Method for preparing (R)-salbutamol

Abstract

The invention relates to a method for preparing (R)-salbutamol. Specifically, L-threonine and a compound 1 are taken as raw materials, a compound 2 is formed under the catalysis of L-threonine transaldolase, the compound 2 is formed into a compound 3 under the catalysis of decarboxylase, on the basis, the compound 3 is taken as a substrate, and the (R)-salbutamol is further synthesized. And the method has the characteristics of low cost of raw materials and auxiliary materials, short process steps, simplicity and convenience in operation, mild reaction conditions and environmental friendliness.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for preparing (R)-salbutamol. Background Technology

[0002] Asthma and chronic obstructive pulmonary disease (COPD) are the most common chronic respiratory diseases. In recent years, the number of patients with asthma and COPD in my country has been increasing. Asthma has now become the second largest respiratory disease in my country, and my country's chronic respiratory diseases are facing a severe situation.

[0003] Asthma is difficult to cure completely; bronchodilators are generally used to relieve or prevent asthma attacks. Commonly used bronchodilators can be divided into six main classes: β2-adrenergic receptor agonists; M-cholinergic receptor antagonists; phosphodiesterase inhibitors; allergy mediator blockers; corticosteroids; and leukotriene receptor antagonists. Salbutamol belongs to the β2-adrenergic receptor agonist class. It selectively stimulates β2-adrenergic receptors in the respiratory system, relaxing the smooth muscles in the airways and achieving a bronchodilation effect, thereby relieving asthma. Salbutamol has a strong and long-lasting bronchodilatory effect with minimal impact on the cardiovascular system, making it a relatively safe and commonly used bronchodilator.

[0004] Salbutamol exists in two configurations: the levorotatory isoform ((R)-salbutamol) and the dextrorotatory isoform ((S)-salbutamol). Studies have shown that the levorotatory isoform binds to β2 receptors 100 times more readily than the dextrorotatory isoform, and its absorption rate in the human body is also significantly higher. Furthermore, the dextrorotatory isoform can cause adverse reactions such as dizziness, headache, and palpitations. The structural formula of levosalbutamol is as follows:

[0005]

[0006] Currently, most synthetic methods for salbutamol employ chemical methods. These methods require expensive catalysts, resulting in high costs, complex reaction steps, long routes, low yields, and severe environmental pollution. CN 116716361A discloses an enzymatic process for the preparation of L-salbutamol, using 2-(tert-butylamino)-1-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl ketone as a substrate, catalyzed by carboxyl reductase to generate (R)-salbutamol, but this method is costly. Research on enzymatic preparations of L-salbutamol is still relatively limited.

[0007] Therefore, there is an urgent need in this field to develop more methods for preparing (R)-salbutamol that have high ee values, low raw material costs, short process steps, simple operation, mild reaction conditions, and are environmentally friendly. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing (R)-salbutamol that has high conversion rate and ee value, low raw material cost, short process steps, simple operation, mild reaction conditions, and is environmentally friendly.

[0009] This invention provides a method for preparing (R)-salbutamol with a conversion rate greater than 95.0%, an ee value greater than 99.0%, low raw material costs, short process steps, simple operation, mild reaction conditions, and environmental friendliness.

[0010] This invention uses 4-hydroxy-3-hydroxymethylbenzaldehyde and L-threonine as raw materials to produce compound 2 under the catalysis of L-threonine aldehyde transoxidase. Compound 2 then undergoes an enzymatic reaction catalyzed by decarboxylase to yield compound 3.

[0011] (R)-4-(2-amino-1-hydroxyethyl)-2-(hydroxymethyl)phenol was isolated and reacted with tert-butane chloride to give (R)-salbutanol.

[0012] In a first aspect of the present invention, a method for preparing compound 2 is provided, comprising the steps of:

[0013]

[0014] (a) In the reaction solvent, L-threonine aldehyde transoxidase is used as a catalyst and pyridoxal phosphate is used as a coenzyme to catalyze the reaction of L-threonine with compound 1 to form compound 2.

[0015] In another preferred embodiment, the L-threonine transaldolase is selected from the group consisting of:

[0016] (a) A polypeptide with an amino acid sequence as shown in any of SEQ ID NO:3-6;

[0017] (b) A polypeptide having the polypeptide function described in (a) formed by replacing, deleting or adding one or more amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residue, of any of the amino acid sequences shown in SEQ ID NO:3-6.

[0018] In another preferred embodiment, the method further includes the step of removing acetaldehyde generated in reaction step (a) using acetaldehyde reductase and acetaldehyde reductase coenzyme.

[0019] In another preferred embodiment, the amino acid sequence of the acetaldehyde reductase is shown in SEQ ID NO:9.

[0020] In another preferred embodiment, the acetaldehyde reductase coenzyme is selected from NADPH, NADH, and NADP. + and NAD +One or more of them.

[0021] In another preferred embodiment, the acetaldehyde reductase coenzyme is NAD. + .

[0022] In another preferred embodiment, the acetaldehyde reductase coenzyme is obtained from a coenzyme regeneration system.

[0023] In another preferred embodiment, the coenzyme regeneration system is selected from a group or combination thereof:

[0024] (1) Formic acid or formate and formic acid dehydrogenase; (2) Glucose and glucose dehydrogenase; (3) Alcohol and alcohol dehydrogenase.

[0025] In another preferred embodiment, the coenzyme regeneration system is formic acid or formate and formic acid dehydrogenase.

[0026] In another preferred embodiment, the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO:11.

[0027] In another preferred embodiment, the nucleotide sequences of the L-threonine aldehyde transoxidase, formate dehydrogenase, and acetaldehyde reductase have all undergone codon optimization.

[0028] In another preferred embodiment, the L-threonine aldehyde transoxidase, formate dehydrogenase, and acetaldehyde reductase may be provided in any form known in the art;

[0029] For example, resting cells, bacterial cells, crude enzyme solution, pure enzyme, crude enzyme powder, immobilized enzyme, free enzyme, and fermentation broth.

[0030] In another preferred embodiment, the reaction solvent is water or a mixture of water and an organic solvent.

[0031] In another preferred embodiment, the concentration of compound 1 in the reaction is 100-500 mM, more preferably 200-400 mM.

[0032] In another preferred embodiment, the molar concentration ratio of L-threonine to compound 1 in the reaction is 1:1 to 2:1.

