85 90 95 Thr Asp Pro Ile Ala Gln Val Pro Leu Asp Asp Val Arg Arg His Phe 100 105 110 Glu Val Asn Thr Ile Gly Pro Leu Val Leu Phe Gln Ser Leu Leu Ala 115 120 125 Leu Leu Thr Lys Ser Ser Ala Pro His Phe Ile Val Val Ser Thr Ile 130 135 140 Ala Gly Ser Ile Ala Ser Met Pro Gln Phe Leu Phe Pro Val Ser Ser 145 150 155 160 Tyr Ala Ile Ser Lys Thr Ala Val Asn Ser Ala Val Val Arg Ile Ala 165 170 175 Val Glu His Pro Asp Leu Asp Ala Phe Val Cys His Pro Gly Val Val 180 185 190 Ser Ser Asp Met Ile Lys Glu Tyr Val Ala Lys Thr Gly Thr Ala Leu 195 200 205 Ser Asp Phe Glu Ser Met Gly Met Ile Thr Pro Glu Glu Ser Ala Ala 210 215 220 Ser Leu Val Lys Leu Phe Asp Gly Ala Lys Lys Glu Thr His Ser Gly 225 230 235 240 Lys Phe Phe Asn Val Asp Gly Thr Phe Leu Pro Trp 245 250 <210> 2 <211> 759 <212> DNA <213> Artificial Sequence <220> <223> Carbonyl reductase gene <400> 2 atgagcagcc cgaccccgaa cgtgtacgtt atcagcggtg cgagccgtgg tattggcttc 60 gcgatcacca gcattctggc gcagcgtgac aacgtgctga tctttgcggg cgcgcgtgat 120 ctgaagagca cccaactgaa cgagctggcg ctgaagagcg gtggcaaagt ggttccggtt 180 aagctggaga gcaccagcgt ggaagatgct gcggcgctgg cgaaagtggt tgaggaaaag 240 gcgggtaaag tggactatgt tctggcggtg gcgggtatca gccagagcac cgatccgatt 300 gcgcaagttc cgctggacga tgtgcgtcgt cacttggaag ttaacaccat cggtccgctg 360 gtgctgtttc agagcctgct ggcgctgctg accaagagca gcgcgccgca ctttattgtg 420 gttagcacca tcgcgggcag cattgcgagc atgccgcaat tcctgtttcc ggtgagcagc 480 tacgcgatca gcaaaccgc ggttaacagc gcggtggttc gtattgcggt ggagcacccg 540 gacctggatg cgttcgtttg ccacccgggt gtggttagca gcgacatgat caaggagtat 600 gttgcgaaaa ccggtaccgc gctgagcgat ttcgaaagca tgggcatgat taccccggag 660 gaaagcgcgg cgagcctggt gaagctgttt gacggcgga agaagaaac ccacagcggt 720 aaattcttta acgttgatgg caccttcctg ccgtggtaa 759

Claims

1. A method for synthesizing chiral amino alcohol compounds, characterized in that, It includes the following steps: In organic solvents and buffer solutions, in the presence of carbonyl reductase and coenzyme, the carbonyl compound shown in Formula II is subjected to the reduction reaction shown below to obtain a conversion solution containing a chiral amino alcohol compound shown in Formula I; the carbonyl reductase is derived from Rhodotorula glutinis (…). Rhodotorula toruloides Carbonyl reductase; ； Among them, R 1 It is (S)-1-phenylethyl and / or (R)-1-phenylethyl; This indicates a chiral carbon, which is in the S configuration or a mixture of S and R configurations; The amino acid sequence of the carbonyl reductase is shown in SEQ ID NO. 1; The organic solvent is one or more of DMSO, DMF, TMBE and methanol; The buffer solution is a phosphate buffer solution, a carbonate buffer solution, a Tris-HCl buffer solution, a borate buffer solution, a glycine buffer solution, a citrate buffer solution, or a MOPS buffer solution.

2. The synthesis method according to claim 1, characterized in that, The carbonyl reductase is an enzyme in free form, lyophilized powder, broken enzyme solution, immobilized enzyme, or enzyme in bacterial cell form.

3. The synthesis method as described in claim 2, characterized in that, The gene sequence encoding the carbonyl reductase is selected from the following group: (a) The sequence shown in SEQ ID NO. 2; (b) a polynucleotide complementary to the sequence defined in (a); or, (c) Any polynucleotide or complementary sequence that has at least 70% sequence identity with the sequence defined in (a); And / or, the carbonyl reductase is a whole-cell lysis enzyme solution of genetically engineered bacteria containing the carbonyl reductase.