[0033] In another preferred embodiment, in the reaction, when the L-threonine transaldolase is calculated based on the mass of the wet bacterial cells used, the mass ratio of the L-threonine transaldolase wet bacterial cells to compound 1 is 1:(50-100), more preferably 1:(60-80).

[0034] In another preferred embodiment, the mass ratio of pyridoxal phosphate to compound 1 in the reaction is 1:10 to 1:1500, more preferably 1:100 to 1:1500.

[0035] In another preferred embodiment, in the reaction, when the acetaldehyde reductase is calculated based on the mass of the wet bacterial cells used, the mass ratio of the acetaldehyde reductase wet bacterial cells to compound 1 is 1:(100-600), more preferably 1:(200-400).

[0036] In another preferred embodiment, the NAD + The mass ratio of compound 1 to compound 1 is 1:(500-2000), preferably 1:(800-1500).

[0037] In another preferred embodiment, the mass ratio of the formate to compound 1 is 1:(1-10), more preferably 1:(1-5).

[0038] In another preferred embodiment, when the formate dehydrogenase is calculated based on the mass of the wet bacterial cells used, the mass ratio of the formate dehydrogenase wet bacterial cells to compound 1 is 1:(8-30), more preferably 1:(10-20), and even more preferably 1:(12-16).

[0039] In another preferred embodiment, in step (a), the pH of the reaction system is 5-9, preferably 6-7.

[0040] In another preferred embodiment, in step (a), the reaction temperature is 20-30°C, more preferably 20-25°C.

[0041] In another preferred embodiment, in step (a), the reaction time is 10-24 h, more preferably 15-20 h.

[0042] In another preferred embodiment, the pH is controlled by a buffer system, preferably a phosphate buffer system.

[0043] A second aspect of the present invention provides a method for preparing compound 3, comprising the steps of:

[0044]

[0045] (a) Compound 2 is decarboxylated using a decarboxylase in a reaction solvent to obtain compound 3;

[0046] (b) Optionally, compound 3 is isolated from the reaction system following step (a).

[0047] In another preferred embodiment, the solvent is water or a mixture of water and an organic solvent.

[0048] In another preferred embodiment, the decarboxylase is dopa decarboxylase.

[0049] In another preferred embodiment, the decarboxylase is derived from Drosophila melanogaster.

[0050] In another preferred embodiment, the amino acid sequence of the decarboxylase is shown in SEQ ID NO: 7.

[0051] In another preferred embodiment, the nucleotide sequence of the decarboxylase has been codon-optimized.

[0052] In another preferred embodiment, the decarboxylase may be provided in any form known in the art;

[0053] For example, resting cells, bacterial cells, crude enzyme solution, pure enzyme, crude enzyme powder, immobilized enzyme, free enzyme, and fermentation broth.

[0054] In another preferred embodiment, the initial concentration of compound 2 is 100-500 mM, more preferably 200-400 mM.

[0055] In another preferred embodiment, when the decarboxylase is calculated based on the mass of the wet bacterial cells used, the mass ratio of the decarboxylase wet bacterial cells to compound 2 is 1:(10-500), more preferably 1:(50-200).

[0056] In another preferred embodiment, the reaction conditions of step (a) include one or more of the following: the pH of the reaction system is 5-9, preferably 7-8; and / or the reaction time is 10-30 h, preferably 15-25 h.

[0057] In another preferred embodiment, compound 2 is obtained by the method described in the first aspect of the invention.

[0058] In another preferred embodiment, in step (b), the separation is performed by recrystallization, preferably using ethanol and water as the solvent.

[0059] In another preferred embodiment, in step (b), the product is precipitated by adjusting the pH of the reaction system, filtered, and optionally recrystallized to obtain the product.

[0060] In another preferred embodiment, in step (b), the separation includes: adjusting the pH of the reaction solution to between 8 and 11 (preferably 9 and 10), stirring, filtering, and recrystallizing to obtain the product.

[0061] In another preferred embodiment, in step (b), the ee value of compound 3 in the reaction system is ≥95%, preferably ≥99%.

[0062] A third aspect of this invention provides a method for preparing (R)-salbutamol, comprising the steps of:

[0063] (a) Compound 2 is prepared by the method described in the first aspect of the present invention, or compound 3 is prepared by the method described in the second aspect of the present invention; and

[0064] (b) The (R)-salbutamol was prepared by further reaction of compound 2 or compound 3;

[0065] (c) Optionally, (R)-salbutamol is isolated from the reaction system following step (b).

[0066] In another preferred embodiment, in step (b), compound 3 reacts with tert-butane chloride to generate (R)-salbutamol.

[0067] In another preferred embodiment, in step (c), the separation includes: after the reaction is completed, adjusting the pH of the reaction system to precipitate the product, filtering it, and optionally recrystallizing it to obtain high-purity (R)-salbutamol.

[0068] In another preferred embodiment, in step (c), the separation includes: adjusting the pH of the reaction solution to between 8 and 12 (preferably 10 and 11) to precipitate the product, filtering, and recrystallizing to obtain the product.

[0069] The fourth aspect of the present invention provides the application of L-threonine transaldehyde enzyme in the preparation of compound 2, compound 3, and (R)-salbutamol, wherein the L-threonine transaldehyde enzyme is the L-threonine transaldehyde enzyme described in the first aspect of the present invention.

[0070] The fifth aspect of the present invention provides the use of a decarboxylase in the preparation of compound 3, (R)-salbutamol, wherein the amino acid sequence of the decarboxylase is shown in SEQ ID NO: 7.

[0071] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0072] The main advantages of this invention are:

[0073] (1) This invention provides a novel method for generating compound 2 from L-threonine and compound 1 as raw materials under the catalysis of L-threonine aldehyde transoxidase, and for generating compound 3 from compound 2 under the catalysis of decarboxylase. Based on this, (R)-salbutamol is further synthesized using compound 3 as a substrate. The method of this invention has a high substrate conversion rate (over 95%) and a high product ee value. It also features low raw material costs, short process steps, simple operation, mild reaction conditions, and environmental friendliness.

[0074] (2) This invention provides a method for preparing (R)-salbutamol with an ee value greater than 99.0%, low cost of raw materials and auxiliary materials, short process steps, simple operation, mild reaction conditions, and environmental friendliness.

[0075] (3) In this invention, 4-hydroxy-3-hydroxymethylbenzaldehyde and L-threonine are used as raw materials to obtain compound 3: (R)-4-(2-amino-1-hydroxyethyl)-2-(hydroxymethyl)phenol by enzyme catalysis under the action of aldolase and decarboxylase. Compound 3 is separated and reacted with tert-butane chloride to obtain (R)-salbutamol. Detailed Implementation

[0076] Through extensive and in-depth research, the inventors have, for the first time, provided a novel method for the formation of compound 2 from L-threonine and compound 1 using L-threonine aldehyde transoxidase, and the subsequent formation of compound 3 from compound 2 under the catalysis of decarboxylase. Based on this method, (R)-salbutamol is further synthesized using compound 3 as a substrate. The method of this invention boasts high conversion rates (over 95%) and ee values, and features low raw material costs, short process steps, simple operation, mild reaction conditions, and environmental friendliness. This invention is based on these findings.

[0077] L-Threonine Aldolasterase

[0078] As used in this article, L-Threonine Transaldolase (LTTA) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that uses L-threonine and aldehydes as substrates to generate β-hydroxy-α-amino acids through reverse aldolase cleavage and aldol addition reactions.

[0079] In this invention, the L-threonine transaldolase can be wild-type or mutant. Furthermore, it can be isolated or recombinant.

[0080] The amino acid sequence of a typical L-threonine transaldolase is shown in any of SEQ ID NO:3-6.

[0081] In this invention, the nucleotide sequence of the L-threonine transaldolase has been optimized using E. coli codons.

[0082] Codon degeneracy refers to the phenomenon that the same amino acid is encoded by multiple codons. Codon optimization is the process of replacing degenerate codons to adapt to the host expression system without changing the encoded amino acid sequence. The deletion or addition of nucleotides usually changes the amino acid sequence.

[0083] The L-threonine transaldolase of the present invention further includes the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences shown in SEQ ID NO: 3-6 within the range of maintaining enzyme activity.

[0084] Based on common knowledge in this field, L-threonine transaldolase can be used in the reaction system in the form of recombinant enzyme resting cells, wet bacterial cells, crude enzyme solution, pure enzyme, or crude enzyme powder, etc.

[0085] decarboxylase

[0086] As used in this article, decarboxylases are a class of enzymes that catalyze the removal of carboxyl groups from organic compounds in the form of CO2. They are widely distributed in living organisms and participate in a variety of important metabolic processes.

[0087] In this invention, the decarboxylase can be wild-type or mutant. Furthermore, it can be isolated or recombinant.

[0088] In a preferred embodiment, the decarboxylase is dopa decarboxylase.

[0089] In a preferred embodiment, the decarboxylase is derived from Drosophila melanogaster.

[0090] The amino acid sequence of a typical decarboxylase is shown in SEQ ID NO: 7. Its encoding gene is shown in SEQ ID NO: 8.

[0091] In this invention, the nucleotide of the decarboxylase is a codon-optimized nucleotide.

[0092] Codon degeneracy refers to the phenomenon that the same amino acid is encoded by multiple codons. Codon optimization is the process of replacing degenerate codons to adapt to the host expression system while keeping the encoded amino acid sequence unchanged.

[0093] The decarboxylase 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: 7 within the range of maintaining enzyme activity.

[0094] Based on common knowledge in this field, wet bacterial cells, crude enzyme solutions, pure enzymes, or crude enzyme powders constructed using the above-mentioned decarboxylases can be used in the reaction system.

[0095] acetaldehyde reductase

[0096] As used in this article, acetaldehyde reductase (GR) belongs to the class of oxidoreductases and has the ability to catalyze the reduction of acetaldehyde to ethanol.

[0097] In this invention, the acetaldehyde reductase can be wild-type or mutant. Furthermore, it can be isolated or recombinant.

[0098] The amino acid sequence of a typical acetaldehyde reductase is shown in SEQ ID NO: 9. Its encoding gene is shown in SEQ ID NO: 10.

[0099] In this invention, the nucleotide sequence of the acetaldehyde reductase has been optimized using E. coli codons.

[0100] Those skilled in the art can obtain mutants of the acetaldehyde reductase nucleotides by appropriately introducing substitutions, deletions, or additions. This invention covers such mutants as long as they retain the activity of converting the byproduct acetaldehyde to ethanol and promoting the forward reaction. While maintaining enzyme activity, the mutants in this invention can be prepared by substituting, deleting, or adding one or more bases to the acetaldehyde reductase nucleotides.

[0101] The acetaldehyde reductase of the present invention also includes an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 9 while maintaining the enzyme activity range.

[0102] Based on common knowledge in this field, the acetaldehyde reductase used in the reaction system can be in the form of: recombinant enzyme resting cells, wet bacterial cells, crude enzyme solution, pure enzyme, or crude enzyme powder, etc.

[0103] Coenzyme

[0104] In this invention, "coenzyme" is an auxiliary factor for enzyme-catalyzed redox reactions and group transfer.

[0105] Typically, in the reaction of compound 1 and L-threonine catalyzed by the aldehyde transoxidase in this invention, the coenzyme is pyridoxal phosphate.

[0106] Typically, in the acetaldehyde reductase-catalyzed reaction of acetaldehyde in this invention, the coenzyme is the reducing coenzyme NADH, NADPH and / or the oxidizing coenzyme NAD. + NADP + .

[0107] In a preferred embodiment, the coenzyme is NAD. + The coenzyme regeneration system consists of formate and formate dehydrogenase. In this invention, formate dehydrogenase and sodium formate dihydrate as co-substrate are preferred.

[0108] In this invention, the formate dehydrogenase can be wild-type or mutant. Furthermore, it can be isolated or recombinant.

[0109] The amino acid sequence of a typical formate dehydrogenase is shown in SEQ ID NO:11. Its encoding gene is shown in SEQ ID NO:12.

[0110] In this invention, the nucleotide of the formate dehydrogenase is a codon-optimized nucleotide.

[0111] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0112] Unless otherwise specified, the reagents and materials used in the embodiments of this invention are all commercially available products.

[0113] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl; adjust the pH to 7.2 with 1 mol / L NaOH.

[0114] LB agar plate: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 18 g / L. pH value 7.2 (adjusted with 1 mol / L NaOH).

[0115] TB liquid culture medium: tryptone 10 g / L, yeast extract 18 g / L, potassium dihydrogen phosphate (KH2PO4) 2.31 g / L, dipotassium hydrogen phosphate (K2HPO4) 16.43 g / L, glycerol 0.4% (v / v).