4. The synthesis method as described in claim 3, characterized in that, When the carbonyl reductase is a whole-cell lysate of genetically engineered bacteria containing the carbonyl reductase, the mass ratio of the carbonyl compound as shown in Formula II to the enzyme solution is 2:

1. And / or, when the carbonyl reductase is a whole-cell lysis enzyme solution of genetically engineered bacteria containing the carbonyl reductase, the concentration of the whole-cell lysis enzyme solution of genetically engineered bacteria is 50 g / L-100 g / L. And / or, the activity of the carbonyl reductase is 100 μ / mg; And / or, the carbonyl reductase is (R,R)-carbonyl reductase.

5. The synthesis method according to any one of claims 1-4, characterized in that, The volume percentage concentration of the organic solvent is 1% to 10%. And / or, the pH of the buffer solution is 7.0-9.0; And / or, the coenzyme is NADH, NADPH, or NAD. + NADP + or a combination thereof; And / or, the molar percentage of the coenzyme to the carbonyl compound as shown in Formula II is 0.01%-1.0%; And / or, the concentration of the coenzyme is 1 mM - 10 mM; And / or, the temperature of the reduction reaction is 10℃-50℃.

6. The synthesis method as described in claim 5, characterized in that, The organic solvent is DMSO; And / or, the volume percentage concentration of the organic solvent is 5%; And / or, the buffer solution is a Tris-hydrochloric acid buffer solution; And / or, the pH of the buffer solution is 8.0; And / or, the coenzyme is NADH or NAD. + or a combination thereof; And / or, the coenzyme comprises 0.1%-0.6% of the carbonyl compound as shown in Formula II; And / or, the concentration of the coenzyme is 5 mM; And / or, when the system of said coenzyme contains NAD + The coenzyme system also includes an enzyme for regenerating the coenzyme and its corresponding cosubstrate. And / or, the temperature of the reduction reaction is 20℃-40℃.

7. The synthesis method according to claim 6, characterized in that, The coenzyme mentioned is NAD. + ; And / or, when the system of said coenzyme contains NAD + When regenerating the enzyme and its corresponding co-substrate, the regenerated enzyme is selected from the group consisting of alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, or combinations thereof. And / or, when the system of said coenzyme contains NAD + When regenerating enzymes and their corresponding co-substrates, the co-substrates are selected from the group consisting of isopropanol, glucose, ammonium formate, or combinations thereof. And / or, when the system of said coenzyme contains NAD + When regenerating the enzyme and its corresponding co-substrate, the molar ratio of the co-substrate to the carbonyl compound shown in Formula II is 1.5:1; And / or, the temperature of the reduction reaction is 25℃-35℃.

8. The synthesis method according to claim 7, characterized in that, When the coenzyme system contains NAD + When regenerating the enzyme and its corresponding co-substrate, the regenerated enzyme and its corresponding co-substrate are glucose dehydrogenase and glucose. And / or, when the system of said coenzyme contains NAD + When regenerating the enzyme and its corresponding co-substrate, the concentration of the regenerated enzyme is 25 mg / L; the enzyme activity of the regenerated enzyme is 500 μ / mg. And / or, when the system of said coenzyme contains NAD + When regenerating the enzyme and its corresponding co-substrate, the concentration of the co-substrate is 90 g / L.

9. The synthesis method according to any one of claims 1-4, characterized in that, The method for synthesizing the chiral amino alcohols includes the following steps: In an organic solvent and Tris-hydrochloric acid buffer solution, controlling the pH at 8.0, and in the presence of a carbonyl reductase and a coenzyme system, a carbonyl compound as shown in Formula II is subjected to the reduction reaction shown, to obtain a conversion solution containing a chiral amino alcohol as shown in Formula I; the carbonyl reductase is derived from Rhodotorula glutinis; the coenzyme system includes NAD+ as an oxidizing coenzyme. + The enzyme used for coenzyme regeneration is glucose dehydrogenase, and its corresponding co-substrate is glucose. And / or, the synthesis method further includes post-processing, which includes the following steps: after the reduction reaction is completed, the bacterial cells are centrifuged, 5% NaOH is added to the supernatant, the precipitated solid is removed by filtration, the remaining conversion liquid is extracted with an organic solvent, washed, dried, and concentrated to obtain the chiral amino alcohol compound as shown in Formula I.

10. A reaction system, characterized in that, The reaction system includes: Organic solvents, buffer solutions, carbonyl reductases, coenzymes, enzymes used for coenzyme regeneration and their corresponding cosubstrates, and carbonyl compounds as shown in Formula II; The organic solvent, buffer solution, carbonyl reductase, coenzyme, enzyme for coenzyme regeneration and its corresponding cosubstrate, and carbonyl compound as shown in Formula II are defined as in any one of claims 1-9.

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