[0116] Substrate conversion was determined by HPLC, as detailed below:

[0117] Molar yield = (Theoretical moles of product / Actual moles of product obtained) × 100%

[0118] Conversion rate = (1 - number of unreacted reactant moles / initial number of reactant moles) × 100%

[0119] ee=([R]-[S] / [R]+[S])×100%=(A [R] -A [S] / A [R] +A [S] )×100%, where A [R] A [S] These are the peak areas for the R and S configurations, respectively.

[0120] Example 1: Preparation of L-threonine transaldase

[0121] L-threonine transaldolase was selected from the company's enzyme library, and its amino acid sequence is shown in Table 1. It was obtained by mutagenesis using conventional methods in the field, based on the L-threonine transaldolase derived from Pseudomonas sp. SK with NCBI accession number WP_170033327.1 (amino acid sequence shown in SEQ ID NO:1, nucleotide sequence shown in SEQ ID NO:2). The codons of the L-threonine transaldolase were optimized, and the coding gene was synthesized. The synthesized coding gene was cloned into the NdeI / BamHI restriction sites of the expression vector pET28a (purchased from Anolun (Beijing) Biotechnology Co., Ltd.) to obtain a recombinant plasmid containing the L-threonine transaldolase gene.

[0122] The synthesized recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells (purchased from Anolun (Beijing) Biotechnology Co., Ltd.) and cultured overnight at 37°C on LB agar plates containing 50 μg / mL kanamycin until single colonies of recombinant Escherichia coli formed on the LB agar plates.

[0123] Single colonies of recombinant *Escherichia coli* were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for 5 h. The colonies were then transferred to TB liquid medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculation rate and cultured at 37°C and 220 rpm until OD500 reached. 600 At a concentration of approximately 0.4–0.6, add IPTG to a final concentration of 0.1 mM and induce culture at 25 °C for 16 h. After culture, centrifuge at 4000 rpm for 20 min, discard the supernatant, and obtain wet cells of L-threonine transaldase.

[0124] Table 1 L-Threonine Aldolasterase Sequence

[0125] Enzyme number amino acid sequence Enz.1 SEQ ID NO:3 Enz.2 SEQ ID NO:4 Enz.3 SEQ ID NO:5 Enz.4 SEQ ID NO:6

[0126] Example 2: Preparation of decarboxylase, acetaldehyde reductase, and formate dehydrogenase

[0127] The preparation of decarboxylase wet cells, acetaldehyde reductase wet cells, and formate dehydrogenase wet cells was performed in accordance with Example 1, which focused on the preparation of L-threonine transaldehydease wet cells. The only difference was the amino acid sequences of the decarboxylase, acetaldehyde reductase, and formate dehydrogenase. Specifically, the amino acid sequence of the decarboxylase is shown in SEQ ID NO:7, and the nucleotide sequence is shown in SEQ ID NO:8; the amino acid sequence of the acetaldehyde reductase is shown in SEQ ID NO:9, and the nucleotide sequence is shown in SEQ ID NO:10; and the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO:11, and the nucleotide sequence is shown in SEQ ID NO:12.

[0128] Example 3: Preparation of (R)-4-(2-amino-1-hydroxyethyl)-2-(hydroxymethyl)phenol

[0129] 4-Hydroxy-3-hydroxymethylbenzaldehyde and L-threonine react with L-threonine aldehyde transoxidase to form compound 2 and the byproduct acetaldehyde. Compound 2 then reacts with decarboxylase to form compound 3, namely (R)-4-(2-amino-1-hydroxyethyl)-2-(hydroxymethyl)phenol. The byproduct acetaldehyde is then converted to ethanol by NADH and acetaldehyde reductase, thus promoting the forward reaction.

[0130]

[0131] (1) Preparation of compound 2: 15.2 g (0.10 mol) of 4-hydroxy-3-hydroxymethylbenzaldehyde (MV: 152.14), 13.1 g (0.11 mol) of L-threonine (MV: 119.12), 10.2 g (0.15 mol) of sodium formate dihydrate, and 300 mL of 100 mM phosphate buffer (pH 6.5) were added sequentially to a 500 mL reaction flask. The reaction system was stirred evenly at 22–25 °C. Then, 15 mg of pyridoxal phosphate and 15 mg of NAD were added. + 200 mg of L-threonine aldehyde transoxidase wet cells, 1 g of formate dehydrogenase wet cells, and 50 mg of acetaldehyde reductase wet cells were added. The pH of the reaction system was controlled between 6.0 and 6.5 using 15% liquid alkali and 20% phosphoric acid. The reaction was stopped after 16 h to obtain a reaction solution containing compound 2. Samples were taken for HPLC analysis. The conversion rate of 4-hydroxy-3-hydroxymethylbenzaldehyde is shown in Table 2.

[0132] (2) Preparation of compound 3: 200 mg of decarboxylase wet cells were added to the reaction solution containing compound 2 obtained in (1), and the reaction was carried out at pH 7.0-7.5 for 20 h to obtain the reaction solution containing compound 3. The conversion rate of compound 2 was more than 95% when sampled and detected by HPLC.

[0133] The pH of the reaction solution containing compound 3 obtained in (2) was adjusted to between 9.5 and 10.0. After stirring for 1 hour, the mixture was filtered and a light yellow solid was collected. The light yellow solid product was recrystallized with ethanol / water to obtain a white solid. The ee value of the white solid was measured after drying and is shown in Table 2.

[0134] Table 2. Conversion rates of different L-threonine aldehyde transoxidases and ee values ​​of compound 3.

[0135] Enzyme number Conversion rate ee value of compound 3 Enz.1 96.8％ 99.5％ Enz.2 96.9％ 99.2％ Enz.3 97.1％ 99.4％ Enz.4 98.5％ 99.5％

[0136] Example 4: Preparation of (R)-salbutamol from Compound 3 as a substrate

[0137]

[0138] In a 500 mL reaction flask, 36.6 g of compound 3, 0.4 g of cuprous chloride, 200 mL of water, and 200 mL of ethanol were added sequentially. The mixture was stirred until homogeneous, and the temperature was controlled at 0–5 °C. 20.4 g of tert-butane chloride was added dropwise, and the mixture was kept at this temperature for 2 h. The ethanol was then distilled under reduced pressure, and 200 mL of water was added. The pH of the reaction solution was adjusted to between 10.5 and 11.0 with 30% alkali solution. The mixture was filtered to obtain an off-white solid, which was then recrystallized from the off-white solid with ethanol / water to obtain a white solid. After drying, 40.9 g of compound 4 was obtained, with a molar yield of 85.4%, an ee value of 99.8%, and a purity of 99.7%.

[0139] Sequence information:

[0140] SEQ ID NO:1

[0141] MNQDKHNAGPLIEWLAQTLDQDYQYRQDTLSLTANENYPSELVRLTSGSTAGA

[0142] FYHCSFPFPVPLGEWHFPEPGQMNEIADDLRGLARRMLGAQAFDWRPNGGSPA

[0143] EQALMLAACKPGEGFVHFAHRDGGHFALEQLASKMGIEIFHLPVDPRSLLIDVA

[0144] KLDDMVRRNPHIRIVILDQSFKLRWQPLAEIRAVLPESCALTYDMSHDGGLILG

[0145] GVFDSPLACGADAVHGNTHKTIPGPQKGYIAFKSAQHPLLVDTSMWVCPHLQS

[0146] NCHAELLPSMWAAFKEMEVFGMAYAQQMVKNAKSLAQQLHELGLEVSGESF

[0147] GFTETHQVHFAVGTLQQALAMCVDSLHAGGIRSTNIEIPGKPGVHGIRLGVQAM

[0148] TRRGMREDDFRQVAGLVADLYFKRTEPARVANRVKELLAGFPLAPLAYSFDGQ

[0149] IDEAHRHLIERGIQR

[0150] SEQ ID NO:2

[0151] ATGAACCAGGACAAACACAACGCAGGTCCGCTGATTGAATGGCTGGCGCAG

[0152] ACCCTGGATCAGGATTATCAGTACCGTCAGGATACCCTGTCTCTGACCGCGA

[0153] ACGAAAACTATCCGTCTGAACTGGTGCGTCTGACCAGCGGCAGCACCGCTG

[0154] GCGCGTTCTATCACTGCTCTTTCCCGTTCCCGGTTCCGCTGGGCGAATGGCA

[0155] CTTCCCGGAACCGGGCCAGATGAACGAAATCGCGGATGACCTGCGTGGTCT

[0156] GGCGCGTCGTATGCTGGGTGCTCAGGCGTTCGATTGGCGTCCGAACGGTGGT

[0157] AGCCCGGCGGAACAGGCACTGATGCTGGCGGCGTGCAAACCGGGCGAAGGT

[0158] TTCGTTCACTTCGCGCACCGCGATGGCGGTCACTTCGCGCTGGAACAGCTGG

[0159] CGTCTAAAATGGGTATCGAAATCTTCCACCTGCCGGTTGATCCGCGTAGCCT

[0160] GCTGATTGACGTTGCGAAACTGGATGATATGGTTCGTCGTAACCCGCACATC

[0161] CGTATCGTGATCCTGGATCAGAGCTTCAAACTGCGTTGGCAGCCGCTGGCGG

[0162] AAATCCGTGCAGTTCTGCCGGAATCCTGCGCTCTGACCTATGATATGAGCCA

[0163] CGACGGTGGCCTGATCCTGGGTGGTGTTTTCGATAGCCCGCTGGCGTGCGGT

[0164] GCAGACGCTGTTCACGGTAACACCCACAAAACCATCCCAGGCCCGCAGAAA

[0165] GGCTATATCGCTTTCAAATCTGCGCAGCACCCGCTGCTGGTTGATACCTCTA

[0166] TGTGGGTTTGCCCGCACCTGCAATCTAACTGTCACGCGGAACTGCTGCCGTC

[0167] TATGTGGGCGGCTTTCAAAGAAATGGAAGTTTTCGGCATGGCGTACGCTCAG

[0168] CAGATGGTTAAAAACGCTAAAAGCCTGGCGCAGCAGCTGCATGAACTGGGC

[0169] CTGGAAGTGTCCGGCGAATCCTTCGGCTTCACCGAAACCCACCAGGTGCACT

[0170] TCGCGGTGGGCACCCTCCAGCAGGCGCTGGCAATGTGCGTTGATTCCCTGCA

[0171] CGCTGGCGGCATCCGTAGCACCAACATCGAGATCCCAGGCAAACCGGGCGT

[0172] GCATGGTATCCGTCTGGGTGTTCAGGCAATGACCCGCCGCGGTATGCGTGAA

[0173] GATGATTTCCGCCAGGTTGCGGGTCTGGTGGCGGACCTGTATTTCAAACGCA

[0174] CCGAACCGGCGCGTGTGGCGAACCGTGTGAAAGAACTGCTGGCGGGCTTCC

[0175] CGCTGGCGCCGCTGGCGTACTCATTCGATGGCCAGATCGACGAAGCGCACC

[0176] GTCACCTGATCGAACGTGGCATCCAGCGT

[0177] SEQ ID NO:3

[0178] MNQDKHNAGPLIEWLAQTLDQDYQYRQDTLSLTASENYPSELVRLTSGSTAGA

[0179] FYHSSFPFPVPLGEWHHPEPGQMNEIADDLRGLARRMLGAQAFDWRPNGGSPA

[0180] EQALMLAACKPGEGFVHFAHRDGGHFALEQLASKCGIEIFHLPVDPRSLLIDVA

[0181] KLDDMVRRNPHIRIVILDQSFKLRWQPLAEIRAVLPESCALTYDMSHDGGLILG

[0182] GVFDSPLACGADAVHGNTHKTIPGPQKGYIAFKSAQHPLLVDTSMWVCPHLQS

[0183] NCHAELLPSMWAAFKEMEVFGMAYAQQMVKNAKSLAQQLHELGLEVSGESF

[0184] GFTETHQVHFAVGTLQQALAMCVDSLHAGGIRSTNIEIPGKPGVHGIRLGVQAM

[0185] TRRGMREDDFRQVAGLVADLYFKRTEPARVANRVKELLAGFPLAPLAYSFDGQ

[0186] IDEAHRHLIERGIQR

[0187] SEQ ID NO:4

[0188] MNQDKHNAGPLIEWLAQTLDQDYQYRQDTLSLTASENYPSELVRLTSGSTAGA

[0189] FYHMSFPFPPLGEWHHPEPGQMNEIADDLRGLARRMLGAQAFDWRPNGGSP

[0190] AEQALMLAACKPGEGFVHFAHRDGGHFALEQLASKCGIEIFHLPVDPRSLLIDV

[0191] AKLDDMVRRNPHIRIVILDQSFKLRWQPLAEIRAVLPESCALTYDMSHDGGLIL

[0192] GGVFDSPLACGADAVHGNTHKTIPGPQKGYIAFKSAQHPLLVDTSMWVCPHLQ

[0193] SNCHAELLPSMWAAFKEMEVFGMAYAQQMVKNAKSLAQQLHELGLEVSGES

[0194] FGFTETHQVHFAVGTLQQALAMCVDSLHAGGIRSTNIEIPGKPGVHGIRLGVQA

[0195] MTRRGMREDDFRQVAGLVADLYFKRTEPARVANRVKELLAGFPLAPLAYSFD

[0196] GQIDEAHRHLIERGIQR

[0197] SEQ ID NO:5

[0198] MNQDKHNAGPLIEWLAQTLDQDYQYRQDTLSLTASENYPSELVRLTSGSTAGA

[0199] FYHCSTPFPVPLGEWHHPEPGQMNEIADDLRGLARRMLGAQAFDWRPNGGSPA

[0200] EQALMLAACKPGEGFVHFAHRDGGHFALEQLASKCGIEIFHLPVDPRSLLIDVA

[0201] KLDDMVRRNPHIRIVILDQSFKLRWQPLAEIRAVLPESCALTYDMSHDGGLILG

[0202] GVFDSPLACGADAVHGNTHKTIPGPQKGYIAFKSAQHPLLVDTSMWVCPHLQS

[0203] NCHAELLPSMWAAFKEMEVFGMAYAQQMVKNAKSLAQQLHELGLEVSGESF

[0204] GFTETHQVHFAVGTLQQALAMCVDSLHAGGIRSTNIEIPGKPGVHGIRLGVQAM

[0205] TRRGMREDDFRQVAGLVADLYFKRTEPARVANRVKELLAGFPLAPLAYSFDGQ

[0206] IDEAHRHLIERGIQR

[0207] SEQ ID NO:6

[0208] MNQDKHNAGPLIEWLAQTLDQDYQYRQDTLSLTASENYPSELVRLTSGSTAGA

[0209] FYHMSPPFPVPLGEWHHPEPGQMNEIADDLRGLARRMLGAQAFDWRPNGGSP

[0210] AEQALMLAACKPGEGFVHFAHRDGGHFALEQLASKCGIEIFHLPVDPRSLLIDV

[0211] AKLDMMVRRNPHIRIVILDQSFKLRWQPLAEIRAVLPESCALTYDMSHDGGLIL

[0212] GGVFDSPLACGADAVHGNTHKTIPGPQKGYIAFKSAQHPLLVDTSMWVCPHLQ

[0213] SNCHAELLPSMWAAFKEMEVFGMAYAQQMVKNAKSLAQQLHELGLEVSGES

[0214] FGFTETHQVHFAVGTLQQALAMCVDSLHAGGIRSTNIEIPGKPGVHGIRLGVQA

[0215] MTRRGMREDDFRQVAGLVADLYFKRTEPARVANRVKELLAGFPLAPLAYSFD

[0216] GQIDEAHRHLIERGIQR

[0217] SEQ ID NO:7

[0218] MSHIPISNTIPTKQTDGNGKANISPDKLDPKVSIDMEAPEFKDFAKTMVDFIAEY

[0219] LENIRERRVLPEVKPGYLKPLIPDAAPEKPEKWQDVMQDIERVIMPGVTHWHSP

[0220] KFHAYFPTANSYPAIVADMLSGAIACIGFTWIASPACTELEVVMMDWLGKMLE

[0221] LPAEFLACSGGKGGGVIQGTASESTLVALLGAKAKKLKEVKELHPEWDEHTILG

[0222] KLVGYCSDQAHSSVERAGLLGGVKLRSVQSENHRMRGAALEKAIEQDVAEGLI

[0223] PFYAVVTLGTTNSCAFDYLDECGPVGNKHNLWIHVDAAYAGSAFICPEYRHLM

[0224] KGIESADSFNFNPHKWMLVNFDCSAMWLKDPSWVVNAFNVDPLYLKHDMQG

[0225] SAPDYRHWQIPLGRRFRALKLWFVLRLYGVENLQAHIRRHCNFAKQFGDLCVA

[0226] DSRFELAAEINMGLVCFRLKGSNERNEALLKRINGRGHIHLVPAKIKDVYFLRM

[0227] AICSRFTQSEDMEYSWKEVSAADEMEQEQ

[0228] SEQ ID NO:8

[0229] ATGAGCCATATCCCTATCAGCAACACCATCCCGACCAAACAGACCGATGGT

[0230] AATGGCAAAGCCAACATTAGCCCGGATAAGCTGGACCCGAAAGTTAGTATT

[0231] GACATGGAGGCCCCGGAGTTTAAAGATTTTGCCAAAACAATGGTTGACTTC

[0232] ATCGCAGAGTACCTGGAAAACATCCGTGAACGCCGTGTTCTGCCTGAAGTTA

[0233] AACCGGGTTATCTGAAGCCGCTGATTCCTGATGCCGCACCTGAGAAGCCTGA

[0234] AAAATGGCAAGATGTGATGCAGGACATCGAGCGTGTTATTATGCCGGGTGT

[0235] GACCCATTGGCATAGCCCTAAGTTTCATGCCTACTTCCCGACCGCAAATAGT

[0236] TATCCGGCCATTGTGGCCGATATGCTGTCAGGTGCAATTGCATGCATCGGTT

[0237] TTACCTGGATCGCAAGCCCTGCATGTACCGAGTTAGAAGTTGTGATGATGGA

[0238] CTGGCTGGGTAAAATGCTGGAACTGCCTGCTGAATTTCTGGCCTGTAGTGGT

[0239] GGTAAAGGTGGCGGTGTGATTCAGGGTACAGCTTCCGAGAGTACCCTGGTG

[0240] GCATTATTAGGTGCAAAGGCCAAAAAGCTGAAGGAGGTTAAGGAGCTGCAC

[0241] CCTGAATGGGATGAACATACCATTCTGGGTAAACTGGTGGGCTATTGTAGCG

[0242] ATCAGGCCCACAGCTCCGTTGAGCGTGCAGGTCTGCTGGGTGGTGTGAAGCT

[0243] GCGTTCCGTGCAGTCCGAGAACCACCGTATGCGTGGTGCAGCACTGGAGAA

[0244] GGCAATTGAACAGGATGTGGCAGAAGGTCTGATCCCATTCTATGCAGTGGT

[0245] GACCCTGGGTACTACCAACTCCTGCGCTTTTGATTACCTGGATGAGTGTGGC

[0246] CCAGTGGGTAATAAGCACAATCTGTGGATTCACGTGGACGCCGCATACGCA

[0247] GGTTCCGCATTCATCTGCCCAGAATATCGCCACCTGATGAAGGGTATTGAGT

[0248] CCGCAGATTCCTTCAACTTCAACCCACATAAGTGGATGCTGGTGAACTTCGA

[0249] TTGCTCTGCAATGTGGCTGAAGGACCCATCATGGGTTGTGAATGCATTCAAC

[0250] GTGGACCCACTGTACCTGAAACATGATATGCAGGGTAGCGCACCAGATTAT

[0251] CGCCATTGGCAAATTCCACTGGGCCGTCGTTTCCGTGCACTGAAGCTGTGGT

[0252] TTGTGCTGCGTTTGTACGGCGTTGAAAACCTGCAGGCTCATATCCGTCGTCA

[0253] TTGCAATTTCGCCAAGCAGTTCGGTGACCTGTGTGTTGCAGATTCTCGCTTTG

[0254] AACTGGCAGCCGAAATCAACATGGGTCTGGTTTGCTTTCGCCTGAAAGGTTC

[0255] TAACGAGCGCAACGAAGCCCTGCTGAAACGTATTAACGGTCGTGGTCACAT

[0256] CCACCTGGTGCCAGCAAAGATTAAGGATGTGTACTTCCTGCGTATGGCCATC

[0257] TGCTCACGTTTTACCCAGTCTGAAGACATGGAGTACAGCTGGAAGGAAGTG

[0258] TCTGCAGCTGCTGATGAAATGGAACAGGAACAG

[0259] SEQ ID NO:9

[0260] MKAAVVTKDHHVDVTYKTLRSLKHGEALLKMECCGVCHTDLHVKNGDFGDK

[0261] TGVILGHEGIGVVAEVGPGVTSLKPGDRASVAWFYEGCGHCEYCNSGNETLCR

[0262] SVKNAGYSVDGGMAEECIVVADYAVKVPDGLDSAAASSITCAGVTTYKAVKL

[0263] SKIRPGQWIAIYGLGGLGNLALQYAKNVFNAKVIAIDVNDEQLKLATEMGADL

[0264] AINSHTEDAAKIVQEKTGGAHAAVVTAVAKAAFNSAVDAVRAGGRVVAVGLP

[0265] PESMSLDIPRLVLDGIEVVGSLVGTRQDLTEAFQFAAEGKVVPKVALRPLADINT

[0266] IFTEMEEGKIRGRMVIDFRH

[0267] SEQ ID NO:10

[0268] ATGAAGGCTGCAGTTGTTACGAAGGATCATCATGTTGACGTTACGTATAAAA

[0269] CACTGCGCTCACTGAAACATGGCGAAGCCCTGCTGAAAATGGAGTGTTGTG

[0270] GTGTATGTCATACCGATCTTCATGTTAAGAATGGCGATTTTGGTGACAAAAC

[0271] CGGCGTAATTCTGGGCCATGAAGGCATCGGTGTGGTGGCAGAAGTGGGTCC

[0272] AGGTGTCACCTCATTAAAACCAGGCGATCGTGCCAGCGTGGCGTGGTTCTAC

[0273] GAAGGATGCGGTCATTGCGAATACTGTAACAGTGGTAACGAAACGCTCTGC

[0274] CGTTCAGTTAAAAATGCCGGATACAGCGTTGATGGCGGGATGGCGGAAGAG

[0275] TGCATCGTGGTCGCCGATTACGCGGTAAAAGTGCCAGATGGTCTGGACTCG

[0276] GCGGCGGCCAGCAGCATTACCTGTGCGGGAGTCACCACCTACAAAGCCGTT

[0277] AAGCTGTCAAAAATTCGTCCAGGGCAGTGGATTGCTATCTACGGTCTTGGCG

[0278] GTCTGGGTAACCTCGCCCTGCAATACGCGAAGAATGTCTTTAACGCCAAAGT

[0279] GATCGCCATTGATGTCAATGATGAGCAGTTAAAACTGGCAACCGAAATGGG

[0280] CGCAGATTTAGCGATTAACTCACACACCGAAGACGCCGCCAAAATTGTGCA

[0281] GGAGAAAACTGGTGGCGCTCACGCTGCGGTGGTAACAGCGGTAGCTAAAGC

[0282] TGCGTTTAACTCGGCAGTTGATGCTGTCCGTGCAGGCGGTCGTGTTGTGGCT

[0283] GTCGGTCTACCGCCGGAGTCTATGAGCCTGGATATCCCACGTCTTGTGCTGG

[0284] ATGGTATTGAAGTGGTCGGTTCGCTGGTCGGCACGCGCCAGGATTTAACTGA

[0285] AGCCTTCCAGTTTGCCGCCGAAGGTAAAGTGGTGCCGAAAGTCGCCCTGCGT

[0286] CCGTTAGCGGACATCAACACCATCTTTACTGAGATGGAAGAAGGCAAAATC

[0287] CGTGGCCGCATGGTGATTGATTTCCGTCAC

[0288] SEQ ID NO:11

[0289] MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETSE

[0290] LDKHIPDADIIITTPFHPAYITKERLDKAKNLKLVVVAGVGSDHIDLDYINQTGK

[0291] KISVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYD

[0292] IEGKTIATIGAGRIGYRVLERLLPFNPKELLYYDYQALPKEAEEKVGARRVENIE

[0293] ELVAQADIVTVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAA

[0294] ALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQ

[0295] TRYAEGTKNILESFFTGKFDYRPQDIILLNGEYVTKAYGKHDKK

[0296] SEQ ID NO:12

[0297] ATGAAAATCGTTCTGGTTCTGTATGATGCGGGTAAACACGCGGCGGATGAA

[0298] GAAAAACTGTACGGCTGTACCGAAAACAAACTGGGCATCGCGAACTGGCTG

[0299] AAAGATCAGGGCCACGAACTGATCACCACCTCTGACAAAGAAGGTGAAACC

[0300] AGCGAACTGGATAAACACATCCCGGATGCTGATATCATCATCACCACCCCGT

[0301] TCCATCCGGCTTACATTACCAAAGAACGTCTGGACAAAGCAAAAAACCTGA

[0302] AACTGGTTGTTGTTGCGGGTGTTGGCAGCGACCACATCGATCTGGATTACAT

[0303] CAACCAGACCGGTAAAAAGATCAGCGTTCTGGAAGTTACCGGCTCTAACGT

[0304] GGTTAGCGTGGCGGAACACGTTGTTATGACCATGCTGGTTCTGGTTCGTAAC

[0305] TTCGTGCCGGCGCACGAACAGATCATCAACCACGATTGGGAAGTTGCGGCA

[0306] ATCGCGAAAGATGCGTACGATATTGAAGGCAAAACCATTGCGACCATCGGT

[0307] GCAGGTCGTATCGGCTACCGTGTGCTGGAACGTCTGCTGCCGTTCAACCCGA

[0308] AAGAACTGCTGTACTACGACTACCAGGCGCTGCCGAAAGAAGCGGAAGAAA

[0309] AAGTTGGCGCGCGTCGTGTTGAAAACATCGAAGAACTGGTTGCACAGGCGG

[0310] ACATTGTGACCGTTAACGCGCCGCTGCACGCAGGCACCAAAGGCCTGATCA

[0311] ACAAAGAACTGCTGTCTAAATTCAAAAAAGGTGCGTGGCTGGTGAACACCG

[0312] CGCGTGGCGCAATCTGCGTTGCGGAGGACGTTGCAGCTGCGCTGGAATCCG

[0313] GCCAGCTGCGTGGTTACGGCGGCGATGTGTGGTTCCCGCAGCCGGCGCCGA

[0314] AAGATCACCCGTGGCGTGACATGCGTAACAAATACGGCGCTGGCAACGCTA

[0315] TGACCCCGCACTATTCCGGTACCACCCTGGATGCGCAGACCCGTTTACGCGGA

[0316] AGGTACCAAAAACATCCTGGAATCTTTCTTCACCGGTAAATTCGATTACCGT

[0317] CCGCAGGATATCATCCTGCTGAACGGCGAATACGTGACCAAAGCGTACGGC

[0318] AAACACGATAAAAAA

[0319] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing compound 2, characterized in that, Including the following steps: (a) In the reaction solvent, L-threonine aldehyde transoxidase is used as a catalyst and pyridoxal phosphate is used as a coenzyme to catalyze the reaction of L-threonine with compound 1 to form compound 2.

2. The preparation method according to claim 1, characterized in that, The L-threonine transaldolase is selected from the following group: (a) A polypeptide with an amino acid sequence as shown in any of SEQ ID NO: 3-6; (b) A polypeptide having the polypeptide function described in (a) formed by replacing, deleting or adding one or more amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residue, of any of the amino acid sequences shown in SEQ ID NO: 3-6.

3. The preparation method according to claim 1 or 2, characterized in that, The method further includes removing acetaldehyde generated in reaction step (a) using acetaldehyde reductase and acetaldehyde reductase coenzyme; wherein the acetaldehyde reductase coenzyme is selected from NADPH, NADH, and NADP. + and NAD + One or more of the following; Preferably, the amino acid sequence of the acetaldehyde reductase is shown in SEQ ID NO:

9.

4. The preparation method according to claim 3, characterized in that, The acetaldehyde reductase coenzyme is obtained from a coenzyme regeneration system, and the coenzyme regeneration system is selected from one or a combination of the following: (1) Formic acid or formate and formic acid dehydrogenase; (2) Glucose and glucose dehydrogenase; (3) Alcohol and alcohol dehydrogenase; Preferably, the amino acid sequence of the formate dehydrogenase is as shown in SEQ ID NO:

11.

5. The preparation method according to any one of claims 1-4, characterized in that, In the reaction solvent, the solvent is water or a mixture of water and an organic solvent; And / or, the initial concentration of compound 1 is 100-500 mM, preferably 200-400 mM; And / or, the initial molar concentration ratio of the L-threonine to compound 1 is 1:1 to 2:1; And / or, when the L-threonine transaldolase is calculated based on the mass of the wet bacterial cells used, the mass ratio of the L-threonine transaldolase wet bacterial cells to compound 1 is 1:(50-100), preferably 1:(60-80). And / or, the mass ratio of pyridoxal phosphate to compound 1 is 1:10 to 1:1500, preferably 1:100 to 1:1500; And / or, when the acetaldehyde reductase is calculated based on the mass of the wet bacterial cells used, the mass ratio of the acetaldehyde reductase wet bacterial cells to compound 1 is 1:(100-600), preferably 1:(200-400); And / or, the NAD + The mass ratio of compound 1 to compound 1 is 1:(500-2000), preferably 1:(800-1500); And / or, the mass ratio of the formate to compound 1 is 1:(1-10), preferably 1:(1-5); And / or, when the formate dehydrogenase is calculated based on the mass of the wet bacterial cells used, the mass ratio of the formate dehydrogenase wet bacterial cells to compound 1 is 1:(8-30), preferably 1:(10-20).

6. The preparation method according to any one of claims 1-5, characterized in that, The reaction conditions include one or more of the following: pH 5-9, preferably 6-7; reaction temperature 20-30℃, preferably 20-25℃; and reaction time 10-24h, preferably 15-20h.

7. A method for preparing compound 3, characterized in that, Including the following steps: (a) Compound 2 is decarboxylated using a decarboxylase in a reaction solvent to obtain compound 3; (b) Optionally, compound 3 is isolated from the reaction system following step (a).

8. The preparation method according to claim 7, characterized in that, The amino acid sequence of the decarboxylase is shown in SEQ ID NO: 7; And / or, the solvent is water or a mixture of water and an organic solvent; And / or, the initial concentration of compound 2 is 100-500 mM, preferably 200-400 mM; And / or, when the decarboxylase is calculated based on the mass of the wet bacterial cells used, the mass ratio of the decarboxylase wet bacterial cells to compound 2 is 1:(10-500), preferably 1:(50-200).

9. The preparation method according to claim 8, characterized in that, The reaction conditions for step (a) include one or more of the following: the pH of the reaction system is 5-9, preferably 7-8; and / or the reaction time is 10-30 h, preferably 15-25 h.

10. The preparation method according to any one of claims 8-9, characterized in that, Compound 2 is obtained by the method described in any one of claims 1-6.

11. A method for preparing (R)-salbutamol, characterized in that, Including the following steps: (a) Compound 2 is prepared by the method according to any one of claims 1-6, or compound 3 is prepared by the method according to any one of claims 7-10; (b) The (R)-salbutamol was prepared by further reaction of compound 2 or compound 3; (c) Optionally, (R)-salbutamol is isolated from the reaction system following step (b).

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